Transcribed by Erica Manion 7/25/2014

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Microbiology - Lecture 13 Clinical and Diagnostic Microbiology II by Dr. Tierno

[Slide 22 of Diagnostic clinical microbiology lecture] Ubiquity of Organisms
[Dr. Tierno] Ok I think we can start. Ok I think we can start, I dont know whats going on outside,
a convention or something?

Yesterday we left off with regard to mouth flora. The point I was trying to make yesterday was that
its important to understand the ecology of each body area. the normal flora that exists in areas of
the body, whether its the skin, the intestines, or the mouth are very useful and can actually help
with product development and offsetting disease. Could you please shut that door? Thank you. So I
was talking about a toothpaste developed based on the ecology of epiphytic growth on plaque on
the surface of teeth. You know, can we close that door so that no one else can come in? Because
yesterday you said at the 50 mark I had to let you go because you had to get to another class. Well,
this is a class. You know, you gotta come on time. Its very disruptive to see people going in and out.
So knowing that organisms are applied on the surface of the tooth in an epiphytic way, layering one
on the other. We can interfere with that, and they added Triclosan, which as I said, may actually be
taken off the market because it mimics hormones, but thats yet to be proven. If it stays on the
market, it is useful in that it prevents the attachment of organisms, so therefore reduces plaque. Of
course organisms have good ways of circumventing the process over time. Another area of ecology
thats important was the invagination of the skin, the vaginal vault. Most of you are familiar with
toxic shock syndrome, Staphylococci, Staphylococcus aureus can be a part of the normal flora of the
vagina. And certain toxigenic strains you know can you guys get here on time? I know you had an
exam or whatever and youre all chit chatting, but if you want to leave at 10:50 Well youre not
gonna leave today at 10:50 I have another class with you afterwards. You know, its very
disruptive. In the vaginal vault, staph aureus can be a part of the normal flora in about 25% of
women. About 3% of strains are toxogenic, and organisms respond to their chemico-physical
environment. The ecological factors I put on the board yesterday, or the screen, are important and
are in play. In fact, when you introduce a synthetic tampon, one lets say that has polyacrylamide
carboxymethylcellulose or one other ingredient - one other ingredient that is synthetic, the
organism responds to the physicochemical environment which is newly created and pumps out
toxins. Whereas if a cotton tampon were used there would be no pumping out of toxin, but the
synthetics have a variety of physico-chemical changes that I wont go into, and will allow that
organism to produce toxin, giving rise to toxic shock syndrome. So thats another example of how
ecology is very important and useful to you.

[Slide #23] - Firmicutes: Bacteroidetes (F:B) Ratio**
In the intestines, we have a balance of about 500 or so organisms. There are 2 main groups, one is
called firmicutes, the gram positive organisms, and the other is the bacteroidetes, gram negative
organisms. Those organisms are in a particular ratio. And that ratio interestingly differs based on
life stages. Infants and the elderly, infants and the elderly seem to have that ratio similar. But the
bulk of people from after infancy to maybe about 70 years old, or 80 years old, it depends, are about
10.9. Look at the difference. And that ratio is very important. We know that people that get
Clostridium difficile colitis and don't respond to antibiotics can be given a stool, or should I say a
normal stool inserted in the rectum to rebalance their flora. Usually it is a relative of the person
who has C. difficile that is not responsive to antibiotic therapy. And this realigns the flora. Of course
the flora would be tested, there is a whole process of cleansing it, just using the bacteria. So, an
enema, for example, of that flora is introduced and there is a rebalancing and its a remarkably high
level of recovery, without antibiotics. So its important to understand ecology. There is a very
significant use of understanding ecology of normal flora in various areas of the body.

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[Slide #24] - Genera in the Colon of 4 Normal Subjects
This is another representation of firmicutes versus the bacteroidates. I just have it here to show you
the so-called normal ranges of flora taken from the transverse colon, the sigmoid, and the rectum
and you can see, these are what we would call normal.

