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Microbiology - Lecture 16 Chemotherapy of bacterial Infections II by Dr. Tierno

[Slide 1] Chemotherapy of Bacterial Infections
[Dr. Tierno] K hello everybody again. This is part two of the chemotherapeutic agent discussion,
primarily on bacteria. I mentioned to you, I think the last time we left off at tetracyclines. But I just
wanted to touch base on this equation to refresh you. Antibiotics are primarily useful for doing
three things which I discussed last time, but I want to summarize it. An infection is related to this
equation, the Theobald Smith equation.

[Slide #2] Biological Role of Antimicrobial Agents
The Theobald Smith approximates infection is mostly a result of the number of organisms that you
are exposed to or are introduced into you, multiplied by their virulence which is expressed by
virulent factors like toxin production as well as adherence ability and things of that nature divided
by the host response. Now antibiotics affect this equation in all three of those instances. One is the
virulence, which includes adherence of the organism to the host, also includes toxicity, and the
number of bacteria. Thats the way it works. Keep in mind that no matter what antibiotic is
administered, you definitely need the help of your immune response. Otherwise the therapy would
be useless to you. Ill give you an example. Let us say there are 2 patients that come into the ER,
both of whom have pneumonia. One man is elderly, he is about 86. He is in the ER being worked up.
He was found to have a Streptococcus pneumoniae. A pneumococcus. Another man is a college
football athlete. He comes into the ER, he has pneumonia. He was found to have the same organism,
Streptococcus pneumoniae, otherwise called pneumococci. And both are treated with penicillin,
because doing an antibiogram, the physicians discovered that these organisms are exquisitely
susceptible to penicillin. They are both given penicillin. Make a long story short, after two days the
elderly man dies, whereas the younger man lives. Both had the same organism, the same
susceptibility, they got the same treatment, but one dies, one lives. The elderly man is
immunocomprimised by definition. If you cant clear your lungs of mucus and cellular debris and
assist the antibiotics from working, you might not survive. If you have an underlying disease the
same thing applies. The very young and the very old succumb to the same situation. By the way,
pneumococci, or not pneumococci, Pneumonia is called the old mans best friend, and by old man, it
is old man or woman. Only because, prior to death, the patient goes into a state of anoxia, so that
the brain is not aware and not functioning, so you sort of die without knowing you are dying. Its
not such a nice characterization, but thats the way it is. So antibiotic therapy is extremely
important.

[Slide #9] No title, figure of bacteria
Let me just give you this one chart to remind you where we were yesterday. These are the
mechanisms by which drugs work. The most popular is interfering with cell wall synthesis, which
we discussed, the beta lactam antibiotics like penicillin and cephalosporin. And we were just
starting on the protein synthesis interference by drugs like the aminoglycosides, which we
discussed. Now we are on tetracycline. And in this case, the protein synthesis interference is either
a 50S inhibitors or a 30S ribosomal segment inhibitor. And thats what we are going to be
discussing, actually representatives of both of those again. Other ways that antibiotics can work,
they can interfere with DNA dependent RNA polymerase, like rifampin, is a characteristic drug,
mostly used for TB but we will touch on that later, and interference with DNA replication, mostly by
interfering with the gyrase enzymes. Two other ways, the last two. Folic acid metabolism, this is
the mechanisms, this is the mechanism of sulfa drugs and another drug called trimethoprim. I
mentioned many of you may have been treated with Septra, or Bactrim, both of which contain those
two drugs. And thereby [?] interfering with the production of folate, folic acid. And lastly we come
to cell wall, strike that, cell membrane interference. And here, there is a group of drugs called the
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polymyxins. They are very toxic, but what they do, they act mostly like a detergent and interfere
with selective entrance of molecules into the cell and out of the cell. And when that selection
Is, that selectivity is eliminated the organism dies. Keep in mind, with gram positive and gram
negative bacteria, both, they have a high internal atmospheric pressure, anywhere from 5 to even as
much as 30 atmospheres inside. So if you interfere with the cell wall, you always have cidality,
because the organism blows up in most cases. Alright. Lets continue with the lecture by getting
back to where we were.

[Scrolling through slides]
We did the aminoglycosides, and I talked about that nuance where you can actually have an
aminoglycoside dependent growth. Keep that in mind.


