Introduction 1

1
INTRODUCTION

Food microbiology is the study of the microorganisms which

inhabit, create or contaminate food. Of major importance is the
study of microorganisms causing food spoilage. However "good"
bacteria such as probiotics are becoming increasingly important
in food science. In addition, microorganisms are vital for the
manufacture of cheese, yoghurt, other fermented foods, bread, beer
and wine.

FOOD SAFETY
Food safety is a major focus of food microbiology. Pathogenic
bacteria, viruses and toxins produced by microorganisms are all
possible contaminants of food. However, microorganisms and their
products can also be used to combat these pathogenic microbes.
Probiotic bacteria, including those which produce bacteriocins
can kill and inhibit pathogens. Alternatively, purified bacteriocins
such as nisin can be added directly to food products. Finally,
bacteriophage, viruses which only infect bacteria, can be used to
kill bacterial pathogens. Thorough preparation of food, including
proper cooking will kill most bacteria and viruses. However, toxins
produced by contaminants may not be heat-labile, and some will
not be eliminated by cooking.

FERMENTATION
Fermentation is one way microorganisms can change a food.
Yeast, especially S. cerevisiae, is used to leaven bread, brew beer
and make wine. Certain bacteria, including lactic acid bacteria,
are used to make yogurt, cheese, hot sauce, pickles and dishes
2 Introductory Food Microbiology
such as kimchi. A common effect of these fermentations is that the
food product is less hospitable to other microorganisms, including
pathogens and spoilage-causing microorganisms, thus extending
the food's shelf-life.
Some cheese varieties also require mold microorganisms to
ripen and develop their characteristic flavors.
Foodborne Pathogens
Foodborne pathogens are the leading causes of illness and
death in less developed countries killing approximately 1.8 million
people annually. In developed countries foodborne pathogens are
responsible for millions of cases of infectious gastrointestinal
diseases each year, costing billions of dollars in medical care and
lost productivity. New foodborne pathogens and foodborne
diseases are likely to emerge driven by factors such as pathogen
evolution, changes in agricultural and food manufacturing
practices, and changes to the human host status. There are growing
concerns that terrorists could use pathogens to contaminate food
and water supplies in attempts to incapacitate thousands of people
and disrupt economic growth.
Enteric Viruses
Food and waterborne viruses contribute to a substantial
number of illnesses throughout the world. Among those most
commonly known are hepatitis A virus, rotavirus, astrovirus, enteric
adenovirus, hepatitis E virus, and the human caliciviruses
consisting of the noroviruses and the Sapporo viruses. This diverse
group are transmitted by the fecal-oral route, often by ingestion of
contaminated food and water.
Protozoan Parasites
Protozoan parasites associated with food and water can cause
illness in humans. Although parasites are more commonly found
in developing countries, developed countries have also experienced
several foodborne outbreaks. Contaminants may be inadvertently
introduced to the foods by inadequate handling practices, either
on the farm or during processing of foods. Protozoan parasites
can be found worldwide, either infecting wild animals or in water
and contaminating crops grown for human consumption. The
Introduction 3
disease can be much more severe and prolonged in
immunocompromissed individuals.
Mycotoxins
Molds produce mycotoxins, which are secondary metabolites
that can cause acute or chronic diseases in humans when ingested
from contaminated foods. Potential diseases include cancers and
tumors in different organs (heart, liver, kidney, nerves),
gastrointestinal disturbances, alteration of the immune system,
and reproductive problems. Species of Aspergillus, Fusarium,
Penicillium, and Claviceps grow in agricultural commodities or
foods and produce the mycotoxins such as aflatoxins,
deoxynivalenol, ochratoxin A, fumonisins, ergot alkaloids, T-2
toxin, and zearalenone and other minor mycotoxins such as
cyclopiazonic acid and patulin. Mycotoxins occur mainly in cereal
grains (barley, maize, rye, wheat), coffee, dairy products, fruits,
nuts and spices. Control of mycotoxins in foods has focused on
minimizing mycotoxin production in the field, during storage or
destruction once produced. Monitoring foods for mycotoxins is
important to manage strategies such as regulations and guidelines,
which are used by 77 countries, and for developing exposure
assessments essential for accurate risk characterization.
Yersinia Enterocolitica
Yersinia enterocolitica includes pathogens and environmental
strains that are ubiquitous in terrestrial and fresh water ecosystems.
Evidence from large outbreaks of yersiniosis and from
epidemiological studies of sporadic cases has shown that Y.
enterocolitica is a foodborne pathogen. Pork is often implicated as
the source of infection. The pig is the only animal consumed by
man that regularly harbours pathogenic Y. enterocolitica. An
important property of the bacterium is its ability to multiply at
temperatures near to 0°C, and therefore in many chilled foods. The
pathogenic serovars (mainly O:3, O:5,27, O:8 and O:9) show
different geographical distribution. However, the appearance of
strains of serovars O:3 and O:9 in Europe, Japan in the 1970s, and
in North America by the end of the 1980s, is an example of a
global pandemic. There is a possible risk of reactive arthritis
following infection with Y. enterocolitica.
4 Introductory Food Microbiology
Vibrio
Vibrio species are prevalent in estuarine and marine
environments and seven species can cause foodborne infections
associated with seafood. Vibrio cholerae O1 and O139 serovtypes
produce cholera toxin and are agents of cholera. However, fecal-
oral route infections in the terrestrial environment are responsible
for epidemic cholera. V. cholerae non-O1/O139 strains may cause
gastroenteritis through production of known toxins or unknown
mechanism.
Vibrio parahaemolytitucs strains capable of producing
thermostable direct hemolysin (TDH) and/or TDH-related
hemolysin are most important cause of gastroenteritis associated
with seafood consumption. Vibrio vulnificus is responsible for
seafoodborne primary septicemia and its infectivity depends
primarily on the risk factors of the host. V. vulnificus infection has
the highest case fatality rate (50%) of any foodborne pathogen.
Four other species (Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis,
and Vibrio furnissii) can cause gastroenteritis. Some strains of
these species produce known toxins but the pathogenic mechanism
is largely not understood. The ecology of and detection and control
methods for all seafoodborne Vibrio pathogens are essentially
similar.
Staphylococcus Aureus
Staphylococcus aureus is a common cause of bacterial
foodborne disease worldwide. Symptoms include vomiting and
diarrhea that occur shortly after ingestion of S. aureus-
contaminated food. The symptoms arise from ingestion of preformed
enterotoxin, which accounts for the short incubation time.
Staphylococcal enterotoxins are superantigens and, as such, have
adverse effects on the immune system.
The enterotoxin genes are accessory genetic elements in S.
aureus, meaning that not all strains of this organism are enterotoxin-
producing. The enterotoxin genes are found on prophage, plasmids,
and pathogenicity islands in different strains of S. aureus.
Expression of the enterotoxin genes is often under the control of
global virulence gene regulatory systems.
Introduction 5
Campylobacter
Campylobacter spp., primarily C. jejuni subsp. jejuni is one of
the major causes of bacterial gastroenteritis in the U.S. and
worldwide. Campylobacter infection is primarily a foodborne
illness, usually without complications; however, serious sequelae
such as Guillain-Barre Syndrome occur in a small subset of infected
patients. Detection of C. jejuni in clinical samples is readily
accomplished by culture and non-culture methods.
Listeria Monocytogenes
Listeria monocytogenes is Gram-positive foodborne bacterial
pathogen and the causative agent of human listeriosis. Listeriae
are acquired primarily through the consumption of contaminated
foods including soft cheese, raw milk, deli salads, and ready-to-
eat foods such as luncheon meats and frankfurters. Although L.
monocytogenes infection is usually limited to individuals that are
immunocompromised, the high mortality rate associated with
human listeriosis makes L. monocytogenes the leading cause of
death amongst foodborne bacterial pathogens. As a result,
tremendous effort has been made at developing methods for the
isolation, detection and control of L. monocytogenes in foods.
Salmonella
Salmonella serotypes continue to be a prominent threat to
food safety worldwide. Infections are commonly acquired by animal
to human transmission though consumption of undercooked food
products derived from livestock or domestic fowl. The second half
of the 20th century saw the emergence of Salmonella serotypes
that became associated with new food sources (i.e. chicken eggs)
and the emergence of Salmonella serotypes with resistance against
multiple antibiotics.
Shigella
Shigella species are members of the family Enterobacteriacae
and are Gram negative, non-motile rods. Four subgroups exist
based on O-antigen structure and biochemical properties;
S. dysenteriae (subgroup A), S. flexneri (subgroup B), S. boydii
(subgroup C) and S. sonnei (subgroup D). Symptoms include mild
to severe diarrhea with or without blood, fever, tenesmus, and
6 Introductory Food Microbiology
abdominal pain. Further complications of the disease may be
seizures, toxic megacolon, reactive arthritis and hemolytic uremic
syndrome. Transmission of the pathogen is by the fecal-oral route,
commonly through food and water. The infectious dose ranges
from 10-100 organisms. Shigella spp. have a sophisticated
pathogenic mechanism to invade colonic epithelial cells of the
host, man and higher primates, and the ability to multiply
intracellularly and spread from cell to adjacent cell via actin
polymerization. Shigellae are one of the leading causes of bacterial
foodborne illnesses and can spread quickly within a population.
Escherichia Coli
More information is available concerning Escherichia coli than
any other organism, thus making E. coli the most thoroughly
studied species in the microbial world. For many years, E. coli was
considered a commensal of human and animal intestinal tracts
with low virulence potential. It is now known that many strains
of E. coli act as pathogens inducing serious gastrointestinal diseases
and even death in humans. There are six major categories of E. coli
strains that cause enteric diseases in humans including the:
(1) enterohemorrhagic E. coli, which cause hemorrhagic colitis
and hemolytic uremic syndrome,
(2) enterotoxigenic E. coli, which induce traveler's diarrhea,
(3) enteropathogenic E. coli, which cause a persistent diarrhea
in children living in developing countries,
(4) enteroaggregative E. coli, which provoke diarrhea in
children,
(5) enteroinvasive E. coli that are biochemically and genetically
related to Shigella species and can induce diarrhea, and
(6) diffusely adherent E. coli, which cause diarrhea and are
distinguished by a characteristic type of adherence to
mammalian cells.
Clostridium Botulinum and Clostridium Perfringens
Clostridium botulinum produces extremely potent neurotoxins
that result in the severe neuroparalytic disease, botulism. The
enterotoxin produced by C. perfringens during sporulation of
Introduction 7
vegetative cells in the host intestine results in debilitating acute
diarrhea and abdominal pain. Sales of refrigerated, processed
foods of extended durability including sous-vide foods, chilled
ready-to-eat meals, and cook-chill foods have increased over recent
years. Anaerobic spore-formers have been identified as the primary
microbiological concerns in these foods. Heightened awareness
over intentional food source tampering with botulinum neurotoxin
has arisen with respect to genes encoding the toxins that are
capable of transfer to nontoxigenic clostridia.
Bacillus Cereus
The Bacillus cereus group comprises six members: B. anthracis,
B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B.
weihenstephanensis. These species are closely related and should
be placed within one species, except for B. anthracis that possesses
specific large virulence plasmids. B. cereus is a normal soil
inhabitant and is frequently isolated from a variety of foods,
including vegetables, dairy products and meat. It causes a vomiting
or diarrhoea illness that is becoming increasingly important in the
industrialized world. Some patients may experience both types of
illness simultaneously. The diarrhoeal type of illness is most
prevalent in the western hemisphere, whereas the emetic type is
most prevalent in Japan. Desserts, meat dishes, and dairy products
are the foods most frequently associated with diarrhoeal illness,
whereas rice and pasta are the most common vehicles of emetic
illness.
The emetic toxin (cereulide) has been isolated and
characterized; it is a small ring peptide synthesised non-
ribosomally by a peptide synthetase. Three types of B. cereus
enterotoxins involved in foodborne outbreaks have been identified.
Two of these enterotoxins are three-component proteins and are
related, while the last is a one-component protein (CytK). Deaths
have been recorded both by strains that produce the emetic toxin
and by a strain producing only CytK. Some strains of the B. cereus
group are able to grow at refrigeration temperatures. These variants
raise concern about the safety of cooked, refrigerated foods with
an extended shelf life. B. cereus spores adhere to many surfaces
and survive normal washing and disinfection (except for
hypochlorite and UVC) procedures. B. cereus foodborne illness is
8 Introductory Food Microbiology
likely underreported because of its relatively mild symptoms, which
are of short duration.
CARNOBACTERIUM: POSITIVE AND NEGATIVE EFFECTS
IN THE ENVIRONMENT AND IN FOODS
The genus Carnobacterium contains nine species, but only C.
divergens and C. maltaromaticum are frequently isolated from
natural environments and foods. They are tolerant to freezing/
thawing and high pressure and able to grow at low temperatures,
anaerobically and with increased CO2 concentrations. They
metabolize arginine and various carbohydrates, including chitin,
and this may improve their survival in the environment.
Carnobacterium divergens and C. maltaromaticum have been
extensively studied as protective cultures in order to inhibit growth
of Listeria monocytogenes in fish and meat products. Several
carnobacterial bacteriocins are known, and parameters that affect
their production have been described. Currently, however, no
isolates are commercially applied as protective cultures.
Carnobacteria can spoil chilled foods, but spoilage activity shows
intraspecies and interspecies variation. The responsible spoilage
metabolites are not well characterized, but branched alcohols and
aldehydes play a partial role.
Their production of tyramine in foods is critical for susceptible
individuals, but carnobacteria are not otherwise human pathogens.
Carnobacterium maltaromaticum can be a fish pathogen, although
carnobacteria are also suggested as probiotic cultures for use in
aquaculture. Representative genome sequences are not yet available,
but would be valuable to answer questions associated with
fundamental and applied aspects of this important genus.
Carnobacteria are ubiquitous lactic acid bacteria (LAB) isolated
from cold and temperate environments. More importantly from a
practical viewpoint, they also frequently predominate in a range
of foods, including fish, meat, and some dairy products. In this
regard, they have been extensively studied in the last two decades
as protective cultures, to inhibit pathogenic and spoilage
microorganisms, and as potential spoilage bacteria in chilled
seafood and meat products. In addition, owing to their presence
in the aqueous environment, their importance as fish pathogens
Introduction 9
or probiotic cultures in the aquaculture industry has been
examined.
The genus Carnobacterium currently consists of nine species,
but only two of these, Carnobacterium divergens and C.
maltaromaticum (formerly C. piscicola) are frequently encountered
in the environment and in foods (Table 1). This review concerns
the importance of these two species in dairy, meat and fish products
and in the aquatic environment. The taxonomy and methods for
isolation and identification of carnobacteria are not described. It
is, however, important to note that older studies frequently used
carbohydrate fermentation patterns to distinguish between C.
divergens and C. maltaromaticum. These methods are now
considered less reliable than molecular methods, and results relying
on phenotypic criteria must be interpreted with caution.
DISTRIBUTION IN THE NATURAL ENVIRONMENT AND
FOODS
Natural Environment
Among the nine reported species of Carnobacterium, only two
species, C. divergens and C. maltaromaticum, are frequently isolated
from various sources. Four species, C. alterfunditum, C. funditum,
C. gallinarum and C. mobile, have been isolated a few times from
at most three sources. Three species, C. inhibens, C. pleistocenium
and C. viridans, have only been isolated from one source, but it
should be noted that Carnobacterium spp. related to, for example,
C. inhibens have been described. Carnobacterium spp. appear to
have both the temperate and polar aquatic environments as habitats
including live fish, marine sponges, Antarctic lakes, Arctic and
Antarctic sea water as well as the deep sea, aquous alkaline tufa
columns from Greenland, and freshwater habitats from the
temperate clima zone, including a Sphagnum pond and rivers in
the northwest region of Spain.
Although C. maltaromaticum and/or C. divergens have been
isolated from tropical fish products, including smoked surubim, a
Brazilian tropical freshwater fish, and from vacuum-packed tuna
caught in the Indian Ocean and processed in Sri Lanka, it cannot,
at least in the latter case, be excluded that this is due to
contamination associated with repackaging in Denmark.
10 Introductory Food Microbiology
The presence of carnobacteria has also been demonstrated in
the terrestrial environment, including a field treated with whey,
Canadian winter soil, permafrost ice, a compost pile, a collapsed
horse manure pile, the larval midgut of a moth species, and other
sources. It appears that the temperate/polar aquatic and terrestrial
environments are both natural habitats.
Carnobacterium divergens and C. maltaromaticum possess
traits that may play a role in their survival in these surroundings.
One study has reported that a Carnobacterium sp. soil isolate
related to C. maltaromaticum survived 48 serial freeze-thaw cycles
better than Escherichia coli and an Enterococcus sp. soil isolate,
but worse than a few other soil isolates, including an Acinetobacter
sp. Indeed, these organisms may survive freezing for considerable
periods of time, as witnessed by the isolation of a Carnobacterium
sp. preserved in a permafrost ice wedge for 25 000 years. Additional
data on the abilities of carnobacteria to grow at low temperatures
and to survive frozen storage is available for food products. Also,
a cold-active b-galactosidase from C. maltaromaticum and a cold-
adapted alanine dehydrogenase from a Carnobacterium sp. related
to C. alterfunditum have been reported. Some carnobacterial isolates
originate from natural high-pressure habitats, and additional data
on resistance to pressure have been reported for high-pressure-
processed foods. It has not been described how other physical/
chemical parameters such as salt content, atmosphere and pH
affect survival and growth of these organisms in the natural
environment.
With respect to energy-yielding substrates, one species, C.
maltaromaticum, expresses chitinase activity. This may facilitate
adherence to and survival on zooplankton as suggested for
enterococci, and also explain the isolation of this species from the
midgut of the larval stage of a species of moth. Interestingly, the
arginine deiminase pathway is expressed by the most frequently
encountered species, C. divergens, C. gallinarum, C.
maltaromaticum and C. mobile but not by C. inhibens or C. viridans.
Arginine is not a substrate that results in growth of C. alterfunditum
and C. funditum, and information is not available for C.
pleistocenium. This amino acid may represent an additional energy
source for growth and survival when carbohydrates are scarce,
Introduction 11
and it may offer protection against acid stress, as described for
Enterococcus faecalis and related species. Finally, carnobacteria
are able to catabolize a range of carbohydrates, although there are
considerable interspecies and intraspecies heterogeneities.
Examples of sources of such carbohydrates include animals
(e.g. chitin and, to some extent, lactose, although the targets of the
lactose hydrolytic activity shown by carnobacteria may instead be
byproducts of plant sugar polymers and saccharides adsorbed to
humic acid substances in the soil), plants (e.g. salicin and sucrose),
fungi (e.g. chitin and trehalose), prokaryotes (N-acetylglucosamine),
and living organisms in general (ribose). The importance of these
abilities for growth and survival in the environment deserves
further study. The genome sizes of C. alterfunditum, C. divergens
and C. pleistocenium strains and a Carnobacterium sp. AT7 deep
sea isolate have been estimated to be 2.9, 3.2, 3.2 and 2.4Mb,
respectively, and for the AT7 isolate, the data are reported in a
database described by Liolios (2006). These sizes are relatively
large in comparison to many other LAB, suggesting that
carnobacterial genomes may encode a range of genes that makes
them well adapted to deal with environmental challenges.
Even if carnobacteria are well adapted to temperate or polar
aqueous environments, this does not always provide improved
ability to survive as compared to other bacteria. Thus, the actual
abilities of strains of C. divergens and C. maltaromaticum isolated
from a Sphagnum pond to survive in water from this source were
not improved as compared to related gram-positive bacteria
originating from other sources. At present, therefore, the precise
mechanisms by which Carnobacterium spp. persist in the natural
environment and their underlying genetics are not known. Finally,
it should be noted that the environments from which carnobacteria
can be isolated are not always as extreme as they appear at first
sight. Thus, Carnobacterium spp. isolated from Lake Vanda,
Antarctica were found at a depth of 61 m, where the temperature
was c. 15-20 1C.
Dairy, Fish and Meat Products
High concentrations of bacteria (4106-107 CFUg_1) in food
are typically required before their activity is sufficient to influence
12 Introductory Food Microbiology
the sensory properties of a product. For this to happen, occurrence
and growth kinetics are the key parameters, and databases,
including ComBase, are available to determine the effect of food
storage conditions and product characteristics on growth of specific
bacteria. However, information on carnobacteria has not yet been
included in such databases.
Carnobacterium maltaromaticum and another Carnobacterium
sp. have been isolated from milk, and they, in addition to C.
divergens, have been detected in soft cheeses, including mold-
ripened brie, mozzarella, Camembert and other types. The high
concentration of C. divergens in the curd of mozzarella, exposed
to 10-37 1C during processing, is in agreement with the maximum
growth temperature (40 1C) of this species.
The occurrence of Carnobacterium in dairy products (and
other foods) is most likely underreported. This is due to the common
use of acetate containing media, particularly MRS agar or Rogosa
agar, for enumeration of LAB. Growth of Carnobacterium is
inhibited by acetate, and both MRS and Rogosa agar media
significantly underestimate concentrations in food.
Carnobacterium divergens and C. maltaromaticum are present
in seafood and are able to grow to high concentrations in different
fresh and lightly preserved products.
Studies of naturally contaminated products suggest which
storage conditions and product characteristics select for
carnobacteria as compared to the other bacteria present in seafood.
For chilled fresh seafood, we have found no reports where C.
divergens and C. maltaromaticum dominated the microbial
communities in aerobically stored products, but this has been
reported for modified atmosphere-packed (MAP) coalfish, cod,
pollack, rainbow trout, salmon, shrimp, swordfish and tuna.
After frozen storage (_20 to _30 1C for 5-8 weeks), C. divergens
and C. maltaromaticum seem to be particularly prominent in
chilled MAP fish, as has been reported for cod, garfish, salmon,
and tuna. In addition to freezing, these carnobacteria are relatively
resistant to high-pressure processing and are found in high
concentrations in vacuum-packed and chilled squid mantle and
cold-smoked salmon previously treated with 200-400MPa for 15-
Introduction 13
20 min. In vacuum-packed coldsmoked or sugar-salted ('gravad')
seafood with 3-7% NaCl in the water phase and a pH of 5.8-6.5,
high concentrations of C. divergens and C. maltaromaticum are
particularly common, as reported for halibut, rainbow trout,
salmon, surubim, and tuna.
High concentrations of C. maltaromaticum are also reported
in salted lumpfish roe, and it has been isolated from frozen, smoked
mussels. Finally, for cooked seafood, high concentrations of C.
maltaromaticum have been detected in MAP shrimp after storage
at 2-8 1C. Both C. divergens and C. mobile were isolated from
cooked and brined MAP shrimps (with NaCl, benzoic acid, citric
acid and sorbic acid) after storage at 2-8 1C. Clearly, carnobacteria
are common in chilled fresh and lightly preserved seafood, but at
higher storage temperatures (15-25 1C), other species, including
Enterococcus spp., more frequently dominate the spoilage microbial
community of seafood.
Carnobacterium divergens and C. maltaromaticum are able to
grow in meat products at temperatures as low as 2 to _1.5 1C, and
they are frequently predominant members (up to 50% C. divergens
and up to 26% C. maltaromaticum of the gram-positive or LAB
isolates obtained) of the microbial community of raw meat (beef,
pork, lamb, and poultry).
The two species are found irrespective of whether products
have been stored aerobically, vacuum packaged, or subjected to
modified atmospheres, including gas compositions of CO2/N2 (%)
ranging from 10 : 90 to 80 : 20. One study demonstrated the
presence of C. divergens in beef stored in air but not in MAP beef
with increased concentrations of O2 (20-40%) in addition to CO2
(40%). The growth of C. funditum is impaired by oxygen, and it
will be of interest to study further whether addition of O2 to the
gas composition of modified atmospheres consistently inhibits
growth of carnobacteria, as has been observed for some other
bacteria.
Further studies are needed in order to determine whether
differences in the presence of carnobacteria in meat are due to
variations in storage conditions or variations in contamination
levels at the processing plants. The source of carnobacteria in
meat products is most probably the processing plant, as these
14 Introductory Food Microbiology
organisms have not been isolated from the gastrointestinal system
or skin of chicken, cattle, pigs or sheep, except for one unpublished
study. Thus, the source of contamination of broiler carcasses by C.
divergens and C. maltaromaticum was shown to be the air in the
processing plant and not incoming broiler chickens. In fact, two
studies confirmed that C. divergens and C. maltaromaticum present
in the raw material were eliminated or reduced in number by
cooking of a processed meat product, ham.
Carnobacterium divergens and C. maltaromaticum have,
however, been detected in a variety of processed meat products,
including the cured pork product bacon, a Danish processed pork
product ('rullep_lse'), various Spanish processed meat products,
cooked poultry meat, pressure-treated (408-888MPa) chicken, and
irradiated pork and chicken. On a few occasions, C.
maltaromaticum has even been isolated from fermented sausages;
as noted by Hammes & Hertel (2003), this contrasts with the usual
nonaciduric habitats of carnobacteria. Other Carnobacterium spp.
isolated from processed meat products include C. gallinarium
(irradiated chicken), C. mobile (cooked turkey) and C. viridans
(vacuum-packed Bologna sausage). Although these organisms are
frequently isolated from processed meat products, it has been
suggested that they are rarely present in high numbers, and that
terminal spoilage of cooked, cured meats is primarily caused by
aciduric LAB.
Finally, a Carnobacterium sp. has also been isolated from egg
contents, and it was shown that this isolate had a similar ability
to penetrate the eggshell and contaminate the whole egg as various
other gram-positive and gram-negative bacteria.
Transmission routes for carnobacteria from the natural
environment into food-manufacturing plants and further on to
dairy, fish and meat products are not known to any extent.
Knowledge on this topic is essential to evaluate if species and
intraspecific clusters that differ in spoilage ability (as discussed
later) also have different colonization abilities. Such knowledge
would also illuminate the feasibility of using the production of
metabolites, especially tyramine, by C. divergens and C.
maltaromaticum as an index of microbial spoilage of specific fish
and meat products.
Introduction 15
FUNCTIONAL PROPERTIES OF CARNOBACTERIA
Bacteriocins and Antimicrobial Properties
Carnobacteria with the ability to produce antimicrobial
peptides, bacteriocins, are commonly encountered in foods. The
characterized carnobacterial bacteriocins belong to class I and
class II [e.g. Drider et al. (2006) for bacteriocin nomenclature]. So
far, only one class I bacteriocin (lantibiotic), UI149, has been
reported to be produced by Carnobacterium. In contrast, ten amino
acidsequenced bacteriocins belong to class II, and most of these
more specifically to class IIa, which comprises small pediocin- like
peptides. The inhibition spectrum of carnobacterial class IIa
bacteriocins includes Listeria, and antimicrobial activity is exerted
by pore formation, dissipation of membrane potential, and leakage
of internal low molecular weight substances.
Class IIa bacteriocins are ribosomally synthesized as inactive
prepeptides that are modified by posttranslational cleavage of the
N-terminal peptide leader at a doubleglycine site in order to release
mature and active cationic peptides. Carnobacterial class IIa
bacteriocins have similar amino acid sequences, with a
YGNGV(X)C(X)4C motif (X denotes any amino acid) near the N-
terminus of the mature peptide. Two cysteine residues form a
disulfide bond in the N-terminal region, and there is an
amphipathic a-helix near the C-terminus. The N-terminal region
is relatively hydrophilic and conserved, whereas the C-terminus
is hydrophobic and diverse. To establish the structure-activity
relationships of carnobacterial bacteriocins, the structures of
carnobacteriocin B2 and divercin V41 were modified. Thus, amino
acid substitutions closer to the N-terminus in carnobacteriocin B2
drastically reduced or eliminated antimicrobial activity, whereas
this was not so for substitutions close to the C-terminal part.
Divercin V41 contains a second disulfide bond located in the C-
terminal region.
When the C-terminal region of divercin V41 was separated
from the N-terminus by endoproteinase Asp-N, only the C-terminal
fragment was active. After trypsin cleavage next to lysine at position
42 or disulfide reduction, the C-terminus lost its inhibitory activity.
These results suggested that both hydrophobicity and folding
imposed by this second disulfide bond were essential for
16 Introductory Food Microbiology
antilisterial activity of the C-terminal hydrophobic peptide.
Chemical oxidation of tryptophan residues by N-bromosuccinimide
showed that these residues were crucial for inhibitory activity, as
modification of any one of them rendered divercin V41 inactive.
The three-dimensional structure of carnobacteriocin B2, its
precursor and the corresponding immunity protein have been
solved by nuclear magnetic resonance. These are, at present, the
only reported carnobacterial protein structures.
Carnobacterial class II bacteriocins that contain a
doubleglycine- type leader peptide are transported by a dedicated
ATP-binding cassette (ABC) transport system. In contrast, divergicin
A does not require a secretion protein, as the transport depend on
the general cellular secretion (sec) pathway. The genes involved in
carnobacterial bacteriocin production are generally clustered in
operons. In the simplest case, corresponding to the sec-dependent
divergicin A, the bacteriocin operon is composed of only the
structural gene followed downstream by the gene encoding an
immunity protein that protects the cell from is own bacteriocin.
Production of class IIa bacteriocins requires four genes as a
minimum, including genes encoding bacteriocin, immunity protein,
ABC of transport protein and its membrane-bound accessory
protein. Genes for bacteriocin production may be encoded on the
chromosome or on plasmids. Carnobacterium maltaromaticum
LV17 produces at least three class II bacteriocins.
Carnobacteriocins A and B2 are encoded on different but
compatible plasmids, pCP49 (72 kb) and pCP40 (61 kb), respectively.
The carnobacteriocin BM1 structural gene and its immunity gene
are localized on the chromosome. Activation and export of
carnobacteriocin BM1 depend on genes located on plasmid pCP40
encoding carnobacteriocin B2. For carnobacteriocins BM1 and B2,
a peptide-pheromone dependent quorum-sensing mode caused by
the bacteriocin itself or an autoinducer peptide is involved in the
regulation of bacteriocin production by two- and three-component
signal transduction systems.
The components of this regulatory system consist of an
induction factor, a histidine protein kinase, and a response
regulator. Four carnobacterial bacteriocin operons including genes
for immunity proteins and regulatory proteins involved in
Introduction 17
bacteriocin expression and secretion have been sequenced. External
parameters also affect bacteriocin production. Thus, increasing
concentrations of NaCl (2-7%) reduced production of A9b/B2
bacteriocin, increasing the temperature from below 19 to 25 1C
inhibited production of piscicolin 126, and reducing the pH from
6.5 to 5.5 inhibited production of carnobacteriocins BM1 and B2.
Acetate (A9b bacteriocin) or the presence of a bacteriocin-sensitive
strain belonging to the same genus induced bacteriocin production.
It is clear that, in order to apply carnobacterial bacteriocins for the
biopreservation of foods, detailed knowledge of the factors that
influence production of the bacteriocin is necessary.
Finally, it should be noted that the bacteriocinogenic activity
may be lost, as shown for a C. divergens divercin V41- producing
strain that retained only partial activity after being subjected to
spray-drying. Interest in applying carnobacterial bacteriocins in
foods and feeds has been directed towards inhibiting Listeria
monocytogenes and spoilage microorganisms, to extend the shelf-
life of lightly preserved seafood. These attempts have generally,
but not always [e.g. regarding prevention of spoilage], met with
success, although diversity in sensitivity of the target organism
has been observed.
However, the occurrence of resistant L. monocytogenes target
organisms has led to the suggestion that bacteriocin-negative LAB
may be more suitable for practical use as bioprotective agents
against L. monocytogenes in ready-to-eat foods. Indeed, L.
monocytogenes is inhibited by carnobacterial cultures that do not
produce bacteriocins, and this is partly due to glucose depletion.
One study also reported a successful application of cultures with
no demonstrated bacteriocinogenic activity for extension of the
shelflife of vacuum-packed cold-smoked salmon. This effect was
not, however, observed for a C. divergens strain that was unable
to produce the class IIa divercin V41.
Resistance of L. monocytogenes to the class IIa divergicin M35
was probably due to modification of the cell wall fatty acid
composition. Listeria monocytogenes strains resistant to the class
IIa divercin V41 also showed substantial differences in protein
expressions as compared with the wild type strain. A s54-
dependent PTS permease of the mannose family, which belongs to
18 Introductory Food Microbiology
the phosphotransferase system (PTS), is responsible for sensitivity
of L. monocytogenes to class IIa bacteriocins. These results
suggested that EIIt Man encoded by the mptACD operon might be
a target molecule for class IIa bacteriocins. Recently, two genes
coding for a glycerophosphoryl diester phosphodiesterase (GlpQ)
and a protein with a putative phosphoesterase function (Pde)
were identified as also being involved in the class IIa sensitivity
of En. faecalis. Finally, it is important to note that the susceptibility
of the target strain is affected by various environmental conditions
such as NaCl and pH.
Another cause of concern when using carnobacteria for
biopreservation is that C. divergens and C. maltaromaticum both
produce tyramine, as discussed further below. Therefore, it has
been suggested that a C. divergens strain in which the tyrosine
decarboxylase gene is inactivated by mutagenesis could be used
as a protective culture to prevent growth of L. monocytogenes in
cold-smoked salmon. Currently, no carnobacterial culture,
bacteriocin producing or not, is commercially applied for protection
against the growth of L. monocytogenes or other bacterial pathogens
or spoilage organisms in food.
Effect of Catabolic Activities on Sensory Characteristics and Safety
of Foods
As shown in Table 4, the catabolic activities of carnobacteria
may result in sensory spoilage of inoculated fish and meat products.
Whether this is so for cheese products is not clear. In naturally
contaminated products, it seems other members of the bacterial
community are typically more important with regard to sensory
effects, including spoilage.
It is of interest that spoilage was enhanced if moderate-spoilage
strains of C. maltaromaticum were inoculated with nonspoilage
Vibrio sp. strains or the moderate-spoilage organism Brochothrix
thermosphacta into cold-smoked salmon that was subsequently
vacuum-packaged. However, this was not so for combinations of
C. maltaromaticum and Photobacterium phosphoreum in this
product. In addition, a spoilage synergy effect was observed for
combinations of B. thermosphacta and C. divergens, C.
maltaromaticum or C. mobile in MAP shrimp.
Introduction 19
We will first focus on catabolic reactions with carbohydrates
and/or organic acids as substrates and with pyruvic acid as an
intermediate metabolite. Respiration might occur in the presence
of hematin, as shown for C. maltaromaticum, and, indeed, this
species consumes substantial proportions of oxygen during
exponential growth under aerobic conditions. Carnobacterium spp.
are, however, considered to be homofermentative organisms that
produce lactic acid from glucose.
The presence of the glycolytic pathway in C. divergens has
been demonstrated. It can be debated whether carnobacteria should
instead be considered facultative or atypical heterofermentative
organisms, as C. divergens and C. maltaromaticum are able to
utilize ribose and gluconic acid as substrates for growth, and may
produce acetic acid, formic acid, and CO2 as end-products of some
secondary decarboxylation/ dissimilation reactions of pyruvic acid.
Indeed, carnobacteria were initially described as heterofermenters.
Acetic acid production by C. divergens and C. maltaromaticum
can be substantial during growth in laboratory media under aerobic
conditions or in MAP shrimp, and quantitatively it can exceed
lactic acid production. Production of acetic acid by C.
maltaromaticum is also increased relative to lactic acid if glucose
is substituted by ribose. Furthermore, C. maltaromaticum can
produce large amounts of ethanol from glucose and ribose during
growth in a shrimp extract under anaerobic conditions.
Acetoin can be generated by C. maltaromaticum from pyruvic
acid, and this reaction is also found for C. divergens and C.
gallinarum but not for C. alterfunditum, C. funditum, C. inhibens
and C. viridance, according to reactions in the Voges-Proskauer
test. Carnobacterium mobile gives variable reactions, and
information is not available for C. pleistocenium.
The factors affecting acetoin production are not well known,
but production is increased by resting cells of C. maltaromaticum
in the presence of hematin. In addition, two out of four strains of
C. divergens and C. maltaromaticum isolated from mozzarella
cheese were reported to be able to metabolize citric acid, but the
potential sensory role of this reaction, e.g. by production of acetoin
and diacetyl, was not examined.
20 Introductory Food Microbiology
Production of diacetyl and 2,3-pentanedione by C.
maltaromaticum during growth in cold-smoked salmon resulted
in a butter-like odor but not in spoilage. In conclusion, even, if
carbohydrate catabolism by carnobacteria appears to result in a
diverse number of metabolites, these have generally a limited effect
on the sensory attributes of foods. H2O2 may be produced by C.
divergens, and formation of this compound by C. viridans has
been associated with spoilage in the form of green discoloration
of cured Bologna ham.
Whether carnobacteria possess proteolytic activities of potential
importance for taste in products such as cheese is not known, and
this deserves further investigation. In contrast, metabolites resulting
from the degradation of amino acids certainly cause sensory effects
in foods. Generation of branched alcohols and aldehydes (2-
methyl-1-butanal, 2-methyl-1-butanol, 3-methyl-1-butanal, 3-methyl-
1-butanol, 2-methylpropanal, and 2-methylpropanol) by
transamination, decarboxylation and reduction of the amino acids
valine, leucine and isoleucine appears to be particularly important.
Production of some of these compounds, such as 3-methyl-1-
butanal, is strain or species dependent. Production by a C.
maltaromaticum strain of 3-methylbutanal, 3-methylbutanol and
3-methylbutanoic acid from leucine was, in general, increased at
pH values of 6.5 or higher and in the presence of increased
concentrations of a-ketoisocaproic acid and glucose. This strain
affected the odor and reduced the leucine content of a sausage
mince, but a clear causal relationship was not demonstrated.
Production by C. maltaromaticum of alcohols and aldehydes from
valine, leucine and/or isoleucine resulted in a malty, green aroma
in skimmed milk and shrimp, and has also caused spoilage of
cured ham. The association between the presence of these
compounds and spoilage is, however, not always straightforward,
as production of 3-methyl-1- butanal and/or 3-methyl-1-butanol
in shrimp by certain C. maltaromaticum strains did not result in
malty off-flavors.
Production of NH4 from arginine as a result of its catabolism
catalyzed by the arginine deaminase pathway may, in theory,
cause spoilage of shrimp or squid that contain high levels of free
arginine. Carnobacterium maltaromaticum and C. divergens but
Introduction 21
not C. mobile were able to produce NH4 in MAP shrimp, but this
was not the cause of spoilage of this product. Production of indole
from the amino acid tryptophan is also a potential cause of spoilage,
although C. divergens, C. maltaromaticum and C. mobile are all
unable to carry out this reaction in standard laboratory media. It
has, however, been observed that C. divergens and strains
belonging to a major phenotypic cluster of C. maltaromaticum
were able to use tryptophan as a substrate during growth in MAP
shrimp. Whether this resulted in generation of indole was not
examined.
Production of tyramine from tyrosine is the only known
metabolic reaction by Carnobacterium spp. that constitutes a cause
of concern regarding food safety. Not all carnobacterial species
possess this ability, as C. mobile does not produce tyramine during
growth in shrimp, and variation exists among different strains
and phenotypic clusters of C. divergens and C. maltaromaticum
for amounts of tyramine produced. Tyramine production by one
C. divergens strain was at its maximum during the stationary
growth phase and at a low initial pH in the presence of 0.6%
glucose, whereas NaCl (10%) was inhibitory. Regardless of strain
variation and the effects of environmental parameters, tyramine
production by C. divergens and/or C. maltaromaticum has been
reported in a range of foods, including meat (up to 28 mg kg_1),
a meat-fat mixture (up to 121 mg kg_1), cold-smoked salmon (up
to c. 370 mg kg_1), frozen and thawed salmon (up to 40-60 mg
kg_1), and shrimp (up to 20-60 mg kg_1).
These levels have no adverse effects on most consumers, but
for sensitive individuals, e.g. with reduced monoamine oxidase
activity due to medication or hereditary deficiency, very little
tyramine can cause migraine headaches, and an intake of no more
than 5mg of tyramine per meal has been recommended. Typical
consumption of fish and meat products is 50-150 g per meal. As
described above, C. divergens and/or C. maltaromaticum are able
to form c. 20-370 mg tyramine kg_1 in these products,
corresponding to c. 1-55 mg of tyramine per meal. Consequently,
tyramine formation by carnobacteria in specific foods can represent
a hazard for sensitive individuals who might suffer migraine
headaches. This risk for sensitive individuals can be minimized
22 Introductory Food Microbiology
by restricting their consumption of fish and meat to products that
are newly processed, i.e. that are not close to the declared shelf-
life expiry day.
Growth of Carnobacterium spp. in food products may result
in accumulation of a range of volatile alcohols, ketones,
hydrocarbons, and other compounds. The metabolic reactions
leading to these products have not been studied in detail for
Carnobacterium, in contrast to the situation for other LAB, but
may be a result of NAD1-generating reactions or nonenzymatic
reactions. Several ketones have characteristic odors, but whether
they contribute to spoilage off-flavors as a result of carnobacterial
metabolic activity is not well understood. With regard to lipids, C.
maltaromaticum has been reported to hydrolyze tributyrin, a
triglyceride with butyrate as fatty acid, but it is not known whether
this lipase activity contributes to flavor changes of dairy, fish and
meat products. Carnobacterium divergens and C.maltaromaticum
were not able to limit oxidation of linoleic acid during growth.
This indicates that these species may not prevent quality
deterioration of meat products by lipid oxidation. Finally, C.
maltaromaticum has been shown to cause a softer texture of salmon
fillets when inoculated in high numbers.
It is worth noting that interspecies and intraspecies differences
exist regarding carnobacterial spoilage capacity. Laursen et al.
(2006) showed that C. mobile and a minor phenotypic cluster of
C. maltaromaticum did not spoil MAP shrimp, in contrast to what
was seen with C. divergens and the major phenotypic cluster of
C. maltaromaticum. It would be of interest to examine whether
similar heterogeneities exist for Carnobacterium spp. growing in
other dairy, fish and meat products. As environmental parameters
readily affect the metabolism of carbohydrates and amino acids,
it is not surprising that product-specific and storage-specific
conditions also play important roles. The ability of two C.
maltaromaticum strains to relatively rapidly spoil meat during
growth under aerobic conditions at 7 1C but not or only after a
long storage period in vacuum packs at 2 1C serves as an example.
Carnobacteria as Pathogenic Organisms and/or Probiotic Cultures
Two human clinical cases caused by opportunistic infection
with C. maltaromaticum and a Carnobacterium sp. have been
Introduction 23
described. Two clinical isolates of C. divergens have also been
obtained. Carnobacteria are not known members of the human
gastrointestinal microbial community, unlike several other LAB.
The exceptions are an unpublished study reporting an uncultured
Carnobacterium sp. clone from cow rumen and two studies
demonstrating the presence of carnobacteria in pig and horse
effluent-impacted environments. Nevertheless, it is safe to conclude
that the presence of Carnobacterium spp. in food does not present
a risk factor for human illness, except for the production of the
biogenic amine, tyramine, as discussed previously. Neither do
Carnobacterium spp. appear to present a risk for nocosomial
infections in hospitals and similar institutions, although they
have, in one instance, been isolated from contaminated blood
plasma.
The presence of virulence factors in carnobacteria is not well
documented. Thus C. maltaromaticum strains isolated from
diseased fish lacked hemolytic and phospholipolytic activities.
Carnobacterium viridans shows, however, b-hemolytic activity on
sheep blood agar. Although several isolates of carnobacteria
produce bacteriocins, none of these compounds has been reported
to exert a cytolytic activity as shown for the En. faecalis class I-
related bacteriocin cytolysin. Regarding chemotherapeutic agents,
fish pathogenic strains of C. maltaromaticum were resistant to
several of the agents widely used in aquaculture, such as
oxytetracycline, quinolones, nitrofurans and potentiated
sulfonamides, but sensitive to erythromycin. This species is not
resistant to lysozyme from salmonid eggs, which supports the
idea that it is not vertically transmitted, at least in this fish species.
Finally, it should be noted that two clinical isolates of C. divergens
have been reported to possess a gene encoding a new class of
penicillinase. It will be of interest to further explore the distribution
of this enzyme among carnobacterial species as well as its
functionality.
It has been documented that some strains of C. maltaromaticum
are pathogenic for several fish species, including Australian
salmonids, carp, rainbow trout, striped bass and channel catfish,
and salmon. Pathologic effects vary, and include, for example,
septicemia, peritonitis, exophthalmia, accumulation of ascitic fluid,
24 Introductory Food Microbiology
and hemorrhages. In most (but not all) instances, the virulence
appears, however, to be low and only causes problems in fish
undergoing severe stress, e.g. due to spawning. It is, therefore, not
surprising that C. maltaromaticum and C. maltaromaticum-like
strains are also found in the intestine or gills of healthy fish,
including Arctic charr, Atlantic cod, Atlantic salmon, brown
bullhead, trout, and various freshwater fish.
The presence of other carnobacterial species in healthy fish
has also been reported, including C. divergens, Atlantic salmon,
Atlantic cod and wolffish, trout, and freshwater fish, C.
alterfunditum-like [rainbow trout], C. funditum-like [Arctic charr
and Atlantic cod], C. gallinarum [Atlantic cod], C. inhibens
[Atlantic salmon], C. mobilelike [Arctic charr and Atlantic cod],
and Carnobacterium spp. [Arctic charr and carp]. Clearly, further
research is necessary to clarify under what circumstances
Carnobacterium spp. may be pathogenic for fish and whether the
infective capability is species and clone specific.
The reported pathogenicity of C. maltaromaticum has not
hindered research into the possibility of using other carnobacteria
as probiotic cultures in aquaculture, including Carnobacterium
sp., C. alterfunditum, C. divergens, and C. inhibens. Isolates of
these species, in addition to C. maltaromaticum, exhibit inhibitory
activity towards bacterial fish pathogens. Carnobacterium
divergens and C. maltaromaticum enhance the cellular and
humoral immune responses and cytokine expression ratios of
rainbow trout. Use of carnobacteria as probiotics leads to increased
survival of the fish in some instances [larvae of cod fry and
Atlantic salmon fry, rainbow trout, and salmon], but not in others
[larvae of cod fry, and rainbow trout]. One study showed that in
vitro exposure to C. divergens did not reverse the effects of bacterial
pathogens, although these were alleviated. Additional research is
necessary to validate the usefulness of carnobacteria as probiotic
cultures.
Carnobacterium maltaromaticum has also been reported to be
pathogenic for the fruit fly, Drosophila melanogaster, upon injection
into the thorax. This specific case of pathogenesis is probably best
understood as a result of opportunistic infection, although C.
maltaromaticum (but not C. divergens, C. gallinarum, C. inhibens,
Introduction 25
C. mobile or C. viridans) exerts chitinolytic acitivity, a feature that
can be perceived as targeting the chitin-containing exoskeleton of
insects. Indeed, insects may serve as a host for this species.
Genomics
The complete chromosomes of many LAB species have now
been sequenced. Currently, 19 complete genome sequences of
streptococci and 18 complete genome sequences of the
nonpathogenic LAB representing 14 species from the order
Lactobacillales are available. However, no genome sequence or
physical genetic map is available for Carnobacterium. The LAB
have relatively small genomes for nonobligatory bacterial parasites
or symbionts, ranging from c. 1.6 to c. 3Mb. For C. divergens and
C. pleistocenium, the genome size was estimated to 3.2Mb, and for
C. alterfunditum pf4T, it was estimated to be 2.9Mb. At the time
of writing (March 2007), there is only one Carnobacterium genome
sequencing project. Carnobacterium sp. AT7, a piezophilic strain
isolated from the Aleutian trench at a depth of 2500m, is being
sequenced for the Moore Foundation Marine Microbial Genome
Sequencing Project with a grant to the J. Craig Venter Institute. The
data already available indicate that the Carnobacterium sp. AT7
genome contains 2.4Mb and encodes 2388 proteins.
Carnobacterium sp. AT7 is closely related to C. alterfunditum and
C. pleistocenium.
To date, knowledge on the genes and DNA sequences of
Carnobacterium is comparatively sparse, concerning mostly
bacteriocin-related genes in the species C. divergens and C.
maltaromaticum and 16S rRNA and 16S-23S rRNA gene intergenic
spacer sequences. Genes involved in some important carnobacterial
metabolic traits have been sequenced. The genetic information
obtained is, for most sequences, derived from just one strain, and
may therefore be strain specific, as shown for a number of L.
monocytogenes genes.
Clearly, our knowledge of important phenotypes of strains
and species of Carnobacterium would benefit from the availability
of a complete genome sequence for a strain representative of C.
maltaromaticum, which is the species with most importance for
the food and aquaculture industries. The most extensive study on
26 Introductory Food Microbiology
phenotypic variation of C. maltaromaticum revealed that the
majority of strains studied belonged to one phenotypic cluster
(cluster H). Thus, in our opinion, a suitable candidate for a whole
genomic sequence could be either C. maltaromaticum LMG 22901
(isolated from pork meat), LMG 22899 (isolated from cod) or LMG
22898 (isolated from salmon), which all belong to this phenotypic
cluster. LMG 22901 does not harbor plasmids, and therefore
appears to be a prime candidate for such an endeavor.
CONCLUDING REMARKS
We have come some way towards understanding important
aspects of the presence of carnobacteria in foods and the
environment, particularly concerning the genetics of carnobacterial
bacteriocins and their application. However, there are important
areas where knowledge on these bacteria is limited. These include
the following:
(1) Distribution and Quantitative Microbial Ecology:
Carnobacterium spp. have not been reported from a number
of habitats, such as plants, fermented vegetable foods and
the gastrointestinal system of birds and mammals,
including humans, that have otherwise been associated
with several genera and species of LAB. This may, in some
instances, be due to faulty methodology for detecting
carnobacteria. Generally, however, our quantitative
understanding is limited regarding factors that influence
inactivation, survival or growth of carnobacteria in various
natural environments. In foods, more detailed information
is available, but mathematical models for quantitative
prediction of processing, product and storage effects on
growth and metabolic activity, including bacteriocin and
tyramine formation, awaits further development together
with recording of responses in databases. In addition,
further studies are needed to elucidate mechanisms
underlying the relationship between environmental
parameters and kinetic responses of carnobacteria.
(2) Routes of Food Contamination: To reduce the negative effects
of carnobacteria in foods (spoilage metabolites and
tyramine), further information on routes of contamination
Introduction 27
is desirable. In fact, very little research has been performed
so far concerning this aspect of carnobacteria in food.
(3) Metabolic Activity: It is known that carnobacteria, or at
least C. maltaromaticum and C. divergens, have the capacity
to produce a wide range of metabolites in chilled food.
Further studies on the importance of these metabolites
with respect to positive and negative sensory attributes of
various foods are, however, needed.
(4) Diversity: We are beginning to appreciate how interspecies
and intraspecies variation determine the positive and
negative effects of the presence of carnobacteria in food
and the environment, but we still do not understand how
such variation affects, for instance, their survival in food-
processing plants and contamination of foods. The
significance of such variation for their potential as fish
pathogens also needs to be substantiated. This is also the
case for their role as spoilage organisms in meat and fish
products, with the exception of a few products such as
vacuum-packed smoked salmon and cooked MAP shrimps.
(5) Genomics: Representative genome sequences would offer a
very valuable road map in order to answer many of the
outstanding questions associated with this important
genus.
Staphylococcus Aureus Growth Boundaries: Moving towards
Mechanistic Predictive Models Based on Solute - Specific Effects
The formulation of shelf-stable intermediate-moisture products
is a critical food safety issue. Therefore, knowing the precise
boundary for the growth-no-growth interface of Staphylococcus
aureus is necessary for food safety risk assessment. This study
was designed to examine the effects of various humectants and to
produce growth boundary models as tools for risk assessment.
The molecular mobility and the effects of various physical
properties of humectants, such as their glass transition
temperatures, their membrane permeability, and their ionic and
nonionic properties, on S. aureus growth were investigated. The
effects of relative humidity (RH; 84 to 95%, adjusted by sucrose
plus fructose, glycerol, or NaCl), initial pH (4.5 to 7.0, adjusted by
28 Introductory Food Microbiology
HCl), and potassium sorbate concentration (0 or 1,000 ppm) on
the growth of S. aureus were determined. Growth was monitored
by turbidity over a 24-week period. Toxin production was
determined by enterotoxin assay. The 1,792 data points generated
were analyzed by LIFEREG procedures, which showed that all
parameters studied significantly affected the growth responses of
S. aureus. Differences were observed in the growth-no-growth
boundary when different humectants were used to achieve the
desired RH values in both the absence and the presence of
potassium sorbate. Sucrose plus fructose was most inhibitory at
neutral pH values, while NaCl was most inhibitory at low pH
values. The addition of potassium sorbate greatly increased the
no-growth regions, particularly when pH was <6.0. Published
kinetic growth and survival models were compared with boundary
models developed in this work. The effects of solutes and differences
in modeling approaches are discussed.
Olsen et al. reported that Staphylococcus aureus was involved
in 42 documented outbreaks of food-borne poisoning in the United
States, with 1,413 cases and 1 death occurring from 1993 to 1997.
S. aureus has been estimated to cause approximately 185,000
illnesses, 1,750 hospitalizations, and 2 deaths per year in the
United States, all from consumption of contaminated foods. Food-
borne staphylococcal poisoning, caused by the ingestion of one or
more preformed toxins in food contaminated with S. aureus, is one
of the most prevalent causes of gastroenteritis worldwide. Recently,
powdered milk produced in Taiki, Hokkaido, Japan, was linked
to contaminated dairy products that made more than 14,700 people
in Japan ill in June and July of 2000. Health authorities determined
that dry skim milk powder was contaminated with staphylococcal
enterotoxin A.
Scott related the relative vapor pressures (rvp's) of foods to the
thermodynamic activity of water, using the definition aw = p/po,
where aw is the water activity as derived from the laws of
equilibrium thermodynamics, p is the vapor pressure of the sample,
and po is the vapor pressure of pure water. He showed a clear
correlation between the rvp of the growth medium and the rate of
S. aureus growth. In the field of food science, the general acceptance
and application of this concept relating the rvp's of foods to a
Introduction 29
minimum aw for microbial growth began with the review by Scott.
The important point is that the equality of aw with p/po is based
on the assumption of the existence of thermodynamic equilibrium
and that the value is true only for specified conditions of
temperature and pressure. Although dilute solutions obey the
laws of equilibrium thermodynamics, the same is not necessarily
true for intermediate-moisture systems and is even less likely for
low-moisture systems, the properties of which are determined
mainly by slow reaction rates. The concept of aw then becomes
meaningless, because the measured rvp of water is no longer the
same as the equilibrium vapor pressure. The equation aw = p/po
therefore applies only in the range 0.95 <aw <1.0, where equilibrium
is established rapidly. With many foods, this assumption of
thermodynamic equilibrium is violated, so the appropriate term is
rvp or relative humidity (RH) rather than aw.
For the past several decades, in the food science literature, the
phenomenon denoted by the term "aw" has been widely used to
predict microbial growth as well as the relationship between many
common food deterioration reactions and aw. Although RH is a
better indicator of food stability and safety than the water content
of a system, it is not always a reliable predictor. For example,
several authors have reported that the growth boundaries for
various genera of microorganisms differ depending on the type of
humectant used to depress RH rather than the absolute value of
RH. The literature on microbial osmoregulation via compatible
solutes has also shown that the type of humectant, rather than the
absolute value of RH, is critical.
In recent years, increasing evidence based on glass transition
theory has shown that molecular mobility (Mm) may be an attribute
that deserves further attention, as it is related to many important
diffusion-limiting properties of foods. This is because the
temperature at which a viscous liquid changes to a "glass" (glass
transition temperature [Tg]), which governs Mm, is a solute-specific
property that is inversely linearly related to the measured RH of
a system. Although the use of RH has served the food industry
well for the past several decades since Scott introduced the concept
of aw in 1953, further study to understand these solute-specific
effects on microbial growth should continue.
30 Introductory Food Microbiology
Microbial growth models are typically developed when the
objective is to understand the responses of microorganisms when
part of the range of conditions studied permits growth to occur.
Such models can describe the increase in numbers with time
(kinetic models), the conditions allowing growth or no growth
(boundary models), or the chance of growth (probabilistic models).
Kinetic growth models are typically based on growth curves over
a range of conditions and can allow growth rates to be predicted.
In the 1980s, the United Kingdom Ministry of Agriculture, Fisheries,
and Food and the U.S. Department of Agriculture (USDA) funded
programs to develop kinetic models for the growth, survival, and
death of food-borne pathogens, including S. aureus. This work
resulted in suites of models which are currently available. NaCl
was the humectant chosen to control the RH in these models, and
the resulting kinetic models do not allow the growth-no-growth
boundary to be estimated.
The statistical effects and interactions of RH, initial pH, and
potassium sorbate concentration on the boundary for S. aureus
growth were modeled in this study by using the approach
described previously. Additionally, the concepts of Mm and the
effects of various physical properties of humectants, such as the
Tg, membrane permeability, and ionic and nonionic properties of
solutes, on S. aureus growth were investigated by controlling the
RH with sucrose-fructose (50:50, wt/wt), glycerol, or NaCl.
MATERIALS AND METHODS
Preparation of Bacteria and Media
Five S. aureus strains were used as a cocktail in this study:
ATCC 13565, ATCC 14458, and ATCC 27664, from the American
Type Culture Collection, Manassas, Va.; D-2, from Toxin
Technology, Inc., Sarasota, Fla.; and A-100, from the U.S. Army
Natick Laboratories, Natick, Mass. A cocktail of strains was chosen
for use because it helps to overcome the variability between strains
and increase the chance of detecting the fastest growth at different
points in the experimental matrix. These benefits of using a cocktail
help to create a fail-safe model without repeating the experiment
many times with different strains. All strains showed typical growth
on Baird-Parker agar (Difco, Detroit, Mich.) plates and were
Introduction 31
coagulase positive. Their identification as S. aureus was confirmed
by the use of a Riboprinter. Stock cultures were grown overnight
at 37°C in brain heart infusion (BHI) broth (Difco), suspended in
solution containing a 2:1 BHI broth-glycerol solution, and stored
at -80°C until needed.
For this study, BHI broth was reconstituted with the
appropriate ratio of water to sucrose-fructose (50:50, wt/wt) or to
NaCl to achieve the desired RHs for the study (84, 88, 92, or 95%).
Appropriate amounts of 10% stock solutions of potassium sorbate
(Hoechst, Frankfurt, Germany) were added to achieve a final
concentration of 1,000 ppm. The pH was adjusted to 4.5, 5.0, 6.0,
or 7.0 by using 0.1 or 1.0 N HCl. The final RH was determined by
using an Aqualab CX-2 aw meter. The RHs of the media measured
at ambient temperature differed by no more than ±0.003% relative
to those measured at 37°C. Each of the 64 broths was filter sterilized
and stored in screw-cap tubes at 4°C until needed.
Bioscreen Microtiter Plate Preparation, Incubation, and
Measurement
The five stock cultures were removed from -80°C storage, and
one loopful of each was transferred separately into 9 ml of BHI
broth. The inoculated broths were incubated for 18 h at 37°C. The
five cultures were individually adjusted to optical densities (ODs)
at 530 nm of 0.750 to 0.780 (measured with a Perkin-Elmer model
35 spectrophotometer) with 0.1% sterile peptone to achieve a
concentration of approximately 108 CFU/ml. Diluted cultures (10
ml) were transferred into a single sterile test tube and vortexed to
create the S. aureus cocktail used in our experiments. Three 1/10
dilutions with sterile 0.1% peptone water were used to make a
final working cocktail with a concentration of approximately 105
CFU/ml. This working cocktail was kept on ice until it was used
to inoculate the various media used in the experiments. Samples
(1 ml) of the working cocktail were taken, serially diluted, spread
plated onto Baird-Parker agar plates, and incubated at 37°C for 48
h, and cells were counted to determine their initial concentrations.
Each of the 64 broths (10 ml) was aseptically transferred to a
sterile tube, and the S. aureus cocktail (100 µl) was added to the
broth to achieve an initial concentration of approximately 103
32 Introductory Food Microbiology
CFU/ml. The inoculated broths (400 µl) were aseptically
transferred into sterile 100-well microtiter plates, with eight replicate
wells per medium. The outer wells of the microtiter plates were
filled with uninoculated medium to act both as a partial moisture
barrier and as uninoculated controls. Each plate contained media
at only one RH level. A piece of sterile thermaseal film was placed
on top of the open wells under the lid during incubation. The
plates were placed on a rack in a sealed plastic container that
contained the appropriate saturated salt slurries needed to
maintain the environmental RH and thereby ensure the stability
of the RHs of the media. The saturated salt solutions used were
as follows: ZnSO4 · H2O (83% RH at 37°C), KNO (89% RH at
37°C), KPO4 (93% RH at 37°C), and K2Cr2O7 (96% RH at 37°C).
The containers were closed and incubated at 37°C. Plates were
periodically removed and shaken for 60 s, and the ODs of the
1,024 individual wells were measured for up to 24 weeks by using
the wideband filter on the Bioscreen C system (Labsystems Oy,
Helsinki, Finland). The experiments were repeated on separate
dates. A total of 1,024 wells were monitored.
Data Collection
The S. aureus population was considered to have grown when
the Bioscreen OD measured with a wideband filter (ODwb)
increased from an initial reading between 0.200 and 0.220 to an
ODwb of 0.350 or higher. The time to growth (TTG) was determined
by calculating the geometric mean of the time of the last
measurement that showed no growth (ODwb < 0.350) and the first
time point that showed growth (ODwb > 0.350). In instances of no
growth, TTG was censored at the final measurement time of 168
days. A threshold ODwb value of 0.350 was used to score wells
with S. aureus growth (ODwb ³ 0.350) and wells with no growth
(ODwb < 0.350). This threshold value was determined as discussed
in detail by Stewart et al. (40). In brief, ODwb measurements were
compared with plate counts, and an ODwb of 0.350 was identified
as the lowest ODwb value at which an increase in turbidity
regularly correlated to an increase in plate counts.
OD measurements were transferred from the Bioscreen C ASCII
file to a DOS text editor and then imported into Statistica software
Introduction 33
for initial analysis. Data were transferred to a Microsoft Excel
spreadsheet to determine TTG and then to SAS software for
modeling by the LIFEREG procedure.
An additional data set was also included in the development
of the model. This data set included information on TTG for the
same S. aureus cocktail obtained by an identical experimental
procedure, with the exception that the humectant used to achieve
the desired RH of the BHI broth was glycerol, a glass-forming
membrane-permeable solute. The experimental design of Stewart
et al. included three levels of potassium sorbate (0, 500, and 1,000
ppm). A total of 768 data points from the glycerol data set were
included in the data analysis.
Statistical Analysis
The TTG data along with a censoring indicator, the percent
RH, the pH, the potassium sorbate level, and the humectant type
(sucrose-fructose, glycerol, or NaCl) were input data for the
construction of the growth boundary model. RH, pH, and
potassium sorbate values were normalized [(RH - 89.75)/4.15],
[(pH - 5.625)/0.96], [(sorbate - 500)/500] prior to model development.
The TTG results from replicate test wells were averaged before
modeling to minimize the effect of measurement error. The SAS
LIFEREG procedure was used to develop predictive models of the
natural logarithm of TTG (ln TTG) as a function of the factors
percent RH, pH, preservative level, and humectant type. The
resulting model can easily be transformed into a regular time
scale. It should be noted that the type of humectant is considered
a categorical variable (i.e., nonnumeric) in the SAS procedure. In
growth modeling, there are often conditions under which no
growth occurs; therefore, TTG may be missing or censored. Under
these conditions, ordinary least-squares regression is not applicable
and special procedures are required.
The LIFEREG procedure can accommodate such censored data
and uses maximum-likelihood estimation methods to find
regression coefficients. Further details of this method of analysis
are given by Allison (1). The LIFEREG procedure works best with
many replicates in which the intervals between growth parameters
are equally spaced, with approximately 50% of conditions limiting
34 Introductory Food Microbiology
growth. The ranges of the parameters studied (RH, pH, and
preservative levels) were chosen to satisfy these requirements.
Toxin Production
Toxin production for experimental conditions close to the
growth-no-growth boundary was measured by the VIDAS staph
enterotoxin assay. The VSEA is a qualitative enzyme-linked
fluorescent immunoassay performed in an automated mini-VIDAS
instrument. The media from the microtiter plates were removed for
assay after the final ODwb measurements were taken (24 weeks).
The broth from each of the eight replicate wells was removed via
pipette, combined into two microcentrifuge tubes, and centrifuged
for 10 min. The supernatants were combined, and the pH values
were adjusted with 1 N NaOH to 6.0 to 8.0. Samples (0.5 ml) were
placed into the appropriate wells in a test strip, and the assays
were run in the mini-Vidas. Positive and negative controls were
run with each set of test strips. The VSEA is sensitive to 1 ng of
toxin/ml of sample.
RESULTS
Model Development and Diagnostics
Models were developed from the 1,792 data points generated.
The models included all three main effects: percent RH, pH, and
potassium sorbate concentration ("sorb" in the equations); their
quadratic effects (RH x RH = RH2; pH x pH = pH2; and sorb x
sorb = sorb2); and the three two-factor interactions (RH x pH, RH
x sorb, and pH x sorb). The impact of the humectant type on the
main effects and interactions was assessed by including indicator
variables in the model.
These main effects, quadratic effects, and two-factor interactions
are collectively referred to as the factors. SAS LIFEREG allows the
user to specify the error distribution to account for the variation
in TTG not explained by the regression model. For model
development purposes, Weibull, lognormal, and loglogistic
distributions were considered. All three distributions gave very
similar models (the regression coefficients were similar in sign
and magnitude), and we selected the loglogistic distribution model,
which has been used in previous work.
Introduction 35
Model development is an iterative process. The first analysis
of the data created models which were all-inclusive; this allowed
for the determination of those factors which had a significant
effect on the growth of S. aureus. All of the main effects, quadratic
effects, and two-factor interactions were significant (P < 0.005),
except for the potassium sorbate concentration quadratic term and
the interaction between RH and potassium sorbate concentration.
The insignificant factors were dropped, and the data were
reanalyzed to develop the final models. SAS LIFEREG outputs a
table of regression coefficient estimates and approximate chi-square
distribution P values for each factor in the model. The relative
importance of each factor can be judged by its P value: factors with
low P values have the greatest influence on and are most predictive
of the TTG.
The resulting model equation for the use of sucrose-fructose as
the humectant was as follows (where TTG is measured in days,
RH is a percentage, and the potassium sorbate concentration is
measured in parts per million): ln TTG = 4.46 - (5.90 x RH) - (2.62
x pH) + (1.42 x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH
x pH) - (0.94 x pH x sorb).
The resulting model equation for the use of glycerol as the
humectant was ln TTG = 2.24 - (3.86 x RH) - (2.29 x pH) + (1.00
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
x pH x sorb).
The resulting model equation for the use of NaCl as the
humectant was ln TTG = 3.15 - (3.59 x RH) - (3.81 x pH) + (1.64
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
x pH x sorb).
From the model equations, the TTG contour plots for sucrose-
fructose, glycerol, and NaCl were created. The contour plots were
created by generating a grid of evenly spaced points in the
experimental design space of pH, RH, and potassium sorbate
concentration. With a grid of 2,268 points, a predicted TTG was
calculated for each point and humectant by using the appropriate
model shown above.
The grid of points and predicted TTG values was input into
the SAS procedure "proc gcontour". Diagnostic methods to check
36 Introductory Food Microbiology
for departures from model assumptions can be used in a manner
that is similar to their use in ordinary regression analysis. However,
when nonnormal distributions are being fitted, an appropriate
standardization of residuals must be used (25). The SAS reliability
procedure was used for residual analysis. A probability plot of
standardized residuals was examined, and no unusual patterns
or anomalies were detected.
Model Development and Diagnostics
Models were developed from the 1,792 data points generated.
The models included all three main effects: percent RH, pH, and
potassium sorbate concentration (“sorb” in the equations); their
quadratic effects (RH x RH = RH2; pH x pH = pH2; and sorb x sorb
= sorb2); and the three two-factor interactions (RH x pH, RH x
sorb, and pH x sorb). The impact of the humectant type on the
main effects and interactions was assessed by including indicator
variables in the model. These main effects, quadratic effects, and
two-factor interactions are collectively referred to as the factors.
SAS LIFEREG allows the user to specify the error distribution to
account for the variation in TTG not explained by the regression
model. For model development purposes, Weibull, lognormal, and
loglogistic distributions were considered. All three distributions
gave very similar models (the regression coefficients were similar
in sign and magnitude), and we selected the loglogistic distribution
model, which has been used in previous work.
Model development is an iterative process. The first analysis
of the data created models which were all-inclusive; this allowed
for the determination of those factors which had a significant effect
on the growth of S. aureus. All of the main effects, quadratic effects,
and two-factor interactions were significant (P < 0.005), except for
the potassium sorbate concentration quadratic term and the
interaction between RH and potassium sorbate concentration. The
insignificant factors were dropped, and the data were reanalyzed
to develop the final models. SAS LIFEREG outputs a table of
regression coefficient estimates and approximate chi-square
distribution P values for each factor in the model. The relative
importance of each factor can be judged by its P value: factors with
low P values have the greatest influence on and are most predictive
of the TTG.
Introduction 37
The resulting model equation for the use of sucrose-fructose as
the humectant was as follows (where TTG is measured in days,
RH is a percentage, and the potassium sorbate concentration is
measured in parts per million): ln TTG = 4.46 - (5.90 x RH) - (2.62
x pH) + (1.42 x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH
x pH) - (0.94 x pH x sorb).
The resulting model equation for the use of glycerol as the
humectant was ln TTG = 2.24 - (3.86 x RH) - (2.29 x pH) + (1.00
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
x pH x sorb).
The resulting model equation for the use of NaCl as the
humectant was ln TTG = 3.15 - (3.59 x RH) - (3.81 x pH) + (1.64
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
x pH x sorb).
From the model equations, the TTG contour plots for sucrose-
fructose, glycerol, and NaCl were created. The contour plots were
created by generating a grid of evenly spaced points in the
experimental design space of pH, RH, and potassium sorbate
concentration. With a grid of 2,268 points, a predicted TTG was
calculated for each point and humectant by using the appropriate
model shown above. The grid of points and predicted TTG values
was input into the SAS procedure “proc gcontour”. Diagnostic
methods to check for departures from model assumptions can be
used in a manner that is similar to their use in ordinary regression
analysis. However, when nonnormal distributions are being fitted,
an appropriate standardization of residuals must be used. The
SAS reliability procedure (SAS Institute, Inc.) was used for residual
analysis. A probability plot of standardized residuals was
examined, and no unusual patterns or anomalies were detected.
MODEL PREDICTIONS AND COMPARISON WITH
PUBLISHED WORK
As conditions became increasingly unfavorable for growth,
the contour lines drew closer together, indicating that conditions
were approaching those that do not allow growth. As the RH or
pH of the system decreased, a corresponding increase in TTG was
seen and the no-growth area of the contour plot increased in size.
The addition of potassium sorbate at low pH values (4.5 to 5.5)
38 Introductory Food Microbiology
dramatically changed the contour plots in the low-pH area of the
plot. The effect of potassium sorbate decreased as the pH value
approached 7.0.
Differences were observed in the growth-no-growth boundary
when different humectants were used to achieve the desired RH
values in both the absence and the presence of potassium sorbate.
The use of sucrose-fructose as the humectant resulted in the greatest
delay of S. aureus growth under the highest pH and RH combination
(i.e., pH 7.0 and 88% RH) either with or without potassium sorbate.
Even with the addition of potassium sorbate at 1,000 ppm at pH
7.0, glycerol and NaCl were less effective in delaying S. aureus
growth than sucrose-fructose with no added preservative. At pH
values above ca. 5.3 and in the absence of potassium sorbate, a
rank order was observed, with NaCl being the least inhibitory,
followed by glycerol, and with sucrose-fructose being the most
inhibitory; by contrast, at pH values below ca. 5.3, NaCl showed
dramatically increased inhibition.
With the addition of potassium sorbate at 1,000 ppm, the same
rank order occurred at pH values above ca. 6.1. It has generally
been accepted that the limits for the growth of S. aureus are 85%
RH when NaCl is used as the humectant and 89% when glycerol
is used.
Our models showed the limiting RH for growth to be
approximately 88% (pH 7.0) when sucrose-fructose was used, 86%
when glycerol was used, and 85% when NaCl was used. The
limiting RH with glycerol as the humectant was markedly different
from that reported by Marshall et al., who found that the growth
limits were 89 and 86% RH when glycerol and NaCl were used
as the humectants, respectively.
This disagreement may be due to strain variation or to the fact
that only quarter-strength BHI broth was used in the work by
Marshall et al. while full-strength BHI broth was used in our
model system. Whatever the reason, this result suggests that if
glycerol makes a large contribution to the humectant content of a
food product, it would be wise to consider the target RH carefully.
Additionally, there is published literature that describes the
ability of S. aureus to grow in various food systems, mainly meats
Introduction 39
and cheeses. Unfortunately, these data cannot be used for model
validation, as RH was not reported and/or other parameters not
included in our models (such as oxygen concentration or
temperature) were varied. Additionally, the pH and/or ingredient
lists of the products used in these studies were not reported.
NaCl was most inhibitory at pH values below ca. 5.3 in the
absence of potassium sorbate and at pH values below ca. 6.1 in the
presence of potassium sorbate at 1,000 ppm. For example, no growth
was seen even at conditions of 95% RH. This finding disagrees
with predictions made by the USDA’s pathogen modeling program
(PMP) version 5.1, which indicates that at 95% RH and pH 4.5,
S. aureus will grow from 103 to 107 CFU/ml in 1.65 days.
Comparison of the sucrose-fructose and glycerol models developed
in this study with other published work is difficult, as the majority
of previously published studies have used NaCl to control the RH
of the system.
As discussed previously, differences in the physical properties
of humectants can lead to various responses, even when RH is
constant. The differences in modeling approaches also make
comparisons difficult. Neither the Food Micro Model of the United
Kingdom Ministry of Agriculture, Fisheries, and Food nor PMP
was created from data sets containing many replicated experiments
where the bacteria were subjected to stressed conditions. There is
also the danger of extrapolation beyond the conditions under which
data were actually collected. The PMP gives a prediction for growth
under the conditions of pH 4.5 and 95% RH (with NaCl as the
humectant), although data were not collected for this combination
of parameters. A full factorial design was utilized for the boundary
models presented in this paper, and the models were constructed
by using interpolation only.
Enterotoxin Assays
When the final ODwb was close to 0.350, the samples were
tested for enterotoxin. Assays were also conducted for all samples
with combinations of 84% RH and pH 7.0 or 84% RH and pH 4.5.
A total of 94 assays were run, and in every case where the data
were scored as “no growth,” the toxin assay results were negative
for the presence of toxin, while in every case where the data were
40 Introductory Food Microbiology
scored as “growth,” the toxin assay results were positive for the
presence of toxin. This further supports the choice of an ODwb of
0.350 as an appropriate cutoff value for TTG in the individual
wells and greatly increases confidence in the practical value of the
models.
DISCUSSION
Using RH without considering the effect of the type of
humectant has often led to difficulties in predicting the stability
and microbial safety of intermediate-moisture foods. For example,
S. aureus is highly salt tolerant and has been reported to grow at
RHs as low as 85% in NaCl concentrations up to 25% (wt/wt);
however, RH values limiting growth are typically higher when
humectants other than NaCl are used to control RH.
Notermans and Heuvelman showed that the RH limiting S.
aureus growth varied depending on whether sucrose (with which
it was reported that an aw of 0.90 limited growth) or NaCl (with
which it was reported that an aw of 0.87 limited growth) was used
as the humectant.
Marshall et al. reported that the level of inhibition of the growth
of S. aureus by glycerol at neutral pH was about 10% greater than
that caused by NaCl in the rvp range (reported as aw) from 0.96
to 0.90.
Clearly, the choice of humectant used to depress the RH in a
food system is critical and its effects on microbial survival and/
or growth depend not only on the RH but also on the chemical and
physical properties of the humectant, as well as the biological
effects of the humectants on the microorganisms.
As stated previously, although RH is a better indicator for
food stability and safety than water content, it is not always a
reliable predictor of stability. As described by Fennema, increasing
evidence has shown that the Tg and Mm may be important means
to explain many diffusion-limiting properties of food.
Interest in Mm was first generated by Luyet and associates in
the United States as well as by Rey in France, who showed
relationships between Mm and biological materials, while basic
concepts relating nonequilibrium Mm in synthetic amorphous
Introduction 41
polymer systems were formulated by Ferry. The importance of the
role of glassy states in various sugar-containing foods was
described by White and Cakebread, who suggested that these states
played an important role in the stability and processability of
many food systems.
The basic principles that apply to synthetic amorphous
polymers, as described by Ferry and others, also apply to the
behavior of glass-forming foods. Foods of this type include starch-
containing products like pasta and bread, boiled confections, protein-
based foods, intermediate-moisture foods, and dried, frozen, or
freeze-dried foods.
The RH and Mm approaches to food stability are
complementary, not contradictory. RH, referred to as aw, focuses
on the availability of water in a food matrix, while Mm defines this
availability in terms of microviscosity and diffusibility, with the
latter being dependent on the properties of water as a plasticizer
to depress the Tg of the food matrix.
Humectants with various characteristics were employed in
this study specifically to begin to explore the influence of various
physical properties of humectants, such as their Tgs, on microbial
growth under osmotically stressed conditions. When glass-forming
solutes are used to achieve desired RH values in systems, the
underlying Tg and the relaxation behavior of the glasses become
the controlling parameters of the system. Within each glass-forming
system, Tg is inversely linearly related to the measured RH of the
system.
However, across different glass-forming solute systems, Tg
increases as the weight-average molecular weight of the solutes
increases. As Tg increases, the viscosity of the system increases,
which in turn decreases the mobility of molecules in the system
(including the mobility of water molecules). This relative decrease
in mobility between glass-forming systems may partially explain
the varied responses of microorganisms in systems with matching
RH values but in which different humectants were utilized to
achieve the targeted RH.
The humectants used in this study included sucrose-fructose
(non-membrane-permeable glass-forming solutes with a reference
42 Introductory Food Microbiology
Tg of approximately -32°C), glycerol (a membrane-permeable glass-
forming solute with a reference Tg of approximately -65°C), and
NaCl (a non-glass-forming ionic solute which may affect various
ion channels in the cell).
The limits of microbial growth cannot be predicted by the RH
of the system alone, as seen by comparison of the growth
boundaries developed with the three types of humectants used in
this study. Instead, the RH of the system should be considered in
conjunction with the physical properties of the solutes and their
effects on the biological systems. Ionic solutes, such as NaCl, may
place both ionic and osmotic stresses on the cell. Of the three
humectants studied, NaCl was the most inhibitory below pH
values of ca. 5.3 in the absence of potassium sorbate.
The no-growth region of NaCl was expanded to pH values
below ca. 6.1 with the addition of potassium sorbate at 1,000 ppm.
This exaggerated interaction between NaCl, low pH, and potassium
sorbate may suggest that the Na+ proton antiport may be the
critical ion channel involved.
These data suggest that growth inhibition may be due to
overwhelming ionic stress. The membrane permeability of solutes
such as glycerol may influence the degree of osmotic stress
experienced by the cell.
Additionally, the influence of the Tg of the solute on Mm
directly affects the degree of osmotic stress a specific solute (e.g.,
sucrose-fructose or glycerol) places on the cell. Sucrose-fructose
with a higher Tg than that of glycerol (and, therefore, a larger
constraint on Mm than that of glycerol) was most inhibitory at pH
values above ca. 5.3 or 6.1 in the absence or presence of potassium
sorbate, respectively.
Glycerol is also membrane permeable, and therefore, the cell
may experience less osmotic stress than when sucrose-fructose
(membrane impermeable) is utilized. Physiological effects on S.
aureus cells have been shown to differ when various solutes are
used to cause osmotic stress. Cellular transport systems responsible
for the uptake of compatible solutes by S. aureus during osmotic
stress have been shown to respond differently to the stresses caused
by NaCl, sucrose, and glycerol.
Introduction 43
Additionally, the effects of osmotic stress on S. aureus cell size
also differ depending on whether NaCl, sucrose, or glycerol is
used as the humectant. Although these models are empirical in
nature, they demonstrate that the limits of growth based on RH
would be different when various humectants were used to control
RH, so comparing studies based on RH alone may lead to false
conclusions.
These results also allow us to begin to explore the possibility
of moving towards a mechanistic approach to creating predictive
models. Further work in this laboratory to study solute-specific
effects on cellular energy (ATP levels) is ongoing in an effort to
continue to work towards a better understanding of the
mechanism(s) of bacterial inactivation.
Three statistical models describing the growth boundaries for
S. aureus with respect to RH controlled by three types of humectants,
pH, and preservative were developed, and results were confirmed
by toxin assays. Building on previous work that has established
regions of growth and no growth, these mathematical models
predict actual TTG as a function of the humectant type, RH, pH,
and preservative level.
These predictions can be used to develop much more detailed
and informative boundary surfaces. Good agreement between all
three of these growth boundary surfaces and PMP growth kinetic
models was found in the relatively unstressed regions. Growth
boundary predictions were outside the confidence limits of the
PMP growth kinetic model under more stressful conditions, even
when NaCl was used as the humectant.
Kinetic models and growth boundary models can be used as
complementary tools for the development of microbially safe
products with short and long shelf lives. These models will allow
product developers to visualize the “safe space” for the formulation
of shelf-stable intermediate-moisture foods by employing these
preservation factors, will allow microbiologists to assess risks
more effectively over a wide range of products, and will ultimately
allow the consumer to have greater assurance of food safety.
The use of sucrose-fructose as the humectant results in the
greatest delay of S. aureus growth when the highest pH and RH
44 Introductory Food Microbiology
(i.e., pH 7.0 and 88% RH) are used either with or without potassium
sorbate. NaCl is the most inhibitory humectant at pH values below
ca. 5.3 in the absence of potassium sorbate and at pH values below
ca. 6.1 in the presence of potassium sorbate at 1,000 ppm. Solute-
specific effects on bacterial growth were evident, as growth
boundaries differed at the same RH values when different
humectants were used.
Over the almost 50 years since Scott first published his work
on the water relations of S. aureus, measurement techniques and
the fields of physical chemistry, microbial physiology, and microbial
modeling have progressed significantly. Scott was well aware that
aw would not necessarily be adequate to describe all of the
properties of solutions that influence microbial growth, metabolism,
and survival. He suggested that, with further work to fill some of
the obvious gaps in our knowledge, we would discover whether
aw could be replaced by something more meaningful. With careful
experimentation and understanding, we will continue to fill the
gaps and allow the food industry to produce the highest-quality
products while ensuring food safety.
Role of Ctc from Listeria Monocytogenes in Osmotolerance 45
2
ROLE OF CTC FROM LISTERIA
MONOCYTOGENES IN
OSMOTOLERANCE
Listeria monocytogenes is a food-borne pathogen with the ability
to grow under conditions of high osmolarity. In a previous study,
we reported the identification of 12 proteins showing high
induction after salt stress. One of these proteins is highly similar
to the general stress protein Ctc of Bacillus subtilis. In this study,
induction of Ctc after salt stress was confirmed at the
transcriptional level by using RNA slot blot experiments. To explore
the role of the ctc gene product in resistance to stresses, we
constructed a ctc insertional mutant. No difference in growth was
observed between the wild-type strain LO28 and the ctc mutant
either in rich medium after osmotic or heat stress or in minimal
medium after heat stress. However, in minimal medium after
osmotic stress, the growth rate of the mutant was increased by a
factor of 2. Moreover, electron microscopy analysis showed impaired
morphology of the mutant grown under osmotic stress conditions
in minimal medium. Addition of the osmoprotectant glycine betaine
to the medium completely abolished the osmotic sensitivity
phenotype of the ctc mutant. Altogether, these results suggest that
the Ctc protein of L. monocytogenes is involved in osmotic stress
tolerance in the absence of any osmoprotectant in the medium.
INTRODUCTION
Listeria monocytogenes is a food-borne pathogen widely
distributed in the environment. This microorganism is of particular
46 Introductory Food Microbiology
concern in the food industry because of its ability to survive and
frequently to grow under a wide range of adverse conditions used
to preserve food such as low temperature, low pH, and high
osmolarity. Growth of L. monocytogenes has been reported at NaCl
concentrations as high as 10%.
Most bacteria cope with elevated osmolarity in the environment
by intracellular accumulation of compatible solutes, called
osmolytes. Among the compatible solutes efficient in L.
monocytogenes, two quaternary amines, glycine betaine and
carnitine, are the most effective. The accumulation of these
osmoprotectants in L. monocytogenes occurs through osmotic
activation of their transport from the medium rather than through
de novo synthesis. Accumulation of glycine betaine and carnitine
occurs via at least two glycine betaine transporters encoded by the
betL gene and the gbu operon and one carnitine transporter encoded
by the opuC operon. A betL knockout mutant and a mutant of gbu
obtained by transposition were significantly affected in their abilities
to accumulate glycine betaine and were unable to withstand
concentrations of salt as high as the isogenic parent strain can
withstand. Similarly, a mutant with an insertional inactivation of
opuCA was defective in the uptake of carnitine and had impaired
growth at high osmolarity. Proline has been identified as an
osmolyte for L. monocytogenes. The proline transport mechanism
has not been characterized yet. However, the proBA operon, coding
for the enzymes that catalyze the two first steps of proline
biosynthesis, has recently been identified. Disruption of this operon
significantly reduced the growth of the corresponding mutant at
high salt concentrations. However, little information is available
concerning other mechanisms that take place in L. monocytogenes
to enable the organism to cope with osmotic stress, especially
when osmolytes are not available in the environment.
In a previous study, by proteomic analysis and mass
spectrometry or N-terminal sequencing, 12 proteins showing high
induction after a salt stress were identified. One of these proteins
is similar to the Ctc protein of Bacillus subtilis, a general stress
protein which belongs to the L25 family of ribosomal proteins. In
B. subtilis, the ctc gene is induced in response to osmotic, heat, and
oxidative stress and glucose limitation (14, 41). Regulation of the
Role of Ctc from Listeria Monocytogenes in Osmotolerance 47
expression of the ctc gene of B. subtilis occurs via the sB RNA
polymerase subunit. The ctc promoter was one of the first sB-
dependent promoters identified and for this reason has been
extensively studied. It is the best-characterized sB-dependent
promoter and has become the promoter of choice in nearly all
investigations of sB regulation. In contrast to the wealth of
information regarding the ctc promoter, the function of the ctc
product itself in B. subtilis is less clear and seems to be dispensable.
Only reduced sporulation efficiency at high temperatures has been
observed in a ctc null mutant.
To investigate the function of the Ctc protein in L. monocytogenes,
especially with regard to the stress resistance of the bacterium, we
analyzed the sequence of the corresponding gene and inactivated
it by insertional mutation. Physiological studies indicate that the
Ctc protein facilitates growth in minimal medium under conditions
of high osmolarity and in the absence of an osmoprotectant. This
is the first time that a role has been assigned to Ctc, which belongs
to a family of unknown proteins.
MATERIALS AND METHODS
Bacterial Strains and Plasmids
L. monocytogenes LO28, a clinical isolate, was obtained from P.
Cossart (Institut Pasteur, Paris, France). Bacterial plasmids were
propagated in Escherichia coli TG1. Plasmid pHT315 was used as
a cloning vector for sequencing, and plasmid pAUL-A was used
for gene disruption.
Culture Media and Stress Conditions
Cells were grown on complex culture media: brain heart
infusion (BHI) broth or agar (Difco Laboratories, Detroit, Mich.). A
chemically defined minimal medium called Improved Minimal
Medium, or IMM, was also used, but pyridoxal, which is not
necessary for growth, was not added. Different stress conditions
used for growth rate or microscopy experiments were induced
according to the following procedure. Overnight cultures of strain
LO28 or the ctc mutant were used to inoculate fresh medium at an
initial optical density at 600 nm (OD600) of 0.05. For heat stress,
BHI medium or IMM was inoculated with a preculture grown in
48 Introductory Food Microbiology
the same medium, and the cultures were incubated with shaking
at 45°C. For osmotic stress, either BHI medium with or without
5.5% NaCl (final concentration, 6%) was inoculated with a
preculture grown in BHI medium, or IMM with or without 3.5%
NaCl or with 0.6 M xylose was inoculated with a preculture grown
in IMM. Betaine (1 mM) or carnitine (1 mM) was added to IMM
containing NaCl when required. The cultures were incubated with
shaking at 37°C. Growth rate experiments were performed with a
Microbiology Reader Bioscreen C (Labsystems, Helsinki, Finland)
in 100-well sterile microplates, each well containing 300 µl of
culture medium. The OD600 was monitored. Experiments were
performed at least in duplicate and were repeated independently,
twice for heat stress experiments and three times for osmotic stress
experiments.
Antibiotics were used at the following concentrations:
ampicillin at 100 µg ml-1 for E. coli, and erythromycin at 5 µg ml-
1
and rifampin at 200 µg ml-1 for L. monocytogenes.
Cloning and Sequencing
Plasmids were prepared using the Plasmid Midi kit (Qiagen,
S.A., Courtabuf, France). Bacterial chromosomal DNA was isolated
as described previously. Restriction endonucleases, T4 DNA ligase,
and Taq polymerases were used as recommended by the
manufacturer (Roche Molecular Biochemicals, Mannheim,
Germany). DNA restriction fragments were purified from agarose
gels by using the QIAquick gel extraction kit (Qiagen).
Oligonucleotides were synthesized by MGW-Biotech. Plasmids
were introduced into E. coli by standard methods, while for L.
monocytogenes, electroporation was achieved as described
previously.
DNA sequencing was performed with the BigDye terminator
cycle sequencing ready reaction kit (Applied Biosystems, Courtabuf,
France), and sequences were analyzed with an automatic DNA
sequencer (ABI Prism 310 genetic sequencer; Applied Biosystems).
Searches for sequence homology were performed with the FASTA
program. Sequencing of the ctc gene of L. monocytogenes LO28 was
performed as follows. A 1,185-bp chromosomal DNA fragment
containing the ctc gene was amplified by PCR using primers OD1
Role of Ctc from Listeria Monocytogenes in Osmotolerance 49
and OD2 with incorporation of two restriction sites, HindIII and
BamHI. These primers were designed using the complete nucleotide
sequence of L. monocytogenes strain EGDe. The PCR product was
digested with the HindIII and BamHI restriction enzymes and was
cloned into similarly digested plasmid pHT315. Inserts of two
plasmids were sequenced.
Construction of a Ctc Insertional Mutant
The ctc gene was insertionally inactivated by a simple
recombination event using the temperature-sensitive suicide vector
pAUL-A. A 435-bp internal ctc fragment was PCR amplified from
chromosomal DNA with primers OD3 and OD4 with the
incorporation of two restriction sites, HindIII and BamHI. The
purified PCR product was digested with HindIII and BamHI, ligated
into similarly digested plasmid pAUL-A, and then transformed
into E. coli. The resultant plasmid pAUL-CTC was used to create
a knockout in ctc by homologous recombination with the L.
monocytogenes chromosome as described previously. In the resulting
mutant, 135 bp in the 3' end of ctc was deleted. Southern blot
analysis and PCR (data not shown) confirmed the authenticity of
a single integration of pAUL-CTC in the chromosome of L.
monocytogenes strain LO28. The stability of the pAUL-A insertion
was confirmed by PCR analysis of cultures grown in BHI medium
with or without erythromycin selection at 37°C.
RNA Isolation and Analysis
RNA samples were prepared from 10 ml of mid-logarithmic-
phase cells (OD600, 0.5) grown in BHI medium or IMM, 30 min
after the addition (or not) of NaCl (3.5% in IMM and 5.5% in BHI
medium). After the shock, cells were harvested by centrifugation at
7,000 x g for 4 min at 4°C. Cells were treated with rifampin and
RNAprotect Bacteria reagent (Qiagen) and centrifuged at 7,000 x
g for 10 min at 4°C. The pellet was resuspended in 400 µl of buffer
S (10% glucose, 12.5 mM Tris [pH 7.6], 66 mM EDTA, 0.5% sodium
dodecyl sulfate) containing 10 mg of lysozyme ml-1, 200 µg of
proteinase K ml-1, and 200 µg of rifampin ml-1. After the addition
of glass beads, cells were subjected to mechanical disruption. RNA
was purified by a Trizol extraction, two chloroform-isoamyl alcohol
extractions, and an isopropanol precipitation. The final RNA pellet
50 Introductory Food Microbiology
was dissolved in water, treated with DNase, and quantified
spectrophotometrically before storage at -70°C. The integrity and
the relative concentrations of RNA samples were checked by
agarose gel electrophoresis and ethidium bromide staining.
The mRNA of ctc was semiquantitatively analyzed by a slot
blot technique as follows. RNA samples were treated at 65°C in
20% formaldehyde for 15 min. The samples were then vacuum
blotted with a Bio-Rad slot blot apparatus onto positively charged
nylon membranes. RNA was cross-linked to the membrane by UV
radiation. Transcription of ctc was monitored using an intragenic
digoxigenin (DIG)-labeled probe generated by PCR with primers
OD3 and OD4. Detection of the labeled probe was mediated by
addition of an anti-DIG alkaline phosphatase-conjugated enzyme
and CDP-star substrate (Roche Molecular Biochemicals). Light
emission was captured by standard autoradiography (Hyperfilm;
Amersham Biosciences). A 16S rRNA probe was used to control
the loading uniformity of RNA extracted under different conditions
of stress.
Electron Microscopy
Stationary-phase-grown bacteria were processed for
observation by scanning electron microscopy (SEM). Bacteria were
fixed for 1 h with 4% glutaraldehyde in a 0.2 M cacodylate buffer.
Cells were dehydrated using a graded ethanol series (70, 95, and
100% ethanol, three times for 10 min each time) and subjected to
an acetone dehydration series of 30, 50, 70, and 100% acetone for
10 min each. One drop was spread on a microcover, coated with
gold in an Emscope SC500, and observed with the Philips SEM
505.
RESULTS
Sequence Analysis of the Ctc Gene of L. Monocytogenes LO28
Primers designed by using the sequences surrounding the ctc
gene (lmo00211) of L. monocytogenes strain EGDe, whose genome
has been entirely sequenced, were used to amplify the ctc gene of
strain LO28 by PCR. The PCR yielded one band, and the nucleotide
sequence of this DNA fragment revealed 100% identity between
the ctc sequences of strains LO28 and EGDe.
Role of Ctc from Listeria Monocytogenes in Osmotolerance 51
The ctc gene starts at an ATG codon, 9 nucleotides downstream
of a potential ribosome binding site (5' GGAG 3'), and ends with
a TAA stop codon. The deduced polypeptide contains 207 residues
with a calculated molecular weight of 22,654 (pI 4.14). Only one
amino acid differs between the Ctc sequences of L. monocytogenes
LO28 and Listeria innocua CLIP 11262 (Lys201® Asn substitution
in L. innocua Ctc protein). Homology searches revealed a significant
degree of similarity with the Ctc protein of B. subtilis (42% identity),
the equivalent protein, TL5, from Thermus thermophilus (36%
identity), and all members of the Ctc protein family identified
during different genome-sequencing projects. Like those of other
Ctc proteins, the N-terminal part of the L. monocytogenes Ctc protein
presents similarities with members of the 50S ribosomal protein
L25 family, for instance, 28% identity with the L25 protein of E.
coli, RplY.
A putative sB-dependent promoter was found 45 bp upstream
from the ATG start codon (5' GTTT-N15-GGGTAG 3') based on a
comparison with the s B-dependent consensus promoter (5'
GTTT[15/16 nt]GGGTAA3'). A stem-and-loop structure
(ATGGAAGATTCGCA TTTGTT TGCGATATCTTCCAT; DG = -77
kJ mol-1) followed by a T track is located 16 bp downstream from
the TAA stop codon of the ctc gene. This palindromic structure is
a putative transcriptional terminator. Analysis of the regions
surrounding the ctc gene in strain EGDe revealed an upstream
gene (lmo00210) transcribed in the opposite direction and encoding
a putative lactate dehydrogenase (67% identity with Lactobacillus
casei lactate dehydrogenase). A gene (lmo00212) encoding a protein
with no significant similarities with any of the proteins recorded
in the databases is located downstream of the ctc gene and in the
same direction of transcription. This gene is separated from the ctc
gene by the putative terminator mentioned above.
Transcriptional Analysis of the Ctc Gene
In a previous study, the Ctc protein was identified as a protein
showing high induction after salt stress. Moreover, as described
above, we found a putative sB promoter in front of the ctc gene, and
B
transcription is strongly induced after an osmotic upshift.
Therefore, semiquantitative analysis of the ctc gene transcription
52 Introductory Food Microbiology
was carried out using slot blots with RNA extracted from cultures
of strain LO28 grown in rich or minimal medium before or after
an osmotic shock. Similar results were obtained in both media.
Under growth conditions without added NaCl, constitutive
expression of ctc was observed. Thirty minutes of exposure to NaCl
(3.5% in IMM and 5.5% in BHI medium [final concentration, 6%])
resulted in an increase in the level of transcription.
Role of Ctc in Osmotic and High-temperature Adaptation
To investigate the function of the ctc gene of L. monocytogenes
LO28, it was mutated by insertional mutation by using the pAUL-
A plasmid, a temperature-sensitive suicide vector, as described in
Materials and Methods. Phenotypic analysis did not show any
difference between the ctc mutant and the wild-type strain with
respect to the aspect of colonies, catalase, hemolysis on blood agar,
and metabolic profiles, as determined by using API-CH50
microplate assays.
The stress tolerance of the ctc mutant was compared with that
of strain LO28. Because the Ctc protein was primarily identified
as a protein induced by an osmotic upshift, we first examined
whether the absence of Ctc would have any effect on the ability
of L. monocytogenes to grow under conditions of high osmotic
strength in a rich (BHI) or minimal (IMM) medium. Growth rates
(doubling times) were found identical for the wild-type strain and
the ctc mutant in BHI medium (55 ± 5 min). No significant difference
was found between the growth rates of the two strains after the
addition of NaCl to BHI medium (85 ± 10 min for the wild-type
strain and 110 ± 20 min for the ctc mutant) or in IMM without
added NaCl (100 ± 15 min for the wild-type strain and 125 ± 15
min for the ctc mutant). However, in IMM supplemented with 3.5%
NaCl, the growth of the ctc mutant was significantly impaired.
The growth rate reached 620 ± 140 min, whereas it was 270
± 15 min for the wild-type strain. In order to test if the sensitivity
of the ctc mutant was linked to salt stress or osmotic stress, growth
was performed in IMM supplemented with 0.6 M xylose. An
average increase of 95% for the growth rate of the ctc mutant was
obtained. The ability of L. monocytogenes to survive high salt
concentrations is attributed mainly to the accumulation of
Role of Ctc from Listeria Monocytogenes in Osmotolerance 53
compatible solutes such as glycine betaine or carnitine. In order to
test if the ctc mutation still had an effect in the presence of
osmoprotectants, we added 1 mM glycine betaine or carnitine to
IMM supplemented with 3.5% NaCl. Addition of glycine betaine
nearly allowed strain LO28 and the ctc mutant to recover the
growth rate of strain LO28 cultivated in IMM without NaCl. The
effect of the addition of carnitine was intermediate, but no
significant difference between the growth rates of the two strains
was observed in this situation.
In order to test if the Ctc protein was involved in general stress
tolerance, the growth of the ctc mutant versus LO28 was measured
under heat stress conditions at 45°C in BHI medium and IMM.
Under these conditions, the growth of the mutant was similar to
that of the wild-type strain. This suggests that the role of Ctc in
stress tolerance is restricted to osmotic tolerance.
The Morphology of the Ctc Mutant is Impaired during Stationary
Phase in Minimal Medium Containing NaCl
The wild-type strain LO28 and the ctc mutant were
subsequently examined using photonic microscopy during the
exponential and stationary phases of growth at 37°C in BHI medium
and IMM with or without NaCl. No difference in morphology was
observed between the two strains during growth in BHI medium
with or without NaCl or in IMM without NaCl (data not shown).
This was confirmed by transmission electron microscopy (TEM)
using negative staining and by SEM by examining 48-h-grown
cultures (data not shown). In these three different media, bacteria
appeared as small rods measuring 1 to 2 µm in length. In contrast,
in IMM containing 3.5% NaCl, the ctc mutant displayed a different
morphology than the wild-type strain as observed by photonic
microscopy, TEM (data not shown), and SEM. After 48 h of growth
in this medium, the morphology of strain LO28 was characterized
by a rod shape with a variable size ranging between 1 and 4 µm.
Approximately 1% of the cells displayed a bent rod shape. The ctc
mutant also displayed a rod shape with a variable size ranging
between 1 and 6 µm, but 80% of the cells had a bent or twisted rod
shape. The morphology of the ctc mutant did not differ significantly
from that of the LO28 strain when 1 mM glycine betaine was
added to the medium (data not shown).
54 Introductory Food Microbiology
We have shown that the ctc gene is involved in the resistance
of L. monocytogenes to high osmolarity in the absence of
osmoprotectants such as glycine betaine and carnitine in the
medium. Thus, a ctc insertional mutant grew twice as slowly as the
wild-type strain LO28 under conditions of high osmolarity (0.6 M
NaCl or xylose) in minimal medium.
Moreover, the morphology of the ctc mutant was impaired in
this growth condition. Whereas the morphology of the wild-type
strain LO28 was characterized by a rod shape, the ctc mutant
morphology was characterized by a bent or twisted rod shape
under osmotic stress conditions. When glycine betaine or carnitine,
known to be the most efficient osmoprotectants in L. monocytogenes,
was added to this medium, the growth of the mutant became
identical to the growth of its isogenic parent strain. This can
explain why the growth rates of the mutant and the wild-type
strain were identical in rich medium (BHI) supplemented with
5.5% NaCl. The BHI medium contains carnitine, which is relatively
abundant in some mammalian tissues. The role of ctc in stress
tolerance seems to be restricted to osmotic stress tolerance, since no
difference between the wild-type strain and the ctc mutant was
observed under conditions of growth at high temperatures in rich
or minimal medium.
Few genes involved in salt stress tolerance have been identified
in L. monocytogenes until now. Survival of L. monocytogenes at high
salt concentrations is attributed mainly to the accumulation of
three osmoprotectants, glycine betaine, carnitine, and proline.
Independently of genes involved in the transport or biosynthesis
of osmoprotectants, two genes encoding proteins of the Clp family
have been identified, clpC and clpP. Inactivation of these genes
confers a general stress sensitivity phenotype, including salt stress
sensitivity, on the corresponding mutants. These genes are known
to encode general stress proteins, chaperones assisting the proper
folding, refolding, or assembly of proteins and proteases processing
those that cannot be refolded. A recent study identified relA, a gene
encoding a (p)ppGpp synthetase, as a gene involved in
osmotolerance. The authors showed that (p)ppGpp is involved in
the growth of L. monocytogenes under high osmotic pressure and
that the intracellular accumulation of (p)ppGpp is probably
Role of Ctc from Listeria Monocytogenes in Osmotolerance 55
controlled by mechanisms distinct from accumulation of compatible
solutes. The last gene which has clearly been associated with
osmotolerance in L. monocytogenes is sB. The absence of sB impaired
the ability of L. monocytogenes to use glycine betaine or carnitine as
an osmoprotectant and impaired the transport of glycine betaine.
The transport of carnitine has not been studied. A potential sB-
dependent promoter has been identified upstream of the betL gene
and the opuC operon. This suggests that sB plays a key role in
osmotolerance of L. monocytogenes via regulation of the expression
of two major osmoprotectant transport systems. We have identified
a putative sB-dependent promoter upstream of the ctc gene. In
contrast to that of B. subtilis, the L. monocytogenes ctc gene does not
seem to belong to an operon. Moreover, expression of the ctc gene
is strongly induced by an osmotic upshift, like that of the sB gene.
Taken together, these observations suggest that the ctc gene may be
regulated, at least in part, at the transcriptional level by sB. This
emphasizes the role of sB, which is probably a key regulator of
osmotolerance in L. monocytogenes in the presence or absence of
compatible solutes in the environment.
Currently, the function of the Ctc proteins is unknown. Our
results suggest that the Ctc protein of L. monocytogenes belongs to
a novel system utilized by this bacterium to adapt to an osmotic
upshift in the absence of an osmoprotectant. This is the first time
that a role has been assigned to the Ctc protein, whose gene is
widely distributed in bacterial genomes. The product of the ctc
gene of L. monocytogenes, like other ctc gene products, presents
similarities in its N-terminal part with the 50S ribosomal L25
protein and consequently belongs to the L25 ribosomal protein
family. According to the COG database, which compares the protein
sequences encoded in 43 complete genomes, representing 30 major
phylogenetic lineages, no L25 homologue is present in the archaeal
genomes, but L25 homologues are present in nearly all eubacterial
genomes.
L25 homologues are found in all gram-negative bacteria and
in all gram-positive bacteria except Lactococcus lactis, Streptococcus
pyogenes, Mycoplasma pneumoniae, and Mycoplasma genitalium. The
sequence homologies observed between the Ctc proteins and the
L25 proteins include many conserved residues, which the 5S rRNA-
56 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 57

