Transcribed by Jacqueline Heath 11.24.14

Infectious Diseases [11 and 12] Intro to Gram negatives and Enterics, E. coli by Dr. Boylan

[Boylan] Ok good afternoon, all you gram negative bacteria lovers. So, up to this point we have covered most of the important gram positive bacteria. If you want to briefly review, there are only two gram positive cocci and gram positive rods. The gram positive cocci were the staphylococci and the streptococci. We talked about aureus and pyogenes and all a few other species in each of those two genera. We dial then to the gram positive rods. The two major genera were the bacillus and the clostridium genera. Each of those gram positive rods that form spores. The big difference is the bacillus are all aerobic and clostridium are all anaerobic. We talked about tetanus and botulism and c. diff and other infections caused by those bacteria. And the other gram positive rods are, let’s see, corynebacterium causes diphtheria, listeria causes a food poisoning, and actinomyces. So those are the bacteria that causes lumpy jaw. So try to remember that category. Write those down in different columns and try to remember what’s in the members of those particular groups. There are no gram positive spirochetes. There are some gram negative spirochetes. We’ll talk about a couple of them later. Dr. Courier will give the next conference in our course on spirochetes. She’ll give some case histories of spirochetal infections. Syphilis and lyme disease, primarily. So today we are going to get into the gram negatives.

[1] Introduction to the gram negative bacteria: Enterics, coliforms and E. Coli[Boylan] Mainly we’re going to spend some time in the GI tract. Not just today, but in some lectures coming up by some guest lecturers. I hope to be here next week to introduce one of our new guest lecturers – I mean new for you – Dr. Bruce Hannah. He is a compatriot really of Dr. Tierno’s, and Dr. Hannah has two sons who are recent graduates of our dental school, so maybe he’ll tell you a little about them and what they’re doing today. Within the last ten years I think they have both graduated. He also was in charge of the clinical microbiology lab at Belleview for many years, so he really knows his stuff too. So he’ll be coming up next week. I think I should be here, but I’ll be away the following week in Chicago myself. But don’t worry, we’ll take care of things before that. So, the gram negative bacteria. An introduction to them. Looking first at the bacteria called the enterics and the coliforms, and e. coli. We’ll spend a lot of time on e. coli. The enterics, first of all. As their name indicates, those are bacteria that grow in our enteric tract – in our gut. Enterobacteriaceae, enteracium, enteric, enterotoxin, something to do with our gut. The coliforms… Within that group of enterics, as we’ll see it’s a major, big group, within the enterics, there are coliforms, and those are the enterics that are normally residents of our gut. They are ordinarily found there, even in a healthy person. E. coli is one of the coliforms. The other enterics that are not coliforms are still gram negative enteric bacteria, but they are considered to be pathogenic all the time. If they are found in our gut, these other ones I’ll mention later, they must be undergoing some infection

at that time or are harboring a dangerous bacteria, like salmonella, at that time. We’ll break them down.

[2] Enterics Introduction to Gram-negative bacteria[Boylan] Just a little bit about the gram negative bacteria that are of course ordinarily found in our gut. Some are found in our mouth in dental plaque in our gingival sulcus, and in a couple weeks I’ll tell you about the periodontal bacteria, which are also gram negative bacteria primarily, the ones that cause periodontal disease, gram negative anaerobic rods. But lets look first at those found in the intestine. So we have a microbial flora of the intestinal tract. The small intestine – not too many there, actually. That area in the small intestine is still influenced by the acidity of the stomach. So of the ones that are there – the bacteria that are there – the genera would be the enterococcus and often the lactobacilli are there, near where the stomach leads into the small intestine. So those that are there can withstand the low pH. The large intestine – that’s where we have the most complex microbiota of our body. The predominant bacteria in the large intestine are anaerobes – strict anaerobes. And the two major genera are shown there. Each makes up maybe 30, at least 30, percent – those two: bacteroides fragilis and fusobacterium fusiformis. Strict anaerobic bacteria make up the bulk of the microbiota of the gut. Ordinarily not pathogenic at all, not even enterics really, but they’re there and members of the gut. Each of those two bacteria, when they’re in the gut – no problem. Once they get, if they get out of the gut, so some perforation in our intestines, if they get into other sites in the body, they can cause serious infections too. Gun shot wounds, stabbing. Remember the pope years ago had a terrible infection – previous pope – after he was shot because some of those gut microbiota got into his pleural cavity. So some of those bacteria got out of the site where they are ordinarily found. Then we have in the large intestine also, facultative bacteria that can grow with or without oxygen. Most of these are called facultative anaerobics. There are facultative anaerobes and facultative aerobes. Facultative anaerobes. This name indicates that they prefer lack of oxygen to grow. Facultative anaerobes will grow very well with a little bit of oxygen. So they do grow well with oxygen, essentially. Facultative anaerobes. These facultative bacteria like e. coli. And they are called the enterics, the ones we are going to talk about, and members of the family enterobacteriaceae. So the family genus species. Family enterobacteriaceae, the enterics.

[3] Enterics[Boylan] The properties of the enterics. They are gram negative, short rods, facultative anaerobes. Are they spore formers? No. No gram negatives are spore formers at all – just clostridium and bacillus as you know by now. We’ll talk about how you differentiate them on the next slide. But let’s break down the enterics. Members of this large family enterobacteriaceae are the coliforms and the pathogens, as shown here. Coliforms, like e. coli, klebsiella pneumoniae, proteus vulgaris, and I should say, there are many other coliforms as well. We are going to restrict ourselves to these because these are some of the ones that do cause sometimes serious infections in humans, especially e. coli. But the other two we’ll

see can cause some unusual and very often life threatening infections as well. Klebsiella pneumoniae and proteus vulgaris. Many different serotypes of e. coli, and we’ll talk about why that is the case. Serotypes – how we can identify them using serological tests – that is tests based upon their antigenic makeup. The enterics that are pathogens, not coliforms, when they’re in the body they are in the gut, that’s where they prefer to grow, but they are not ordinarily there. Salmonella typhi causes typhoid fever, salmonella enteritidis, food poisoning, shigella dysenteriae, which causes dysentery, a very serious type of gastroenteritis, or diarrhea. And yersinia pestis, that causes bubonic plague, a pneumonic plague. Of these listed here, you think, which does not seem to belong at all, is yersinia pestis. The others cause – most of them cause gastroenteritis in one form or another, and then when they leave that area of the enteric gut can form other types of infections. But yersinia pestis – why is it included among the enterics? Well because when you analyze it and look at its biochemical properties and its makeup and the various biochemical tests you run, it seems to fit in with the others listed here, in that family enterobacteriaceae. Even though it causes plague. So a little bit about that at the end of the lecture today as well.

[4] Enterics – Differentiation[Boylan] How do you differentiate the gram negative short rods in the lab? Well you can’t do it by gram stain alone. They’re all gram negative. They all look pretty much alike. Not that many are longer than others or more curved than others. So you have to find other ways to identify them in the lab, to differentiate among them. One of the couple of ways you do it are listed here. First of all, lactose fermentation. Among the enterics, one of the big tests you do originally, right off the bat when you get them in culture in a lab is to see whether or not they ferment lactose. Some of them ferment lactose, others do not. So that’s the first sort of big biochemical test to put them in different groups. Lactose fermenters and non-lactose fermenters. And then you grow them on selective or differential medium. Once again, somebody has a GI infection, a gastroenteritis, you want to identify the potential pathogen. This bacteria is not going to be the only one present in fecal matter, so you want to try to separate it from all the others – the other coliform, the other bacteria that are there that you know are not causing the infection. So try to find a medium to grow these bacteria in the lab that will allow you to grow them and not the other bacteria, or reduce the growth of the other bacteria. So once again, that’s a selective medium. Select for the growth of some bacteria – the ones you’re interested in, and suppress the growth of others that probably are not causing the infection. So, selective medium to separate what you what from all the other hundreds of genera that may be present in the gut. Then you use a differential medium, which means of course that when these bacteria do grow on the selective medium, they do form colonies, often you can differentiate among them by the appearance of the different colonies that they form. Are they pink, are they red, colorless, have a metallic sheen, all different properties. So often you can tell by the type of media you use to grow the bacteria on, the type of colony they form, you can differentiate – that’s an e. coli, that’s probably shigella, or maybe it’s a salmonella, but you can begin to process right away by the types of colonies they form on selective and differential media.