[Slide #30] Tetracyclines
Alright, now we are on the tetracyclines. The tetracyclines are a relatively large molecule and have
many different permutations. Each of these Rs represent a different add-on molecule. The idea
here is that, sometimes with the parent molecule like tetracycline, if its exposed, if bacteria are
exposed to it long enough, they may develop a method to circumvent the activity of the drug. And
most of these antibiotics that in the last 60 to 70 years that have been used, are coming nearly
unusable because antibiotic drug resistance is so rampant, and the use of drugs is a major problem
in the medical field because people use them like cough drops rather than careful administration of
antibiotics. We also use it in the animal industry and agricultural industry so that pieces of meat
that you handle, and even the vegetables actually, since there is cow manure and other manures on
the vegetable crops and can contain some highly resistant organisms.
So now, if you manipulate those Rs you can get different molecules of tetracyclines or different
permutations of tetracyclines. Two of the best, believe it or not, are the doxycycline and the
minocycline. Two of the best functioning tetracyclines. And in fact, it is doxycycline that is used if
you are exposed to Lymes disease, early on and a tick has been discovered on you and has bitten
you. Especially if it is an izotes [?] tick, and in nymphal stage they will administer two doxycycline
capsules and that is about all you would need to stave off Lymes disease. But, if

[Slide #31] No title
If Lymes disease occurs, it becomes a significant problem to eradicate, and there may even be
chronic forms, but that is another story. Now, here is where tetracycline interferes with the 30S
ribosomal RNA unit. In fact, the transfer RNA, the transfer RNA is interfered with so that the
growing chain is interfered with or becomes non-sensical.

[Slide #32] Tetracyclines
And in fact, because the tetracycline molecule binds reversibly, as opposed to covalently, it is
usually a static drug. It usually indicates a static drug if it is a reversible link. And here, the site of
action of all tetracyclines, the 30S ribosomes, but its binding is reversible and it prevents
polypeptide synthesis. So these are the categories of things you must knowfor this group of drugs.
They are static.
Now, they are broad spectrum in that they do effect gram positive organisms as well as gram
negative organisms. They are fairly good at some gram positive organisms, and as I say, the more
we are using them, and it seems like tetracycline is being overused now at a remarkable frequency,
that there is some problems arising with resistance.
This group of drugs is interfered with by many things, many food stuffs. Especially dairy, antiacids,
and iron. Tend to inactivate the tetracycline. So if you are taking tetracycline you have to do it
without food, you do it on an empty stomach. It also can cause depression of bone if you are
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younger than 8. Although in some cases, as I mentioned, Lymes disease , even in children, they may
give those two doxycycline capsules too, so there are exceptions. But in general you should be
careful because it causes mottling of the teeth. In fact, when tetracycline was one of a few drugs
back in the 50s, many individuals were given tetracycline and had teeth that were stained and
many dentists didnt have the current way of capping those or whatever you would call it, putting
a layer on it, and nowadays of course that is possible. So there are many people that went around
with mottled teeth.
Now, this group of drugs, as well as a few other groups which I will talk about now, can get inside
the eukaryotic cell. A drug like penicillin, ampicillin, cephalosporins, can not get inside your cells,
your eukaryotic cells. This is an exception. Tetracyclines can get in your cells. If you have bacteria
that are intracellular, at some point you can use tetracycline if you have susceptibility. You cannot
use a penicillin because it doesnt go in eukaryotic cells. There are two, or three other main classes
that get inside eukaryotic cells.

The second group is macrolides, like erythromycin. You may have heard of the Z pack. Azithromax,
Azithromycin. Were going to discuss that but that is the second group that can get inside your
eukaryotic cell to affect a killing of a particular organism that is intracellular.

Another group is the quinolones like cipro, ciprofloxacin. You may have heard of that drug during
the anthrax attacks. You might have been very young, and maybe you read about it, or maybe I
doubt if youve been on the drug. Ciprofloxacin. The quinolones is the third group
Then you have the sulfa drugs that can get inside the cell as well as act outside the cell. That is very
important when you are dealing with organisms like Chlamydia trachomatis, which is an
intracellular bacterium. Keep that in mind.