L25 structure confirmed to be involved in the rRNA-protein

binding interaction, thereby confirming that these two groups of
proteins are strongly related. It is highly probable that the Ctc
proteins are associated at least in their N-terminal parts with the
ribosome and bind the 5S rRNA. Ribosomes have been implicated
as sensors of heat and cold shock in E. coli. Recent results implicated
the ribosome as a possible mediator of the activity of Obg, an 3
essential GTP-binding protein, and the stress induction of sB,
suggesting that ribosomes are also general sensors in B. subtilis. We
can hypothesize that the ribosome is also a sensor of salt stress,
IDENTIFICATION OF LISTERIA
at least in L. monocytogenes, through the activity of Ctc. Further MONOCYTOGENES GENES
investigations will be required to clarify the function of Ctc in L.
monocytogenes and in other bacteria.
The capacity of Listeria monocytogenes to tolerate salt and
alkaline stresses is of particular importance, as this pathogen is
often exposed to such environments during food processing and
food preservation. We screened a library of Tn917-lacZ insertional
mutants in order to identify genes involved in salt and/or alkaline
tolerance. We isolated six mutants sensitive to salt stress and 12
mutants sensitive to salt and alkaline stresses. The position of the
insertion of the transposon was located in 15 of these mutants. In
six mutants the transposon was inserted in intergenic regions, and
in nine mutants it was inserted in genes. Most of the genes have
unknown functions, but sequence comparisons indicated that they
encode putative transporters.