One of the ones we use a lot for the enterics is called MacConkey’s medium, and it has in it some various dyes, some various bio salts, in other words it has selective agents in it that will allow for the growth of most of the enterics so that you can more easily pick them out. Because an e. coli infection, uh, the enterics are really only present at much less than 1%. Even though we hear about e. coli all the time, it’s present at a very low percentage of the total population of the enteric bacteria – well less than 1%. So you want to kill the other 98 point whatever percent and hope you increase the growth of e. coli if it’s causing infection on selective differential media. So MacConkey’s has some dyes, salts, that allow for the growth of these enterics and not other bacteria that could be present in the gut. And then you have a biochemical screen, that is you test these different bacteria in various media to see whether or not they have a positive or negative response to the test, and we’ll show you on the next slide – uh that will come up a couple slides from now. But biochemical screen, biochemical testing can be run. A lot of testing can be run to identify enterics, but I just want to mention four more. They’ll come up in a couple slides.

[5] Lactose fermenters and non-fermenters: MacConkey’s medium[Boylan] So MacConkey’s is a selective and differential medium, as you can see here on the left side we have one of them on the left side in this media that has a lot of lactose in it, and so whatever bacteria is on the left side of the plate broke down, were lactose fermenters, and they formed colonies or showed growth – it is sort of a pinkish red. So lactose fermenters, because of the dyes in the media, when they grow they incorporate the dye and they have this pink red appearance to them. On the right there, these bacteria were grown on MacConkey’s but they didn’t ferment lactose – so yeah they still grew, but they didn’t ferment lactose, so they remain colorless or often just transparent, almost like you can see right through them, but they’re not pink or red if they’re not lactose fermenters.

[6] Biochemical Screen[Boylan] Here’s an example of a biochemical screen. Once again, you have these tables of about thirty different tests, and all the enterics across the top, and you match them up. Basically you’re asking the question – when you grow these bacteria in these media, is it a positive or a negative? It is a yes or no response? Yes, no, yes no. And you get a whole number of different yes no results from the tests that you run, and that helps you identify the genus and species often of the bacteria. These are all for yes no tests. They help identify the bacteria very simply by color change –a color change. These are called – these four here – the IMViC tests – shown across the top there. Four commonly used tests to identify the members of the enteric group of bacteria. The first one here is called… So IMViC standing for Indole, M for methyl red, Vi for Voges-Proskauer (threw the “i” in there just to make this pronounceable), and the c stands for Citrate. In the first test here on the top left, you have two tubes, and initially they were exactly the same sterile some kind of broth, and on the left you inject, inoculate one type of bacteria, and on the right another. And if you see a difference, then you incubate these tubes overnight as you do in all four cases here, come back the next day and look for a color change, or some color

change that will occur when you add something to the media. So as we did here, on the left there –both of them actually had a little bit of solution, you can see kind of on the top, that ring, it’s a solution that has some alcohol in it and some other things, but the thing is, you can see on one it’s still yellowish on the left and it’s red on the right. The red on the right means it’s a positive Indole test. So right away you know – indole test positive. What does indole positive test mean? It means that those bacteria on the right there grew in a medium and they were able to break down the amino acid tryptophan. Remember tryptophan? They produce this enzyme called tryptophamase. They break down tryptophan producing indole. When indole is formed, the reaction in the test tube will be red. If they don’t form indole, that means that they don’t break tryptophan, they don’t have trypthophamase, and there is no change to the color of the solution put on top there. So yes, no, the one on the left is no, the one on the right is yes for positive indole test, they produced indole. Another one is the M – methyl red. Once again, some of these enterics will grow in a medium rich in carbohydrates, and when they do that they form a lot of acids, like pyruvic acid, lactic acid. So those with a positive methyl red test are those that grew well and produced a lot of acids. They fermented the sugars that were in the medium, creating a low pH, and you add methyl red to them the next day and if it goes red, if you add a couple drop of methyl red dye and the tube stays red, it is a positive methyl red test. Those bacteria form a lot of acid on the left methyl red negative test – they did not form a lot of acid. They grew, maybe, but they didn’t form enough acids to change the color of that indicator to form a red color. Ok, so that’s the methyl red test. A third test is the Voges-Proskauer test shown here, and once again it’s a simple color test, color change. You inoculate the broth with the bacteria, incubate them overnight, come back the next morning, take them out of the incubator, add a reagent, as you do with the indole test, and look for a color change. And it doesn’t show up as well on the slide here, but if it’s a positive test, once again you see a red ring around the top. That’s a positive Voges-Proskauer test. The bacteria in the tube on the left – negative voges-proskauer. So, anybody remember what the component responsible for the positive test is here? We briefly mentioned it when we, way back in the microbiology course, talked about bacteria that have mixed fermentations, and during one pathway fermentation reacts pathway, one of the intermediates in the pathway was something called acetoin. So- the point being here, some enterics will carry out this metabolic pathway and acetoin will be formed during that pathway. Other enterics will not carry out that pathway, they’ll never produce acetoin, which is the key component of this test, they would be negative. And the fourth IMViC test, shown here on the right, is citrate. Citrate utilization. This is kind of an interesting test, it’s a little bit different from the others we talked about where often the other tests are indicated by, show a color because of a lower pH or a pH in the acidic range. But the citrate test is shown here, you have two tubes, and these tubes, I think Dr. Succine might have mentioned it one time – it’s a media you make in the lab, agar medium, you know an agar medium usually used on petri plates, and that solidifies a nice flat plate. Well these tubes here, before we talk about the test, is they made the media up, it had agar in it, they sterilized it, while it was still hot they put the media in a test tube, and then as it cools you put the tube at a 45 degree angle. And then when it solidifies it’s a nice long slant of solid medium,

and then you can streak your bacteria on this slant, and that’s what happened here. If you look at the top of this tube here, you can see it’s kind of clear, but maybe about halfway up the tube it’s a slant. Very thick but at the bottom, and then you get thinner as you go up. But anyhow, that’s a slant, solid medium. In this medium, there is a bacteria. The only source of carbon for the bacteria to grow on is citrate, so in other words they don’t use any amino acids in this medium, they don’t get any other sugars like glucose, sucrose, any sugar at all. Only citrate, or only citric acid. Some of the enterics will grow with citrate, others will not. That’s they key once again, yes or no. And as it turns out that the actual color of the medium when you make it up, the citrate medium, is green. I’ll tell you, the dye in the medium is called Brom thymol blue. That color indicator is green at neutral pH. However, if bacteria will grow on the citrate in the medium in the tube, they will actually raise the pH a little bit- become slightly alkaline when citrate is broken down. When that happens, that green color, brom thymol blue at neutral pH, becomes blue. So a positive test means citrate was used, pH increased slightly to alkaline pH, changing the color of the dye from green to blue, and the blue is a positive test. So those are just examples of four fairly simple tests to run. You just have to inoculate the tubes, stick them in the incubator, come back the next day, sometimes you can just look at them like the two on the right. The first and the third you have to add some reagent to them, but within seconds you know. So the IMViC tests. For e. coli, we know the IMViC tests would be listed as positive, positive, negative, negative. E. coli would produce indole and it would create a very acidic pH in the medium, but it would not be positive form the voges-proskauer test, meaning it wouldn’t produce acetoin, and it is unable to grow with citrate as the sole source of carbon. Other enterics that look just like e. coli, you run these tests you get different patterns. There’s another one we often confused with e. coli that is found a lot in nature, but we run these IMViC tests we can easily distinguish it from e. coli because this other one is negative, negative, positive, positive. Just the opposite four results from e. coli. Some are plus, minus, plus minus. You get the idea. But this is one of the ways we help identify these enterics. Not by the gram stain alone – impossible. But we run some biochemical tests, these are four we can use.