[Slide #25] - Acquisition and Nature of Oral Flora
Now, what is the acquisition of oral flora. Its interesting, we used to think the placenta was sterile.
Yesterday I told you that we now know lungs and bladder and other organs are not sterile. Well,
neither is the placenta. And we used to think the first exposure to microbes was coming down the
birth canal of the mother. And if it was a caesarian section, was being handled by the mother with
her flora. We now know the mothers birth canal is the second, actually, exposure. The first
exposure is the placenta. And the placenta has been found to have mouth flora of the mother. So
that the babys initial flora is mothers flora, its maternal. In fact that was done by one of the
researchers here at the dental school, the original work. And now we know why that mouth flora is
in a child. Then the birth canal is the second exposure as you come down the vaginal vault you are
picking up vaginal flora which has a lot of flora also in the mouth. And gut flora. The gut flora comes
from whats called a fecal squirt at the time the child comes down the vaginal vault. And of course
you get exposure to the mothers skin, which is rich in flora. And breast milk has a diverse
population. In fact, its rich, its not sterile as we once thought it was. It just adds another layer of
microorganisms. So that is where the organisms are introduced into a baby.
The oral cavity, we used to think was sterile at birth, its very low population depending on the child
and how the child aspirates any fluids. But right after birth it is colonized. In three to five days the
buccal - and just to show you the specificity of things, the buccal epithelia allow attachment of
Streptococcus salivarius. This is one of the alpha-? (inaudible), the Streptococcus viridans group.
And actually, with, there are others too. With the eruption of teeth, thats six to nine months, I dont
know how that one got there, nine months, Strep mutans and sanquis now attach. And with the
creation of the gingival crevice, anaerobes start to occur. Now its interesting, an edentulous
person, an edentulous person will lose the sanquis and mutans. Thats how specific those are to the
teeth. Even synthetic teeth, they will attach. If you take them out, there is a loss of the the sanquis
and mutans. They will reappear, of course, with dentures, and there is a decrease in anaerobes in
the mouth of an edentulous person. But with the gingival crevice of course you have fusobacteria,
porphyromonas, prevotella, (I hope those are right, they arent listed on the slide and Im not sure
how to spell it, sorry!) and others. So thats the way we develop and as we have puberty you refine
the flora because of the hormones that a person has.

[Slide #26] - Table 8.16 Bacterial species detected in the gingival crevice
Heres an example of the types of organisms that can be in the gingival crevice. There are many
others, and now we have methods other than growing organisms. We can use PCR and microbiomic
research to identify organisms. So we get a better handle on the organisms that are present. So all
of the current books will be changing over time.

[Slide #27] - Oral Bacterial Colonization
Heres an example of the epiphytic growth of organisms. There is a very complex interrelationship.
Here is the tooth surface, pellicle which leads to the first attachment of organisms. And these
organisms, interfacing one with another, attaching, each having a specific role, a very complex thing
which I think you have a course in oral microbiology. But just to give you an appreciation of the
interrelatedness of these organisms, they arent just there, they have purpose, both
immunologically and other purposes. But right now we are just looking for diagnostic
microbiology.

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[Slide #28] - Normal Flora Quantitative Analysis
I talked about the quantities of organisms which vary. Even though the skin, lets take the skin,
would be 10
4
organisms, 10
5
depending upon the area, maybe as low as 10
2
. In other words, 100
bacteria. Various areas of the skin have different numbers of bacteria. Even the numbers vary,
besides the types of organisms. If you have oily skin, you tend to have different organisms than if
you have dry skin, if you are very acidic, certain areas of the skin are about 5.5. But in certain areas
like the buttocks, its more like 6.5 The hair or the head, I dont have to worry [because he is bald]
but it is a different pH. So you get the picture. The numbers of organisms and the variety of
organisms may change at different sites, so its important to know the ecology and the mechanisms
of ecology.
Tissue trophism. There are certain organisms that have a predilection for particular sites and thats
for example the adherence to specific the mutans adhering to teeth. Another thing would be the
skin. You can only have organisms that live on the skin, I said its acidic, 5.5, so you can only have an
organism that is acidophilic, that is lipophilic, because we have oils on the skin, that has protein and
proteinases, enzymes that degrade proteins of the skin, and can grow aerobically, facultatively, or
anaerobically. So thats why we have staph, which enjoy those environments. We also have
Propionibacterium which can range anaerobic, to facultative, to anaerobic. We also have a wide
variety of similar types of organisms that reside in that area of the skin or on the skin because of the
physicochemical environment. If there is only one take away point here, it is that. The
physicochemical environment determines what organisms live where on the body in both
health and disease. There is one other area of the body and as dentists you may come across this,
that has 10
12
colony forming units, or bacteria per gram of material. It is something that if you are
examining a patient during a procedure you should be aware of. There is something called a food
lith. Who here has tonsils? Ok. 1 in 4 of you, so there are many of you, have this situation where you
have a food lith in the back of the tonsil that grows to the size of a lentil or a pea and eventually
sloughs off if you were to take that food pith and squeeze it you would find that it has a very
anaerobic smell, and it is 10
12
colony forming units or bacteria per gram of material. So when you
are probing, it would be difficult sometimes to pick that food lith with forceps. It should be a blunt
edge forceps. Because if you poke that area of the skin that individual can have a whopping
infection, so you have to use a blunt edge to remove it, you dont want them to choke on it. But it is
perfectly harmless. You swallow it and it is fine. Your intestinal bacteria handle it, and in part some
of those organisms are found in the intestines. The idea is you dont want to aspirate on that, you
dont want someone choking on it so it should be removed. And that is the third area of the body
with 10
12
bacteria per gram, which is like a paste of bacteria. It is very dense, and those are mostly
anaerobic.