INTRODUCTION
Listeria monocytogenes is a food-borne pathogen that is widely
distributed in the environment. This microorganism is of particular
concern in the food industry because of its ability to survive, and
frequently to grow, under a wide range of adverse conditions used
to preserve food, such as low temperature, low pH, and high
osmolarity, or used to clean and sanitize equipment, such as high
pH. Growth of L. monocytogenes has been reported at NaCl
concentrations as high as 10% and at pHs as high as 9.
There is little information on the mechanisms that allow this
bacterium to cope with alkaline environments. Knowledge
concerning the mechanisms used by gram-positive bacteria for
adaptation and growth at alkaline pHs comes mainly from studies
58 Introductory Food Microbiology
of alkaliphilic strains of Bacillus species, such as Bacillus halodurans
C-125 or Bacillus pseudofirmus OF4. There is strong evidence that
monovalent cation-proton antiporters are essential for maintaining
a neutral cytoplasmic pH and, therefore, for growth under alkaline
conditions. In addition, the acidic cell wall polymers teichuronic
acid and teichuronopeptides contribute to pH homeostasis. These
wall macromolecules may provide a passive barrier to ion flux and
elevation of the cytoplasmic buffering capacity at highly alkaline
growth pHs. In Bacillus subtilis, monovalent cation/proton
antiporters also seem to be important since the mrpA gene encoding
an Na + /H + antiporter and the tetA(L) gene encoding a
multifunctional tetracycline-metal/H+ antiporter that also exhibits
monovalent cation/H+ antiport activity are involved in Na+-
dependent pH homeostasis.
Most bacteria cope with elevated osmolarity in the environment
by intracellular accumulation of particular osmolytes called
compatible solutes. These compatible solutes act in the cytosol by
counterbalancing the external osmolarity, thus preventing water
loss from the cell and plasmolysis without adversely affecting
macromolecular structure and function. Compatible solutes can be
either transported into the cell or synthesized de novo. Survival of
L. monocytogenes at high salt concentrations is attributed mainly to
the accumulation of three compatible solutes: glycine betaine,
carnitine, and proline. Accumulation of glycine betaine and
carnitine occurs via two glycine betaine transporters encoded by
the betL gene and the gbu operon and one carnitine transporter
encoded by the opuC operon. Disruption of these genes reduced
the osmotolerance of L. monocytogenes. Both betL and opuC have
putative sB-dependent promoters. The absence of sB impaired the
ability of L. monocytogenes to use glycine betaine or carnitine as a
compatible solute.
Proline transport has not been characterized yet. However,
disruption of the proBA operon (proline biosynthesis-encoding
operon) reduced the growth of the corresponding mutant at high
salt concentrations. Little information is available concerning other
mechanisms that L. monocytogenes uses to cope with salt stress,
especially when compatible solutes are not available in the
environment. Two genes, clpC and clpP, encoding a ClpC ATPase
Identification of Listeria Monocytogenes Genes 59
and a ClpP serine protease, respectively, have been identified.
Inactivation of these genes conferred a general stress sensitivity
phenotype, including sensitivity to salt stress, to the corresponding
mutant. In a recent study workers identified relA, a gene encoding
a (p)ppGpp synthetase, as a gene involved in osmotolerance via
a mechanism different from the mechanism involving accumulation
of compatible solutes. It has also been shown that the general
stress protein Ctc of L. monocytogenes is involved in osmotolerance
in the absence of any compatible solutes in the environment.
In order to obtain a better understanding of the mechanisms
involved in salt and alkaline tolerance, we used a library of
transposon insertional mutants of L. monocytogenes LO28 to isolate
mutants with decreased NaCl and/or alkaline tolerance. We
succeeded in identifying different mutants that exhibit less
resistance to salt and/or alkaline stress than the parental strain
and characterized the genes interrupted.
MATERIALS AND METHODS
Bacterial Strains
The L. monocytogenes strains used were LO28, a clinical isolate
of serotype 1/2C, and a library of Tn917-lacZ mutants of strain
LO28. Bacterial plasmids were propagated in Escherichia coli strain
TG1.
Culture Media and Stress Conditions
Cells were grown on complex culture media, including brain
heat infusion (BHI) broth or agar (Difco Laboratories, Detroit, Mich.).
Screening of the library of Tn917-lacZ mutants for sensitivity to
salt and alkaline stresses was performed as follows. Wells of
microplates containing 100 µl of BHI medium with erythromycin
(BHI-erm) were inoculated with the different mutants. The
microplates were incubated at 37°C overnight and subsequently
used to transfer the mutants, after 1:1 dilution with BHI-erm, onto
agar plates with a replicator. The plates used contained BHI-erm
with or without 5.5% NaCl (final concentration, 6%) and BHI-erm
adjusted to pH 8.6 with NaOH. The growth of the mutants was
recorded after 48 h. Mutants selected after this first step were used
to perform liquid growth experiments with a Microbiology Reader
60 Introductory Food Microbiology
Bioscreen C (Labsystems, Helsinki, Finland) in 100-well sterile
microplates, and each well contained 300 µl of culture medium, as
follows.
Overnight cultures of Tn917-lacZ mutants in BHI-erm were
used to inoculate different media (BHI-erm with or without 5.5%
NaCl and BHI-erm with the pH adjusted to 8.5) at an initial optical
density at 600 nm of ~0.1. The cultures were incubated with shaking
at 37°C. The optical density was monitored at 600 nm. Experiments
were repeated independently at least twice. The LO28 strain was
used as a control and was inoculated into BHI medium lacking
erythromycin. For more detailed physiological characterization of
mutants sal5 and sal11, liquid growth experiments were performed
by using the same procedure except that the pH of BHI medium
was adjusted to 8.5 with a glycine-NaOH-NaCl buffer. Growth
experiments were also performed in BHI medium supplemented
with 7% KCl or 15% xylose and were repeated independently at
least four times. Growth curves were fitted by using a modified
Gompertz equation, and the generation time was calculated by
using nonlinear regression with the Statistica statistical software
(Statsoft, Tulsa, Okla.).
Antibiotics were used at the following concentrations: 100 µg
of ampicillin ml-1 for E. coli and 5 µg of erythromycin ml-1 for L.
monocytogenes.
Identification of Transposition Target
Inverse PCR was used to amplify the DNA fragment next to
the region downstream from the Tn917-lacZ chromosomal
insertions. Bacterial chromosomal DNA was isolated as described
previously. Chromosomal DNA was digested with HindIII for
mutants sal1, -2, -5, -6, -11, -17, -22, and -23 and mutants sl7, -10,
-13, -14, and -25 and with NdeI for mutants sl12 and sal21 and was
subsequently circularized by self-ligation. The region downstream
from the Tn917-lacZ insertion was amplified by using primers
RG7 (5'-ATTCCGTCTGAAGCAGTGGT-3') and RG9 (5'-
GAACGCCGTCTACTTACAAG-3') for HindIII-digested DNA and
primers RG9 and RG11 (5'-GAATCACGTGTCCCTTTGCG-3') for
NdeI-digested DNA. Amplification products were sequenced either
directly or after they were cloned into the pGEM-T plasmid
Identification of Listeria Monocytogenes Genes 61
(Promega France, Charbonnières, France), a 3' T-end vector
specifically designed for cloning PCR fragments, by following the
manufacturer’s specifications. DNA sequencing was done with a
BigDye terminator cycle sequencing Ready Reaction kit (Applied
Biosystems, Courtaboeuf, France). The reactions were performed
with unlabeled primers and fluorescent dideoxynucleotides, and
then the reaction mixtures were analyzed with an automatic DNA
sequencer (ABI Prism 310 genetic sequencer; Applied Biosystems).
Blast sequence homology analyses were performed by using the
National Center for Biotechnology Information network service.
The primers used for the sequence were RG1 (5'-
CCCACTAAGCGCTCGGG-3'), RG7, and RG9. Oligonucleotides
were synthesized by MGW-Biotech (Courtaboeuf, France).
RESULTS
Selection of Salt and Alkaline Stress-sensitive Mutants
A library of approximately 2,500 Tn917-lacZ insertion mutants
was screened for salt and/or alkaline stress sensitivity. Each mutant
was grown on BHI-erm plates with or without 5.5% NaCl or with
the pH adjusted to 8.6. Twenty-three mutants showed a growth
delay on at least one of the two stress media. Six mutants seemed
to be affected under salt stress conditions, nine mutants seemed to
be affected under alkaline stress conditions, and eight mutants
seemed to be affected under both conditions. The phenotypes of
the mutants were further confirmed in liquid growth experiments
by comparing the growth curves to that of the LO28 wild-type
strain. Five mutants were removed after this second step. The
growth of two mutants sensitive to both stresses was impaired in
BHI medium, and the alkaline sensitivity phenotype of three
mutants was not confirmed. Finally, we isolated six mutants
sensitive to salt stress and 12 mutants sensitive to both salt stress
and alkaline stress. The designations of mutants that were sensitive
only to salt stress begin with sl (for salt sensitivity locus), and the
designations of mutants that were that were sensitive to salt stress
and alkaline stress begin with sal (for salt and alkaline sensitivity
locus). Southern hybridization with HindIII-digested chromosomal
DNA and, when required, EcoRI-digested chromosomal DNA with
a digoxigenin-labeled DNA probe specific for the Tn917-lacZ
62 Introductory Food Microbiology
transposon revealed that the 18 mutants contained a single copy
of the transposon and that the loci corresponded to 18 independent
insertion loci (data not shown). The 18 remaining mutants were
kept for further characterization.
Identification of the Transposition Target
The inverse PCR method enabled us to clone and sequence the
downstream transposon-chromosome junctions of 15 mutants. We
did not succeed in cloning the junctions of three mutants, sal3, -
4, and -19. The sequences were compared with the complete
genome sequence of L. monocytogenes strain EGDe. In nine mutants,
the transposon was inserted into open reading frames, whereas it
was inserted into intergenic regions in six mutants.
Genes Encoding Proteins with an Identified Function or a Putative
Function
In the sl12 mutant, the transposon was inserted into the mutS
gene, which is involved in DNA mismatch repair, whereas in
mutants sal1 and -11 and sl14 it was inserted into transporters or
putative transporters. In sal1, the transposon was inserted into the
ATPase subunit of an ATP binding cassette transporter, and in
sal11 it was inserted into the permease subunit of another ATP
binding cassette transporter. In sl14, the transposon was inserted
into the mdrL gene, which encodes a multidrug efflux transporter
of the major facilitator superfamily. If the orientation of the genes
and the presence of putative terminators were taken into account,
the phenotypes of mutants sal1 and -11 and sl14 could not be
linked to a polar effect of the mutation on downstream genes.
However, the phenotype of mutant sl12 could be linked to a polar
effect of the mutation of a downstream gene, mutL coding for a
DNA mismatch repair protein or lmo1405 coding for a putative
antiterminator regulatory protein.
Genes Encoding Proteins with Unknown Functions
In sl7 and sal22, the transposon was inserted into the lmo1432
gene at positions separated by 29 bp. The deduced protein encoded
by the lmo1432 gene is specific to Listeria. In sal2 and -17, the
transposon was inserted into two genes, lmo1443 and lmo2232,
respectively, which encode proteins having unknown functions in
Identification of Listeria Monocytogenes Genes 63
other organisms. lmo1443 has orthologues in different gram-
positive bacteria, and lmo2232 has a paralogue (lmo2399) and five
orthologues in B. subtilis yhdP, yrkA, yqhB, yugS, and yhdT and in
other eubacteria. Considering the orientation of the genes and the
presence of putative terminators, the phenotypes of these four
mutants could not be linked to a polar effect of the mutation on
downstream genes. This was not the case for sal5, in which the
transposon was inserted into lmo0992, a gene of unknown function
located upstream from lmo0991 which encodes a protein with
similarities to NtpJ of Enterococcus hirae, a K+/Na+ transporter.
In these five mutants, similarity searches could not assign a
putative function to the genes interrupted, but a search for
transmembrane domains with the DAS program revealed that all
corresponding proteins have putative transmembrane domains.
Intergenic Regions
For six mutants, the position of the insertion of the transposon
was located in intergenic regions.
The physiology of two mutants, sal5 and sal11, which were
identified as sensitive to salt and alkaline stresses during the
screening analysis, was characterized by quantifying the growth
of these organisms in different media.
Physiology of the sal5 and sal11 Mutants in Response to Osmotic
and Alkaline Stresses
Growth of the sal5 and sal11 mutants was examined by using
BHI medium, BHI medium supplemented with 5.5% NaCl, 7%
KCl, or 15% xylose, and BHI medium with the pH adjusted to 8.5.
For the growth experiments under osmotic stress conditions, the
concentration of each of the three solutes (NaCl, KCl, and xylose)
was approximately 1 M. The results showed that the phenotype
identified during the screening analysis was confirmed. Both
mutants were sensitive to NaCl stress and alkaline stress. However,
the sensitivity was far more pronounced with the alkaline stress
conditions; under these conditions the growth rate (doubling time)
of both mutants was approximately 300 min and was fivefold
greater than the growth rate of the wild-type strain. Under NaCl
osmotic stress conditions, the growth rates of mutants sal5 and
64 Introductory Food Microbiology
sal11 were 1.4- and 1.3-fold greater, respectively. Moreover, both
mutants were also sensitive to KCl stress (but to a lesser extent)
and slightly sensitive to xylose stress.
DISCUSSION
We isolated six Tn917-lacZ insertional mutants of L.
monocytogenes strain LO28 sensitive to salt stress (sl mutants) and
nine mutants sensitive to both salt and alkaline stresses (sal
mutants) and located the positions of the insertions of the
transposons in the genomes of the different mutants. We used the
complete genome sequence of L. monocytogenes strain EGDe to
rapidly identify the positions of the insertions of the transposons.
We sequenced 15 fragments corresponding to a total of 6,249
nucleotides and found only 4 nucleotides which differed in strains
LO28 and EGDe. These results suggest that the DNA sequences of
these two strains are very similar and justify utilization of the
complete genome sequence of L. monocytogenes strain EGDe to
study the L. monocytogenes strain LO28 genome.
On the basis of the functions or putative functions of the genes
disturbed by insertion of the transposons, we classified the mutants
in four categories.
Genes with known Functions Directly Linked to Salt Stress
In mutants sal6 and sl10, the transposon was inserted in front
of the gbu operon. This operon, which is very similar to the opuA
operon of B. subtilis, encodes an ATP-dependent transporter
belonging to the ATP binding cassette transporter superfamily and
is involved in the transport of glycine betaine. A mutant with this
operon inactivated was found to be salt sensitive. Ko and Smith
also identified sequences with significant similarities to the sA-
type -35 and -10 promoter recognition sequence TTGTGT-N15-
TATTGC. In the sl10 mutant, the transposon was located between
the putative promoter and the coding DNA sequence of the gbu
operon. In this mutant, the promoter of the gbu operon is separated
from the gbu operon by the transposon.
Consequently, the gbu operon cannot be transcribed. The salt
stress sensitivity of the sl10 mutant confirmed the results of Ko and
Smith. This result provides good validation of our screening
Identification of Listeria Monocytogenes Genes 65
protocol. In sal6, the transposon was inserted 109 bp upstream
from the putative promoter of the gbu operon. This result indicates
that regions upstream from the putative promoter are probably
involved in the transcription of this operon. In B. subtilis, two
promoters have been identified in front of the opuA operon, and we
hypothesize that this is also the case for L. monocytogenes.
Surprisingly, the sal6 mutant was found to be sensitive to salt and
alkaline stresses. However, the alkaline phenotype is weak. Using
two-dimensional electrophoresis, we identified the GbuA protein,
the ATPase subunit of the Gbu transporter, as a protein induced
by salt stress. In L. monocytogenes, expression of the sB factor is
strongly induced by salt stress, and consequently, expression of
the genes under the control of this sigma factor is also induced by
salt stress. However, no sB promoter recognition sequences are
present in front of the gbu operon, indicating that another salt
induction pathway is present in L. monocytogenes.
Genes with known Functions not Directly Linked to Salt Stress
In mutants sl12 and sl14 the transposon was inserted into the
mutS and mdrL genes, respectively. The mutSL locus of L.
monocytogenes is involved in both mismatch repair and homologous
recombination. The mdrL gene encodes a multidrug efflux
transporter and is involved in the efflux of ethidium bromide. In
both cases, no salt stress sensitivity of the mdrL or mutSL mutant
has been mentioned previously; thus, this is the first time that
these two loci have been associated with salt stress. MdrL extrudes
different toxic components, and we hypothesize that this transporter
also extrudes Na+, perhaps in a nonspecific manner.
GENES ENCODING PUTATIVE TRANSPORTERS
In mutants sal1 and sal11, even though the function is unknown,
sequence comparisons indicated a putative function. In both
mutants, the transposons were inserted into genes encoding
domains of two distinct ATP binding cassette transporters. In sal1,
the gene interrupted, ykpA, encodes the ATP binding protein
domain of the transporter, and in sal11 the gene interrupted, lmo668,
encodes the permease protein domain. ykpA has orthologues in
gram-positive bacteria. The corresponding protein has been
identified in Staphylococcus aureus as an immunodominant antigen,
66 Introductory Food Microbiology
and antisense ablation of the ykpA gene led to a growth-inhibiting
effect.
The lmo668 gene is located downstream from its putative
associated ATP binding protein. Lmo668 has very significant
similarities with Yadh of gram-negative bacteria. In these cases it
is highly probable that ykpA and lmo0668 encode transporters, but
the transported solutes remain unknown. The transporters
probably extrude Na+ and/or H+, perhaps in a nonspecific manner.
Growth experiments with mutant sal11 indicated that the lmo0668
gene is probably involved in pH homeostasis because the sal11
mutant was highly sensitive to alkaline pH, moderately sensitive
to NaCl stress, and slightly sensitive to KCl stress and xylose
stress. The differences observed among the effects of the three
osmolytes, NaCl, KCl, and xylose, which were added at
concentrations of approximately 1 M to the medium, were probably
due to the fact that NaCl tends to be more stressful than KCl and
the fact that xylose provides only one-half the osmotic stress of
NaCl and KCl.
The last mutant in which expression of genes encoding putative
transporters are disturbed is mutant sal5. lmo0992, the gene
interrupted by the transposon, belongs to an operon consisting of
five genes. The first gene, lmo0989, encodes a putative
transcriptional regulator of the MarR family. The next three genes,
lmo0990, lmo0991, and lmo0992, encode proteins whose functions
are unknown but which are putative integral membrane proteins.
Lmo0991 and Lmo0992 are very similar to YkoY of Bacillus
megaterium, B. subtilis, and Bacillus anthracis. The last gene, lmo0993,
encodes a protein with similarity to the NtpJ protein of E. hirae
(33% identity). The ntpJ gene encodes a component of the KtrII
uptake system. NtpJ mediates K+ and Na+ cotransport.
The growth of an ntpJ-disrupted mutant is impaired at pH 10
in K+-limited medium. The phenotypes of an ntpJ-disrupted mutant
and the sal5 mutant are similar; the sal5 mutant is highly sensitive
to alkaline pH in BHI medium. Thus, the phenotype of the sal5
mutant is probably due to a polar effect of the mutation on lmo0993,
the ntpJ-like gene. In E. hirae, the ntpJ gene is the last gene of an
operon (ntp) encoding a V-type Na+-ATPase, which is important
for Na+ extrusion at high pH. The NtpJ K+/Na+ uptake system
Identification of Listeria Monocytogenes Genes 67
functions with the V-type Na+-ATPase at high pH and/or high
Na+ concentrations in order to eliminate sodium ions and to drive
potassium ion uptake. We found no similarities between the
sequences of lmo0990, lmo991, and lmo992 and the sequences of
the ntp genes encoding the V-type Na+-ATPase, and further work
is needed to check if there is an analogy of function among the
genes.
Genes with Unknown Functions
For mutants sal2, sl7, sal22, and sal17, the function of the gene
interrupted is completely unknown, but the three genes interrupted
encode proteins with putative transmembrane domains. The
transposon was inserted into the same gene, lmo1432, in mutants
sl7 and sal22. This interrupted gene seems to be specific to the
genus Listeria. In sal2 the interrupted gene seems to be specific to
gram-positive bacteria, whereas in sal17 it seems to be specific to
eubacteria. In mutants sal21 and sl25 the transposon was inserted
in front of putative operons encoding proteins with unknown
functions.
We were not able to identify sA-type or sB-type promoter
recognition sequences in front of these operons. However, we
hypothesize that in these two cases transcription of the operons
was eliminated by insertion of the transposon in the promoter
region and that the phenotypes of the mutants are linked to at least
one of the genes of the operons.
Finally, the salt sensitivity phenotype of mutant sl13 and the
salt and alkaline sensitivity phenotype of mutant sal23 are difficult
to explain because in sl13 the transposon was inserted between
two convergent genes and in sal23 it was inserted between the end
and the terminator of the lmo1140 gene.
We did not identify the relA gene during our screening
procedure. This gene encodes a (p)ppGpp synthase and was recently
identified during a screening procedure very similar to our
procedure. However, this is not surprising because a library of
transposons cannot be exhaustive. Moreover, we did not succeed
in identifying the positions of insertion of the transposons in three
mutants, and we cannot exclude the possibility that one of these
three mutants corresponds to a relA mutant.
68 Introductory Food Microbiology
We were interested in studying salt stress because it is one of
the most commonly used methods for food conservation, and we
were interested in studying alkaline stress because of the alkaline
nature of most of the detergents and some of the chemical sanitizers
used to clean and sanitize equipment in food processing plants.
Little information is available concerning the physiology of L.
monocytogenes in response to alkaline stress. Growth of food factory
isolates was reported at pHs as high as 9, and this pathogen has
been shown to be resistant to storage at pHs up to 12. It has also
been shown that alkaline pH induces cross-protection of L.
monocytogenes against heat. Moreover, there is no information
concerning the mechanisms that take place in L. monocytogenes in
order to cope with alkaline stress. It was interesting to study these
two stresses in parallel because a combination of salt stress and
alkaline stress is more effective in decreasing the survival of L.
monocytogenes than an individual type of stress. It is also known
that homeostasis of Na+ and H+ ions is tightly linked, and in most
cell membranes there are proteins that couple the fluxes of the two
ions via the Na+/H+ antiporter or the Ntp complex, for example.
During the screening procedure used in this study we identified
few genes involved in Na+ and/or alkaline stress tolerance. Further
work is needed to investigate the function of these genes, but the
fact that they encode putative integral membrane proteins indicates
that they probably encode ion transporters.
CELL-WALL PROTEINASES PRTS AND PRTB HAVE A
DIFFERENT ROLE IN STREPTOCOCCUS THERMOPHILUS /
LACTOBACILLUS BULGARICUS MIXED CULTURES IN MILK
The manufacture of yoghurt relies on the simultaneous
utilization of two starters: Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation
usually takes place between the two species, which often results
in enhanced milk acidification and aroma formation compared to
pure cultures. Cell-wall proteinases of Lactococcus lactis and
lactobacilli have been shown to be essential to growth in milk in
pure cultures. In this study, the role of proteinases PrtS from S.
thermophilus and PrtB from Lb. bulgaricus in bacterial growth in
milk was evaluated; a negative mutant for the prtS gene of S.
thermophilus CNRZ 385 was constructed for this purpose. Pure
Identification of Listeria Monocytogenes Genes 69
cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant
were made in milk as well as mixed cultures of S. thermophilus and
Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant
was associated with several strains of Lb. bulgaricus, including a
PrtB-negative strain.
The pH and growth of bacterial populations of the resulting
mixed cultures were followed, and the Lactobacillus strain was
found to influence both the extent of the benefit of Lb. bulgaricus/
S. thermophilus association on milk acidification and the magnitude
of S. thermophilus population dominance at the end of fermentation.
In all mixed cultures, the sequential growth of S. thermophilus then
of Lb. bulgarius and finally of both bacteria was observed. Although
proteinase PrtS was essential to S. thermophilus growth in milk in
pure culture, it had no effect on bacterial growth and thus on the
final pH of mixed cultures in the presence of PrtB. In contrast,
proteinase PrtB was necessary for the growth of S. thermophilus,
and its absence resulted in a higher final pH. From these results,
a model of growth of both bacteria in mixed cultures in milk is
proposed.
Streptococcus thermophilus is a thermophilic lactic acid bacterium
(LAB), widely used as a starter to produce fermented dairy
products. It is generally used in association with other micro-
organisms, in particular with Lactobacillus delbrueckii subsp.
bulgaricus (Lb. bulgaricus) for the manufacture of yoghurt. For this
application, the fast-growing capacity of these bacteria in milk is
crucial to enable intense and rapid acidification of milk. LAB are
fastidious micro-organisms, which have in particular several amino
acid auxotrophies. Most S. thermophilus strains are stimulated by
the supply of two to five amino acids, whereas lactobacilli require
between three and 14 amino acids.
The optimal growth of LAB in milk thus depends on their
proteolytic system, which hydrolyses milk caseins into peptides
and amino acids. The cell-wall proteinases of LAB are of major
importance in this process, as they are responsible for the first step
of casein breakdown. They belong to the same multi-domain
proteinase family and show significant homologies, even though
differences in specificity, bacterial anchor and domain organization
have been described. The cell-wall proteinase of Lactococcus lactis
70 Introductory Food Microbiology
(PrtP), which is very frequent in this species, has been extensively
studied. In milk, Lc. lactis PrtP-negative strains only reach 10% of
the cell densities observed with PrtP-positive strains. In S.
thermophilus, the presence of a cell-wall proteinase, PrtS, recently
characterized, is less common than in Lc. lactis. In this species,
high cell-wall proteinase activities are associated with high milk-
acidifying capacities. In Lb. bulgaricus, the cell-wall proteinase, PrtB,
is also essential for optimal growth in milk; a proteinase-negative
strain reaches only 22% of the final biomass of a proteinase-positive
strain when grown in milk.
In yoghurt, S. thermophilus and Lb. bulgaricus are grown in
association, which often results in a positive interaction. This
relationship, called protocooperation, has a beneficial effect on
growth of both species and on acid and aroma production. S.
thermophilus indeed produces pyruvic acid, formic acid and CO2,
which stimulate the growth of Lb. bulgaricus. In turn, Lb. bulgaricus
produces peptides and amino acids that stimulate S. thermophilus
growth.
In the present study, we wished to determine the role of the
cell-wall proteinases PrtS from S. thermophilus and PrtB from Lb.
bulgaricus in bacterial growth in milk. We therefore constructed a
negative mutant for the cell-wall proteinase gene (prtS gene) of S.
thermophilus CNRZ 385, which was recently sequenced and
characterized in our laboratory. The latter mutant was used to
study the role of PrtS on the growth of S. thermophilus in pure
culture in milk. We also took advantage of the availability of a
PrtB-negative mutant of Lb. bulgaricus to evaluate the role of cell-
wall proteinases PrtS and PrtB on growth and thus on acidification
in S. thermophilus/Lb. bulgaricus mixed cultures.
METHODS
Strains of S. thermophilus and Lb. bulgaricus were grown in
three different media: reconstituted skim milk (Nilac Low Heat
Milk powder, NIZO) heated for 10 min at 95 °C, supplemented
with yeast extract (3 g l-1; Difco) when required, M17 medium
supplemented with 20 g lactose l -1 , and MRS medium
supplemented with 20 lactose g l-1 and acidified at pH 5·2,
supplemented with streptomycin (Sigma) (2 mg ml-1) when required.
Identification of Listeria Monocytogenes Genes 71
The Escherichia coli strain was grown at 37 °C on Luria-Bertani
(Difco) medium with shaking, in the presence of erythromycin
(Ery) (150 µg ml-1) when required.
Stock cultures of each strain of S. thermophilus and Lb. bulgaricus
were prepared after growth at 42 °C on skim milk, supplemented
with yeast extract for proteinase-negative strains, from overnight
skim milk cultures, supplemented with yeast extract when required.
The pH was then measured, and bacterial numbers were estimated
by plating, with an automatic spiral plater, appropriate dilutions
of the culture on agar medium: M17Lac was used for specific
enumeration of S. thermophilus cells, and MRSLac pH 5·2,
supplemented with streptomycin when required, for specific
enumeration of Lb. bulgaricus cells.
For S. thermophilus strains and before dilution, chains of cells
were disrupted for 30 s in a mechanical blender. After 48 h
incubation at 42 °C in anaerobic jars, cells were enumerated with
the EC1 colony counter software. At the end of culture, bacteria
were directly frozen in liquid nitrogen and kept at -80 °C.
Growth rates of S. thermophilus 385 and 385-PrtS strains were
determined in M17 at 42 °C using a Microbiology Reader Bioscreen
C (Labsystems) in 100-well, sterile, covered microplates. Each well,
containing 200 µl M17Lac, was inoculated at 1% with overnight
M17Lac cultures of S. thermophilus and covered with one drop of
paraffin oil. The optical density was measured at 600 nm every
20 min, after gentle shaking. The apparent growth rate (µmax) was
defined as the maximum slope of semi-logarithmic representation
of growth curves, assessed by optical density measurements.
Mixed cultures of S. thermophilus and Lb. bulgaricus strains
were performed at 42 °C by inoculating skim milk with 5x106
c.f.u. ml-1 of stock cultures of each strain. For proteinase-negative
strains, cells from stock culture were washed three times in 50 mM
Tris buffer (pH 7) before inoculation to avoid peptides and/or
amino acids being supplied in the mixed culture. Every 20 min, the
pH of the culture was measured, and bacteria were enumerated as
described above. Total bacterial populations were estimated by
addition of data from enumerations of each bacterial species on
specific medium to the others, as indicated above.
72 Introductory Food Microbiology
Proteinase Assay
The PrtS proteinase phenotype of S. thermophilus strains was
determined on bacterial colonies in two ways. First, bacteria were
grown on FSDA medium (Fast Slow Difference Agar) (Huggins &
Sandine, 1984 ). This milk-based agar medium made it possible to
differentiate bacteria exhibiting slow or limited growth in milk
from those exhibiting rapid growth; in particular, bacteria
possessing a cell-wall proteinase activity appeared as white,
opaque, rounded colonies, whereas proteinase-negative colonies
were small, flat and translucent.
Second, bacteria from an overnight skim milk culture were
diluted and plated on agar skim milk plates (cell culture dishes,
35 mm in diameter). After 24-48 h incubation at 42 °C in anaerobic
jars, colonies were covered by a solution containing Tris buffer
(50 mM, pH 7), a chromogenic substrate of proteinase PrtS (Suc-
Ala-Ala-Pro-Phe-ßNA, 10 mg ml-1; Novabiochem), 10 mg ml-1 Fast-
Garnet (GBC, Sigma) and 10-50 mM CaCl2. PrtS-positive clones
appeared as red colonies, whereas PrtS-negative clones remained
white.
Proteinase activity was measured on cellular suspensions
using [14C]casein as the substrate according to the method of
Monnet et al. (1987) , modified as follows. Cell suspensions were
prepared from 4 ml overnight M17 cultures; cells were recovered
by centrifugation (20 min, 8000 g, 4 °C) and washed three times in
Tris buffer (50 mM, pH 7). The last pellet was suspended in 150 µl
Bistris buffer (50 mM, pH 6·5) containing 10 mM CaCl2. Fifty
microlitres of cell suspension was incubated with 50 µl of 14C
casein solution (0·1%) at 37 °C for 15, 60 and 120 min. Enzyme
reactions were stopped by the addition of 100 µl TCA (12%), left
for 30 min at room temperature and centrifuged for 2 min at 10000 g,
and the radioactivity was then measured in the supernatants.
Protease activity corresponded to the percentage of casein
hydrolysis in 10 min.
DNA Manipulations and Sequencing
Total DNA Preparation
Total DNA of S. thermophilus CNRZ 385 was prepared as
described by Pospiech & Neumann (1995).
Identification of Listeria Monocytogenes Genes 73
Preparation of Electrocompetent Cells of S. thermophilus and Lc.
lactis
Electrocompetent cells of S. thermophilus CNRZ 385 and Lc.
lactis MG1363 were prepared according to the method of Holo &
Nes (1989) , modified as follows. From an overnight culture in
M17Lac, a culture was performed at 37 °C (S. thermophilus) or at
30 °C (Lc. lactis) by 1% inoculation of M17Lac containing DL-Thr
(100 mM) for S. thermophilus or Gly (1·5%) for Lc. lactis until the
OD600 reached 0·6-1. Cells were collected by centrifugation at 5000
g for 5 min and washed four times in 0·5 M sucrose/10% glycerol
solution. They were then resuspended in 10% glycerol/30%
PEG2000 solution for S. thermophilus or in 0·5 M sucrose/10%
glycerol solution for Lc. lactis and immediately frozen in liquid N2
and stored at -80 °C.
DNA Sequencing
The Sanger method of DNA sequencing was carried out on
double-strand DNA plasmids and on PCR products with the
BigDye Terminator cycle sequencing ready reaction kit (370A DNA
sequencer, Applied Biosystems).
Construction of a Negative Mutant for PrtS
A 3776 bp PCR product containing part of the prtS gene was
amplified using oligonucleotides 1 (5' CAT CAC GGA AAG TCT
AGG 3') and 2 (5' AAC GTA TTG ATA CTT ACC 3') from total
DNA of S. thermophilus CNRZ 385 strain. Streptococcal DNA
(100 ng) was added to a PCR mixture containing 2·5 U of Taq
polymerase (Quantum Appligene) and 0·26 µM of each
oligonucleotide (Life Technology). After 5 min of denaturation at
94 °C, 30 cycles of 30 s annealing at 50 °C and 3 min of elongation
at 72 °C were carried out using a Perkin-Elmer DNA thermal cycler
(model 480). The amplified fragment was purified from 0·7%
agarose gel with the QIAquick gel-extraction kit (Qiagen). It was
then ligated to pCR-XL-TOPO vector (Invitrogen) and cloned by
transformation of electrocompetent TOP10 E. coli cells (Invitrogen)
according to the manufacturer’s protocol. The recombinant vector,
pCR-XL-TOPO-DprtS-1, was purified with QIAprep Spin Miniprep
Kit (Qiagen) from the recombinant cells and digested with BsgI
(New England Biolabs).
74 Introductory Food Microbiology
A 5·4 kb fragment containing the TopoXL vector and part of
the prtS gene was then purified from 0·7% agarose gel with
QIAquick gel extraction kit (Qiagen) and blunt-ended with T4
polymerase 3'® 5' exonuclease (Life Technologies) according to the
supplier’s protocol. It was then circularized by self-ligation with
Fast-link DNA ligation kit (Epicentre Technology); the resulting
plasmid, pCR®-XL-TOPO-DprtS-2, was produced by
transformation of electrocompetent TOP10 E. coli cells and purified
as described above. It was then digested with NotI and SpeI
(Eurogentec), and the resulting 2·078 kb fragment was purified as
already described above. The 2·078 kb fragment (~200 ng) was
ligated to pGhost9 vector (1~00 ng) (Maguin et al., 1996 ), digested
with NotI and SpeI. The ligation mix was used to electrotransform
100 µl of electrocompetent cells of Lc. lactis MG1363, as described
by Holo & Nes (1989) . Recombinant clones were selected on
M17Lac Ery plates after incubation at 28 °C. The recombinant
vector, pG+h9::DprtS, was purified as described above, and 20 µg
was used to transform electrocompetent cells of S. thermophilus
CNRZ 385, as previously described (Garault et al., 2000 ). Integration
of pG+h9::DprtS into the streptococcal chromosome was performed
as described by Garault et al. (2000) with the following modification:
to induce chromosomal integration of the plasmid, the culture was
diluted and plated on M17Lac Ery plates.
Finally, the mutant for PrtS was obtained by successive
incubations of the culture containing the chromosomal integration
at 37 °C to favour the excision of the pGhost9 plasmid.
PrtS is Essential to S. thermophilus Growth in Milk
S. thermophilus PrtS-negative mutant construction. We have
described here for the first time the construction of a targeted
negative mutant for S. thermophilus cell-wall proteinase PrtS. This
mutant of S. thermophilus CNRZ 385 was constructed by gene
replacement using a truncated copy of prtS gene cloned in pGhost9
plasmid. DNA sequencing confirmed that this copy was inserted
at the prtS locus and that pGhost9 was subsequently excised,
resulting in a truncated prtS gene. As expected, the truncated gene
was deprived of part of the signal sequence, all the pro-region
(removed after maturation of the protein in the parental strain),
and almost all of the region encoding the catalytic domain of the
Identification of Listeria Monocytogenes Genes 75
enzyme. Only the region encoding the six C-terminal amino acids
among the 495 constituting the catalytic domain (PR domain) was
still present in the mutant and did not include the sequence
encoding the residues involved in the catalytic activity of the
proteinase. Furthermore, protein exportation signals were no longer
present in the mutant; the signal sequence was truncated, and the
expected peptide cleavage site was excised.
Cell-wall proteinase activity of the wild-type and PrtS-negative
mutant of S. thermophilus. Using two different methods, we checked
that the S. thermophilus PrtS-negative mutant lacked cell-wall
proteinase activity. First, using 14C-labelled casein as a substrate,
we observed that cell suspensions of the PrtS-positive strain were
capable of hydrolysing casein (12·5% of total casein hydrolysed
within 10 min), whereas PrtS-negative cells had no detectable
caseinolytic activity. Second, we set up a rapid test on colonies
using a chromogenic substrate of PrtS. Three strains of S.
thermophilus were used: the proteinase-negative strain CNRZ 302
as negative control and the two proteinase-positive strains CNRZ
385 and CNRZ 703, which have a high cell-wall proteinase activity.
After growing on milk agar plates, colonies were covered with a
solution containing the substrate Suc-A-A-P-F-ßNA, Fast-Garnet
and different concentrations of CaCl2, the latter being an activator
of PrtS proteinase. Whatever the CaCl2 concentration (10, 20 or
50 mM), colonies of strains 703 and 385 rapidly became red,
whereas those of the negative strain 302 remained white. Using
this test, we confirmed that the mutant strain was PrtS-negative,
as colonies remained white even after several hours of contact
with the substrate solution. This test functioned on milk plates but
not on rich medium M17 plates for strain 703, which confirmed a
probable regulation of prtS expression by the growth medium as
already observed for this strain. This test will be useful to screen
for S. thermophilus PrtS-negative strains in milk and also for PrtS-
deregulated strains in M17.
Growth characteristics of the wild-type and PrtS-negative
mutant of S. thermophilus. By comparing the phenotypes of the
parental strain 385 and its PrtS- mutant on FSDA, and their growth
curves in liquid M17 and milk, we showed that proteinase PrtS
was essential to the growth of S. thermophilus in milk.
76 Introductory Food Microbiology
The PrtS- mutant, plated on FSDA, appeared as flat and
translucent colonies, as expected for PrtS- bacteria, whereas the
PrtS+ parental strain appeared as white, opaque, rounded colonies.
In M17, both strains had similar growth curves with a µmax
of 0·89 and 0·85 h-1 for the parental strain and the mutant strain,
respectively. In milk, streptococcal growth was determined
indirectly by pH measurement. Growth of the PrtS- mutant was
severely impaired in milk, as indicated by the reduced acidification
of milk by this strain compared to the parental strain 385. For the
PrtS- strain, milk acidification, and thus bacterial growth, was
restored to the same extent as that for the wild-type strain, after the
addition of yeast extract to milk.
The Lactobacillus strain influences the extent of the positive
effect of S. thermophilus/Lb. bulgaricus association.
Mixed cultures of S. thermophilus and Lb. bulgaricus were made
using two different strains of Lb. bulgaricus. To choose the last two
strains, we first determined the effect of the co-culture of Lb.
bulgaricus strains with the S. thermophilus CNRZ385 strain on milk
acidification, compared to the pure culture of Lb. bulgaricus. Among
the three strains of lactobacilli tested, the effect of adding strain
385 on the acidification was greatest with strains Lb. bulgaricus 397
and 1038; indeed, for these two Lactobacillus strains, the addition
of the Streptococcus highly enhanced the acidification rate compared
to the Lb. bulgaricus strain alone. Furthermore, the positive effect of
the bacterial association on milk acidification was more intense for
strain 1038, as, for this strain, the acidification rate and the final
pH were higher and lower, respectively, in the mixed culture than
in the pure culture. In contrast, addition of S. thermophilus strain
385 had no significant effect on milk acidification by Lb. bulgaricus
strain 752. Thus, strains 397 and 1038 of Lactobacillus were kept for
the following study. In addition, a proteinase-negative mutant of
strain Lb. bulgaricus CNRZ 397 was available and was used for the
following experiments.
In mixed cultures, proteinase PrtS has no effect on final pH and
bacterial populations, but PrtB affects both.
The effect of proteinases PrtS and PrtB on acidification of
mixed cultures and bacterial populations was estimated by
Identification of Listeria Monocytogenes Genes 77
measuring the final pHs, final total bacterial populations and final
individual populations of cultures performed with a strain of S.
thermophilus PrtS+ (strain 385) or PrtS- (strain 385-PrtS) and a strain
of Lb. bulgaricus PrtB+ (strains 397 and 1038) or PrtB- (strain 397-
PrtB).
The presence of proteinase PrtS had no effect either on the
final bacterial populations or on the final pHs of mixed cultures
involving PrtB+ Lactobacillus strains. The final total populations
were always similar in the presence or not of proteinase PrtS: 1·39
and 1·36x109 c.f.u. ml-1, respectively, for cultures involving Lb.
bulgaricus strain 1038, and 7·05 and 6·27x108 c.f.u. ml-1, respectively,
with strain 397. The absence of any differences in final total
populations corresponded to similar final individual populations
of S. thermophilus and Lb. bulgaricus, regardless of the presence of
PrtS: 1·3x109 c.f.u. ml-1 for strains 385 and 385-PrtS and 8·9x107 c.f.u.
ml-1 for strain 1038 for mixed cultures involving strain 1038,
5·5x108 c.f.u. ml-1 for strains 385 and 385-PrtS and 1·1x108 c.f.u. ml-
1
for strain 397 in mixed cultures involving strain 397. This
correlated well with the similar final pH obtained: 4·72 and 4·85 for
mixed cultures involving, respectively, strain 1038 and strain 397.
It is noteworthy that both the final total bacterial populations
and the acidification rates varied according to the Lactobacillus
strain associated with S. thermophilus strain 385. The final total
population when using Lactobacillus strain 1038 (1·38x109 c.f.u. ml-
1
) was twice as high as that of strain 397 (6·67x108 c.f.u. ml-1),
because the Streptococcus populations were more than twice as
high with strain 1038, Lactobacillus populations remaining constant.
Final pHs were not significantly different, but the time required to
reach these pHs was shorter for mixed cultures, including strain
1038, than those including strain 397 (4·66 h with strain 1038
versus 5·66 h with strain 397).
In contrast, the presence of proteinase PrtB affected both the
final bacterial populations and the final pHs. Final total bacterial
populations were threefold higher in the presence of PrtB than in
its absence (7·05x108 versus 2·8x108 c.f.u. ml-1). This resulted from
higher final populations of S. thermophilus in the presence of PrtB
(6·1x108 versus 2·06x108 c.f.u. ml-1) and led to a significantly better
acidification in the presence of PrtB (final pH 4·86 versus 5·42).
78 Introductory Food Microbiology
In our conditions of inoculation (Streptococcus/Lactobacillus ratio
of 1:1), S. thermophilus was systematically predominant in the total
final populations, regardless of the strain of Lactobacillus and the
presence of proteinases PrtS and PrtB. The magnitude of this
predominance depended on the Lb. bulgaricus strain used: with
strain 1038, S. thermophilus populations were 15-fold higher than
Lactobacillus populations and fivefold higher with strain 397, when
PrtB was present. This predominance was less marked in the
absence of PrtB, as the S. thermophilus populations were threefold
lower (6·1x108 c.f.u. ml-1) than populations reached in the presence
of PrtB (2·06x108 c.f.u. ml-1).
Variation of individual populations of S. thermophilus and Lb.
bulgaricus throughout mixed cultures in milk.
Proteinase PrtS had no significant effect on the variation of pH
and of individual populations throughout the culture and,
regardless of the mixed culture considered (except that involving
strain PrtB-), the variation of these two parameters remained similar.
Fig. 4a gives an example of this variation (a mixed culture made
of S. thermophilus 385 and Lb. bulgaricus 1038); for mixed cultures
including Lb. bulgaricus strain 397, we observed the same behaviour.
During the first 60-90 min, which corresponded to the first
acidification phase, S. thermophilus grew exponentially, whereas
Lb. bulgaricus did not grow significantly. Then, as the pH remained
constant, the streptococcal population stabilized for about 60-
90 min, whereas Lb. bulgaricus started to grow regularly and
continuously. Finally, during the last 2 or 3 h of fermentation,
when the acidification rate was the highest, both the Lactobacillus
and the Streptococcus grew regularly.
In contrast, proteinase PrtB was clearly involved in the
variation of bacterial populations and of pH, as demonstrated
with mixed cultures involving strain 397-PrtB. In fact, regardless
of the presence of PrtB, the first two phases of acidification
corresponding to the sequential growth of S. thermophilus and Lb.
bulgaricus were similar. However, during the third acidification
phase, the growth of S. thermophilus slowed down in the absence
of PrtB, the bacterial populations remaining almost constant during
the last 2 h of fermentation. This reduced growth resulted in a
Identification of Listeria Monocytogenes Genes 79
reduced acidification rate and an increased final pH (pH 5·42 in
the absence of PrtB and 4·86 in the presence of PrtB).
DISCUSSION
The present work aimed at evaluating the role of proteinase
PrtS from S. thermophilus in the growth in milk of S. thermophilus
in a pure culture. We also determined the effect of the presence of
both PrtS and PrtB from Lb. bulgaricus on S. thermophilus/Lb.
bulgaricus mixed cultures. For this purpose, we constructed a
targeted negative mutant of proteinase PrtS from S. thermophilus
CNRZ 385 and performed pure cultures of S. thermophilus and
mixed cultures with Lb. bulgaricus in milk.
In milk, the extent of the beneficial effect of the S. thermophilus/
Lb. bulgaricus association varies.
We observed that the effect of the co-culture of Lb. bulgaricus
strains with S. thermophilus strain 385 on the acidification of milk,
and thus the benefit of the bacterial association, depends on the
strain of Lb. bulgaricus used. In fact, with Lb. bulgaricus strain 752,
we did not obtain a marked beneficial effect of the association with
S. thermophilus 385 as already observed by several authors with
other strains. In contrast, mixed cultures of strains 1038 and 397
resulted in a higher acidification than pure cultures. Acidification
was higher with strain 1038 than with strain 397 due to higher S.
thermophilus populations, Lb. bulgaricus populations being similar.
These higher S. thermophilus populations probably resulted from a
better peptide and/or amino acid supply by one Lactobacillus strain
compared to the other as these nitrogen compounds are growth-
limiting for S. thermophilus in milk.
The two strains of Lb. bulgaricus thus probably differ in their
proteolytic potential, which is in agreement with the differences
observed in the final quantities of free amino acids and free NH2
groups in the supernatants of mixed cultures including these two
strains (data not shown). Some authors have also reported a
variability in the Lb. bulgaricus proteolytic potential. This variability
could be related to the presence of one cell-wall proteinase in Lb.
bulgaricus, which is the case of strain 397, or of two proteinases,
as reported for other strains.
80 Introductory Food Microbiology
PrtS is essential to the growth of S. thermophilus in milk in
pure culture but not in mixed culture.
Proteinase PrtS is essential to the growth of S. thermophilus
growth in milk as its PrtS- mutant was unable to grow efficiently
in milk until a nutritional complement [yeast extract or
bactotryptone (data not shown)] was added. This indicated that
proteinase PrtS was involved in nitrogen supply to the cell, via
casein hydrolysis, which is consistent with data previously
obtained with cell-wall proteinases of other lactic acid bacteria.
However, we demonstrated here that proteinase PrtS had no
significant effect on the growth of S. thermophilus in mixed cultures
in milk with Lb. bulgaricus; the growth of the parental strain 385
and of the PrtS- mutant in mixed culture was similar when Lb.
bulgaricus proteinase PrtB was present. This indicates that
assimilable nitrogen compounds necessary for S. thermophilus
growth are supplied by PrtB, as confirmed by the fact that the
absence of PrtB led to lower streptococcal populations. Furthermore,
as the streptococcal population was higher in the presence of PrtB
than in the presence of PrtS, we can assume that PrtB is more
efficient in the supply of peptides to S. thermophilus than PrtS. This
can be explained by a more active proteinase PrtB compared to
PrtS, as previous studies reported that the global proteolytic
activities of Lb. bulgaricus strains were 25-70 times higher than that
of S. thermophilus strains. We cannot rule out the possibility that
PrtS and PrtB have different substrate specificity, which leads to
the production of different peptides, some being more assimilable
than others. Indeed, PrtS is capable of hydrolysing MS-Arg-Pro-
Tyr-pNA, a substrate also hydrolysed by lactococcal proteinase
PrtP but not by PrtB. Furthermore, when comparing the substrate-
binding region of proteinases PrtS and PrtB, in particular the
residues 138, 166, 748, which have been identified as being directly
involved in substrate specificity in lactococci, we noticed that they
are totally different in PrtS (Thr, Ala, Asp) and PrtB (Gly, Val, Thr).
Model of growth of S. thermophilus associated with Lb.
bulgaricus and effect on acidification.
In all the mixed cultures performed in milk, we observed the
sequential development of S. thermophilus and then of Lb. bulgaricus,
which is in agreement with previous studies. Recently, the growth
Identification of Listeria Monocytogenes Genes 81
of S. thermophilus in pure culture in milk has been characterized,
in particular with regard to nitrogen nutrition; it consists of two
exponential growth phases, interrupted by a non-exponential
growth phase. From these latter results and those of the present
work, we propose the following model of growth of S. thermophilus
in mixed cultures with Lb. bulgaricus with three S. thermophilus
growth phases corresponding to three acidification steps.
During the first acidification step, characterized by a small
decrease in pH (<0·5 pH units), S. thermophilus grows exponentially,
whereas Lb. bulgaricus does not grow; S. thermophilus is thus
responsible for this first acidification, as first observed by Pette &
Lolkema (1950c ). The preferential growth of S. thermophilus can be
explained first by the fact that S. thermophilus has fewer nutritional
requirements than lactobacilli in milk. In particular, S. thermophilus
requires few amino acids and is capable of synthesizing branched-
chain amino acids; its growth can probably be supported by free
amino acids and peptides present in milk, as previously
demonstrated in pure culture, regardless of the presence of PrtS. In
contrast, Lb. bulgaricus is much more demanding from a nutritional
point of view than S. thermophilus (Letort, 2001 ); its optimal growth
relies on the supply of essential factors (CO2, pyruvate, formate)
produced by S. thermophilus. Second, in our study, mixed cultures
were performed at 42 °C, a temperature more favourable for S.
thermophilus, whose optimal growth temperature ranges between
40 and 45 °C, versus 45-50 °C for Lb. bulgaricus.
Then, the S. thermophilus exponential growth pauses and,
concomitantly, the acidification, while Lb. bulgaricus begins to grow
slowly and regularly until the end of fermentation. This pause
probably corresponds to depletion of amino acids and peptides in
milk, due to their consumption by S. thermophilus, as shown recently
by Letort et al. (2002) in pure culture, and the absence of
compensatory production by cell-wall proteinases. These authors
actually demonstrated that proteinase PrtS synthesis starts in the
middle of this phase and is maximal during the second exponential
growth phase in pure culture. Concerning the growth of Lb.
bulgaricus, we assume that as S. thermophilus reaches a high cellular
density during its first growth phase, it probably produces enough
growth-stimulating factors to favour the growth of Lb. bulgaricus.
82 Introductory Food Microbiology
Finally, during the following acidification phase, which leads
to a high pH decrease (about 1·5 pH units), Lb. bulgaricus continues
to grow; at the same time, S. thermophilus starts a second exponential
growth phase. We suggest that this acidification results not only
from the growth of Lb. bulgaricus but also from that of S. thermophilus.
This acidification phase is indeed greatly improved by the addition
of S. thermophilus to a Lb. bulgaricus culture; furthermore, in the
absence of PrtB, acidification is reduced, while only S. thermophilus
populations significantly decrease. The growth of S. thermophilus
probably occurs because of the utilization of peptides produced by
PrtS (when PrtB is absent) but also mainly by PrtB. No differences
in the growth of S. thermophilus were observed in the presence or
absence of PrtS when PrtB was present, and S. thermophilus
populations were significantly reduced in the absence of PrtB, i.e.
when PrtS was the sole source of peptide production.
In conclusion, we have determined the role of cell-wall
proteinases PrtS and PrtB in the growth of S. thermophilus and Lb.
bulgaricus in mixed cultures. We have shown that PrtB is involved
in the optimal growth of S. thermophilus, whereas PrtS does not
play a significant role when PrtB is present. Studies of the effect
of these proteinases on the free amino acid and peptide contents
as well as on the aroma profiles of mixed cultures are in progress.
As precursors, amino acids are involved in the formation of aroma
in dairy products, and variations in their composition can affect
aroma development. However, the different pH values observed in
the present study at the end of fermentation, when varying the
presence of proteinase PrtB, can modify the yoghurt flavour.
Experimental Validation of Low Virulence in Field Strains 83
4
EXPERIMENTAL VALIDATION OF
LOW VIRULENCE IN FIELD STRAINS
Several reports have described Listeria monocytogenes strains
which were nonpathogenic or weakly pathogenic, but little is
known about these low-virulence strains. We found that 9 field L.
monocytogenes strains were hypovirulent and 17 were avirulent,
based on the number of mice contaminated and the colonization
of their spleens after subcutaneous inoculation. All these strains
possessed the known virulence genes. We have now assessed the
low virulence of these strains in other assays before determining
how they differ from virulent strains. We have shown that the low-
virulence strains exhibited a phenotypic stability and were not a
mixture of virulent and avirulent bacteria. They did not recover
virulence after many passages in mice and colonized the spleens
of mice more poorly than virulent strains after i.v. inoculation.
Their lethal capacities, determined by 50% lethal dose (LD50), were
lower than those of virulent strains. Like Listeria innocua, 14 of 17
avirulent strains had no LD50 and were eliminated by the lymph
nodes after subcutaneous inoculation. The virulent, hypovirulent,
and avirulent strains were always significantly different, whatever
the tests of virulence used, confirming the importance of these low-
virulence field strains in identifying the proteins involved in
virulence.
INTRODUCTION
Listeria monocytogenes organisms are ubiquitous gram-positive
bacteria. They are widespread in the environment and have been
isolated from many sources, including soil, sewage, decaying
vegetation, and food. They are responsible for human diseases
84 Introductory Food Microbiology
characterized by meningitis, meningoencephalitis, septicemia,
abortion, and gastroenteritis. Through contaminated food, bacteria
reach the gastrointestinal tract and can translocate the intestinal
barrier to infect lymph nodes. Then, through lymph and blood, a
fraction of the bacteria reach the spleen and liver. Apoptosis,
neutrophils, and phagocytic cells contribute to the rapid clearing
of the bacteria before complete abolition by the specific immune
response. In some cases, such as the immunocompromised host,
bacteria multiply unrestrictedly in the hepatocytes from which
they disseminate through blood to the brain and placenta. Although
L. monocytogenes is also present in the environment and is probably
frequently ingested by humans, listeriosis is very rare. The incidence
is very low, around two to eight sporadic cases annually per
million people in Europe and the United States. If we exclude the
susceptibility of the host, another reason for this conflicting evidence
may lie in the variability of virulence in the L. monocytogenes strains.
Serotypes of L. monocytogenes could also be linked to the level of
virulence, as only three serotypes (1/2a, 1/2b, and 4b) have been
implicated in human cases. However, no bacterial genes related to
the serotype have yet been found.
Studies using different assays have shown that virulence varies
from one strain of L. monocytogenes to another. The mouse assays
are extremely sensitive assays for evaluating the pathogenicity of
L. monocytogenes by the systemic route. The immunocompromised
mouse model has shown a considerable difference in the 50%
lethal doses (LD50s) of virulent and nonvirulent strains. In the
same way, subcutaneous (s.c.) inoculation of immunocompetent
mice is very sensitive and specific, depending on the clinical
origin of the strains. Tissue culture assays, i.e., cytopathogenic
tests, have also been developed to distinguish pathogenic and
nonpathogenic L. monocytogenes strains. Certain genetic or
phenotypic markers have been linked to the virulence of the strains.
In our previous paper, the virulence of L. monocytogenes strains
was evaluated with a plaque-forming (PF) assay on HT-29 cells,
followed by s.c. injections of immunocompetent mice. We found 26
low-virulence field L. monocytogenes strains identified as
hypovirulent or avirulent. All these strains possessed the known
virulence genes and exhibited the same growth in nonselective
media by a bioscreen study. However, the cause of the low virulence
Experimental Validation of Low Virulence in Field Strains 85
is presently unknown. As a preliminary step toward understanding
the cause of this low virulence, it seemed important to know
whether low virulence is a stable character over time which cannot
be enhanced after in vivo passages. It was also important to know
whether these strains are also attenuated after intravenous (i.v.)
inoculation and whether their lethality is modified.
MATERIALS AND METHODS
Listeria Strains: The Listeria strains used and their
characteristics are given in Table 1 . Virulence was estimated by
the method of Roche et al. Different studies allowed the detection
of 9 hypovirulent strains and 17 avirulent strains. To analyze
these strains, 13 virulent L. monocytogenes strains and 1 Listeria
innocua strain were added as control strains. The strains were
maintained in storage medium at 4°C. For analysis, they were
cultured in brain heart infusion broth (3 ml) at 37°C for 8 h. BHI
agar (BHIA; Difco) slopes were then seeded and incubated overnight
at 37°C. The colonies were suspended in 2 ml of phosphate-buffered
saline (PBS) (pH 7.3), standardized turbidimetrically, and diluted
appropriately for each test.
Cell Line and Culture Conditions: The human adenocarcinoma
cell line HT-29 (no. 85061109; European Collection of Animal Cell
Cultures, Salisbury, United Kingdom) between passages 27 and
67 was used. Cells were grown in 75-cm2 plastic tissue culture
flasks in Dulbecco’s modified Eagle’s medium with glucose (4.5 g/
liter) (Invitrogen) supplemented with 10% (vol/vol) fetal calf serum
(Invitrogen) and 2 mM L-glutamine (Invitrogen). Antibiotics (100
IU of penicillin per ml and 100 µg of streptomycin per ml; Sigma,
Saint-Quentin Fallavier, France) were routinely added to the culture
medium except for the virulence assays. Cells were maintained in
a humidified incubator (at least 90% relative humidity) at 37°C
under 5% (vol/vol) CO2.
Phenotypic Stability: An initial PF assay was done on four
strains to demonstrate that hypovirulence was not the result of
two populations of bacteria, one infecting the cells and other unable
to infect the cells. The bacteria forming plaques were collected and
used for a second PF assay. The first PF assay was performed with
confluent monolayers of HT-29 cells in six-well tissue culture
86 Introductory Food Microbiology
plates. Cells were infected with 5 log CFU (105 CFU) per well
suspended in Dulbecco’s modified Eagle’s medium for 2 h at 37°C.
Incubation was continued for a further 1.5 h with 100 µg of
gentamicin (Sigma) per ml in the culture medium. Each well was
then overlaid with an agarose gel containing 0.48% indubiose in
culture medium supplemented with 10 µg of gentamicin per ml.
The same medium was then added to prevent cell starvation, and
incubation was continued for 3 days. The cells including bacteria
around plaques were recovered and lysed, and the bacteria were
used for a new PF assay (15). Bacteria maintained in the storage
medium were also tested. The results are expressed as the number
of plaques obtained for 7 log CFU deposited per well.
Virulence Recovery after in Vivo Passages: Spleen colonization
by five strains was monitored during 10 successive passages in
mice, and a PF assay was performed at the end of the experiment.
Groups of five 7-week-old conventional Swiss female mice (Iffa-
Credo, Saint-Germain-sur-l’Arbresle, France) were injected s.c. in
their left hind footpad with 6 log CFU suspended in 50 µl of PBS.
Each inoculum was checked by a viability count on tryptic soy
agar (TSA) plates (Bio-Mérieux, Marcy l’Etoile, France). The plates
were incubated for 48 h at 37°C. Mice were killed 3 days after
injection. Their spleens were removed aseptically, pooled, and
homogenized. Aliquots of each homogenate were used to assess
the bacteria in the spleens or to prepare the inoculum for the
passage in the next mouse.
The spleen colonization assessed on TSA plates is expressed
as the number of log CFU per homogenate (homogenates from the
spleens from the five mice in the group were pooled). The inoculum
for the next passage was prepared by incubating 100 µl of
homogenate in BHI broth at 37°C for 32 h (step enrichment). The
incubated homogenate was then seeded on BHIA slopes and
incubated for 17 h at 37°C. The strains isolated after passage 10
were compared to bacteria maintained in the storage medium in
a PF assay. The results are expressed as the number of plaques per
7 log CFU deposited per well.
Determination of Lethal doses in Mice: DBA/2 breeder mice
were purchased from Iffa-Credo. The mice were kept in the animal
house of the laboratory in level 2 containment facilities, and the
mice reproduced. Groups of six 8- to 10-week-old female mice were
Experimental Validation of Low Virulence in Field Strains 87
inoculated s.c. in their left hind footpad with 50 µl of bacteria
suspended in PBS. The inocula contained approximately 1 to 9 log
CFU for the virulent strains, 2 to 9 log CFU for the hypovirulent
stains, and 4 to 9 log CFU for the avirulent or nonpathogenic
strains. Each inoculum was checked by counting viable cells after
incubation on TSA plates for 48 h at 37°C. Mice were observed
every day for 15 days, and all deaths were recorded. All the mice
remaining on day 15 were killed. The LD50s were calculated using
a probit dose-response model, considering a log transformation of
dose rates and the total number of mice that died. The percentage
of mice that died in the three groups were also analyzed by logistic
regression.
i.v. Injection: Female Swiss mice (6 to 9 weeks old) (Iffa-Credo)
were injected with hypovirulent and avirulent strains. The mice
were kept under controlled conditions (humidity, temperature, food
delivery, and stress) during the experiments. Bacteria (4.5 log CFU)
were suspended in 0.5 ml of PBS and injected i.v. The mice were
killed with carbon dioxide, 2 days after inoculation. Their spleens
were removed and homogenized in PBS using a glass homogenizer
with a loose-fitting pestle. Triton X-100 was added to a final
concentration of 0.001%, and dilutions were made immediately.
Four mice were used for each strain. The viable bacteria in the
inoculates and spleens was counted on Columbia agar. Results
were compared by analysis of variance and analyzed by the Tukey-
Kramer multiple comparison method.
Kinetics of Colonization: Kinetics of colonization were analyzed
for four strains. Groups of five 6-week-old conventional Swiss
female mice (Iffa-Credo) were injected s.c. in their left hind footpad
with 4 log CFU suspended in 50 µl of PBS. The mice were killed
1, 24, 48, and 72 h after injection.
The left and right popliteal lymph nodes, lumbar lymph nodes,
spleen, liver, and lungs were removed from each mouse aseptically.
Samples were homogenized, and the homogenates were diluted in
BHI broth. Appropriate dilutions were plated onto TSA plates and
incubated at 37°C for 48 h. Viable bacteria were counted. The mean
log CFU per organ was calculated only for samples with bacteria.
Homogenates were kept overnight in BHI at 37°C for enrichment.
Enriched homogenates in which Listeria strains were not detected
were isolated on TSA plates and further incubated overnight at
88 Introductory Food Microbiology
37°C. The results are expressed as the number (log) of CFU per
organ.
RESULTS
Phenotypic Stability: All the low-virulence strains studied
were cloned. We checked the possibility that low virulence could
be the result of expression of two phenotypes, one of which could
infect cells while the other could not. If this was the case, a small
subpopulation of bacteria would be able to form plaques and if we
recovered these bacteria, the number of plaques in a second test
should be higher. Among the low-virulence strains, we used the
four strains that produced few plaques (strains BO34, CR282, 449,
and 442). Indeed, in order to observe a possible increase or decrease
in plaque number, we could not choose strains forming a high
number of plaques or no plaque at all. We found no difference in
the virulence of the bacteria recovered from the plaques and the
control bacteria under the conditions we used, confirming that all
bacteria within the population have the same level of virulence.
Lack of Recovery of Virulence after in Vivo Passages: In order
to analyze a possible recovery of virulence in low-virulence strains
after 10 passages in mice, we chose the three strains that infected
three of five mice (436, BO34, and 464) and the two strains that
infected one of five mice (SO205 and BO43). As these strains were
hypovirulent, we pooled the five spleens in order to increase the
chance of recovering bacteria. The numbers of bacteria recovered
from the spleens of mice infected with strains 436, 464, and BO34
were fairly constant during the experiment. The numbers of bacteria
found in the spleens of mice infected with strains SO205 and BO43
were equal to the threshold of detection, because they were not
directly recovered from spleen homogenate but were recovered
after 1 and 10 passages, respectively, in spleen homogenates only
after 32 h of growth in BHI medium. After four passages in vivo,
the SO205 strain was not recovered from the spleens of the five
mice, despite the enrichment step. Thus, this value was below the
threshold. The number of plaques of bacteria recovered from the
spleens after 10 passages was then compared to the number of
plaques of bacteria maintained in storage medium. The bacteria
that had been passaged in vivo formed 10 times fewer plaques
than the control bacteria.
Experimental Validation of Low Virulence in Field Strains 89
Lethality Study: Under our study conditions, L. monocytogenes
strains were virulent, hypovirulent, or avirulent, depending on
their ability to colonize the spleens of mice. In order to know
whether these strains exhibited the same lethality, the numbers of
mice dying were recorded during the 15 days after inoculation
with increasing doses of Listeria strains. DBA/2 mice were chosen
for these study because they are more sensitive to L. monocytogenes
than the Swiss mice. Some LD50s were calculated from very few
observations due to the few deaths caused by some strains. For
some strains, none of the mice died after 15 days or there were not
enough deaths to calculate an LD50. In that case, maximal injected
doses are used and indicate the difference in the virulence of the
three groups (virulent, hypovirulent, and avirulent strains). An
LD50 could be calculated for only 3 of the 17 avirulent L.
monocytogenes strains; they were between 8.7 and 9.3 log CFU. The
14 other strains were not lethal. The LD50s could be determined
for only five of the nine hypovirulent L. monocytogenes strains (8.3
to 9.0 log CFU). The LD50s for the 13 virulent strains were 4.1 to
7.9 log CFU. We also compared the percentages of dead mice in the
three groups by logistic regression. The difference was highly
significant (P < 0.0001), depending on the injected dose. The
difference between the hypovirulent and avirulent strains was
also highly significant (P < 0.0001), with the hypovirulent strains
being more lethal after injection of 9 log CFU.
i.v. injection: Differences in spleen colonization and lethal
capability were observed after s.c. injections. Bacteria were injected
i.v. to determine whether their virulence was modified when the
mode of inoculation changed. Only 4 of the 13 virulent strains
were tested, and their mean virulence ranged from 5.8 to 7.2 log
CFU per spleen homogenate, with a mean of 6.58 log CFU for the
4 strains. The mean virulence of the hypovirulent strains was
lower than that of the virulent strains (4.0 to 6.3 log CFU per
spleen homogenate; mean, 5.07 log CFU). The avirulent strains
included 11 strains with a virulence of 3.5 to 5.2 log CFU per
spleen homogenate, with a mean of 2.93 log CFU. The numbers of
CFU recovered for six avirulent strains were below the threshold
for the four mice (2 log CFU per spleen homogenate). The percentage
of mice infected by the hypovirulent and virulent strains (100% of
51 mice) was significantly different from those infected by the
90 Introductory Food Microbiology
avirulent strains (42% of 60 mice). Analysis of variance of the
means of log CFU indicates a highly significant difference (P <
0.0001) between the three groups of strains. Analysis of the pairwise
differences in mean numbers of bacteria per spleen homogenate
was obtained by the Tukey-Kramer multiple comparison method.
All the different values compared, namely, the values for
hypovirulent and virulent strains, hypovirulent and avirulent
strains, and virulent and avirulent strains, were statistically
significant. All the simultaneous 95% confidence intervals by the
Tukey method exclude zero.
Rate of Colonization: The rates of colonization by the virulent
L. monocytogenes strain EGDe, the hypovirulent strain BO34, the
avirulent strain 442, and L. innocua BUG499 were measured to
determine how fast the bacteria spread in mice after s.c. inoculation
in their left hind footpad. Bacteria of the virulent strain were found
in all the organs studied 1 h postinoculation. The number of mice
infected and the degree of infection increased with time, so that all
the mice were infected on day three. The hypovirulent strain spread
more slowly than the virulent strain did. Bacteria were recovered
in the spleens only on and after the second day and only in two
or three of the five mice. The liver was never infected. The avirulent
strain infected only the lymph nodes. The bacteria spread from the
left popliteal lymph nodes to the lumbar lymph nodes but not to
the spleen or liver, suggesting that the lymph nodes were sufficient
to eliminate the bacteria. No bacteria were found in the blood, even
after enrichment, but the lymph nodes were more severely infected
than those of mice injected with the L. innocua strain.
DISCUSSION
It is difficult to detect and characterize low-virulence L.
monocytogenes strains for several reasons. They grow at the same
rate as virulent strains on nonselective media (i.e., TSA and BHIA),
but detecting low-virulence strains on some selective media is
problematic. Indeed, some of these strains could be detected on
Palcam medium only after 3 days of growth and were generally
not detected or poorly detected on Rapid L’mono medium.
Moreover, the low virulence of these strains is often ascertained by
a single test, and there is no standard, well accepted method for
identifying and defining low-virulence L. monocytogenes strains,
Experimental Validation of Low Virulence in Field Strains 91
although several virulence assays have been described. We recently
developed a virulence assay based on a PF assay and the s.c.
infection of mice. It allowed the detection of 26 low-virulence field
strains of L. monocytogenes. However, it is important to better
characterize this low virulence before undertaking genetic
characterization of the strains. In 1987, Pine et al. reported that the
ATCC 35152 strain contains nonvirulent, nonhemolytic colonies
that originated as spontaneous variants from the hemolytic parent
strain. We therefore looked for such nonphenotypic stability, as
any uncertainty could raise questions about the reliability of all
data obtained with such strains. The bacteria recovered from and
around a plaque formed the same number of plaques (of the same
size) as the parent bacterial culture, showing that low-virulence
strains did not consist of a mixture of virulent and avirulent
bacteria.
Waseem et al. demonstrated that passaging the L. monocytogenes
NCTC 7973 strain increases its virulence in rabbits, as evaluated
by the recovery of viable bacteria from the infected organs. In the
same way, Wirsing von Koenig et al. have shown that mice became
more resistant after many passages as evaluated by LD50s. Thus,
it is possible that the virulence of our strains also increases after
in vivo passages. Our data clearly show that bacteria conserved in
storage medium do not increase their virulence during successive
subculturings. The number of Listeria per spleen after 10 passages
in mice was the same as those obtained at the first passage. In
addition, the virulence of the strains after 10 passages was not
increased over that of their parent strains and was even diminished
for some strains.
We also confirmed the low virulence of these strains by several
classical tests. The lethality and the bacterial load of organs were
therefore used as criteria of pathogenicity after both s.c. and i.v.
inoculation. We did not use the oral inoculation route in mice,
because the results were less reproducible. Moreover, it is not
representative of human infection because mouse enterocytes do
not express the same E-cadherin that human enterocytes do,
according to the observations of Lecuit et al. Virulent strains have
LD50s from 1 x 104 to 8 x 107 CFU, and the spleens are heavily
colonized after both s.c. and i.v. inoculation. The hypovirulent and
avirulent strains were much less virulent than the virulent strains
92 Introductory Food Microbiology
in all the assays used. The hypovirulent strains were close to
avirulent strains in term of lethality, with an LD50 greater than 2
x 108 CFU. Mice inoculated with 109 CFU of L. innocua or avirulent
strains were only lethargic and had ruffled for the first few days
after infection and they recovered soon afterwards. However, the
hypovirulent strains colonized the organs better than the avirulent
strains, particularly the spleen. Taking into account the capacity
of the strains to colonize host organs, our results suggest that the
avirulent strains spread better than the nonpathogenic L. innocua
strain BUG 499 because L. innocua did not colonize the lumbar
lymph nodes after inoculation into the footpad.
The rate of infection after s.c. inoculation suggested that the
virulent strains spread quickly to heavily infect all the internal
organs and lymph nodes. The lumbar lymph nodes and liver seem
to play a key role in the elimination of hypovirulent strains injected
into the hind footpad, whereas the lymph nodes alone are sufficient
to eliminate avirulent strains and L. innocua. These data agree with
studies showing that Listeria bacteria are cleared rapidly from the
lymph nodes by CD8+ T cells and from the bloodstream by the
neutrophils and Kupffer’s cells in the liver.
Thus, our data show that all the L. monocytogenes strains
screened by our PF assay are low-virulence strains and that this
low virulence is not an artifact. Although the virulence of the
avirulent L. monocytogenes strains is very similar to that of L. innocua,
the rates at which they colonize organs are different. They also
have the same in vitro and in vivo phenotypes as the strains
attenuated by deletion of actA/plcB and are no danger to human
health. However, the hypovirulent L. monocytogenes strains have a
low but real virulence that is confirmed by the clinical origin of
two strains. All the virulence assays used (LD50, spleen colonization
after i.v. inoculation or s.c. inoculation in their left hind footpad)
clearly showed significant differences between the three levels of
virulence established by our virulence assays. All these strains
have the main known genes of virulence, but in-frame mutations
could decrease their virulence. Genetic and phenotypic analyses of
hypovirulent and avirulent L. monocytogenes strains are now in
progress in our laboratory.
The Effect of Inoculum Size on the Lag Phase 93
5
THE EFFECT OF INOCULUM SIZE
ON THE LAG PHASE
The effect of inoculum size on population lag times of Listeria
monocytogenes was investigated using the Bioscreen automated
microtitre plate incubator and reader. Under optimum conditions,
lag times were little affected by inoculum size and there was little
variation between replicate inocula even at very low cell numbers.
However, in media containing inhibitory concentrations of NaCl,
both the mean lag time and variation between replicate inocula
increased as the inoculum size became smaller.
The variation in lag time of cells within a population was
investigated in more detail by measuring the distribution of
detection times from 64 replicate inocula containing only one or
two cells capable of initiating growth. The variance of the lag time
distribution increased with increasing salt concentration and was
greater in exponential than in stationary phase inocula. The number
of cells required to initiate growth increased from one cell under
optimum conditions to 105 cells in medium with 1.8 M NaCl.
The addition of spent medium from a stationary phase culture
reduced the variance and decreased lag times. The ability to initiate
growth under severe salt stress appears to depend on the presence
of a resistant sub-fraction of the population, although high cell
densities assist adaptation of those resistant cells to the
unfavourable growth conditions by some unspecified medium
conditioning effect. These results are relevant to the prediction of
lag times and probability of growth from low numbers of stressed
cells in food.
94 Introductory Food Microbiology
INTRODUCTION
The lag phase of the microbial growth cycle represents the
time period needed for bacteria to adapt to a new environment
before cell multiplication commences. Recent work modelling the
behaviour of bacteria in foods has shown that the lag phase is
more difficult to predict than is the specific growth rate. This
implies that certain relevant variables affecting lag time are not
taken into account, particularly the physiological state of the
inoculum and the inoculum size.
The physiological state of cells will be affected by their previous
growth environment and by exposure to stress conditions. Cellular
injury caused by heating freezing or drying can extend the lag
time considerably and also increase its variability. Starved cells
may also have very extended lag phases. The temperature history
of the inoculum culture and its growth conditions have a profound
effect on lag. Some mathematical models used in predictive food
microbiology contain a parameter related to the physiological state
of the inoculum but it has not been possible so far to measure
physiological state directly in terms that are useful for predicting
behaviour. This may become possible eventually with developments
in methods for measuring single cell metabolic activity.
Two classes of inoculum size effect on population lag may be
envisaged (a) cooperative or inhibitory effects of high cell
concentrations or (b) statistical effects at low cell concentrations
arising from the variability in individual lag times. There is little
specific information about the possible effects of cell–cell
interactions on lag time although cell signalling has been shown
to affect the emergence of cells from dormancy and the lag time of
populations in biofilms. The effect of substances produced in
culture supernatants on microbial growth was reviewed by
Kaprelyants et al. (1999)
Mathematical treatments of the statistical effects of inoculum
size on population lag were provided by Baranyi (1998) and
Baranyi and Pin (1999). These authors showed that as the cell
number in the inoculum decreases, the population lag increases
by an amount that depends on the distribution of individual lag
times and the maximum specific growth rate. The mathematical
analyses predict that under optimum growth conditions, this
The Effect of Inoculum Size on the Lag Phase 95
statistical effect would only be expected at inoculum levels of
below about 102–103 cells. McKellar and Knight (2000) have also
presented a model of the lag phase of Listeria monocytogenes that
takes into account the variability of individual cells.
There have been relatively few experimental tests of the effect
of inoculum size on lag, but available data suggests that the effects
are small in broth cultures under optimum growth conditions and
also in food. However more recently, it has been shown that the
lag time of L. monocytogenes was extended in stressed cells when
the inoculum size was small. Stephens et al. (1997) also showed
that the length of the lag phase is not only very variable in heat
injured cultures of low cell density, but also on average, longer
than those of high cell density.
The purpose of this study was to investigate the effect of
inoculum size on the population lag time of L. monocytogenes
under conditions of salt stress and to assess the heterogeneity in
lag times of inocula containing very low cell numbers. During the
course of this work, an effect of population size on the probability
of initiating growth became evident.
MATERIALS AND METHODS
Organism and Culture Conditions
L. monocytogenes NCTC 11994 was used throughout these
experiments. It was kept on glass beads at “70 °C and maintained
on tryptone soya agar slopes at 5 °C. Stationary phase inocula
were prepared by inoculating 20 ml tryptone soya broth and
incubating overnight at 37 °C. To produce an exponential phase
inoculum, 0.1 ml of a stationary phase culture was inoculated into
20 ml TSB (pre-warmed to 37 °C) and incubated until the optical
density at 680 nm (OD 680 ) reached 0.15 measured in a
spectrophotometer with a light path of 1 cm. Viable counts were
measured by using spread plates. Tenfold dilutions of culture
were made in Maximum Recovery Diluent (Oxoid) and duplicate
50 µl samples spread on TSA plates.
Bioscreen
The effect of inoculum size on the variation in the population
lag was investigated by preparing tenfold serial dilutions of
96 Introductory Food Microbiology
exponential and stationary phase cultures in either TSB, TSB with
1.2 M NaCl or TSB with 1.6 M NaCl.
Ten replicate 300µl inocula from each dilution were placed in
the wells of a microtitre plate and the plates were then incubated
at 37 °C in the Labsystems Bioscreen C. The increase in turbidity
at 600 nm was monitored automatically every 10–30 min for 2–12
days. The plates were shaken between measurements.
Detection times obtained from the outermost wells of the
microtitre plates were generally longer than those from the other
wells, particularly when the medium contained a high salt
concentration. On further investigation, we found that for long
incubations (greater than 7 days), there was a decrease in the
volume of media in these outer wells of up to 10%. We therefore
discarded the results obtained from all the outer wells in all
experiments. So, for the initial experiments, there were only eight
replicates rather than 10 per dilution.
To investigate variability in the length of the lag phase at very
low inoculum levels, cultures were diluted to a level that gave
growth in approximately 50% of 64 wells. According to the Poisson
distribution, 70% of positive wells would then contain a single
cell and 24% two cells. Three salt concentrations, 0, 1.2 and 1.6 M,
were investigated.
The possible effect of medium conditioning (alteration of some
physicochemical aspect of the growth medium by the previous
growth of a culture) or cell signalling was checked by preparing
dilutions of log phase cells using 50:50 spent broth/fresh TSB,
with added NaCl as before. The spent broth was prepared by
filtering a 24-h culture through a 0.2-mµ pore size nylon membrane
filter (Nalgene, Milton Keynes, UK). All experiments were repeated
at least twice.
Probability of Growth
The effect of inoculum size on the probability of initiating
growth in a culture was investigated by incubating 40 replicates
of a few critical inoculum levels, taken around the growth/no-
growth inoculum size boundary at three salt concentrations, 0, 1.7
and 1.8 M.
The Effect of Inoculum Size on the Lag Phase 97
Mathematical Treatment of Data
Data from the Bioscreen were analysed using the approach of
Baranyi (1998). Detection time is converted to a corresponding
‘physiological state’ parameter (a) that is an expression of the
‘readiness of cells to grow’ using the following transformation
exp ( − µma x T det )
α=
r
where µmax is the maximum specific growth rate, Tdet is the detection
time, r is the ratio between the initial and the detected concentration
reached at Tdet.
RESULTS
The Effect of Inoculum Size on Lag Time
To test whether there was an inoculum size effect, tenfold
serial dilutions of cultures were made in TSB containing different
levels of NaCl, and the detection times recorded. Assuming that
specific growth rate is unaffected by inoculum size, the difference
in detection time between consecutive dilutions should be constant
and proportional to the maximum specific growth rate (µmax)
provided there is no effect of inoculum size on population lag
times. Hence, a plot of detection time versus the logarithm of the
inoculum size should give a straight line. If, on the other hand
population lag times were affected by inoculum size, a deviation
in linearity would be expected. Under optimum growth conditions,
i.e. in TSB with no added NaCl, detection times were indeed
linearly related to log inoculum size. The slopes of the
experimentally determined lines were close to those predicted
from the growth rate estimated from viable count data from cultures
growing in the wells. The specific growth rates estimated from the
detection times in Fig. 1a were 1.12 and 1.22 h”1 which is close to
a growth rate of 1.27 h”1 estimated from viable count data from
cultures growing in the wells. There was little variability in the
detection times of replicate inocula at each inoculum level although
slight scatter was observed at the lower inoculum levels (1–10
cells). Under these conditions, there was no marked effect of
inoculum size on population lag and no difference in the response
of exponential and stationary phase cells.
98 Introductory Food Microbiology
As conditions became more stressful and growth rate
decreased, i.e. in TSB containing 1.2 M NaCl, there was greater
variation in lag between replicates particularly for the exponential
phase inoculum. The variability increased with decreasing
inoculum size especially below 104 cells per well. The trend of the
scatter was towards longer detection times rather than being
distributed around the expected detection time. The net effect was
that as inoculum size decreased, the mean detection time deviated
more from that predicted assuming that lag time and growth rate
remain constant. Assuming that the maximum growth rate is
independent of inoculum size, the observed deviation represents
an increase in lag at low cell numbers. An additional effect was
that, with exponential phase cultures, no growth was seen from
inocula containing less than 10 cells. Cultures inoculated with
stationary-phase cells showed a linear relationship between
inoculum size and detection time, except at very low cell numbers,
where detection times were longer than predicted and fewer wells
showed growth.
The results from experiments with TSB containing 1.6 M NaCl
show a continuation of this trend, such that the relationship
between inoculum size and mean detection time is no longer
linear. Less than 50% of the cultures grew when inoculated with
104 cells, and none grew with inocula of less than 103 cells,
irrespective of the growth phase. Exponential phase inocula gave
consistently longer detection times than stationary phase inocula.
The detection time data were transformed according to the
method of Baranyi and Pin (1999) to yield values. If the lag times
of the individual cells are not affected by the inoculum size, then
these physiological state values should be scattered around a
constant, independently of inoculum size, but with increasing
variance as the inoculum size decreases. When data from stationary
phase cells inoculated into 1.2 M NaCl were transformed in this
way, values for stationary phase cells did scatter around a constant,
except at the lowest inoculum level, indicating that inoculum size
did not influence readiness of cells to grow except at the lowest
inoculum level.
However, with exponentially growing cells, the values deviated
progressively from the constant showing that inoculum size affected
The Effect of Inoculum Size on the Lag Phase 99
lag by a mechanism separate from the statistical effect due to the
inherent lag time distribution.
The distribution of detection times was studied in more detail
for very small inocula. Sixty-four replicates were prepared at
inoculum levels that gave growth in approximately half the
replicates. The average number of cells per inoculum required to
give growth in half the replicates was determined independently
by plate counting.
For TSB containing 0, 1.2 or 1.6 M NaCl, the mean inoculum
sizes were 1.2, 5.0 and 2000 cells, respectively. However, since
only half the wells actually showed growth, the effective mean
inoculum size according to the Poisson distribution would have
been 0.7 cells per well in each case. The data were normalised by
setting histogram bin size equal to the doubling time for each salt
concentration, hence, the increase in variability in detection times
shown in the figures was not simply an effect of growth rate. In
TSB alone, there was very little variation in detection times (and
hence lag times) between inocula of approximately one cell.
In medium containing 1.2 M NaCl, there was a pronounced
difference between the responses of exponential and stationary
phase cells. Stationary phase cells showed relatively little variation
in detection times a slight tailing towards longer detection times.
The range between shortest and longest detection time was
equivalent to about five doubling times. Under the same conditions,
exponential phase cells showed a much larger variation, with the
longest detection times being approximately twice that of the
shortest (a range of about 22 doubling times). A similar effect was
observed at the higher salt concentration of 1.6 M.
Effect of Inoculum Size on the Ability to Initiate Growth
When cells were inoculated into TSB with no added salt, the
percentage of wells showing growth was close to that predicted
from the Poisson distribution, showing that growth was possible
from single cells. However, with increasing salt concentration, the
number of cells required to initiate growth in 50% of the wells
increased to more than 103 cells in media containing 1.7 M NaCl
and around 106 cells the presence of 1.8 M NaCl.
100 Introductory Food Microbiology
Effect of Spent Medium
To further investigate the cause of the inoculum size effects,
log phase cells were inoculated into fresh TSB mixed with an
equal volume of spent broth. The hypothesis was that inoculum
size effects (a shortened lag phase or increased probability of
growth at high cell density) might be caused either by carry-over
of substances with the inoculum, or the presence of a signal
chemical produced during the lag phase that may also be present
in medium from stationary phase cultures. Spent medium had no
effect at NaCl concentrations of 1.2 M or less.
However, with 1.6 M NaCl detection times were shortened
with inocula of 3000 cells per inoculum or less, and the variability
between samples reduced. A simple ANOVA was run on the
detection times measured at the two lowest inoculum levels, where
the variabilty of the lag times of individual cells has the biggest
effect on the population lag. An F-test showed that the probability
that the shorter avarage lag times were observed only by chance
was less than 10"3. The standard deviation of the detection times
decreased by more than 50% for both inoculum levels, confirming
the significance of the spent medium effect.
DISCUSSION
Use of the Bioscreen
The Bioscreen proved to be a useful means of producing large
quantities of growth curve data. It has been used for a number of
different applications such as determining bacterial growth rates,
studying the effect of different conditions on growth, growth
inhibition studies, enumeration of bacteria from food samples,
and comparison of pre-enrichment media for resuscitation of
injured salmonellae. However, the data produced need to be
analysed with care. The spurious readings obtained from the
outermost wells were most obvious when the cells were grown
with high NaCl concentrations under conditions that required
long incubation times before growth became detectable. This
probably resulted in evaporation from the outermost wells, a feature
that limits the incubation times that can be employed in these
studies.
The Effect of Inoculum Size on the Lag Phase 101
THE EFFECT OF INOCULUM SIZE ON LAG TIME
Our basic assumption was that the maximum specific growth
rate (µmax), within the physiological range of any given organism,
is independent of cell history and is uniquely determined by its
environment. This is shown by the global constancy of reported
µmax values for particular organisms grown under the same
conditions. A recent report by Membré et al. (1999) describing an
effect of inoculum pre-treatment on growth rate of L. monocytogenes
is an exception to this rule, though the effect was very small.
Therefore, for any set of conditions, a plot of the logarithm of
inoculum size versus detection time should yield a straight line
whose slope is proportional to specific growth rate, provided lag
time is constant and unaffected by inoculum size. If the units on
the abscissa are Ln inoculum size, the slope will equal 1/µmax, if
the units are log2 inoculum size, the slope will be equal to doubling
time. From the above, a deviation from linearity would imply that
population lag was not independent of inoculum size.
The lag time of a population is less than the average lag time
of individual cells because cells with shortest lags begin to multiply
soonest and their descendants dominate the population
numerically. This effect was analysed by Baranyi (1998) who
derived a mathematical relationship between population lag and
the distribution of individual cell lag times and growth rate. This
analysis revealed that as cell numbers in the inoculum increased
from 1 to 103, the scatter of lag times from replicate inocula
decreased. The greatest variation in lag should thus occur in
inocula containing low numbers of cells from populations that
had a wide scatter of individual cell lag times. Any variation in
lag time between replicate inocula will be reflected in a
corresponding variation in detection times.
Because plots of inoculum size versus detection time were
essentially linear for growth in TSB, we conclude that, under
optimum conditions, there was no effect of inoculum size on
population lag, except that due to statistical variation which was
noticeable only at low cell numbers (100 cells per well or less). Our
results obtained under optimum conditions agree with the work
of Jason (1983) who found that lag and growth rate of Escherichia
coli were independent of inoculum size. Duffy et al. (1994b) also
102 Introductory Food Microbiology
concluded from viable count data that the lag times of L.
monocytogenes in Listeria Selective Broth, Palcam broth or a mixture
of beef mince and broth, were unaffected by inoculum size.
Although an increase in lag time was observed with decreasing
inoculum size this was statistically insignificant due to the wide
scatter of the data.
Under the more exacting growth conditions provided by TSB
containing 1.2 or 1.6 M NaCl, there was a wider scatter of detection
times as inoculum size decreased, and the mean value diverged
increasingly from that predicted assuming a constant lag.
This was particularly noticeable with exponential phase
inocula. Exponential phase cells are generally more fragile and
susceptible to injury than those in the stationary phase and this
may account for their longer and more variable lag times following
osmotic shock. Transformation of the data according to Baranyi
and Pin (1999) showed that inoculum size had an effect on lag
that was separate from the statistical effect caused by the
distribution of lag times of single cells, i.e. there was a cooperative
population effect.
An effect of inoculum size on lag has been shown previously
with L. monocytogenes inoculated into broth at pH 5.9 and an
incubation temperature of 14 °C, designed to simulate ripening of
Camembert cheese. The biggest inoculum size effect was seen
when cells were grown at 30 °C then held for 4 weeks at 4 °C
before inoculation into broth at 14 °C, when an inoculum of 103
cells gave a lag of less than 21 h compared with 7.7 days for an
inoculum of 10 cells. An extension of lag time and an increase in
variability between replicate inocula has also been reported for
heat-injured cells of Salmonella typhimurium. This is similar to the
effect described here with salt-stressed cells of L monocytogenes.
Effect of Inoculum Size on the Probability of Initiating Growth
Under optimum conditions a single cell was able to initiate
growth in TSB but, in the presence of high salt concentrations,
much larger inocula were needed. A similar observation was made
by Razavilar and Genigeorgis (1988) who found that the number
of cells of L. monocytogenes required to initiate growth at 30 °C was
unaffected by NaCl concentrations up to 1.37 M. However, at
The Effect of Inoculum Size on the Lag Phase 103
concentrations of 1.71 to 2.05 M NaCl, at least 105 cells were
required for growth to occur, comparable with an inoculum size
of 103 with 1.7 M and 106 with 1.8 M NaCl that we observed at
37 °C.
Statistical Explanations of Population Size Effects
Studies with inocula containing predominantly single cells
suggested that the increase in mean population lag time at low
cell number under stressful growth conditions could have been
explained simply by the corresponding increase in scatter in the
lag times of single cells. In unstressed populations the variation
of lag times was only about ±2 doubling times. Part of that variation
can be accounted for by variation in the number of cells per well
as predicted from the Poisson distribution.
Since the number of wells showing growth was around 50%,
about 70% of positive wells would contain one cell and 24%
would contain two cells. The detection times of wells containing
two cells would be shorter by one generation time than those
containing only a single cell. Under stressful conditions, the scatter
increased to about ±10 doubling times implying that the variability
in detection time was largely due to greatly extended lags rather
than different numbers of cells in the wells.
The question nevertheless arises whether inoculum size effects
on lag time and the probability of initiating growth can be
explained solely by the distribution of some resistance attribute
within the population. In broth containing 1.2 M NaCl, a wide
scatter of lag times was seen even with inocula containing 3000
cells, a number which, from the theoretical results of Baranyi
(1998), should have ensured that the lag times of replicates would
have converged close to the population mean.
However, a comparison of the dilution at which wells showed
no growth with the known number of cells inoculated, showed
that the number of cells able to initiate growth was much less than
the number present in the wells, and this may therefore bring the
effective inoculum size within the range where statistical effects
become apparent. The same argument applies with cells in 1.6 M
NaCl except the fraction of cells able to initiate growth was even
smaller.
104 Introductory Food Microbiology
Cooperative Effects on Lag and the Ability to Initiate Growth
An alternative explanation for the inoculum size effects is that
some kind of conditioning of the culture medium or chemical
signalling is required before growth can occur under stressful
conditions. There appears to be a critical threshold cell density
below which growth initiation is not possible and this threshold
is related to the severity of the culture conditions.
Cells may produce a chemical or physicochemical change in
situ, or there may be carry-over of substances from the inoculum.
The addition of spent medium to TSB containing 1.6 M NaCl
shortened detection times, and decreased lag time variability, but
no change was recorded in the probability of initiating growth. A
requirement for specific signal molecules in recovery from stress
has been reported in some bacteria. Cells of Nitrosomonas europaea
recovered from starvation and commenced growth sooner when
cells were present in a biofilm.
Acylated homoserine lactones were shown to reduce the length
of the lag phase in a concentration dependent manner, suggesting
that these signal molecules responsible for this cooperative effect.
In the Gram positive Micrococcus luteus a peptide signal molecule
that is necessary for the revival of dormant cells has been isolated
from culture medium.
The growth stimulatory effects described here may also involve
signal molecules, but a non-specific conditioning effect could also
explain the phenomenon. For example, the protective effect of high
cell densities on stressed bacteria may be due to non-specific
effects such as leakage of magnesium or other unspecified material
from dead and injured cells that protects the remaining cells.
Oxygen tension and redox potential are other examples of non-
specific factors that can markedly effect the recovery of stressed
cells.
The results reported here support the view that the ability to
initiate growth under severe salt stress depends on the presence
of a resistant sub-fraction of the population, but that high cell
densities appear to assist the adaptation of those cells to the
unfavourable growth conditions by some unspecified medium
conditioning effect.
The Effect of Inoculum Size on the Lag Phase 105
RELEVANCE TO GROWTH IN FOOD
The results presented here predict that, under inhibitory
conditions, the mean lag time of cells that were sparsely distributed
in food would be appreciably longer than that indicated from
broth experiments with large inocula. Mackey and Kerridge (1988)
found that lag times of salmonellae growing in minced beef at
temperatures between 10 and 35 °C were much more variable than
the corresponding growth rates. Considering all data, there were
no statistically significant differences between the lag times of
large and small inocula (40 or 104 cells g”1, respectively) but at the
lowest most stressful temperature measured (10 °C), the lag time
was 50% longer (60 h compared with 40.6 h) for the lower inoculum
level.
The results presented here also suggest that, under inhibitory
conditions, the probability that a pathogen could initiate growth
in any single food item containing very low numbers of cells
would be less than that suggested by growth studies in broth
inoculated with thousands of cells. The exact probability value
per pack would depend on the distribution of resistance within
the population and the actual number of cells present per pack.
The overall probability that growth would occur at least in some
packs would be the same as that calculated from broth studies
with large inocula. Any reduction of lag time by cooperative or
conditioning effects would depend on cell concentration and
proximity; for example whether cells were present as microcolonies
or single cells. Predicting the behaviour of bacteria in food is often
based on experiments with initial inocula in excess of 103 cells
ml”1. From our work, it would appear that beneath this level of
inoculation, the lag phase becomes longer and the probability of
growth is less. This may have significance in estimating risk and
calculating safe storage times for foods.
106 Introductory Food Microbiology
6 investigations which described the growth of micro-organisms in
MODELLING THE GROWTH OF
LISTERIA MONOCYTOGENES IN
DYNAMIC CONDITIONS
A recurrent neural network for the prediction of Listeria
monocytogenes growth under pH and aw variable conditions was
developed. The use of this model offered the possibility to take into
account the consequences of the variations of the factors on L.
monocytogenes growth. The effects of solutions, such as NaCl, acetic
acid and NaOH, and their interactions on the response of L.
monocytogenes cells were studied. Furthermore, the results showed
the capacity of the recurrent neural network to predict growths
carried out in different experimental conditions without using
those used for its elaboration.
INTRODUCTION
Predictive microbiology combined the knowledge of bacterial
growth responses over a range of conditions with the power of
mathematical modelling to enable predictions of growth.
Mathematical modelling techniques can help to predict how food
preservation systems may affect growth kinetics. Most mathematical
models developed to simulate the growth of micro-organisms in
relation to environmental conditions, were elaborated from data
coming from growth carried out in constant conditions of
environmental factors, such as aw or pH. But, food-manufacturing
processes can decrease the pH of food, produce organic acids, e.g.,
pickling or fermentation, or reduce water activity, e.g., by addition
of an agent such as sodium. These processes are extensively used
Modelling the Growth of Listeria Monocytogenes... 107
as mechanisms to prevent microbial growth and to ensure food
safety. Therefore, it is important to understand and to be able to
predict the responses of micro-organisms, more particularly
pathogenic micro-organisms as Listeria monocytogenes, in the
presence of variable environmental factors. In recent years, several
the presence of temperature changes, were carried out. When
building these dynamic models, these authors made the hypothesis
that the transposition of results obtained from constant conditions
to variable conditions was possible. Cheroutre-Vialette et al. (1998)
demonstrated the importance of taking into account the variations
of environmental factors, which can occur during a food processing
and induce a stress situation for the micro-organisms. The objective
of this work was to produce a dynamic model that predicts the
growth of L. monocytogenes as a function of fluctuating conditions
of acid pH, alkaline pH and concentration of NaCl. To this end,
an alternative approach to conventional methods of microbial
growth predictions, that is, a recurrent multilayer neural network
approach, was used.
MATERIAL AND METHODS
Strain and Medium
L. monocytogenes 14 (serotype 4b), obtained from an industrial
environment, was used throughout the study. All growth
experiments were conducted in a tryptic meat broth (TMB). TMB
regulated at pH=7 and aw=1 was called standard medium.
Experimental Procedure
An automated turbidimeter was used to follow the growth of
the strain in the micro-titer plates. The working volume in each
well of the micro-titer plate was 400 l. Optical density (O.D.) was
read at a wavelength of 600 nm. According to the procedure
expoused by Cheroutre-Vialette et al. (1998), for all experiments
three conditions were studied: standard, limiting and shock
conditions. Under standard and limiting conditions, bacteria were
grown in TMB adjusted to the desired aw and pH values. The
osmotic variation was achieved by the addition of NaCl according
to Chirife and Resnik (1984). Acetic acid and NaOH (Prolabo)
108 Introductory Food Microbiology
were added to adjust low and high pH respectively. The shock
condition was defined as follows: the bacteria were grown in
standard medium until the beginning of the exponential phase
and were then shocked by the abrupt addition of shock solutions.
These shock solutions were prepared in order to obtain a final
value similar to those indicated for the limiting conditions. The
temperature was 20°C. For each combination, six growth repetitions
were carried out.
Experimental Design
The data of L. monocytogenes 14 growths at 20°C were taken
from an experimental study using a combination of two central
composite designs and a factorial design. The ranges of the different
environmental parameters were as follows: NaCl: 0–8%; acid pH:
5.6–7.0 or alkaline pH: 7.0–9.5. Each factor was studied at five
levels and for each combination, the shock and limiting conditions
were studied. At least 50 experiments were performed according
to 25 growths in shock condition and 25 growths in limiting
condition.
Additional Experiments
A two-litre fermentor SET2M was used. O.D. of the growth
was measured with a spectrophotometer at 600 nm. The protocol
was similar to that in Bioscreen C adjusting the different volumes.
Growths in shock conditions carried out in the fermentor allowed
the study of the osmotic shock effects by continuous addition
mode. Seven hundred and fifty milliliters of standard medium
were inoculated. At the beginning of exponential phase, 250 ml of
osmotic solution giving a final concentration of 8% NaCl were
added using a peristaltic pump. The addition mode of shock
solution in Bioscreen C was by steps of 2%.
Data Analysis
Averages of the O.D. were calculated for the six repetitions of
inoculated media and for the four repetitions of non-inoculated
media. The data were then analysed using the procedure described
by Bégot et al. (1996). Four quantities were calculated at time t:
• (O.D.i)t, the mean of the O.D. of the 6 repetitions of
inoculated media for each combination;
Modelling the Growth of Listeria Monocytogenes... 109
• (O.D.ni)t, the mean of the O.D. of the 4 repetitions of non-
inoculated media for each combination;
• (DO.D.)t=(O.D.i)t–(O.D.ni)t;
• Yt=log10[(DO.D.)t/(DO.D.min)] where DO.D.min was the
lowest DO.D. value above the detection threshold.
Recurrent Neural Network (RNN) Model
Neural network (NN) maps inputs to outputs. Supervised
neural networks are capable of learning from previous examples
through iteration without the requirement of a priori knowledge
of the relationships between the process variables. NNs are made
of a number of simple, highly connected processing elements (PE)
called neurones. The architecture of a NN describes how the NN
is constructed from layers of PEs. Each PE receives inputs from
other PEs or from the outside. The PEs in the input layer only
transfer scaled inputs to the appropriate PEs in the hidden layer
through weighted connections. Each PE of the hidden layer and
output layer calculates the weighted sum of its inputs and passes
the result through a transfer function. Most often a non-linear
sigmoid transfer function is employed. The NN is iteratively trained
by presenting to it representative exemplar input/output vectors.
The weights of the neural connections are adjusted in order to
minimise a cost function equal to the mean square of the output
error. The weights are usually randomly initialised. It has been
shown that one hidden layer of neurones is sufficient to
approximate any continuous non-linear function, although more
complex networks may be employed in special applications.
The distinction between feedforward and recurrent multilayer
NNs is the following: for the feedforward multilayer NNs an
element of a given layer can only receive information from previous
layers; on the contrary, for recurrent multilayer NNs, the values
calculated by the NN can be fed back to the NN input layer or any
previous layers. The difference in structure induces a difference in
training: recurrent multilayer NNs have to be trained on a sequence
of predictions, whereas training feedforward multilayer NNs
involves only instantaneous predictions. Training a recurrent
neural network (RNN) is more time-consuming but it is able to
provide better results than a feedforward NN when simulating the
110 Introductory Food Microbiology
evolution of phenomena with time. RNNs trained to learn dynamic
evolutions of a process are more constrained and are forced to find
a greater stability as well as a representation of the dynamic links
between control and process variables. Therefore, RNNs provide
a better prediction confidence than feedforward multilayer NNs
that only predict the outputs at a fixed horizon. Furthermore, this
technique is more interesting in an advanced control perspective.
In the present study, a recurrent multilayer structure which
contains one input layer, one hidden layer and one output layer,
was used. The architecture of the RNN is presented in Fig. 2. It
was designed to contain:
1. in the input layer, five input parameters: Yt–Dt, Yt–2.Dt,
Yt–3.Dt, pHt–Dt and NaClt–Dt (%)
2. in the output layer, one output parameter: Yt which
represented the predicted response.
A priori, there are no rules for the choice of the hidden
structure. It is determined empirically: the structure that gives the
best results is chosen. The optimum neurone number of the hidden
layer was iteratively determined by developing several RNNs that
vary with the size of the hidden layer (3 to 10 neurones were
tested) and simultaneously observing the change in the mean
square of the output error.
This was carried out with the training and testing data. Six
neurones in the hidden layer was determined as the best structure.
The sigmoid function f(x)=1/(1+exp(-x)) was chosen as an
activation function for each neurone. The RNN was trained by
iteration using a repeated presentation of representative exemplar
input/output vector pairs.
The weights of the neural connections, initially chosen
randomly, are adjusted by a non-linear optimisation technique:
the quasi-newtonian formula of Shanno (1970) in order to minimise
a cost function equal to the mean square of the output error. The
learning base was used to adjust the weights, the testing base to
provide over-learning during weights optimisation, and the
validation base for validation of results. At least, 60% of experiments
were included in the learning and testing bases, the last 40% were
in the validation base. In order to ensure the validity of the study,
Modelling the Growth of Listeria Monocytogenes... 111
the growth results carried out in fermentor or in Bioscreen C with
various modes of shock exposure were included into the validation
base.
RESULTS
Growth Predictions of the Validation Base
The analysis of the growth predictions in the limiting
conditions showed that the RNN represented satisfactorily the
experimental data whatever the conditions tested (alkaline–osmotic
or acid–osmotic). The different characteristics of the L. monocytogenes
response, i.e. induction of a lag time and growth recovery different
to those observed in the new environment, were taken into account
by the RNN whatever the combination, alkaline–osmotic or acid–
osmotic. Furthermore, RNN was able to predict the effect of the
type of shocks and their combinations. There was a good agreement
between the experimental growth and the prediction.
Growth Predictions of the Additional Experiments
The previous results of the validation base demonstrated the
ability of the RNN to predict growths under shock conditions
when the pH and aw transitions were carried out abruptly by one
step. The objective of additional experiments was to investigate
the capacity of the RNN to represent the response of L.
monocytogenes cells shocked in exponential phase with 8% NaCl
added by repeated steps of 2% NaCl or continuously during a
duration of one or four generation times. The effects of adding 8%
NaCl in 7.7 h (four generation times). The extrapolation to new
experimental conditions (mode of shock exposure) was made with
a good agreement by the RNN. It confirmed that the RNN has the
capacity to predict growths carried out in different experimental
conditions from those used for its elaboration.
DISCUSSION
These results obtained at variable conditions showed that
neural networks can effectively be used to study the complex
effects of fluctuating environmental conditions on micro-organism
behaviour. Such dynamic model could follow the microbial impact
of different steps associated with production, distribution and
112 Introductory Food Microbiology
retailing of a food and so could be an important support to HACCP
and food safety systems.
Growth of Listeria monocytogenes as a function of dynamic
environment at 10°C and accuracy of growth predictions with
available models.
A combination of a factorial design and two central composite
designs was used to assess quantita-tively the effects and
interactions of water activity (1-0.95) and pH (5.6-9.5) variations
on the growth of Listeria monocytogenes in a meat broth at 10°C. At
inoculation or at the beginning of the exponen-tial phase, the cells
were exposed to the addition of NaCI and acetic acid or NaCI and
NaOH.
The effects of abrupt fluctuating conditions on the generation
and lag times were analysed using turbidity mea-surements. The
data indicated that the cells exposed to osmotic and acid or alkaline
variable conditions from the time of inoculation were less affected
than cells exposed at the beginning of the exponential phase. In
this last case, a lag phase could be induced and the growth
recovery was different from those expected in the new environment.
Generation time values were estimated by three available predictive
models which describe the effects of temperature, salt concentration
and pH on L. monocytogenes growth to highlight the potential
problems of variable conditions.
The aim of predictive microbiology lies in predicting the growth
kinetics of micro-organisms at a given moment during food
processing and so predicting the evolution of food during storage,
manufacturing or preservation accidents. One of the great interests
of predictive micro- biology is to optimize the shelf life of a high
risk food product by the use of appropriate predictions.
Mathematical models have been developed to simulate the growth
of micro-organisms in relation to given environ-mental conditions
and can be classified by the microbiological event studied, the
modelling approach used or the variables considered.
Most of these models were elaborated from data acquired
un-der constant environmental conditions as poly-nomial models,
e.g. Food micromodel, Pathogen modeling program, which
expressed bacterial growth as a function of factors such as pH,
Modelling the Growth of Listeria Monocytogenes... 113
water activity (%NaCl), temperature. But envir-onmental factors,
such as temperature, Aw, or pH which are often the most important
factors governing microbial behaviour in food, may vary extensively
during food processing, throughout the complete production and
dis-tribution chain of a food product. Indeed, a food product is
exposed to environmental var-iations during certain stages in a
process, e.g. food cooling, natural product acidification or product
brining. In this way, it is important to be able to quantify
undesirable micro-organism growths, like Listeria monocytogenes,
in envir-onmentally variable conditions.
The purpose of this study was to determine the effects of the
combined alkaline-osmotic and acid-osmotic conditions, constant
or vari-able during time, on the growth of a L. monocy-togenes
strain at low temperature. Considering the temperatures likely to
be used for refriger-ated storage (0-10°C) or for working meat
pro-duct premises, the temperature of the study was established
at 10°C.
NaCl, acetic acid and NaOH were used to regulate water
activity, acid pH and alkaline pH respectively. As the capacity of
Bioscreen C for studying growth kinetics under variable conditions
was ever established, this material was used in this study.
Moreover, with the objective to illustrate the potential pro-blems
associated with the modelisation of growth under fluctuating pH/
Aw, conditions, we proposed to analyse the accuracy of the
gen-eration time predictions with available models and
characterize these predictions with our ex-perimental data.
Materials and Methods
Strain and Media
Listeria monocytogenes 14 (serotype 4b, ob-tained from industrial
environment) was used throughout the study. Stock cultures were
maintained in TSA agar slopes and stored at 4°C.
For preparation of inocula, cultures on TSA were subcultured
in a meat medium (MM) which contained meat peptone 10 gl-1,
yeast extract (Dif-co) 5 gl-1 and glucose 5 gl-1. The culture medium
for growth studies was a tryptic meat broth (TMB). It was buffered
with a K2HPO4-KH2PO4 (Merck) 0.1 mol l-1 solution in proportion
1:1 (v/v) to pH 7.0.
114 Introductory Food Microbiology
Monitoring of Bacteria/growth
An automated turbidimeter was used to follow the growth of
L. monocytogenes 14 in the micro-titer plates. Optical density (OD)
was read at a wavelength of 600 nm.
Experimental Procedure
The strain was incubated on TSA agar slopes for 7 h at 37°C
and then transfered to MM that was incubated in a rotary shaker
waterbath for 20 h at 20°C, by which time the strain had reached
the stationary phase. A proportion of the culture was inoculated
in various media to give a con-centration of about 3.107 cfu ml-1,
in order to be above the detection threshold of the Bio-screen C.
The viable number of bacteria was confirmed by TSA plate counts.
Figure. Experimental plan showing the combination of osmotic and acid
stresses using NaCI and acetic acid respectively ((D); and the combination
of osmotic and alkaline stresses using NaCI and NaOH respectively (Q).
The points of the factorial design are represented by n. The experiment
numbers are indicated in italics.
For all experiments, three conditions were studied: standard,
limiting and shock conditions. Under standard and limiting
conditions, bacteria were grown in TMB or in TMB ad-justed to
the desired AR, and pH values. For the inoculated TMB medium,
300 µl were dispensed in each of six wells and the same vo-lume
of non-inoculated medium was dispensed in each of four wells in
order to determine the OD of the growth medium and to detect
Modelling the Growth of Listeria Monocytogenes... 115
possi-ble contamination. The well numbers were con-sidered as
replicates. The shock exposures were arranged as follows: 300 µl
of inoculated and 300 µl of non-inoculated TMB were dispensed
in each of six and four wells, respectively. An aliquot (100 µl) of
concentrated ( x 4) shock so-lutions or standard TMB was added
when the OD was near 0.2, corresponding to the begin-ning of the
exponential phase. The osmotic shock was achieved by the
addition of NaCl (Prolabo) according to Chirife and Resnik (1984).
Acetic acid was added to adjust acid pH. The effect of high pH
stresses was studied with the presence of NaOH (Prolabo). The
time which elapsed before adding the shock solutions to 90 wells
was about approximately 10 min. These plates were placed in the
Bioscreen C previously adjusted to 10°C.
Experimental Design
The growth of L. monocytogenes 14 at 10°C in the presence of
salt (0-8%), corresponding to Aw value (1-0.95) and in different
values of acid pH (5.6-7.0) or alkaline pH (7.0-9.5) was stu-died by
using an experimental design. This design was a combination of
two central com-posite designs and a factorial design. Each factor
was studied at five levels and for each combination, the shock and
limiting conditions were studied. At least 50 experi-ments were
performed.
Data Analysis
Averages of the OD were calculated for the six repetitions of
inoculated media and for the four repetitions of non-inoculated
media. The data were then analysed using the procedure
1. (ODi)t, the mean OD of the six repetitions of inoculated
media;
2. (ODni)t, the mean of the OD of the four repetitions of non-
inoculated media;
3. (DOD)t=(ODi)t-(ODni)t;
4. log10[(ÄOD)t/(ÄODmin)] where ÄODmin was the lowest
DOD value above the detection threshold.
As indicated by Cheroutre-Vialette et al. (1998), a modified
Gompertz equation (Zwietering et al. 1990) was used to fit the
growth curves log10[(DOD)t/(DODmin)]=f(t). Growth parameters
116 Introductory Food Microbiology
such as A (logarithmic increase of population), LT (lag time) and
µ (maximal growth rate) were determined by non-linear regression
with STAT-ITCF statistic software. GT (generation time) was derived
from µ using the relatio: GT=log10(2)/µ.
In standard and limiting conditions, the time of inoculation
was taken as zero time when considering the growth curve. In
shock conditions, the time of the addition of the shock solution
was taken as zero time in the calculation of growth parameters.
Model Predictions
The ratio of predicted and observed generation times was
calculated for the studied combinations (predicted generation time/
observed generation time). One complementary measure, proposed
by Ross (1996) as simple index of the performance of models in
predictive microbiology, was also evaluated. This value, termed
the ‘bias factor, may be interpreted as the average ratio of the
predicted and observed values and is defined as follows:
where y predicted is the predicted generation time, y observed
is the observed value, and n is the number of observations used
in the calcu-lation. Perfect agreement between predictions and
observations will lead to a bias factor of 1.
Results
Growth kinetics of L. Monocytogenes
The abrupt addition of 8% NaCl has a marked effect on the
growth recovery of the micro-organism compared to the effect
produced by the presence of NaOH to alkaline pH 9.5. The growth
curve asso-ciated to the combined alkaline-osmotic shock solutions
differed from those of uncombined shocks suggesting an interactive
effect of NaOH and NaCl at 10°C. The effects of acid shock (pH
6.3), osmotic shock (8% NaCl) and combined acid (pH 6.3) - osmotic
(8%) shocks at 10°C on the exponential phase of L. monocy-togenes
14 growth. The acid-osmotic variable conditions have particu-larly
affected the growth recovery of the strain. Indeed, the organic acid
and the solute pre-sented an interactive effect on the growth of
micro-organism.
In shock condition, L. monocytogenes 14 responded
instantaneously to small changes of pH and Ate,. Small acid and
Modelling the Growth of Listeria Monocytogenes... 117
acid-osmotic variations involved an unusual growth curve as
shown by Fig. 3. Indeed, consecutively to these shocks, the growth
of micro-organism contin-ued and a rupture of the sigmoid curve
was seen before the growth recovery. The Gompertz equation
fitting left this phenomenon out of account and considered the
growth recovery cor-responding to the generation time values. In
exchange, large environmental conditions dur-ing exponential
phase (i.e. shock condition) of the micro-organism induced a lag
phase before the growth recovery. This lag phase was more
pronounced especially as the concentrations of shock solutions
were higher in case of alkaline-osmotic shocks. This induced lag
period was really observed and cal-culated when a high
concentration of NaCl, e.g. 6.8 and 8%, was added with the organic
acid in the culture.
Some growths were particularly affected by the exposure to
the combined shock solutions in exponential phase. Indeed, no
increase of op-tical density during the experimental period, i.e. 21
days, underlying no increase of population, was noted for the
combination pH 5.6 and 4 or 8% NaCl.
An adaptation period to the new environ-ment (temperature,
pH and water activity) was observed in limiting condition, i.e.
when the shock solutions were present at the begin-ning of the
culture. This period reached 12 days if the cells were placed in
presence of higher concentrations of NaCl and alkaline pH, 8%
and 9.5, respectively (data not shown). An experimental condition
(pH 5.6, 8% NaCl) caused no increase of optical density during the
experimental period. The generation times calculated with the
Gompertz equation revealed that growth re-covery obtained in
limiting conditions was dif-ferent to those observed in shock
conditions. Indeed, the shock condition pre-sented a generation
time value higher than calculated in limiting condition whatever
the environmental variation.
In conclusion, two principal pheno-mena were observed when
the bacteria sub-mitted to abrupt change of pH and Aw (i) large
environmental variations induced a lag phase following the
exposure to shock solutions, and (ii) the growth continued with
a generation time value different from that observed before the
change.
118 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 119

