BOHOLST,

Kristine Joy MICROBIOLOGY

GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester


Aerobic Nonspore-Forming Gram-Positive Bacilli: Corynebacterium, Listeria, Erysipelothrix, Actinomycetes

Corynebacterium diphtheria
Corynebacteria are 0.51 m in diameter and several micrometers long. Characteristically, they possess irregular
swellings at one end that give them the "club-shaped" appearance
Irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes
(metachromatic granules) that give the rod a beaded appearance.
Individual corynebacteria in stained smears tend to lie parallel or at acute angles to one another. True branching
is rarely observed in cultures.

Pathogenesis
C diphtheria
The principal human pathogen of the genus Corynebacterium
the causative agent of respiratory or cutaneous diphtheria
occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers.
It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or
in skin abrasions, and those that are toxigenic start producing toxin

Pathology
Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial
inflammatory response.
The necrotic epithelium becomes embedded in exuding fibrin and red and white cells, so that a grayish
"pseudomembrane" is formedcommonly over the tonsils, pharynx, or larynx. The regional lymph nodes in the neck
enlarge, and there may be marked edema of the entire neck.
. The diphtheria bacilli within the membrane continue to produce toxin actively. This is absorbed and results in distant
toxic damage, particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle, liver, kidneys,
and adrenals, sometimes accompanied by gross hemorrhage. The toxin also produces nerve damage, resulting often in
paralysis of the soft palate, eye muscles, or extremities.

Clinical Finding
In the respiratory tract:
1. Sore throat and fever usually develop
2. Prostration and dyspnea soon follow because of the obstruction caused by the membrane. This obstruction may
even cause suffocation if not promptly relieved by intubation or tracheostomy. Irregularities of cardiac rhythm
indicate damage to the heart.
3. Later, there may be difficulties with vision, speech, swallowing, or movement of the arms or legs. All of these
manifestations tend to subside spontaneously

Diagnostic Laboratory Tests
Dacron swabs
from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are
administered. Swabs should be collected from beneath any visible membrane. The swab should then be placed in
semisolid transport media such as Amies. Smears stained with alkaline methylene blue or Gram stain show beaded rods
in typical arrangement.
Specimens should be inoculated to a blood agar plate (to rule out hemolytic streptococci), a Loeffler slant, and a
tellurite plate (eg, cystine-tellurite agar or modified Tinsdale's medium) and incubated at 37C. In 1218 hours,
the Loeffler slant may yield organisms of typical "diphtheria-like" morphology. In 3648 hours, the colonies on
tellurite medium are sufficiently definite for recognition of C diphtheria
BOHOLST, Kristine Joy MICROBIOLOGY
GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester

Treatment
Treatment with antitoxin is mandatory when there is strong clinical suspicion of diphtheria. From 20,000
100,000 units are injected intramuscularly or intravenously after suitable precautions have been taken (skin or
conjunctival test) to rule out hypersensitivity to the animal serum. The antitoxin should be given on the day the
clinical diagnosis of diphtheria is made and need not be repeated. Intramuscular injection may be used in mild
cases.
Antimicrobial drugs (penicillin, erythromycin) inhibit the growth of diphtheria bacilli. Although these drugs have
virtually no effect on the disease process, they arrest toxin production. They also help to eliminate coexistent
streptococci and C diphtheriae from the respiratory tracts of patients or carriers.

L. Monocytogenes
Is important as a cause of a wide spectrum of disease in animals and humans.
It is capable of growing and surviving over a wide range of environmental conditions.
It can survive at refrigerator temperatures (4C), under conditions of low pH and high salt conditions.
It is able to overcome food preservation and safety barriers, making it an important foodborne
pathogen.

Morphology and Identification
L. monocytogenes is a short, gram-
positive, non-spore-forming rod.
It is catalase positive and has a tumbling
end motility at 22-28C but not at 37C.

Culture and Growth Characteristics:
It grows well on media such as 5% sheep blood
agar on which it exhibits the characteristic small
zone of hemolysis around and under colonies.
The organism is a facultative anaerobe and is
catalase positive, and motile. Listeria produces
acid but not gas from utilization of a variety of
carbohydrates.

Antigenic Classification:
Serologic classification is done only in reference laboratories and is primarily used for epidemiologic studies.
There are 13 known serovars based on O (somatic) and H (flagellar) antigens.
Serotypes 1/2a, 1/2b, and 4b make up more than 95% of the isolates from humans.
Serotype 4b causes most of the foodborne outbreaks.

