Transcribed by David Landsman 7/31/14 4

Microbiology Lecture 18 Antibiotics Lab by Dr. Tierno

Slide 14 Relationship between zones of inhibition and MIC
[Dr. Tierno] - Ok I Guess we can start. Last time we left off with the, lets see, ah yes, the
regressive analysis curve. Chooo. Oops sorry about that. Let me just shut that off.

Now this is not meant for you to glean information concerning your susceptibility pattern, I
had a couple of questions the last time students wondering how theyre supposed to glean
information. Youre not, this was just to show you there was a relationship between
measuring the zone size around the disk on the Kirby-Bauer method and the MIC method,
which is tube dilution method, or you could do an MIC with an E test, or you could do an
MIC with the Vitech II, roman numeral 2. Any of those would be where you get your
information from. Youre not going to be getting it from this curve. Alright?

Slide 15 Bacteriostatic versus bacteriocidal antibiotics
Now, the concept of bacteriostasis versus bacteriocidal, meaning death to organisms, can
lead you to be confused about the use of certain drugs. This curve takes a specific aliquot of
time, its not an infinite amount of time, its just a small snapshot, and its showing you the
action of two antibiotics: tetracycline, which you know is a bacteriostatic drug and a
penicillin because it affects the cell wall is a bacteriocidal drug. And time is represented as
well as the colony count, the number of organisms, the log of the number of organisms. You
can see the control, if there were no antibiotics, the control growth would occur ascending
up to the time youre gonna begin your plateau growth, we are just taking this snapshot
and trying to understand the difference between bacteriostasis and bacteriocidal activity.
Now from this you can glean that the bacteria have stopped growing, this aliquot of time
doesnt show any death to organisms and what this does at this particular point in time, it
allows your immune response to come and clean up. Whereas you might have been losing
the race between the growth of the organism and your defence, now you have some time
afforded by the action of the antibiotic for your immune response to catch up and start
working for your benefit. But, it is not to stay if you extend the line further we would not
get death. Just think of sulfur drugs, its the quintessential static drug, remember 50% of
the population would pick up sulfur and 50% would pick up the right substrate, the para-
aminobenzoic acid. Therefore those that pick up the sulfur would eventually die off over
time. But you still have 50% of the population left. So of that, 50% will pick up PABA and
50% will pick up the sulfur so there can be death over time. But by definition, it holds in
check the further growth of the microorganism. Thats our working definition. Bactericidal
activity like penicillin, if you recall beta-lactam antibiotics work on the cell wall. There is a
high internal pressure, atmospheric pressure, inside the bacteria, anywhere from 5 to 30
atmospheres -- compared to the 1 atmosphere that we have here in the ambient air. So if
you put a nick in that cell wall, it will explode the bacteria or cause the explosion of the
bacteria and it will die, because you have disrupted vital functions of the organism. So
cidality, when you administer the drug and all both drugs were administered at about just
slightly less than the effect that it causes, so that you do get a certain number of organisms -
-remember you are dealing with population dynamics, you are not dealing with an
organism or two or three, you are dealing with large numbers of these bacteria. So you are
going to start to see death. It is not immediate, stark, complete, but you will start to see
Transcribed by David Landsman 7/31/14 4
over time the organisms dying off. Now the question always arises --is that clear? Fairly
straight forward. By the way, some organisms can be bactericidal and bacteriolytic, in other
words they can lyse the cell. So if you take an example of this is blood, in saline, you can see
the opacity of the blood, its pinkish, but if you add blood to water you can see a clearing
because the blood cells are lysed in water, its hypo-osmotic conditions. So not all
bactericidal drugs are bacteriolytic, for example aminoglycosides theyre cidal but they
interfere with protein synthesis so they dont lyse the cell. Just keep that in mind. Now,
before I get to that one, oh you already saw it. The question comes to mind, if you were ill
and you had lets say a bacteremia, or another type of infection, would you think a
bactericidal drug would be better than a bacteriolytic drug? Anybody?