[7] Serological tests[Boylan] Ok, how about another way to differentiate among the enterics - E. coli and others. We can run various serological tests based upon the antigens present on the surface of the bacteria. The antigens present. We are going to mention just three types of antigens that we use in the lab to identify enterics. First, these tests we are going to use to identify these enterics serologically are all called agglutination tests. Meaning that if these bacteria we are going to study are positive for these tests, they will agglutinate – they will form big globs of each other and then settle to the bottom of the test tube. So agglutination if they are positive. Why would they be positive? Well let’s look first at the top one of the antigens – the “o” antigen. So these agglutionation, these serological tests, are based upon either the o antigens, the k antigens, or the h antigens. And with just looking at these three types of antigens, these enterics can have, there are probably thousands of different serotypes. But they are very very useful in identifying the bacteria, and it’s very important to that

that often during some epidemics or outbreaks of these. What enteric is it? Oh it’s an e. coli. But which e. coli is it? Well let’s not just identify it biochemically, but serologically as well. Maybe we can track down the source of the infection. So serological identification tests are very very important often, especially when there are outbreaks of these e. coli diseases we’ll talk about. So the o antigen is found in the LPS, or endotoxin, same thing. What’s the real name of this particular component? LPS – what does LPS stand for? Lipopolysaccharide. Here it is on the upper right here, the gram negative cell wall, and you can see on top the o antigens there. Remember the LPS has the lipid part, the toxi (?) part, and it has two different polysaccharide parts: the core, but then the o antigens that stick up like whiskers on the surface of the bacterial cells. And that’s what our immune systems see when they come in contact with these bacteria. They see the carbohydrates and the o antigen. And the o antigens vary, there can be variations dependent upon the sugars in them, and how they link to each other, and the side groups on these sugars. So o antigens are the polysaccharide antigens found on the outer surface of the gram negative cell wall, in the outer membrane up on the top there. They are repeats, 3 or 4 sugars, those same 3 or 4 sugars again, and again and again kind of like if you had 4 different types of ice cream on your cone, chocolate, vanilla, strawberry, pistachio, chocolate, they repeat over and over again. Those are the o antigens. Every bacteria have different types of ice creams, different types of sugars in their o antigen. Oligosaccharide units. K antigens are found in the capsule. Polysaccharides of the capsule. Remember we know about the pneumococcus can have over 80 different antigenic types. It’s not an enteric of course, but based on the capsular composition, different sugars and combinations of them. And finally the third type of antigen we can look at to identify these bacteria serologically, is the h antigen. The h antigen. And those are found in the flagella of the bacteria. So the point I want to make is only those bacteria that are motile, only those enterics that are motile, that are flagellated, will have an h antigen. An example I want to show is this e. coli O157:H7. That’s the one you read about a lot in the news lately. That’s the one that can cause so many cases of gastroenteritis, and we’ll talk about it more later. In the news lately, outbreaks of severe bloody diarrhea. But why is it called O157:H7? Because of the antigens. The O antigen it has is 157. I’m not sure what those sugars are, but it means it’s O antigen it has a combination of sugars, 3 or 4 repeated over and over again, that are identified as 157. Another e. coli might be O122 or 65 or whatever. But once again, it refers to those O antigens. And the H7 means that the flagella antigen it has, so it must be a mobile bacteria, the flagellar antigen, is called H7. So once again, the first two, the O antigen and K antigen are polysaccharides, or sugars, whereas the H antigen is a protein, and it’s found in the flagella. We know the flagella are made up of protein. And even within the flagella, there can be different types of amino acids, different types of flagellar proteins. At least 7. So that’s how it gets a specific fingerprint classification. O157:H7. One other one I just, when we got this out, I did eliminate from that discussion, so you can forget about the Vi antigen. I didn’t follow up, I cut that out on my slides on salmonella. But there are other antigens as well. So serological tests. We have all these different antigens- hundreds, if not thousands. We have the antibodies stockpiled in our refrigerators. We have antibodies against O157:H7. So you add the bacteria, add the antiserum,

and see if it agglutinates. If the antibody against O157:H7 agglutinates the bacteria. If it does, that identifies it as such, that particular strain. We have antibodies against O and H and K serotypes of all these different bacteria. We can identify them very specifically, much more specifically the strains of these bacteria, the strains or serotypes, than simple biochemical tests. Very powerful types of identification tools, these tests.

[8] LPS[Boylan] In the bacteria we find the LPS, one of the most interesting biological molecules that we know about, lipopolysaccharide. I found something in a book recently, you can read this over later, but look at all the effects of having LPS produced by these gram negative bacteria during the course of a gram negative infection. LPS, that outer membrane component of the gram negatives, can induce macrophages to do a number of things. So macrophages ingest these gram negative bacteria causing infection, and the LPS in the bacteria or even by itself, can induce macrophages to produce inflammatory cytokines, like interleukin 1, tissue necrosis factor alpha, and some others as well. So cytokines help stimulate our acquired immune system to come into play and help fight off the infection. But also, the fever. We’ve mentioned a few times that the fever induced by LPS is caused by the lipid portion, lipopolysaccharide, remember lipid A. Lipid A causes a fever. What happens is that our macrophages ingest the gram negative bacteria and the LPS inside the macrophages induce the macrophages to produce the cytokines, especially interleukin 1. I have two others listed here, there are others, but interleukin 1 I want to focus on that. Cause that’s the one that these activated macrophages ingesting the bacteria, LPS induces interleukin 1, that gets out into the blood stream, and it goes to our hypothalamus. And they hypothalamus…how do we get fever? The hypothalamus is kind of the thermostat of the brain. It regulates our body temperature, and Interleukin 1 is that one that convinces the hypothalamus that it should elevate the body temperature up to where we have a fever. A fever, as you know, helps us fight off the infection. We don’t want to have a big increase, but even a couple degrees, 3 degrees, we are responding to the infection. The infectious agent. The fever is helping us fight off the infection. It slows down the growth of the bacteria at elevated temperature. At the same time, the fever stimulates our immune system, our cellular immunity especially comes into play and even our acquired immunity, humoral immunity, to produce antibodies. So it stimulates our immune system to help fight off the infection. But fever can also cause hypotension, a low blood pressure, vasodilation, it increases the permeability of the vessels, that’s not good. It can cause coagulation, DIC – dissemination intravascular coagulation, which clots form in the blood, especially peripheral circulation, fingers, toes. That’s going to interrupt the flow of blood in those sites, so the sites become necrotic. Coagulation of the blood, fibrin clots. And even endotoxic shock. If we have an overwhelming gram negative infection especially, shock can lead to shut down of our essential organs – the liver, the pancreas, the lung, everything shuts down when there’s a lot of LPS. So these are very important molecules that have a lot of different effects. Most of them are not good for us, of course, as you see here. Well, fever would be, fever is good, helps us, unless it’s an excessive fever, but if it’s a prolonged

gram negative infection, lower blood pressured, hypotension, coagulation, shock, can be the agent responsible for the death of the people who have these gram negative infections we’ll talk about.