Polynomial Models Predictions The predictions were more especially conser-vative as the
The aim of this part was not to com-pare polynomial models, culture conditions were severe, i.e. low pH and high salt
but to show the position of their predictions with regard to variable concentration. Few plots were above the identity line indicating
environmental conditions. The range of predicted values was wide. that the predicted parameters were higher than the observed values
Considering the limiting condition, the ratio calculated was in in limiting conditions. This was observed for growth predictions
general less than 1.0, indicating that the pre-dicted value was associated to combined alkaline pH-osmotic conditions or
inferior to those observed. uncombined osmotic conditions. In cases of growth predictions
under low pH (pH < 6.3), the predictions values were in general
Table : Generation times, expressed in h, observed in shock or limiting three or four times be-low the observed values and might be 14
conditions. In brackets: asymp-
times inferior in presence of low pH and higher salt concentration,
Totic 95%
confidence i.e. pH 5.8 and 6.8% or pH 6.3 and 8%. Considering the shock
interval condition, the trend of models to have ‘fail safe’ predictions were
Experiment pH % NaCI Generation times confirmed and increased. All the points were below the identity
number line, representing predictions which were shorter than the observed
Limiting Shock generation time. These results were confirmed by the bias factors
conditions condition
which maybe interpreted as the geometric mean ratio of predicted
2 5. 6 0 26.6 (±0.4) 64 (±2)
2
and observed generation times. The bias factors calculated in
2 6. 3 0 9.6 (±0.1) 14.0 (±0.2) shock condition showed, on average, 1.5-fold greater bias than
1 5. 8 1. 2 19.9 (±0.3) 68 (±6) those observed in limiting condition.
5
1 6. 8 1. 2 6.8 (±0.1) 9.2 (±0.3) Discussion
4
5 5. 6 4 120 (±1) NI For several years, it was largely demonstrated that refrigeration
8 6. 3 4 19.9 (±0.3) 41(±1) in conjunction with other factors, e.g. salt or acid, may severely
9 6. 3 4 20.6 (±0.3) 39 (±1) retard or prevent the growth of micro-organisms. As L.
1 5. 8 6. 8 190 (±2) 215 (±3) monocytogenes is common in the food proces-sing environment and
9
1 6. 8 6. 8 10.2 (±0.1) 18.0 (±0.3) as a food product is often exposed to environmental variations, it
8 is important to evaluate the adapta-tion of this bacteria to variable
2 5. 6 8 NI NI conditions of environmental factors as pH and Aw at low
5
tem-perature. A previous study, Cheroutre-Vialette et al. (1998)
1 6. 3 8 56.8 (±0.6) 129 (±2)
1 has reported that the generation times calculated for the growths
20 (standard 7. 0 0 5.1(±0.1) of L. monocy-togenes in shock and limiting conditions under variable
condition) pH or Aw conditions at 20°C were sig-nificantly different.
3 7. 0 4 6.3 (±0.1) 9.5 (±0.1) Moreover, an additional lag time in shock condition could be
2 7. 0 8 9.3 (±0.1) 17.9 (±0.2)
4 observed. The results obtained in the present study car-ried out at
a: Growth parameter was calculated according to zero time considered as the low temperature and under com-bined pH-Aw conditions were in
time of inoculation. agreement with the conclusions established in this pre-vious study.
b: Growth parameter was calculated according to zero time considered as the

moment of shock.
The aim of this paper was to introduce more advances into the
NI: No increase in optical density during 21 days. field of predictive microbiology, by specifying the microbial
120 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 121

behavior under dynamic environment of pH and water activity investigations of bacterial growth pre-dictions with changes in
and collecting experimental data. In this way, the use of Bioscreen temperature by Zwie-tering et al. (1994) andVan Impe et al. (1995),
C allowed to rapidly ex-plore the microbial impact of varying pH or with changes in pH by Rosso (1995) admitted the possible
and Aw conditions. In recent years, the interest in developing transposition of results obtained from constant conditions to
dynamic mathematical models that describe the growth of variable condi-tions. Considering our results, such postulate is
microorganisms in the presence of environmental factor variations not suitable to take into account the physio-logical response of
has increased. With the objective to take into account the effects micro-organisms.
of temperature variations on microbial growth, Baranyi et al. (1993) The results of this study highlighted the pro-blems associated
observed that the abrupt transitions could lead to adjustment with the variable conditions and the available models established
periods, i.e. microbial cultures, when shifted abruptly from one in con-stant conditions. The results obtained with the predictions
temperature to another, may exhibit a transient growth rate before estimated with the models (FMM, PMP, Lm14) are not surprising
assuming the growth rate expected at the new temperature. taking into con-sideration that models were developed with strains
Table : Comparison of model predictions of the data of different origins, in media of various compositions and in
PMP FMM Lm14
different conditions of growth. For example, the pH was adjusted
Limiting Shock Limiting Shock Limiting Shock
with HCl in PMP or Lm14, lactic acid in FMM. Acetic acid was
na 15 14 15 14 9 8
Ratiob average 0.53 0.33 0.55 0.34 0.62 0.43
used for pH regulation in our growth experiments and it was well
Ratio minima 0.07 0.06 0.05 0.05 0.08 0.12 estab-lished that this organic acid presents a higher inhibitory
Ratio maxima 1.15 0.60 1.19 0.63 1.25 0.82
activity on L. monocytogenes. As can be pointed out by this study,
Bias factor 0.40 0.25 0.39 0.25 0.50 0.33
a: n ˆ number of observations used in the calculation.
the predictions of these models based on data generated under
constant conditions can reliably predict growth under fluctuating
b: ratio ˆ predicted generation time/observed generation time.
conditions of pH and Aw. But, a systematic over-prediction was
revealed under dynamic environment. Under a dynamic
Our data under higher pH and/or Aw, variations confirmed environment, the bias factor showed that the recovered growth
the pre-sence of adjustment period which was repre-sented by an rates caused much larger generation times than those under
induced lag phase. When these environmental changes were constant conditions. In consequence, the structure of these models
smaller, an unusual growth curve was observed. This seemed to be inappropriate to take into account the microbial
char-acteristic raised some questions about its origin. This could response to variable en-vironmental conditions.
be assigned to the material used, the Bioscreen C. The lateral
agitation of the plates in Bioscreen C could have been insufficient Moreover, the induced lag phase observed in dynamic
for homogenous distribution of the compound in the medium. At environment of pH and Aw could not be integrated by these models.
the moment of shock (acid or acid-osmotic), a time is needed for The necessity to determine a new way to model changing pH
the repartition of the shock solutions in the well. A study of values, varying Aw or other food paramenters was underlined.
Jorgensen et al. (1995) has also reported that the cells of L. Neuronal networks could represent a new approach to take into
monocytogenes incubated in media containing NaCl became rapidly account the varia-bility of cell response in variable conditions and
longer than cells grown without salt. Such morphological changes produce a dynamic model. A previous study provided a good
could also lead to variations in the turbidity of the medium. prediction of L. monocytogenes growth under variable conditions.
Such dynamic models could follow the microbial impact of the
The experiments of the present study showed also that the different steps associated with the production, distribu-tion and
recovered growth rate after the change of pH and/or Aw was retailing of a food.
usually less than those associated to the new environment. Other
122 Introductory Food Microbiology
EFFECTS OF PH OR AW STRESS ON GROWTH OF LISTERIA
MONOCYTOGENES
The growth of three strains of Listeria monocytogenes at 20°C in
a meat broth of different pH or water activity was investigated. At
inoculation or at the beginning of the exponential phase, cells
were exposed to stress by the addition of NaOH or NH4+, acetic
acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or
an aw of 0.950 or 0.965, respectively.
The effects of the exposure to stress on the generation and lag
times of each strain were analysed by turbidity measurements for
cultures in micro-titer plates. Results were confirmed by conducting
the same experiments in a fermentor, except for the maximal
population reached. The three strains showed similar behaviour.
Cells were able to overcome the alkaline stress rapidly whereas
acid and osmotic shocks induced important changes of the growth
parameters. Cells exposed to acid or osmotic conditions from the
time of inoculation were less affected than cells exposed at the
beginning of the mid-exponential phase.
Listeria monocytogenes, a psychrotrophic bacteria, is considered
as an important food-borne pathogen. It can persist and grow at
low pH, high pH, low water activity and at refrigeration
temperatures.
Work on predictive microbiology has been carried out in order
to improve the shelf life and safety of foods. Predictive models of
microbial growths were set out with respect to the main controlling
factors of the environment such as temperature, pH, and water
activity.
These factors were often considered constant during growth.
But, environmental factors, particularly the temperature, may vary
extensively throughout food processing. To take the environmental
variations during time into account, different authors have
improved predictive models and proposed dynamic models which
describe growths at varying temperature profiles.
In this study, we report the effects of water activity and pH
shifts on the growth of three strains of L. monocytogenes using
different osmotic solutes (NaCl, KCl), organic acid (acetic acid)
and bases (NaOH, NH4+).
Modelling the Growth of Listeria Monocytogenes... 123
Monitoring of Bacterial Growth
An automated turbidimeter (Bioscreen C, Labsystem, Labsystem
France SA, Les Ulis, France) was used to follow the growth of
Listeria strains in the micro-titer plates. Optical density (O.D.) was
read at a wavelength of 600 nm.
A two-litre fermentor SET2M was used. The O.D. of the growth
was measured with a spectrophotometer (UV-160A, Shimadzu,
Japan) at 600 nm.
Microorganisms
The strains used by Bégot et al. (1997) in the studies with
Bioscreen C were chosen for this work: L. monocytogenes CLIP
19804 isolated from meat products, L. monocytogenes 14 obtained
from the food processing environment and L. monocytogenes CA
Wisconsin associated with a cheese-borne listeriosis outbreak. All
were serotype 4b which was involved in the recent epidemics in
France. L. monocytogenes 14 strain was also used in the fermentor
studies. Stock cultures were maintained on TSA agar slopes and
stored at 4°C.
Media
For preparation of inocula, cultures on TSA were subcultured
in meat medium (MM) which contained meat peptone 10 g/l,
yeast extract (Difco) 5 g/l and glucose 5 g/l.
The culture medium for growth studies was a tryptic meat
broth (TMB) (Fournaud et al., 1973). It was buffered with a
K2HPO4–KH2PO4 (Merck) 0.1 mol/l solution in proportion 1:1 (v/
v) and adjusted to pH 7.0 with NaOH (40 g/l).
Types of Experiment
For all experiments, three conditions were studied: standard,
limiting and shock conditions. Under standard and limiting
conditions, bacteria were grown in TMB or in TMB adjusted to the
desired aw or pH values. The following additions were made to
TMB: NaOH (solution of 200 g/l; Prolabo) 12.2 g/l or NH4+ (Merck)
7 g/l to obtain pH of 9.0, acetic acid (Carlo Erba, Nanterre, France)
4.1 g/l to obtain a pH of 5.6, 80 g/l of NaCl (Prolabo) or 80 g/
l of KCl (Prolabo) (Jakobsen et al., 1972) to obtain aw=0.95 and a
124 Introductory Food Microbiology
pH of 7.0 or aw=0.965 and pH of 7.0, respectively. The shock
conditions were defined as follows: the bacteria were grown in
standard TMB until the beginning of the exponential phase and
were then exposed to the pH and aw values described above.
Growth in Microplates
Each strain was incubated on TSA slopes for 7 h at 37°C and
then transferred to MM that was incubated in a rotary shaker
waterbath (Aquatron, Infors, Switzerland) for 18 h at 20°C, by
which time growth of all strains had reached the stationary phase.
A proportion of the culture was inoculated in the various
media to give a concentration of about 5.107 CFU/ml in order to
be above the detection threshold of the Bioscreen C. For the
inoculated TMB medium, 300 l were dispensed in 6 wells and the
same volume of non-inoculated medium was dispensed in 4 wells
in order to determine the O.D. of the growth medium and to detect
possible contamination. The well numbers were considered as
replicates. The same procedure was used for the pH and aw
adjusted TMB. The shock exposures were arranged as follows: 300
l of inoculated and 300 l of non-inoculated TMB were dispensed
in 6 and 4 wells, respectively. An aliquot (100 l) of concentrated
(×4) stock solution or standard TMB was added when the O.D.
was near 0.2. The delay in adding the solutions to 70 wells was
approximately 7 min. These plates were incubated in the Bioscreen
C previously adjusted to 20°C.
Growth in Fermentor
The protocol was similar to that in Bioscreen C except for the
fermentor volume with additions adjusted accordingly. The
additions lasted about 10 min. The experimental conditions were
20°C with a shaking speed of 100 rpm and an aeration regulated
at 0.2 litre of sterile air per litre of medium per min.
Data Analysis
Averages of the O.D. were calculated for the six repetitions of
inoculated media and for the four repetitions of non-inoculated
media. The data were then analysed using the procedure described
by Bégot et al. (1996). Four quantities were calculated at time t:
Modelling the Growth of Listeria Monocytogenes... 125
1. (O.D.i)t, the mean of the O.D. for the 6 repetitions of
inoculated media;
2. (O.D.ni)t, the mean of the O.D. for the 4 repetitions of non-
inoculated media;
3. (DO.D.)t=(O.D.i)t–(O.D.ni)t;
4. log10 [(DO.D.)t/(DO.D.min)] where DO.D.min was the lowest
DO.D. value above the detection threshold.
A modified Gompertz equation (Zwietering et al., 1990) was
used to fit the growth curves log10 [(DO.D.)t/(DO.D.min)]=f(t).
Growth parameters such as A (logarithmic increase of population),
L (lag time) and (maximal growth rate) were determined by non-
linear regression with STAT-ITCF software. Tg (generation time)
was derived from µusing the relation:
log10 (2)
Tg =
µ
In standard and limiting conditions, the time of inoculation
was taken as zero time when considering the growth curve. In
shock conditions, the time of the addition of the shock solution
was taken as zero time in the calculation of growth parameters.
RESULTS
The growth curves of L. monocytogenes 14 in the presence of
acetic acid and NaCl can be seen in Fig. 1a and b, respectively.
When the shock treatments (acid and osmotic) were applied after
the beginning of growth of L. monocytogenes in standard media,
lags were shorter, but generation times increased, as compared
with cells grown in limiting media. This phenomenon was
particularly pronounced for the acetic acid example.
In comparison with growth in standard media, growth of the
strain was not really affected by the presence of NaOH and NH4+
whatever the treatment, limiting or shock conditions. The growth
parameter values, i.e. generation and lag times, confirmed that
cells adapted rapidly to the alkaline upshift to pH 9.0 with NaOH
or NH4+. Indeed, these values calculated in alkaline media were
consistent with those found in standard media. Under acid and
osmotic conditions, cell growth was more affected. Media
126 Introductory Food Microbiology
containing acetic acid was the most severe culture condition for
the growth of the three strains. Under limiting conditions, although
the presence of acetic acid did not cause increasing lag times,
generation times were at least 3.5 times greater compared to the
values in standard media. The solute used to control aw can
influence the growth pattern of the microorganism. At the tested
concentration (80 g/l), L. monocytogenes which is also considered
as a salt-tolerant microorganism, tolerated KCl better than NaCl.
Indeed, the generation and lag times in limiting and shock
conditions, were higher in the presence of NaCl 80 g/l. This
influence of NaCl can be due to a larger aw effect.
Generally L. monocytogenes 14 and 19804 had similar
generation times whereas L. monocytogenes CA had longer
generation times under limiting conditions. Variations in lag time
were noted among the strains in all tested conditions.
Generation and lag times of cultures in the fermentor are
presented in Table 3. The values of generation times were similar
to those obtained in Bioscreen C, but large variations in lag phases
were noted. The results obtained from the fermentor culture
experiments confirmed the observations made with the Bioscreen C.
DISCUSSION
In previous studies about growths in non-variable conditions,
the interest in using the Bioscreen C for analysis of microbial
growth parameters and its good reproductivity was established.
Our study confirmed these observations. The results obtained from
the experiments carried out in the automated turbidimetric system
were in agreement with those obtained in the fermentor. The increase
in the number of L. monocytogenes was underestimated in the
Bioscreen C as compared to the fermentor. The homogeneous
aeration and agitation which were controlled in the fermentor
might be a possible explanation for this observation. Nevertheless,
the differences found in the generation times between the two
methods were not significant. This suggests, therefore, that the
Bioscreen C is suitable for studying the growth of bacteria under
variable conditions. A high concentration of cells is required in
the Bioscreen C for a detectable change in absorbance, but
Buchanan and Phillips (1990) and Dalgaard et al. (1994)
Modelling the Growth of Listeria Monocytogenes... 127
demonstrated that the growth kinetics of L. monocytogenes were
unaffected by the size of the initial inoculum i.e., the generation
and lag times derived from the Gompertz equation were not affected.
The L. monocytogenes strains showed similar responses
following aw or pH stresses. Variations were generally larger in
lag times than in generation times. There was very little difference
in generation times between L. monocytogenes strains 14 and 19804
for most of the experiments. Any major variations were observed
with the most severe culture conditions, i.e., in the presence of
acetic acid. L. monocytogenes strain CA was more affected by the
different stress conditions and in particularly under limiting
conditions. These findings are consistent with those of
Papageorgiou and Marth (1989) who also reported that L.
monocytogenes CA was less salt tolerant than strains in whey and
skimmed milk containing 12% NaCl.
In recent years, the interest in developing dynamic
mathematical models that describe the growth of microorganisms
in the presence of temperature variations, has increased. When
building dynamic models to predict bacterial growth with change
in temperature, two hypotheses have been formulated: (i) the
bacteria show no additional lag phase due to a change in
temperature during the exponential phase, and (ii) growth
continues immediately at the specific growth rate associated with
the temperature post-shift. More recently, for the elaboration of
dynamic models, Van Impe et al. (1995) and Rosso (1995) accepted
as a postulate, that variable environmental conditions do not
induce stress conditions on a microbial population, i.e., they
considered that the microflora responds instantaneously to pH
(Rosso, 1995) or temperature changes (Van Impe et al., 1995 and
Rosso, 1995). Our tests carried out with the three L. monocytogenes
strains showed that growth under modified osmotic or acid
environmental conditions in exponential phase did not agree with
these hypotheses. Since 1992, Van Impe et al. (1992) have
recognized the need to take into account the previous history of
a product, and therefore the previous history of a strain which
could have an important impact on the lag time prediction of a
microorganism. It is also necessary to quantify the ability of a
microorganism to grow under variable conditions. This could
128 Introductory Food Microbiology
allow the optimal combination of temperature, pH, aw and other
factors with the purpose of increasing the shelf life of a given
product.
VALIDATING THE USE OF GREEN FLUORESCENT-
MARKED ESCHERICHIA COLI O157
Monitoring bacterial kinetics in food is of great importance in
food safety. The targeted micro-organism has to be identified
accurately among competitive flora. Using green fluorescent protein
(GFP)-transformed strains is a possible answer to such issues.
However, quantitative studies require that this transformation does
not alter the micro-organism behaviour: parent and transformed
organisms were thus compared.
Three Escherichia coli O157:H7 strains were transformed using
a GFP-plasmid expressing. Parent and transformed strains were
compared according to their genetic characteristics and serotypes.
Growth ability was also assessed in constant and fluctuating
temperature profiles. Cardinal values of pH, water activity and
temperature were computed. No differences were observed between
parent and transformed strains for all these experiments. The
plasmid was satisfactorily maintained within transformed strains
throughout the studies. Growth was eventually monitored in beef
meat.
Although Escherichia coli is a common inhabitant of the human
and animal gastrointestinal tract, several pathogenic types of the
species are able to cause a variety of human diseases. First
recognized as a human pathogen in 1982, E. coli O157:H7 [Shiga
toxin-producing E. coli (STEC)] has since been associated with
outbreaks of food-borne illness in countries across the world, with
many foods identified as vehicles of infection. The main virulence
factor of STEC is the ability to form cytotoxic exotoxins. Two major
categories of Shiga toxins have been distinguished, Shiga toxin 1
(stx1) and Shiga toxin 2 (stx2). The other virulence factors are the
property of producing attachment-effacement lesions and the
presence of an entero-haemolysin gene.
Given the severity of the illness associated with E. coli O157:H7,
it is necessary to provide the food industry with procedures for
preventing its presence and controlling its growth. The aim of
Modelling the Growth of Listeria Monocytogenes... 129
predictive microbiology lies in predicting growth kinetics of micro-
organisms at a given moment during food processing and thus
predicting the evolution of food during storage, manufacturing or
preservation accidents. Rosso et al. (1993, 1995) proposed, in their
cardinal approach, a model in which the maximum microbial
specific growth rate ( max) is a function of temperature or pH. The
cardinal values of the considered strain are then determined. As
an example, for temperature values, the following parameters are
considered: T min (the temperature below which growth is no
longer observed), T max (the temperature above which no growth
occurs) and T opt (the temperature at which the maximum specific
growth rate max equals its optimal value opt). The same procedure
can be applied to pH and water activity a w. The minimal and
maximal values are important factors to food preservation, and
the optimal value is a crucial parameter in predictive microbiology.
In this approach, for a given strain, opt is a food-dependent
parameter.
The development of sensitive methods for monitoring bacteria
in foods, especially in presence of natural contaminating flora, is
of marked importance. Indeed, models have to be validated and it
is usually more appropriate to collect new data in food to have a
good validation. In this way, the objective of this work was firstly
the construction of E. coli O157:H7 strains marked with the green
fluorescent protein (GFP) gene carried by a plasmid. The GFP from
the jellyfish Aequorea victoria, a versatile 27-kDa protein, has proved
to be valuable as a tool for studying a variety of biological questions,
e.g. to study gene expression, to determine protein distribution or
to tag a cell lineage in vivo, in situ and in real time. The GFP emits
green light when excited with ultraviolet (u.v.) radiation and no
substrate or cofactor is required for fluorescence.
Ajjarapu and Shelef (1999) used GFP-bearing E. coli O157:H7
in ground beef and were able to monitor its evolution despite the
presence of a background microflora. It should yet be verified that
using GFP-transformed strains instead of nontransformed strains
is a right way, i.e. transformed strains and parent strains should
behave in a similar manner. Fratamico et al. (1997) found no
differences according to morphological or biochemical criteria or
using PCR. In a second part of the present work, the influence of
130 Introductory Food Microbiology
temperature on the growth kinetics of both GFP-E. coli strains and
their parental strains was assessed in liquid medium. GFP
expression was evaluated during growth. Cardinal values of pH,
a w and temperature of GFP or parental strains were determined
comparatively. Furthermore, experiments at varying temperature
profiles were carried out in order to simulate temperature-changing
conditions (food processing, food storage, etc.). The results obtained
in liquid medium were complemented by experiments in food.
Strains and Media
Three E. coli O157:H7 strains were used in this study. One of
the strains was isolated from bovine faeces and will be referred to
as strain 1, the other two were of clinical origin and will be
referred to as strains 2 and 3. These strains were transformed with
a plasmid vector pBAD-GFPuv (BD Biosciences - Clontech Inc.,
Palo Alto, CA, USA), adapted for visualization under u.v. light.
Stock cultures were maintained at 80°C in cryobank.
All strains were resuscitated before use by inoculation into
10 ml of brain-heart infusion (BHI) broth (Oxoid Ltd, Basingstoke,
Hampshire, UK), followed by incubation at 37°C for 8 h. The
subculture and culture medium was BHI (Oxoid) supplemented
with yeast extract (Oxoid) 3 g l 1 and glucose 2 g l 1. The BHI
supplemented medium was sterilized by filtration (0·20 m pore
size). This will be referred to as modified BHI.
The count medium was plate count agar for the E. coli strains.
Plates were incubated at 37°C for 24 h.
Construction and Identification of GFP-expressing E. coli
Escherichia coli strains were transformed with pBAD-GFPuv
by the calcium chloride method. The pBAD-GFPuv plasmid vector
contains an arabinose-induced promoter and an ampicillin
resistance gene. Transformants were selected by plating on PCA
supplemented with arabinose 2 mg ml–1 and ampicillin 50 µg ml–
1
(PCA + Ara + Amp). Colonies on PCA + Ara + Amp, that were
induced by arabinose and ampicillin resistant, were fluorescent
under u.v. light. Stability of fluorescence was monitored. Indeed,
the percentage of fluorescent colonies before and after
experimentations was also examined throughout the present study.
Modelling the Growth of Listeria Monocytogenes... 131
Parent and transformed strains were compared according to
their genetic characteristics (stx genes), serotypes and biochemical
characteristics. Detection of stx genes was performed with
degenerate primers ES149 and ES151 as described by Read et al.
(1992). These primers amplified a conserved sequence of stx 1 and
stx 2 genes. For DNA preparation, 1 ml of an overnight sample
broth (BHI) was centrifuged at 12 000 g for 3 min. The pellet was
washed in PBS buffer (pH 7·4; Sigma). The DNA from pelleted
cells was released by boiling and purified using Instagen DNA
purification matrix. Ten microlitres of the DNA were used as
template for PCR detection of stx genes. The conditions for the
PCR amplification were the same as those described by Uyttendaele
et al. (1998).
Serotyping of the different strains was performed for O157
and H7 determination with Difco antisera.
Parent or transformed strains were biochemically confirmed
by using the API 20E test strips.
Growth Kinetics as Function of Constant or Dynamic Temperature
in CARY 100
Behaviour of parent and GFP-expressing strains in function of
temperature was compared in modified BHI. Parent and
transformed strains were grown simultaneously in a
spectrophotometer CARY 100. Three replicates were carried out
for each condition.
After resuscitation (in modified BHI, 8 h at 37°C), the parent
or GFP-expressing strains were subcultured in modified BHI and
in modified BHI + ampicillin (50 g ml–1), respectively at the defined
temperature until the beginning of exponential phase. A 0·5% of
this inoculum, corresponding to inoculum size of 106 CFU ml–1,
was then transferred to the culture medium (modified BHI) for
growth assessment. Growth was monitored in constant
temperature conditions at 37, 20 and 10°C. Moreover, growth
evaluation was carried out in dynamic temperature conditions as
follows: the strains were first placed at a start temperature until
beginning of exponential phase and then, a new temperature was
applied. Studied conditions were: 37°C followed by 20°C, 37°C
followed by 10°C, 20°C followed by 10°C and 10°C followed by
132 Introductory Food Microbiology
20°C. The temperature gradient was usually achieved in <15 min.
In order to check fluorescence, plate counts (PCA + Ara) were
performed at the beginning and at the end (cells in stationary
phase) of every experiment.
Determination of Cardinal Values of pH, a w, Temperature in
Bioscreen C
The methodology was defined in the framework of the French
Predictive Microbiology Project, Sym’Previus as described by
Membre et al. (2002). The detection time method was used to
calculate max for different conditions of temperature (tested range:
8-45°C), pH (adjusted by HCl: pH 4-7) or a w (regulated by NaCl:
0·5-8%) in a Bioscreen C. Briefly, this method is based on the fact
that successive binary dilutions produce growth curves that are
shifted from each other by one-generation time value.
The obtained values of µmax allowed determination of cardinal
values (T min, T opt, T max, pHmin, pHopt, pHmax, a w min, a w opt, a w
) and µopt with Rosso secondary model. Cardinal values a w max
max
were set to 1. Cardinal values pHmax were chosen so that pH
model would be symmetric, i.e.:
pHopt = (pHmin + pHmax)/2
pHmax = 2pHopt – pHmin.
The cardinal values of pH, a w and temperature were obtained
in modified BHI for the parent strain 2. For the transformed strain
2, these cardinal values were determined in modified BHI + Ara.
The determination of fluorescence percentage was studied as
described before.
Experiments in Food
In order to evaluate the potential utilization of GFP strain in
food product, growth of GFP-transformed strain 2 was monitored
in raw ground beef at 10°C. The strain was first subcultured in
modified BHI + Amp at 37°C for 8 h; a second subculture was
conducted in the same medium at 10°C for 4 days.
The suspension was diluted to produce an inoculation level
in beef of ca 2·5 103 CFU g 1. Inoculated meat was divided into
10 g aliquots and placed in sterile bags.
Modelling the Growth of Listeria Monocytogenes... 133
A growth curve was produced with 10-15 points and repeated
once. On each measurement time, one bag was opened for
enumeration. Meat was placed in 90 ml of tryptone salt broth, and
then homogenized for 1 min. Dilutions were made in tryptone salt
broth to obtain appropriate levels. Plating was made on PCA for
total bacterial count, on PCA + arabinose (0·2%) (PCA + Ara) for
visualization of the GFP strain under u.v. light, and on
PCA + arabinose (0·2%) + ampicillin (50 g ml –1 )
(PCA + Ara + Amp) as a selective medium allowing expression of
fluorescence.
Statistical Analysis of Data
Analysis of variance, regressions or confidence intervals
calculations were performed using software S-Plus 2000. Graphical
outputs were also produced with Microsoft Excel 2000.
The primary model chosen in this study was the model of
Rosso (1995). It was used for constant and dynamic temperature
studies and for food experiments, in order to calculate primary
model parameters (mainly lag time and growth rate max). A cardinal
values model was chosen as secondary model.
RESULTS
Construction and Identification of GFP-expressing E. coli
pBAD-GFPuv was successfully introduced into the three E.
coli O157:H7 strains. Colonies of the transformed and parent strains
had identical morphological characteristics on PCA + Ara + Amp
(for GFP strains) and PCA (for parent strains), except that colonies
of transformants appeared green under u.v. visualization, whereas
those of the parent strains remained nonfluorescent.
The comparison of the parent and transformed strain genetic
characteristics, i.e. detection of stx genes, showed that virulence
genes were present after the plasmidic transformation. Furthermore,
no serotypic (i.e. O157:H7) or biochemical difference was recorded
between parent and transformed strains.
Stability of fluorescence was also monitored. The
transformants were cultured at 37°C in the nonselective liquid
medium, i.e. without ampicillin. The plasmid was maintained in
more than 60% of colonies after 13 days of overnight cultures.
134 Introductory Food Microbiology
Growth Kinetics as Function of Constant or Dynamic Temperature
GFP-expressing strains were compared with their respective
parent strains according to growth rate and lag time values
(calculated from Rosso primary model) under various temperature
conditions. Correct agreement between the strain results was
observed.
Loss of GFP expression was also examined for transformed
strains. Colonies plated on PCA + Ara were counted, and another
count was made under u.v. light in order to evaluate the proportion
of fluorescent colonies. The percentage of nonfluorescent colonies
corresponded to plasmid loss within the bacterial population.
Mean plasmid loss percentage was under 10% in all the cases.
The maximal range of observed plasmid loss was 0-21%. These
observations indicated of a correct GFP expression, and therefore
a conservation of the plasmid at whatever the environmental
temperature conditions. A constant temperature of 10°C gave less
favourable results, but experiments were longer (5 days). Strains
seemed to keep their fluorescence better in dynamic conditions
(rapid temperature changes) of growth.
Cardinal values of a w, pH, Temperature
Cardinal values of a w, pH and temperature for strain 2 are
indicated in Table 2 with their confidence intervals. As shown by
these results, the confidence intervals between the parent and the
transformed strains are overlapping. This trend confirmed the
previous observations, showing no differences between the parent
strain and the GFP-expressing strain behaviour. It must be noted
that the pHmax was estimated by a symmetric model and no
experiment was conducted to alkaline value. This could explain
the fluctuation of calculated pHmax values.
The loss of GFP-expression for transformed strain 2 was
evaluated for all tested conditions of a w, pH and temperature. The
means of the plasmid loss percentage, as well as the minimal and
maximal percentages, were calculated and indicated in Table 3.
Whatever the environmental conditions, strain 2 exhibited a good
conservation of the plasmid. In general, the plasmid loss was
below 10%. This value could be more pronounced for the most
severe culture conditions, e.g. in presence of the highest salt
Modelling the Growth of Listeria Monocytogenes... 135
concentration, the lowest acid pH or the highest temperature. But
the percentage never exceeded 33%.
Growth in Beef Meat
The potentiality of use of GFP-expressing strain in food
product was evaluated. A challenge-test with the transformed
strain 2 was carried out in raw ground beef at 10°C. High counts
of a competitive flora, up to 105 CFU ml–1, were initially found in
meat, which represented particularly hard condition to follow the
growth of a specific E. coli strain. The total flora was counted on
PCA, and GFP strain on PCA + Ara and PCA + Ara + Amp (as a
selective medium). The evolution of the GFP strain was easily
distinguished from competitive flora thanks to its fluorescence: its
survival could be monitored throughout the experiment. The results
obtained in the two plating media were similar.
DISCUSSION
Using GFP transformation allowed for successful applications
in assessing various biological issues. In the present work, the
construction and evaluation of E. coli strains carrying the GFP-
expressing plasmid pBAD-GFPuv are reported.
The presence of this plasmid does not affect the intrinsic
characteristics of the E. coli strains, i.e. the serotypic or biochemical
traits and virulence genes. No significant behaviour difference
between marked strains and parent strains could be found, as
indicated by overlapping confidence intervals. A major limitation
of the GFP marker is the small number of studies on the influence
of environmental conditions on the GFP expression, and more
particularly, for a plasmid marker.
Our results have demonstrated the stability of the marker in
unfavourable environment, i.e. in presence of acid, salt and in
cooling/heating environment. Indeed, the study of the transformant
strain cardinal values showed that the GFP persists in large ranges
of pH values (4·0-7·0), NaCl concentrations (0·5-8%) and
temperature (8-45°C). Furthermore, the plasmid stability was
confirmed in dynamic conditions of temperature, where the extent
of variation could reach 27°C. It is well known that this factor may
vary extensively throughout food processing. The stress induced
136 Introductory Food Microbiology
by the abrupt change of temperature did not increase the plasmid
loss.
Most foods are complex systems with heterogeneous microbial
populations. The introduction of the GFP marker provides a simple
method to differentiate between inoculated strains and natural
competitive flora of the product. Indeed, in the experiments on
food described above, we could rapidly distinguish individual
species within the natural flora of the product. GFP labelling in
micro-organisms makes it a marker of choice for ecological studies.
Furthermore, the stability of the plasmid in the absence of selection
obviates the need for the addition of antibiotics to these systems
in short-time experiments (14 days in the tested food experiment).
In laboratory medium, GFP-labelled strain could be detected on
the basis of green fluorescence, even after 13 days of inoculation.
This observation is in accordance with the previus study of
Bloemberg et al. (1997), who examined the stability of a GFP-
containing plasmid in Pseudomonas spp.
Marking cells with GFP expressed from a plasmid provides
the flexibility to transform a wide range of strains. Indeed, the
construction of such strains is relatively easy.
Predictive food microbiology has been focusing on modelling
the microbial responses to food environment, in the interest of
food safety and for avoiding spoilage. Predictive models have
been regularly published to describe the growth of a strain or a
mixture of strains as a function of the environmental factors of
food. A good way of validating a model is to compare its prediction
to real data obtained from food products. The standard method of
data collection is the total viable count, which is a very labour-
intensive method. In this way, the use of GFP-marked strain of
providing a useful system for monitoring the evolution of a defined
micro-organism and even developing among competitive ones,
presents strong advantages.
GROWTHS KINETICS COMPARISON OF CLINICAL AND
SEAFOOD LISTERIA MONOCYTOGENES ISOLATES IN ACID
AND OSMOTIC ENVIRONMENT
Comparison of pathogenic bacterial strains of clinical origin
with strains of the same species isolated from the environment
Modelling the Growth of Listeria Monocytogenes... 137
may be a valuable tool for microbial risk assessment, especially for
foodborne pathogens. Thus, a number of Listeria monocytogenes
strains responsible for human cases of listeriosis, in relation to the
consumption of contaminated seafood, have been compared with
“natural” L. monocytogenes strains isolated from similar seafood
products. Complete factorial designs were used to assess
quantitatively the growth abilities of four clinical and four seafood
isolates of L. monocytogenes placed in various environmental
conditions. The cells were submitted to acid and osmotic stress as
they were in stationary phase (constant condition) or in
exponential phase (dynamic condition). The effects and interactions
of pH (5–7) and NaCl concentration (0.5–8% v/v) were studied at
two growth temperatures (10 and 20 °C). Growth parameters (lag
and generation times calculated with Gompertz equation) were
used to compare the behavior of the strains with respect to the
conditions of culture. The results indicated an overall weak effect
of acid stress alone, whereas osmotic stress clearly affected bacterial
growth and a synergic effect between these two factors was
observed. Clinical strains displayed better adaptation than seafood
strains in stationary phase, however, this difference was not verified
in exponential phase. Low temperature (10 °C) usually confirmed
the observations at 20 °C, and the differences between clinical and
food strains were more pronounced. Finally, a classification of the
eight strains, based on the collected data, showed three groups: (i)
seafood strains, (ii) three clinical strains and (iii) the last clinical
strain, alone due to its high resistance to adverse conditions.
Listeria monocytogenes assumed public health significance as a
result of its presence in foods linked to several outbreaks of
listeriosis in Europe and North America. Most cases of human
listeriosis occur in immunocompromised individuals, pregnant
women and the elderly.
L. monocytogenes is a widespread microorganism that has been
isolated from a variety offoods including fish. Although several
kinds of food products have been incriminated as source of
sporadic or epidemic listeriosis cases, the involvement of fish and
fishery products is still very rare. In 1992, two perinatal listeriosis
cases, which occurred in Auckland, New Zealand, were associated
to the consumption of smoked mussels. Mussels were also involved
138 Introductory Food Microbiology
in cases occurred in Sydney, Australia. An outbreak caused by
rainbow trout was reported in Sweden.
The prevalence of L. monocytogenes in naturally contaminated
seafood was studied by Jorgensen and Huss (1998): the highest
prevalence was found in cold-smoked fish (34–60%), while the
lowest was found in heat-treated and cured seafood (4–12%). L.
monocytogenes has been found in smoked salmon in several studies.
Ben Embarek (1994) reported a contamination rate of between 0%
and 75%, with an overall prevalence of 10%, in cold-smoked
salmon samples.
In fresh as well as in several lightly preserved seafoods
including cold-smoked salmon, none of the processing steps
eliminate L. monocytogenes. Furthermore, cold-smoked fish products,
which are typically consumed without cooking, are among the
ready-to-eat foods of particular concern due to the lack of a heat
inactivation step during processing. The ability of a microorganism
to survive in various foods depends on several combined
parameters in the foods such as temperature, pH, salt concentration
(water activity). A range of seafoods, particularly the lightly
preserved products (<6% salt concentration, pH>5) such as smoked
fish products (hot and cold smoked), lightly salted products (brined
cooked shrimp) or marinated products, are capable of supporting
the growth of L. monocytogenes (Huss et al., 2000). Moreover, L.
monocytogenes is able to survive or grow at refrigeration
temperatures. The pathogen growth was observed in vacuum-
packed smoked salmon stored at 10 or 2 °C. It is important to note
that cold smoking is conducted at temperatures below 30 °C, most
frequently 19–22 °C, for 2 to 3 h and, therefore, provides a possible
environment for bacterial growth.
Several studies have shown that the virulence of individual L.
monocytogenes strains may differ. Furthermore, Datta (1994)
demonstrated that the pathogenicity of this microorganism can be
affected by various substrate factors, i.e. in foods. There are fewer
published systematic studies in which growths of clinical and
food strains of L. monocytogenes under unfavourable conditions
(found in industrial environment) were compared. The comparison
of pathogenic bacterial strains of clinical origin to strains of the
same species isolated from environment may be a valuable tool for
Modelling the Growth of Listeria Monocytogenes... 139
microbial risk assessment, especially for foodborne pathogens.
Therefore, it was of interest to evaluate and compare the capacity
of growth of clinical and food strains, in order to determine whether
there was a relationship between strain origin and the growth
potentialities in function of environmental factors.
Consequently, the aim of this work was to investigate the
growth behaviors of four L. monocytogenes strains responsible for
human cases of listeriosis in relation to consumption of
contaminated seafood and four L. monocytogenes strains isolated
from seafood products, in function of temperature, pH and salt
concentration. Furthermore, the effects of the pH and salt
concentration variations on the growth of the strains were taken
into account, with the objective to simulate the variations which
can occur during food processing and induce a stress situation for
the microorganism.
Monocytogenes Strains and Media
Experiments were carried out with eight L. monocytogenes
strains. Four were clinical strains associated to fish or fish products
and four were isolated from seafoods.
Stock cultures were maintained at “80 °C in cryobank. All
strains were resuscitated before use by inoculations into 10 ml of
Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, Hampshire,
UK), followed by incubation at 37 °C for 8 h.
The subculture and culture medium was BHI (Oxoid)
supplemented with yeast extract (Oxoid) 3 g l”1, glucose (Prolabo,
Fontenay sous bois, France) 2 g l”1 and buffered with a K2HPO4–
KH2PO4 0.1 mol l”1 solution in proportion 1:1 (v/v) to pH 7.0.
Monitoring of Bacterial Growth
An automated turbidimeter was used to follow the growth of
L. monocytogenes strains in the micro-titer plates. Optical density
(O.D.) was read at a wavelength of 600 nm. The working volume
in each well of the micro-titer plate was 400µl.
Experimental Procedure
The ability of the eight L. monocytogenes strains to grow in
osmotic and acid environment was determined as described by
140 Introductory Food Microbiology
Cheroutre-Vialette et al. (1998). After resuscitation, strains were
subcultured (1%) in the supplemented BHI 18 h at 20 °C, by which
time the strains had reached stationary phase. 0.5% of this
inoculum was then transferred in the culture medium for the
growth studies. The concentration of cells in the culture medium
was about 107 cfu ml”1, in order to be above the detection threshold
of the Bioscreen C. The viable number of bacteria was confirmed
by PCA plate counts (AES). Plates were incubated for 24 h at 37
°C and enumerated.
For all experiments, three conditions were studied: control,
constant and dynamic conditions. Under control condition, cells
in stationary phase (after subculture) were grown in culture
medium BHI, without addition of salt and acid. In constant
conditions, bacteria in stationary phase were grown in culture
medium adjusted to the desired aw and pH values. The osmotic
variation was achieved by the addition of NaCl (Merck). A solution
5 M of HCl (Merck, 32%) was added to regulate low pH. The
dynamic condition was defined as follows: the bacteria were grown
in culture medium (control condition, i.e. without addition of
NaCl and HCl) until the beginning of the exponential phase and
were then shocked by the abrupt addition of shock solutions.
These shock solutions (osmotic and acid) were prepared in order
to obtain final values similar to those indicated for the constant
conditions. An aliquot (100 l) of concentrated (×4) shock solutions
was added when the O.D. was near 0.2, corresponding to the
beginning of exponential phase.
For each combination, five growth repetitions were carried
out.
Experimental Design
A factorial design was used to assess quantitatively the effects
and interactions of NaCl and pH (HCl) conditions on the growth
of L. monocytogenes clinical or seafood strains at two temperatures.
The combinations of the following conditions were used:
Temperature (°C): 10, 20 °C
pH: 5, 6, 7
NaCl (% v/v): 0.5, 4, 8
Modelling the Growth of Listeria Monocytogenes... 141
For each combination, the constant and dynamic conditions
of pH and NaCl concentration were studied. At least, for each
strain, 36 experiments were performed according to 18 growths in
constant condition and 18 growths in dynamic condition.
Statistical Analysis of Data
Experimental data were statistically analysed by the use of S-
PLUS 2000 software.
Averages of the O.D. were calculated for the five repetitions of
inoculated media and for the two repetitions of non-inoculated
media. The data were then analysed using the procedure described
by Bégot et al. (1996). Four quantities were calculated at time t:
• (O.D.i)t, the mean of the O.D. of the five repetitions of
inoculated media for each combination;
• (O.D.ni)t, the mean of the O.D. of the two repetitions of non-
inoculated media for each combination;
• (DO.D.)t=(O.D.i)t–(O.D.ni)t;
• Yt=log10[(DO.D.)t/(DO.D.min)], where DO.D.min was the
lowest DO.D. value above the detection threshold.
As indicated by Cheroutre-Vialette et al. (1998), a modified
Gompertz equation was used to fit the growth curves Yt=f(t).
Growth parameters, A (logarithmic increase of population), l(lag
time) and µ(maximal growth rate), were determined by non-linear
regression (S-PLUS software). GT (generation time) was derived
from musing the relation: GT=log10(2)/m.
In control and constant conditions, the time of inoculation
was taken as zero time when considering the growth curve. In
dynamic condition, the time of addition of the shock solution was
taken as zero time in the calculation of the growth parameters.
To summarize the similarities in behavior of the strains, a
hierarchical clustering method was used. A global data set,
including all the conditions of the experimental design, was built.
Lag and generation times for the 36 treatments (two temperatures,
three pH, three NaCl concentrations, static and dynamic
conditions) were used as variables measured on the eight strains.
Data were standardized for each variable. The first stage of this
method is the computation of a distance measure between the
142 Introductory Food Microbiology
individuals. Each individual is considered as an initial cluster.
The two “closest” clusters are merged in a bigger one at each step
of the process. The distance between clusters is calculated using
the average distance method: the distance between clusters A and
B is the average of the distances between the points of A and the
points of B. The algorithm stops when there is only one cluster left.
The distances between merged clusters are retained: this will allow
the program to produce a graph in the form of a classification tree.
Each node in the tree appears at a height equal to the distance at
which the clusters were merged.
Growth of L. monocytogenes Strains in Constant Acid and Osmotic
Environment
At 20 °C in control condition (i.e. in culture medium BHI), all
the strains, clinical or food isolates, presented similar growth
parameters, i.e. lag and generation times.
The pH had negligible effect on the different strains. Indeed,
whatever the strain origin, the calculated lag and generations
times at pH 6.0 were similar to those obtained with the control
growths. The growth parameters values of a clinical strain, Lm-
C4, and a food strain, Lm-E4 in control condition and in acid
constant condition (pH 6.0 and pH 5.0). The growths at pH 5.0
have shown longer lag times. On the other hand, at 20 °C, the
strains were more sensitive to NaCl. The presence of salt (8%)
induced longer lag phase and increased the generation time by a
factor 2.5, on average, compared to the corresponding control
condition. All the results obtained at 20 °C showed that no
differences were observed between clinical and seafood strains
when testing acid environment alone or osmotic environment alone.
On the other hand, at this growth temperature, it was observed
differences between strains when the two factors were considered
simultaneously. On the whole, the calculated generation times of
the clinical strains were lower than those of food strains for
pHd”6.0 combined with 4% or 8% NaCl. Among the clinical
strains, Lm-C1 was less affected by the combined acid and osmotic
conditions, more particularly in the most severe culture condition.
Variations in lag times were noted among the strains in the tested
conditions.
Modelling the Growth of Listeria Monocytogenes... 143
At refrigerated temperature (10 °C), the strains were more
sensitive to low pH. The interactive effect of the factors pH and
concentration of NaCl on the L. monocytogenes growth were more
pronounced at 10 °C than at 20 °C. Furthermore, the differences
between clinical and seafood strains were more underlined,
especially as the culture conditions were severe, i.e. low pH and
presence of NaCl. No food strains could grow in medium regulated
to pH 5.0 supplemented to 4% of NaCl, whereas the growth
recovery of the whole clinical strains could be observed. The ability
of Lm-C1 to grow in severe conditions was confirmed by these
results. No strains were able to grow, i.e. no increase of optical
density was observed during the experiment duration (14 days),
at 10 °C, pH 5.0, 8% NaCl.
Comparison with Growths in Dynamic Conditions
In dynamic condition, L. monocytogenes strains were subject to
abrupt variations of pH and salt concentration during the
beginning of exponential phase at 20 or 10 °C. The stress induced
by these variations could induce a lag phase, i.e. an adaptation
period to new environment. The duration of this period was
variable and no relation between this period value (lag time) and
the strain origin or the environmental factors could be established.
The generation times associated to growth recovery
consecutively to the environmental variation was different from
those calculated in constant condition. For example, in Table 4,
the values of ratios of GT calculated in dynamic condition and GT
in constant condition for strains Lm-F4 and Lm-C4 are indicated.
In most cases, the growth recovery of the L. monocytogenes strains
exposed to environmental variations in exponential phase, i.e.
dynamic condition, showed a generation time longer than in
constant condition, when the strains were in stationary phase.
The conditions of culture in correlation with the presence of NaCl
induced the higher ratios, i.e. the generation time observed in
dynamic condition was longer than in constant condition.
The better adaptation of clinical strains, as compared to food
strains observed in constant condition was not established in
dynamic condition. The parameters values of the strains were
comparable. It must be noted that, contrary to results in constant
conditions, all the strains were capable to grow in the condition
144 Introductory Food Microbiology
pH 5.0, 4% NaCl, 10 °C. At this temperature, no growths were
observed at the most severe condition, pH 5.0, 8% NaCl, as shown
in constant condition.
Among the clinical strains used in this work, two strains, Lm-
C3 and Lm-C4, were implicated in the same outbreak, but isolated
from different patients and had different clonal types. Therefore,
it was interesting to analyze their characteristics of growths. At 20
°C, Lm-C3 and Lm-C4 had a similar behavior in front of the
studied factors and their variations. The growth parameter values,
i.e. lag and generation times, did not show significative difference.
As shown in Fig. 4 for 10 °C, few differences could be observed
whatever the studied condition. In constant condition, at pH 6.0–
8% NaCl or pH 5.0–4% NaCl, Lm-C4 presented longer lag and
generation times, e.g. at the last combination the lag time of Lm-
C4 was elongated of 17 h compared to Lm-C3.
Classification of the Different Strains
The data base collected in this present work allowed the
constitution of a classification of the different strains, with the
objective to verify the global behavior of the clinical and food
strains. The result, confirmed the previous conclusions. Three
groups were created. One of the groups was constituted by all the
seafood L. monocytogenes strains (Lm-F1, Lm-F2, Lm-F3, Lm-F4), a
second one contained three of the clinical L. monocytogenes strains
(Lm-C2, Lm-C3, Lm-C4). This means that the food strains were in
general “closer” to each other than to the clinical strains. The
particular behavior of the clinical strain Lm-C1 underlined in our
results was confirmed by this classification, since it was isolated
from the other ones. Indeed, as indicated by the experimental
results, this clinical strain presented a better adaptation to
unfavorable environment of temperature, acid pH and salt
concentration.
DISCUSSION
It is well known that L. monocytogenes is a microorganism
which is able to survive, and frequently grow, under a wide range
of adverse conditions such as low temperature, low pH and high
osmolarity. In this work, significant differences in growth kinetics
were found between clinical and seafood strains in acid and/or
Modelling the Growth of Listeria Monocytogenes... 145
osmotic environment, at different temperatures. Indeed, the clinical
strains, in stationary phase, were revealed to be more resistant to
these environmental conditions, in comparison to environmental
strains. These results are in accordance with the study of Dykes
and Moorhead (2000) who examined the acid stress response of
clinical or meat L. monocytogenes strains and demonstrated the
ability of clinical strains to survive an acid shock. Likewise, Avery
and Buncic (1997) focused on another environmental factor, the
temperature, and showed that 15 clinical strains of L. monocytogenes
had higher resistance to the effects of unfavorable storage
conditions, compared with 15 meat strains.
Our results confirmed that more investigations were necessary
to allow the evaluation of the growth characteristics of clinical
strains, compared to food strains, in particular in conditions found
in the food environment, with the objective to improve the risk
assessment. Eight strains were studied in the present work. The
behavior of a larger number of strains (implicated in the main
listeriosis cases in the world or associated with different foods) in
function of different environmental factors should now be explored
to consolidate our conclusions. Indeed, when foodborne listeriosis
is considered, it could be hypothesized that a high resistance of
some L. monocytogenes strains to environmental factors found in
foods or industrial environment may contribute to the particular
capability of certain strains to cause illness and, consequently, to
become clinical strains. The influence of environmental factors
(such as temperature, acid, etc.) on the expression of virulence
markers was also highlighted.
The experiments of the present study, comparing the strains
behavior in constant and dynamic condition, showed differences
on the calculated generation times. This observation is in agreement
with the previous studies of Cheroutre-Vialette et al. (1998) and
Cheroutre-Vialette and Lebert (2000a). They observed that cells of
L. monocytogenes isolated from meat products or food processing
environment submitted to environmental variations of pH/aw in
exponential phase were usually more sensitive than cells in
stationary phase. It was largely demonstrated that, phenotypically,
bacteria in stationary-phase growth are more thermotolerant, acid-
resistant and are better equipped to survive osmotic stress (Hill
and Rees). Nevertheless, in this work, for a studied combination
146 Introductory Food Microbiology
(pH 5.0, 4% NaCl, 10 °C), the food strains were able to overcome
the stress in exponential phase but not in stationary phase (this
experiment was repeated three times with different inoculum).
The presence of L. monocytogenes in seafoods has been
examined by several authors. The prevalence of this pathogen can
be associated to the light preservation process (salting, etc.), the
extended shelf life at refrigerated temperatures, capable of
supporting the growth of L. monocytogenes, the consumption
without further cooking. Therefore, it is important to evaluate the
adaptation of this microorganism in the food processing
environment which could include variations (temperature, salting,
acidification, etc.) during time. Mathematical modeling techniques
are gradually becoming recognized and accepted as powerful
tools for predicting the effects of food-preservation systems on
bacterial growth kinetics. The applicability of available predictive
models to fish products, considering the products characteristics,
was approached. Recently, several studies have demonstrated the
significative interest to take into account the dynamic condition in
the field of predictive microbiology, and to collect experimental
data in this way.
With the objective to introduce more advances into the field of
predictive microbiology and increase the safety of products, studies
allowing correlations between clinical strains characteristics (such
as growth kinetics, cell physiology, e.g. stress condition, expression
of pathogenicity in function of particular environment found in
food) prove to be necessary. Another prospect is to study the
genetical background of environmental strains in order to select
the ones that may be most representative of the whole natural
population. The knowledge of typing data (serotype, ribotype,
pulse field gel electrophoresis) for such strains and their
comparison to the corresponding epidemiological data should
provide a useful tool for the Listeria risk assessment.
VARIABILITY OF THE RESPONSE OF 66 LISTERIA
MONOCYTOGENES AND LISTERIA INNOCUA STRAINS TO
DIFFERENT GROWTH CONDITIONS
The growth of 58 strains of Listeria monocytogenes and eight
strains of Listeria innocua iso-lated from meat products (68%) and
Modelling the Growth of Listeria Monocytogenes... 147
industrial sites (23%), were compared in four con-ditions of
temperature, water activity (aw) and pH. Temperatures ranged
from 10-37 °C, pH from 5·6-7·0 and aw from 0·96-1. Growths were
performed in a meat broth with an auto-mated turbidimeter
(Bioscreen C, Labsystem). Growth curves were fitted using the
Gom-pertz function, and growth parameters were calculated. The
differences between strains in lag phase duration were much
greater than in growth rate. The greatest differences occurred at 10
C, pH 7 and aw 0·96 : lag time values ranged from 4 h to 4 days.
the Listeria population was separated into five groups, according
to the lag time and maximal growth rate values using clustering
analysis. The majority of the strains isolated from industrial sites
were grouped together and showed faster growth than the others
in the four con-ditions studied. The serotype or the nature of the
meat from which the strains were isolated did not influence growth.
The variability observed among strains raises questions about the
consequences in quantitative risk assessment and about the
construction of models in pre-dictive modeling.
Listeria bacteria are widespread in the environment. Among
the different species, Listeria monocytogenes and Listeria innocua are
the two most commonly isolated from food processing. L.
monocytogenes can cause the death of both the very young and
immunocompromised individuals. Most cases are traced to the
contamination of raw or processed foods with L. monocytogenes.
Listeriosis is therefore a major threat to human health.
Listeria monocytogenes can grow at low temperatures, low pH
and low water activity (aw); they are therefore able to survive and
multiply in a wide range of food products.
Work on predictive microbiology has been carried out in an
attempt to improve the shelf life and safety of food. Predic-tive
models of microbial growths are set out with respect to the main
controlling factors of the environment such as temperature, pH
and water activity. Despite variations in growth among strains
numerous models have been constructed using only one or a
small pool of strains. In this work, we compared the growth of 66
strains of L. monocytogenes and L. innocua, isolated from meat, meat
prod-ucts and industrials sites. Strains were clus-tered according
to their growth in a broth medium in four conditions of temperature,
148 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 149