[Slide #29] - Chart
Lets start to identify a disease process in a patient for which you would take a culture and submit it
to a lab for identification and for an antibiotic susceptibility test being done on it. The following
events take place during an infection. All infectious diseases require an encounter of the organism,
bacteria, virus, or whatever microbe with the host. In some way you have to contact the organism.
It doesnt matter how but you have to make that contact. It enters the host, whether its oral,
through the skin, or some other way. And then organisms that are pathogenic, remember we define
pathogenicity in terms invasive ability - invasiveness via enzymes and toxigenicity, production of
toxins and other chemicals that are toxic to the body, which allows the organism to really raise
havoc. Then there is damage, and now you have a host response. Your immune system usually
comes in to play. People who are immunocompromised have a bigger problem. Then the outcome.
Remember that see saw? That was the picture, the microbe vs the host. Thats going on in your body
every day. Either you win, or the host wins. Now all these steps require breaching defenses, in
other words your last defense other than the skin and lets say indirect, well, lets just say the last
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defense is your immune response and leave it at that.

[Slide #30] - Identification
Now once you have an infection you take a culture. This is the classic way cultures are being
identified. This is culture dependent technology. In other words you have to grow an organism, and
generally it takes anywhere from a few days to maybe as much as five days to identify an organism.
Thats useless to you in your practice because youre not getting information real time. So you are
empirically treating. You may change your therapy, but initially you have to empirically treat an
infection, you cant let a patient leave with an infection. And that requires the identification of the
organisms and the simultaneous biogram, or the antibiotic susceptibility panel, which will tell you
the drugs you can use or cant use based on the organisms resistance pattern. The first thing a
laboratory would do would be to take the swab that you send, they would probably put it in a
diluent and put on a slide to look at it. They will look at the stained preparation, microscopic
morphology. Is it a coccus? Is it a bacillus? Is it a spore forming bacillus? Is it a spiral organism or
curved bacillus? There are many morphological characteristics that are identified by simple
microscopy. To make matters a little more complicated, and make them more specific, a gram stain
is usually performed. Dr. Gram split two groups of organisms. One group, gram positive, blue
staining. The other group, gram negative, red staining.
E. coli for example, is a gram negative bacillus. Staph aureus is a gram positive coccus. So you can
help identify, or begin the identification of an organism by the gram stain and the microscopic
morphology. There are characteristics of some organisms, for example and E. coli swims, its motile.
A protista swims, its motile. A Klebsiella is non motile. It just sits there, brownian motility, in other
words movement, not motility. So it is non motile. Shagella, non motile. Shigella, non motile.
Salmonella, motile. So motility is an important characteristic. As is the flagella arrangement - of a
peritrichous, having flagella all around them. There is a stain you can do. Or of a lophotrichous,
having a cluster on one end of the organism, of flagella. So its important. Is it tumbling motility?
Some organisms actually tumble under the microscope. Like Listeria would be a tumbling, motile
organism. Then you plate them onto a battery of media. This is the old, classic way, and is still being
used today. Not everybody has molecular methods that give real time processing and real time
identification of organisms, which actually should occur within two hours of delivery of the
specimen. That would be ideal. So you look, when you plate them out onto a battery of media, to be
able to grow different types of organisms. The colonial morphology, the colonies they grow specific,
different types of growth occur. They could be convex, concave, they could be pigmented, non
pigmented, all sorts of types of colonies. They could be mucoid, could be [triclosan?], could be flat,
could be raised, so you get the picture. Colonial morphology is important.
Pigmentation. Pseudomonas aeruginosa is green on plates of agar. Pseudomonas is a common
organism, very highly antibiotic resistant, and it is ubiquitous, it is in water. In your kitchen sink, in
your drain, if you swab it, you will get Pseudomonas aeruginosa out of there. So these ubiquitous
organisms and luckily we have a good immune response and we have flora that competes with
other intruders, the transient flora that comes in, and for the most part we are ok. So pigmentation
is important. Staph aureus is yellow. Staph epidermis, another skin Staph is white in color. No
pigment.
Growth requirements are important. Does an organism grow on a petri dish? Artificial media, like
bacteria do, or does it require living cells for its growth like viruses do? Then comes that large
group of biochemical physiologic characteristics. Does it metabolize glucose? And glucose is the
sugar that is used for most differentiation or initial differentiation of fermentative vs oxidative
organisms. Some organisms like Pseudomonas require oxygen. Other organisms truly ferment, so
they dont need oxygen. For example, Pseudomonas aeruginosa is oxidative, it doesnt grow
anaerobically, or it doesnt grow well anaerobically, whereas E. coli will grow either way, with or
without. Glucose determination is important for the metabolism. What are the end products of
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fermentation? Is it an acid and gas, or just an acid, or just the gas? And is it anaerobic vs aerobic vs
requiring carbon dioxide tension? In other words extra carbon dioxide. Oral flora sometimes
requires extra carbon dioxide for growth. And the same is so for the various enzymes, catalase for
example. Staph aureus is catalase positive, in Streptococcus pyogenes is catalase negative. And
catalase is an enzyme that splits hydrogen peroxide into water and oxygen effervescence. You can
see the oxygen if you add a drop to the colony of Staph. And there are other enzymes. Urease,
splitting urea, like some organisms can do. And there are other methods in the classic diagnostic
criteria, like serology, DNA probes, and molecular techniques. Im gonna touch on some of those in
a minute. But this gives you a general overview of identification.