[9] E. Coli – the colon bacillus[Boylan] Let’s look at e. coli. It’s called the colon bacillus. It’s found in the colon. I guess that’s the name it deserves. Maybe it doesn’t appreciate it, but that’s how it got its name. Coli – it’s found in the colon, the large intestine. The general properties, well we’ve already gone through those. The things that distinguish it – we know now that it’s a small gram negative rod, IMViC tests, we know the results of those tests on e. coli. It’s called the colon bacillus because of all these bacteria we are talking about that are in the gut, the coliforms and others, e. coli should only be found in the gut. It should not be found what we call outside the gut, or free living, out there in nature. It shouldn’t be found in streams or vegetation or soil. It really shouldn’t be, and if it is found there, that means one thing – that material has been fecally contaminated. Many other coliforms that look very much like e. coli we find them free living. They can survive outside the gut for a long time in soil, streams, etc. But not e. coli. So if e. coli is present in your drinking water or your pools or oceans after a storm, that means that water has somehow been fecally contaminated. So you look for e. coli when you tests these waters – oceans, streams, lakes, whatever, if they may be fecally contaminated, you look for e. coli. The colon bacillus, because that should only be found, whether it’s human waste or animal waste, it’s from some animals of waste. It’s the one that’s used for water tests. There are over 700 different serotypes based upon primarily it’s O antigens, but also it’s K I think. There are 4 principle types of infections caused by e. coli: urinary tract infections, bacteremia leading to sepsis, neonatal meningitis, and intestinal (diarrheal) diseases, also known as gastroenteritis. So here we see the gram negative rods, short rods there, red color. And we see that most of these e. coli have a lot of pili all over the surface often, the pili, that’s what helps them adhere to our tissues. Sometimes tenaciously to our tissues. The pili make them look ugly, but they are able to adhere and not be voided from our system they stick so well.

[10] E. coli- virulence factors[Boylan] So this is another key, very important point right here I think. We talk about e. coli and it is a member of our gut. Microbiota, all of us. Why is it that it can cause so many different types of infection, as was on the previous slide? A harmless bacteria, what gives it the ability to do so many terrible things if we come in contact with them? Well, it comes down to what we see here. We have many different serotypes of e. coli. The ones we have in our gut are harmless to us – our body is used to them, but these other serotypes of e. coli are a result of the particular plasmids they have or the particular bacteriophage genes they harbor. In contrast to the chromosomal genes, pretty much the same in all e. coli. What makes them non pathogenic, causing gastroenteritis, or sepsis, or urinary tract infections, depends upon the serotype of e. coli you’re looking at. And these different serotypes, once again, are based upon the genes in their plasmids or bacteriophage genes they harbor. Let’s go back to bacteriophage genes for a second. Remember what type of

bacteriophages survive in as part of the bacterial chromosome, whether they’re lytic or temperate? We talked about the two types way back then. Temperate, right? So some bacteriophages, these that are DNA-phages, they’re viruses that infect bacteria, and often when they infect them, they become part of the bacterial chromosome. They inject their DNA into the chromosome of the bacteria. So when they are like that, well here’s a bacteria chromosome that has a relatively small piece of bacteriophage genome it’s carrying, that particular piece of DNA from the phage is called the prophage. The prophage is the DNA of the phage when it’s integrated into the bacterial chromosome. (?) So okay here’s this prophage in the chromosome, and most of it’s genes are involved with prophage replication later on, it’s living there and part of the chromosome for a long time perhaps, maybe generation after generation, but sooner or later, these prophage genes say I’ve had enough of this, I want to break free and I want to produce more and more bacteriophage progeny, right? So the thing is, most of the genes in the prophage are involved with replication of the phage. But there are also a few cases where these prophage have some extra genes they carry around with them, and the genes code for toxins. They code for toxins, they code for invasins. They code for other things that make the bacteria pathogenic. So these e. coli, if they didn’t have the prophage, they would be non-pathogenic if it didn’t have plasmids either. But if they have certain prophage, some of them carry genes, and when the prophage in the chromosome are activately encoding toxins, enzymes that make the bacteria, e. coli, pathogenic. Same thing with plasmids. If the e. coli harbor certain plasmids, as we’ll see, those plasmids have the genes to make them stick better, to make them more toxic, etc., invade better, that’s where they’re found. So that’s why we have so many e. coli, a commensurate harmless member of our gut bacteria, can turn out to be pathogenic. It has genes from these two sources. What are the virulence factors of e. coli? Of course most of the enterics have some or all of them. The pili, also known as the fimbriae, enterotoxins, the toxins they produce while they are living in the gut that act in our gut tissue, invasive factors help them spread, endotoxins, we saw how that can contribute to infections in the previous slide, a capsule, anti-phagocytic, and one other component many of these enterics have is called the siderophore, or siderophores. These are proteins in their cell membrane that are called iron chelating compounds. What these do, the siderophores, is any available iron that may be available in their environment – they want to grasp it, they want to use it. There are very very low concentrations of iron in our tissue where the e. coli and other bacteria replicate. But whatever they have access to, they want it. They need iron to grow. These e. coli and other enterics – they need iron to grow. And so the advantage would be to have these siderophores to latch on to or bind to iron and then use it for their own purposes. So it is a very good virulence factor as well

[Student] Can it cause iron deficiency? [Boylan] It’s not a major cause of that I don’t think. Maybe over the course of an overwhelming infection, but we don’t need that much either, but for bacteria it is essential. So I don’t think it causes, I’m not sure if it’s involved with iron deficiency anemia or anything like that, but theoretically if it’s a massive infection it could. But that’s not usually the case. We’ll talk about the infections it does cause.

[11] E. coli and UTI’s[Boylan] Ok, lets talk about e. coli and urinary tract infections. E. coli is the most common cause of UTI’s, right off the bat. And it causes UTI’s – that is, infections of the urethra, the bladder, the ureters, and even the kidney, by the ascending route. The ascending route – in other words, it enters through the urethral opening and makes its way up to the bladder and further up possibly to the kidney. What do we mean by ascending – that’s the way we get these infections, as opposed to say the blood bourne route. So these UTI’s are not usually a result of bacteria being in the blood getting to these sites, but the ascending route, going up through the urethra and progressing to it. So the source – what do you think the source of these e. coli that cause urinary tract infections is? Some outside exogenous source, or our own endogenous bacteria? It’s our own bacteria. So with urinary tract infections, the principle is it’s almost always caused by our own e. coli. What does that mean? That means that urethral openings become contaminated with e. coli from our fecal, from our anal opening, shall we say. And also, this is why women have a much higher percentage of UTI’s than males. The bacteria don’t have that far to go, maybe an inch, inch and a half. Whereas in men, it’s 8 inches or something like that. So women have more urinary tract infections. And it is a result, most of the time, of infection by their own e. coli that contaminates the urethral opening. So serotypes causing them are their own endogenous serotypes.