Slide 16 Antibiotic activity: cidal vs static
It turns out that in testing, cidality and stasis, it turns out when a classic study, there were
several major studies, there are hundreds of reports to support this. But the 1980 McCabe
study is the big one, he had 612 bacteremias, thats blood infections, bacteria are in the
blood stream. That were treated with cidal drugs and static drugs, half and half. The
outcome was similar as far as death was concerned. People survived just as readily with a
static drug as they would with a cidal drug. So it really doesnt matter, personally you may
think well you want a cidal drug, there are times when cidality is bad. For example, women
who suffer toxic shock syndrome, which is a toxin mediated disease, if they get a beta-
lactam antibiotic, where the cell bursts open the cell staphylococcus aureus bursts open--
itll release whatever toxin is in its cell compartment. And if you do that times tens of
thousands or millions of bacteria, you have a bolus effect of extra toxin. Whereas if you use
a protein synthesis interfering agent like clindamycin you save a life because it keeps that
toxin in the cell, in the staph, and it allows your body to engulf the staphylococcus without
releasing toxin. So I just wanted to do a weighing of cidality or toxicity, certainly in
intensive care units I think that is where we see the greatest use of cidal drugs. Nobody
wants to take any chances.

Now there are 4 documented where cidality is a must. There is no competition with the
static drugs. The first was pyogenic meningitis, bacterial meningitis, this was done by
people at the VA hospital here a while ago. Doctors Rahal and Simberkoff, they did this
study in 82, and the reason you need a cidal drug is because phagocytic blood cells cannot
trap and engulf bacteria in a meningeal space. You really need a chemical agent that can kill
bacteria if those agents are taken up by the bacterium. And then later you can eradiate the
debris and thatll be cleaned up by the body. So thats one exception. And the other is a
study done in 1995 by Sande et al and because there is a lack of inflammatory response at
the site of infection, endocarditis would require a cidal drug, in fact they usually require 2
drugs at the same time. And one of the older regimens was beta-lactam plus
aminoglycoside, but there are others, also in cases of immunosuppression because of the
lack of an immune response, and thats an obvious one, for example HIV patients, patients
who have cancer, patients who are treated with chemotherapeutic agents for that cancer,
people who have blood problems, blood diseases of various types, where there immune
response is literally diminished significantly. So you need always a cidal drug. And last is
osteomyelitic processes. Osteomyelitis, bone infections require cidal drugs. Is that ok? You
get it?
Transcribed by David Landsman 7/31/14 4
Slide 17
Now, there are times when you have to treat because of severity of infection with 2
antibiotics. I mentioned endocarditis, would be one of those examples. And you say to
yourself, I wonder if these two drugs are going to be better than either one alone and the
combined effort will be synergistic, a greater or enhanced effect, or might one antagonize
the other. There is a laboratory study that you can do and you can request what is called a
synergy-antagonism study so that you can tell whether your regimen is efficacious in that
patient and whether indeed you do have synergy. Now lets take synergy first, there are
two different antibiotics on a disc agar plate and that plate --this is a very simple test, there
are more complicated tests done in tubes. But very simply, you take antibiotic A and B and
you put a lawn of the bacteria you are trying to kill and you incubate for eighteen hours
overnight and you look for a zone of inhibition which you can clearly see under the A,
under the B and somewhat under the C and certainly under the D. But we have to explain
those, they are different, remarkably different, and they give us a hint as to the functionality
of the combination. Now lets look at synergy. If you take the two drugs and put them next
to each other and you see a bridge between the two zones of inhibition where you have no
antibiotic present but the miniscule amounts of A and B in that center area are sufficient to
have an enhanced effect so you can see the zone of inhibition is increased. As opposed to
example A, where both are effective, you can see the susceptibility pattern of A and B, you
have a zone of inhibition around both drugs. So this is not synergy because theres no
enhanced effect. But you can still treat a patient if you get a pattern like A. Its called
additive effect, in other words youre hitting an organism with two mechanisms of
defeating a cell and that is a very good thing also, besides the synergistic. So additive or
synergistic you can treat. Now there are occasions when synergy is more or less a last
resort in treating the patient. This is a synergy. This is rarely used, but I will tell you, we
have at Tisch, as well as other medical centers, bugs, mainly and namely klebsiella
pneumoniae resistant to every single drug known presently on the formulary. Totally
resistant, called PAN resistant. The opposite of that would be PAN sensitive or susceptible,
meaning youre susceptible to just about every drug. Well PAN resistant bugs are very
difficult to treat. In many cases we dont even get that synergy, this D example. But they
treat it anyway with two drugs or three drugs in hopes of an in vivo activity, whereas in
vitro we dont see it, and these patients either die or get better on their own, they are
isolated from other patients to prevent the spread of those highly antibiotic resistant bugs
and klebsiella pneumoniae, you will hear in your practice later on, is one of those bugs, its a
ubiquitous organism, its on vegetables, its in soil and in the past was easy to treat, now it
has carbapenemases which endow the organism with ability to kill off or inactivate
imipenem, carbapenem drugs which are very powerful drugs. So, we might get a pattern
like this. So that if you were to test both of these drugs individually on an agar plate you
would look and say this is resistant to A and resistant to B. But, putting them together you
can see some little bit of synergy, that believe it or not is enough to allow a clinician to treat
with that drug in these special cases. And there are very special drugs that are used. One I
mentioned to you is polymyxin - that is rarely used because of the side effects - but they use
that one plus a very expensive tetracycline called tigercycline, well talk about efflux
mechanisms. This antibiotic is not easily effluxed out of a cell. Well talk about that on
Monday when we talk about antibiotic resistance. Now we come to C, antagonism. Here you
could see sort of a kidney bean shape or coffee bean shape. What this is showing is that you
Transcribed by David Landsman 7/31/14 4
have antagonism, you have a truncated zone. Zones are usually circular because this
chemical radiates out from the center and it should be a round zone but instead you see a
truncated flattened where the two antibiotics merge. Those drugs are said to be
antagonistic, they cannot be used together because you get a lesser effect than the better of
the two drugs so we opt to go with the better drug with the zone size and other parameters.
You follow? So this is another thing you can order from a laboratory during your practice if
you ever came to use multiple, uh two drugs. Which is not unusual.