pH and aw. Consequences on the construction of models are Bioscreen C. For each strain, eight successive wells of the same
discussed. column were filled. The last two wells received the same volume
of non-inoculated medium in order to determine the growth medium
MATERIALS AND METHODS optical density (OD) and controlled any possible contamination.
Strains Ws methodology is simi-lar to that used by Be got et al. (1996).
Fifty-eight strains of L. monocytogenes and eight strains of L. Experimental Design
innocua were studied: 45 strains were isolated from meat and meat
A Plackett Burman design (Plackett and Bur-man 1943) was
products, 15 from meat and dairy plants (materials, floors, walls)
used to test the effects of three factors: temperature, pH and water
four were involved in outbreaks. Five of 13 serotypes of L.
activity. Each factor was studied twice at two levels: high and low
monocytogenes were represented: 1/2a, 1/2b, 1/2c, 4b, 4d. Listeria
(Table 3). While a fac-torial design would have required eight
cultures were stored on tryptic soy agar slopes at 4 C.
experiments, four were sufficient with a Plackett Burman design.
Media
Curve Fitting
Culture inocula were prepared in a meat medium (MM): meat
OD data were transferred from the Bioscreen C to Excel
peptone (Merck) 10 g l 1, yeast extract (Difco) 5 g l 1, glucose
software (Microsoft Windows) and transformed for each
(Merck) 5 g l 1. The pH was adjusted to 7·0 with NaOH (Prolabo)
measurement. Four quantities were calculated at time t: (ODi)t, the
1 mol l 1. The medium was autoclaved at 120 C for 20 min.
average of the OD of eight replicates; (ODni)t, the average of the
Growth experiments were carried out in a tryptic meat broth OD of the non-inoculated medium
(TMB) which was steril-ized by filtration derived from tryptone
(DOD)t=(ODi)t (ODni)t
meat agar (GTV5p, Fournaud et al. 1973): meat extract (Merck) 10
g l 1, proteose peptone (Merck) 10 g l 1, tryptone (Difco) 5 g l 1, Log10[(DOD)t/DODmin]
glu-cose 5 g l 1. The medium was buffered with a K2HOP4-KH2PO4 where DODmin was the lowest DOD value above the detection
(Merck), 0·1 mol l 1 solution in proportion 1:1 (v/v) and adjusted threshold.
to pH 7·0 with NaOH 1 mol l 1. aw was set by adding NaCl In the linear range of the Bioscreen C ( DOD<1’2) (Begot et al.
(Merck) in accordance with Chirife and Resnik (1984). 1996), growth curves were fitted using the modified Gompertz
Tryptone sodium chloride medium (TSC) (tryptone 1 g l 1, equation:
NaCl 8·5 g l 1, pH 7·0) was used for dilutions, and TSA and APT Table : Serotypes, food sources and laboratory references of
agar (Difco) were used for plate counts and agar slope cultures. tested strains of Listeria
Growth Species Serotype Food source
Growths were measured by optical density in an automated a
1 Listeria monocytogenes P1 1/2c Pig carcass
turbidimeter, Bioscreen C. All strains were inoculated in MM and
2 L. innocua 2138 a 6a Minced meat
incubated for the same period of time (17 h) at 30 C. All cultures a
3 L. monocytogenes 24631 1/2b Rib of lamb
were therefore in stationary phase, thus avoiding disparities in
the subsequent growth kinetics. Nine millilitres of TMB were 4 L. monocytogenes 3670 a 1/2a Minced meat
inoculated with the subculture. The inoculum size was confirmed 5 L. monocytogenes 2141 a 1/2c Minced meat
a
by plate counts. The inocu-lated TMB was dispensed aseptically 6 L. monocytogenes 27795 1/2a Minced meat
in 300 µl volumes into honeycomb microplates (10 10) of the 7 L. monocytogenes 4133 a 1/2c Minced meat
150 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 151

8 L. monocytogenes 28423 a 1/2c Minced meat 46 L. monocytogenes 17e 1/2b Industrial sites
9 L. monocytogenes 2143 a 1/2a Minced meat 47 L. monocytogenes 18e 4b Industrial sites
10 L. monocytogenes 5602 a 4b Pig shoulder 48 L. monocytogenes 29e 4b Industrial sites
11 L. monocytogenes 880030b 1/2a Meat products 49 L. monocytogenes 39e 4b Industrial sites
12 L. monocytogenes 880390b 4b Meat products 50 L. monocytogenes 41 e
4b Industrial sites
13 L. monocytogenes 880398b 1/2c Meat products 51 L. monocytogenes 42 e 1/2c Industrial sites
14 L. monocytogenes 890307b 4b Meat products 52 L. monocytogenes 44 e 4b Industrial sites
15 L. monocytogenes 890313b 4d Meat products 53 L. monocytogenes 70e 4b Industrial sites
16 L. monocytogenes 890467b 4b Meat products 54 L. monocytogenes 99e 4b Industrial sites
17 L. monocytogenes CLIP 19908c 1/2c Meat products 55 L. monocytogenes Lo 28f 1/2a Human isolate
18 L. monocytogenes CLIP 19910c 1/2a Meat products 56 L. monocytogenes CDC Atlanta 9g 4b Milk
19 L. monocytogenes CLIP 19532c 1/2c Meat products 57 L. monocytogenes CA Wisconsing 4b Cheese
20 L. monocytogenes CLIP 19534c 1/2c Meat products 58 L. monocytogenes OH Wisconsing 4b Cheese
21 L. monocytogenes CLIP 19536c 1/2c Meat products 59 L. monocytogenes Scott A Wisconsing 4b Human isolate
22 L. monocytogenes CLIP 19712c 1/2a Meat products 60 L. innocua CLIP 20719 g
Meat
23 L. monocytogenes CLIP 19734c 1/2a Meat products 61 L. innocua CLIP 20728g Poultry
24 L. monocytogenes CLIP 19802c 4b Meat products 62 L. innocua CLIP 20741g Meat
25 L. monocytogenes CLIP 19804c 4b Meat products 63 L. innocua CLIP 20595g Meat
26 L. monocytogenes CLIP 19884c 1/2c Meat products 64 L. innocua CLIP 20600g Poultry
27 L. monocytogenes CLIP 19887c 1/2b Meat products 65 L. innocua CLIP 12511g Poultry
28 L. monocytogenes 925331d 1/2a Chicken 66 L. innocua CLIP 12512g Poultry
29 L. monocytogenes 925318d 1/2b Guinea-fowl Strains of Listeria were donated by:
30 L. monocytogenes 925321d 1/2c Sausages a
Dr Nicolas (Regional Laboratory of Haute-Vienne, France).
31 L. monocytogenes 925222d 1/2a Chicken b
Dr Marly (INRA of Nouzilly, France).
32 L. monocytogenes 925228d 4b Chicken c
Dr Rocourt (Pasteur Institute, France).
33 L. monocytogenes 925267d 1/2a Sausages d
Dr Courtieu (National Center of Listeria references, France).
34 L. monocytogenes 925257d 1/2c Minced meat e
Dr Jolivet (SOREDAB, France).
35 L. monocytogenes 925261d 1/2c Minced meat f
Dr Cossart (Pasteur Institute, France).
36 L. monocytogenes 925253d 1/2a Sausages g
Dr Richard (National Institute of Agronomic Research of Jouy-en-Josas,
37 L. monocytogenes 925201d 1/2a Sausages France).
38 L. monocytogenes ATCC 19111 1/2a Poultry
39 L. monocytogenes ATCC 19115 4b Human ⎛ N ⎞ ⎛ ( ∆OD )t ⎞
L og10 ⎜ ⎟ = L og10 ⎜⎜ ⎟⎟ (1)
cerebrospinal fluid ⎝N0 ⎠ ⎝ ∆ODmin ⎠
40 L. monocytogenes 1
e 4b Industrial sites
41 L. monocytogenes 4e 1/2b Industrial sites ⎛ ⎛ µ.e ⎞⎞
42 L. monocytogenes 10e 1/2a Industrial sites = A . exp ⎜ − exp ⎜ (L − t ) + 1 ⎟ ⎟
⎝ ⎝ A ⎠⎠
43 L. monocytogenes 13e 1/2b Industrial sites
44 L. monocytogenes 14e 4b Industrial sites where A is the logarithmic increase of bac-terial population, L, the
45 L. monocytogenes 16e 4b Industrial sites lag time, µ, the maxi-mal growth rate and t, the time.
152 Introductory Food Microbiology
Growth parameters were determined by non-linear regression
with Statitcf software (Technical Institute of Cereals and Fodders,
France). Generation time (T) was derived from the maximal growth
rate (2):
T = [Log10(2)]/m
Table: Conditions of culture incubation and growth
measurements of Listeria strains in anautomated turbidimeter
(Bioscreen C)
Temperature 37 C 10 C
Number of wells 100 200
Wavelength 600 nm
Measurement time 30 h 240 h
4t 5 min 60 min
Shaking frequency 30 s per 1 min
Shaking intensity medium
Statistical Analysis
Two techniques were used to compare the growth of strains.
Lag times (L) and maxi-mum growth rates ( ) were both considered.
Average and standard deviation were calcu-lated for both variables.
Each of the 66 values were then normalized: the average was
sub-tracted and the result divided by the stan-dard deviation.
Each strain was depicted in a space of 2x4=8 dimensions.
Principal component analy-sis (PCA) was used to reduce the
dimension of the sample space. The method looks for the unique
subspace of a given dimension which explains the major part of
the variance in the original data. This analysis was performed
using Statitcf software. A cluster analysis was used and calculated
with Statitcf software. Classification is achieved through successive
aggre-gations of individuals and then groups of individuals. The
principle is to regroup indi-viduals according to their distance
from the centre of gravity for the set of experimental points. Two
individuals or two classes were aggregated when their fusion
involved a minimal increase of the intra-class dispersion. The
average value of each parameter was then calculated for each
group and an overall average was calculated for the 66 strains.
Modelling the Growth of Listeria Monocytogenes... 153
Table: Plackett Burman design to study the effect of
temperature, aw, pH on the growth of 66Listeria strains
Conditions Name aw Temperature( C) pH
1 010 0·96 37 5·6
2 100 1·00 10 5·6
3 001 0·96 10 7·0
4 111 1·00 37 7·0
Results
The logarithmic increase of popu-lation, A, was maximal in
condition 111. Par-ameter A was higher in both conditions of pH
7·0 than those of pH 5·6.
The mean, below and above which there is an equal number
of strains, was below the average value for the parameters
considered in all conditions. A proportion of 50-60% of the strains
was under the average for each parameter.
Large variations in lag time and gener-ation time were noted
among the strains in all conditions. The largest differences in lag
time among strains were recorded in con-dition 001. In this
condition, the values were multiplied by a factor 25 from the
minimum to the maximum value, taking the value from 4 h to 4
days. Larger variations among lag times were found successively
in condition 001, 100, 010 and 111.
The more severe the growth conditions, the larger the number
of disparities found in lag time. Fewer differences were found in
generation time. In condition 100, generation times tripled from
the minimal to the maximal value. In the other conditions,
generation times doubled when comparing the slowest and
fastest strain for each condition. Slight variations were found in
parameter A which corresponds to the logarithm increase of the
population density. We observed that strains exhibiting the longest
generation time of the entire population did not show the maximal
lag time.
For example L. monocytogenes ATCC 19115 which has the
longest generation time in condition 001, showed a lag time of
50·8 h whereas the longest lag time was 97·9 h. Similarly, the
154 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 155

strain exhibiting the shortest generation time did not show the Cluster Analysis
shortest lag time. Results were identical in all conditions tested. The cluster analysis divided the population into five groups.
Table: Effect of temperature, aw and pH on the growth of 66 Group 1 (10 strains) was clearly separated from other groups.
Listeria strains It was comprised of strains which grew faster than the overall
average in all the conditions studied. The majority (66%) of the
Parameter aw Temp. pH Min. Max. Average Mean
strains belonging to this group originated from industrials sites.
A001 1·2 1·9 1·5 1·5
Group 2 (15 strains) was characterized by strains which grew
T001 0·96 10 7·0 8·9 20·1 13·3 13·0 more slowly than the overall average, which is in opposition to
L001 3·9 97·9 31·8 28·2 group 1. L. innocua was not present.
A010 1·1 1·6 1·4 1·4 Group 3 (28 strains) was intermediate. Strains exhibited lag
T010 0·96 37 5·6 1·3 2·9 1·7 1·7 times and maximal growth rates around the overall average. It is
L010 1·2 10·2 4·0 3·4 located at the centre of the PCA figures. All origins were represented.
A100 0·8 1·8 1·3 1·3 Group 4 (eight strains) clustered which grew more slowly
T100 1·00 10 5·6 8·4 24·2 12·9 11·8 than the overall average except for condition 100 where they grew
L100 2·4 40·2 17·4 16·6 faster. No epidemiological strains of L. monocytogenes were present
A111 1·1 1·9 1·7 1·8 in this group.
T111 1·00 37 7·0 0·5 0·9 0·7 0·7 Group 5 (five strains) was composed of strains which grew
L111 0·2 2·6 0·9 0·6 more slowly than the average of the strains, except for condition
001 where they grew faster. 80% of group 5 strains were L. innocua.
The variability of the growth response was expressed by the L. monocytogenes Scott A, which has been widely used to set up
following parameters: logarithm increase of population density models, belonged to this group.
(A), generation time (T) and lag time (L). T and L were expressed
The nature of the meat they were isolated from had no effect
in hours, A was an increase of Log10.
on the cluster analysis:
Strains were separated according to their ability to grow in
Table: Comparison of the generation times (h) and lag times (h)
each condition by using the variables L and µ. We chose three
averages calculated on the 66 Listeria strains and on each
axes which explained 68% of the initial information. Axis 1
group defined by the cluster analysis
separated strains according to the values of parameters L and µ.
Lag time and maximal growth rate were negatively and highly 10 C aw 0·96 37 C aw 0·96 10 C aw 1·0037 C aw 1·00
corre-lated in all conditions. pH 7·0 pH 5·6 pH 5·6 pH 7·0
T001 L001 T010 L010 T100 L100 T111 L111
Strains exhibiting fast growth (low lag time and high maximal
growth rate) were isolated from strains which grew slowly (high Overall average 13·3 31·8 1·7 4·0 12·9 17·4 0·7 0·9
lag time and low maximal growth rate). The second axis scaled Group 1 10·7 16·2 1·5 2·3 9·7 9·2 0·6 0·5
the conditions from worst to best: 001-100-010-111. Strains Group 2 13·9 44·8 1·9 6·6 13·5 23·2 0·6 0·6
separated again according to growth conditions. Axis 3 segregated Group 3 13·8 36·0 1·6 3·2 12·1 18·7 0·7 0·7
condition 001 from the others. Strains that grew well in this Group 4 13·3 28·2 2·1 4·2 10·8 10·1 0·8 1·8
condition, where temperature and water activity were low, were
Group 5 10·2 6·5 1·8 4·0 19·1 20·1 0·7 1·3
clustered.
156 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 157

Table: Distribution of the serotypes of L. monocytogenes strains difference between both strains is seven days and is reached in
in the five groups defined by the cluster analysis condition 001, when the inoculum is 1 cfu ml 1. Such results can
have great consequences in quantitative risk assessment. Listeriosis
Group 1/2a 1/2b 1/2c 4b 4d L. innocua Non- Total causes illness in certain categories of the population
determined
(immuno-compromised, old or pregnant people) but the conditions
1 2 3 1 3 0 1 0 10 of foodborne outbreaks are still not well known. The infectious
2 4 1 5 3 0 1 1 15 dose is supposed to be high but the minimal dose causing illness
3 8 2 5 11 1 1 0 28 depends on the vulnerability of the person who ingested the food,
4 1 0 3 3 0 1 0 8
the type and quality of the food, the level of the pathogen in the
food and the virulence of the strain. As a consequence, such
5 0 0 0 1 0 4 0 5
unknown data linked to the variability of the growth response
Total 15 6 14 21 1 8 1 66 show the difficulties than can be faced in estimating the probability
each group was composed of strains of diff-erent serotypes. of occurrence of listeriosis.
In our study, the strains were clustered according to their
Discussion
growth ability in the four conditions. They were not grouped
This study highlighted differences in growth among L. round their serotype nor the nature of the meat from which they
monocytogenes strains. Larger variations between the 66 strains were isolated. However it is interesting to note that group 1 was
were shown in lag times rather than in generation times. in majority composed of strains isolated from industrial sites.
Particularly wide variations were observed in condition 001 (10 C, These strains grew faster than the others in all the conditions
aw 0·96, pH 7·0) where lag times extended from 4 h to 4 days. tested. This suggests that they are accustomed to growing in a
These results tally with those of Barbosa et al. (1994) who reported wide range of harsh conditions (e.g. high osmolarities, low
that among 39 strains of L. monocytogenes, lag times ranged from temperatures). Studies have recently shown the ability of Listeria
1·5-2·9 days at 10 C, pH 7·2. Lag time is known to be the time to adapt their cellular physiology to harsh environments. Most of
required for the organ-isms to adapt their cellular components to the L. innocua studied were found in group 5, and grew more
the new environment. As a consequence, it is affected by the slowly than the average population, except in condition 001 (10
difference between subculture and culture condition, the C, aw 0·96, pH 7·0). These results concord with those of Barbosa
physiological state of the inoculum and by the strain. Indeed, in et al. (1994) but not with studies where modified or selective
our study, as we were careful to homogenize the conditions of the media were used. Listeria monocytogenes Scott A was also clustered
sub-cultures so as to avoid disparities in the sub-sequent growth in group 5; in condition 001, it has the fastest lag time (3·9 h) and
kinetics, the differences we obtained in the growth parameters the fastest generation time (9·4 h) of all the 66 strains. These
were attributed to the nature of the strain. results do not tally with those of Bar-bosa et al. (1994) who studied
Such variability in lag times added to the variability in this strain among 39 other strains and showed that it had the
generation times characterizes strains with slow or fast growth. longest lag time at 4 and 10 C. Other strains that were the causes
As the Inter-national Commission on Microbial Specifi-cation for of epidemic out-breaks were found in different groups: L.
Foods set the toler-ance limit in food at <100 L. monocytogenes per monocytogenes OH Wisconsin was clustered in group 1
gram at the point of consumption, it was possible to calculate the (characterized by fast growth), L. monocytogenes CDC Atlanta in
time needed to reach this limit in the four conditions studied for group 2 (slow growth) and L. monocytogenes CA Wisconsin, in
both ‘slow’ and ‘fast’ strains as the inoculum varied. The maximal group 3 (average growth).
158 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 159

Table: Prediction of time (in days) necessary to reach a limit We have shown the importance of studying a large number of
level of 102 cfu ml 1 as the inoculum varied from 1-10 cfu ml1 strains of various origins before constructing models to assess
strain variability. However, other factors should also be considered
Conditions aw Temp. pH Strains 1 cfu ml 1 10 cfu ml 1 to produce effective mod-els, for example inoculum level,
001 0·96 10 7·0 Fast 2,6 1,2 interactions between bacteria, interactive effects between strain
Slow 9,6 6,5 and product as shown by Rosenow and Marth (1987) and
010 0·96 37 5·6 Fast 0,4 0,2 comparison of growth in liquid media to growth on solid media.
Slow 1,2 0,8 Growth of Pseudomonas fluorescens and Pseudomonas fragi in a
100 1·00 10 5·6 Fast 2,4 1,1 meat medium as affected by pH (5.8 – 7.0), water activity (0.97
– 1.00) and temperature (7 – 25°C)
Slow 8,2 4,5
111 1·00 37 7·0 Fast 0,2 0,1 A total of 59 strains of Pseudomonas, isolated from meat products,
were grown in micro-titer plates in a meat medium over a range
Slow 0,4 0,2
of pH (5.8–7.0), aw (0.97–1.00) and temperature (7–25°C). Growths
The time was calculated in the four conditions of growth for a ‘slow’ were performed in a meat broth with an automated turbidimeter
‘ strain and a ‘fast’ ‘ strain. (Bioscreen C, Labsystem, France). The growth curves obtained in
Such results point out the difficulties in choosing a strain to this study did not have sigmoidal shapes making it impossible to
build a predictive model. Indeed numerous studies have involved calculate growth parameters using the Gompertz equation. The
the testing of one or a cocktail of three or five strains. L. medium was weakly oxygenated in the micro-titer plates and
monocytogenes Scott A was fre-quently used. The results of Barbosa reached 0%-dissolved oxygen at the beginning of the exponential
et al. (1994) along with those presented here raise some questions phase. Strains were separated into two groups: P. fragi and P.
as to predictive models: is it wise to construct models with only fluorescens. Strains of P. fragi had shorter lag times than those of
one strain? If so, which strain? That with an average, fast or slow P. fluorescens. The impact of such results is interesting in that these
growth? If not, how can we integrate the growth variability in could assist to explain the succession of flora that is observed
mod-els? If the fastest strain were considered, the model would during the processing of meat: P. fluorescens is the dominant bacteria
always predict a shorter lag time and lower growth rate than among Pseudomonas spp. at the beginning of a slaughter line and
those found for the majority of strains, and would thus always P. fragi becomes dominant during the chilling process.
provide safer growth estimates. But in this case, the industry Many flora of spoilage-bacteria have an effect on the shelf-life
could not support the costs involved in reducing the shelf life of of refrigerated food products. The main flora responsible for spoilage
a product. Furthermore, there is nothing to guarantee that a faster in fresh meat and milk products during aerobic storage however
strain will not be found in the future. For public health safety, the are the Pseudomonas species. These are dominant in poultry meat,
slowest strains should be avoided. In order to take the variability pork, and beef and lamb. In milk and cream the major spoilage
of the strains into account, two ways are proposed: the first bacteria have been identified as P. fluorescens and P. fragi. Widders
approach is the production of two predictive models using two et al. (1995) reported that the average contamination on whole
strains that are charac-terized by a fast and a slow growth; this carcasses at 4°C peaked at 3.90 log10 cfu/cm2 and 4.54 log10 cfu/
would result in giving a growth response interval. The second cm2 on meat surfaces at the same temperature. P. fragi and P.
approach would be a model integrating first the study of a fluorescens cause deterioration in quality of meat and milk products
strainhaving an average behavior in a large range of experimental due to the production of extracellular proteases and lipases at low
conditions, and second the results of this study. temperatures. Off-odours occur in milk when the population of
160 Introductory Food Microbiology
Pseudomonas spp. reaches 2.2×106 to 3.6×107 cfu/ml or on meat
when the population reaches 107 to 108 cfu/cm2. P. fragi strains
produce fruity and putrid odours on beef and have a deleterious
effect on the colour of meat stored at 1°C, resulting in a green and
slimy appearance.
Although many studies have been carried out on the occurrence
and levels of contaminations in meat products, there has been
little work on a comparison of relative growth of Pseudomonas
species and on the growth variations among these strains. The
aim of this study therefore was to compare the growth rates of
strains of P. fragi and P. fluorescens in a selected media over a
range of pH, water activity (aw) and temperature, and to attempt
to describe the resulting growth curves.
Strains
A total of 59 strains of Pseudomonas were selected from bacterial
counts made from meats of different origin. All were isolated from
or grown on Pseudomonas Agar Base supplemented with
Pseudomonas CFC Supplement (Oxoïd). The strains were identified
as P. fluorescens or P. fragi using standard tests. This involved:
growing on Plate Count Agar at 4°C, and a range of tests including
Gram stain, oxidase and catalase reaction, mobility, production of
acid in the presence of maltose and cellobiose, degradation of
gelatine and production of pigment on King B medium. The strains
were stored at 4°C on PCA slants and transferred monthly.
Media
Two media were used for successive subcultures — these
were PCA slants and meat medium (MM) which contained meat
peptone (Merck) 10 g/l, yeast extract (Difco, OSI, Maurepas, France)
5 g/l, and glucose (Prolabo, Fontenay sous Bois, France) 5 g/l. The
pH was adjusted to 7.0 by NaOH (40 g/l). The culture medium
was a tryptic meat broth (TMB) (Fournaud et al., 1973) and was
buffered with a K2HPO4-KH2PO4 (Merck), 0.1 mol/l solution to
adjust the pH. Water activity was controlled by adding NaCl
(Merck) according to Chirife and Resnik (1984). An aliquot of 0.5
ml/l of anti-foam ANBIO 15 (1% v/v) was added for growth in
a fermenter.
Modelling the Growth of Listeria Monocytogenes... 161
Materials
An automated turbidimeter was used to follow the growth of
Pseudomonas spp. in the micro-titer plates. Optical density (OD) of
the growth was measured on a spectrophotometer. OD was read
at a wavelength of 600 nm. The threshold of detection for the
Bioscreen C, and for the spectrophotometer, were determined at
6.0×106 cfu/ml and 1.0×107 cfu/ml, respectively. The range where
OD was proportional to the bacterial population extended from
0.010 to 0.8 OD for both apparatus.
A 2-l fermenter was used with pH monitored by a heat-
sterilisable electrode. Dissolved oxygen was measured directly in
the fermenter with an oxygen probe and the zero calibration
determined with an oxygen-free medium provided by Setric Génie
Industriel. A full-scale reading was obtained by saturation of the
medium with air supplied continuously to the culture.
Inoculum
Each strain was incubated on PCA slants at 25°C for 8 h and
then transferred to MM that was shaken in a rotary shaker
waterbath for 17 h at 25°C, by which time growth of all the strains
had reached the stationary phase. A proportion of the culture was
inoculated in TMB to give a concentration of about 107 cfu/ml in
order to be above the detection threshold of the spectrophotometer
and the Bioscreen C. The viable number of bacteria was determined
on PCA plates using standard microbiological practice immediately
after inoculation of the growth medium. Viable numbers for growth
in Bioscreen C ranged from 6.9–7.1 log10 cfu/ml and from 7.1–7.3
log10 cfu/ml for growth in fermenter.
Growth Experiments
Growth of the strains was obtained on micro-titer plates using
the protocol developed by Bégot et al. (1996): for each strain, 300
l of inoculated TMB was dispensed in eight successive cuvettes in
the micro-titer plates. Some cuvettes were filled with non-inoculated
TMB and were used as controls. OD was measured at 600 nm.
The working volume of the fermenter was 1 l. The stirring
speed was regulated to 70 rev./min. Prior to inoculation the
medium was saturated with oxygen, so that growth began at a
162 Introductory Food Microbiology
level between 90 and 95% of dissolved oxygen. Air could be
supplied at the following volumetric rates: 0, 0.24, 0.40 and 0.80
l/l of medium/min. A fraction of the fermenter volume was
periodically removed to measure OD, however, no more than 10%
of the working volume was pipetted during growth.
Growth Conditions
A fractional factorial design for each of the environmental
variables, aw, temperature, pH at three levels, gave nine conditions
for growth for each of the 59 Pseudomonas strains. These growth
conditions are given in Table 2. Growth in the fermenter was
carried out with P. fragi 103 in condition E111.
Growth Curve Treatments
Growth data obtained as a function of OD versus time were
transferred from Bioscreen C to Excel software (Microsoft Windows)
and transformed for each measurement. Four quantities were
calculated at time t:
1. (ODi)t, the OD average for the eight replicates
2. (ODni)t, the OD average for the non-inoculated TMB
3. (DOD)t=(ODi)t”(ODni)t
4. f(t)=Log10[(DOD)t/DODmin] where DODmin was the
lowest DOD value above the detection threshold.
The function f(t) was used to calculate two parameters for the
59 strains in the nine conditions: the maximal growth rate, Vmax,
and the lag time, L. The maximum growth rate of all the growth
rates Vmax, was calculated for each time tn (Eq. 2). L corresponded
to the intersection of the x-axis and the tangent in Vmax:
f (t n +1 ) − f (t n −1 )
V tn = (1)
t n +1 − t n −1
Vmax = maximum (Vtn) (2)
Statistical Analysis
Average, mean, minimum and maximum were calculated for
the two variables L and GT. Strains were classified according to
their ability to grow in the different conditions. A cluster analysis
was carried out with STAT-ITCF software.
Modelling the Growth of Listeria Monocytogenes... 163
RESULTS
Strains of both species grew at 4°C on PCA, were Gram-
negative, motile, gave positive oxidase and catalase reactions. P.
fluorescens strains produced fluorescent pigments and gelatinase,
P. fragi strains did not. P. fragi strains produced acid in the
presence of maltose and cellobiose, P. fluorescens strains did not.
As shown in numerous studies, a decrease in temperature, pH
or aw increased the duration of both lag and generation times. Fig.
1 shows for most growth conditions, growth was characterised by
an unusually shaped growth curve, i.e. the lag phase was clearly
visible with the growth phase generally reaching its maximal
growth rate at the beginning of this phase. After the maximal
growth rate, there was a break in the curve leading to a decreasing
phase which did not reach a steady stationary phase, even after
long periods of incubation (up to 45 h in condition E111). These
growth curves are thus characterised by an inflection point situated
not long after the end of the lag phase and by a non-symmetrical
shape. The two parameters GT (generation time) and L (lag time).
It is seen that the interval between minima and maxima increased
as the conditions of pH, aw and temperature moved away from the
most favourable condition E111. It is to be noted that the gap
between minima and maxima increased more for L than for GT.
P. fragi 103 rapidly metabolised dissolved oxygen when no air
was supplied, i.e. there is a break in growth as the dissolved
oxygen level reached 10% 1.5 h after inoculation. The time needed
to reach 10% of the saturated value for dissolved oxygen was 1.5,
1.5, 2.5 and 3.5 h when the rates of air were respectively 0, 0.24,
0.4 and 0.8 l per l of medium per min. Zero percent dissolved
oxygen was reached 1 h following these periods. Growth curves
in these four conditions are shown in Fig. 3. For 2 h, growth
curves are similar, then they separated and rose faster as the air
supply increased. The growth curve obtained in Bioscreen C was
close to that obtained in the fermenter at a flow rate of 0.24 l per
l of medium per min. It assumed confidently that conditions of
aeration in the Bioscreen C cuvettes are similar to this type of
aeration.
The classification carried out on all the strains revealed two
main groups. The first was comprised of all the P. fragi and four
164 Introductory Food Microbiology
P. fluorescens namely 51, 11.6A, M2L3 and P×10. The second
comprised the remaining P. fluorescens. Statistical values for all
strains. L, averages, means, minima and maxima for each group.
It can be seen that shorter lag times were obtained for the first
group. Between each group differences were greater in conditions
E133 (aw 1.00, 7°C, pH 5.8), E232 (aw 0.985, 7°C, pH 6.4), E331 (aw
0.97, 7°C, pH 7.0) related to the lowest temperature.
DISCUSSION
The growth curves obtained in this study did not have
sigmoidal shapes making it impossible to calculate growth
parameters with the Gompertz equation which is usually used by
authors. A steady stationary phase was rarely obtained and for
long experiments a biofilm could be seen on the top of the medium
in the cuvettes. In addition to possible insufficient shaking of the
micro-titer plates in Bioscreen C, it was supposed that the aeration
was also too weak. Growth in the fermenter confirmed this and
highlighted that the aeration in the cuvettes was equivalent to 0.24
l of air per l of medium per min. These conditions of aeration
played too important a role for aerobic bacteria and became a
limiting factor for the growth of the Pseudomonas spp. A possible
explanation for the unexpected shape of the growth curves is that
the combined effect of the depletion of oxygen and the change in
the metabolism: indeed, in some cases, Pseudomonas spp. can use
nitrate as an alternate electron acceptor and can grow
anaerobically.
For many studies carried out in milk and milk products with
Pseudomonas spp. the generation time was the main parameter
for comparison. Cox and MacRae (1988) reported that P. fragi
grew faster than P. fluorescens in both U.H.T. and raw milk at 4°C.
In meat products, few studies have compared the behaviour of the
species. This work carried out on 59 strains of Pseudomonas has
shown small differences between calculated generation times that
are confirmed by results of Pooni and Mead (1984) who worked
on strains isolated from poultry products and grown in heart
infusion broth. These authors showed that pigmented and non-
pigmented Pseudomonas made little difference in average generation
times. The differences in lag time observed between both species
are important. For the 59 strains separated into the two groups,
Modelling the Growth of Listeria Monocytogenes... 165
only four strains of P. fluorescens were in the P. fragi group. These
were characterised by shorter lag times in condition E111 (25°C,
aw 1, pH 7). This explains their presence in the first group. The P.
fragi group had on average shorter lag times than P. fluorescens.
These differences have been observed on a great number of strains
and in a wide range of temperature, pH and aw; e.g. average L was
two-fold shorter in the P. fragi group at 7°C.
These results could help to explain the changes in the ecology
of Pseudomonas flora shown by different authors who have studied
levels of contamination and areas of isolation during meat
processing and retailing. Lahellec and Colin (1981) observed the
spoilage strains in poultry: pigmented strains represented the
major population isolated in processing plants, but during storage
non-pigmented strains outgrew the former. On beef, lamb and
pork, studies have shown the predominance of P. fluorescens from
the slaughter line to the chilling process. P. fluorescens is known
to be largely present in the environment (floor, water), on animals
(hide, skin) or also in water and surfaces in meat factories. On
cutting lines and during storage and retailing, P. fragi was then
found as the dominant flora on meat. Drosinos and Board (1995)
studied the evolution of the bacterial flora in minced lamb stored
aerobically at 4°C and showed that P. fragi outgrew P. fluorescens.
They tried to explain this succession of flora by differences in the
metabolism of both species. In our study shorter lag times observed
for P. fragi strains could be linked to their ability to adapt better
to new environmental conditions compared to P. fluorescens.
Our findings make it possible to choose a limited number of
representative strains from the 59 Pseudomonas — so as to diminish
the number of experiments needed to study these few strains with
more precision in broth or on surface tissue of meat. A ‘low’,
‘quick’ or ‘average’ strain characterised by a slow, rapid or average
growth in all or few conditions of the experimental design, could
then be chosen in both species. Further studies should be
undertaken: to substantiate the differences in lag times between
the ‘average’ strains of both species and compare their generation
times in better conditions of aeration; to study a ‘low’ strain and
a ‘quick’ strain and observe the variability in their growth
responses; or to compare the growth parameters obtained in broth
and on the surface tissue of meat.
166 Introductory Food Microbiology
DIVERGICIN 750, A NOVEL BACTERIOCIN PRODUCED BY
CARNOBACTERIUM DIVERGENS 750
Divergicin 750, a bacteriocin produced by Carnobacterium
divergens 750, preferentially inhibited the growth of strains of
Carnobacterium and Enterococcus. Selected strains of Listeria
monocytogenes and Clostridium perfringens were also inhibited.
The bacteriocin was purified to homogeneity by ammonium sulfate
precipitation and sequential S-Sepharose, hydrophobic interaction
and reversed phase chromatography. The complete amino acid
sequence was determined by Edman degradation. The peptide
consisted of 34 amino acid residues. The calculated M(r) from the
peptide sequence, 3447.7, agreed well with that obtained by mass
spectrometry. Divergicin 750 did not show any sequence
similarities to other known bacteriocins. The plasmid-located
structural gene encoding divergicin 750 (dvn750) was cloned and
sequenced. The gene encoded a primary translation product of 63
amino acids with a deduced M(r) = 6789.4 which is cleaved
between amino acid residues 29 and 30 to yield the mature
bacteriocin. Lactic acid bacteria produce a variety of com- pounds
with antimicrobial activity. Some of these are proteins or peptides
and are termed bacteriocins. Bacteriocins from lactic acid bacteria
are cur- rently being divided into four classes. Class II bacteriocins
are small and have little post-translational modifications. They
are usually heat stable, hydrophobic and often act on the cell
membrane of susceptible target cells. Bacteriocins are commonly
secreted by a dedicated transport and maturation system. The
bacteriocins usually inhibit the growth of closely related species.
Some bacteriocins also inhibit the growth of pathogens and spoilage
organ-isms and may thus be of interest for enhancing food safety
and food hygiene.
Carnobacteria grow at low temperature, produce relatively
little acid and volatile compounds and they generally also have
low proteolytic and lipolytic activity. Little is known about
bacteriocins from carnobacteria. Some bacteriocin-producing
carnobacteria have been identified. We have previously purified
and characterised piscicolin 61 from Carnobacterium piscicola LV61.
C. piscicola LV 17 produced three bacteriocins, termed
carnobac-teriocin A, BM1 and B2 of which carnobacteriocin A
was identical to piscicolin 61. Carnobacteriocin BM 1 and B2
Modelling the Growth of Listeria Monocytogenes... 167
show significant sequence similar-ity and contain the -YGNGVXC-
motif typical of bacteriocins active against Listeria. Recently,
diver-gicin A, produced by C. dicergens LV 13 was char-acterised.
It constitutes an interesting exception in being a small class II-like
bacteriocin which uses the cells’ sec system for translocation. A
lanthion-ine-containing bacteriocin, carnocin UI49 has been
purified as well. Here we describe the purification, characterisation
and cloning of divergicin 750, a novel bacteriocin from C. diuergens
750.
Growth of Bacteria
Thirty-seven strains of carnobacteria were screened for
production of bacteriocin. Carnobac-terium dicergens 750 from the
laboratory stock of Bundesanstalt fur Fleischforschung, Kulmbach.
Ger-many, produced divergicin 750. For production of bacteriocin,
cells were grown at 25°C overnight in cMRS medium containing
Peptone proteose no. 3 (10 g l-1 ), yeast extract (5 g l-1), sucrose (20
g l-1 ), K HPO4 (2 g l-1 ), (NH4)2 citrate (2 g l -1 ), MgSO4 (0.02 g l-
1
), MnSO4 (0.02 g l-1 ). pH was adjusted to 8.5 with NaOH and the
medium auto-claved for 20 min. In growth kinetic experiments C.
diuergens 750 was grown in D-MRS broth adjusted to initial pH
6.6 and pH 8.2. The broths were inoculated at a 0.1% level of
overnight cultures and incubated for 48 h at 25°C. At appropriate
time intervals the number of colony-forming units and bacteriocin
activity in the culture supernatant was determined. Growth of C.
dicergens 750 in D-MRS broth of different pH values was monitored
using an automated turbidometer, BIOSCREEN C. 10 µl of a ten-
fold diluted 24-h culture were added to honeycomb wells
con-taining 190 µl D-MRS broth of pH 4.5, 5.0, 6.0. 7.0, 8.0, 9.0,
and 10.0. Al! inoculations were done in triplicate and incubation
was for 48 h at 30°C. After 14 h and 38 h, samples were withdrawn
from a second honeycomb plate and tested for bacteriocin activity.
To test the bacteriocin for pH stability, fractions of culture
supernatants were adjusted to pH values between 2 and 11 with
5 M NaOH or 2 N HCI. After 1 h of incubation at 20°C, residual
activ-ity was examined by use of the agar spot assay.
Bacteriocin Activity Assays
Bacteriocin activity was quantified by using serial dilutions of
the bacteriocin in the agar spot test as described previously. One
168 Introductory Food Microbiology
arbitrary unit (AU) was defined as the reciprocal of the highest
dilution yielding a definite zone of inhibition on the indicator
lawn. Alternatively, bacteriocin activity was quanti-fied in a
microliter plate assay system by measuring the growth of the
indicator organism in two-fold dilutions of bacteriocin in medium
as described previously.
When necessary, bacteriocin fractions were adjusted to pH 6.5
and sterilised by filtration through Millipore filters (0.22 µm) prior
to activity measurements. One bacteriocin unit (BU) was defined
as the amount of bacteriocin which inhibited growth of the
indicator organism by 50% as compared to a control culture
without bacteriocin. Unless otherwise stated C. dicergens L66 was
used as an indicator strain.
Purification and Analysis of Divergicin 750
Bacteriocin was purified essentially as described previously.
In short, a 2-1 culture of C. diuer-gens 750 was grown to the
stationary phase and cells were removed by centrifugation. The
bacteriocin pre-sent in the supernatant fraction was concentrated
by ammonium sulfate precipitation and subjected to ion exchange
chromatography (S-Sepharose), hydropho-bic interaction
chromatography (Octyl-Sepharose) and reversed phase FPLC
(Pharmacia). The purified bacteriocin was stable in 40% (v/v) 2-
propanol containing 0.1% trifluoroacetic acid at - 20°C.
Amino acid sequencing and mass analysis of puri-fied
divergicin 750 were done as described previously.
Recombinant DNA Techniques
Basic cloning techniques were used. The probes were end-
labelled with 32 P using a terminal transferase end-labelling kit.
Plas-mid DNA from C. divergens 750 was digested with various
restriction endonucleases and the resulting fragments were
separated on agarose gels, blotted onto nylon filters and hybridised
with a divergicin 750-specific degenerate oligonucleotide probe.
Two partly overlapping fragments, a 2-kb EcoRI frag-ment
and a 0.2-kb Mbol fragment, hybridising to the oligonucleotide
probe, were purified by excising them from a low-melting-point
agarose gel and subse-quently employing the MagicTM clean-up
Modelling the Growth of Listeria Monocytogenes... 169
system. Cloning was done in Escherichia coli DH5 a using the
cloning vector pGEM-7Zf( + ) (Promega). Positive clones were
identified by colony hybridisation. The cloned DNA fragments
were se-quenced completely on both strands using the Seque-nase
version 2.0 DNA sequencing kit (Amersham) and a primer walking
strategy. Computer analyses were carried out on an IBM personal
computer employing the DNASIS sequence analysis program and
on a UNIX computer employing the GCG programme package.
RESULTS
Production and Inhibition Spectrum of Divergicin 750
Growth of C. divergens 750 at different initial pH values of the
growth media was measured. The bacteria grew well at high pH
while growth was significantly retarded at pH 5. Bacteriocin was
pro-duced over a wide range of pH values and could be detected
even down to pH 5. It appeared that diver-gicin 750 was produced
in the late exponential phase of growth as the cells entered the
early stationary phase and remained fairly stable in the growth
super-natant after production.
When C. divergens 750 was grown at different temperatures,
maximum production of bacteriocin occurred at 25°C . Bacteriocin
production was de-tected down to growth at 4°C. At 37°C no
activity was observed. Consequently, cells were grown at initial
pH 8.5 and 25°C to enhance the yield of bacteriocin.
Table: Inhibition spectrum of purified divergicin 750
Indicator bacteria Number of sensitive/number tested
Carnobacterium dicergens 1 / 1
Carnobacterium piscicola I / 1
Carnobacteriumgallinarum 0/1
Enterococcus faecalis 2/2
Enterococcus faecium 0/
Lactobacillus sake 0/1
Bacillus cereus 0/1
Brochothrix thermosphacta 0/1
Clostridium butvricum 0/1
170 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 171