[Slide #33] Chloramphenicol
Now, chloramphenicol actually, erythromycin and lincosins have a similar, or actually the same
binding site in some cases, and we will discuss them together as a group. The mechanism is the
same for each group. What characterizes chloramphenicol is the nitrobenzene ring, which you can
see up there nicely. Chloramphenicol inhibits peptide bond formation. Unfortunately,
chloramphenicol, or chloromycetin as it is called, is a very rough drug. Its not given as a first line
drug because of its side effects. It has significant side effects, and actually has been involved in
deaths of people who had suppression of the bone marrow, aplastic anemia. So, you have to reserve
this drug for unusual circumstances where all else is failing. Chloromycetin, as they say, the class is
nitrobenzene antibiotics. The site of action is 50S ribosomal subunit.

[Slide #34] Chloramphenicol
Again, reversibly. So that tells you immediately, if it is binding reversibly, it is a static drug. Well
talk about stasis vs lytic and cidal at the next lecture. Were talking about antibiotics in general.
Um, it is broad spectrum, it does cover gram negative as well as gram positive drugs. And it used be
used a lot to treat salmonella back in the 60s, and thats where all these cases of bone marrow
products came up. It can be given orally and parenterally. Remember the aminoglycosides? You
cant take them orally. You can only take them parenterally. Well, this drug you can take both
ways, by mouth or by injection. Another good thing about chloro, if you have a whopping anaerobic
infection, it might be useful to use this drug. But as a class it is reserved as a last resort type
antibiotic when all else fails.

[Slide #35] Erythromycin
Now the other member that is intracellular is erythromycin. A macrolide antibiotic, and there are
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various permutations of this drug, by changing the molecules, as you can see, I think I have a chart
no I dont. Well, changing configuration of some of these molecules, OH groups, CH3 groups and H
groups, you can add on and make the drugs more functional and overcome resistance or make it
more difficult for the bacterium to develop resistance.

[Slide #36] Erythromycin
This particular drug is static but has a unique property. If you give it at very high concentrations, it
can be cidal to some organisms. So thats one of the unique things about erythromycins. It does
cause a lot of side effects, unfortunately, like intestinal side effects because it kills a lot of organisms
in the gut, and that leads to some problems. The newer drugs, azithromax or azithromycin, and
clairithromycin, they are two new derivatives of the macrolides. They have lower doses

and half life, and less, a greater half life, and less GI problems. So, if youve ever been on a Z pack
youve had a macrolide. And it is broad spectrum yes, but the gram negatives are specialty gram
negatives that get affected. Remember I mentioned chlamydia, which is an intracellular bug, and
that is why it is used for chlamydia. Rikettsia is the same thing. There intra-cellular organisms that
some point, and this drug is very useful. Same with Hemophilus, Legionella and mycoplasma. The
mycoplasma are cell wall-less bacteria. And a Mycoplasma pneumoniae, can cause pneumonia in
people. And Legionella, of course you may be familiar with Legionella or pneumonia, and
bartonella, you may not be as familiar with that but these are specialty gram negatives, and this
particular drug is useful because of the intracellular activity. Thats its only claim to fame.

[Slide #37] Lincomycin and Clindamycin
Now, lincomycin and clindamycin, which is a chlorinated lincomycin, are two extremely good drugs.
Unfortunately, the lincomycins have many side effects

[Slide #38] Clindamycin
Lincosamides have side effects. For example, they can give rise to Clostridium difficile
pseudomembranous enterocolitis. When this drug first came out, I was testing it, actually for use
on hemodialysis patients. And unfortunately, we did not recognize the fact that these people
developed diarrhea from the drug. We looked at a large group of people and only a few of them
developed diarrhea, profuse diarrhea that we had to take them off the study. Well, they actually
had pseudomembranous enterocolitis, and we were assuming, because they were on hemodialysis,
that it was just an anomaly, they are on dialysis, theres fluid imbalances. So as Louis Pasteur said,
chance favors the prepared mind, our mind was not prepared to see that. We were doing a study.
So sometimes, inadvertently, you may overlook things, but of course I learned my lesson from that.
This is also a static drug that can be cidal in high concentrations. Because of its widespread use,
against C. difficile - strike that. Widespread use, it gave rise to many bouts of C .difficile in people, so
now it is used only to treat anaerobic infections. It actually treats very well an anaerobic infection.
Its one of the primary drugs that is used for an anaerobic infection. Its also useful in Toxic Shock
Syndrome. If you remember the slide that I showed you at the beginning, the first lecture on
antibiotics, you saw that this particular drug reduced toxin to just about zero and yet didnt
interfere with growth of the organism. So even that would be useful staving off a whopping toxic
shock infection.