[Slide #31] - Image
Here are some slides just for your purview. This is a gram positive, take my word it doesnt look
blue, but gram positive diplococci from a lung specimen. And this is a white cell, a pus cell,
polymorphonucleated cell. PMNs. And usually you see the pus response because these organisms,
these phagocytic blood cells come to engulf the organism and destroy it. What do you think that is?
[Student response: Tuberculosis?] No, no. Thats an acute infection, gram positive diplococcus, its a
Streptococcus pneumoniae.

[Slide #32] - Image
Heres that same organism from that sputum to which we added an antisera which blows up the
capsule. So you dont need a molecular method here. Right from the sputum from the patient, 10
minute test, you add polyvalent antisera, and it will explode the capsule and you can identify
Pneumococcus on a slide. Not every test requires sophisticated, expensive testing.

[Slide #33] - Image
Now this comes from the penis of the male. Lots of pus cells, and inside the pus cells are diplococci,
gram negative, coffee bean shape or kidney shaped diplococci. What might that be? Gonorrhea. You
could hazard a guess.

[Slide #34] - Image
This comes from a skin lesion, I cant hear you. This comes from a skin lesion and its gram positive
cocci in pairs, tetrads, and clusters. What might that be? Staph aureus. Right.

[Slide #35] - Image
This is another type of gram negative, very fat and a lot of mucous present and white cells. This is a
Klebsiella.


[Slide #36] - Image
And here, from the vaginal vault, a culture of a very large coccal form, but it is really a budding
yeast. What might that be? With hyphae, pseudohyphae. This is a pseudohyphae. And this is the
bud on the yeast cell, heres a bud, and heres another bud. And you can see the little buds here and
there. That is a Candida albicans. Thrush.

[Slide #37] - Microbial Isolation and Identification
So it gives you an idea that you can identify an organism from a smear, at least know what it most
likely is. You have to confirm the identity. So not every test, what Im telling you, will require
sophisticated days for identification. For confirmation, yes. We have to confirm that was a Candida
albicans. But there are some other tests you can do. But with the Pneumococcus, ten minutes you
can identify it. Now usually, to ascribe a name to an organism, you have to deal in pure culture. So
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youre streaking out on a plate something called streak dilution. You want to get that streak all over
so you get colonies at the end of the streak that are isolated. So you can pick them and sub culture
them, and identify. And we identify organisms and then of course we do an antibiogram, or an
antibiotic susceptibility test by four media.