Slide 18 Schlichter test
Now we come to a situation, lets say youre in a transplant unit and you have very sick
patients. Patients who are getting multiple organs, patients who are on antifungals, who are
on all sorts of immunologic boosting drugs, and multiple antibiotics, not just 2, maybe 3,
maybe 4 antibiotics plus antifungals, antivirals. How can you assess the effect of all of that
in a patient? There is no test, really that can adequately deal with those combinations.
However, Dr. Schlichter got the bright idea many years ago. What if I take a sample of the
patients own blood with all these drugs in it and react it to the patients own bug to see if
its killing the bug. That might be a way to asses for so many drugs in an in vivo condition
you really have no clue whats happening. So what he devised was taking a aliquot of blood
and spinning it down using the serum and reacting it to a 2 fold serial dilution of that
serum, with a diluent, and then adding the patients bug to every single tube. And of course
you have a control positive and control negative. Well this is what the Schlichter test is. It is
sometimes called the serum-cidal level. You can also do this for cerebrospinal fluid, CSF
fluid, but it is commonly called a Schlichter test or the serum-cidal level. Theres another
rule of thumb, and the rule of thumb is: if you can get a titer of greater of 1 to 8 or higher,
its greater than 1 to 8 (thats why my mind was saying dont say that) 1 to 8 or higher
dilution of that blood and get kill of 99.9% of bacteria, which by the way is the definition of
cidality. So if you can - through logs [??] - get cidality, 99.9% of the population killed, then
at a dilution of 1 to 2 or higher dilution then it is said to be efficacious [he says 1 to 2 but it
may be wrong, a few sentences later he says 1 to 8] thats the rule of thumb. So, it is a very
simple test and it works. In fact, many of the disease people at NYU when they request a
test, we get dilutions of 1 to 512. In other words you can dilute that blood 1 to 512 and still
get cidality, so they hit em hard and hit em well. Now if you get less than 1 to 8 you are not
doing that well and its considered not good, you may have to try to eliminate a drug and do
other tests, individual antagonism and synergy studies and try to come up with a different
formulation or try to cut some drugs. So each plate here is representing what you cant
visibly see, thats really stasis, then you subculture to a plate, those that show no growth,
and you can see you had growth 1 colony, 2 colonies, thats still 99.9% killed because you
are starting with a high level -- 10
8
colony forming units per millimeter and this tube, 1 to
32 is showing greater than 110% growth. So the cidal level, serum-cidal level will be this
one, Schlichter test, 1 to 16, so the treatment is efficacious, its greater than or equal to 1 to
8. You follow? A very simple test but its done very often.