Pathogenesis and Immunity:
L monocytogenes enters the body through the gastrointestinal tract after ingestion of contaminated foods such
as cheese or vegetables.
The organism has several adhesin proteins (Ami, Fbp A, and flagellin proteins) that facilitate bacterial binding to
the host cells and that contribute to virulence.
It has cell wall surface proteins called internalins A and B that interact with E-cadherin, a receptor on epithelial
cells, promoting phagocytosis into the epithelial cells.
After phagocytosis, the bacterium is enclosed in a phagolysosome, where the low pH activates the bacterium to
produce listeriolysin O. This enzyme lyses the membrane of the phagolysosome and allows the listeriae to
escape into the cytoplasm of the epithelial cell.
BOHOLST, Kristine Joy MICROBIOLOGY
GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester

The organisms proliferate, and ActA, another listerial surface protein, induces host cell actin polymerization,
which propels them to the cell membrane. Pushing against the host cell membrane, they cause formation of
elongated protrusions called filopods.
These filopods are ingested by adjacent epithelial cells, macrophages, and hepatocytes, the listeriae are
released, and the cycle begins again. L monocytogenes can move from cell to cell without being exposed to
antibodies, complement, or polymorphonuclear cells.
Iron is an important virulence factor. Listeriae produce siderophores and are able to obtain iron from transferrin.
Immunity to L monocytogenes is primarily cell mediated, as demonstrated by the intracellular location of
infection and by the marked association of infection with conditions of impaired cell-mediated immunity such as
pregnancy, AIDS, lymphoma, and organ transplantation.
Immunity can be transferred by sensitized lymphocytes but not by antibodies.

Clinical Findings:
2 forms of perinatal human listeriosis:
1. Early onset syndrome (granulomatosis infantiseptica) infection in utero and is disseminated form of the
disease characterized by neonatal sepsis, pustular lesions, and granulomas containing L monocytogenes in
multiple organs. Death may occur before or after delivery.
2. Late onset syndrome - development of meningitis between birth and the 3rd week of life; caused by serotype
4b.

Adults can develop Listeria meningoencephalitis, bacteremia, and (rarely) focal infections.
Meningoencephalitis and bacteremia occur most commonly in immunosuppressed patients, in whom Listeria is
one of the more common causes of meningitis.
In immunocompetent individuals, illness may not occur after ingestion of contaminated food or patients may
develop a symptomatic febrile gastroenteritis. This develops after an incubation period of 648 hours.
Symptoms include fever, chills, headache, myalgias, abdominal pain, and diarrhea.
Illness is usually self-limiting in 13 days; most clinical laboratories do not routinely culture for Listeria from
routine stool samples. The diagnosis of listeriosis rests on isolation of the organism in cultures of blood and
spinal fluid.
Spontaneous infection domestic and wild animals. In ruminants, it may cause meningoencephalitis with or
without bacteremia. In smaller animals, there is septicemia with focal abscesses in the liver and heart muscle
and marked monocytosis.
Many antimicrobial drugs inhibit Listeria species in vitro.
Clinical cures have been obtained with ampicillin, erythromycin, or intravenous trimethoprimsulfamethoxazole.
Cephalosporins and fluoroquinolones are not active against L monocytogenes.
Ampicillin plus gentamicin is often recommended for therapy, but gentamicin does not enter host cells and may
not help treat the Listeria infection.
Trimethoprimsulfamethoxazole is the drug of choice for central nervous system infections in patients who are
allergic to penicillin.

Erysipelothrix Rhusiopathiae
Erysipelothrix rhusiopathiae is a gram-positive bacillus that produces small, transparent glistening colonies. It
may be -hemolytic on blood agar. On Gram stains, it sometimes looks gram negative because it decolorizes
easily.
The bacteria may appear singly, in short chains, randomly, or in long nonbranching filaments. The colony
morphology and Gram stain appearance vary depending on the growth medium, incubation temperature, and
pH.
Erysipelothrix is catalase, oxidase, and indole negative. When Erysipelothrix is grown on triple sugar iron (TSI)
agar, hydrogen sulfide is produced, turning the TSI butt black.
BOHOLST, Kristine Joy MICROBIOLOGY
GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester

E rhusiopathiae is distributed in land and sea animals worldwide, including a variety of vertebrates and
invertebrates.
It causes disease in domestic swine, turkeys, ducks, and sheep. The most important impact is in swine, in which
it causes erysipelas.
In humans, erysipelas is caused by group A -hemolytic streptococci and is much different from erysipelas of
swine. People obtain E rhusiopathiae infection by direct inoculation from animals or animal products.
The most common E rhusiopathiae infection in humans is called erysipeloid. It usually occurs on the fingers by
direct inoculation at the site of a cut or abrasion (and has been called seal finger and whale finger).
After 27 days incubation, pain, which can be severe, and swelling occur. The lesion is raised, and violaceous in
color. Pus is usually not present at the infection site, which helps differentiate it from staphylococcal and
streptococcal skin infections.
Erysipeloid can resolve after 34 weeks or more rapidly with antibiotic treatment.
Additional clinical forms of infection (both rare) are a diffuse cutaneous form and bacteremia with endocarditis.
Erysipelothrix is highly susceptible to penicillin G, the drug of choice for severe infections. The organism is
intrinsically resistant to vancomycin.

ACTINOMYCETES
large, diverse group of gram-positive bacilli, tendency to form chains or
filaments, related to the corynebacteria
include multiple genera of clinical significance such as Mycobacteria and
saprophytic organisms such as Streptomyces
As the bacilli grow, the cells remain together after division to form
elongated chains of bacteria (1 m in width) with occasional branches
It is rudimentary in some actinomycetesthe chains are short, break
apart after formation, and resemble diphtheroids
Others develop extensive substrate or aerial filaments (or both); or
fragment into coccobacillary forms
Categorized on the basis of the acidfast stain
Truly positive acid-fast organisms, weakly positive genera include Nocardia, Rhodococcus, and a few others of
clinical significance.
Streptomyces and Actinomadura, two agents that cause actinomycotic mycetomas, are acid-fast stain negative
R equi may appear to be a bacillus after a few hours of incubation in broth, but with further incubation, the
organisms become coccoid in shape
R equi occasionally cause infections such as necrotizing pneumonia in immunosuppressed patients with
abnormal cell-mediated immunity
R equi is present in soil and in dung of herbivores and occasionaly cause disease in cattle, sheep, and swine and
can cause severe lung infections in foal
Species of Rhodococcus also frequently produces pigmented colonies after 24 hours of incubation that range
from salmon pink to red, present in the environment but rarely cause disease in humans
The organisms are generally weakly acid-fast positive when stained by the modified Kinyoun method.

NOCARDIOSIS
genus Nocardia continues to undergo extensive taxonomic reclassification
New species continue to be recognized, and at least 30 species have been implicated as causes of human
infections
The pathogenic nocardiae, similar to many nonpathogenic species of Nocardia , are found worldwide in soil and
water.
Nocardiosis is initiated by inhalation of these bacteria.
BOHOLST, Kristine Joy MICROBIOLOGY
GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester

The usual presentation is as a subacute to chronic pulmonary infection that may disseminate to other organs,
usually the brain or skin.
Nocardiae are not transmitted from person to person

Morphology and Identification
Nocardia species are aerobic and grow on a variety of media.
Over the course of several days to 1 week or more, they develop heaped, irregular, waxy colonies.
Strains vary in their pigmentation from white to orange to red. These bacteria are gram-positive, catalase-
positive, and partially acid-fast bacilli.
They produce urease and can digest paraffin. Nocardiae form extensive branching substrates and aerial
filaments that fragment after formation, breaking into coccobacillary cells.
The cell walls contain mycolic acids that are shorter chained than those of Mycobacteria. They are considered to
be weakly acid fast, that is, they stain with the routine acid-fast reagent (carbol-fuchsin) and retain this dye
when decolorized with 14% sulfuric acid instead of the stronger acid-alcohoL decolorant.
The species of Nocardia are identified primarily by molecular methods such as 16S rRNA gene sequencing and
restriction fragment length polymorphism (RFLP) analysis of amplified gene fragments such as hsp or secA.