Primary, enrichment, selective, and differential media As the names imply, primary would be
most organisms growing on a blood plate, lets say. I used to have a demonstration where I would
bring blood plates but its really wasting your time. Another type of media is enrichment. Lets say
you want to grow a very fastidious organism, you might have to enrich the agar with some factors
like for Haemophilus influenzae, which is a gram negative bacillus that causes flu and many other
respiratory problems as well as other types of infections. So you may have to up the anty by putting
some enrichment into the media to grow the organism. Another is selective. Selective agar will
usually select, as the name implies, for one type or a certain group of organisms. That is selective.
MacConkey agar for gram negatives actually is a selective agar, because it selects for the gram
negatives. And I have information for the agars for you. A differential agar can also be selective.
Most agars may have both names. You can have a selective agar like MacConkey which grows gram
negatives, yet it can be differential because some gram negatives are red in color, showing lactose
utilization, like E. coli, and others are colorless on the agar, like a Salmonella would be, lactose
negative. So you can have dual designations. This is the type of agar that is used in classic, clinical
microbiology.

[Slide #38] - Chart
And these are some examples of that. MacConkey, what you need to know is MacConkey. Thayer-
Martin is a good example of enrichment and selective. Thayer-Martin grows gonorrhea very well.
And so it can be enriched to get the growth of gonorrhea, and it is selective as the names imply. And
MacConkey is very important. It is one of those that are used. Then you get a differential and
mostly selective, but its still selective even though it is moderately selective, Hektoen-Enteric agar
which will grow gram negative enteric bacteria and can differentiate Salmonella and Shigella on
that agar. Sabouraud dextrose agar is selective for yeast and fungi. Yeast is a type of fungus

[Slide #39] - Was culture taken properly?
You always have to understand whether a culture was taken properly. A culture result is no better
than the culture you submit to the laboratory. If you know you missed the area or took a bad
culture, dont even waste your time to send it to the laboratory. Retake it. It is very important to
get a representation of the area that you need. Then submit it to the lab. And even though that is
done, as you know now, certain organ systems are positive and we cant get anything to grow from
them. Like the placenta or the bladder. Generally speaking, there is a flora intrinsic to that area.
Even the lungs. You can only assay them by non-growth technologies of microbiomic research. So
keep in mind that the culture methods employed doesnt guarantee everything present. But for the
most part, most of the pathogens will grow and you look for the prominent organism in a particular
culture if you have an overt infection. And you have to understand different agars will give you
different ratios of organisms. So interpretation isnt gonna be your job, its gonna be the job of the
laboratories to interpret the growth. But they may appear - some have a better chance of growing if
they are in mixed culture than others. So you always look at the gram stain to see what the ratio of
organisms are in a culture. You dont have to worry too much about it but you should be aware of
it. In vivo and in vitro situations are distinctly different, but we still have a ballpark understanding
of whats causing the infection. Does the isolation of an organism indicate pathogenicity? No. You
can isolate normal flora, it may be a virus causing the infection. You get organisms, you know the
normal flora of the area. So thats why its important to understand what normal is, to understand
whats abnormal. And if you dont have any bacterial pathogens you have to look at fungi and
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perhaps viruses. Just keep that in mind.

[Slide #40] - Characteristic/Organism
These are some characteristics of some organisms on various agar. The most commonly used agar
is blood agar. And hemolysis - and blood agar is the primary media that grows most things, but it is
also differential. It can differentiate among the streptococci. The green strep, or the alpha strep, like
the viridans strep and even Strep pneumoniea is green. Meaning there is greening around the
colony, a green zone around the colony on a red plate, a blood plate. Whereas beta is a clearing
around the colony. Like a group A beta-hemolytic Streptococcus. And some strains, most strains of
Staphylococcus aureus can also be hemolytic. They would exemplify the beta, or clear, area around
the colony.
As far as pigmentation, know that Staph aureus is yellow. Serratia marcescens is actually maroon in
color, call it red. Any shade of red is red in bacteriology. And the Pseudomonas aeruginosa is green.
If you know those I am happy. Some organisms can swarm across the plate, they dont give you
distinct colonies. I have a picture of that, the protists are guilty of that. And there are other things
like Hemophilus tend to satellite because they need enrichment. So they tend to grow around Staph
and Strep species, they satellite around Staph, because Staph is providing nutrients to them if we
dont. And many organisms like Strep pneumoniea, the Pneumococci are mucoid, and so is the
Klebsiella, very mucoid, and it is that organism that is raising havoc in hospitals across America.