Clostridium perfringens 1 / 1 Purification of Divergicin 750

Clostridium sporogenes 0/ 1
Purification stage Fraction Total Specific Yield
Listeria innocua 0/2 volume protein activity
Listeria icanocii 0/1 (ml) (mg) (BU mg-I X 10-4) (%)
Listeria monocytogenes 1 /5 Culture supernatant 2000 120 1.6 100
Listeria seeligeri 0/1 (NH4)>SO4 conc. (Fr I) 200 90 4.2 190
Listeria welshimeri 0/ 1 S-Seph. chrom. (Fr))) 50 21 0.2 2.5
Propionibacterium acnes 0/ t Octyl-Seph. chrom. (FrIIl) 9 1.4 100 70
Staphylococcus aureus 0/4 Rev. phase FPLC (FrIV) 2 0.07 1000 35
Streptococcus mutans 0/ 1
Inhibition was tested by using the purified bacteriocin (2048 AU ml-1) Cloning and DNA Sequencing of the Divergicin 750 Structural Gene
in the agar spot test assay described in Materials and methods. Hybridisation of a degenerate oligonucleotide probe to plasmid
Some lactic acid bacteria are known to produce more than one DNA from C. dirergens 750 gave one strong hybridisation signal
bacteriocin. To rule out interference from other inhibitory to a 2-kb EcoRI fragment and a partly overlapping 0.2-kb Mbol
substances, purified divergicin 750 was used in determination of frag-ment. The EcoRl and Mbol fragments were cloned and
sequenced. The gene encoded a primary translation prod-uct of 63
the inhibition spectrum. Divergicin 750 preferably inhibited strains
amino acids with a calculated Mr = 6789.4. Cleavage of this peptide
of Carnobacterium and Ente-rococcus. Selected strains of Clostridium
between amino acid residue 29 and 30 would give the mature
perfringens and Listeria monocytogenes were also inhibited.
divergicin 750. Uncertain amino acid residue determinations in
Purification of Dirergicin 750 posi-tions 1, 6, 11 and 26 of divergicin 750 were corrobo-rated by
deduction from the sequence of the cloned gene. Downstream of
Divergicin 750 was purified by ammonium sulfate
the structural gene, a region of dyad symmetry was found.
precipitation, sequential cation exchange, hydropho-bic interaction
and reversed-phase chromatography. The purified bacteriocin DISCUSSION
appeared homogeneous when subjected to FPLC-reversed phase
Divergicin 750 is a low molecular mass, hy-drophobic and
chromatography. Overall, a more that 600-fold increase in specific
basic peptide and thus shares charac-teristics with other
activity was observed (BU mg-1 protein). The purified bacteriocin
bacteriocins of lactic acid bacte-ria. Divergicin appeared to be
had an activ-ity of approx. 10 000 BU µg -1 protein.
produced only in the late exponential phase of growth . This is in
The complete amino acid sequence of the purified bacteriocin contrast to many other bacteriocins, for example sakacin A from
was determined by Edman degradation. The sequence was L. sake Lb706 and sakacin P from L. sake Lb674, which are
identical to that deduced from the structural gene after cloning. constitutively synthesized. Nothing is known about the mechanism
Divergicin 750 consisted of one polypeptide chain of 34 amino of regulation of divergicin 750. The bacteriocin was purified by a
acid residues. No unusual amino acid residues such as lanthionins procedure simi-lar to that used for other bacteriocins. During
were found. The calcu-lated Mr of 3447.7 was almost identical to purification a drop in activity was observed after elution from the
the value of 3448.5 Da obtained by PDMS. The extra 1 Da in the S-Sepharose column, whilst the activity was regained after the
mass analysis probably originated from protonation of the next purification step. The reason for this apparent drop is
polypeptide during desorption. unknown. The molecular mass of divergicin 750 as calcu-lated
from the deduced amino acid sequence was very close to that
172 Introductory Food Microbiology
determined by PDMS and is consistent with the proposed amino
acid sequence. Taken together with the information that no
lanthion-ins were detected upon amino acid composition anal-ysis,
divergicin 750 appeared not to be subjected to extensive post-
translational covalent modifications and thus belonged to the
class II bacteriocin group. No sequence similarities of divergicin
750 with other proteins encoded by sequences in the EMBL and
GenBank sequence databases were found. Divergicin 750 thus
differs from other bacteriocins of the lactic acid bacteria.
A general mechanism of action for the class II bacteriocins has
been suggested. In this model the bacteriocin molecules are thought
to form am-phiphilic a-helices that combine to make a pore in the
cell membrane of susceptible cells. Nothing is known about the
mechanism of action of divergicin 750. The region encompassing
amino acid residues 7-28 may be able to form an amphiphilic a-
helix and may indicate a similar mode of action for divergicin 750.
Divergicin 750 is processed from a longer pri-mary translation
product. The N-terminal 29 amino acid residues are cleaved off
adjacent to a glycine doublet to yield the mature product. This
leader sequence, although unusually long, shows typical
signatures of bacteriocin leader peptides of the dou-ble-glycine
type, for example a hydrophilic re-gion at positions - 8 to - 11
flanked by hydropho-bic amino acid residues. Other bacteriocins
of lactic acid bacteria with this type of leader invariably have a
dedicated secretion system for their export. Indeed, a putative
open reading frame starting ap-prox. 1 kb downstream of the
structural gene showed a high degree of similarity to bacteriocin
ABC ex-porter genes such as sapT. This indicates that dvn750 may
be part of a larger gene cluster involved in production and secretion
of divergicin 750. Bacteriocins and bacteriocinogenic strains may
be used by the food industry as additives to prevent outgrowth of
pathogens and spoilage organisms, giv-ing an enhanced control
over production processes. The study of the structure of bacteriocins
is an important step in establishing knowledge about the parts of
the molecule that are responsible for the inhibitory activity.
Subsequently, it may be possible to increase the inhibitory spectrum
and enhance the activity by changing specific amino acid residues
in the bacteriocin molecules.
Epidemiological Typing of Bacillus spp Isolated from Food 173
7
EPIDEMIOLOGICAL TYPING OF
BACILLUS SPP ISOLATED FROM
FOOD
Biotypes, fatty acid profiles, and restriction fragment length
polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB
gene) were compared for 62 isolates of the Bacillus cereus group.
Eleven isolates originated from various foods, and 51 isolates
were obtained from pasteurized milk which had been processed
by two different dairies. The isolates were clustered into 6 biotypes,
10 fatty acid groups, or 7 PCR-RFLP clusters. Isolates with
mesophilic or psychrotrophic characteristics were preferentially
distributed into specific fatty acid or PCR-RFLP groups (P = 0.004).
Unique fatty acid clusters were predominantly found in milk
samples of each dairy (P < 0.0001), suggesting that certain dairy
plants may harbor plant-specific B. cereus which might constantly
contribute to postpasteurization contamination.
Improving microbial safety and extending the shelf life of
pasteurized milk and related products have always been an
important concern to the dairy industry. A major factor limiting
realization of these goals is microorganisms surviving the
pasteurization process and/or contributing to postpasteurization
contamination. Bacillus cereus, Bacillus thuringiensis, and Bacillus
mycoides are frequently found in pasteurized milk, causing
spoilage because of the production of lipases and proteases. They
can also exhibit a health risk to the consumer since they produce
enterotoxins. It has been proposed to merge B. cereus, B.
thuringiensis, and B. mycoides into one single species. DNA-DNA
174 Introductory Food Microbiology
hybridization experiments and pulsed-field gel electrophoresis of
chromosomal DNA demonstrated a very close genetic relationship
among the three species. Furthermore, the 16s rRNA sequences of
these species are more than 99% similar. In the present article, B.
cereus, B. thuringiensis, and B. mycoides are, therefore, not
distinguished and are referred to as B. cereus.
Many dairy scientists believe that contamination of pasteurized
milk with Bacillus spp. results from spores that were present in
the raw milk and survived the pasteurization process. However,
other data in the literature suggest that postpasteurization
contamination might significantly contribute to Bacillus counts in
pasteurized milk. This controversy can partly be related to the lack
of adequate typing methods for B. cereus. Ternstro¨m et al. described
an attempt to use numerical phenotypic analysis for
characterization of dairy Bacillus spp. However, the authors did
not show any relationship between Bacillus isolates from raw and
pasteurized milk. Va¨isa¨nen et al. used fatty acid analysis and
phages to type dairy B. cereus isolates. Although they found both
techniques useful for typing of B. cereus, the authors did not
systematically compare B. cereus isolated throughout the
processing lines of single dairies.
Thus, their data do not allow conclusions about the origin of
B. cereus found in pasteurized milk. The objective of the present
study was to evaluate the feasibility of three typing techniques for
B. cereus. The techniques chosen were biotyping, analysis of cellular
fatty acids (CFA), and restriction fragment length polymorphisms
of PCR products (PCR-RFLP). To allow direct comparison, the
three techniques were applied to a defined collection of B. cereus
strains. Because a large proportion of the Bacillus strains studied
were isolated from pasteurized milk, additional epidemiological
data for dairy Bacillus spp. were also acquired.
Bacterial Strains
A total of 62 isolates of B. cereus were used. Eleven of these
isolates, originating from food-borne B. cereus outbreaks and from
randomly collected milk samples, were obtained from our culture
collection. The remaining 51 isolates were cultured from 23 retail
cartons (250 ml and 500 ml) of pasteurized milk that had been
Epidemiological Typing of Bacillus spp Isolated from Food 175
processed by two different dairies. Isolation procedures followed
the methods described in the Association of Official Analytical
Chemists’ Bacteriological Analytical Manual, except that B. cereus
selective agar supplemented with polymyxin B and egg yolk
emulsion was used as selective plating medium.
Growth of B. cereus at 8°C
Growth curves for the 62 B. cereus isolates were determined
with the multiwell photometric plate reader Bioscreen C. Bacteria
were grown for 18 h in brain heart infusion broth at 30°C and
were diluted in fresh brain heart infusion broth to an optical
density at 600 nm (OD600) of 0.005, and the suspensions were
used to inoculate the multiwell plate of the Bioscreen C. The
turbidity development (OD600) of the suspended bacteria at 8°C
was recorded for 144 h. Growth curves (i.e., turbidity development
curves) were subsequently generated from the OD readings by
using the computer software BIOLINK.
Preliminary experiments had shown that growth velocity at
8°C can be used to divide mesophilic and psychrotrophic B. cereus
strains by recording the detection time for each strain. The detection
time is de- fined by the BIOLINK software as the time at which an
OD of 0.05 is reached. The detection time of psychrotrophic B.
cereus isolates is less than 100 h, whereas it is considerably
higher than 100 h for mesophilic isolates.
Biochemical Properties
Twenty-one biochemical variables of the 62 B. cereus isolates
were determined by using the VITEK Jr. system according to the
manufacturer’s instructions. From the biochemical patterns
obtained, a binary profile was established for each isolate. The
distances between these profiles were calculated by Jaccard’s index
[d = 1 - c/(p + q + c)], where c is the number of variables present
in both strains, and p and q are the numbers of variables present
in each strain.
A dendrogram was constructed from the resulting distance
matrix by using the unweighted pair-group method with arithmetic
averages (UGPMA). Calculations were made with the computer
programs MacDendro and MacMul.
176 Introductory Food Microbiology
PCR-RFLP
It has been demonstrated that the lecithinase and
sphingomyelinase genes (cerAB) of B. cereus display genetic
variations among different isolates. This diversity and its possible
application for typing of B. cereus were further explored in the
present study. A PCR product encompassing a 1.5-kb fragment of
the cerAB gene was used to assess the genetic diversity of the 62
B. cereus isolates at that locus. PCR amplifications with primers
specific for cerAB were performed as described previously. Ten-
microliter aliquots of each amplicon were digested for 2 h with 10
U of the following restriction endonucleases: MnlI, HgaI, Sau96I,
PvuII, and BstXI. Reaction conditions were set as recommended by
the manufacturers of the enzymes. RFLP profiles were analyzed
by electrophoresis on 1.5% agarose gels stained with ethidium
bromide. A binary matrix of the profiles was used to calculate the
distances between strains by Jaccard’s index. A dendrogram was
constructed from the distance matrix by UGPMA as described
above.
Analysis of Whole-cell Fatty Acid Profiles with the MIDI System
Fatty acid methyl esters of B. cereus were analyzed by using
the Microbial Identification System. The analysis was conducted
according to the instructions given by MIDI (10). Briefly, isolates
were grown on Trypticase soy agar (BBL) for 24 ±1 h at 28±1°C.
At that time, 45 ±1 mg (wet weight) of cells was harvested from
single colonies. Total fatty acids of the bacteria were converted to
fatty acid methyl esters which were subsequently separated and
quantified with a Hewlett-Packard 5890 series II gas liquid
chromatography column equipped with a flame ionization detector.
A dendrogram based on the fatty acid profiles was established for
137 B. cereus isolates (62 isolates described in this study plus 75
B. cereus isolates isolated from various foods) by using the MIS
library generation software. This program calculates a similarity
coefficient based on the Euclidean distances (ED) between pairs of
isolates.
Clustering of the isolates is determined by UGPMA. The
phenotypic expression of relative amounts of fatty acids within
bacterial cells depends on various factors, which include medium
Epidemiological Typing of Bacillus spp Isolated from Food 177
composition, growth temperature, and growth rate. To determine
an ED value that is not affected by such variations in CFA and
that could therefore be used to obtain a reproducible grouping of
the B. cereus isolates within the dendrogram, the variability of the
fatty acid profile analysis was assessed by repeated testing of B.
cereus ATCC 14579. Repeated testing was conducted during a 3-
week period in the following manner. Two separate batches of
medium plates were prepared independently and stored at 4°C.
During each week, the test strain was subcultured daily onto fresh
Trypticase soy agar plates.
The second, fourth, and sixth subcultures were analyzed. Each
of the subcultures tested was streaked onto duplicate plates of
both batches of media. Each preparation of fatty acid methyl esters
was split into two subsamples, and the fatty acid profiles were
analyzed separately with the MIS.
ED were calculated for the following datum pairs: (i) duplicate
readings of 33 identical sample preparations, (ii) sample
preparations from 33 identical subcultures streaked onto duplicate
plates, (iii) sample preparations from 33 identical subcultures
grown on two different batches of media, (iv) 43 pairs of sample
preparations obtained from common subcultures grown during
each week (e.g., second, fourth, or sixth subculture obtained in the
1st and 2nd, 2nd and 3rd, or 1st and 3rd weeks), and (v) forty-
four pairs of sample preparations obtained from serial
subcultures grown during each week (e.g., second and fourth,
fourth and sixth, or second and sixth subcultures obtained in each
week).
In this study, identical subcultures of the B. cereus isolates
were used to generate the fatty acid profiles. The critical value
observed with two batches of media (ED, 6.7) could, therefore, be
used to group the 137 B. cereus isolates, resulting in 11 fatty acid
clusters. Although the MIS was originally designed for species
identification, it has been suggested that it may also be valuable
for epidemiological typing of bacteria. Consequently, the datum
library generated with 137 B. cereus isolates was used to evaluate
the relationships of the 62 B. cereus isolates. The fatty acid profiles
of these isolates were attributed to 10 (Mi 2 to Mi 11) of the 11 B.
cereus fatty acid groups.
178 Introductory Food Microbiology
Correlations
Comparison of growth characteristics and fatty acid profiles
of the 62 isolates showed that psychrotrophic isolates were
preferentially (P , 0.0001) grouped into fatty acid clusters Mi 2 to
Mi 6, whereas mesophilic isolates belonged to fatty acid groups
Mi 7 to Mi 11. A similar tendency (P 5 0.004) was perceived when
growth temperature and PCR-RFLP groups were compared. No
association could be observed among biotypes and growth
characteristics, PCR-RFLP groups, or fatty acid groups. Evaluation
of the fatty acid groups for the 51 B. cereus isolates from pasteurized
milk revealed interesting tendencies. A majority of the isolates
found in samples processed by dairy A belonged to the subgroup
Mi 5, whereas isolates found in milk from dairy B belonged to
groups Mi 6 and Mi 8 (P , 0.0001). Va¨isa¨nen et al. exploited
phage typing, minimum growth temperature, and analysis of CFA
for differentiation of dairy B. cereus isolates.
They found a correlation of fatty acid composition and
minimum growth temperature; however, they did not examine the
fatty acid data with regard to further typing of the isolates. Our
results confirm the observations regarding composition of CFA
and ability to grow at low temperatures. Additionally, we found
indications that psychrotrophic B. cereus strains with a specific
CFA profile were predominantly found in pasteurized milk
processed by one dairy, while they were less frequently found in
milk from the other processing plant. This plantspecific distribution
of B. cereus in pasteurized milk suggests that the strains may
originate from common reservoirs within dairy plants or within
certain farms supplying raw milk. This research was funded by
the Ontario Food Quality and Safety Research Fund, by the Dairy
Farmers of Ontario, and by the Natural Sciences and Engineering
Research Council of Canada. H.S. was supported by a fellowship
from the Commission for the Advancement of Young Scholars,
Zu¨rich, Switzerland.
RAPID ASSESSEMENT OF THE QUALITY OF POULTRY
CARCASSES
An assay has been developed whereby it is possible to assess
microbial concentrations in poultry carcass rinses within 10
Epidemiological Typing of Bacillus spp Isolated from Food 179
minutes. The test is a modification of one previously developed for
determining raw milk quality. In the latter test, milk samples were
treated with a detergent to lyse non-microbial cells present in the
milk. Microbial cells were removed by filtration and the ATP
present in the cells extracted and assayed using the luciferase-
luciferin reaction. A number of problems were encountered when
this protocol was applied to chicken carcass rinses. The primary
problem was a quenching effect on the light emission by the
luciferase reaction caused by the presence of particulate and lipid
material in the rinse water. The quenching effect was substantially
reduced by incorporating a pre-filtration step and an enzyme
treatment prior to filtration through the bacteria-retaining filter.
Using the modified assay, an excellent correlation (r=0.9) was
obtained when the ATP count obtained on 150 chicken rinse
samples was compared with plate counts carried out on the same
rinse washes.
The test can be used to make a rapid assessment of poultry
carcass quality, based on selective cut-offs predetermined by the
processor. If this level of count is exceeded, the carcass can be
subjected to a heat treatment before sale. The accuracy of the
prediction was high. Work was undertaken to compare methods
used to enumerate bacteria in chicken carcass rinses. The methods
studied included plate count (by the Spiral plater), ATP
bioluminescence, impediometry (using the Bactometer), HGMF,
and turbidiometry. This enabled comparison of the repeatability
and accuracy of the various methods. Only the ATP
bioluminescence assay and HGMF correlated well with plate counts
for chicken carcass rinses and the repeatability of these test methods
was excellent.
The ATP method was used to monitor critical control points
in the poultry processing plant. The CCP’s studied included the
scald, the prechill and the chill tank waters. It was shown that the
ATP test could be used to give a reliable indication of the microbial
content of process waters during production and results could be
obtained within 10 minutes. The microbial load in the scald tank
water was shown to increase throughout the working day and
this was shown to be due to accumulation of microorganisms
washed from the carcass rather than growth. Levels of microbial
180 Introductory Food Microbiology
contamination of the pre-chill and chill tanks were low in the
plant studied on several sampling days. The microbial load in the
tanks was dependent on flow rate of water through the tanks and
it was concluded that, based on microbial load in the chill tank,
water consumption at the plant could be reduced substantially.
This will enable plant personnel to more efficiently respond to
high microbial loads that may develop during chilling and result
in more economical management of the water supply.
ANTIMICROBIAL COMPOUNDS AND EXTRACELLULAR
POLYSACCHARIDES PRODUCED BY LACTIC ACID
BACTERIA STRUCTURES AND PROPERTIES,
DISSERTATION
In a variety of ecological niches, microorganisms compete
with each other for survival and through evolution form unique
flora. In some food ecosystems, lactic acid bacteria (LAB) constitute
the dominant microflora.
These organisms are able to produce antimicrobial compounds
against competing flora, including food-borne spoilage and
pathogenic bacteria. Under unfavorable environmental conditions
many species of LAB also produce exopolysaccharides (EPSs),
which protect themselves against desiccation, bacteriophage and
protozoan attack.
Lactic acid bacteria provide the major preservative effects in
food fermentation which mankind has practiced for thousands of
years. The primary antimicrobial effect exerted by LAB is the
production of lactic acid and reduction of pH. In addition, LAB
produce various antimicrobial compounds, which can be classified
as low-molecular-mass (LMM) compounds such as hydrogen
peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione),
uncharacterized compounds, and high-molecular-mass (HMM)
compounds like bacteriocins.
Among bacteriocins so far characterized, nisin is best defined,
and the only purified bacteriocin produced by LAB that has been
approved for use in food products. A LMM antimicrobial
compound, reuterin, has also been chemically identified, and the
reuterin-producing Lactobacillus reuteri strain has been applied as
a probiotic in dairy products.
Epidemiological Typing of Bacillus spp Isolated from Food 181
The EPSs produced by LAB are either present as a capsule
attached to the cell surface, or secreted into the environment.
Based on their sugar compositions, the EPSs can be divided into
homopolysaccharides, composed of a single type of
monosaccharide, and heteropolysaccharides, containing several
types of monosaccharide. Dextran is the most important
homopolysaccharide, which is a glucan produced, e.g. by
Leuconostoc mesenteroides. The heteropolysaccharides produced by
LAB are generally composed of repeating units of up to eight
monosaccharide residues; the chain length and degree of branching
vary with the producing strains. The rheological properties of the
polysaccharides depend on the monomeric composition, the
number of side chains, the chain length and the charge (neutral
or anionic) of the polysaccharides, as well as the anomeric
configuration of the monosaccharides and the sequence in which
they are arranged. The EPSs produced by LAB may act as
viscosifying agents to improve the texture and consistency of
fermented foods. Since LAB are food-grade microorganisms with
the GRAS status (Generally Recognized As Safe), the use of the
secreted EPSs as natural alternatives to produce all-natural food
products without additives has received increased attention. It
has also been claimed that EPSs isolated from LAB cultures have
antitumor activity.
Lactic acid bacteria are able to produce a large variety of
compounds which contribute to the flavor, color, texture and
consistency of fermented foods. However, the present study focuses
on two potentially important group of compounds, antimicrobial
compounds and EPSs, which differ largely in their chemistry and
functionalities. Attention has been paid to developing methods
suitable for separation and purification of LMM antimicrobial
compounds aiming at inhibition of food-borne spoilage bacteria.
In order to understand the relation between the structures and
rheological properties of the EPSs, a knowledge of their primary
molecular structures is required. In this study, the primary
molecular structures of the EPSs produced by Lb. helveticus strains
have been studied by NMR spectroscopy. The EPSs produced by
several slime-forming Lactococcus lactis ssp. cremoris strains have
been characterized to understand the roles of the EPSs in the
rheology of fermented foods.
182 Introductory Food Microbiology
Antimicrobial Compounds Produced by Lactic Acid Bacteria
Lactic acid bacteria are a physiologically diverse group of
organisms, which can be generally described as Gram-positive,
nonsporing cocci or rods with lactic acid as the major product of
carbohydrate fermentation. Traditionally, LAB comprise four genera
Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. However,
several new genera have been suggested for inclusion in the group
of LAB due to a recent taxonomic revision. The genus Streptococcus
has been reorganized into Enterococcus, Lactococcus, Streptococcus
and Vagococcus. LAB are involved in the fermentation of a range
of milk, meat, cereal and vegetable foods. The antimicrobial
compounds produced by LAB can inhibit the growth of pathogenic
bacteria of possible contaminants in the fermented products. In
the past two decades, there have been many reports on the
bacteriocins produced by LAB. These bacteriocins are of a
proteinaceous nature and they have been grouped into class I,
lantibiotics which are small peptides (e.g. nisin), class II, small
heat-stable peptides, class III, large heat-labile proteins, and class
IV, complex bacteriocins which are not well defined. In the
following text, the LMM antimicrobial compounds produced by
LAB will be discussed.
Organic Acids
Fermentation by LAB is characterized by the accumulation of
organic acids and the accompanying reduction in pH. The levels
and types of organic acids produced during the fermentation
process depend on the species of organisms, culture composition
and growth conditions. The antimicrobial effect of organic acids
lies in the reduction of pH, as well as the undissociated form of
the molecules. It has been proposed that the low external pH
causes acidification of the cell cytoplasm, while the undissociated
acid, being lipophilic, can diffuse passively across the membrane.
The undissociated acid acts by collapsing the electrochemical
proton gradient, or by altering the cell membrane permeability
which results in disruption of substrate transport systems.
Lactic acid is the major metabolite of LAB fermentation where
it is in equilibrium with its undissociated and dissociated forms,
and the extent of the dissociation depends on pH. At low pH, a
Epidemiological Typing of Bacillus spp Isolated from Food 183
large amount of lactic acid is in the undissociated form, and it is
toxic to many bacteria, fungi and yeasts. However, different
microorganisms vary considerably in their sensitivity to lactic
acid. At pH 5.0 lactic acid was inhibitory toward spore-forming
bacteria but was ineffective against yeasts and moulds. It was
possible to grow Aspergillus parasiticus NRRL 2999 in a medium
containing 0.5 or 0.75% lactic acid at pH 3.5 or 4.5. Lindgren and
Dobrogosz (1990) showed that at different pH ranges the minimum
inhibitory concentration (MIC) of the undissociated lactic acid
was different against Clostridium tyrobutyricum, Enterobacter sp.
and Propionibacterium freudenreichii ssp. shermanii. In addition, the
stereoisomers of lactic acid also differ in antimicrobial activity, L-
lactic acid being more inhibitory than the D-isomer.
Acetic and propionic acids produced by LAB strains through
heterofermentative pathways, may interact with cell membranes,
and cause intracellular acidification and protein denaturation.
They are more antimicrobially effective than lactic acid due to
their higher pKa values (lactic acid 3.08, acetic acid 4.75, and
propionic acid 4.87), and higher percent of undissociated acids
than lactic acid at a given pH. Acetic acid was more inhibitory
than lactic and citric acids toward Listeria monocytogenes. Acetic
acid also acted synergistically with lactic acid; lactic acid decreases
the pH of the medium, thereby increasing the toxicity of acetic
acid.
Hydrogen Peroxide and Carbon Dioxide
Hydrogen peroxide is produced by LAB in the presence of
oxygen as a result of the action of flavoprotein oxidases or
nicotinamide adenine hydroxy dinucleotide (NADH) peroxidase.
The antimicrobial effect of H2O2 may result from the oxidation of
sulfhydryl groups causing denaturing of a number of enzymes,
and from the peroxidation of membrane lipids thus the increased
membrane permeability. H2O2 may also be as a precursor for
the production of bactericidal free radicals such as superoxide
(O2 -) and hydroxyl (OH.) radicals which can damage DNA.
It has been reported that the production of H2O2 by Lactobacillus
and Lactococcus strains inhibited Staphylococcus aureus, Pseudomonas
sp. and various psychotrophic microorganisms in foods. In raw
184 Introductory Food Microbiology
milk, H2O2 activates the lactoperoxidase system, producing
hypothiocyanate (OSCN-), higher oxyacids (O2SCN- and
O3SCN-) and intermediate oxidation products that are inhibitory
to a wide spectrum of Gram-positive and Gram-negative bacteria.
Carbon dioxide is mainly produced by heterofermentative LAB.
The precise mechanism of its antimicrobial action is still unknown.
However, CO 2 may play a role in creating an anaerobic
environment which inhibits enzymatic decarboxylations, and the
accumulation of CO2 in the membrane lipid bilayer may cause a
dysfunction in permeability. CO2 can effectively inhibit the growth
of many food spoilage microorganisms, especially Gram-negative
psychrotrophic bacteria. The degree of inhibition by CO2 varies
considerably between the organisms. CO2 at 10% could lower the
total bacterial counts by 50%, and at 20-50% it had a strong
antifungal activity.
Aroma Components
Diacetyl is produced by strains within all genera of LAB by
citrate fermentation. The antimicrobial effect of diacetyl has been
known since the 1930s. It inhibits the growth of Gram-negative
bacteria by reacting with the arginine-binding protein, thus
affecting the arginine utilization.
Jay (1982) showed that Gram-negative bacteria were more
sensitive to diacetyl than Grampositive bacteria; the former was
inhibited by diacetyl at 200 ìg/mL and the latter at 300 ìg/mL.
Diacetyl at 344 ìg/mL inhibited strains of Listeria, Salmonella,
Yersinia, Escherichia coli, and Aeromonas. Since the production of
diacetyl during lactic fermentation is low, e.g. 4 ìg/mL produced
by Lc. lactis ssp. diacetylactis (Cogan 1980), and the acceptable
sensory levels of diacetyl are at 2-7 ìg/mL, its practical use as a
food preservative is limited. However, diacetyl may act
synergistically with other antimicrobial factors and contribute to
combined preservation systems in fermented foods.
Acetaldehyde is produced by Lb. delbrueckii ssp. bulgaricus by
the action of a threonine aldolase, which cleaves threonine into
acetaldehyde and glycine. Since Lb. delbrueckii ssp. bulgaricus and
S. thermophilus in yoghurt cannot metabolize acetaldehyde, it
accumulates in the product at a concentration of about 25 ppm.
Epidemiological Typing of Bacillus spp Isolated from Food 185
Acetaldehyde at 10-100 ppm inhibits the growth of Staphylococcus
aureus, Salmonella typhimurium and E. coli in dairy products.
Fatty Acids
Under certain conditions, some lactobacilli and lactococci
possessing lipolytic activities may produce significant amounts of
fatty acids, e.g. in dry fermented sausage and fermented milk. The
antimicrobial activity of fatty acids has been recognized for many
years. The unsaturated fatty acids are active against Gram-positive
bacteria, and the antifungal activity of fatty acids is dependent on
chain length, concentration, and pH of the medium. The
antimicrobial action of fatty acids has been thought to be due to
the undissociated molecule, not the anion, since pH had profound
effects on their activity, with a more rapid killing effect at lower
pH.
Reuterin and Other Low-molecular-mass Compounds
Reuterin is produced by Lb. reuteri, a heterofermentative species
inhabiting the gastrointestinal tract of humans and animals. It is
formed during the anaerobic growth of Lb. reuteri by the action of
glycerol dehydratase which catalyzes the conversion of glycerol
into reuterin. Reuterin has been chemically identified to be 3-
hydroxypropanal (â- hydroxypropionaldehyde), a highly soluble
pH-neutral compound which is in equilibrium with its hydrated
monomeric and cyclic dimeric forms. The biosynthesis pathway
from glycerol to the three forms of reuterin is shown below.
Methods for Evaluation of Antimicrobial Activity
Among many methods available for evaluation of antimicrobial
activity, the methods described below have been used for
determining the antimicrobial activity of compounds produced by
LAB.
The Agar Diffusion Method
The agar diffusion method has long been used for testing
antimicrobial activity, and it was first used by Fleming in 1924.
The method has been widely used for evaluation of antimicrobial
activity, specially for biologically derived compounds. It includes
agar well diffusion assay and disc assay. In this test, an
186 Introductory Food Microbiology
antimicrobial compound is applied to an agar plate on a paper
disc or in a well. The compound diffuses into agar resulting in a
concentration gradient that is inversely proportional to the distance
from the disc or well. The size of the inhibition zone around the
disc or well is a measure of the degree of inhibition. The results
of the test are generally qualitative. The method requires that the
indicator organisms must grow rapidly, uniformly, and aerobically.
Since highly hydrophobic antimicrobial compounds cannot diffuse
in agar, they are not suitable for tests by this method used an agar
well diffusion assay for testing the antimicrobial activity of Lb.
rhamnosus GG by addition of a 10-fold concentrate of the GG strain
or MRS broth to wells (diameter 4 mm) in agar against various
anaerobic and facultative bacteria. The activity of an antimicrobial
substance produced by Lb. delbrueckii ssp. bugaricus 7994 was
tested quantitatively with a disc assay procedure, using paper
assay discs 12.7 or 6.35 mm in diameter wetted with 30 or 10 ìl
of sample against Pseudomonas fragi and Staphylococcus aureus. The
assay methods used for determination of the antimicrobial activity
of different species of LAB were slightly different with respect to
the sizes of the wells, discs and samples, and the incubation
conditions were dependent on the indicator organisms used.
Several modified procedures based on the agar diffusion method
have also been used for testing antimicrobial activity of LAB.
These procedures include the agar spot test, deferred antagonism
assay, and spot-onlawn assay.
The Agar and Broth Dilution Methods
Agar and broth dilution methods are used as quantitative
methods, suitable for microorganisms with variable growth rate
and for anaerobic, microaerophilic microorganisms. The results
are expressed as MIC, which is the lowest concentration of an
antimicrobial that prevents growth of a microorganism after a
specific incubation period. In this test, an antimicrobial is serially
diluted and a single concentration added to a culture tube or plate
added with nonselective broth or melted agar medium, which is
then inoculated with test organisms and incubated. The MIC is
defined as the lowest concentration at which no growth occurs
(absence of turbidity) in a medium following incubation. The broth
dilution assay has been used for the determination of the
Epidemiological Typing of Bacillus spp Isolated from Food 187
antimicrobial activity of reuterin produced by Lb. reuteri, and the
activity of reuterin was expressed as MIC values or as the maximum
dilutions of the reuterin fraction.
The Automated Turbidometric Assay
A turbidometric assay based on automated systems determines
the effect of a compound on the growth or death kinetics of a
microorganism. It provides information concerning the effect of an
antimicrobial that may cause a delayed lag phase or reduced
growth rate at concentrations below the MIC. Since the bacterial
growth is monitored by measuring the turbidity of the broth
medium, the method demands that the instrument be highly
sensitive. Growth at levels below log 5.0 CFU/ml may not be
detectable. Skyttä and Mattila-Sandholm (1991) described a
quantitative method based on automated turbidometry for assaying
antimicrobial activity, which was expressed as area reduction
percentage values measured under the growth curve. The method
has been used to test the antimicrobial activity of antimicrobial
compounds produced by P. damnosus and P. pentosaceus and Lb.
plantarum.
Exopolysaccharides Produced by LAB
Lactic acid bacteria produce polysaccharides as cell wall
components and storage polymers, and also in many species, as
a capsule or slime. In the dairy industry, the slime-forming LAB
strains have traditionally been used in the production of fermented
milk products, e.g. yogurts, Finnish ‘viili’ and Scandinavian
‘långfil’. It has been generally acknowledged that the secreted
EPSs by LAB play an important role in the rheological behavior
and texture of the products.
Homopolysaccharides
Homopolysaccharides are a group of polysaccharides
composed of one monosaccharide type. Several species of LAB are
able to utilize sucrose as a specific substrate to produce dextrans,
mutans, and levans. Dextrans are a large class of extracellularly
formed glucans produced by the genus Lactobacillus, Leuconostoc,
and Streptococcus, of which Leuc. mesenteroides and Leuc. dextranicum
are the well-known dextran producers. Although each bacterial
188 Introductory Food Microbiology
strain produces a unique glucan, a common structural feature of
all dextrans is a high percentage (up to 95%) of á- 1,6 linkages
with a smaller proportion of á-1,2, á-1,3, or á-1,4 linkages resulting
in a highly branched molecule (Franz 1986). Dextrans are
synthesized outside the cell by dextransucrase, which catalyzes
sucrose to produce D-fructose and D-glucose, and transfers the
latter to an acceptor to form dextran. The reaction is as follows:
sucrose + glucan acceptor dextran or mutans + D-fructose Mutans
are synthesized in a similar way by S. mutans and S. sobrinus.
However, mutans differ from dextrans in containing a high
percentage of á-1,3 linkages, which are attributed to the insoluble
nature of this type of polymers. Some S. salivarius strains are able
to produce fructans of the levan type with 2,6-linked â-
fructofuranoside residues. An extracellular enzyme levansucrase
is involved in hydrolyzing sucrose and transferring D-fructose to
growing fructan chains to form levans: sucrose + fructan acceptor
levan + D-glucose
Another type of homopolysaccharide is the galactan produced
by Lc. lactis ssp. cremoris H414, which is composed of a branched
pentasaccharide repeating unit as shown below.
A Pediococcus strain produced a â-D-glucan with a trisaccharide
repeating unit. Lactobacillus spp. G-77 has been shown to produce
a 2-substituted- (1’!3)-â-D-glucan, identical to the EPS produced
by P. damnosus 2.6. Lactobacillus spp. G-77 also produced a á-D-
glucan composed of a trisaccharide repeating unit. Recently, van
Geel-Schutten et al. (1998) reported for the first time the production
of a fructan by Lb. reuteri strain LB121 with raffinose as a sugar
substrate; this strain also produced both a glucan and a fructan
on sucrose.
Heteropolysaccharides
A wide range of LAB strains can produce
heteropolysaccharides, which are composed of repeating units.
The monosaccharide compositions of these EPSs are mostly
galactose and glucose, and also small amounts of rhamnose,
fructose, mannose, and galactosamine. In comparison with the
homopolysaccharides, the production of heteropolysaccharides
by LAB is much lower (60 to 400 mg L-1). Generally, the
Epidemiological Typing of Bacillus spp Isolated from Food 189
heteropolysaccharides are synthesized intracellularly at the
cytoplasmic membrane utilizing sugar nucleotides as precursors
for the assembly of polysaccharide chains.
Lactobacillus
The ability of lactobacilli to produce EPSs has been recognized
for many years. In 1968, Kooiman first reported the structure of a
heteropolysaccharide produced by a Lb. brevis strain isolated from
kefir grains. This polysaccharide consists of a hexasaccharide
repeating unit with D-galactose and D-glucose in the molar ratio
1:1. In the last decade, a number of heteropolysaccharides produced
by the Lactobacillus species have been investigated.
Lb. helveticus strains produce several EPSs with varying
repeating units, though all containing galactose and glucose. The
EPS produced by Lb. helveticus 776 has hexasaccharide repeating
units containing D-galactose and D-glucose. The EPS produced by
Lb. helveticus TY1-2 consists of heptasaccharide repeating units
with Dgalactopyranosyl and D-glucopyranosyl, and 2-acetamido-
2-deoxy-D-glucopyranosyl residues. Recently, Stingele et al. (1997)
showed that the EPS produced by Lb. helveticus Lh59 had an
identical primary molecular structure as the one produced by Lb.
helveticus TN-4, a presumed spontaneous mutant of the strain
TY1-2. This polymer is composed of a tetrasaccharide backbone
with a lactosyl side-chain, and the molar ratio of D-galactose and
D-glucose is 1:1.
Robijn et al. (1995b) reported the primary molecular structure
of a viscous EPS produced by Lb. sake 0-1 which was isolated from
fermented meat products. The EPS consists of a pentasaccharide
repeating unit of glucose, rhamnose, and glycerol phosphate. The
threedimensional structure of this polymer has been further studied
by molecular mechanics calculations. The helics generated by a
polysaccharide builder program are highly extended, with either
2-fold or 3- or 4-fold right-handed chiralities. Grobben et al. (1997)
showed that Lb. delbrueckii ssp. bulgaricus NCFB 2772 produced an
EPS made up of galactose, and small quantities of glucose and
rhamnose, and another EPS that, according to Sikkema and Oba
(1998), was similar to the structure of the EPS produced by Lb.
delbrueckii ssp. bulgaricus rr. The enzymes involved in the production
190 Introductory Food Microbiology
of the sugar nucleotides of strain NCFB 2772 have been analyzed,
and based on this analysis a biosynthetic pathway for the EPS has
been proposed. Growth of the strain in a fructose-based medium
led to the absence of the enzyme activities for the synthesis of the
rhamnose nucleotide, and accordingly no rhamnose was present
in the polysaccharide produced. The EPSs produced by Lb.
acidophilus LMG9433, Lb. kefiranofaciens K1, and Lb. paracasei 34-1
have also been structurally evaluated, the repeating units being
pentamers, hexamers and tetramers, respectively. Recently, Lb.
rhamnosus strain C83 has been shown to produce an EPS composed
of a pentasaccharide repeating unit with a linear structure. This
strain, as well as Lb. casei CG11 and Lb. sake 0-1, produced more
EPSs at lower temperatures, whereas several other Lactobacillus
strains produced more EPSs at higher temperatures (compared
with the optimum temperatures of growth).
Lactococcus
Among lactococci, only the slime-forming Lc. lactis ssp. cremoris
strains have been investigated. These strains producing EPSs play
a role in the proper consistency of the fermented milk. The sugar
components of the EPSs are most frequently galactose, glucose,
and very often rhamnose. Nakajima et al. (1992a) reported a
phosphate-containing heteropolysaccharide, named ‘viilian’,
produced by Lc. lactis ssp. cremoris SBT 0495 which was isolated
from a Finnish ‘viili’ starter culture. The EPS consists of the
following repeating unit:
Lc. lactis ssp. cremoris B40 has also been found to produce a
phosphopolysaccharide with an identical repeating unit as shown
above. Lc. lactis ssp. cremoris strain LC330 appeared to produce
concurrently two EPSs; an anionic EPS composed of galactose,
glucose, rhamnose, glucosamine and phosphate, and a neutral
EPS containing galactose, glucose and glucosamine with branched
terminal galactose moieties. The mechanism for the biosynthesis
of the EPS by Lc. lactis ssp. cremoris has not been investigated in
detail. Recently, Oba et al. (1996) proposed a biosynthetic pathway
for the production of ‘viilian’ by strain SBT 0495 with the following
steps: preparation of the membraneembedded lipid carrier;
incorporation of the first monosaccharide with the phosphate on
C-1; assembly of the intact repeating unit.
Epidemiological Typing of Bacillus spp Isolated from Food 191
The biosynthesis of the EPSs produced by Lc. lactis strains is
generally associated with a plasmid. Transferring mucoidity
plasmids from Lc. lactis ssp. cremoris ARH87 and MS to nonmucoid
Lc. lactis strains proved the latter to be mucoid. van Kranenburg
et al. (1997) described a novel 12 kb EPS gene cluster located on
a 40 kb plasmid, which was essential for the EPS synthesis of Lc.
lactis ssp. cremoris NIZO B40. Introduction of the EPS gene cluster
from S. thermophilus Sfi6 to a non-EPS-producing Lc. lactis MG1363
produced an EPS with a different structure from the EPS of the
native host (Stingele et al. 1999). The absence of the GalNAc residue
in the EPS of Lc. lactis MG1363 was probably caused by the lack
of a UDP-N-acetylglucosamine C4- epimerase activity.
Streptococcus
S. thermophilus strains are used in combination with Lb.
delbrueckii ssp. bulgaricus strains in yoghurt starters. The EPSs
produced by several S. thermophilus strains have been found to
have similar or identical primary molecular structures. Doco et al.
(1990) first reported the structure of an EPS produced by ropy S.
thermophilus strains CNCMI 733, CNCMI 734 and CNCMI 735,
which consisted of a tetrasaccharide repeating unit of D-galactose,
D-glucose, and N-acetyl-D-galactosamine in a molar ratio 2:1:1.
An EPS with an identical repeating unit structure has been reported
to be produced by S. thermophilus Sfi6. Lemoine et al. (1997) showed
that the EPSs produced by S. thermophilus Sfi12 and Sfi39 had
molecular masses greater than 2 x 106, and both yielded a slimy
texture rather than a thickened one in yoghurt. However, they had
different sugar compositions and structures; the former consisting
of a hexasaccharide repeating unit of galactose, glucose and
rhamnose, and the latter a tetrasaccharide repeating unit of
galactose and glucose.
Recently, Faber et al. (1998) showed that S. thermophilus Rs
and Sts produced EPSs of identical repeating units, but they had
different molecular masses, resulting in a difference in viscosity in
their milk cultures. Bubb et al. (1997) also showed that the EPS
produced by S. thermophilus OR 901 had a similar repeating unit
to the one of the strains Rs and Sts; all being branched
heptasaccharide repeating units of D-galactose and L-rhamnose
in the same molar ratio: 5:2.
192 Introductory Food Microbiology
Isolation and Structural Elucidation of EPSs
Isolation of EPSs
EPSs produced by LAB can be isolated by precipitation with
trichloroacetic acid (TCA) to remove proteins from the culture
media, and subsequently by ethanol and/or acetone to precipitate
polysaccharides. Gel filtration or ion exchange chromatogrphy is
often used for further purification of EPSs. Robijn et al. (1995a,
1995b, 1996a, 1996b) purified EPSs produced by several
Lactobacillus strains by gel filtration with different columns (Sephacel
500, Sephacryl S-500, and Superrose-6).
Gel filtration techniques were also used for purification of the
EPSs produced by Lc. lactis ssp. cremoris H414, S. thermophilus
Sfi16, and S. thermophilus CNCMI 733. Since many EPSs are
negatively charged, they can be bound to an anion exchanger.
This technique has been used for purification of the anionic EPSs
produced by Lc. lactis ssp. cremoris B40, and Lb. helveticus TY1-2
and TN-4. Marshall et al. (1995) showed that Lc. lactis ssp. cremoris
strain LC330 produced at the same time both neutral and anionic
EPSs in the medium; these two EPSs were effectively separated
and purified by anion exchange chromatography.
Sugar and Methylation Analyses
In the analysis of monosaccharide compositions of EPSs
produced by LAB, the EPSs are hydrolyzed with trifluoroacetic
acid (TFA), reduced and acetylated, and the acetate derivatives are
analyzed with GC. For the methylation analysis, monosaccharides
are usually derivatized into partially methylated alditol acetates,
which are introduced into the EI source of MS from a GC, or GLC
interface.
The substitution pattern of the monosaccharides can be
determined by comparing their fragmentation pattern with
reference EI-MS spectra. Yamamoto et al. (1994, 1995) studied the
substitution pattern of the monosaccharides in the EPSs produced
Lb. helveticus TY1-2 and TN-4 by GLC-MS of the methylated and
acetylated sugar residues. The EPS produced by Lb. helveticus 766
was analyzed by GLC-MS on DB-1 of the partially methylated
alditol acetates.
Epidemiological Typing of Bacillus spp Isolated from Food 193
Nuclear Magnetic Resonance Spectroscopy
NMR spectroscopy relies on the interaction of radio-frequency
electromagnetic radiation with magnetically active nuclei in a
strong magnetic field. The radio frequencies used range from 200
to 800 MHz, corresponding to magnetic fields from 4.7 to 18.8
Tesla. 1H and 13C are the spin-active nuclei most frequently
encountered in carbohydrates. 1H and 13C NMR spectroscopy,
including one- (1D) and two-dimensional (2D), is a powerful tool
for structural studies of carbohydrates, which also include
polysaccharides produced by LAB. 1D 1H NMR spectroscopy can
be used for rapid identification or to check the purity of a
polysaccharide sample. Signals in the anomeric region (about 4.3-
5.5 ppm) of the spectrum and the coupling of the anomeric protons
(JH1,H2) may provide useful information about the number of
residues in a repeating unit, and the anomeric configuration,
respectively.
Yamamoto et al. (1995) recorded the 500 MHz 1H NMR
spectrum of the EPS produced by Lb. helveticus TN-4 in D2O at 70
oC, and found six signals in the anomeric region with nearly
equal integrated intensities, suggesting there was a hexasaccharide
repeating unit for this EPS. A study on the EPS produced by Lb.
helveticus Lh59, by 400 MHz 1H NMR spectroscopy in Me2SO-d6
at 80 oC produced four anomeric protons signals in a molar ratio
1:2:2:1, which also indicated a hexasaccharide repeating unit. The
1H NMR spectrum of the EPS produced by Lb. paracasei had four
doublets in the anomeric region, and the coupling constants
(JH1,H2) of these signals (8.3 Hz, 7.7 Hz, 7.3 Hz, 7.7 Hz) were of
the pyranoid ring form with all the residues in the â configuration.
Since the natural abundance of 13C is very low (1.1% relative
to 12C), the peak intensity of 13C has to be enhanced in 1D 13C
NMR spectroscopy by using a large number of pulses, by taking
advantage of the nuclear Overhauser effect (NOE), or by using
distortionless enhancement by polarization transfer (DEPT)
experiments. The values of 13C chemical shift and 13C-1H
coupling (1JC,H) provide structural information of the
polysaccharides. Robijn et al. (1995a) found six signals in the
anomeric region of the 13C NMR spectrum of the EPS produced
by Lb. helveticus 766, confirming the suggested hexasaccharide
194 Introductory Food Microbiology
repeating unit by 1H NMR spectroscopy. Based on the C-1 chemical
shifts in the 13C NMR spectrum of the EPS produced by Lb.
rhamnosus strain C83, Vanhaverbeke et al. (1998) assigned two
downfield signals (ä 110.12, 107.67) to two residues having â
configuration, and three signals at ä 99.73, 99.99 and 100.73 to
three residues having á configuration.
The 1D NMR techniques are often used for the assignment of
signals in the anomeric region. For detailed assignment for the
spin system of sugar residues, 2D techniques are needed. These
techniques include 1H,1H-correlated spectroscopy (COSY), total
correlation spectroscopy (TOCSY), homonuclear Hartmann-Hahn
spectroscopy (HOHAHA), gradient selected heteronuclear single
quantum coherence (gHSQC), gradient selected heteronuclear
multiple-bond correlation (gHMBC) experiment, and nuclear
Overhauser effect spectroscopy (NOESY). Bubb et al. (1997) used
a TOCSY experiment to further assign two signals of anomeric
protons in the 1H NMR spectrum of the EPS produced by S.
thermophilus OR 901. By means of 2D COSY, HOHAHA, NOESY,
and 13C-1H HMQC (heteronuclear multiple quantum coherence),
Robijn et al. (1996a) assigned almost all 1H and 13C resonances
in 1D 1H and 13C NMR spectra of the EPS produced by Lb.
paracasei 34-1. The sequence of the monosaccharide residues in a
repeating unit can be established by 2D NOESY and HMBC
experiments. The former experiment gives information about the
interresidue linkage from observation of the NOE between anomeric
protons and the protons at the substituted positions of
neighbouring sugar residues. The latter experiment gives rise to
crosspeak between proton and carbon atoms that are long-range
scalar coupled. Faber et al. (1998) used 2D NOESY together with
HSQC-NOE experiments to determine the sequence of the sugar
residues in the EPS produced by S. thermophilus Rs and Sts. The
monosaccharide sequence in the EPS produced by Lb. paracasei 34-
1 was established by 2D NOESY experiments.
Rheological Characterization of EPSs
Solution Viscosity
Viscosity ç is defined as the ratio between shear stress and
shear rate. The intrinsic viscosity [ç], which measures the
Epidemiological Typing of Bacillus spp Isolated from Food 195
hydrodynamic volume of a molecule, is obtained by extrapolating
the Huggins equation to zero concentration: çsp /c = [ç] + k’[ç]2c,
where çsp is specific viscosity, c is polymer concentration, and k’
is a constant for a series of polymers of different molecular mass
in a given solvent. çsp /c is also defined as the reduced viscosity
çred. For ionic polysaccharides in aqueous solutions, the value of
çred increases with decreasing concentration, showing a
polyelectrolyte effect. The behavior of the polyelectrolytes is
influenced by intrachain Coulombic interaction, ionic strength,
pH and specific counterions. Oba et al. (1999) suggested that in a
strain sweep test at very high dilution of the EPS produced by Lc.
lactic ssp. cremoris SBT 0495, the higher cross-over frequency of the
EPS in 0.1 M NaCl compared to that in pure water was due to the
polyelectrolyte effect of this EPS. van den Berg et al. (1995) showed
that over a wide range of shear rates, the viscosity of a 1% solution
of the EPS produced by Lb. sake 0-1 decreased with increasing
shear rates, indicating a shear-thinning behavior, and the viscosity
was comparable to that of xanthan gum.
Dynamic Viscoelasticity
In response to an applied stress, polysacharides may show a
viscoelastic behavior, i.e. a combination of truely viscous flow and
perfectly elastic response. In a dynamic test, the polysaccharide
sample is subject to sinusoidal shear oscillation with a wide range
of frequencies (0.01-300 Hz). The relative magnitudes of G’ (storage
modulus) and G” (loss modulus) vary with the state of the
polysaccharide. For entangled solutions, where there is a greater
contribution from the viscous element, G’ is low. When frequency
decreases, there is a crossover in G’ and G”, and they flow as high
viscosity liquids at very low frequencies. For gel systems, G’ and
G” are parallel, with G’ > G” and largely frequency independent.
Oba et al. (1999) showed that in a dynamic and steady shear
measurement the aqueous solutions of the EPS produced by Lc.
lactis ssp. cremoris SBT 0495 behaved as an entangled solution but
not as a weak gel. Nishinari (1997) reported the frequency (0.01
- 10 ù/radÅ”s-1) dependence of G’ and G” for 1-3% solutions of
gellan gum, an EPS produced by Pseudomonas elodea.
The 1% (0-30 °C) and 2% (30 °C) solutions had a typical dilute
solution behavior with G” > G’. The 2% (15 °C, 25 °C) and 3% (30
196 Introductory Food Microbiology
°C) solutions, however, had a concentrated solution behavior with
a crossover of G’ and G” and G’ > G” at higher frequencies.
Gelation occurs at 3% at 0-25 °C with G’ and G” being slightly
frequency dependent.
Gelling Properties
Gels are defined as loose three-dimensional networks with
structures ranging from homogeneous solutions (enthalphy-driven
aggregations) to heterogeneous rigid porous systems. Clark and
Farrer (1995) described the mechanisms of gel formation in three
main classes, firstly by point crosslinking with covalent bonds,
secondly by chain association driven by changes in temperature,
pH and ionic strength, and the presence of small molecules and
specific counterions, and thirdly by particle aggregation. Many
polysaccharide gels are formed by thermoreversable physical
associations, involving Coulombic, dipole-dipole, van der Waals,
charge transfer, and hydrophobic and hydrogen bonding
interactions, as well as double-helix formation and aggregation.
Gels can be characterized to be strong or weak based on their
response to deformation. At large deformations, strong gels will
rupture and fail, while weak gels flow without fracture, and show
recovery of solid character. Xanthan gum, the EPS produced by
Xanthomonas campestris, forms a weak gel, with large deformation
fluid properties, but it also forms strong gel under extreme
conditions.
Rheological behaviors of EPSs in Fermented Milk
The use of EPS-producing LAB strains may improve the
rheological properties of fermented milk. The gel structure and
viscosity of the products are affected by the gel formation
conditions, as well as the amount and the type of the EPSs
produced. Hammelehle et al. (1998) showed that fast warming
rates (20-50 °C) during acidification increased the gel firmness
and storage modulus, and decreased the syneresis of a milk gel.
Skim milk fermented by ropy EPS-producing strains exhibited
similar rheological properties, and had greater viscosity than skim
milk fermented by non-ropy strains. Ropy EPS-producing strains
also increased the viscosity of yoghurt when compared to yoghurt
made with non-ropy cultures, and improved the texture of quarg.
Epidemiological Typing of Bacillus spp Isolated from Food 197
As described ealier, S. thermophilus Rs and Sts produced EPSs of
the same structure, but had different viscosities in the milk cultures
due to their different molecular masses.
The rheological behavior of the polysaccharides is also related
to their three-dimensional structure. In addition to the viscosifying
effect of the polysaccharides, the interactions between the EPSs
and the milk proteins, e.g. caseins, also play a role. Studies of a
yogurt gel with a scanning electron microscopy showed that the
cells were attached to the protein coagulates by a network structure
consisting of polysaccharide filaments. The microorganisms and/
or the EPSs that they produce may affect the protein aggregation,
thereby affecting the physical properties of the milk gel. A recent
study showed that the rheological properties of stirred yoghurt
were affected by the type of EPSproducing strains used, suggesting
an effect due to the interaction between the polymer and milk
proteins. Hess et al. (1997) proposed a model for shear-induced
degradation of the microstructure of EPS-producing yogurt. Since
the associations of EPS with bacterial cells or casein micelles are
stronger than the associations between the casein micelles, an
increase in shear will first disrupt the casein micelle network that
is not associated with EPS, subsequentely the associations between
the cells and EPS, and then the portion of the casein network that
is associated with EPS.
Applications in Foods and Health Aspects
Antimicrobial Compounds as Natural Food Preservatives
The quality of most foods deteriorates during storage. In
addition to physical, chemical and enzymatic factors which may
alter the sensory characteristics, the microbiological changes in
foods may bring about a wide range of spoilage reactions, including
food poisoning. Therefore, it is of significance to inhibit the growth
of spoilage microorganisms in foods. Due to a strong demand for
natural and minimally processed foods, there has been a growing
interest in the use of antimicrobial compounds produced by LAB
as a safe and natural way of food preservation.
In addition to nisin which has been widely used in foods,
another antimicrobial compound that has been proposed for use
in food preservation is reuterin produced by Lb. reuteri. Addition
198 Introductory Food Microbiology
of reuterin to ground beef was found to inhibit the growth of E.
coli. Surface treatment of herring with a mixture of Lb. reuteri and
glycerol significantly improved the shelf-life of the product. Lb.
reuteri has been commercially used in combination with
Bifidobacterium infantis and Lb. acidophilus in sweet and fermented
milk under the trade name BRA-mjölk.
Antimicrobial compounds can be applied to foods either as
purified chemical agents, or as viable cultures in the case of
fermented products. Novel purified antimicrobial compounds
require data to substantiate their lack of toxicity in order to obtain
approval for their use in foods. Traditional fermented products
that naturally contain antimicrobial compounds have been
consumed for centuries, and starter cultures with selected
antimicrobial properties may be used to replace those used in
traditional fermented foods. However, problems may arise with
respect to retaining the flavor and texture of the products.
EPSs in Food Applications
Polysaccharides may function in foods as viscosifying agents,
stabilizers, emulsifiers, gelling agents, or water-binding agents.
The majority of the polysaccharides used in foods are of plant
origin. Most of them are chemically or enzymatically modified in
order to improve their rheological properties, e.g. cellulose, starch,
pectin, alginate and carrageenan. Therefore, their use is strongly
restricted. EPSs of microbial origin have unique rheological
properties because of their capability of forming very viscous
solutions at low concentrations and their pseudoplastic nature.
The EPSs produced by foodgrade LAB have been considered as a
new generation of food thickeners to improve the rheological
properties of foods. Dextran is the first industrial polysaccharide
produced by LAB. It was discovered in 1880 in sugar cane or beet
syrups where dextran was found to be responsible for the
thickening and gelation of the syrups. Due to their structural
differences, some dextrans are water soluble and others are
insoluble. Dextran can be used in confectionary to improve moisture
retention, viscosity and inhibit sugar crystallization. In gum and
jelly candies it acts as a gelling agents. In ice cream it acts as a
crystallization inhibitor, and in pudding mixes it provides the
desirable body and mouth feel. In addition, dextran has also been
Epidemiological Typing of Bacillus spp Isolated from Food 199
used as blood plasma extenders and as the basic component of
many chromatographic stationary phases.
Xanthan gum is the second microbial EPS which was approved
for use in foods in 1969. Although it is produced by the plant-
pathogen Xanthomonas campetris, Sutherland (1998) described
xanthan as the “benchmark” product with respect to its importance
in both food and nonfood applications, which include dairy
products, drinks, confectionary, dressing, bakery products, syrups
and pet foods, as well as the oil, pharmaceutical, cosmetic, paper,
paint and textile industries. The production of xanthan is relatively
inexpensive because of the high conversion of substrate (glucose)
to polymer (60-70%). According to Becker et al. (1998), xanthan in
solutions exhibits a high viscosity at low concentrations and strong
pseudoplasticity, and it is stable over a wide range of pHs,
temperatures and ionic strengths.
Another probiotic Lb. reuteri also produced an antimicrobial
compound with a wide spectrum of activities. Studies on bacterial
adhesion showed that capsular polysaccharide might promote the
adherence of bacteria to biological surfaces, thereby facilitating the
colonization of various ecological niches. The EPSs were found to
be present in adherent biofilms; the EPSs might function as initial
adhesion, and permanent adhesion compounds. As well as live
bacteria (probiotics) which can improve intestinal balance to
promote health, dietary carbohydrates may function as prebiotics,
beneficially affecting the colonic microflora. These dietary
carbohydrates include polysaccharides of plant origin (resistant
starch, â-glucan, cellulose, inulin), oligosaccharides (fructo-, gluco-
, malto-, xylo- and soybean oligosaccharides), and lactose
derivatives. There have been no reports of the use of EPSs produced
by LAB as prebiotics. Although milk fermented with an EPS-
producing strain Lc. lactis ssp. cremoris SBT0495 had cholesterol
lowering activity, the mechanism is unknown.
Oda et al. (1983) reported an antitumor EPS produced by Lb.
helveticus ssp. jugurti. The antitumor activity of the EPS was tested
against ascites Sarcoma-180 by injecting the EPS preparation
intraperitoneally. Mice given a 20 mg kg-1 dose for nine succesive
days had an increased life span value of 144%, and a value of
greater than 233% corresponding to a 40 or 80 mg kg-1 dose. The
200 Introductory Food Microbiology
authors concluded that the antitumor activity of the EPS might be
based on its host-mediated actions. In order to understand the
antitumor activity, the effect of the EPSs or the EPS-producing cells
on the immune system has been investigated. Forsén et al. (1987)
showed that cell surface materials, possibly lipoteichoic acids, of
Lc. lactis ssp. cremoris T5 produced Tcell mitogenic activity in
human lymphocytes. The slime produced by Bifidobacterium
adolescentis had immunomodifying effects on mouse splennocytes.
Kitazawa et al. (1992) showed that the slime-forming Lc. lactis ssp.
cremoris KVS20 had antitumor activity, and the slime contained
strong B-cell dependent mitogenic substances.
AIMS OF THE STUDY
One of the aims was to study the antimicrobial compounds
produced by dairy lactic acid bacteria, particularly the low-
molecular-mass compound inhibitory toward various spoilage and
pathogenic bacteria in foods. Another aim was to study the
extracellular polysaccharides produced by dairy lactic acid bacteria
in view of the role of the exopolysaccharides in the improvement
of the texture and consistency of fermented foods. The specific
aims of the study were:
1. To separate, purify and identify low-molecular-mass
antimicrobial compounds produced by the lactic acid
bacterial strains, and to study the antimicrobial properties
of these compounds.
2. To isolate exopolysaccharides produced by the lactic acid
bacterial strains, to evaluate the primary molecular
structures of the exopolysaccharides, and to study the
rheological properties of the viscous exopolysaccharides.
MATERIALS AND METHODS
Antimicrobial Compounds Produced by LAB (I, II)
Bacterial Strains and Growth Conditions
All bacterial strains used in the study of antimicrobial
compounds were obtained from Valio Ltd, Research and
Development Service, Helsinki, Finland. The bacterial cultures
were maintained at -80 °C in glass beads and they were subcultured
Epidemiological Typing of Bacillus spp Isolated from Food 201
twice before use. The LAB strains examined for producing
antimicrobial compounds were grown in MRS, KCA, and whey
permeate or whey media, and incubated at 30 °C or 37 °C. Food
spoilage bacteria and also LAB strains were used as indicator
organisms for antimicrobial tests.
Separation and Purification of Antimicrobial Compounds
After the growth of the LAB strains under proper conditions,
cells in the culture broth were filtered, and the cell-free broth was
concentrated 10-fold by lyophilization. The concentrate was then
precipitated stepwise by ethanol from 30 to 97.5% with intermediate
centrifugation (30 min, 22 000g, 4 °C). The precipitates obtained
from each addition of ethanol and/or the final supernatants
showing antimicrobial activity were further purified by
chromatographic methods.
Gel filtration was performed using a Bio-Rad Econo System.
The sample (100 mg) was loaded onto a column (75 x 1.5 cm) on
Bio-Gel P-2 polyacrylamide gel (M=100-1800, -400 mesh, Bio-Rad)
eluted with 0.05 M ammonium acetate (NH4OAc) at a flow rate of
10 ml h-1, and the eluant was monitored at 280 nm. The active
fractions were collected, lyophilized, and subjected to anion
exchange chromatography using a Bio-Rad Econo system with a
column (25 x 1.5 cm) on weakly basic Fractogel TSK DEAE-650(S)
gel. Elution was carried out at a flow rate of 1.0 ml min-1 using
a stepwise elution program: fractions 1-30 with water; fractions
31-65 with 0.04 M NH4OAc adjusted to pH 5.5 with acetic acid
(AcOH); fractions 66-90 with 0.5 M NH4OAc adjusted to pH 5.5
with AcOH.
A fraction was collected every four minutes with monitoring
at 254 nm. The active fractions (except fractions containing lactic
acid) from anion exchange chromatography were further purified
by RP-HPLC using a model 600 E multisolvent delivery system
equipped with a Baseline 810 software. The mobile phase, 0.02 M
NH4OAc containing 1% AcOH (pH 3.80), was used after filtration
through a membrane filter (pore size, 0.2 ìm). Elution was performed
isocratically from a Spherisorb S5 C8 column (250 x 4.6 mm, Phase
Separations Ltd, Chester, England), fitted with a C8 precolumn
(Millipore) at a flow rate of 0.75 ml min-1, and at 40 °C for 30 min.
202 Introductory Food Microbiology
A fraction was collected each minute. The absorbance was
monitored at a range of wavelengths from 190 to 300 nm at an
interval of 5 or 10 nm.
Identification of Antimicrobial Compounds
1H and 13C NMR measurements were carried out on a Bruker
AM 400 WB spectrometer (Karlsruhe, Germany), operating at 400.1
MHz for 1H. Spectra were recorded with sample solutions in
H2O/D2O (90/10) at ambient temperature and referenced to
sodium 3-trimethylsilyl- [2,2,3,3-2H4]propanoate.
The electron impact (EI) and fast atom bombardment (FAB)
mass spectra were recorded on a Jeol SX-102 double-focusing
spectrometer.
EI: The sample was injected into the direct probe and the
solvent (water) evaporated. The probe was inserted into the ion
source (250 °C). The filament was heated at a rate of 16 °C/min
up to 300 °C/min, the ionization current being 300 mA. The
ionization energy was 70 eV and the accelerating voltage 10 kV.
The spectra were recorded over the range 10-500 m/z. Calibration
was based on PFK (perfluorokerosin, positive ion mode).
FAB: The sample was introduced on the target plate directly
into the ion source (40 °C) in a glycerol matrix. The target was
bombarded with xenon atoms having a maximum of 6 kV energy.
The acceleration voltage of generated ions was 10 kV. The spectra
were recorded at a scan range of 0-800 m/z. Calibration was based
on solid CsI (cesium iodide, positive ion mode).
Antimicrobial assay
The agar diffusion method was performed using a disc test
and a spot test. The disc test was performed according to a
procedure developed by Pulusani et al. (1979) with some
modifications: 10 ml of the melted agar medium was seeded with
100 ìl of an 18 ± 2 h old broth culture of the test organism in a
sterile petri dish. When the soft agar had hardened, an antibiotic
test disc (diameter 6 mm, Schleicher & Schuell) was placed on the
agar surface, and 22 ìl of the sample was spotted onto the disc.
After incubation for 20 ± 2 h at the appropriate temperature for
each organism tested, the diameter of the inhibition zone around
Epidemiological Typing of Bacillus spp Isolated from Food 203
the disc was measured. The spot test was done by spotting the
liquid sample (3 ìl) directly onto the surface of the solidified,
seeded agar medium, and the diameter of the inhibition zone was
measured after incubation. Turbidometric assays were performed
using a Bioscreen C automated turbidometer equipped with a
Biolink software. The growth of indicator organisms in broth
(300 ìl) containing antimicrobial compounds was studied in plates
(100 wells). Each well was inoculated with 100 ìl broth culture
(grown overnight) of the test organism diluted to 106 to 107 CFU
ml-1. The optical density was measured automatically at 30 min-
interval, using a wideband filter (405-600 nm), and the plates were
shaken at 3 min-interval for 20 s. The growth curves were
determined from the turbidity data.
EPSs Produced by LAB (III, IV, V)
Bacterial Strains and Growth Conditions
The LAB strains examined for producing EPSs and their
growth conditions are shown in Table 4. The source and methods
of maintainance of these strains were the same as described above
for the LAB strains examined for producing antimicrobial
compounds in this study.
Isolation of EPSs
For the isolation of the EPS produced by Lb. helveticus Äki4
grown in MRS broth, bacterial cells were filtered from the medium,
and the cell-free supernatant was concentrated 10- fold by
lyophilization. The concentrate was fractionally precipitated with
ethanol from 40 to 95% with intermediate centrifugation. The
polysaccharide precipitated at 40% ethanol was washed, and
dissolved in water. After filtration through a syringe filter (0.8 ì/
0.2 ìl), it was freeze-dried. The crude polysaccharide (20 mg) was
purified by anion-exchange chromatography with a column (25 x
1.5 cm) on Fractogel TSK DEAE-650(S) gel using a Bio-Rad Econo
system. The column was eluted at about 60 ml h-1 first with water
for 80 min, and subsequently with 0.06 M NH4OAc adjusted to
pH 5.5 with AcOH for 120 min. A fraction was collected every
eight minutes with monitoring at 254 nm, and the presence of
sugar was tested with a Molish reagent.
204 Introductory Food Microbiology
For the isolation of the EPSs produced by other LAB strains
grown in milk or whey medium, proteins and cells were initially
precipitated by addition of 4% (w/v) TCA (Merck) to the culture,
and the mixture was stirred for 2 h. After centrifugation (35 min,
22 000 g, 4 °C), the supernatant was collected and filtered. Cold
ethanol was then gradually added to the cell-free supernatant
from one to two, and three volumes of the supernatant with
intermediate centrifugation. The EPS precipitated was washed
and dissolved in water. The aqueous solutions of EPS were filtered,
and then extensively dialyzed against water overnight at 4 °C
with two changes of water, and finally lyophilized.
The purity of the EPS material was examined by gel filtration
using a column (75 x 1.5 cm) of Bio-Gel P-30 polyacrylamide gel
(exclusion limit 40 000 daltons, 100-200 mesh). The sample (1 mg)
was loaded onto the column and eluted with 0.05 M NH4OAc
with UV monitoring at 280 nm. To check the ionic nature of the
EPSs, anionexchange chromatography of the EPS solutions
(~1 mg mL-1) was performed using a column (25 x 1.5 cm) of
weakly basic Fractogel TSK DEAE-650(S) gel. Elution was carried
out at 1.1 mL min-1; first with water for 2 h, and subsequently
with NH4OAc from 0.1 to 0.5 M using a linear increasing gradient.
Structural Elucidation of EPSs
GC analysis of alditol acetates was performed on a HP-5
fused silica column (0.20 mm x 25 m) using a temperature program
of 180 °C for 1 min followed by 3 °C min-1 to 250 °C. Hydrogen
was used as the carrier gas. The column was fitted to a Hewlett-
Packard model 5890 series II gas chromatograph equipped with
a flame ionization detector. GLC-MS analysis was performed on
a Hewlett-Packard model 5970 mass spectrometer equipped with
an HP-5MS fused silica column (0.2 mm x 25 m). A temperature
program of 170 °C for 3 min followed by 3 °C min-1 to 250 °C was
used with helium as the carrier gas.
In sugar analysis, the EPS samples were hydrolyzed with 2 M
TFA at 120 °C for 2 h. After reduction with sodium borohydride
(NaBH4) and acetylation, the samples were analyzed by GC. The
absolute configuration of the sugars present in the EPSs was
determined essentially as devised by Leontein et al. (1978) but
Epidemiological Typing of Bacillus spp Isolated from Food 205
with (+)-2-butanol. Methylation analysis was performed according
to Hakomori (1964) using sodium methylsulfinylmethanide and
iodomethane in dimethyl sulfoxide. The methylated compounds
were recovered by use of Sep-Pak C18 cartridges using the method
of Waeghe et al. (1983). The purified methylated sample was then
hydrolyzed (2 M TFA, 120 °C, 2 h), reduced, and acetylated.
The partially methylated alditol acetates were analyzed by
GLC-MS. NMR spectra of solutions in D2O were recorded at 65 °C
and pD 5.5, using a Jeol GSX- 270, Jeol Alpha-400, or Varian Inova
600 or 800 MHz instrument. Chemical shifts are reported in ppm
relative to sodium 3-trimethyl-(2,2,3,3-2H4)propanoate (äH 0.00)
or acetone (äc 31.00) as internal references, or dioxan as an external
reference. Data processing was performed using standard Jeol
software, VNMR software, or Felix 2.3 (Biosym/MSI, San Diego,
CA, USA). 1H,1H-COSY, relayed COSY, double-relayed COSY,
TOCSY, 13C,1H-COSY, gHSQC (Wilker et al. 1993) and HMBC
(Bax and Summers 1986) experiments were used to assign signals
and performed according to standard pulse sequences. For inter-
residue correlations, 2D NOESY experiments with mixing times of
300 and 400 ms (III), or 75 and 150 ms (IV), and HMBC experiments
with 60 and 90 ms (III), or 45 and 90 ms (IV) delays for the
evolution of long-range couplings were used.
Rheological Measurements of the EPSs Produced by Lc. lactis ssp.
Cremoris Strains
The viscosities of the dilute solutions of EPS at concentrations
of 0.01 up to 0.1 g dL-1 were measured at 25 °C with an Ubbelohde
capillary viscometer (536 13/Ic, SCHOTT35 GERÄTE, Hofheim,
Germany), which allows the determination of the flow times with
an accuracy of 0.03 s. The aqueous solutions of EPS with or
without the addition of salt were prepared by dissolving a
measured amount of EPS in 0.1 M sodium chloride (NaCl) solution
or in deionized water. Sample dilution to the various required
concentrations of EPS was done directly in the viscometer. After
about 5 min for temperature equilibration, flow times were taken,
and each flow time was reproduced six times. The reduced viscosity
and intrinsic viscosity of the EPS solutions were calculated from
the collected data.
206 Introductory Food Microbiology
The effect of temperature, pH and salts on the rheological
behavior of the EPS solutions (1%, w /v) was studied with a
Bohlin VOR rheometer (Bohlin Instruments Ltd, England) using
concentric cylinders (C14) with a gap of 0.5 mm between the
upper and lower geometries. The viscometry measurements were
performed at 5, 25, 40 or 60 °C, with increasing shear rates up to
291 s-1 in 29 steps. The pH of the EPS solutions was adjusted with
lactic acid to 4.0, 5.0 and 6.5. The EPS solutions containing salts
were prepared by addition of EPS to 0.1 M NaCl or 0.1 M calcium
chloride (CaCl2) solutions. The oscillation measurements were
performed for the EPS (1%, w/v) in aqueous and salt solutions,
and in skim milk at a frequency sweep from 0.01 to 15 Hz in 19
steps. For every temperature-dependent measurement a thermal
equilibration time of about 60 min was used.
RESULTS AND DISCUSSION
Antimicrobial Compounds Produced by LAB (I, II, unpublished
results)
Separation, Purification and Identification of Antimicrobial
Compounds
After ethanol precipitation of the cell-free cultures of the LAB
strains, the obtained precipitates and the final supernatants were
tested for antimicrobial activity. The supernatants containing
antimicrobial activity were subjected to chromatographic separation
and purification. The active fractions appeared in a relatively
narrow part of the chromatogram on gel-filtration on Bio-Gel P-2
of the supernatants. Anion exchange chromatography of these
active fractions resulted in two different ranges of active fractions
(52-54 and 58- 63), fractions 58-63 containing lactic acid. Further
purification by RP-HPLC of the fractions 52- 54 gave rise to one
major peak at retention time 4.96 min and two small peaks at 3.65
and 5.78 min. The absorption maximum of the antimicrobial
compound was at 215 nm. Fractions of these peaks were collected
and tested for antimicrobial activity. Only the fraction of the peak
at 4.96 min was found to contain antimicrobial activivity.
Identification of this active fraction by both NMR (1H and 13C)
and MS (EI and FAB) spectra indicated that the antimicrobial
compound was 2-pyrrolidone-5-carboxylic acid (PCA), also known
Epidemiological Typing of Bacillus spp Isolated from Food 207
as pyroglutamic acid. Among the twenty LAB strains examined,
thirteen Lactobacillus and five Pediococcus strains were found to
produce PCA under the growth conditions used in this study.
Four Lactobacillus strains produced, in addition to PCA, also HMM
antimicrobial compounds, as indicated by the presence of
antimicrobial activity in the precipitates obtained from ethanol
precipitation. Since these HMM compounds exhibited a rather
narrow range of activity they were not subjected to further studies.
On the basis of the results of this study, it seems that many LAB
strains, particularly Lactobacillus strains, are able to produce PCA.
Since LAB produce relatively large amounts of lactic acid,
being antimicrobially active, it is important to remove the lactic
acid in order to find other antimicrobial compounds, especially
those of low molecular mass. In the separation and purification
procedures developed in this study, both PCA and lactic acid
were present in the culture supernatant obtained from ethanol
precipitation, and they also appeared in almost the same range of
fractions after gel filtration. However, complete separation of lactic
acid was achieved by anion exchange chromatography based on
a gel matrix (Fractogel TSK) suitable for separation of biomolecules.
Previously, size exclusion HPLC was used to separate lactic acid
from LMM antimicrobials, but all the fractions obtained were
found to contain antimicrobial activity because the mobile phase
(sodium phosphate) used was antimicrobially active.
Niku-Paavola et al. (1999) reported the separation of lactic
acid by gel filtration on Sephadex G-10 with water as an eluent
and found several LMM antimicrobial compounds produced by
Lb. plantarum. Although there were some reports on the separation
and purification of LMM antimicrobial compounds produced by
LAB, the techniques for seperation of lactic acid appeared not to
be clearly demonstrated. In addition, there were reports on the use
of a neutralization technique to eliminate the antimicrobial effect
of lactic acid, but lactic acid could not be separated with this
technique, and the activity of acidic antimicrobials might be
suppressed due to neutralization. PCA is a natural constituent of
foods of plant origin, including vegetables and fruits, and
fermented soybean and cereal products. Among LAB strains, only
S. bovis has previously been shown to produce PCA by conversion
208 Introductory Food Microbiology
of glutamine. PCA can also be synthesized by heating glutamic
acid using a dehydration process. It has been reported that PCA
is able to increase cerebral blood flow and decrease the resistance
of brain vessels, which result in enhanced brain metabolism, i.e.
increased glucose uptake and utilization by cerebral tissues and
decreased brain lactate dehydrogenase activity. Other biological
functions of PCA are related to its presence as an amino-terminal
residue in many biologically significant peptides and proteins,
e.g., eisenine, LH-RH (luteinizing hormone releasing hormone),
and TRH (thyrotropin releasing hormone).
The Antimicrobial Activity of 2-pyrrolidone-5-carboxylic Acid
Produced by LAB
Although LAB are able to produce a variety of antimicrobial
compounds, the present study, for the first time, reports that the
production of a certain cyclic amino acid (PCA) is involved in the
antimicrobial action of LAB. PCA has been considered, following
the identification of reuterin, to be another well identified LMM
antimicrobial compound produced by LAB.
The antimicrobial assay of PCA by agar diffusion method
showed that PCA at 2% inhibited Bacillus subtilis, E. coli, Enterobacter,
Klebsiella, and Pseudomonas strains. The most sensitive strains
Enterobacter cloacae 1575, Pseudomonas fluorescens KJL G, and
Pseudomonas putida 1560-2 were inhibited by PCA at 0.5% and
1.0%. Among all bacterial strains tested, the Gram-positive strains
such as LAB strains, and several Listeria and Staphylococcus strains
were not inhibited by PCA. An Enterococcus faecalis strain was
moderately inhibited by 0.5% PCA in BHI broth at 37 °C. It seemed
that Gramnegative bacteria were more sensitive to PCA than the
Gram-positive ones. This is in agreement with previous studies
which showed that organic acids (lactic and acetic acids) were
more inhibitory toward Gram-negative bacteria than Gram-positive
ones. PCA was heat stable and the antimicrobial activity did not
change after heat treatments (63 °C 30 min, 72 °C 15 s or 121 °C
20 min). Although raising the pH of the PCA in water solutions
reduced the antimicrobial activity, and completely destroyed the
activity at pH 3.8-4.0, the antimicrobial activity of PCA seemed to
be not solely due to the pH effect, but other factors may be possibly
involved. The turbidometric assay of antimicrobial activity showed
Epidemiological Typing of Bacillus spp Isolated from Food 209
that PCA in broths was inhibitory against the test organisms at
pH 5.0-5.89. At pH values when 1% PCA showed no activity, 2%
PCA was still active. Earlier studies showed that neutralization
caused the loss of the antimicrobial activity of Lb. acidophilus, since
many inhibitory substances produced by lactic cultures were
relative stable in an acidic pH. In addition, the antimicrobial
activity of PCA, like other organic acids, may also be dependent
on the undissociated form of the acid.
PCA was found to be less inhibitory than lactic acid. At the
same concentrations, lactic acid was more active than PCA against
several indicator organisms tested. During the course of this study,
we also observed that the production of PCA varied with strains
and it was generally small when compared with the amount of
lactic acid produced. However, the significance of PCA is that, in
addition to its wide spectrum of antimicrobial activity, it is also
involved in many important biological functions as discussed
above. Among the PCA-producing LAB strains of this study, Lb.
rhmnosus GG was found to produce relative large amounts of PCA.
Lb. rhamnosus GG has also been reported to produce a microcin-
like LMM antimicrobial compound, and the strain has been
commercially applied in the production of probiotic milk products,
e.g. Gefilus in Finland. Considering the potential applications of
PCA, e.g. as PCA-producing starters in food preservation, further
studies are needed on the optimal production of PCA by LAB
strains, the antimicrobial effects of PCA in foods and the effective
PCA concentrations, as well as the sensory quality of foods when
PCA is used.
EPSs Produced by LAB
Of the thirteen LAB strains examined, except for Lb. fermentum
G.1.2.1, Lb. rhamnosus LC705 and Lc. lactis ssp. cremoris SEPH 11
which did not produce EPSs, ten strains produced neutral or
anionic EPSs of varying amounts when they were grown under
the conditions of this study.
Previous studies have shown that the production of EPS is
growth-associated, and it is influenced by medium compositions,
culture temperature and pH. Gamar et al. (1997) showed that
variations in carbon sources in the growth medium resulted in
210 Introductory Food Microbiology
different yields of the EPS produced by Lb. rhamnosus strain C83.
Addition of glucose or sucrose to the milk medium stimulated the
EPS production and modified the sugar composition of the EPS
produced by Lb. casei ssp. casei NCIB 4114.
Several authors have also reported that more EPSs could be
produced by different LAB strains at lower temperatures, or with
limited nitrogen sources. Although bacteria grow well, and more
cells may be obtained under optimized growth conditions,
unfavourable culture conditions may stimulate EPS production by
the cells as a form of bacterial self-protection. Therefore, these
factors discussed above need to be considered for optimizing EPS
production by the LAB strains in this study.
EPSs Produced by Lb. Helveticus Strains
Lb. helveticus Äki4 grown in MRS broth, and strains Lb161
and K16 grown in skim milk were found to produce EPSs.
Viscometric measurements by Ubbelohdetype capillary viscometer
showed that at 0.5% (w/v) EPS in aqueous solutions, the EPSs of
strains Lb161 and K16 were viscous, but the EPS of strain Äki was
not viscous. The primary molecular structures of the EPSs produced
by strains Äki4 and Lb161 have been studied by sugar and
methylation analyses, and 1D and 2D NMR spectroscopy.
Structural Elucidation of the EPSs Produced by Lb. Helveticus Aki4
(III) and Lb161 (IV)
The EPS produced by strain Äki4 was shown to be composed
of a hexasaccharide repeating unit containing one terminal â-D-
galactose, one 4- substituted á-D-galactose, one 6-substituted â-D-
galactose, one 4-substituted â-D-glucose, one 3,6-substituted â-D-
galactose and one 6-substituted â-D-glucose residue. The
assignment of the spin systems for the six sugar residues was
performed using 2D homo- and heteronuclear techniques. Each
spin system with a specific sugar residue and substitution pattern
was identified from the JH,H values, indicating the anomeric
configuration, and from the 1H and 13C NMR chemical shifts.
The sequence of the sugar residues was determined using 2D
NOESY and HMBC experiments. The structure of the repeating
unit of the EPS is as follows:
Epidemiological Typing of Bacillus spp Isolated from Food 211
The EPS produced by strain Lb161 was shown to be composed
of a heptasaccharide repeating unit containing two terminal â-D-
glucose, one 2,3-substituted á-D-glucose, one 3- substituted á-D-
galactose, one 4-substituted á-D-glucose, one 3,4-substituted â-D-
galactose and one 3-substituted â-D-glucose residue. The
assignments of the spin systems for the seven sugar residues were
performed using 2D homo- and heteronuclear techniques. Each
spin system with a specific sugar residue and substitution pattern
was identified on the basis of the JH,H values and the 1H and 13C
NMR chemical shifts. The sequence of the repeating unit was
established using 2D NOESY and HMBC experiments with the
following structure:
Structural Variations Among the EPSs Produced by Lb. Helveticus
Strains
As shown above, the repeating units of the EPSs of Lb helveticus
Äki4 and Lb161 are a hexamer and a heptamer made up of a main
chain branched by one and two side chains, respectively. Structural
studies on the EPS of strain K16 showed that the repeating unit
of the EPS was composed of six sugars: one terminal D-galactose,
one terminal D-glucose, one 4- substituted D-galactose, one 4-
substituted D-glucose, one 2,4-substituted D-glucose and one 4,6-
substituted D-glucose.
Comparing with the so far published structures of the EPSs of
other four Lb. helveticus strains, the common structural feature of
the EPSs produced by all these Lb. helveticus strains is that the
EPSs contain either a hexa- or heptasaccharide repeating unit of
D-galactose and D-glucose. The EPS produced by Lb. helveticus
strain TY1-2 contains N-acetyl-D-glucosamine in addition to D-
galactose and D-glucose. Rhamnose and other monosaccharides
which have been found in the EPSs produced by many LAB
strains are not present in the EPSs of the Lb. helveticus strains.
Since the rheological properties of polysaccharides are closely
related to their primary molecular structures and threedimensional
structures, it would be of interest to study the relation between
structures and functional properties of the EPSs produced by LAB
strains within the same species. The difference in the viscosities
of the EPSs of the Lb. helveticus strains of this study is probably
due to their different primary molecular structures (linkage and
212 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 213