Of course, if you increase the concentration, you dont need to give sub lethal doses, you can give
lethal doses, you will kill off the organism as well as stop its toxin production. I didnt add that here
but it is very useful. Its one of the primary drugs used if they suspect, if your clinician suspects
toxic shock syndrome. If a woman comes into the ER who is mensruating, using a tampon, and has a
constellation of symptoms, they will administer this drug in addition to fluids. So, this lincosin
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group is very important and as I say the clindamycin is a 7 chloro-deoxy lincosin. Very similar to
the parent drug.


[Slide #39] Figure 18.15
Now, the next category that were talking about are the sulfa drugs. They interfere with the folate
synthesis. And in fact if you look at the sulfanilamide molecule and compare it to the PABA, para-
aminobenzoic acid, which is important in the metabolism of folates, you can see how similar they
are, they are called analogs. Just like penicillin. Look at how similar penicillin was to the amino
acid, alanine. And other drugs were analogs that we discussed.

well this is another analog, sulfur is an analog of para-aminobenzoic acid. And so the organism
picks it up approximately half the time. There is an anticipated frequency of pick up 50% of the
time. Youre dealing with population dynamics, remember, not one organism. We are dealing with
multitudes. So, over time the population, each population that remains will pick up the drug 50%.
So over time it will dwindle down the population and eventually effect its stasis or cure.


[Slide #40] Sulphonamides
Sulfa drugs are a very large group in fact. As I said, they are analogs of PABA, and they interfere
with competitive metabolic inhibition by preventing the conversion of para-aminobenzoic acid
(PABA) to dihydropteroic acid. They do this by adhering to enzymes, much like the transpeptidase
enzyme that penicillin adheres to. This also adheres to enzymes. Not only is folic acid interfered
with but also purines and pyrimidines from the same metabolic pattern. So they interfere with the
production of purines and pyrimidines.
And they are static in nature. And next lecture, in the lecture after this we are gonna talk about
stasis vs cidality. Which is better. Would you rather have a static drug, or would you rather have a
cidal drug, a drug that kills, or a drug that holds in check all things. All populations. It is broad
spectrum, it actually interferes with some parasites like pneumocystis carinii, and also some
protozoa parasites, plasmodium in this particular case. Its intracellular, again. And it is synergystic
with trimethoprim. Thats why Septra and Bactrim use this particular drug, these particular drug
combinations to fight infection, because they effect the pathway in two separate areas. They can
give rise to some side effects. Generally speaking they are minor. You can control the nausea and
the vomiting with administration of fluids. You have to take a lot of fluids with this drug.


[Slide #41] Figure 11-15 Structure of sulfonamide and trimethoprim
And here is where you can see the action of the sulfa drug and the action of the trimethoprim. They
are interfering with the synthetase enzyme and the reductase enzyme, trimethoprim reductase, the
sulfonomide synthetase enzyme

[Slide #42] Trimethoprim (an antifolic acid agent)
Trimethoprim is also used alone. Its not used in the United States alone, but in Europe it is
commonly used alone. So in case, you should be aware of that anyway. So trimethoprim can be used
without sulfa as a, interfering with the reductase enzyme it adheres to. It is a static drug classically
unless, if you are using it alone you are using it in higher concentrations, therefore it can be cidal.
So these are the exceptions to the static-cidal rule. This is the third exception. Its broad spectrum,
although mainly it is mixed with sulfur in this country and that is the way it would be used, because
of the synergistic activity. It has the same adverse effects, nausea, vomiting. You need to take fluids
with it to make sure that the liver is cleansing the system of the excess drug, so the water facilitates
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the elimination of the drug.