[Slide #41] - Chart: Examples of Specific Enzymes which Aid in the Identification of Bacterial
Species
And these are, the example, if you use catalase, understand that Strep is negative, Staph is positive,
produces catalase.

[Slide #42] - Red and Yellow Test Tubes
Im going to show you some pictures. Heres the Staph aureus, the color yellow, Staph epidermidis is
not.

[Slide #43] - Test tubes Showing Different Sugar Fermentation
Sugar fermentation you can see on the left is acid only, next to the left is acid and gas, there is an
inverted tube there. So if the organism is producing acid and gas it will fill up the tube which is
upside down. And of course there is a negative, no reaction and an uninoculated. That tells you
sugar fermentation.

[Slide #44] - Citrate Utilization
This is sometimes used. Various organisms can utilize citrate and turn the agar, which is green, to
blue because of a change in pH. So they can utilize citrate and they produce byproducts that change
the pH.

[Slide #45] - Gelatin Utilization
Gelitinase, you know gelatin is a protein, and that gelatin is semisolid. If you put organisms that
have gelatinase, an enzyme that breaks down that protein, you will liquify it. And that is what this
is showing. Serratia marcescens liquifies, and Salmonella typhimurium does not.

[Slide #46] - Urea
Its just an example of some of the methods that are used in classic microbiology. And here is urea.
Urea agar is uninoculated on the right. Urea is negative, meaning it does not have the ability split
urea and break down to ammonia, which changes the pH to alkaline, or red. Some organisms that
do that are called urease positive. Protists is one of the organisms that is urease positive.
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[Slide #47] - Catalase
Here is the catalase reaction, oxygen on a Staph.

[Slide #48] - Motility test
And something called a motility test. Instead of looking under a microscope, you actually can have a
tube that you stick a needle in and see if you have migration from the line of inoculation, as
exemplified on the right here. That stab mark, you can see there was movement. That is another
way to do motility.

[Slide #49] - No title
And then finally you have something called an antibiogram, where you put the organisms across a
plate and you are dropping disks, each one representing different antibiotics.

[Slide #50] - No title
And you get zone sizes. This will be a separate discussion of the antibiotics laboratory discussion. I
think thats the following week.

[Slide #51] - Lab test result chart
Then you come up with a characterization. Dont worry about this chart. Its just meant to show you
there are many parameters of identification of different organisms based on the tests I showed you.
The idea is you take a sum total of those tests and come up with an identification of the organism.
This identification is a diagnostic, or clinical identification. Sometimes, its not absolute and
sometimes you may have to use serology or DNA probes or other confirmatory tests. But in the
main, this is useful and has been useful in the past.

[Slide #53] - Manual API systems
Now here is a way to utilize microtubes, these are very tiny tubes. This strip is about this big,
smaller than a ruler. Each one with different cupules that will give you the same reaction as you
would do in a big tube, in individual tubes. Dont worry about the name, just know API is one of the
names. The API for gram negative identification.

[Slide #54] - Automated Clinical Microbiology
We currently at Tisch have an apparatus called the VITEK 2. The VITEK 2 actually, automatically
does about 36 tests in those little cards. The card is about as big as a pack of cigarettes. And the
card is inserted into the machine. The machine will grow at it, look at it hourly and identify the
organism automatically and give rise to an antibiogram. Thats all you kneed to know on that one,
the VITEK.

[Slide #55] - PNA Fish
Now there is something called PNA fish. Peptide nucleic acid. You know that DNA molecules have a
charge, so you cant put a probe together with a DNA molecule, because they will move away from
each other. So thats what this test does. Instead of a charge, it has a polyamide or peptide
backbone that is non-charged, like the next diagram shows.

[Slide #56] - PNA Fish Figure
If you put two charges together here, you get movement. And you cant get identification. PNA fish
technology fluorescent in situ technology allows you to put a probe which is non-charged, which
mimics a DNA probe. And yet you can put them together right on a slide and make an identification
in a couple of hours.
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[Slide #57] - DNA vs PNA Hybridization
So Ill leave it at that, thats all you need to know. There is electrostatic repulsion which is
overcome by using a Polyamide probe rather than using bits of DNA.