degree of branching), molecular masses, three-dimensional lowered in the order of pH 6.5 > pH 5.0 > pH 4.0. The higher
structures and chain-chain interactions of the polysaccharides in viscosity of EPS in 0.1 M CaCl2 solutions than that in 0.1 M NaCl
solutions. solutions is due to different interactions between cations and
polyelectrolyte chains, Ca2+ being more effective than Na+ in this
EPSs Produced by Lc. Lactis ssp. Cremoris Strains and their
respect. Changing Na+ into Ca2+ ion was observed also to increase
Rheological Characterization (V)
the storage modulus of the sample. Samain et al. (1997) compared
The growth of Lc. lactis ssp. cremoris strains (ARH53, ARH74, the effect of salts on the viscosity of a gel-forming EPS produced
ARH84, ARH87, B40) in skim milk at 25 °C for 18-20 h resulted by Alteromonas sp. strain 1644 and showed that the divalent
in very viscous cultures as compared to the Lb. helveticus strains magnesium cation was more effective than the monovalent sodium
of this study. The EPSs (164-263 mg L-1) produced by these ‘viili’ cation.
strains were separated and purified from the cultures by treatment
The aqueous solutions of the EPS (1%, w/v) of strain ARH53
with 4% TCA and ethanol precipitation. The EPSs were shown to
behave viscoelastically. As shown in the oscillation measurements
be anionic in nature by anion exchange chromatography, and
at 5, 25 and 40 °C, the EPS solutions behaved as a viscoelastic
they contained the same monosaccharides rhamnose, glucose and
fluid at lower frequencies with G” > G’, and showed a predominant
galactose in similar molar ratios. The combined evidence from
elastic character (G’ > G”) at higher frequencies. The drastic decrease
sugar analysis and NMR spectroscopy indicates that the primary
in viscosity of the EPS aqueous solution with increasing shear rate
molecular structures of these EPSs are identical or closely related
also clearly shows the non-Newtonian behavior (shear-thinning)
to that from Lc. lactic ssp. cremoris SBT 0495. The underestimation
of the solution.
of the ratio of galactose resulted from incomplete release by acid
hydrolysis. The galactose residues were involved in the In skim milk to which the EPS of strain ARH53 was added,
phophodiester linkage in the polysaccharide and it was hard to the viscosity was much higher as compared with the EPS in
release them. aqueous solutions at 5, 25 and 40 °C. At shear rates lower than
9.21 s-1, an upward shift in viscosity was observed at 40 °C. The
In dilute aqueous solutions (up to 0.1 g dL-1), the EPSs
viscosity was higher at 40 °C than at 25 °C, and even higher than
produced by Lc. lactis ssp. cremoris strains exhibited a polyelectrolyte
at 5 °C at shear rates lower than 0.37 s-1. In the oscillation
effect. At very low concentrations ( 0.02 g dL-1), an increase in
measurements, at 5 °C there is a moderate frequency dependence
reduced viscosity with a decrease in concentration was observed.
of G’ with a crossover of G’ and G” at the lower end of the
In the presence of 0.1 M NaCl, the reduced viscosities of all the
frequency range, suggesting the formation of a weak gel. At 25 °C
EPS solutions were significantly lowered, and the polyelectrolyte
the system exhibited a viscoelastic behavior with a crossover of G’
effect disappeared as indicated by the straight lines of the Huggins
and G” at about 0.8 Hz. Increasing temperature to 40 °C led to the
plot. The intrinsic viscosities of the EPSs were therefore obtained
formation of a strong gel, as indicated by G’ » G”, and the parallel
by a linear extrapolation of the plots to zero EPS concentration.
curves of G’ and G” with only a slight frequency dependence, and
Since the EPS produced by strain ARH53 gave the highest intrinsic
the value of G’ being 198 Pascals at 15 Hz. The interactions
viscosity (19.62 dL g-1), this EPS might possess a relatively large
between EPS and casein micelles of skim milk were possibly
molecular size than the other Lc. lactis ssp. cremoris strains studied.
involved in the gelation. Hess et al. (1997) proposed that in yoghurt
At a higher concentration (1%, w/v), the viscosities of the aqueous
the interactions between EPS and casein micelles are stronger
solutions of EPS produced by strain ARH53 were temperature, pH
than those between the casein micelles. Intercalation of the EPSs
and salt dependent. At the same pH (4.0, 5.0 or 6.5), increasing
into the casein matrix was also involved in the improvement of the
temperature from 5 °C to 60 °C caused a decrease in viscosity. At
texture of quarg using ropy cultures. It seemed that the interactions
higher temperatures (40 °C and 60 °C), the viscosity was clearly
214 Introductory Food Microbiology
between the EPS and casein micelles increased at higher
temperatures as observed in this study.
The production of EPSs by Lc. lactic ssp. cremoris strains has
been reported earlier. However, the rheological properties of these
EPSs were poorly understood. Recently, Oba et al. (1999) reported
the viscoelastic properties of the EPS produced by strain SBT 0495.
It is of interest that the rheological behavior of this EPS in aqueous
solutions, e.g. polyelectrolyte effects and viscoelasticity, is similar
to that of the EPS of strain ARH53. This is propably due to their
similar or identical structures as shown in this study. The similarity
in rheological behavior was also noticed among the EPSs produced
by the five ‘viili’ strains of this study. The difference in viscosity
of the EPSs in dilute, as well as in concentrated solutions was
possibly due to their different molecular masses.
It has previously been known that slime production is an
important factor contributing to the special slimy characteristic of
Finnish fermented milk ‘viili’. The increase in viscosity and gelling
at 40 °C resulted from the addition of the EPS produced by Lc.
lactis ssp. cremoris demonstrated in this study is of interest with
respect to the possible use of the EPS to improve the rheological
properties of milk products, for instance, yoghurts with
fermentation at about 42 °C. Considering the potential applications
of the EPSs, future work on the optimization of the EPS production
and further physical characterization is required.
SUMMARY AND CONCLUSIONS
Lactic acid bacteria are able to produce a large variety of
compounds which give fermented foods their characteristic flavor
and color, and also impart improved safety and rheology to the
foods. In this study, the production of antimicrobial compounds
and extracellular polysaccharides by LAB strains obtained from
the dairy industry has been investigated in order to find the LAB
strains with potential applications in foods.
Studies on the twenty dairy LAB strains from Lactobacillus,
Lactococcus, Pediococcus and Streptococcus showed that eighteen
strains produced a LMM antimicrobial compound, 2-pyrrolidone-
5-carboxylic acid (PCA). Separation and purification of PCA from
the growth media were achieved by ethanol precipitation and
Epidemiological Typing of Bacillus spp Isolated from Food 215
chromatographic methods. The technique of anion exchange
chromatography developed in this study was essential for effective
separation of PCA from lactic acid, facilitating the identification
of PCA by NMR and mass spectrometry. The chromatographic
procedure based on this technique can be used for separation and
purification of other LMM antimicrobial compounds. To our
knowledge, this is the first report of a cyclic amino acid, PCA,
which is involved in the antimicrobial action of LAB. PCA can be
considered as a well identified LMM antimicrobial compound
following the identification of reuterin produced by Lb.reuteri.
PCA was shown to be inhibitory toward many food-borne
spoilage bacteria such as Bacillus subtilis, Enterobacter, E. coli,
Klebsiella and Pseudomonas. In antimicrobial tests against different
organisms, the Gram-negative spoilage bacteria were found to be
more sensitive to PCA than the Gram-positive ones. Heat treatment
of PCA did not change its antimicrobial activity. Although PCA
was active under acidic conditions, it appeared that the activity
was not solely due to the pH effect. Regarding the mechanism of
antimicrobial action of PCA, more study is needed on the mode of
action of PCA on sensitive bacterial cells and the MIC value, as
well as the biosynthesis of PCA in different species of LAB. In
addition, further investigation on a wide range of LAB strains is
needed in order to find out whether production of PCA is a
common characteristic of LAB.
During the course of the studies on the antimicrobial
compounds, we gradually precipitated HMM antimicrobial
compounds from culture supernatants by ethanol, while at the
same time following how polysaccharides were precipitated. The
first EPS, which was precipitated at 40-60% ethanol, was found
to be produced by Lb. helveticus Äki4 grown in MRS broth. NMR
spectroscopic studies of the EPS showed that it was composed of
a hexasaccharide repeating unit of D-galactose and D-glucose in
a molar ratio of 2:1, and the main chain was branched with a side
chain of D-galactose. Although there were relatively large amounts
of the EPS (~500 mg L-1) produced by Lb. helveticus Äki4, this EPS
was found to be not viscous.
In the screening study of LAB strains producing viscous EPSs,
another Lb. helveticus strain, strain Lb161 grown in skim milk was
216 Introductory Food Microbiology
found to produce a viscous EPS. The EPS was isolated by the
treatment of the growth medium with TCA to precipitate the
proteins first, and subsequently by ethanol precipitation to obtain
the polysaccharide. The primary molecular structure of the EPS
has been elucidated by NMR spectroscopy. The EPS was shown
to consist of a heptasaccharide repeating unit of D-galactose and
D-glucose in a molar ratio 2:5 with two side chains, each consisting
of a D-glucose residue.
Further studies on slime-forming Lc. lactis ssp. cremoris strains
showed that growth of the strains ARH53, ARH74, ARH84, ARH87
or B30 in skim milk resulted in very slimy cultures. By using
basically the same methods as for the EPS of Lb. helveticus Lb161,
polysacharides of varying amounts were isolated from these
cultures. Sugar analysis and NMR spectroscopy showed that the
EPSs had a rather similar or probably identical structure to the
one reported earlier. Rheological studies showed that the EPSs in
dilute aqueous solutions behaved as polyelectrolytes, and the EPS
of strain ARH53 gave the highest intrinsic viscosity. Further
characterization of the EPS of strain ARH53 showed that the
viscosity of the EPS in concentrated solutions was dependent on
the temperature, pH and ionic strength of the solutions. In skim
milk, addition of the EPS of strain ARH53 resulted in a clear
increase in viscosity and a gel was formed at 40 °C.
In our future studies of EPSs, we have planned to continue the
study of the EPSs produced by LAB, as well as bifidobacterial
strains of food origins, and strains from the human intestine,
aiming at applications in food, functional food or clinical products.
Although the present study provides data on the structures and
rheological properties of EPSs, further studies are needed in order
to understand the structure-function relations of the EPSs, the
interaction of polysaccharides and proteins (e.g. casein), and the
roles of EPSs in their adhesion interaction with EPS-producing
strains in the human intestine.
Modelling the Growth Boundary of Staphylococcus Aureus 217
8
MODELLING THE GROWTH
BOUNDARY OF STAPHYLOCOCCUS
AUREUS
Knowing the precise boundary for growth of Staphylococcus
aureus is critical for food safety risk assessment, especially in the
formulation of safe, shelf-stable foods with intermediate relative
humidity (RH) values. To date, most studies and resulting models
have led to the presumption that S. aureus is osmotolerant.
However, most studies and resulting models have focused on
growth kinetics using NaCl as the humectant. In this study, glycerol
was used to investigate the effects of a glassforming nonionic
humectant to avoid speci? c metabolic aspects of membrane ion
transport. The experiments were designed to produce a growth
boundary model as a tool for risk assessment.
The statistical effects and interactions of RH (84 to 95%
adjusted by glycerol), initial pH (4.5 to 7.0 adjusted by HCl), and
potassium sorbate (0, 500, or 1,000 ppm) or calcium propionate (0,
500, or 1,000 ppm) on the aerobic growth of a ? ve-strain S. aureus
cocktail in brain heart infusion broth were explored. Inoculated
broths were distributed into microtiter plates and incubated at
37°C over appropriate saturated salt slurries to maintain RH.
Growth was monitored by turbidity during a 24-week period.
Toxin production was explored by enterotoxin assay. The 1,280
generated data points were analyzed by SAS LIFEREG procedures,
which showed all studied parameters significantly affected the
growth responses of S. aureus with interactions between RH and
pH. The resulting growth/no growth boundary is presented.
218 Introductory Food Microbiology
INTRODUCTION
Staphylococcus aureus is one of the leading causes of foodborne
illness and is ranked as one of the most prevalent causes of
gastroenteritis worldwide, even though the occurrences of this
foodborne disease are grossly underreported. S. aureus was
determined to be the etiological agent in 367 (19.6%) of 1,869
documented bacterial foodborne outbreaks in the United States.
Approximately 25 major outbreaks of Staphylococcus food poisoning
occur annually in the United States. S. aureus has been estimated
by the Centers for Disease Control and Prevention to cause 185,060
illnesses, 1,753 hospitalizations, and 2 deaths per year in the
United States, all of which are via consumption of contaminated
foods.
Because of its prevalence as a food poisoning organism, S.
aureus has been extensively studied to determine the physical and
chemical parameters that affect its growth and toxin formation. S.
aureus is ubiquitous to the mucous membranes and skin of warm-
blooded animals. It is a poor competitor with other bacteria and
is easily destroyed by cooking temperatures; however, its toxins
can survive heat processing equivalent to that given to low-acid
canned foods. Staphylococcal food poisoning frequently occurs
when food is contaminated after cooking by a person carrying the
organism, and subsequently the food is temperature abused for
several hours, which allows for toxin production before
consumption. It is the ingestion of the toxin that causes the
foodborne disease.
S. aureus is highly salt tolerant and has been reported to grow
at relative humidities (RHs) as low as 85% in NaCl concentrations
up to 25% (wt/wt) (11). The RHs limiting growth are typically
higher when humectants other than NaCl are used to control RH
(11). Notermans and Heuvelman (19) reported that at a water
activity of 0.98 to 0.93, sucrose was more favorable for growth
than NaCl, but at a water activity of 0.87, NaCl was more favorable.
Marshall et al. reported that glycerol inhibition of the growth rate
of S. aureus was about 10% greater than that caused by NaCl at
water activity levels between 0.96 and 0.90. Empirical
microbiological models can be designed to predict how
microorganisms relate to the environment.
Modelling the Growth Boundary of Staphylococcus Aureus 219
Predictive models are typically broadly applied for the
screening of products and/or processes before challenge or shelflife
studies. However, it must be remembered that models are only
tools to be used to help design products with food safety concerns
addressed at the beginning of the development process and that
there must be validation of the system through challenge or shelf-
life studies with the real product.
Both the U.S. Department of Agriculture Pathogen Modeling
Program (PMP) and the Food MicroModel (FMM) developed in the
United Kingdom through the Ministry of Agriculture, Fisheries
and Food include S. aureus growth kinetic models. The PMP models
the effects of temperature, initial pH, NaCl concentration, and
sodium nitrite concentration in a broth system on aerobic and
anaerobic growth kinetics of the organism.
The FMM models the growth responses as affected by NaCl
concentration, pH, and storage temperature in a model broth
system. The PMP also includes a model for the survival of S. aureus
in nongrowth conditions, which was developed from a model
broth system that includes the effects and interactions of pH
controlled by lactate buffer, lactic acid concentration, NaCl
concentration, sodium nitrite concentration, and varying
temperatures.
For all of the above-mentionedmodels, NaCl was the humectant
chosen to control the RH in the system, and the resulting models
are kinetic in nature and, therefore, do not allow the boundary for
growth/no growth to be estimated. In contrast to kinetic modeling,
probability modeling focuses on determining if the microorganism
of concern will or will not grow, in other words, determining the
growth/no growth interface.
This becomes increasingly important when pathogens are of
concern, because the rate of growth may be less important than the
fact that the organism is present and may be able to grow to an
infectious dose or produce toxins.
In this study, the statistical effects and interactions of
RH controlled by glycerol, initial pH, and potassium sorbate or
calcium propionate on the boundary for S. aureus growth were
modeled
220 Introductory Food Microbiology
MATERIALS AND METHODS
Organisms and Media Preparation
The . ve S. aureus strains used to create the bacterial cocktail
used in this study were S. aureus ATCC 13565 (American Type
Culture Collection, Rockville, Md.), which produces staphylococcal
enterotoxin A (SEA), ATCC 14458 (SEB), and ATCC 27664 (SEE);
D-2 (SED) obtained from Toxin Technology, Inc. (Sarasota, Fla.);
and A-100 (SEA) obtained from U.S. Army Natick Labs. All strains
showed typical growth on Baird-Parker agar plates and were
coagulase positive. Their identi. cation as S. aureus was con- . rmed
by use of a Riboprinter. Stock cultures were grown overnight at
37°C in brain heart infusion broth, suspended in a 2:1 broth-
glycerol solution, and stored at -80°C until needed. The BHI broth
was reconstituted with the appropriate ratio of water to glycerol
to achieve the desired RHs for the study (84, 88, 92, or 95% RH).
Appropriate amounts of 10% stock solutions of potassium sorbate
or calcium propionate were added to achieve . nal concentrations
of 500 or 1,000 ppm. The pH was adjusted using 0.1 or 1.0 N HCl
(J. T. Baker, Inc., Phillipsburg, N.J.; pH 4.5, 5.0, 6.0, or 7.0). Final
RH was determined using the Aqualab CX-2 water activity meter.
It was determined that the RH of the media did not change more
than 60.003% when measured at ambient temperature versus 37°C.
Each of the 80 broths was . lter sterilized and stored in screw-
capped tubes at 4°C until needed.
Bioscreen microtiter plate preparation, incubation, and
measurements.
The . ve stock cultures were removed from -80°C storage, and
one loopful was transferred into 9 ml of BHI broth, separately. The
inoculated broths were incubated for 18 h at 37°C. The . ve cultures
were individually adjusted to an optical density (OD) at 530 nm
of 0.750 to 0.780 (Perkin-Elmer 35 spectrophotometer) with 0.1%
sterile peptone to achieve a concentration of approximately 108
CFU/ml. Two milliliters of each diluted culture was transferred
into one sterile test tube and vortexed to create the S. aureus cocktail
used in the studies. Three 1:10 dilutions with sterile 0.1% peptone
water were used to make a . nal working cocktail with a
concentration of approximately 105 CFU/ ml. This working
Modelling the Growth Boundary of Staphylococcus Aureus 221
cocktail was kept on ice until used to inoculate the various media
used in each experiment. A 1-ml sample of the working cocktail
was taken, serially diluted, spread plated onto Baird-Parker agar
plates, incubated at 37°C for 48 h, and counted to determine initial
concentrations of cells.
Ten milliliters of each of the 80 broths as described above was
aseptically transferred to sterile tubes, and 100 ml of the S. aureus
cocktail was added to the broth to achieve an initial concentration
of approximately 103 CFU/ml. The inoculated broth (400 ml) was
aseptically transferred into sterile, 100-well microtiter plates in
eight replicate wells per medium. The outer wells of the microtiter
plates were . lled with uninoculated medium to act as a partial
moisture loss barrier and as uninoculated controls. Each plate
contained media at one RH level only. A piece of sterile Thermaseal
. lm was placed on top of the open wells, and the lid was also in
place during incubation. The plates were placed on a rack in a
plastic sealable container, which had the bottom . lled with
appropriate saturated salt slurries to maintain the environmental
RH to ensure RH of the media in the plates remained as stable as
possible. The saturated salt solutions used were as follows: ZnSO4
· H2O (83% RH @ 37°C), KNO (89% RH @ 37°C), KPO4 (93% RH
@ 37°C), and K2Cr2O7 (96% RH @ 37°C). The containers were
closed and placed into a 37°C incubator. The plates were
periodically removed from incubation, and the OD of the 640
individual wells was measured for up to 6 months using the
wideband . lter on the Bioscreen C system (Labsystems Oy, Helsinki,
Finland). The experimentswere repeated on separate dates. A total
of 1,280 wells were monitored.
Data Collection
The S. aureus population was considered to have shown growth
when the Bioscreen wideband OD (ODwb) measurement increased
from an initial reading of 0.200 to 0.220 to 0.350 or higher. Time
to growth (TTG) was determined by calculating the geometric
mean of the time of the last measurement that showed no growth
(ODwb <0.350) and the . rst time point that showed growth (ODwb
>0.350). In instances of no growth, TTG was censored at the . nal
measurement time of 168 days.
222 Introductory Food Microbiology
OD measurements were made and transferred from the
Bioscreen C ASCII . le to a DOS text editor and then imported into
Statistica software for initial analysis. Data were then transferred
to a Microsoft Excel spreadsheet to determine TTG and then to
SAS for modeling using LIFEREG.
Statistical Analysis
The TTG data along with a censoring indicator and the RH,
pH, and potassium sorbate or calcium propionate levels were
input data for construction of two separate models: one based on
the potassiumsorbate data set and one based on the calcium
propionate data set. These two data sets used a common block of
data generated from the experimental conditions, which had no
preservatives added. The SAS LIFEREG procedure was used to
develop predictive models of ln TTG as a function of the factors
RH, pH, and preservative level. By default, the procedure . ts a
model to the log of the dependent variable.
The resulting model can easily be transformed to a regular
time scale. In growth modeling, there are often conditions where
no growth occurs; therefore, TTG is sometimes censored. Under
these conditions, ordinary least-squares regression is not applicable,
and special procedures are required. The LIFEREG procedure can
accommodate such censored data and uses maximum likelihood
estimation methods to . nd regression coef. cients. When creating
models with the LIFEREG procedure, there is a need to have many
replicates of conditions equally spaced out about the matrix, with
approximately 50% of the conditions allowing and 50% not
allowing growth for best-. tting purposes. Therefore, the ranges of
conditions studied (RH, pH, and preservative levels) were chosen
to satisfy these requirements.
Toxin Production
Toxin production for experimental conditions close to the
growth or no growth border was explored by enterotoxin assay
using the VIDAS Staph Enterotoxin Assay. The VIDAS Staph
Enterotoxin Assay is a qualitative enzyme-linked • uorescent
immunoassay performed in the automated mini-VIDAS instrument
(bioMerieux, Inc., Hazelwood, Mo.). After the . nal ODwb
measurements were completed (24 weeks), the media from the
Modelling the Growth Boundary of Staphylococcus Aureus 223
microtiter plates were removed for assay. The broth from each of
the eight replicate wells was removed via pipet and combined into
two microcentrifuge tubes. The samples were spun down for 10
min, and the supernatant from the two tubes was combined. The
pH of the supernatant was adjusted with 1 N NaOH to a pH of
6.0 to 8.0. A half a milliliter of the sample was placed into the
appropriate well in a test strip, the strips were placed into the
mini-VIDAS, and the assays were run. A positive and negative
control were run with each set of test strips. The test is sensitive
to 1 ng of toxin per ml of sample.
RESULTS
A threshold ODwb value of 0.350 was used to score wells
with S. aureus growth (ODwb $ 0.350) versus wells with no growth
(ODwb, 0.350). This threshold value was determined in two ways.
First, ODwb measurements were compared with plate counts, and
it was determined that an ODwb reading of 0.350 was
approximately the . rst ODwb value where an increase in turbidity
regularly correlated to an increase in plate counts, indicating the
approximate ODwb where the instrument was sensitive enough to
accurately determine an increase in cell numbers by a change in
turbidity. Second, the data were analyzed to determine TTG in
each well using threshold ODwb values for growth of 0.300, 0.350,
and 0.400, and models were created and compared. The models
created with the threshold value set at 0.300 did not make
biological sense. We suspect that this was because this ODwb
corresponded to a level that is lower than the sensitivity of the
instrument to measure an increase in cell numbers. When the
threshold ODwb value was raised to 0.400, the models were similar
to those created with the TTG data when the threshold ODwb of
0.350 was used; however, the TTG data when the threshold ODwb
of 0.350 was used gave a slightly more conservative model. For
these reasons, the models were developed with TTG data obtained
by making the ODwb of 0.350 or more the lower limit for
determining that growth had occurred in a particular well.
The models included all three main effects: RH (rh), pH (ph),
and potassium sorbate (sorb) or calcium propionate (cal), their
quadratic effects (RH·RH 5 rh2, pH·pH 5 ph2, and potassium
224 Introductory Food Microbiology
sorbate·potassium sorbate 5 sorb2 or calcium propionate· calcium
propionate 5 cal2), and the three two-factor interactions (rh ·ph, rh
·sorb, ph·sorb or rh ·ph, rh ·cal, ph· cal). These main effects, quadratic
effects, and interactions are collectively referred to as the factors.
LIFEREG outputs a table of regression coef. cient estimates and
approximate chi-square distribution P values for each factor in the
model. The relative importance of each factor can be judged by the
P value: factors with small P values are most in• uential and
predictive of the TTG. LIFEREG also allows the user to specify the
error distribution to account for the variation in TTG not explained
by the regression model. For model development purposes, Weibull,
lognormal, and log-logistic distributions were considered. It was
found that all three distributions gave very similar models; the
regression coef. cients were similar in sign and magnitude. Since
the three distributions are not all from the same class of
distributions, it is not possible to formally test for goodness of . t
using likelihood ratio tests. However, since all models produced
similar regression equations, we selected the log-logistic
distribution model, because it had the largest sample log likelihood.
The model development process is an iterative process. The .
rst analysis of the data created models that were all inclusive; this
allowed for the determination of those factors that had a signi.
cant effect on the growth of S. aureus. All of the main effects,
quadratic effects, and two-factor interactions were signi. cant for
each model (P <0.005) except for the interaction between RH and
potassium sorbate in the potassium sorbate model and the calcium
propionate quadratic term in the calcium propionate model. For
this reason, in each model the insigni. cant factor was dropped
and the data were reanalyzed to develop the . nal models. The
resulting model equation from the calcium propionate data was
2.250 to 3.703· rh - 1.488·ph + 0.418· cal + 1.572· rh2 + 0.343·ph2
+ 0.814· rh·ph - 0.130·rh · cal - 0.221·ph·cal, and the resulting
model equation from the potassium sorbate data was 2.624 to
3.938·rh - 2.421·ph + 0.907· sorb + 1.638· rh2 + 0.887·ph2 - 0.190·
sorb2 + 0.903·rh ·ph - 0.756·ph·sorb. From the model equations, the
TTG contour plots for calcium propionate and for potassium
sorbate were created. For diagnostic purposes, plots of ln predicted
time to growth versus ln observed time to growth were examined.
Modelling the Growth Boundary of Staphylococcus Aureus 225
Plots of residuals versus main effects were also examined, and no
unusual patterns or anomalies were detected.
There was a slight difference in the curvature of the contour
lines in the plots for the media with no preservatives data for each
model, despite the fact that the same data for the no preservative
condition were used for developing each model. This difference
occurs because two distinctly different mathematicalmodels were
produced by the analysis, and when different models are used to
predict TTG at a zero preservative level, they will produce slightly
different predictions. It should be noted that the differences are
small and mainly in the low pH areas of the plots. In practice, the
target product formulation would be well away from the growth
boundary to allow for inherent process variation.
All of the models make good biological sense. As conditions
become more and more unfavorable for growth, the contour lines
are closer together, indicating conditions are approaching those
that do not allow growth of the organism. As the RH or pH of the
system decreases, a corresponding increase in TTG is seen, and
the no growth area of the contour plot increases in size. The
addition of potassium sorbate at low pH (pH 4.5 to 5.5)
dramatically changes the contour plots with steep curvature in the
low pH area of the plots. The no growth boundary with respect
to RH is increased from approximately 89% to approximately 93%
when 1,000 ppm of potassium sorbate is present at pH 4.5. This
effect is not as evident when calcium propionate is present; the no
growth boundary with respect to RH only increased approximately
1.5% from 88 to 89.5% when 1,000 ppm of calcium propionate is
present at pH 4.5. There is little change in the boundary with
respect to RH when the system is at pH 7.0 with the addition of
either preservative studied.
Various regions on the contour plots generated by the models
were explored with enterotoxin assays for all conditions where
the . nal ODwb measurement was borderline. Also, assays were
conducted for all samples with treatments at 84% RH, pH 7.0, and
84% RH, pH 4.5, although all ODwb measurements for treatments
at 84% RH were less than 0.350. The results are indicated by a
plus sign (toxin present) or minus sign (no toxin present) on the
contour plots. A total of 71 assays were run, and in every case
226 Introductory Food Microbiology
where the data were scored as ‘‘no growth,’’ the toxin assay
results were negative for toxin, and in every case where the data
were scored as ‘‘growth,’’ the toxin assay results were positive for
the presence of toxin. This further supports the choice of an ODwb
of 0.350 as the correct cutoff value for time to growth in the
individual wells.
DISCUSSION
Comparison of the models developed in this study with other
published work is dif. cult, because most previously published
studies have used NaCl to control the RH of the system, whereas
this study has used glycerol. The limits of S. aureus growth, or the
growth of any microorganism, cannot be predicted by the RH of
the system alone; instead, both the RH of the system and the
physical properties of the humectant used should be considered.
For example, NaCl is a non–glass-forming ionic humectant that
has specific effects on membrane transport systems. Therefore,
both the osmotic and ionic stresses placed on bacterial cells by
NaCl could affect the ability of those cells to grow. Glycerol is a
nonionic glass former that passively permeates the cell membrane.
Therefore, glycerol would not have a direct effect on ion transport
systems. Unlike NaCl, glycerol forms an aqueous glass. The glass
transition temperature (Tg) is a specific characteristic for each
glass-forming compound. The influence of a solute’s Tg on
molecular mobility directly affects the amount of osmotic stress a
specific solute places on cells. For these reasons, it should be
expected that the limits of growth based on RH would be different
when various humectants are used to control RH, and, therefore,
comparing studies based on RH alone could lead to false
conclusions.
The differences in modeling approaches also make
comparisons dif. cult. Both the FMM and PMP have very few
kinetic curves generated in which cells would be under stressed
conditions, the most dif. cult conditions in which to obtain kinetic
information. Also, there are few true replicates in these areas, with
most of the experimental design similar to a central composite
design in which most of the replicates are in the middle of the
design. Kinetic models also model the mean value, which, due to
Modelling the Growth Boundary of Staphylococcus Aureus 227
natural biovariability in the bacterial population, can be
meaningless under stressed conditions. The LIFEREG procedure
used to create the boundary models handles this by . tting the
distribution to the error term, hence allowing probabilities to be
calculated. This is useful for risk assessment purposes and allows
the model to be easily used in Monte Carlo simulations. One
consequence of modeling this way is that there is a need to have
many replicates equally spaced about the matrix, with
approximately 50% of the conditions allowing and 50% not
allowing growth for best-. tting purposes. The log time to growth
is used to normalize the variance.
It has generally been accepted that the limits for growth of S.
aureus are 85% RH when NaCl is used as the humectant and 89%
when glycerol is used. Our models, based on glycerol, show the
limiting RH for growth to be approximately 86% (at pH 7.0),
which disagrees with work published by Marshall et al., who
found the growth limits were 89 and 86% RH when glycerol and
NaCl were used as the humectants, respectively. This disagreement
could be due to strain variation or to the fact that only quarter-
strength BHI broth was used in the work by Marshall et al.,
whereas full-strength BHI broth was used in our model system.
Shapero et al. reported growth of S. aureus at 88% RH on tryptic
soy agar plates with RH adjusted with glycerol but did not conduct
experiments with the system at any lower RH values.
Studies with sucrose (a glass former that is not cell membrane
permeable and has its own Tg) used as the humectant showed
growth at 90% RH but not at 87% RH, and Scott reported growth
at 88 but not at 86% RH. Broughall et al. developed models for
growth of S. aureus in UHT milk, with RH adjusted by the addition
of glucose (a glass former that is not cell membrane permeable and
has a different Tg). They found growth at 88% RH but did not
explore the RH boundary further, because the additional glucose
caused the system to become saturated. There is published literature
that describes the ability of S. aureus to grow in various food
systems, mainly meats and cheeses, but most of these systems
used NaCl as the RH depressant and, therefore, cannot be used
to validate the models created with glycerol. Often in experiments
conducted with food systems, the pH of the product is not reported,
228 Introductory Food Microbiology
nor is the list of ingredients published, which also leads to dif.
culty when trying to compare data from various published studies.
The effectiveness of potassium sorbate to inhibit growth was
considerably greater compared with calcium propionate, which
was especially noticeable at low pH. Potassium sorbate and
calcium propionate have some key similarities that may lead to
the belief that they should act similarly as preservatives; potassium
sorbate has a pKa value of 4.74 and a molecular weight of 150.2,
whereas calcium propionate has a pKa value of 4.87 and molecular
weight of 186.2.
Both have been reported in the literature to act as weak acids;
the molecules remain undissociated at low pH, diffuse through
the bulk lipids of the cell membrane, then enter and dissociate in
the cytoplasm, where the pH is close to neutral. It is the dissociated
molecules that decrease the internal pH of the cell, which may
prevent growth by perturbing metabolism. Recent studies have
shown that sorbic acid does have inhibitory effects in the
undissociated form, suggesting not only that it may act as a weak
acid but also that it may have a secondary mode of action associated
with membrane-resident sorbic acid that acts as an uncoupler,
which may disable active transport. It has also been reported that
for S. aureus the minimum inhibitory concentration of propionic
acid is much greater than sorbic acid. This suggests that the levels
of calcium propionate used in this study that are in line with the
typical use levels in foods may have been too low to observe the
inhibitory effect of calcium propionate on S. aureus growth.
In summary, two models describing the growth boundaries
for S. aureus with respect to RH controlled by glycerol, pH, and
preservative were developed. The growth boundaries were explored
with toxin assays. For the . rst time, these models describe the
growth/no growth boundaries for S. aureus with respect to RH
controlled by glycerol and pH in addition to the preservatives
potassium sorbate and calcium propionate. These models will
allow product developers to visualize the ‘‘safe space’’ for the
formulation of shelf-stable intermediate moisture foods using these
preservation factors, will allow microbiologists to assess risks
more effectively over a wide range of products, and will ultimately
allow the consumer to have greater assurance of food safety.
Modelling the Growth Boundary of Staphylococcus Aureus 229
Molecular methods for detection of probiotics and intestinal
microbiota and evaluation of Lactobacillus brevis as a potential
probiotic dietary adjunct.
The human gastrointestinal (GI) tract harbours an extremely
complex microbiota mainly composed of fastidious anaerobic
organisms. Because these microbes have a profound impact on
host’s health, modulation of microbiota with probiotic bacteria
has been proposed. However, the mechanisms of action of both
the GI microbes and the proposed probiotics remain obscure. To
gain more information, molecular identification methods for
members of the GI microbiota and the probiotic strains are needed.
Quick, robust methods are necessary for obtaining an overall image
of changes in GI microbiota, and more sophisticated methods are
needed for following up selected species or strains.
In this study, new methods were tested for their applicability
in monitoring GI microbiota and probiotic strains. Strain-level
genetic labelling without introduction of foreign DNA was
demonstrated with Lactobacillus helveticus CNRZ32 by inserting
a site containing silent mutations into the chromosomal pepX
gene. Intact phenotypic properties of the mutated strain were
confirmed with a peptidase assay. The mutated and wild-type
strains could be detected from faeces and milk with the help of
specific primers. Thus, the labelling method could be used for
specific marking of industrial or probiotic strains in a ‘food-grade’
manner provided that a suitable target gene and genetic
transformation tools are available.
For analysis of GI microbes and selected intestinal or dairy
lactic acid bacteria, several species- or group-specific
oligonucleotide primers and probes were designed and tested
with three different techniques. A polymerase chain reaction -
enzyme-linked immunosorbent assay (PCR-ELISA) application
with simultaneous utilisation of multiple species- or group-specific
oligonucleotide probes was suitable for detection of predominant
members present in a mixed bacterial population. Sensitivity of
the method was improved by using primers selective for the genus
Bifidobacterium. Comparison of dot blot hybridisation and real-
time PCR demonstrated the superior properties of realtime PCR for
detection and quantification of bacterial ribosomal DNA targets.
230 Introductory Food Microbiology
The final part of this study comprised the evaluation of two
Lactobacillus brevis strains ATCC 8287 and ATCC 14869T as
supplements in dairy products. The L. brevis strains showed
promising in vitro antagonistic properties towards selected
potentially harmful microbes and were suitable as supplementary
strains in yoghurt, producing no undesirable side effects on the
quality or preservation of products. A small-scale feeding study
demonstrated the survival of L. brevis ATCC 8287 in the human
GI tract, indicating, together with the favourable antagonistic
properties, that this strain could be a candidate for use as a
probiotic supplement in dairy products.
Molecular methods have facilitated culture-independent
studies of gastrointestinal (GI) tract microbes. The GI microbiota
is mainly composed of anaerobic organisms and moreover, direct
molecular approaches have confirmed the abundance of
uncultivable microbes in intestinal samples. The value of molecular
methods for studying GI tract microbes is therefore immense.
GI microbiota can be influenced by probiotic bacteria. The
probiotics, defined as “live microbial food supplements which
beneficially affect the host by improving the intestinal microbial
balance", have been proposed to possess several advantageous
properties, such as antagonistic actions, production of
antimicrobial substances, modulation of immune responses and
an impact on the metabolic activities of the gut.
Probiotic bacteria seem to hold great promise for treatment of
gastrointestinal disorders, yet further studies are required to create
a more scientific basis for the probiotic action. Furthermore, as no
single strain is likely to have all the aforementioned beneficial
properties, screening of new strains for probiotic potential is
necessary.
Although several applications, such as dot blot hybridisation,
fluorescence in situ hybridisation (FISH), denaturing gradient gel
electrophoresis (DGGE) or thermal gradient gel electrophoresis
(TGGE), and polymerase chain reaction (PCR) with species- or
group-specific primers, are available for the detection and
semiquantitative analysis of GI microbiota, improvements are
required, especially in relation to sensitivity, cost and quantification
power of the methods. This is fundamental for better understanding
Modelling the Growth Boundary of Staphylococcus Aureus 231
of the GI tract microbiota and the effects of probiotic bacteria on
these microbes.
REVIEW OF THE LITERATURE
Intestinal Microbiota
An extremely complex microbiota which has a profound impact
on host’s health. The normal gut bacterial population of an adult
is estimated to comprise more than 400 species, with a
predominance of obligate anaerobes. The total number of microbes
present in one gram of intestinal content varies from less than 103
microbes in the stomach to 104 – 107 microbes in the small intestine
and 1010 – 1012 microbes in the colon. Indeed, the quantity of
microbes present in the intestine (about 1014) exceeds 10-fold the
total number of all human cells. Until recently, analysis of intestinal
bacteria has mainly been based on cultivation-dependent methods.
Culture-independent studies have however confirmed that only a
fraction of the organisms present in faeces are cultivable, therefore,
the results obtained by cultivation are likely to be biased.
Generally, Bacteroides, Eubacterium, Clostridium, Ruminococcus,
Peptococcus, Peptostreptococcus, Bifidobacterium and Fusobacterium are
reported to constitute the majority of microbiota. Molecular analyses,
however, suggest that most faecal bacteria belong to a few
phylogenetic lineages composed of organisms from several genera.
Bacteria related to Bacteroides, Prevotella and Porphyromonas seem
to represent one-third of bacteria present in faeces, whereas
Clostridium leptum subgroup and Clostridium coccoides groups both
account for approximately one-fifth of the faecal bacterial
populations. However, besides being an extremely diverse
microbial ecosystem, the intestinal microbiota appears to be unique
for each individual. The normal microbiota of the GI tract works
as a barrier against pathogens, contributes to degradation of some
food components, stimulates the host immune system, and
produces certain B vitamins, enzymes and short-chain fatty acids.
The microbes can also metabolise potentially carcinogenic
substances and drugs in either a beneficial or a disadvantageous
way. However, the role and action of individual microbial species
or groups present in the GI tract are poorly known. Starting at
birth, the microbiota develops in a successional manner. The first
232 Introductory Food Microbiology
colonising microbes include bifidobacteria, enterobacteria,
clostridia, enterococci and ruminococci. Recent studies have
suggested the importance of the type of colonising bacteria on the
development of the gut immune system. A role in the development
of food allergies has been postulated for the intestinal microbiota
due to the occurrence of an adult-like bifidobacterial species
composition in the intestine of allergic infants. Significant structural
changes occur in the microbiota with age, including reduction of
bifidobacteria as well as an increase in the diversity of Atopobium
cluster species present in faeces. Nutritional aspects, however,
have a profound effect on the composition of the intestinal microbial
population.
The Probiotics
Probiotic bacteria have been defined as “live microbial food
supplements which beneficially affect the host by improving the
intestinal microbial balance." Probiotic bacteria are increasingly
utilised in human food as well as in animal feed products.
However, composition of the intestinal microbiota is poorly known,
which hinders understanding of the probiotic functions. A probiotic
strain should be of host origin, non-pathogenic, technologically
suitable for industrial processes, acid- and bile-fast, adhere to the
gut epithelial tissue, persist in the gastrointestinal tract for short
periods, produce antimicrobial substances, modulate immune
responses and influence the metabolic activities of the gut. The
properties of the strain should be well documented. Although
some criteria, such as the non-pathogenic status, technological
suitability and careful documentation of the probiotic effects of a
microbial strain, are invariably required, no single strain is likely
to carry all of the abovementioned properties. Moreover, probiotic
properties are considered strain-specific, and results obtained with
one strain cannot therefore be claimed for another, even a closely
related strain. Microbes used in probiotic products include strains
from several Lactobacillus and Bifidobacterium species, Enterococcus
faecalis and Enterococcus faecium, Lactococcus lactis, Leuconostoc
mesenteroides, Pediococcus acidilactici, and Sporolactobacillus inulinus,
Streptococcus thermophilus, as well as Bacillus cereus, Escherichia coli,
Propionibacterium freudenreichii, Saccharomyces cerevisiae and
Saccharomyces boulardii. However, lactobacilli and bifidobacteria
Modelling the Growth Boundary of Staphylococcus Aureus 233
are most common in probiotic products designed for human use;
probiotic properties of these bacteria are also the best studied.
Lactobacilli belong to the lactic acid bacteria, which comprise
a diverse group of Gram-positive bacteria, most typically
represented by non-sporing, catalase-negative, devoid of
cytochromes, non-aerobic but aerotolerant, fastidious and acid-
tolerant cocci or rods producing lactic acid as the major end-
product during the fermentation of carbohydrates. Being of
fastidious nature, lactic acid bacteria require a rich environment
for growth, such as decaying plant material, food products and a
mammalian gastrointestinal tract or vagina. The genus Lactobacillus
is heterogeneous, containing species with 32-53% G+C of the
chromosomal DNA arranged into three groups based on differences
in sugar metabolism caused by the presence or absence of fructose-
1,6-diphosphate aldolase and phosphoketolase. Although
possessing some phenotypical features common for lactic acid
bacteria, the genus Bifidobacterium is actually related to the
Actinomycetes branch, having a high chromosomal G+C content.
Sugar metabolism of bifidobacteria differs from that of lactic acid
bacteria; the bifidobacteria lack aldolase and glucose-6-phosphate
dehydrogenase, and hexose sugars are exclusively degraded by
the fructose-6-phosphate pathway characterised by fructose-6-
phosphate phosphoketolase. Bifidobacteria are predominant
members of the human intestinal microbiota, with bacterial counts
of 109-1011 per gram of stool, with B. bifidum, B. longum, B. infantis,
B. breve, B. adolescentis, B. angulatum, B. catenulatum, B.
pseudocatenulatum, and B. dentium reported as human isolates.
Several positive effects have been proposed for probiotic
lactobacilli and bifidobacteria. Antagonism towards intestinal
pathogens has been demonstrated for probiotics. Alleviation of
diarrhoea is a well-documented characteristic of some strains, and
particularly the ability of Lactobacillus rhamnosus GG to shorten the
duration of acute rotavirus diarrhoea has been established.
Stimulation of the gut immune system by probiotic strains has
been reported, and induction of cytokine profiles has been shown
to be strain-dependent. Alleviation of allergic reactions in the
gastrointestinal tract has also been suggested for some strains:
using a mouse model, Murosaki et al. (1998) showed that heat-
234 Introductory Food Microbiology
killed Lactobacillus plantarum L-137 suppressed production of
antigenspecific IgE by stimulating the production of IL-12. In
addition, Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-
12 have been demonstrated to relieve symptoms of atopic eczema.
Administration of L. rhamnosus GG has been shown to enhance
the production of IL-10, acting thus as an anti-inflammatory
mediator in atopic children.
Consumption of probiotics has also been observed to lower
activities of harmful faecal enzymes. Some animal models have
even suggested the ability of probiotic strains to reduce the incidence
of cancer. Finally, probiotics may have significance for alleviation
of intestinal disturbances. For example, ingestion of a mixture of
lactobacillar and bifidobacterial strains together with a Streptococcus
salivarius ssp. thermophilus was observed to help maintain remission
status in ulcerative colitis patients.
Although lactobacilli and bifidobacteria are stated as GRAS
(generally recognized as safe) organisms due to their long usage
and non-pathogenic status, safety issues have been investigated.
Isolation of these organisms from infections has been reported
with a low incidence, and in most cases, bifidobacteria and
lactobacilli were associated with immunocompromised patients.
One major concern among lactic acid bacteria is, however,
resistance to vancomycin, especially within the genus Enterococcus
where the resistance has been shown to be transferable.
Vancomycin resistance has been reported for lactobacilli by several
authors, but it is generally considered to be an intrinsic property.
Vancomycin resistance expressed by the probiotic strain L.
rhamnosus GG has been studied in detail.
Strain GG was not observed to transfer vancomycin resistance
or receive other resistance elements from enterococci, nor were any
genes resembling enterococcal vancomycin resistance genes
found in Lactobacillus GG. Similarly, Klein et al. (2000) reported
no indications for the presence of the vanA gene cluster, the vanB
gene or the vanC gene from five L. reuteri strains or L. rhamnosus
GG, suggesting that the vancomycin resistance of the strains
studied is unrelated to the acquired resistance in the Enterococcus
species.
Modelling the Growth Boundary of Staphylococcus Aureus 235
Genetic Labelling of Lactic Acid Bacteria
Insertion of an extra DNA label into a target strain genome
provides a way to monitor the specific strain in various
environments. However, introduction of foreign DNA is often
required, and in many cases, genetic elements producing a
phenotypic change are used to distinguish the marked strain.
Genetic elements encoding resistance to an antibiotic have been
utilised for labelling of lactic acid bacteria, but the increasing
spread of antibiotic resistance factors between bacterial species or
genera make such approaches unsuitable for strains intended for
human or animal use. Therefore, usage of various ‘food-grade’
markers has been suggested. Labelling of lactic acid bacteria with
a plasmid-encoded green fluorescent protein gene placed under
an inducible promoter has been reported for Lactococcus lactis and
Lactobacillus plantarum, whereas Allison and Klaenhammer (1996)
suggested the use of a native Lactobacillus gene encoding immunity
to Lactacin F as a food-grade genetic marker. A similar role has
also been demonstrated for ltnI conferring immunity to lacticin
3147. Site-specific integration of desired genetic elements into
bacterial chromosomes through phage attachment sites has been
described for Lactococcus lactis, Lactobacillus delbrueckii and
Lactobacillus plantarum.
Legislation as well as consumer acceptance limit the usage of
genetically engineered organisms, and therefore, genetic changes
introduced to target organisms must be carefully considered.
Maguin et al. (1996), for example, combined insertion sequence
ISS1 with a thermosensitive replicon, enabling a high frequency of
random insertion (about 1%) with Lactococcus, Enterococcus and
Streptococcus thermophilus, while efficient excision of the plasmid
generated stable mutants with no foreign markers, leaving only a
single ISS1 copy at the mutated site. However, the smallest
detectable alteration introduced to a target organism is changing
one or a few bases in a gene-coding sequence without affecting the
amino acid sequence of the corresponding gene product. In
principle, such a mutation could be caused by natural processes
due to the degeneracy present in the genetic code.
Silent mutations have been utilised for modification of a
Lactococcus lactis subsp. cremoris strain plasmid-encoded proteinase
236 Introductory Food Microbiology
prtP gene by in vitro mutation of the third positions of four adjacent
codons, thus providing a genetic label with no phenotypic effects.
Although technically more demanding, genetic marking should,
however, preferably be directed to gene replacement on a
chromosomal locus to ensure maximal stability of the alterations
introduced.
Utilisation of Nucleic Acid Based Methods for Identification and
Monitoring of Bacteria in Population Samples
Utilisation of Methods Independent of Prior Knowledge on
Sequence Data: Cloning and sequencing of the 16S ribosomal
DNA (rDNA) pools in a population sample provides a method for
obtaining sequence-level information on uncultivable bacteria
abundantly present in various parts of the gastrointestinal tract.
Suau et al. (1999) analysed the sequence of 284 16S rDNA clones
derived from one faecal sample and classified the clones into 82
molecular species, using 98% similarity criteria for a species.
Importantly, only 24% of the molecular species were derived from
a described organism. Similarly, comparative 16S rDNA sequence
analysis of the intestinal bacterial community in pigs revealed
only a 17% fraction of previously described organisms among a
total of 375 phylotypes. In a large-scale study of subgingival plaque
samples, including analysis of a sequence of 2522 16S rDNA
clones, 347 species was observed among the clones studied, which
correlated well with a previous estimate of expected species
diversity in the oral cavity. Thus, the direct cloning approach
seems to give a good idea of the total microbiota present in a
complex population. It also facilitates determination of species-
level differences between bacterial populations, as shown by the
discovery of novel phylotypes and species not previously
associated with childhood caries by comparing of the oral
microbiota of a healthy subject and a subject with early childhood
caries.
Approaches based on cloning are, however, rather tedious
and are not optimal for analysis of large numbers of samples. Like
all PCR-dependent methods, construction of clone libraries may
be prone to biasing, possibly leading to falsification of the library
structure. Nevertheless, with the help of cloning, design of
phylogenetically relevant oligonucleotide probes is enabled.
Modelling the Growth Boundary of Staphylococcus Aureus 237
Denaturing Gradient Gel Electrophoresis (DGGE) and Thermal
Gradient Gel Electrophoresis (TGGE)
Denaturing gradient gel electrophoresis (DGGE) and thermal
gradient gel electrophoresis (TGGE) have become popular methods
for analysis of microbial populations present in various habitats
such as water ecosystems, microbial fermentations, and the GI
tract. These methods allow separation of nucleic acid molecules
based on their size and sequential differences. Thus, a population
of DNA molecules, such as the 16S ribosomal RNA (rRNA) or
PCR-amplified 16S rDNA, can be studied and predominant
members of the population identified via sequencing of isolated
nucleic acid bands. Sensitivity of gradient gel electrophoresis is
affected by the choice of PCR primers used. For example, utilisation
of universal 16S primers limits the sensitivity to detection of 1%
subpopulations. By contrast, utilisation of species- or group-
specific primers may allow detection and identification of bacteria
representing a minority of the total population.
Drawbacks of gradient gel electrophoresis include difficulty of
comparisons between individual gels, requirement of careful
adjustments, and the need for sequencing to confirm identities of
bands seen in a gel. Furthermore, some bacteria may remain
unidentified due to low resolution of DNA bands. With both
DGGEand TGGE-based methods, sequencing is required for correct
identification of individual bands seen in the gel. As stated by
Schmalenberger et al. (2001), intraspecies operon heterogeneities
may significantly contribute to genetic profiles in microbial
community analysis, as amplification of one bacterial DNA may
yield several separate bands, which can then be wrongly
interpreted as high microbial diversity. Depending on the 16S
binding universal primer pairs used, single-strand conformation
polymorphism analysis (SSCP) revealed an average of 1.7 – 2.3
bands per pure cultured bacterial organism.
Terminal Restriction Fragment Length Polymorphism (T-RFLP)
Terminal restriction fragment length polymorphism (T-RFLP)
is based on endonuclease digestion of PCR-amplified DNA and
capillary electrophoresis analysis of the terminal restriction
fragment (TRF) containing a fluorescent label. Terminal restriction
238 Introductory Food Microbiology
patterns have been used to analyse marine bacterioplankton
communities as well as faecal bacteria. The method is, however,
limited by the choice of primers, which can, with their different
affinities, dramatically change the patterns observed, while another
problem is the TRF length overlap by phylogenetically distant
bacteria.
Analysis of Community DNA Profiles
Analysis of total bacterial community structure can be
accomplished by measurement of guanosine-cytosine profiles of a
population DNA sample with DNA reassociation and density
gradient centrifugation. Density gradient fractionation of
community DNA enables analysis of interesting fractions by
cloning of PCR-amplified DNA pools, followed by sequencing.
Density gradient centrifugation has been applied in analysis of
intestinal microbial guanosinecytosine profiles. However, because
this approach requires sophisticated and expensive equipment, it
is out of reach for most research laboratories.
Utilisation of Specific Oligonucleotide Primers or Probes
Hybridisation: Hybridisation offers a means for direct semi-
quantitative monitoring of population samples. A very high
sensitivity for detection of DNA targets can be obtained with
radioactively labelled probes. However, because of its abundance
in bacterial cells, rRNA provides for a more attractive target for
hybridisation studies. Indeed, hybridisation assays targeting rDNA
have been verified as 10-fold less sensitive than assays for rRNA.
Dot blot hybridisation with rRNA – targeted probes has been used
for semiquantitative analysis of ruminal microbes and intestinal
microbiota. Comparison of hybridisation results of a specific probe
with a universal probe enables assessment of the target bacterial
proportion present in a sample. At best, detection of a 0.1 – 0.01%
rRNA subpopulation has been reported, corresponding to
approximately 107 target cells if 1011 bacteria are considered to
be present in a gram of faeces. Use of radioactive isotopes, especially
in large-scale and long-term analyses, is, however, complicated by
the short half-lives of labels. Nonradioactively labelled probes are
more convenient, but their sensitivity is limited. Preenrichment of
the target DNA by polymerase chain reaction can be used to
Modelling the Growth Boundary of Staphylococcus Aureus 239
enrich the target DNA. For example, 0.1 pg of B. distasonis or B.
thetaiotaomicron DNA (approximately 10-20 cells) or 0.01 pg of B.
vulgatus DNA (1-2 cells) applied to a PCR was sufficient to produce
a positive hybridisation signal with specific digoxigeninlabelled
probes.
An additional factor of concern for filter hybridisations is the
need to label each probe separately and to perform hybridisation
reactions after optimisations in separate vessels. However,
Ehrmann et al. (1994) described a reverse dot blot hybridisation
with several membrane-bound oligonucleotide probes for
identification of lactic acid bacteria present in mixed populations.
Similarly, Becker et al. (2002) used simultaneous detection of PCR-
amplified rDNA of 23 oral bacterial species or groups with
oligonucleotide probes.
In the fluorescence in situ hybridisation (FISH) method, the
bacterial cell samples to be studied are immobilised on microscope
slides and made permeable for fluorescently labelled
oligonucleotides with subsequent microscopic observation of the
hybridisation signal intensities.
FISH has been used for monitoring faecal microbiotas, and
detection of a 1 – 0.1% bacterial subpopulation has been obtained.
Automated analysis of fluorescent signals has also been utilised
to facilitate objective interpretation of results. The major problem
with FISH applications is the different penetration of probes in
bacteria with various cell wall types, resulting in a possible
underestimation of Gram-positive bacteria.
Polymerase Chain Reaction (PCR): Amplification of target
nucleic acids with PCR is an effortless method for detection of
target DNA from various samples. In principle, the detection limit
for a PCR assay, based on usage of one or two oligonucleotide
primers specific for the target bacteria, is the presence of one copy
of the amplified DNA region. Although achievement of such an
extremely sensitive assay is unlikely, the PCR remains a very
powerful detection method, typically requiring a minimum of 10-
20 target copies for successful amplification. The high sensitivity
is also somewhat problematic, as a simple aerosol contamination
may lead to false amplification.
240 Introductory Food Microbiology
PCR-based detection of rRNA genes has been used for direct
detection of various bacteria present in faeces and pathogens from
clinical samples. Partial or full amplification of the 16S rDNA
with primers specific for a genus or a few closely related genera
has also been reported. In addition, utilisation of 16S-23S intergenic
sequences as targets of speciesspecific primers for different lactic
acid bacteria has been described (Tilsala-Timisjärvi and Alatossava,
1997). Good sensitivities have been reported for the PCR detection
assays of faecal bacteria. For example, PCR detection sensitivity of
five ruminococcal species with species-specific primers from faecal
samples spiked with the target species did not markedly differ
from the observed detection limit of 4-100 cells from pure cultures.
Although a highly potent detection method, results obtained
by conventional endpoint PCR should not be considered to be
directly quantitative. PCR may lead to differential amplification of
target templates originally present in equal amounts. In addition,
very low template concentrations may generate random fluctuations
in priming efficiency of population DNA samples with universal
primers, leading to a bias in the end-product concentration.
Knowledge of the rRNA gene copy numbers and genome sizes of
bacteria in a mixed DNA sample has been observed to be
insufficient in predicting the final product ratio of a PCR
amplification. Suzuki and Giovannoni (1996) also noticed that the
accumulation of end products during mixed-template PCR caused
biasing of the various end-product ratios towards a 1:1 situation,
which was hypothesised to be caused by an increase in the
homologous template hybridisation, decreasing the efficiency of
primer annealing and subsequent amplification.
Some improvements in the reliability of PCR quantification
have been obtained by competitive PCR approaches. Hahn et al.
(1995) created a quantitative PCR assay based on post-
amplification differentiation of the internal standard and the
sample DNA by selective restriction analysis and digoxigenin-
based colorimetric detection. Quantitative detection of
Mycobacterium tuberculosis PCR products was performed by enzyme-
linked immunosorbent assay by comparison of hybridisation
results with two probes and two IS6110 elements derived from
either an internal control or a modified template. With co-
Modelling the Growth Boundary of Staphylococcus Aureus 241
amplification of an internal standard, detection of Clostridium
proteoclasticum was linear between 1 × 104 and 5 × 101 cells, with
a detection limit of 50 fg or 25 cells, whereas detection of Oxalobacter
formigenes from human faecal samples by competitive PCR was
shown to be linear over a range of six logarithmic units, with a
detection limit of approximately 100 genomes.
Polymerase Chain Reaction – Enzyme-linked Immunosorbent
Assay (PCR-ELISA) : Polymerase chain reaction – enzyme-linked
immunosorbent assay (PCR-ELISA) combines utilisation of
polymerase chain reaction for efficient multiplication of the target
DNA and hybridisation with a detection probe to ensure the
specificity of the reaction. Figure 1 summarises the concept of
PCR-ELISA detection. The PCR-amplified products are labelled
with digoxigenin during or after the amplification reaction and
hybridised with the specific biotinylated detection probe. The probe
is immobilised in streptavidincoated microtitre plate wells, and
hybridised DNA products are detected via digoxigenintargeted
antibodies linked with an enzyme capable of producing a
colorimetric or fluorimetric signal when brought together with a
substrate. Usage of PCR-ELISA hybridisation for detection of single
pathogens of clinical importance and spoilage bacteria of food has
been described. In comparison with filter hybridisation, PCR-ELISA
provides more easily standardised reaction conditions by
utilisation of commercially supplied microtitre plates. Other
advantages of PCR-ELISA compared with several other methods
are its relative simplicity and low costs.
Good sensitivities have been reported for PCR-ELISA assays,
and the results can in some cases be interpreted quantitatively.
The limit for detection of Bordetella pertussis was 100 target
organisms, starting from appliance of different target DNA amounts
to PCR reaction. Less than ten Epstein-Barr virus genome copies
added to 750 ng of background DNA were required for a positive
PCR-ELISA result. Similarly, a sensitivity of 5 cfu/ml blood was
obtained for detection of C. albicans and A. fumigatus cells,
corresponding to the sensitivity of Southern blotting using
digoxigenin-labelled oligonucleotides. Fletcher et al. (1998) could
detect Aspergillus fumigatus quantitatively by a PCR-ELISA method
on a log-scale between 100 and 1 pg of target DNA. Detection of
242 Introductory Food Microbiology
Escherichia coli in oysters was reported to be quantitative in the
range of 10-105 cfu/g. Detection of Campylobacter jejuni and C. coli
with PCR-ELISA was shown to be 10- to 100-fold more sensitive
than a gel-based PCR method using the same primers, the smallest
amount of C. jejuni template DNA giving a positive signal in the
assay being 1.5 fg. Some of the described PCR-ELISA applications
can be considered to be quantitative competitive PCR approaches.
Real-time PCR: Real-time PCR is based on on-line
measurement of the amplification reaction, enabling quantification
of the product during the logarithmic phase of PCR. The first and
so far most commonly used real-time PCR approach is the 5´-
nuclease (TaqMan) assay introduced by Holland et al. (1991). The
original assay operates using a radioactively labelled probe that
hybridises in the PCR template region to generate a specific,
detectable signal from the amplification reaction. The detection
probe becomes annealed to one of the DNA strands during the
amplification and is cleaved by the Thermus aquaticus DNA
polymerase 5´-exonuclease activity during the primer extension
step. Lee et al. (1993) further extended the 5´-nuclease assay by
utilisation of doubly labelled oligonucleotide probes for fluorescent
measuring of formation of the specific PCR product during primer
extension. Introduction of an automated detection method enabled
real-time monitoring of the PCR product formation during the
exponential amplification phase. Since the advent of real-time
PCR, several techniques have been introduced, including primers
with fluorescent dyes, molecular beacons, dual probes and
intercalating dyes such as SYBR Green I. Real-time PCR is a
superior technique for quantification of nucleic acids. While
competitive PCR has been demonstrated to be as reproducible and
accurate as real-time PCR, the latter has the benefit of an easier
methodology, reducing the need for sample DNA treatment.
Although real-time PCR is a relatively new technique, several
detection or quantification assays targeting various bacteria have
already been described. Target bacteria include carious dentine
bacteria, Desulfotomaculum from soil, faecal bifidobacterial species,
Helicobacter hepaticus, Staphylococcus aureus, Borrelia burgdorferi sensu
lato, Campylobacter jejuni, Listeria monocytogenes, Rhodococcus
coprophilus and Mycoplasma genitalium. Some applications for
Modelling the Growth Boundary of Staphylococcus Aureus 243
determination of larger bacterial groups have also been published.
Typically, sensitivities of 1 to 100 target genomes per reaction and
linearity ranges of 4 to 8 logarithmic units are obtained in assays
targeted to specific bacteria. Real-time PCR is also utilised as a
rapid diagnostic tool for detection of pathogenic bacterial species
or strains present in a sample. With pathogenic bacteria, the target
of choice for real-time PCR is generally a gene associated with the
pathogenic traits.
Detection of PCR amplicons with specific probes is often
favoured over usage of intercalating dyes due to the former’s better
sensitivity and lack of detection of falsely primed products.
However, SYBR Green I has become popular because of the
possibility to use this intercalating dye in virtually any assay.
Real-time quantitative PCR with SYBR Green I has been reported
to be 10-fold less sensitive than a corresponding TaqMan assay
due to the formation of non-specific products in reactions starting
with small amounts of template DNA. On the other hand, SYBR
Green I has great potential in situations where a diverse target
population is to be detected with PCR. A probe-based methodology
requires a binding site for the probe in the vicinity of one of the
primers; however, such a conserved site is likely to be missing
from a degenerate target DNA population. This should be taken
into account, especially when a bacterial population containing
several hitherto unknown species is studied.
AIMS OF THE STUDY
The aims of this study were to develop molecular methods for
analysis and monitoring of faecal bacterial populations and
putative probiotic strains as well as to characterise the technological
properties of two Lactobacillus brevis strains. The following goals
were set:
1. To create a genetic label by introducing silent mutations to
adjacent amino acid codons of a genomic peptidase gene
of a selected Lactobacillus strain and to confirm the
unchanged phenotype of the strain with an available
peptidase assay.
2. To develop oligonucleotide PCR primers or probes targeting
the 16S ribosomal DNA as species- or group-specific
detection tools.
244 Introductory Food Microbiology
3. To exploit a PCR-ELISA application with multiple
oligonucleotide capture probes for analysis of artificial
mixed DNA samples or faecal DNA preparations.
4. To test suitability of real-time PCR for quantification of
selected intestinal or probiotic bacteria in faecal samples.
5. To evaluate the applicability of two Lactobacillus brevis
strains as supplementary strains with potential probiotic
actions in dairy products.
MATERIALS AND METHODS
Microbial Strains, Plasmids, Human Cell Lines and Culture
Conditions
The microbial strains, plasmids and human cell lines used in
this study have been described in detail in the respective original
publications I-V. Briefly, Lactobacillus helveticus CNRZ32 was chosen
as a model organism for demonstration of genetic labelling (I).
Plasmids pUC19 and pSA3 were used to construct plasmids
pKTH5052 and pKTH5053, respectively (I). The PCR-ELISA
application with universal primers targeting 16S and 23S rDNA
was tested with a set of bacteria including 25 species, that
represented type strains of lactobacilli, bifidobacteria and other
bacteria reported as members of human intestinal microbiota, or
lactobacilli species used in dairy fermentations (II). Extension of
the PCR-ELISA method with bifidobacteria-targeted 16S rDNA
amplification was tested with a set of ten bifidobacterial species
belonging to the human intestinal microbiota, suggested as
potentially harmful oral microbes, or used in dairy products (III).
With real-time PCR, six bacterial strains, each representing a
bacterial species or group, present in the human intestinal tract or
dairy products, were used (IV).
Dairy technological and probiotic properties of two lactobacilli
strains, L. brevis GRL1 (ATCC 8287) and L. brevis GRL62 (ATCC
14869T), were determined using dairy starters, positive or negative
control strains or target strains for testing the antagonistic
properties (V). The human intestinal cell lines Intestine-407 and
CacO2 were used in adhesion studies (V). Culturing of microbes
and human cell lines was carried out using media and conditions
Modelling the Growth Boundary of Staphylococcus Aureus 245
as outlined in the Materials and methods sections of original
publications (I-V).
Basic DNA Techniques
Rapid isolation of genomic DNA from pure cultured bacteria
was performed by cell mill disruption of bacterial samples in the
presence of glass beads, followed by phenol-chloroform extraction
and ethanol precipitation (I, II, III, V). For rapid and effective
purification and isolation of DNA from faecal material, a method
described in Study I was used (I, III). A large-scale DNA isolation
method described by Apajalahti et al. (1998) was used to produce
a sufficient amount of template DNA for real-time PCR (IV).
Wizard Minipreps were used for isolation of plasmid DNA
from E. coli clones (I), whereas the Qiagen Plasmid Protocol Kit
was exploited for isolation of plasmid DNA from L. brevis (V).
Restriction enzyme digestions and ligations were carried out
according to the enzyme manufacturer’s recommendations. The
transformations of L. helveticus cells were performed as described
by Bhowmik and Steele (1993). Dot blot hybridisation was executed
using standard methods (IV). DNA concentrations were measured
with a Versafluor fluorometer (Bio-Rad). DNA sequencing was
performed with an ABI310 DNA sequencer (Applied Biosystems)
using BigDye Terminator chemistry (I).
Design of Oligonucleotide Primers and Probes
Oligonucleotide primers and probes were designed with the
help of published sequence data available in sequence databanks
(I, II, III, IV). Generally, the online Internet tools ClustalW and
Fasta provided by the European Bioinformatics Institute (EBI) were
used for identifying the primers and probes with desired specificity
towards intended target DNA. Oligonucleotides required for
creation of the silent mutation site (I) were planned by utilising the
sequence of the L. helveticus CNRZ32 pepX gene (Accession number
U22900). While creating the mutations, major changes in the codon
usage frequency were avoided to reduce the likelihood of changes
in the expression level of the target gene. The Ribosomal Database
Project was utilised for designing the primers and probes used in
Study IV. In addition, signature oligonucleotides published
246 Introductory Food Microbiology
previously by others were utilised or modified when necessary
(II, III, IV).
Polymerase Chain Reaction (I-V)
For end-point detection or production of DNA templates,
polymerase chain reactions were carried out in reaction conditions
recommended by the manufacturer of Dynazyme DNA polymerase.
In Study I, the PCR conditions used in detection of wildtype and
mutant strains were as follows: the reaction mixture consisted of
50 mM Tris- HCl pH 9.0, 15 mM (NH4)2SO4, 0.1% Triton X-100,
1.8 mM MgCl2, 360 µM of each deoxynucleotide triphosphate, 1
µM of each primer and 0.02 U/µl Dynazyme EXTTM Polymerase,
and 1 µl of template or water. Each sample was subjected to a
primary denaturation cycle at 95ºC for 2 minutes followed by 30
cycles of denaturation at 95ºC for 30 seconds, annealing at 55ºC
for 30 seconds and elongation at 72ºC for 1.5 minutes. The reaction
was terminated at an elongation step for 5 minutes at 72ºC and
followed by incubation at 4ºC. In Study IV, gradient PCR was used
to select optimal annealing temperatures for PCR primer pairs.
Oligonucleotides were synthesised by commercial suppliers.
PCR-ELISA (II, III)
PCR-ELISA was carried out as described in Studies II and III.
Briefly, the PCR products were labelled with the DIG-High Prime
Kit according to the instructions of the manufacturer, and
concentrations of the PCR products were measured with the
Versafluor fluorometer. For calculation of molecular quantities in
the labelled end products, the amount of DNA (ng/µl) was divided
by the length of the PCR product (base pair) multiplied by the
average weight of a base pair (ng/base pair). The hybridisations
were performed in commercial streptavidin-coated microtiter plates
(Labsystems, Finland). Results were analysed by measuring
absorbance at 450 nm.
Real-time PCR (IV)
Real-time PCR was tested with two different chemistries (5´-
nuclease- and SYBR Green I assays) as described in Study IV.
Quantitative PCR was performed with an iCycler iQ and iCycler
Optical System Interface software version 2.3. All PCR reactions
were performed in triplicate, in a volume of 25 µl, using 96-well
Modelling the Growth Boundary of Staphylococcus Aureus 247
optical grade PCR plates and optical sealing tape. Optimal
concentrations for various reaction components were tested for
each primer set and chemistry with a dilution series of genomic
DNA from the target test species. With SYBR Green I chemistry,
effect of the polymerase type on PCR product formation was tested
with a standard polymerase, Dynazyme II, and hot-start
polymerases AmpliTaq Gold® DNA Polymerase, BlueTaq and
FastStart Taq DNA Polymerase. The TaqMan assays were
performed successfully with the Dynazyme II enzyme and therefore
switching to the hot-start enzyme was not considered.
Peptidase Activity Assays (I)
Activity measurement of the PepX was determined from both
the wild-type and mutated L. helveticus strains according to the
method of El Soda and Desmazeaud (1982).
Evaluation of L. brevis Strains as Dairy Adjuncts
In vitro tolerance of the L. brevis strains to low pH, bile salts
and pancreatic fluid was examined as described in Study V.
Antimicrobial properties towards selected food spoilage bacteria
and dairy lactic acid bacteria starters were evaluated by following
the microbial growth in MRS broth supplemented with 10% filter-
sterilized L. brevis GRL1 or GRL62 supernatant at 37ºC for two
days with an automated turbidometer Bioscreen C Analysing
System. Adherence to Caco-2 and Intestine-407 cell lines was
studied as described in Study V. Resistance to selected antibiotics
was tested with disc diffusion and microdilution methods, modified
from instructions given by the antibiotic disc manufacturer and
NCCLS. In vivo feeding trials with four healthy volunteers were
performed to see whether the L. brevis strains could survive the GI
passage. Technological suitability of the strains for dairy processes
was also evaluated as described in Study V.
RESULTS
Strain-specific Genetic Labelling of Lactobacillus Helveticus
CNRZ32 (I)
Silent mutation positions were introduced in adjacent codons
of the Lactobacillus helveticus CNRZ32 chromosomal pepX gene
without altering the amino acid sequence of the gene product,
248 Introductory Food Microbiology
creating a strain-specific tag which enabled direct nucleic-acid
level identification of the changed strain. The mutation site was
introduced into the pepX gene with a PCR primer containing
designed mismatched nucleotides. The target gene was amplified
in two separate reactions to obtain a conserved fragment and a
mutation site-containing fragment, which were ligated together to
form a single mutation site-containing fragment. The mutant gene
was first cloned with pUC19 to E. coli to create plasmid pKTH5052
and further subcloned to pSA3, resulting in the plasmid pKTH5053,
which was then used to transform L. helveticus CNRZ32.
A L. helveticus CNRZ32 transformant strain GRL1021
containing the plasmid pKTH5053 was grown at a restrictive
temperature (45ºC) under antibiotic selection to find clones with
the thermosensitive plasmid integrated into the bacterial
chromosome through homologous recombination between the wild-
type and mutant forms of pepX. An erythromycin-resistant strain,
observed to contain the integrated plasmid, was grown at
permissive conditions (37ºC) for approximately 100 generations to
enable a second homologous recombination event between the
two pepX genes, resulting in excision of the plasmid and one of
the pepX genes. Samples were plated out and colonies that had
lost antibiotic resistance were screened with PCR for replacement
of the wild-type pepX with the mutated pepX. A strain with the
mutated gene was found and designated as GRL1023. Sequencing
was used to confirm the correctness of the integration construct.
The intact phenotypic properties of the GRL1023 were verified by
measuring the activity of the target gene product PepX by utilising
its ability to break down L-glycyl-Lprolyl- p-nitroanilide, with
production of colour. End-point PCR with PCR primers targeting
the mutated or original pepX sequence was successfully used to
detect the wildtype and mutant strains added to faeces or milk.
Suitability of PCR-ELISA for Analysis of Mixed DNA Populations
(II, III)
PCR-ELISA with Universal Primers Targeting the 16S and 23S
Genes (II)
A PCR-ELISA method was tested with 16 oligonucleotide
probes to simultaneously detect under standardised conditions
Modelling the Growth Boundary of Staphylococcus Aureus 249
selected intestinal bacteria, lactobacilli and bifidobacteria. For this
purpose, species- or group-specific oligonucleotide probes for
lactobacilli and selected intestinal bacteria were designed or
adapted from the literature. Specificity of the probes was tested
with 25 species. The 16S and 23S region of these test species was
first PCR-amplified with universal eubacterial primers, followed
by digoxigenin-labelling of the PCR products and addition of 2 ×
1010 labelled molecules to each hybridisation reaction. For
hybridisation, the oligonucleotide probes to be tested were bound
to the microtiter plate wells via biotin-streptavidin linkage. Under
the hybridisation conditions used, the specificity level expected
was obtained with most of the oligonucleotide probes chosen.
Only the differentiation of closely related L. casei, L. paracasei and
L. rhamnosus was partly unsatisfactory. Furthermore, in addition
to its intended target DNA, the F. nucleatum probe, fuso16S, also
recognized E. biforme. Sensitivity of the PCR-ELISA with the probes
tested varied between detection of 6.77 × 107 and 1.29 × 109 PCR-
amplified molecules in a hybridisation reaction. The sensitivity
obtained was somewhat lower than expected, which was at least
partly due to compromises that had to be made in the hybridisation
conditions when using several probes simultaneously.
PCR biasing was studied by combining genomic DNA from
different test species into two mixed sample pools, each with
seven DNA targets, prior to PCR amplification. PCR was also
performed using the pools in the same amplification.
The amplification products were hybridised with 14 different
probes. Most of the probes tested performed well and could detect
their target DNA from mixed samples of both seven and fourteen
species, but the probes for B. adolescentis (ado440, b162), L. brevis
(lab86), L. paracasei (par160), L. plantarum (pla448) and L. curvatus
(cur150) failed to recognize their target DNAs. Failure in the
detection of B. adolescentis, L. paracasei and L. plantarum in particular
could have been due to either bias during multi-template PCR,
where some templates are amplified more efficiently as a result of
differences in the specificity of eubacterial primers, or bias by
chance. Changing of the total template concentrations tested in the
PCR amplification was not observed to affect the hybridisation
results.
250 Introductory Food Microbiology
PCR-ELISA with bifidobacterial primers targeting the 16S genes
(III) The PCR-ELISA method was extended for detection of the
most common Bifidobacterium species in humans and applied to a
feeding trial including administration of Bifidobacterium lactis Bb-
12 and galacto-oligosaccharide -containing syrup as probiotic and
prebiotic preparations, respectively.
Oligonucleotide probes based on 16S rDNA sequences were
designed and tested for specificity and sensitivity with nine
different bifidobacterial species, followed by analysis of faecal
samples. Faecal bifidobacteria were monitored for fluctuations
during and after the feeding trial. Although the bifidobacterial
populations present in faeces were generally consistent between
samples taken from different time points, PCR-ELISA results
suggested that Bifidobacterium longum was partly replaced by B.
lactis Bb-12 during the probiotic feeding. This proposition could
not, however, be verified with statistical methods. Generally, the
bifidobacterial species composition of individual subjects was
consistent and comprised 3-4 bifidobacterial species or groups.
The predominant species were Bifidobacterium longum and B.
adolescentis.
Comparison of Real-time PCR and Dot Blot Hybridisation for
Quantification of the 16S Ribosomal DNA of Target Bacteria
(IV)
Real-time PCR was tested with two chemistries: SYBR Green
I and TaqMan probes. Six detection assays were tested for their
sensitivity in detecting target bacterial DNA from pure and mixed
samples with both chemistries. The same target DNAs were also
quantified with dot blot hybridisation probes. Real-time PCR was
shown to have sensitivity superior to dot blot hybridisation. The
linear range of amplification of pure cultured target DNA varied
between 0.1-1 pg and 1-10 ng of specific target genome, which
corresponds to approximately 20-400 to 2×105–4×106 genomes,
depending on the genome size of the target species. Furthermore,
a 1 pg addition of target DNA to 20 ng of mixed DNA samples
could be detected reliably with both real-time PCR chemistries. A
reconstruction assay with addition of 109, 108, 107, 106 or 105
target bacterial cells to faecal samples confirmed that real-time
Modelling the Growth Boundary of Staphylococcus Aureus 251
PCR could detect addition of 106 cells to one gram of faeces,
provided that the target bacterium was not included in the original
faecal sample. Addition of 109 cells of Bifidobacterium longum to
faeces resulted in no difference compared with control sample,
which had no added bacterial cells, due to the pre-existence of B.
longum in the original sample.
The two chemistries used in real-time PCR were equally
sensitive; however, successful usage of the intercalating dye SYBR
Green I required applying of a hot start polymerase to prevent
formation of primer dimers, whereas with 5´-nuclease chemistry,
a similar sensitivity level was reached with a conventional DNA
polymerase. Furthermore, the 5´-nuclease chemistry was faster to
perform because of its more straightforward incubation protocol,
one run taking approximately 80 min.
Suitability of L. brevis Strains as Dairy Supplements (V)
Two Lactobacillus brevis strains, ATCC 8287 and ATCC 14869T,
were evaluated for their applicability as putative probiotics in
dairy products. The strains tolerated well low pH, bile acids and
pancreatic fluid under in vitro conditions. They also expressed
good in vitro adherence to human CacO2 and Intestine-407 cells.
Adherence of the L. brevis strains was especially strong to the
small intestinal cell line, Intestine 407. In antimicrobial activity
assays, strain ATCC 8287 showed inhibitory properties towards
selected potentially harmful micro-organisms, particularly against
Bacillus cereus. Antimicrobial resistance tests revealed that both L.
brevis strains were resistant to vancomycin as well as to several
other antibiotics, which is, however, typical for the genus
Lactobacillus. The L. brevis strains were unable to acidify milk to
yoghurt but were suitable as supplement strains in yoghurts. This
was demonstrated by producing a set of yoghurt products and
analysing their rheological and sensory properties during a cold
storage period of 28 days. Despite its human origin, L. brevis
ATCC 14869T did not survive the gastrointestinal tract, whereas
L. brevis ATCC 8287 was detected in faecal samples taken during
and immediately after ingestion of the strain. In conclusion, L.
brevis ATCC 8287 was considered a promising candidate for use
as a probiotic adjunct in dairy products.
252 Introductory Food Microbiology
DISCUSSION
Molecular biology tools are needed for better understanding of
the composition and functions of the intestinal microbiota. In this
study, different levels of detection were developed for lactic acid
bacteria and faecal microbiota.
‘Probiotic studies require careful documentation of the
proposed effects. For better monitoring of a potential strain, strain-
specific discrimination could be beneficial in, for example, feeding
studies. Furthermore, specific recognition of industrial strains with
commercial value would help in monitoring the usage of these
strains. Therefore, Lactobacillus helveticus CNRZ32 was chosen as
a model organism for demonstration of genetic labelling. Although
the strain in question does not fulfil the criteria set for a probiotic,
selection of this organism was considered justified for several
reasons. The strain is a starter strain used in the dairy industry,
its proteolytic system has been studied extensively and methods
for its transformation and gene disruption have been developed.
Various ‘food-grade’ markers for lactic acid bacteria have been
suggested, often with introduction of new phenotypic properties.
However, creation of a label without introduction of any
phenotypic changes into the target strain would be more easily
accepted by consumers and legislators. Hertel et al. (1992) labelled
a Lactococcus lactis strain by introducing silent mutations into a
plasmid-encoded gene. By contrast, to ensure the stability of the
created tag, we used a chromosomal gene for labelling of L.
helveticus CNRZ32. As demonstrated in Study I, a silent mutation
site is effortlessly detected from different matrices, such as faeces
or milk, by PCR. Alternatively, hybridisation probes could be
utilised for detection.
Direct detection of target nucleic acids present in population
samples removes the need for cultivation steps. Here, PCR-ELISA,
dot blot hybridisation and real-time PCR were compared for their
suitability for analysis of population samples. Similar to PCRELISA
method described in this paper, DGGE- and TGGE-based
applications have been found to detect approximately 1-10%
subpopulations with PCR-amplified 16S rDNA fragments when
studying faecal samples, suggesting their use for analysis of
predominant microbes of population samples. By using DGGE
Modelling the Growth Boundary of Staphylococcus Aureus 253
with groupspecific 16S rDNA primers, however, characterisation
of diversity of smaller subpopulations has been reached. Similarly,
the bifidobacteria-specific PCR-ELISA could successfully be used
for species-level analysis of faecal bifidobacteria; previous DGGE
analysis results of the same samples by Satokari et al. (2001b)
generally support results obtained with PCRELISA. One drawback
of PCR-ELISA identification has, however, been uncovered. When
results were compared with PCR-ELISA analysis, it soon became
evident that ado440, intended to be specific for B. adolescentis, also
recognized B. ruminantium observed in the faeces of some volunteers.
New sequence alignments confirmed that the ado440 probe had
no mismatches with B. ruminantium 16S rDNA. While the
advantage of DGGE is the possibility for further sequence analysis
and identification of emerging new amplicons in DGGE profiles,
PCR-ELISA seems to be slightly more sensitive to changes in the
relative amounts of target species.
Strain-level studies have confirmed the complexity, uniqueness
and stability of bifidobacterial populations in the GI-tract. PCR-
ELISA results were in accord with earlier reports. The observed
species distribution correlated well with previous European
studies, and species distribution usually remained unchanged
within the samples of each subject. However, reduction of intensities
of B. longum - specific probes was observed frequently in the
groups that ingested the probiotic B. lactis Bb-12, although this
could not be verified with statistical analysis, mainly due to the
small size of the test groups. This suggests that B. lactis Bb-12 may
have partly replaced B. longum during the ingestion period.
Hybridisation allows direct semi-quantitative monitoring of
population samples and thus avoids the problems caused by PCR
bias. Dot blot hybridisation with rRNAtargeted oligonucleotide
probes is a well-established method for studying faecal microbes.
Ribosomal RNA is abundantly present in the bacterial cells, thus
increasing the sensitivity of the dot blot hybridisation to a
reasonable level. However, radioactively labelled probes are
generally needed for successful detection of nucleic acid targets
from population samples. Here, dot blot hybridisation was
performed with rDNA-targeted oligonucleotide probes to quantify
the same starting material as with the real-time PCR applications
254 Introductory Food Microbiology
tested. Dot blot hybridisation could, at its best, detect a 3% DNA
subpopulation from mixed DNA samples. A similar sensitivity
level has been reported by Muttray and Mohn (2000) for
measurement of rDNA:rRNA ratios. Thus, the result was as
expected, being approximately 10-fold less sensitive than that
obtained with rRNAtargeted oligonucleotides. Real-time PCR was
tested as an alternative for a sensitive detection of target bacteria
in faeces. The sensitivity level obtained for the real-time PCR
assays was 200-400 target bacteria in pure cultures or mixed DNA
samples, depending on the genome size of a target bacterium.
Results obtained from artificial DNA mixtures were confirmed by
a reconstruction assay; addition of 106 bacterial cells to faecal
samples containing approximately 1011 bacteria could be detected
with real-time PCR in a quantitative manner. The acquired level,
providing a means for quantifying a 0.01% subpopulation in a
DNA sample, was considered sufficient for studying the
gastrointestinal tract ecology.
Although nucleic acid based detection methods offer a means
to avoid bias caused by cultivation or phenotypic testing of bacterial
isolates, these techniques are not totally devoid of problems. The
main issues for detection of nucleic acids in mixed samples are
isolating DNA from different sources with constant efficiency,
minimising the occurrence of false-positive reactions due to
contamination or poor planning of the primers or hybridisation
probes used, and avoiding negative reactions due to inhibiting
substances. Also, any oligonucleotides used as detection probes or
primers should be considered specific only in relation to the current
sequence data. Several methods for faecal sample treatment for
isolation of DNA have been described. Nevertheless, this step
remains somewhat problematic because of the presence of PCR-
inhibiting substances in the faeces and the overall complexity of
faecal microbial populations. Although over 90% recovery of the
total bacterial DNA present in faeces has been claimed, non-
selective isolation of bacterial DNA from population samples is
difficult to achieve or demonstrate. Commercial kits have recently
become available for isolation of faecal DNA. However, McOrist
et al. (2002) observed differences in the relative efficacy of extraction
of bacterial DNA from faeces with four commercial kits.
Modelling the Growth Boundary of Staphylococcus Aureus 255
Furthermore, the relative sensitivities could not be extrapolated
from DNA extractions performed directly from pure cultures.
Evaluation of possible probiotic properties of a bacterial strain
requires intensive and carefully planned testing of the strain both
in vivo and in vitro. Although L. brevis is not typically used as a
probiotic, Kishi et al. (1996) have shown that oral administration
of live L. brevis ssp coagulans strain significantly stimulated the
host immunity system by increasing IFN-á production in human
subjects. This result cannot, however, be directly extrapolated to
the L. brevis strains studied here. Adherence of lactic acid bacteria
to various components of human intestine has been studied with
the human colon adenocarcinoma CacO2 cell line, Intestine-407
cells derived from human embryonic jejunum and ileum, and
intestinal mucus. Both CacO2 and Intestine-407 cells were used in
the adhesion assays for evaluation of the adhesion ability of L.
brevis. These cell lines offer complementary models for adhesion
studies. When compared with the probiotic L. rhamnosus GG,
adherence of the L. brevis strains was especially strong to the small
intestinal cell line, Intestine 407, suggesting that the small intestine
could be affected with the strains studied. L. brevis GRL62 has also
been shown to have intermediate adhesion properties to human
intestinal mucus.
The two L. brevis strains studied were antagonistic towards
some of the potentially harmful micro-organisms, while they did
not significantly inhibit the growth of yoghurt-starter bacteria. In
a previous study of Koga et al. (1998), L. brevis GRL1 (ATCC 8287),
included in a panel of 41 Lactobacillus strains belonging to L.
gasseri, L. reuteri, L. casei, L. helveticus, L. brevis, L. fermentum and L.
plantarum, weakly inhibited two Vibrio cholerae strains of the eight
strains tested. In the present study, L. brevis GRL1 was shown to
strongly inhibit B. cereus and to some extent also the growth of S.
aureus and other harmful micro-organisms chosen for the assay. In
vitro adhesion assays and testing for tolerance of low pH, bile
acids and pancreatic fluids have often been considered to be good
indicators of the survival of a bacterial strain through the GI tract.
In this study, both L. brevis strains performed well in the in vitro
tests, and survival through the stomach could therefore be
suggested for both strains, especially when simultaneous ingestion
256 Introductory Food Microbiology
of the bacteria with food, resulting in a higher pH in the stomach,
is taken into account. However, L. brevis GRL62 failed to survive
under in vivo conditions. This was surprising as the strain has
originally been isolated from human faeces. In contrast, L. brevis
GRL1, originally isolated from fermented olives, could tolerate the
gastrointestinal conditions and was found to persist in the human
gut.
Although the strains studied were not suitable for fermenting
yoghurt, supplementation of yoghurt with either of these strains
had no negative effects on yoghurt taste, appearance, or
preservation. Therefore, L. brevis GRL1, which was able to survive
in the GI tract, could be considered for use as a supplementary
strain in yoghurt or other dairy products. To date, several
commercial probiotics are already available and many others are
being studied, but no single strain is likely to possess all of the
beneficial properties suggested for probiotics. L. brevis GRL1, shown
to be antagonistic towards Bacillus cereus (this study) and Vibrio
cholerae, could be a valuable addition to the probiotics field. The
surface (S)-layer of this strain is well-characterized and potential
applications for the S-layer have been suggested. Recently, the S-
layer has been shown to mediate the ability of the L. brevis GRL1
strain to adhere to human intestinal, urinary bladder and
endothelial cells. Good adhesion properties could assist in the
competitive exclusion of potentially harmful microbes by L. brevis
GRL1, and usage of this strain as an effective mucosal vaccination
tool because of its binding properties may be warranted. More
studies are, however, required to confirm these hypotheses.
CONCLUSIONS
Creation of a strain-specific nucleic acid tag by labelling of a
chromosomal target gene with a silent mutation and utilisation of
the label for strain-specific detection of the target strain was
demonstrated using Lactobacillus helveticus CNRZ32. Although the
strain in question is not considered to be a probiotic, the method
described here could be used for probiotics. However, this requires
the pre-existence or development of suitable transformation tools
as well as sequence data of a suitable target gene. From the
viewpoint of current legislation and consumer acceptance, the
Modelling the Growth Boundary of Staphylococcus Aureus 257
silent labelling method should be the least unacceptable method
for making genetic alterations to a bacterial strain used in food
processing, especially as the phenotypic properties of the labelled
strain were demonstrated to remain unchanged, thus attesting to
the safety of the method. PCR-ELISA with universal primers was
shown to be suitable for detection of predominant bacteria present
in a mixed population sample. Specificity of the PCR step proved
to be critical for the sensitivity of the method; application of
bifidobacterialspecific 16S primers enabled species- or group-
specific detection of bifidobacteria present in human faeces. The
overall sensitivity of PCR-ELISA matched DGGE approaches, with
similar PCR primer specificities. Furthermore, being technically a
rather simple method, PCR-ELISA is easily transferred between
different laboratories, and could, in principle, be used in a similar
manner as DGGE for analysis of large sample numbers.
Real-time PCR was established to be a superior method for
detection of single bacterial species or larger groups from mixed
DNA samples or faeces. Compared with dot blot hybridisation
with radioactive probes, real-time PCR provided an improved
sensitivity for detection, a means to avoid usage of radioactive
labels, increased speed and volume of sample analysis, an easier
methodology and better reproducibility. Detection of target bacteria
added to faeces prior to the DNA isolation and purification steps
was demonstrated. In future, multiplexing with different labels
and improvements in hardware and software to allow
simultaneous monitoring of a large number of PCR reactions are
likely to make real-time PCR an even more potent tool for
population analyses.
L. brevis GRL1 could be considered to be a potential probiotic
strain. Although several probiotics are already on the market, new
strains with beneficial properties are continually being sought.
The strain in question has a well-characterized S-layer, and offers
promise as a potential mucosal vaccination strain.
258 Introductory Food Microbiology
9 of a foreign substance introduced into the organism. When
GLOSSARY
Acid dyes: Dyes that are anionic or have negatively charged
groups such as carboxyls. Acid fast: Bacteria like the mycobacteria
that cannot be easily decolorized with acid alcohol after being
stained with dyes such as basic fuchsin.
Acid-fast staining: Staining procedure that differentiates
between bacteria based on their ability to retain a dye when washed
with an acid alcohol solution.
Acidophile: Microorganism that has its growth optimum
between about pH 0 and 5.5.
Actinobacteria: Group of gram-positive bacteria containing
the actinomycetes and their high G 1 C relatives.
Actinomycete: Aerobic, gram-positive bacterium that forms
branching filaments (hyphae) and asexual spores.
Aerobe: The descriptive name given to a microorganism that
can grow in conditions where oxygen is present. Such organisms
are capable of growing in normal air, equivalent to 20% oxygen,
or in aerated liquids containing dissolved oxygen. Many aerobes
are equally able to grow in the absence of oxygen. These are
termed facultative anaerobes.
Allele: One of two or more alternative nucleotide sequences at
a single gene locus which occurs on either of two homologous
chromosomes in a diploid organism.
Anaerobe: A microorganism that is capable of growing in the
complete absence of oxygen. Some of these organisms may also be
able to grow in oxygenated conditions (facultative aerobes), whereas
Glossary 259
others cannot tolerate oxygen and are killed when exposed to air.
Such organisms are termed obligate anaerobes.
Antibody: An inducible immunoglobulin protein produced by
B lymphocytes of the immune system, in humans and other higher
animals, which recognizes and binds to a specific antigen molecule
antibodies bind to corresponding antigens they set in motion a
process to eliminate the antigens.
Antigen: Any foreign substance, such as virus, bacterium, or
protein, which after introduction into an organism (humans and
higher animals), elicits an immune response by stimulating the
production of specific antibodies; or any large molecule which
binds specifically to an antibody.
Antimicrobial: A chemical agent that kills microorganisms or
inhibits their growth.
Apoptosis: Programmed cell death, the body’s normal method
of ending the lifecycle of cells through the cellular self-destruction.
When either heritable or somatic cell mutations cause malfunctions
to occur in the apoptotic pathway, uncontrolled cell growth may
proceed unchecked and cancer may result.
Bacterial growth: Can exhibit at least four different phases:
lag phase, growth phase, stationary phase and death phase.
Bacterial strain: Population of bacterial cells all descended
from a single pure isolate.
Base pair (bp): Two complementary nitrogenous bases in a
DNA molecule, such as the nucleotide coupling of adenine with
thymine (A:T) and guanine with cytosine (G:C); also, a unit of
measurement for DNA sequences.
Biofilm: Adherent layer of bacteria and/or other
microorganisms on a solid surface bound together in a bacterially-
derived polysaccharide matrix that is protective for the organisms;
generally occuring at a liquid/solid interface and often developing
into a complex ecological community (e.g., dental plaque bound
tother by dextrans).
Bleaching: The loss of fluorescence usually due to
photochemical reactions.
260 Introductory Food Microbiology
cDNA (complementary or copy DNA): DNA copies
synthesized from a messenger RNA template using the enzyme
reverse transcriptase; the single-stranded copy is often used as a
probe to identify complementary sequences in DNA fragments or
genes of interest.
Chromosome: A single DNA molecule that is the self-
replicating genetic structure within the cell which carries the linear
nucleotide sequence of genes. In humans (or eukaryotes), the DNA
is supercoiled, compacted, and complexed with accessory proteins,
and organized into a number of such structures. Normal human
cells contain 46 chromosomes (except the germ cells, egg and
sperm): 22 homologous pairs of autosomes and the sex-determining
X and Y chromosomes (XX for females and XY for males).
Prokaryotes carry their entire genome on one circular chromosome
of DNA.
Coccus: (singular): Spherical-shaped cell; cocci (plural).
Coliform bacteria (coliforms): Any fermentative (specifically
lactose-fermenting) Gram-negative anaerobic enteric bacilli (E. coli-
like).
Commensal: Organisms existing in or on an animal or human
without causing disease.
Conjugation (or bio-conjugation): The chemical joining of a
biomolecule to another.
Death phase: The death phase occurs when cells are being
inactivated or killed because conditions no longer support growth
or survival. Some environmental factors such as temperature can
cause acute inactivation. Others may cause mild inactivation as
with growth in the presence of organic acids.
DNA (deoxyribonucleic acid): The nucleic acid molecule
consisting of deoxyribonucleotide building blocks that encode
genetic information. The genome of most organisms is contained
in a double-stranded, double-helical form held together with
chemical bonds between each strand of complementary nucleotide
base pairs.
DNA probe: A single-stranded piece of DNA that binds
specifically to a complementary DNA sequence; the probe is labeled
Glossary 261
(e.g., with a fluorescent or radioactive tag) in order to detect its
incorporation through hybridization with DNA in a sample.
Dot blot: A method for detecting proteins by the
specific binding of an antibody or binding molecule to a
sample spot on nitrocellulose paper. The bound sample is
visualized using an enzymatic or fluorimetric reporter conjugated
to the probe.
Doubling time: The time taken for a population to increase in
number by a factor of two.
Enteric (entero-): Relating to the intestine.
Enterotoxin: Proteins produced by bacteria that are either
ingested as pre-formed toxins or are produced by a pathogen that
has colonised the gastro-intestinal tract. Usually the toxin has
specific targets and either disrupts cell function or kills the cell.
Eukaryote: (meaning “true nucleus”) An organism which
possesses a nucleus with a double layer of membrane and other
membrane-bound organelles; includes such unicellular or
multicellular members as all members of the protist, fungi, plant,
and animal kingdoms.
Exons: The segment of a gene present in mature mRNA
transcripts that specify the amino acid sequence of a polypeptide
during translation; exons of a gene are linked together by mRNA
splicing.
Exotoxin: Potent toxic substance formed and released
extracellularly by species of certain bacteria.
Exponential phase: The period in which the cells of a defined
bacterial population are growing and dividing continuously.
Extracellular: Produced, then excreted outside the organism.
Facultative: Ability to adapt and live under various conditions.
Facultative anaerobe: Anaerobe that can survive with or
without oxygen.
Family: Taxonomic level below order and above genus.
Fastidious: Complex nutritional or cultural requirements,
making isolation and culture of a fastidious organism more
difficult.
262 Introductory Food Microbiology
Fermentation: Enzymatic breakdown (catabolism) of
carbohydrates generally in the absence of oxygen.
Fimbriae: Short, hair-like projections or appendages
(organelles) on the outer surface of certain bacteria composed of
protein subunits (pilin) extending outward from the surface that
act as a virulence factor by promoting adherence; formerly known
as pili; fimbria (singular).
Flagellum: whip-like bacterial locomotory (provide motility)
organelles anchored in the cell membranes that are composed of
helically-coiled protein subunits (flagellin); flagella (plural).
FISH (Fluorescence In Situ Hybridization): A technique that
employs fluorescent molecular tags to detect probes hybridized to
chromosomes or chromatin; useful for genetic mapping and
detecting chromosomal abnormalities.
Flow cytometer: Analytical instrument for flow cytometry.
Flow cytometry: Automated analysis of cells or subcellular
components by detection of the fluorescence or light-scatter of
sample fractions passing in narrow-stream droplets through a
laser beam.
Fluorophores: Molecules that produce a fluorescent
emission when irradiated with light at a suitable excitation
wavelength.
Fomite: Inanimate object capable of transmitting infectious
organisms to a host, e.g., soiled clothes, tissues and handkerchiefs,
food processing equipment, dishrags, etc.
Gene amplification: The presence of multiple copies of a gene
or segment of DNA; a mechanism by which proto-oncogenes are
activated in malignant cells. A tumor cell amplifies, or copies,
DNA segments as a result of cell signals or the effects of
environmental insults.
Gene expression: The process by which the encoded
information of the genome is converted into cellular components.
The DNA-coding sequences of expressed genes include those that
are transcribed into mRNA and then translated into proteins, and
RNA that is transcribed from DNA, yet not translated into protein
(i.e., transfer and ribosomal RNAs).
Glossary 263
Gene mapping: A linear map determining the relative position
of genes along a chromosome or plasmid. Distances are established
by linkage analysis and measured in linkage units.
Gene: A nucleotide sequence of DNA that codes for a
protein, or functional or structural RNA molecule; a locus
on a chromosome. The element that determines a trait in an
organism.
Genetic mutation: An alteration in the nucleotide sequence of
a DNA molecule; often from one allelic form of a gene to another
allele alternative.
Genome: The total amount of genetic material in a cell; in
eukaryotes the haploid set of chromosomes of an organism. The
chromosome set is species-specific for the number genes and linkage
groups carried in genomic DNA.
Genomics: The study of genes and their biochemical function
in an organism.
Genotype: The genetic constitution of an organism; or, a
reference to an individual’s particular allele pair at a specific gene
locus in the genome.
Genotyping: Analysis of genotype.
Genus: Taxonomic level below Family and above Species.
Gram-negative or Gram-positive: The classification given to
bacteria according to their staining properties as defined by the
Gram stain procedure.
Growth curve: A graph displaying the behaviour of a bacterial
population over time.
Growth phase: During the growth phase, cells grow
exponentially and at a constant rate. The maximum slope of the
curve is the specific growth rate of the organism. Cell growth is
dependent upon the current environment (nutrients, temperature,
pH, etc.), but is not dependent upon the previous physiological
state. In the field of predictive microbiology, growth rate is
commonly expressed as the change in cell number per time interval.
Growth rate: This is an expression of population increase in
numbers expressed as log10 cfu/hour.
264 Introductory Food Microbiology
Haploid: A cell or individual with a genetic complement
containing one copy of each nuclear chromosome. (Diploid refers
to the condition when a eukaryotic cell possesses two sets of
chromosomes.)
Humectant: A solute that binds free water in a food, reducing
the amount of water available to the microorganisms.
Hybridize (or hybridization): The process where the hydrogen
bonding of complementary DNA and/or RNA sequences forms a
duplex molecule.
Immunoassays: A technique that detects and measures a
specific antigen or biological substance by employing antibodies
(e.g., dot blot, western blot, and ELISA).
in situ hybridization (ISH): Use of a nucleic acid probe to
detect and identify specific complementary sequences of DNA in
chromosomes or RNA in bacteria, eukaryotic cells, and tissue.
in vitro: (“in glass”) Refers to the recreation of biological
processes in an artificial environment such as a test tube.
in vivo: (“in living”) Refers to biological processes within a
living organism or cell.
Inoculum: A medium containing microorganisms to be
introduced into fresh media or food source in an experiment.
Intron: A nucleotide sequence intervening between exons
(coding regions) that is excised from a gene transcript during
RNA processing.
Kinetics: The properties of chemical agents or enzymes in the
efficiency and speed of their action upon a chemical reaction.
Klebsiella: Gram-negative rods occur in human feces and
clinical specimens, soil, water, grain, fruits, and vegetables. Some
species are opportunistic pathogens. Kluyvera: Gram-negative rods
occur in food, soil, sewage, and human clinical specimens. They
are infrequently opportunistic pathogens. Kurthia: Gram-positive
rods are widely distributed in the environment, and are common
in animal feces and meat products.
Lag phase: During the lag phase, cells increase in size but not
in number because they are adapting to a new environment, and,
synthesis and repair are taking place. The length of the lag phase
Glossary 265
depends on the current environment as well as the previous
physiological state of the cells. Cells that are from a very different
environment or are damaged from their previous physiological
state may require more time to adjust. In some foods a lag phase
does not exist which results in cells that are ready for immediate
growth.
Lag time: The initial period in a bacterial population life
when cells are adjusting to a new environment before commencing
growth.
Legionella: Fastidious gram-negative rod is isolated from
surface water, mud, and thermally polluted lakes and streams.
There is no known soil or animal source. It is pathogenic for
humans, causing pneumonia (Legionnaires’ disease) or a mild,
febrile disease (Pontiac fever).
Ligand: The molecule which binds to a protein molecule (e.g.,
receptor). As a ligand binds through the interaction of many weak,
noncovalent bonds formed to the binding site of a protein, the
tight binding of a ligand depends upon a precise fit to the surface-
exposed amino acid residues on the protein.
Listeria: Gram-positive rod widely distributed in the
environment. Some species are pathogenic for humans and animals
(e.g. L. monocytogenes).
Locus: (plural, loci) The specific site of a gene on a genetic
map or chromosome.
Marker (genetic marker): Any genetically derived phenotypic
difference used in the analysis of inheritance patterns or to
differentiate between types of cells. An observable site on a
chromosome that is heritable and can be either a genetically-
expressed region or noncoding segment of DNA (intron).
Maximum population density: Point at which the maximum
number of bacterial cells can exist in an environment.
Meiosis: Process that allows one diploid celll to divide in a
special way to generate haploid cells in eukaryotes.
Mesophile: Microorganism able to grow well between 20°C
and 45°C, having an optima of 30°C to 40°C. Many can sustain
growth at below 10°C albeit very slow growth.
266 Introductory Food Microbiology
Metaphase: Stage in mitosis or meiosis during which
the chromosomes are aligned along the equatorial plane of the
cell.
Methylobacterium: Mostly isolated from water and leaf surface
microflora, and are facultative methylotrophs, that is capable of
growing on one-carbon compounds such as formate, formaldehyde,
and methanol as the sole source of carbon and energy, as well as
on a wide range of multicarbon substrates.
Microaerophilllic environment: Environment with reduced
oxygen concentrations, often below 5%. Carbon dioxide levels
may approach 10%.
Microbacterium: Gram-positive rod is found in dairy products,
sewage, and insects.
Microbial load: Total number of living microorganisms in a
given volume or mass of microbiological media or food.
Micrococcus: Gram-positive cocci occur primarily on
mammalian skin and in soil, but are commonly isolated from food
products and the air.
Microorganism: A living organism too small to be seen with
the naked eye.
Mitosis: Process by which a cell separates its duplicated
genome into two identical halves. It is generally followed
immediately by cytokinesis which divides the cytoplasm and cell
membrane. This results in two identical daughter cells with a
roughly equal distribution of organelles and other cellular
components.
Moraxella: Gram-negative rod is parasitic on the mucous
membranes of humans and other warm-blooded animals.
MRNA (messenger RNA): The RNA molecule, transcribed
from the DNA of a gene, which serves as a template and encodes
the amino acid sequence of a protein.
Multiplexing: Method by which many parameters are tested
and processed simultaneously.
Native microflora: Microorganisms that are normally found
within a food source (often referred to as spoilage organisms).
Glossary 267
Northern blot: Technique used to separate and transfer mRNA
from a gel to a filter in order to identify and locate mRNA sequences
that are complementary to and hybridize with a labeled DNA
probe.
Novosphingobium: Gram-negative rods were originally
included with Sphingomonas (see Sphingomonas).
Nucleic acid: Molecule composed of nucleotide subunits. See
DNA and RNA.
Nucleotide: Basic building block of nucleic acids that is a
monomeric molecule of DNA or RNA composed of: a pentose
sugar (with 5-carbons such as deoxyribose in DNA, ribose in
RNA), an organic nitrogenous base, and a phosophate group.
DNA consists of the four bases: adenine (A), guanine (G), cytosine
(C), and thymine (T); likewise for RNA, except for the substitution
of uracil (U) for T.
Oligonucleotide: (“oligo” means few) A short length of DNA
nucleotides, often used as primers for DNA synthesis or probes for
arrays and ISH/FISH; usually referred to as “oligo(s).”
Opportunistic: Microorganism that will only cause disease in
a patient with a poor or somehow weakened immune system.
Organelle: Microscopic bodies in the cytoplasm of cells that
have distinctive functions (e.g., nucleus, mitochondria,
endoplasmic reticulum, etc.).
Pasteurisation: Mild heat treatment process given to foods.
The process is designed to eradicate potential vegetative pathogens
(not bacterial spores) and reduce other microorganism numbers in
an effort to decrease the rate of spoilage.
Pantoea: Gram-negative rods are isolated from plant surfaces,
seeds, soil, and water, as well as from animals and human clinical
specimens. They are opportunistic human pathogens.
Pathogen: Microorganism associated with disease in man.
pH: Measure of the acidity or alkalinity of a solution, defined
as the - log10 of the hydrogen ion concentration.
Phenotype: Observable manifestation of a genetic trait,
resulting from a specific genotype and its interaction with the
environment.
268 Introductory Food Microbiology
Physical map: Map indicating physical locations on a DNA
molecule such as restriction enzyme recognition sites, RFLPs, and
genes; measured in base pairs (bp).
Predictive microbiology: Area of food microbiology that uses
mathematical models to define growth kinetics of microorganisms
in food.
Primary model: Model that describes changes in microbial
numbers in response to time.
Primer: Short segment of DNA or RNA that anneals to
a single strand of DNA in order to initiate template-
directed synthesis and extend a new DNA strand by the
enzymatic action of DNA polymerase to produce a duplex-
stranded molecule.
Probe: Single-stranded DNA or RNA molecule of specific base
sequence, either radioactively or fluorescently labeled, that is used
to identify the complementary nucleotide sequence by
hybridization to the DNA fragment or gene of interest.
Protein translocation: Spatial movement of protein within a
cell (e.g., from the cytoplasm to nucleus, or into organelles).
Protein: High-molecular weight biological molecule composed
of a polymer of amino acids linked via peptide bonds; may consist
of more than one polypeptide molecule that is folded into complex
shapes such as helices or sheet-like structures. Proteins are encoded
by the specific sequence of DNA nucleotides in a gene and give
rise to the structure, function, and regulation of cells, tissues, and
organs within the body. Protein classes include enzymes,
antibodies, receptors, hormones, and growth factors.
Proteome: Entire protein make-up of a particular organism.
Proteomics: Study of proteins and their biochemical function
in an organism.
Providencia: Gram-negative rods are isolated from human
clinical specimens and from penguins.
Pseudomonas: Gram-negative rod is widely distributed in
nature. Some species are pathogenic for humans, animals, or plants
(e.g. P. aeruginosa).
Glossary 269
Psychrobacter: Gram-negative rod is associated with fish,
processed meat and poultry products. Some strains have been
isolated from pathological specimens from humans and animals.
Psychrotroph: Microorganism able to grow well between 0°C
and 7°C, having an optima of 20°C to 30°C.
Rahnella: Gram-negative rods occur in freshwater. They are
occasionally isolated from human clinical specimens, but are not
considered clinically significant.
Ralstonia: see Pseudomonas.
Raoultella: see Klebsiella.
Rathayibacter: Some of these species are phytopathogens of
terrestial plants. Their main habitats are their respective plant
hosts.
Receptor: Surface-exposed membrane protein on a cell which
binds to a specific ligand molecule with high affinity, in order to
transmit an extracellular signal and trigger intracellular
biochemical events within the target cell.
Reporter: Gene which codes for an easily measured protein
product and is fused downstream of the gene of interest in order
to assess the activity in the region upstream of the reporter gene.
Colorimetric and fluorimetric reporters also can be conjugated to
probes to monitor biological events.
Rhodococcus: Aerobic, Gram-positive actinomycetes. These
widely-occurring organisms are of considerable environmental
and biotechnological importance due to their broad metabolic
diversity and array of unique enzymatic capabilities, plus their
capacity to degrade hydro-carbons. They are able to survive for a
long time in soil. They are the most efficient in oil degradation
and, relatively speaking, the most abundant in soils and marine
environments.
Rhizobium: Group of small, rod-shaped, gram-negative
bacteria, which are able to produce nodules on the roots, or on
some cases the stems, of leguminous plants.
RNA (ribonucleic acid): DNA-like organic molecule that
consists of nucleotide subunits—such as adenine, guanine,
cytosine, and uracil—which contain ribose sugars linked through
270 Introductory Food Microbiology
phosphodiester bonds. Different types of RNA function in the
process of gene expression.
Saprophyte: Microorganism that normally grows on dead
material.
Secondary model: Model that predicts changes in primary
model parameters based on environmental conditions.
Serratia: Gram-negative rods occur in human clinical
specimens, soil, water, plant surfaces, and other environmental
sites, digestive tracts of rodents, and insects. Some species are
opportunistic pathogens.
SNP (Single Nucleotide Polymorphism): Variations in the
sequence of DNA among individuals that are present in humans
with a frequency of about once in every 1000 bases, and useful in
assessing the patterns of inheritance in genetic linkage studies.
Somatic cell mutation: Mutation in a cell that is acquired
during the lifetime of an organism and which cannot be genetically
inherited by offspring.
Somatic cell: Any cell in the body except the germ-line cells
(sperm or egg cells).
Southern blot: Procedure which transfers elecrophoretically
separated DNA fragments on an agarose gel to nitrocellulose
filters for detection by hybridization with a labeled probe
complementary to the sequence of interest; the position on the
filter of the probe, when exposed to x-ray film, appears as a band
on an autoradiogram.
Sphingobacterium: Gram-negative rod found in soil, on plants,
foodstuffs, and in water sources. Sphingobium: Gram negative
rods were originally included with Sphingomonas (see
Sphingomonas).
Sphingomonas: Relatively new genus derived from
Pseudomonas paucimobilis. These organisms are widely
distributed, including having been found in water. Only
Sphingomonas paucimobilis is considered clinically significant,
and has been isolated from a variety of clinical specimens
Spoilage organisms: Microorganisms naturally found within
a food source that cause food spoilage.
Glossary 271
Staphylococcus: This gram-positive coccus is mainly
associated with the skin and mucous membranes of warm-blooded
vertebrates, but they are often isolated from food products, dust
and water. Some species are opportunistic pathogens of humans
and animals, or produce extracellular toxins.
Stationary phase: The stationary phase occurs at the maximum
population density, the point at which the maximum number of
bacterial cells can exist in an environment. This typically represents
the carrying capacity of the environment. However, environmental
factors such as pH, preservatives, antimicrobials, native microflora
and atmospheric composition as well as depletion of growth-
limiting nutrients can affect the maximum population density.
Stenotrophomonas: see Pseudomonas.
Sub-lethal injury: This is cellular damage which results in
disruption of metabolic processes which, under ideal conditions,
is repairable. Such damage must be repaired before normal growth
can recommence.
Tertiary model: Computer software routines that turn the
primary and secondary models into “user-friendly” programs.
Tsukamurella: Gram-positive rods are isolated from soil,
human sputum, and parts of bed bugs. Some strains can be
pathogenic.
Toxins: Compounds produced by a microorganism that are
poisonous to other organisms.
Vegetative cell: The vegetative cell state is the form in which
an organism is able to grow and divide continuously, given
favourable conditions. Unlike endospores, vegetative cells are
relatively poor at surviving environmental stresses such as high
temperature and drying.
Water activity (aW): Water activity is a measure of the amount
of free unassociated water molecules in a system. It runs on a scale
of 0 to 1.0, where pure water equals 1.0. This parameter can also
be expressed as a percentage referred to as the relative humidity.
It is influenced by dissolved solutes and insoluble food components
which act to bind water, thus reducing the available free water
and hence aW value.
272 Introductory Food Microbiology
Weeksella: Gram-negative rod is not known in the general
environment. It is apparently a parasite, saprophyte, or commensal
of the internal surfaces of humans or other warm-blooded animals.
Western blot: A technique which transfers proteins
electrophoretically separated in a polyacrylamide gel to a
nitrocellulose membrane and uses specific antibodies to bind,
locate, and visualize the protein of interest.
Yersinia: Gram-negative rods occur in a broad spectrum of
habitats, including humans, animals, soil, water, dairy products,
and other foods. Some species are pathogenic for humans and
animals; others are opportunistic pathogens, yet others are
nonpathogenic.
Xanthomonas: Most Gram-negative rods are plant pathogens,
or occur in association with plants. X.maltophilia is the only
exception, being an opportunistic pathogen of humans.
Zoonotic: Microorganism normally found in or on animals.
Bibliography 273
BIBLIOGRAPHY
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274 Introductory Food Microbiology Index 275