Slide 19 Antibiotic blood levels
Now, there are times when you have a circumstance when youre dealing with very tough
drugs, rough drugs, lets say colistin [?], polymyxins and lets say an aminoglycoside, these
drugs can cause ototoxycity, nephrotoxicity, a whole bunch of toxicities. Chloramphenicol.
Transcribed by David Landsman 7/31/14 4
Drugs that you know are relatively toxic if given in the wrong dosage. Unfortunately,
sometimes, even with the right dosage, you can have hearing loss especially with
aminoglycosides and other types of effects. And you will without a question, without a
doubt, be sued. Suing unfortunately is a big problem, heh, in America. We have a lot of suits
running around. So for your protection, C.O.A., your protection in addition to the patient
benefit, it behooves you to request a blood level. I do have to make one comment about that
and I want to go back to something else. A blood level that is in the peak and the trough
level. It is called a peak and trough, a blood level, you take two. A peak blood level of the
drug is the drug, maybe within the half hour depending upon the administration of the drug
of administration, you take that blood sample. That will be its high point. Then you wait
just before the time you have to give the next dose, lets say its Q at H [??], so it will be like
about right before that eighth hour, you would take a trough specimen. Theoretically, that
is the lowest amount of drug you should find. Ok. And you do a peak and trough to make
sure that you are within the level, the guidelines, for aminoglycoside. Every single
aminoglycoside has different blood levels that are recommended to be practicing good
dentistry or good administration of antibiotics, you cant go above them without some toxic
effect. So if you dont have a record. The first thing a lawyer will say: well doctor, how did
you know you werent giving too much of this toxic drug, this poison, to your patient, and
you can say well -- to the lawyer -- I have data to support my treatment and to support
what Ive done with this patient. And you have to show that data. So thats where this test
comes in. Antibiotic blood levels. It is a pain in the neck because it involves two tests, and
you are sending it out to the laboratory. But believe me, you have to do it to make sure A,
that the patient is getting the proper dose so that you can kill the organism, B, the patient is
not getting too much so you dont hurt the patient, remember the chemotherapeutic effect,
selective toxicity, you want to kill the organism not the patient -- remember that arsenic
business with salvarsan 6-0-6 -- and thirdly to cover yourself, C.O.A., you need those tests.
Now I mentioned peak and trough for taking blood serum. But I have to go back and tell
you, we do a peak and a trough for the serum cidal level also, the Schlichter test. In other
words, if both the peak and the trough are 1 to 8 or higher, the Schlichter test is said to be
efficacious. Same thing with blood levels, you do a peak and a trough. The
radioimmunoassay is rarely done, it is no longer really done because people are afraid of
radiologic components and its a hazard. Interestingly, for your own edification before I
give you a summation, I worked with Rosalyn Yalow quite a ways back, before she got the
Nobel prize, and she developed radioimmunoassay to detect hormone levels in patients.
She was laughed at by the American medical association when she presented her data. And
they said you cant have antibodies to hormone. In those days, you know this is back in the
late 60s. And she was just so distraught because she knew she was right. And when you
know youre right you want to pursue it. So she went to Europe to present the same data
and they gave her a standing ovation for the data she was presenting. And she said in
America, to try to get this recognized, she came to me. At that time I was working on a
research, I was doing my graduate work, I worked on my doctorate at the VA hospital in the
Bronx. And she came to me and she said Phil, you are the only one who can help me. I said I
dont know if you can help me but Id be happy to. She said you are doing blood levels,
speaking of blood levels, blood levels, and you could help me because I developed a
radioimmunoassay. I will tag the antibiotic to a protein and I will run it through
radioimmunoassay so I said let me look at the procedure. I looked at the whole thing and I
Transcribed by David Landsman 7/31/14 4
said to myself: I think shes batty. But that was just the first thing that came to my head. But
she wasnt, she was right on target. I failed to see how she was going to develop antibodies,
which I shouldve done. And I always try to live up to Louis Pasteurs dictum: chance favors
the prepared mind. And later she called me on it, and she said you know I hoped that you
couldve been here with me. That was such a blow!! Anyway, so I just figured I would tell
you a little story so you have a little time. Everybody get this? Usually its done
colorimetrically or fluorimetrically, these tests.