[Slide #58] - PNA Fish Figure
Heres the way it works. Its another picture. You see on the left, you see how the probe would move
away from the target. On the right there is no electrical force so it tends to anneal and you can
identify,

[Slide #59] - How PNA Fish Works
..using a probe like this, a Staph aureus. It says 2.5 hours, they got it down to 1.5 hours. The
identity.

[Slide #60] - No title
Theres also blood cultures that can utilize this, and actually stain a different color so you can
understand whether you have a Staph.

[Slide #61] - PNA Fish Results
And heres a whole list of other things it can identify. You dont have to know any of these, you just
should know that PNA fish technology can identify an organism on a slide in less than two hours, or
even if you put 2.5 you get full credit.

[Slide #62] - Mass Spectrometry: Cutting Edge Microbial Identification
Now, the cutting edge technology that we have presently is this, MALDI TOF. Matrix Assisted Laser
Desorption/Ionization Time of Flight. And here, this is the way we can identify an organism by
blasting it with a laser, exploding it, and having particles move in an electrical field, and by their
flight identify the organism based on their fingerprint.

[Slide #63] - MALDI - TOF Technology
And here it is. Here is the thing in operation. The laser blasts a particular area on the cell, its a
small slide, by the way, that contains maybe 20 of these tests. You blast it, it comes up a tube which
is ionized, and based on the actual graph you get, you can identify the organism in ten minutes.
Thats absolutely amazing technology. And that is what you are going to be privy to in your run.

[Slide #64] - The Multiplex Film Array
And here is another way you can identify something, in one hour.

[Slide #65] - The Multiplex Film Array For Respiratory Viruses
All of these viruses. It used to be, we would take a swab and do individual tests for each of those
viruses and these bacteria. Mycoplasma pneumoniae, youre gonna get these, Im gonna teach you
those in December or November. Chlamyophila pneumoniae, Bordetella perfussis is a bacterium, and
Legionella pneumophilia, youre going to get a lecture on them. This test takes one swab from the
throat and identifies 23, theres actually one more microbe added - in one hour! Theres no guess
work. Sometimes children may have a Respiratory Syncytial Virus, but if that is negative, we know
it is probably a Metapneumovirus. This does all of them at once on a slide. One swab from a patient
in one hour. There are other systems actually

[Slide #66] - FilmArray GI Panel
that do the go tract in a similar way.
Transcribed by Erica Manion 7/25/2014

10

[Slide #67] - FilmArray STI Panel
They do sexually transmitted infections. Actually, the current panel for STDs has increased by
double, so we have 18 organisms we can identify.

[Slide #68] - FilmArray BioThreat Panel
And these have even been applied to bioterrorism, we have a whole panel that will identify the
most likely bio-terror agents with that product.

[Slide #69] - FilmArray Pipeline
And FilmArray, down the pike will be, a lower respiratory/pneumonia panel, transplant panel,
meningitis, urinary tract, febrile infant, antibiotic resistance panel, wound panel, identified in one
hour. So we are here at a real time microbiology. But in order to appreciate this, you have to know
what is currently done and will likely continue to be done in some labs because of cost. These
machines that I talked about are expensive. The VITEK that identifies organisms and gives you an
antibiogram is a half million, each machine. And institutions need multiple machines. Some of these
are very expensive to run. One viral panel that I showed you, the 22 tests with those bacteria in
addition, costs about $105 to us per test, and you have to add technology time, we have to add the
electricity, the room rent, whatever. There is a certain schedule they have. So that test could wind
up costing the patient $300. So we only use it when we have to and until costs come down, we are
going to continue to be very selective as to how it is used and when it is used. The more serious the
patients condition is, the more the justification occurs.

Thats it for Diagnostic Microbiology. Any questions? Basically im going to tell you, what I test you
on is whats in the notes. I dont trick you. If you know whats in the notes Im happy. How you
know it, how you study, whatever, thats enough for me. Same thing with my lectures in
chemotherapy and antibiotic resistance and the antibiotics lab. Whats there could be on the test.
You can take a break but please be back in 5 minutes. Were going to talk about chemotherapeutic
antibiotics. Thats a two part lecture.