Pathogenesis and Clinical Findings
In most cases, nocardiosis is an opportunistic infection associated with several risk factors, most of which impair
the cell-mediated immune responses, including corticosteroid treatment, immunosuppression, organ
transplantation, AIDS, and alcoholism.
Nocardiosis begins as chronic lobar pneumonia, and a variety of symptoms may occur, including fever, weight
loss, and chest pain. The clinical manifestations are not distinctive and mimic tuberculosis and other infections.
Pulmonary consolidations may develop, but granuloma formation and caseation are rare.
The usual pathologic process is abscess formation (neutrophilic inflammation).
Spread from the lung often involves the central nervous system, where abscesses develop in the brain, leading
to a variety of clinical presentations. Some patients have subclinical lung involvement and present with brain
lesions.
Dissemination may also occur to the skin, kidney, eye, or elsewhere

Diagnostic Laboratory Tests
Specimens consist of sputum, pus, spinal fluid, and biopsy material.
Gram-stained smears reveal gram-positive bacilli, coccobacillary cells, and branching filaments.
With the modified acid-fast stain, most isolates will be acid fast. Nocardia species grow on most laboratory
media.
Serologic tests are not useful.
Molecular methods are required for species-level identification, which is necessary for both epidemiologic and
treatment purposes.

TREATMENT
The treatment of choice is trimethoprimsulfamethoxazole.
If patients fail to respond, a number of other antibiotics have been used with success, such as amikacin,
imipenem, minocycline, linezolid, and cefotaxime.
However, because the susceptibility patterns vary by species, susceptibility testing should be performed to guide
treatment approaches.
Surgical drainage or resection may be required.


BOHOLST, Kristine Joy MICROBIOLOGY
GALABIT, Nikka Valerie PABLO QUEDADO, MD
OLOD, Jude Lester

Emerging Actinomycetes: Gordonia and Tsukamurella
Members of the genera Gordonia and Tsukamurella are modified acid-fast positive bacteria that are becoming
more frequently responsible for opportunistic infections among hospitalized immunocompromised patients.
Gordonia produce orange, wrinkled colonies. On Gram stain, the organisms appear coryneform and do not
branch. When these organisms are recovered from nonsterile sources such as sputum, they may be mistakenly
disregarded as normal flora or contaminants. Tsukamurella species form whitish to orange colonies and on stain
appear as long, straight, sometimes curved rods. Members of both genera are best identified by cell wall fatty
acid analysis or 16S rRNA gene sequencing. These organisms have been associated with a variety of infections,
including postoperative wound infections, catheter-associated bloodstream infections, ear drainage, and
pulmonary infections.
Treatment has been based on anecdotal experiences but does equire removal of catheters and drainage of
abscesses.
For infections caused by Gordonia species, vancomycin, carbapenems, aminoglycosides, fluoroquinolones, and
linezolid have all been used successfully for treatment.
In the case of Tsukamurella infections, in vitro susceptibility has been demonstrated with the aminoglycosides,
sulfamethoxazole, fluoroquinolones, carbapenems, and clarithromycin.

Concept Checks
Several members of the large group of aerobic Actinomycetes are modified acid fast positive, most commonly
Nocardia, Tsukamurella, and Gordonia.
Nocardia species are branching, beaded gram-positive rods found in soil and other environmental sources that
cause systemic disease primarily in immunocompromised patients.
Tsukamurella and Gordonia species are less commonly isolated and are associated primarily
All three groups are best identified after recovery on routinemedia by using molecular methods such as 16SrRNA
gene sequencing.
Trimethoprimsulfamethoxazole is the drug of choice for treatment of Nocardia infections. The use of other
agents should be dictated by results of susceptibility testing.

ACTINOMYCETOMA

Mycetoma (Madura foot) is a localized, slowly progressive, chronic infection that begins in subcutaneous tissue
and spreads to adjacent tissues.
It is destructive and often painless. In many cases, the cause is a soil fungus that has been implanted into the
subcutaneous tissue by minor trauma.
An actinomycetoma is a mycetoma caused by filamentous branching bacteria.
The actinomycetoma granule is composed of tissue elements and gram-positive bacilli and bacillary chains or
filaments (1 m in diameter).
The most common causes of actinomycetoma are Nocardia asteroides, Nocardia brasiliensis, Streptomyces
somaliensis, and Actinomadura madurae. N brasiliensis may be acid-fast.
These and other pathogenic actinomycetes are differentiated by biochemical tests and chromatographic analysis
of cell wall components.
Actinomycetomas respond well to various combinations of streptomycin, trimethoprimsulfamethoxazole, and
dapsone if therapy is begun early before extensive damage has occurred.