Slide The Multiplex PCR
Now, there is a new test I mention to you by the way I did that twice more, with two other
Nobel laureates, but I wont even go into it, I had my chance. Multiplex PCR, its the best
possible test but its too expensive right now. This test not only detects bacteria, just about
any bacteria or viruses in one hour, but also antimicrobial resistance genes by PCR.

Slide Film Array
It is a film array, its a large card which does testing, is easy to set up, it takes a minute or
two to set up. You put it in that machine and you get the result of every single chromosomal
and extrachromosomal gene of resistance. We are going to be talking about that on
Monday, resistance. And identifying that from the bacteria you can say well this drug will
be no good, this drug will be no good, its got beta-lactamases this one that one the other
one maybe about 60 80 different beta-lactamases alone. So you would not use a particular
drug based on this, in an hour, which is pretty good. The test costs about 150$ as opposed
to a Kirby-Bauer which costs less than a dollar, or a Vitech MIC which costs 3$, the actual
test so we can only use it under certain circumstance but it will be the wave of the future,
your future, so the practice of real time microbiology will be yours. Arent you lucky! Now,
lets just rehash where weve been, summarize.

Back to Slide 4 - Antibiotics
For most ordinary purposes you just need a Kirby-Bauer. Many small community hospitals
cannot afford larger machines like the Vitech which costs a half million dollars a unit and
you may need 2 or 3 units. Of course NYU just got 1.2 billion dollars from FEMA, pretty
good, but they had about 3 billion dollars damage when you add up all the material so its a
big loss from hurricane Sandy.