[Slide #43] Quinolones
Now we are coming to another pathway, DNA synthesis. The quinolones are a very large class of
drugs. Ciprofloxacin is probably one of the more used of the quinolones. The same with the
levoquin, is another one that is commonly used. Nalidixic acid, which is an old drug, is actually a
quinolone. But the quinolones were talking about are fluoroquinolones, they have a fluoride
molecule. Thats what differentiates these quinolones, the newer quinolones from the older
quinolones like Nalidixic acid.

[Slide #44] Figure 2: DNA Supercoiling
And here you can see on the gyrase system is interfered with. Supercoiling is one of the things
these fluoroquinolones interfere with, and in fact there are two topoisomerases

[Slide #45] Quinolones
DNA gyrase topoisomerase II, and topoisomerase IV, they are needed for both regulation of the
supercoiling as well as separation of the DNA. So you have both those enzymes involved in
levoquin and moxifloxacin, which is another quinolone, and ciprofloxacin interfere, or inhibits that
gyrase activity. They are cidal because of that, they are broad spectrum, gram positive, gram
negative. They are ok for methicillin susceptible Staph aureus. But if it is methicillin resistant Staph
aureus, its not good. It is shown to clinically fail. It can cause some skeletal abnormalities, therefore
it is not indicated for people under 18 years of age, or for a pregnant woman with a developing
fetus.
When it was first introduced in the 80s, Ciprofloxacin, just to give you an idea of its susceptibility to
one organism. There is an organism called Pseudomonas aeruginosa, its a water borne organism,
youll get that later in the infection course, which is found in most peoples faucets as I mentioned
the last time. It causes a great deal of burn infections as well as significant infections in patients
who have immunocompromised conditions. In the 80s, when it was first introduced, ciprofloxacin,
that is, pseudomonas was 95% susceptible, 95% of the strains were susceptible to this group of
drugs, to quinolones. Currently less than 45% are susceptible to any degree of quinolones. Thats a
pretty significant drop. So that its usefulness against certain hearty organisms has diminished many
others unfortunately because of inappropriate usage. Apparently very few drugs are being
reserved to treat important infections.

[Slide #46] Rifampin
Now, this is rifampin I mentioned that interferes with DNA dependent RNA polymerase. It is a
complex molecule, and it is mostly used for TB. It can be used for Staphylococci, but only with
another drug. This drug can never be used alone, whether it is against TB or against Staphylococci.
And you can manipulate these various R groups to make other types of Rifomycins

[Slide #47] Rifampin (Macrocyclic antibiotics)
Since resistance develops extraordinarily fast, you have to use it with another drug. That is the only
caveat. And yet, this has been useful in treating methicillin resistant Staph aureus which are
community acquired. Which are toxogenic organisms. And they have used successfully Rifampin
with doxycycline of all things. Now doxy is a static drug. This drug is listed as cidal but some people
think it really is static, and it depends on the organism. It is broad spectrum, it does hit the Staph
and Haemophilus influenza which is a gram negative. And nongonacoccal organisms. Neisseria
meningitidis, TB. It is mostly used for TB and now methicillin resistant strains of Staph aureus with
other drugs. Its a macrocyclic antibiotic as you see, a very large molecule. It is a unique class,
aromatic ring system.
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[Slide #48] No title
And rifampin, the messenger RNA up at the top of the grid, thats where it interferes with.

[Slide #49] Polymyxins
Now this is a group of drugs that I had mentioned before, polymyxins. They act less like an
antibiotic than they do a detergent. A disrupter of the cell envelope. They separate the membrane
and the cell wall so that it causes selective permeability to be upset so things go out of the cell ad lib
and into the cell ad lib and therefore it eventually kills the organism because of selective toxicity
loss. And you can see where it fits right in the cell envelope. See how it sits there like two little
detergent molecules. Very similar to a disinfectant. And you can see this is an interesting molecule.
It has many amino acids

[Slide #50] Colistin
In fact there are two types. Polymyxin B and polymyxin E, bth of which are used in medicine today.
It has a lot of side effects so it is now a last resort drug. Unfortunately a lot of the last resort drugs
are becoming first order drugs because of the high degree of antibiotic resistance. This is especially
useful against Pseudomonas aeruginosa. It is a big problem in intensive care units and a big
problem with chronic disease patients as well as burn centers. It is a neurotoxic and nephrotoxic
side effect. It has both neurologic and kidney side effects, major effects, so it has to be used with
caution. And you have to take blood levels for any drug that is toxic in any way to make sure the
patient is not given an over dose of drug. That is extremely important in your practice, because you
want to make sure you are adhering to the rules, so to speak, and you are not giving beyond the
appropriate dose. And it is also for your protection, to protect against the suits. As I said it causes
leakage of the cell envelope and eventually can kill, its a cidal drug because it disrupts the cell
envelope.