[Student] They’re not always endogenous, though, right? Because sometimes you can be in a lake and there can be e. coli there…[Boylan] Yeah, I mean, you might be swimming in fecally contaminated water. [Student] So not always[Boylan] No I wouldn’t say always, but the odds of a much more generous dose of e. coli come from the gut, I think, you know, a larger dose than if you’re swimming. But it could happen, I don’t want to say it always happens that way. It could happen. That’s often how males get it too, from fecally contaminated water, but ordinarily, it is more common in females. It’s going to cause an infection, entering through the urethra, getting to the bladder. And a bladder infection is called cystitis, and that means e. coli can crawl up the ureters to get to the kidney to cause pyelonephritis, which is a very serious infection. Hopefully it never gets that far. How do they do it? You have urine being released from the bladder, you’re voiding, and you’d think you’d be able to rinse out these e. coli before they get a chance to go up that far. But these e. coli often have what are called P fimbriae, those fimbriae we saw on the previous slide, the e. coli are coated with pili, or fimbriae, and somehow it helps them stick to the epithelial cells in the genital tract, urinary tract, and climb up, or hold on. And eventually over a period of time it could go up to the kidney and cause pyelonephritis, and that can lead to obstruction of the kidney, and that’s when it’s very serious. So urinary tract infections, you have burning sensation upon urination, you have to go more frequently, and you feel a bit feverish sometimes as well, so you know when they’re coming on ordinarily, these infections.

[12] E. coli – bacteremia and sepsis

[Boylan] E. coli bacteremia and sepsis. The most common cause also of bacteremia. So we spent a lot of time previously talking about staph aureus and all that it can do. E. coli is another one. It’s a part of our endogenous bacteria. It’s there. It gets out of the site where it’s ordinarily found to cause a lot of these infections. Septicemia, septic shock, can be a rapidly overwhelming infection if they get inside the blood stream and multiply. Endogenous infection, once again, caused by our own strains. UTI’s cause pyelonephritis. Iatrogenic many times – IV lines and catheters as shown there in the picture on the right can be introduced if they are contaminated and they get into the blood stream, they can cause these infections. At risk, often these types of infections occur in people who are hospitalized. I mean, anybody can get it. But who’s at risk? It’s a nosocomial infection, usually, because of catheters, endoscopy that are contaminated with e. coli bacteria from a patient’s own body, they can cause bacteremia, sepsis, shock, and death. So bacteremia, as you know, bacteria in the blood stream. Septicemia, an overwhelming blood infection, also known as sepsis if it really gets out of hand.

[13] E. coli – Neonatal meningitis[Boylan] Here’s an interesting one. We’ll take a break after this one. E. coli meningitis in a newborn. Why would a strain of e. coli from a mother cause a serious life threatening infection like meningitis in a newborn? This is e. coli. A newborn the first three months of life is most susceptible to this type of meningitis. You see the brain, spinal cord meninges around these organs, infected. The main virulence factor of the e. coli causing this is a capsule. Anti-phagocytic capsule. It is an unusual composition of this capsule. It’s called a K1 capsule. Remember that. K1 capsule. And it is composed of something that is not foreign to our body. In other words, our immune system, we saw this once before, our immune system, it responds to foreign invaders. It recognizes something in our body that shouldn’t be there. It does not recognize the material, in this particular in this capsule, of these e. coli, as foreign, so it doesn’t try to destroy the bacteria. It lets them go. It tastes them, but it doesn’t gobble them up. What is this component? It is sialic acid. And the capsule is made up of pure sialic acid monomers that form a large polymer of capsule. So even as a fetus, as the child is developing, sialic acid is a part of our normal tissues. It is especially found in our nerve tissue as we’re developing in utero. So in other words our immune system is recognizing this material as self, so it doesn’t respond to it or try to knock it out, or neutralize it, destroy it. So when the child is born, it’s infected with e. coli maybe from it’s mother, and this e. coli strain has this particular capsule, and it doesn’t try to destroy it. It says ok, this is normal human material. And so that’s why it’s not destroyed and can go on to cause meningitis. That’s why we have the infection. Alright let’s take a break and then we’ll have some more to say about e. coli and a couple other coliforms and enterics.

[14] E. Coli – gastroenteritis[Boylan] And if you see some other students later, and they ask you – what was that lecture on this afternoon, you say – that lecture was full of feces, full of feces. I will not take it personally, ok? Put it that way. Ok let’s get to the other main infection caused by e. coli – diarrhea. Gastroenteritis. Transmission: fecal-oral route. E. coli

lives in our gut, contaminates our hand, after going to the bathroom, we contaminate food or water we touch. Food handlers have to be very careful especially not to transmit it. And a big difference between this and the others I just talked about is this comes from an exogenous source. Generally gastroenteritis is not caused by our own e. coli in our gut. We pick it up from some outside source – contaminated water, vegetables, water, meats, almost anything, a lot of plants sometimes. Spinach, we’ll see, crops grown in large fields, irrigation water irrigating them – and if that water is contaminated with feces from somebody else, often from some farm animal, we then if we then eat that spinach without washing it thoroughly, that can also lead to these types of gastroenteritis. So we’ve seen a lot of that lately. The contaminated water used for irrigation of many types of plants we eat. We are going to talk about two types of e. coli that cause gastroenteritis. You’re in luck, because I used to talk about four. But there are so many of these bacteria coming up that cause gastroenteritis of one form or another that I think just two e. coli is enough. The first one is called ETEC. These are the enterotoxigenic e. coli. So EC always means e. coli, the ET stands for enterotoxigenic. These are the e. coli that elaborate a pretty potent toxin that results in the infection. These e. coli ordinarily cause their damage in the small intestine – the small colon, rather than the large. And the factors that play a role in the infection cause by ETEC are the colonization factors, called CFAI and CFAII, colonization factor AI and colonization factor AII. These are both pili, so that’s what helps these bacteria adhere, and both of the genes for those two types of pili are found on plasmids. And there are two enterotoxins they may produce as well, usually it’s one or the other, but these enterotoxins, the genes for them are also found on plasmids. So these e. coli, the plasmids they harbor carry the genes for either adherence or toxigenicity. Once again, not chromosomal, not on the prophage.

[15] untitled slide[Boylan] Here’s the way we’re probably most familiar. We go away on vacation, and we go away to certain countries, most any country. We’re exposed to some contaminated food or water with e. coli that comes from somebody else – humans, cattle, other animals. How do the enterotoxic e. coli make us sick? Is basically what I’m trying to show on this slide. Up on the upper left there, you’ll see an e. coli with it’s pili, and it is in our gut, and it’s going to stick to our epithelial cells, our brush border, the microvilli of our epithelial cells in our gut, and then it’s going to elaborate the enterotoxin. The enterotoxin gets into the cell, gets to the cell membrane of these cells. And remember, this is happening with probably millions of our gut cells that are infected. And the enterotoxin, what it does, is stimulate the enzyme adenyl cyclase, which is shown down below there. Adenyl cyclase. So the two toxins are LT or ST. LT means that toxin is heat labile, the ST toxin means that that toxin is heat stable. So LT, the toxicity much more lost with just a little bit of heat. Whereas even at high temperatures, ST can still be effective. They both act the same…well, they act the same way, but it’s the LT, the heat labile toxin, that when these e. coli elaborate, this toxin, this enterotoxin, it stimulates adenyl cyclase. As a result of that adenyl cyclase, that enzyme in our membranes, sometimes in our cytoplasm, when it is activated, it builds up the levels of cyclic AMP. That’s the key.