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Ihekoronye, A.I., and Ngoddy, P.O., : Integrated Food Science and INDEX
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A 202, 209, 211, 215, 216,
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Accounts, 4. 219, 230, 231, 232, 233,
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Acid Dyes, 258.
Metting, Jr., F.B.: Soil Microbial Ecology: Applications in Agricultural Acidophile, 258. 239, 240, 241, 242, 243,
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Macmillan Press Ltd., London, 1985. Anaerobe, 258, 261. 257, 258, 259, 261, 262,
Apoptosis, 84, 259. 263, 264, 265, 271.
Roberts, D.: Food Poisoning and Food Hygiene, Edward Arnold Ltd,
Application, 17, 26, 28, 69, 176, Bioscreen, 31, 32, 48, 60, 71,
London, 1987. 84, 93, 95, 96, 97, 100,
229, 244, 257.
Sackler, Warren: Foodservice Cost Control Using Microsoft Excel for 108, 111, 113, 115, 120,
Approach, 30, 43, 97, 107, 112,
Windows. New York: Wiley, 1996. 121, 129, 158, 236, 238, 123, 124, 126, 132, 140,
Shetty, Kalidas: Food Biotechnology, New York, Dekker/CRC Press, 242, 266. 147, 148, 149, 152, 159,
2005. Association, 20, 69, 70, 76, 79, 161, 162, 163, 164, 167,
175, 178, 196, 272. 175, 203, 220, 221, 222,
Sims-Bell, Barbara: Career Opportunities in the Food and Beverage
Atmospheres, 13. 247.
Industry. New York: Facts on File, 1994.
Biotechnology, 61.
Slater, R.J.: Experiments in Molecular Biology, Clifton, Humana Press, B
1986. C
Bacteria, 1, 8, 11, 12, 13, 14,
Sprenger, R.A.: The Food Hygiene Handbook, Highfield Publications, 26, 30, 39, 46, 49, 50, Campylobacter, 5, 242.
Doncaster, 1996. 53, 55, 56, 57, 58, 63, Capacity, 22, 27, 57, 58, 69,
66, 67, 68, 70, 72, 73, 92, 106, 111, 113, 139,
Susan, R.: Biotechnology: An Introduction, Belmont, Thomson/Brooks/
76, 81, 84, 85, 86, 87, 269, 271.
Cole, 2005.
88, 89, 90, 91, 92, 93, Carbohydrates, 8, 10, 11, 19,
Thomas R.: Bountiful Harvest: Technology, Food Safety and the 22, 193, 199, 233, 262.
95, 101, 105, 107, 109,
Environment, Washington DC, Cato Institute, 2003. Carbon Dioxide, 87, 180, 183,
115, 116, 119, 120, 123,
Walden, Richard: Genetic Transformation in Plants, England, Open 124, 125, 127, 128, 130, 184, 266.
University Press, 1988. 141, 146, 148, 160, 162, Carnobacteria, 8, 9, 10, 11, 12,
Wallace, L.: Introduction to Professional Foodservice. New York: 165, 167, 168, 170, 171, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 26, 27,
Wiley, 1996. 173, 176, 177, 178, 180,
181, 182, 183, 184, 185, 166, 167.
Zaccarelli, Herman E.: Food Service Management by Checklist: A Carnobacterium, 8, 9, 10, 11,
Handbook of Control Techniques. New York: Wiley, 1991. 186, 187, 188, 200, 201,
12, 13, 14, 15, 16, 19,
276
20, 21, 22, 23, 24, 25,
26, 166, 169, 170.
Chromosome, 16, 49, 62, 74,
248, 260, 263, 264, 265.
Commission, 156, 178.
Community, 13, 18, 23, 236,
237, 238, 259.
Comparison, 11, 37, 39, 42, 51,
87, 90, 100, 103, 125,
133, 136, 138, 143, 145,
146, 155, 159, 160, 164,
174, 178, 179, 188, 226,
229, 238, 240, 241, 250.
Conditions, 12, 13, 18, 19, 22,
29, 30, 33, 34, 37, 39,
41, 43, 45, 46, 47, 50,
52, 53, 54, 57, 58, 59,
61, 63, 78, 85, 87, 88,
89, 93, 94, 95, 97, 98,
99, 100, 101, 102, 103,
104, 105, 106, 107, 108,
111, 112, 113, 114, 115,
116, 117, 118, 119, 120,
121, 122, 123, 124, 125,
126, 127, 130, 131, 132,
134, 135, 137, 138, 140,
141, 142, 143, 144, 145,
146, 147, 152, 153, 154,
155, 156, 157, 158, 162,
163, 164, 165, 176, 180,
182, 185, 186, 196, 200,
201, 203, 207, 209, 210,
215, 219, 222, 225, 226,
227, 241, 244, 246, 248,
249, 251, 256, 258, 260,
261, 270, 271.
Conjugation, 260.
Conservation, 68, 134.
Constitution, 144, 263.
Construction, 33, 49, 73, 74,
129, 130, 133, 135, 136,
147, 148, 222, 236.
Introductory Food Microbiology
Cultures, 8, 9, 17, 22, 24, 31,
47, 48, 49, 52, 53, 60,
68, 69, 70, 71, 72, 76,
77, 78, 79, 80, 81, 82,
85, 95, 96, 97, 98, 100,
113, 120, 122, 123, 126,
130, 133, 139, 148, 156,
167, 181, 191, 196, 197,
198, 200, 206, 209, 212,
213, 216, 220, 240, 254,
255.
D 165, 181, 184, 218, 233,
Department, 30, 219.
Development, 26, 33, 34, 35,
36, 43, 80, 82, 129, 175,
200, 219, 224, 232, 256.
Diagnostics, 34, 36.
Distribution, 3, 9, 23, 26, 34,
35, 36, 93, 94, 96, 99,
101, 102, 103, 105, 111,
120, 129, 156, 178, 224,
227, 253, 266.
Diversity, 17, 27, 176, 232, 236,
237, 253, 269.
DNA, 25, 48, 49, 50, 60, 61,
62, 64, 72, 73, 74, 131,
168, 169, 171, 173, 174,
183, 229, 233, 235, 236,
237, 238, 239, 240, 241,
242, 243, 244, 245, 246,
247, 248, 249, 250, 251,
254, 255, 257, 259, 260,
261, 262, 263, 264, 265,
266, 267, 268, 269, 270.
E Institute, 25, 37, 151, 152, 245.
Ecology, 4, 26, 165, 254.
Economic Growth, 2.
Ecosystem, 231.
Electron Microscopy, 45, 50, 53,
197.
Index
Emission, 50, 179, 262.
Energy, 10, 43, 202, 266.
Enteric Viruses, 2.
Enterotoxin, 4, 6, 28, 34, 39,
217, 220, 222, 225, 261.
Environment, 4, 8, 9, 10, 11,
14, 26, 27, 46, 55, 57,
58, 59, 83, 84, 94, 101,
107, 111, 112, 113, 119,
120, 121, 122, 123, 135,
136, 138, 139, 142, 143,
144, 145, 146, 147, 156,
263, 264, 265, 266, 267,
271, 272.
Escherichia Coli, 6, 10, 47, 59,
71, 101, 128, 130, 169,
184, 232, 242.
Eukaryote, 261.
Evaluation, 131, 135, 145, 178,
185, 229, 230, 247, 255.
Evolution, 2, 110, 112, 129, 135,
136, 165, 180, 205.
Experiments, 31, 32, 39, 45, 47,
48, 59, 60, 61, 63, 66,
76, 87, 95, 96, 98, 105,
107, 108, 110, 111, 114,
120, 121, 122, 123, 126,
127, 128, 130, 132, 133,
134, 136, 139, 140, 141,
145, 148, 149, 161, 164,
165, 167, 174, 175, 193,
194, 205, 210, 211, 217,
227.
F 157, 158, 159, 161, 264.
Farmers, 178.
Fermentation, 1, 9, 69, 78, 81,
82, 106, 180, 182, 184,
214, 233, 262.
Fermentor, 108, 111, 122, 123,
124, 126.
277
Fluorophores, 262.
Food Contamination, 26.
Food Microbiology, 1, 94, 136,
268.
Food Safety, 1, 5, 21, 27, 43,
44, 107, 112, 128, 141,
172, 222, 223, 233.
Foodborne Pathogens, 2, 137,
139.
G
Genetic Mutation, 263.
Genomics, 25, 27, 263.
I
Identification, 9, 31, 45, 57, 60,
62, 130, 133, 176, 177,
193, 202, 206, 208, 215,
229, 236, 237, 239, 248,
253.
Industry, 9, 29, 44, 46, 57,
128, 158, 172, 173, 187,
214, 252.
Infections, 4, 5, 23, 234.
Information, 6, 10, 12, 19, 25,
26, 33, 46, 47, 57, 58,
61, 68, 94, 109, 154, 172,
187, 193, 194, 226, 229,
236, 260, 262.
Injection, 24, 86, 87, 89.
Inoculum, 86, 87, 93, 94, 95,
96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 127,
131, 140, 146, 148, 156,
Institutions, 23.
Instruments, 206.
Integration, 49, 74, 235, 248.
Interests, 112.
Isolation, 5, 9, 10, 49, 165, 175,
278
192, 203, 204, 234, 245,
254, 257, 261.
L Microbiology, 1, 48, 59, 71, 94,
Laboratory, 19, 21, 43, 70, 86,
92, 136, 149, 151, 167.
Legislation, 235, 256.
Listeria Monocytogenes, 5, 8, 17,
45, 57, 83, 93, 95, 106,
107, 112, 113, 122, 136,
137, 146, 147, 149, 157,
166, 170, 183, 242.
Literature, 29, 38, 174, 227,
228, 231, 249.
Low Virulence, 6, 83, 84, 85,
88, 90, 91, 92.
M Mycotoxins, 3.
Majority, 26, 39, 147, 155, 157,
158, 178, 198, 231.
Management, 180.
Manufacture, 1, 68, 69.
Measurement, 31, 32, 33, 44,
76, 133, 149, 152, 162,
195, 206, 221, 225, 238,
242, 247, 254, 259.
Mechanism, 4, 6, 43, 46, 59,
99, 171, 172, 184, 190,
199, 215, 262.
Media, 12, 19, 21, 30, 31, 32,
34, 47, 52, 53, 59, 60,
61, 63, 70, 84, 90, 93,
96, 99, 100, 108, 109,
113, 114, 115, 120, 121,
123, 124, 125, 126, 130,
135, 139, 141, 148, 157,
159, 160, 169, 177, 192,
201, 214, 220, 221, 222,
225, 244, 264, 266.
Methodology, 26, 132, 149, 242,
243, 257.
Introductory Food Microbiology
Methylobacterium, 266.
Microbacterium, 266.
Microbial Ecology, 26.
106, 112, 116, 119, 122,
129, 132, 136, 146, 147,
263, 268.
Microorganisms, 1, 2, 8, 17,
29, 30, 40, 52, 130, 133,
137, 183, 189, 194, 195,
197, 198, 200, 211, 212,
232, 272.
Microplates, 48, 59, 60, 71, 124,
148.
Molecules, 41, 104, 172, 182,
196, 228, 237, 249, 262,
271.
N 160, 161, 165, 221, 227,
Natural Environment, 9, 10, 11,
14.
Network, 61, 106, 107, 109,
197.
O 107, 109, 111, 112, 113,
Observations, 55, 89, 91, 116,
126, 134, 137, 178.
Opportunistic, 22, 24, 264, 267,
270, 271, 272.
Organelle, 267.
Organisms, 6, 10, 11, 14, 17,
18, 19, 22, 27, 63, 69,
83, 101, 106, 107, 112,
119, 121, 128, 129, 136,
172, 180, 182, 184, 186,
201, 203, 209, 215, 220,
229, 230, 231, 234, 235,
236, 241, 251, 255, 258,
259, 260, 262, 266, 269,
270, 271.
Index
Osmotolerance, 45, 54, 55, 58,
59.
P 41, 43, 44, 55, 60, 69,
Pathogens, 1, 2, 3, 4, 5, 6, 8,
18, 24, 27, 30, 137, 139,
166, 172, 219, 231, 233,
240, 241, 264, 267, 270,
271, 272.
Performance, 116.
Plants, 11, 13, 14, 26, 27, 68,
148, 165, 173, 178, 268,
269, 270, 272.
Population, 6, 32, 69, 77, 78,
80, 88, 93, 94, 95, 97,
100, 101, 102, 103, 104,
105, 116, 117, 122, 125,
127, 134, 141, 146, 147,
151, 153, 154, 155, 157,
229, 231, 232, 236, 237,
238, 240, 243, 252, 253,
254, 257, 259, 261, 263,
265, 271.
Predictions, 37, 39, 43, 106,
116, 118, 119, 121, 225.
Preparation, 1, 30, 31, 72, 73,
113, 123, 131, 177, 190,
199, 220.
Prevention, 17, 218.
Production, 3, 4, 8, 14, 16, 17,
19, 20, 21, 23, 28, 34,
70, 80, 81, 82, 111, 113,
121, 158, 159, 160, 167,
169, 172, 173, 179, 180,
183, 184, 187, 188, 189,
190, 199, 208, 209, 210,
214, 215, 217, 218, 222,
230, 234, 246, 248, 255,
259.
279
Products, 1, 3, 5, 7, 8, 9, 10,
11, 12, 13, 14, 18, 19,
20, 21, 22, 27, 28, 39,
73, 82, 123, 136, 137,
138, 139, 145, 146, 147,
148, 150, 159, 160, 164,
173, 174, 180, 181, 182,
184, 185, 187, 189, 196,
198, 199, 207, 209, 214,
216, 219, 228, 230, 232,
233, 240, 241, 243, 244,
246, 249, 251, 256, 264,
266, 269, 271, 272.
Project, 25, 132, 245.
Protection, 11, 18, 68, 210.
Proteins, 7, 16, 25, 45, 46, 47,
51, 54, 55, 56, 62, 63,
66, 67, 68, 83, 166, 172,
182, 192, 197, 204, 208,
216, 260, 261, 262, 268,
272.
Protozoan Parasites, 2.
R
Relations, 44, 216.
Research, 24, 27, 151, 178, 200,
238.
Resolution, 237.
RNA, 45, 47, 49, 50, 52, 237,
253, 260, 262, 263, 264,
266, 267, 268, 269, 270.
S
Salmonella, 5, 102, 184, 185.
Species, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 19,
20, 21, 22, 23, 24, 25,
26, 58, 68, 70, 71, 128,
136, 138, 147, 149, 159,
160, 163, 164, 165, 166,
280 Introductory Food Microbiology Introductory Food Microbiology 281

173, 174, 177, 180, 182, 72, 81, 87, 94, 102, 105,
185, 186, 187, 189, 211, 107, 108, 112, 113, 117,
215, 229, 230, 231, 232, 119, 120, 121, 122, 127,
233, 234, 235, 236, 237, 128, 129, 130, 131, 132,
239, 240, 242, 243, 244, 133, 134, 135, 136, 137,
247, 249, 250, 253, 257, 138, 139, 140, 142, 143,
261, 263, 264, 265, 268, 144, 145, 146, 147, 149,
CONTENTS
269, 270, 271, 272. 152, 153, 154, 159, 160,
Spoilage, 1, 2, 8, 13, 14, 17, 162, 163, 164, 165, 166,
18, 20, 21, 22, 26, 27, 177, 178, 196, 202, 204, Preface
136, 159, 164, 170, 171, 205, 206, 209, 212, 213, 1. Introduction 1
177, 178, 185, 186, 189, 216, 218, 219, 220, 226,
202, 205, 220, 246, 254, 248, 260, 263, 271. 2. Role of Ctc from Listeria Monocytogenes in
272. Treatment, 97, 101, 125, 179, Osmotolerance 45
Stress Conditions, 45, 47, 53, 54, 198, 212, 215, 216, 230,
59, 61, 63, 94, 127. 242, 254, 267. 3. Identification of Listeria Monocytogenes Genes 57
Symptoms, 4, 5, 8, 234.
V 4. Experimental Validation of Low Virulence in
T Validation, 39, 64, 83, 110, 111,
Field Strains 83
Technology, 30, 73, 74, 220. 129, 219. 5. The Effect of Inoculum Size on the Lag Phase 93
Temperature, 11, 12, 17, 29, Velocity, 175.
31, 39, 46, 49, 52, 57, Viruses, 1, 2. 6. Modelling the Growth of Listeria
Monocytogenes in Dynamic Conditions 106
‰‰‰
7. Epidemiological Typing of Bacillus spp Isolated
from Food 173
8. Modelling the Growth Boundary of
Staphylococcus Aureus 217
9. Glossary 258
Bibliography 273
Index 275
 INTRODUCTORY
FOOD MICROBIOLOGY