Now, so Kirby-Bauer disc agar diffusion method on a petri dish looking for zones of
inhibition is an easy test to be done in most laboratories but it takes 18 hours so you have
to empirically treat until you get back your susceptibility if you are using that test. And you
get SLR reporting, susceptible, intermediate or resistant. Now lets move to the MIC test.
Minimum inhibitory concentration. Thats a tube dilution, macro tube or micro tube. Some
of the micro tubes are micro titer plates. You can see the little wells instead of big tubes,
you save on media and you save in general money when doing that test. And or the Vitech.
Thats that little card I showed you with 36 little holes or wells which can have different
antibiotics and generate a curve and susceptibility pattern. Thats minimum inhibitory
concentration. When would you use that? Why couldnt you use Kirby-Bauer for
everything? Well, there are times when you have infections that are quite recessed in the
body in different organs, the prostate or the gallbladder and you have to know a few things
Transcribed by David Landsman 7/31/14 4
more before you treat. With a drug based on Kirby-Bauer you have to know what the
concentration of drug would be at the site. And you want to know what the minimum
amount of drug is necessary to kill the bacterium. Therefore you can figure out, based on
the MIC data, youve got an MIC of 1 lets say, the manufacturer tells you 4 micrograms are
found at the site, and you know that the if using an antibiotic such as beta-lactam you need
4 times the MIC to kill the organism so that 4 times 1 is 4 you can use it, it is efficacious. So
under those circumstances you may need an MIC. An E test is a modified MIC using a strip
on a Mueller-Hinton plate, the same plate that we do the Kirby-Bauer on, but the strip has
gradations of antibiotics on it and you get an elliptical zone size and you look at the
meniscus to determine what the MIC is. Thats an alternative to the MIC test. It is an MIC,
but a different type called an E test. Of course it is not 2 fold dilution. There are many more
in between steps. Another modification would be the microtiter plate, thats a micro
method of doing MIC. You have macrotube and you have microtube. Now also, MBC can be
generated after the MIC is gleaned. You know that there are organisms that are said to be
tolerant meaning that they elongate. And the reason they elongate, I might not have
mentioned it the other day, is because the lytic enzymes or autolysins are not produced.
The organism is just shutting that down so that it is not going to explode. It is sort of a
defence, different than resistance, it is not a resistance, just turning off of cellular
machinery so the organism elongates and but doesnt get eradicated. And if you take the
drug away the organism can come back. Remember the pneumococcus is notorious for
doing this, and there are other organisms that can likewise do it. So tolerance is important.
To know tolerance, what would you need? An MBC, minimum bacteriocidal concentration,
because you want to know whether the organism is being killed. So you take the MIC tubes,
which involves visualization, the last tube visually that has the lowest amount of drug that
can inhibit bacteria. you take all those tubes that have no apparent growth, subculture
them to a petri dish of agar to look for growth, and if you find growth then you have to do
the lowest amount of drug that kills all the bacteria as your MBC. And usually thats one
tube off, the same tube, one tube off, no more than 2. But if you have 4, 5, 6, 8 or 10 tubes,
between an MIC value and an MBC value, then the organism is said to be tolerant and you
change the drug. Thats tolerance. Now we also know that we can have the laboratory do
antagonism and synergistic studies, and you saw the importance of them. This is not just an
exercise to see how drug dynamics works. This is useful information, patients lives have
been saved. So either drug works more effective than the two combined, in other words
greater value than the additive effect of the two. Or you can have additive which is not an
enhancement but you have two ways of killing a bacterium, lets say one is a cell wall active
drug and the other is protein synthesizing drug, youre not hitting the same bug all the
time. Remember you have population dynamics so it works. And then you can also have an
antagonism where you have two drugs being less effective than the greater of the two
drugs alone. And then of course when youre treating with multiple drugs in a complex
situation such as transplantation when you have patients on antifungals, who are on
steroids, who are on all sorts of drugs in addition to multiple antibiotics, the Schlichter test
(the serum-cidal level) is the most effective test to identify efficacious application
combination of drugs, especially when there are many. And you know there is a rule of
thumb, 1 to 8 dilution or higher, that still kills the bug, is said to be the serum-cidal tider.
And you do that for peak, the high point of the drug and the trough, the low point of the
drug, and make sure you get 1 to 8 or higher. Then, after the Schlichter test you are in the
Transcribed by David Landsman 7/31/14 4
clear, you just have to worry about the patient getting the proper level of drug if you are
using toxic drugs by doing blood levels, antibiotic blood levels, those are done
colorimetrically or fluorimetrically and you know the reasons you need to do that if you
deal with those drugs. And I did touch on all of these words, you should be familiar with
these concepts because thats the bulk of the laboratory aspect of antibiotic susceptibility
generation. Lastly I showed you the PCR, thats not commonly used because its too
expensive. But it will be used in your lifetime, in your professional lifetime, and it will be to
your benefit, because the practice of real time microbiology is the goal. In other words,
generating information within two hours after the submission of a test, thats real time
microbiology, and thats coming to you. It is here basically but a little expensive and we
judiciously use testing to reduce cost. Any questions?

[Student]: In the scope of our licence, would we be able to do blood levels?

[Dr. Tierno] - You will send the person to a laboratory, you are fully able to go to a medical
laboratory, I dont want to say use NYU Langone but thats a good one. You go to the
outpatient center, you write your prescription, what you need to have done for your patient
and it goes right to the outpatient laboratory at Langone, or Columbia, or Sinai, or private
labs, there are many of them. Of course you have to know your lab and know the work that
they do and come to conclusions as to whether you trust them. Certainly going to university
hospitals is the mecca of labs. You are not going to personally draw the blood, the patient
would have to go. Its like doing a glucose tolerance test, you are not going to do that,
clinicians dont do it they send the patient to a lab. And they are gonna sit there for a few
hours, they drink the stuff and the technician takes blood every half hour or whatever and
reports back.