Cylic AMP. We have very small quantities of cyclic AMP floating around in our cells. It’s essential for our growth of our cells and should be maintained at a certain low level. But when the level of cyclic AMP is increased, it can lead to dire consequences of these cells, as we’ll see. So it’s stimulation of the enzyme adenyl cyclase, leading to the production of more cyclic AMP in our cells, what happens next? The enzyme cyclic AMP, you can see what happens here, on the top right of the slide. (?) You can see, the overall result is- look what’s being lost from these cells as a result of cyclic AMP being built up. You lose water, not just water, but chloride ions, sodium, potassium, and bicarbonate ions are lost from these cells as a result of stimulation by the cells of enterotoxin, cyclic AMP, increases cyclic AMP levels. So water is lost, all these electrolytes are lost, the gut fills up with fluids, we have diarrhea. We have gastroenteritis. And that’s what flows from the body of this guy here for example. And also, he’s vomiting to try to get rid of this – your body is trying to get rid of the toxin from both sides, from your mouth and rear. So diarrhea as well as vomiting occur. It’s your body responding to this toxin and it’s consequences. The other one, the ST, does the same thing only it stimulates guanyl cyclase. So LT stimulates adenyl cyclase, ST stimulates guanyl cyclase, but they both lead to increase of cAMP or gAMP, and that leads to loss of electrolytes, loss of ions, loss of salts, and water. So it’s not just the water, it’s the loss of salts. They call it an electrolyte imbalance. The electrolytes our cells need to survive are lost as well. The incubation period is about three days or so. Two or three days after you eat the food or drink the water, you’re gonna get sick, and then you can be sick for three or four days, really. Just enough to ruin your vacation if you’re on a week vacation. So hypersecretion of water and electrolytes – chloride, sodium, potassium, bicarbonate. It’s called traveler’s diarrhea, because you get it often when you’re in other countries. Mild to explosive watery diarrhea, nausea, and vomiting. I want to point out here this diarrhea is watery, no blood. As a result of the ETEC strain we’re talking about here causing this. And depending upon what country you visit, it is also known as Montezuma’s revenge, you know, I think that’s mexico I guess. Dehli belly in New Dehli. Casablanca crud. There are so many beautiful names given to this disease. I think when Mexicans come here and go to San Francisco and get sick, they are exposed to these new e. coli from some people, they call it the San Francisco Fire. I used to say, when I didn’t have these other terms written out, I used to say Montezuma’s revenge and Dehli belly. I remember looking at a students notes one time, he had Dehli spelled “Deli”, so I thought, no, it doesn’t come from being at a delicatessen. Dehli belly means from New Dehli, India. Once again, it happens worldwide. Outside strains of e. coli.

[16] Enterohemorragic E. coli – EHEC (aka STEC)[Boylan] The other one of e. coli, EHEC, enterohemorrhagic e. coli. This can be much more dangerous in the long run than ETEC. It’s an infection not of the small intestine, but the large intestine. And here is our friend again the e. coli O157:H7, most frequently found to cause it. The toxins here destroy mucosal cells in the lumen of the large intestine. They destroy the cells. So here it’s not just loss of water, it’s destroying the cells. And there will be bleeding as well. So you’re going to have bloody diarrhea. That’s why I wanted to give these two examples of different types

of diarrhea – one watery the toxin, this one actually destroys the cells in the gut. Copious bloody diarrhea, abdominal cramps, usually no fever and no inflammation. So there are some unusual features of this one. This is the one you read about a couple of times every year, oftentimes because of contaminated water reaching plants and other things that we eat or fecally contaminated food from cattle or animals. At risk: children and the elderly. The first case of this was found maybe 15 years or so ago now in a fast food restaurant in Washington. A jack in the box, I think it was called, but many many people came down with this unusual bloody diarrhea after eating hamburgers there, but the hamburgers were undercooked. They came from cattle who themselves were carriers of this particular strain of e. coli, O157:H7. They got sick, and actually some elderly people died from it. So you can see, it can be quite serious infection.

[17] EHEC cont’d[Boylan] Primary reservoir is cattle. Transmission: fecal-oral route, as most of these gastroenteritis infections are. Undercooked hamburgers, unpasteurized milk from cattle, contaminated well water, and apple cider. So just think of cattle fecal matter getting to a well water, getting to… manure from cattle in these fields, it can get rained on, it can wash the manure all over the field and anyplace down the hill from there. And what happened a few years ago… but also apple cider. Why apple cider? Oftentimes they pasteurize it but they don’t really heat it well enough to kill the bacteria. Well, what happned was, there was a fair in upper New York state, and they served apple cider there. And a number of children especially for some reason, some adults too, got very sick and had EHEC infections. And they found out the apple cider had not been pasteurized, or not adequately pasteurized to kill the bacteria, but what happened, the apple cider. When you use apples for apple cider, you use the apples that fall to the ground from the tree. If you want to sell nice pristine apples, you want to pick them. I’ve done that one day in my life, that’s the hardest job I’ve ever had. To pick apples from a tree and try to make enough money to make it worthwhile. It didn’t work out. So I did it just once. But I know not to pick the apples that fall to the ground, because they’re bruised. So we’re gonna take those bruised apples and use those to make apple cider. But they also had cattle roaming there in the apple orchards as well, defecating, and so the apples that were lying on the ground were contaminated with feces. They used them for apple cider, they were not pasteurized. I don’t know if pasteurization would really kill this bacteria, but they weren’t adequately – the cider wasn’t heated enough to kill the bacteria, so another outbreak of gastroenteritis caused by this bacterium. The exotoxins they produce are shiga-like toxins. You don’t know what that means yet, but we will later. Shiga-like toxins mean the toxins these EHEC strains produce are those toxins that are like the ones found in the genus shigella, which is coming up after a few more slides. So these are very potent toxins. Much more so than the LT and ST we saw before. Shigella. These are toxins these e. coli derive from their gram negative enteric friends the enterics. Somehow or other these toxins from the shigella bacteria, which cause dysentery, those toxins in shigella were transferred to the e. coli. They picked them up, and now they produce these potent toxins just like the ones in the bacteria that cause dysentery. They are encoded by phage, not plasmids,

called Stx 1 and 2, the shigella-like toxins. Shigella toxins 1 and 2. Very dangerous, potent toxins. We’ll see what effect they have.

[18] Shiga-like toxins[Boylan] So these toxins, how do they work? They destroy the microvilli of the intestinal mucosa. Toxins enter the blood, damage the blood vessels, increase platelet aggregation leading to formation in the bloodstream of thrombi, and they damage the kidneys primarily due to blockage of blood flow in the kidneys. So overall, as a result, these toxins lead to often kidney failure.

[19] EHEC cont’d[Boylan] Also they can produce hemorrhagic colitis. Colitis – inflammation of the colon with hemorrhaging, bleeding. Abdominal cramps, copious bleeding, diarrhea, still no fever. So the cramping too. I mean, you have diarrhea, you have vomiting, but the cramping, the gas that’s produced, that’s what really really causes a lot of pain. That’s why people with staph aureus, food bourne infections, and these too, if there’s a lot of gas being formed, cramping can really get you, can lay you low for, in this case, many hours. In addition to that, it can also lead to hemorrhage uremic syndrome, the most serious manifestation of EHEC, mostly in children. Potentially life threatening. The red blood cells are destroyed, so hemolytic, and acute renal failure. So this is a very, very serious gram negative e. coli infection. Why? Because the toxins they produce. Where do they get the toxins from? Neighboring enteric shigella. And it can lead to all these dangerous situations and potentially lethal outcomes. Colitis of the colon, kidney failure, death. Prevention: thoroughly cooking ground beef, avoid drinking unpasteurized milk and unpasteurized cider as well I guess. So in general, once again, it’s found in cattle primarily, cattle feces that can contaminate a whole bunch of different things, and eventually get to you through the food you eat or the cider or water you drink.

[20] EHEC cont’d[Boylan] A couple other things. EHEC is now called STEC for shiga toxin producing e. coli. There is one very useful diagnostic test in the lab to identify EHEC or STEC. They do not ferment sorbitol. Somebody found that out just a few years ago. Other e. coli, other strains, can ferment sorbitol. It’s another sugar alcohol, you add that to media, have it be the only carbon source, the only source of energy. Most e. coli can break down sorbitol. This strain, the EHEC strain, O157:H7, cannot ferment sorbitol. So you see maybe it’ll grow on the plate, but it won’t change the color, it won’t break it down, change the pH. So that’s a key thing to remember about this strain. It really helps a lot in identifying it. Treatment: fluid therapy, intravenously if necessary. Try gatorage. Remember you’re losing water, losing electrolytes, and with HUS you might need blood transfusions, kidney dialysis might be necessary. That’s happened quite often, especially in the very young and very old. Also the immunocompromised of course. Very serious infections.