[Slide #51] Amphotericin
Now, there are just a couple of drugs that I wanted to touch base on. Which actually interfere with
fungi. Now, you are familiar with and have used quite ordinarily in many patients that have chronic
conditions, in transplant centers. So youre going to be using, in those cases, antifungals as well as
antibacterials. Now, what I was talking about was primarily for antibacterials, you should be aware
of these couple of drugs that are used against fungi. Amphotericin, amphotericin B, a polyene
antibiotic, is selectively toxic to fungi. Unfortunately it does have some side effects in humans.
There is nothing that is totally non injurious to the body. If you take drugs for a long period of time,
even if it just upsets the flora, you cant be on drugs for months or years (with some exceptions, TB
is one of them).

[Slide #52] Amphotericin
Now amphotericin B is a polyene antibiotic. You may have heard of Nystatin. Its actually in this
class. Many women have it prescribed for candida albicans, thrush infections of the vaginal vault.
The cite of action, it binds to ergosterol, which can actually create holes in the membrane. Now we
have cholesterol in our cells, mammalian cells. In fungal membranes they contain sterols, but not
cholesterol. They contain ergosterol, zymosterol
enzymosterol. And this is where the action of this drug is selective. It uses the selective toxicity
ratio that we talked about last time. It is selective for fungi because we dont have ergosterol and
zymosterol in our membranes. So it works at causing a membrane hole or pore. At high
concentration, however, a side effect is, it can bind to cholesterol even though we dont have
ergosterol, and that is its main function, at disrupting that in fungi. We do have cholesterol which is
a similar molecule, similar sterol, so that enough of it may interfere with cholesterol. And that is its
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biggest side effect, in using this particular drug. This is one of the most importantly used
antifungals. When other antifungals fail, this is still the one that is used, and it is a cidal drug.

[Slide #53] Azoles
Another group of drug, because once you make a hole in the membrane and the wall, remember
youre getting loss of selective entrance and exit for the cell. Now azoles are a whole group of
antifungals. Imidazole is one of the most common. Imidazole therapy. Ketoconazole and
micronazole are the other two most common azoles.

[Slide #54] Azoles
But azoles in general interfere with the cytochrome P450 dependent reaction, the same thing,
ergosterol is interfered with, but it leads to methyl sterols which are less stable than ergosterol,
And are subject therefore to leakage when they make their envelope, it is subject to leakage, the
membrane. As opposed to putting a hole in it which is lethal, this, you take the drug away, you have
fungi that survive and could reproduce. So it is static drug. And unfortunately people who have
liver problems may have to avoid use of these particular drugs because they are eliminated by the
liver and taking too much of a dose can raise havoc in the body if you have a liver problem of any
kind.

[Slide #55] Microscopy images
Heres a control, heres the way it works, these azoles. There is a yeast growing, a Candida albicans.
At one hour, at four hours, at seven hours, it is developing all these pseudomycelia, which actually
continue the infection. But if you give a small dose of ketoconazole, you can see its not going
through its normal sequence and its not developing normal hyphae. You give a higher amount and
you can actually have death occur to the organisms. So it does work and it is used often. There are
other drugs, I just wanted to touch on these two.

[Slide #56] Diagram
These are the classes of other drugs. You dont really have to know these, be aware of them except
for the two I mentioned, the azoles and the amphoteracin. The azole we already discussed and the
polyene weve discussed. You do have other drugs like the candins that interfere with the cell wall
formation, the beta-D-glucan production. So you get a pore [in the?] cell wall and that can kill the
organism. You have five fluorocytosene which can interfere with protein synthesis as well as DNA
synthesis if the fungus picks it up. But thats just there for your edifice. Lets see, I think thats about
it. Remember its not the drug only that gives rise to a cure. The patient has to have a good immune
system operating to affect a cure and resolution of an infection.
Alright, any questions? Take about a five or six minute break and then we will continue.