[21] Salmonella, Shigella and opportunistic coliforms

[Boylan] Ok let’s get out of the e. coli, away from e. coli for a while, and go on to others. Salmonella and shigealla are indeed enterics, but when they are found in our gut, it is indication that there is some infection going on, because they are not coliforms.

[22] Salmonella[Boylan] Infection caused by salmonella is called salmonellosis. Identification: well, it is motile. I want you to remember the difference between salmonella and shigella as far as motility is concerned. These two bacteria have often similar types of infection sometimes, and you have to run some tests in a lab that will distinguish between these two genera, salmonella and shigella. Well salmonella are motile, shigella are non-motile. Biochemical tests: probably the most useful one for salmonella identification, and helps distinguish it from shigella and other bacteria, is that these bacteria, the salmonella, produce hydrogen sulfide, H2S, when they grow. Hydrogen sulfide, when they grow, and they grown on media, you have some proteins in there, peptides, and as you know there are some amino acids in peptides that have sulfur in them, like cysteine, methionine, right? So these bacteria, the salmonella, are able to break down the amino acids that have sulfur in them and form H2S – hydrogen sulfide. Shigella will not do that. And what a salmonella does, the media they have devised to determine whether or not the bacteria produce H2S, when they do produce H2S, they form a little blackening – the media turns black. So H2S test, the bacteria grow on the media, if the bacteria produce H2S, it turns black. Salmonella does this, and it’s one of the few that do, so it’s a very important test to identify that particular gene of salmonella. That’s about all for the biochemical tests. Antigenically, Kauffmann-White scheme is used to identify salmonella. Over 2000 serotypes, and s. typhi, the one that causes typhoid fever, there’s only one serotype. The other 1999 are all variants of the serotype salmonella enteritidis, the salmonella that causes food poisoning. And if you read the literature you can be very confused about the naming of this particular species and this particular genus. Let’s stick with this: typhi, typhoid fever bacterium, one serotype. All the others, salmonella enteritidis, found in the enteric tract, food poisoning. Salmonella reservoir: (?), cattle. There’s something unusual to say about the reservoir I believe…. Ah, in addition, the salmonella and shigella are both lactose negative. They are non lactose fermenters. Salmonella, shigella, non lactose fermenters. Ok. Transmission: fecal oral route, because again, these grow in the gut. That’s the way they’re transmitted. Reservoirs: yeah, uh, cattle, sheep, but another unusual reservoir for these bacteria, the salmonella, are pet turtles. And I mention that for two reasons. We had to prohibit the… we no longer allow pet turtles from other countries to come to the United States. I don’t know what it is, but the kids used to have these pet turtles, and whether it was the feed they used to use for them, but something in the turtle food they used, had salmonella bacteria that caused food poisoning, and even typhoid fever. So pet turtles is one of the…and believe it or not, that was a question on jeopardy years ago. Not that many people got that right. But I guess those of you who had pets and turtles as a kid, you probably would have known that they are no longer admitted to this country. Fecal oral route.

[23] Typhoid fever[Boylan] Let’s talk about typhoid fever. We have that one species that causes typhoid fever and all the other serotypes of the other species cause food poisoning- gastroenteritis. Transmission by the five F’s. While most of these GI disturbances are indeed transmitted by bacteria by the five F’s. What are the five F’s? Well the big one is feces. I mean, fecal oral transmission. But feces leads to transmission by other sources. F’s I have for fingers, food, fluids, and flys as well, cause flys often land on contaminated food and then land on your food. So the five F’s, but all because whatever material it was, the water, the fluids, the food, was contaminated with the bacteria salmonella. Incubation period for typhoid fever: 5 to 21 days. So that’s quite a period. Once again depending upon the dose you’re exposed to. Symptoms: typhoid fever is also called enteric fever, meaning it’s a continuous fever. The fever stays high all during the course of the infection. Many other infections that have fevers associated with them, the fever ebbs and flows, there are peaks and valleys over a period of time. It might be high for a day, low for a couple. High, low. But that’s the thing about typhoid fever: the fever remains high for days or week, depending upon how long you have it. It invades and replicates in cells of the peyer’s patches, the M cells. Inflammation of the small intestine occurs, and you get a bacteremia. It spreads to many organs. Once again, in contrast to the next thing we’re going to discuss, shigella, that causes dysentery, shigella do not invade the blood stream. Shigella bacteria coming up next do not cause a bacteremia. Salmonella that cause typhoid fever cause a bacteremia. As a result of that, there are bacteria in the blood stream, and they are circulating in our blood stream, and they have a pre-deliction, or a affinity, or a tropism for the organs of our reticulo endothelial system – the RES. Liver, spleen, lungs, other organs as well. And they adhere to them. Then you have rose spots, hence the name typhoid. Rose spots all over the body have red, maybe flat red macules, or slightly raised pimple like papules, all over the body. And that’s how it got its name typhoid fever – typhoid means like typhus. We haven’t discussed typhus, but typhus is an infection where you have a rash all over your body as well. We knew about typhus since napoleon’s time or before, caused by a protozoan. So typhoid fever is a bacterial infection, has rose spots that look like those of typhus, so typhoid. Fever, lethargy, delirium. Here’s typhoid Mary. Lab diagnosis: well, you can get a sample from any…from urine, from feces, from blood, from biopsies. This bacterium grows all over your body, in all systems of your body, not just in the gut. Do the H2S test as well, look for gram negative bacteria. H2S, it’s going to be lactose negative. Treatment and control: well, treatment. There are some antibiotics you can use. They had some new ones out that they use I believe. Actually you can use beta lactams and some of the ciprofloxacin you can use. That’s unusual that penicillin and some of the beta lactams can be used to treat typhoid fever. But in those people who have typhoid fever and it’s not treated with any antibiotic, it has a mortality rate of about 15%. So it’s pretty bad, pretty serious. Carriers. What happens with this bacteria is that many people can become carriers, well a certain percent, a small percent. Typhoid Mary was one of the carriers of typhoid fever bacteria, and she lived….I think I told you this story before. She lived in New York City, 3rd and 33rd, and a couple other places. I remember one time this slide, or mentioning it to the class, she lived up town, on 3rd

and 33rd, and I hear a big yelp from a couple students because that’s where they were living at that time – in an apartment building on 3rd and 33rd. I’m sure it probably wasn’t the same building that typhoid Mary lived in. She moved around New York. She was a cook. She came from Ireland, that’s how she made her living. She was single. That’s all she knew how to do to support herself, and she became a carrier of the typhoid fever bacteria in her gallbladder. It was said that her gallbladder was completely covered, infiltrated with typhoid fever bacteria, salmonella typhi. The thing is she had never been sick with typhoid fever. She harbored the bacteria, she was a carrier in her gallbladder, but never had typhoid fever. So there were some outbreaks of typhoid fever in homes where she served as a cook. A couple people died. She kept moving on though, she would work for a while and then, oh some people are getting sick in this place where I’m working so I better move on to some other location. She worked in hospitals, she worked in restaurants, but also private homes. So after a couple of outbreaks of typhoid fever in the early part of the last century, they tracked her down. And I should say that the typhoid fever was pretty rampant in New York City at that time, but it wasn’t just that she had it and nobody else did. She had it, but what gained her notoriety was that when they finally tracked her down, they wanted to bring her back and check her out, and maybe even quarantine her for a while, she put up a big fight with the police. Got engaged in a fight, and I think a photographer from the paper was there and took a picture of that. So she was known as typhoid Mary. So they said, ok, well look at your gallbladder, it has all this bacteria in it, and we will allow you to go back and work as a cook if you permit us to remove your gallbladder. And she said no, she won’t do that. She said she was never sick with typhoid fever a day in her life, never a fever at all. And they said, well ok we’ll let you go under two conditions. One that you promise never to serve as a cook again in a household and two that you report back to us every few months. And she said ok. Guess what? She disappeared. She didn’t come back. And she continued to work as a cook, because that was the only way she could make a living. And so there were a couple more outbreaks of typhoid fever, they tracked her down again after really a long period because she kept moving on, and finally she spent the last 20 or so years of her life in quarantine because she wouldn’t allow her gallbladder to be removed. Some island up on the east river, one of the brothers islands up there near where I guess you get to the manhattan Bronx border, way up on Roosevelt Island, where she was quarantined for 20 years or so of her life. And so, that was it. Once again, it wasn’t because she became notoriety because that incident she was involved with, not because she was the only one. Typhoid fever was prevalent throughout New York City way back in the early part of the last century and before that. Immunization – there are indeed three vaccines, different types of effectiveness, but it’s pretty rare. We don’t usually get vaccinations against typhoid fever. It’s fairly rare. People that work with these bacteria in a lab setting routinely would get the vaccinations.

[24] Food poisoning[Boylan] Food poisoning, gastrogenteritis. There is a very large dose of bacteria needed. And we are going to see some stories in the paper in the next couple of weeks caused by this bacterium because the main source of the bacteria are poultry

– chicken and turkeys. Cattle as well. But what happens is food handlers, or somebody preparing the turkey does not heat the turkey enough or handles the turkey before heating it, gets the bacteria on his or her fingers, goes to the bathroom, touches that or other food you’re going to serve at thanksgiving dinner, and the guests all get sick. You’ll read about an outbreak of 20 people at that gathering, or 7 people in a family come down with salmonellosis, and it’s always the same thing. They didn’t clean the turkey, rinse it, make sure they didn’t contaminate other foods they were working with that day, or didn’t go to the bathroom and clean up properly. It’s often very embarrassing to the host or the cook who provided others with that turkey and also food poisoning. The thing you need is a large dose. No bacteremia here, as opposed to salmonella typhi. Incubation period is a matter of a couple days is all. Symptoms, general symptoms of cramping, diarrhea. Recovery takes a few days. At risk are the usual people with compromised immune systems: the eldery, young. Avoid eggs and avoid raw uncooked chicken, things like that could be sources of infection. Undercooked chicken, undercooked turkey, raw eggs, poultry. Usually, I should say, I’m sorry let’s go back. You eat the food, within about two days you get sick, and you have this illness for about two or three more days. Usually you’re ok after that. Wash hands, avoid antacids. Well, avoid antacids. How could that help to prevent? How do antacids compound the problem? Well you can see up here that a large dose is needed, maybe thousands and thousands of these salmonella enteritidis that cause these food bourne infections. Why? Because as they pass through – you eat the food, as the bacteria passes through your stomach, the pH, the acidity of your stomach, will destroy most of the bacteria so only a few survive. But enough to pass through and cause the diarrhea. However if you elevate the pH of the stomach by taking antacids, making it a little more alkaline, then the bacteria have a greater chance of passing through because it isn’t quite as acidic. They’re not destroyed by the high concentration of hydrogen. So some people do that when they feel sick, they take antacids with an upset stomach. Well that can possibly increase your chances of getting infection by these bacteria. You’re going to allow the dose of the bacteria to remain fairly large and pass through.

[25] Shigella[Boylan] Shigella. Dysentery, of those three different species. Lab ID tests: lactose non-fermenters, non-motile, does not produce hydrogen sulfide. Causes dysentery – bacillary dysentery, as opposed to viral or other types. Maybe more than any other infection in the history of human kind has this disease affected the outcome of the world today. Dysentery is an infection only of humans, you don’t get it from animals, but it can spread very rapidly, and in contrast to the previous bacteria we just discussed, you only need a very very small dose, maybe even under 200. So the key is a small dose of these bacteria is all you need. They are more resistant to the acidity of the stomach. The food poisoning caused by salmonella enteritidis you need thousands of bacteria. So very very infectious bacterium. So only humans. What I mean by affected history is so many times in the course of battles in European countries especially in the middle ages and later, whole armies would be infected with these bacteria. And once they were… dysentery is very terrible, it’s like EHEC. Remember because that’s where EHEC got their toxins from – this bacteria,

the shigella toxins, the shiga toxins. So these toxins are shiga toxins. In e. coli they’re shiga-like toxins, but e. coli, this is where they got them from. They cause cramping and pain, and diarrhea, and vomiting, and what happens… and once again, bacteria only of humans, but these soldiers fighting these battles would go to streams to defecate there, bathe there, use the stream water to wash their food, and they would get the infection. And the invading army would see an army that was on the ground, writhing in pain, unable to defend themselves, hoping they would shoot them actually, they were in such pain. And so many times, poor sanitary conditions, as you would find in the course of these battles, would lead to contamination of water that would find its way eventually. They didn’t know about bacteria in those days. So many battles, the outcome was changed because of illnesses among the soldiers fighting the battle on one side. Ok, so incubation – a couple days. But when you’re sick with this dysentery, it’s very serious and very incapacitating. Invasion of lamina propria, ulcers produced, severe and bloody. So, as with the STEC we saw we e. coli, these are the same way. They damage the epithelial cells, the microvilli, and cause bleeding and consequent symptoms of serious GI infections. I want to throw in a couple others now.

[26] Other coliforms: UTI – urinary tract infections[Boylan] Getting back to coliforms. We talked about a couple pathogens – salmonella and shigella. Here are some bacteria once again that are coliforms, that are part of our normal microbiotia, not ordinarily causing infections. Here are some that cause urinary tract infections. They are opportunists. They take advantage of some other malady or some other infection or condition in the body to cause these UTI’s. Serratia marcescens. This is the one that causes nosocomial infections. We used to work with this one in the lab all the time because it forms this red pigment. When you grow this bacterium at room temperatures in the lab, it forms a red pigment. When you put the plate of these bacteria in the incubator and grow it at, say, 37 degrees, our body temperature, it does not produce the pigment. It loses the ability to produce the red pigment. So it was a test where you could see a bacteria could change a property they have based upon environmental conditions. Environment changes, temperature changes. At a low temperature they produce the pigment, elevate the temperature, something happens to the enzymes involved in pigment production, temperature rises, and they no longer can produce it. Bring the temperature back down to 20 degrees or room temperature, and it forms the red pigment. So it never changes its genotype, but change the temperature, change the property it has based upon the condition. And actually in the old days, even in the labs, micro labs, students who took microbiology, the used to give them some serratia marcescens after it grew for a while at room temperature, and you could see in a while it got in the blood stream, you could see the blood vessels would be redder as a result of these bacteria. Of course after a while they would lose the pigmentation, but if you give somebody this bacteria, if you inject them in them or you have them eat them, some would get in the blood stream and you would see it, especially in the gingiva. Of course that was never a good idea, and we don’t do that anymore. Proteus vulgaris and proteus mirabilis- these are bacteria that are swarmers. They are so motile, members of the genus proteus, that they put a little

drop on the center of a petri plate, and within a matter of hours you will see they swarm over the whole plate. They are tremendously fast spreaders. They swarm. Extreme motility. And you put a little drop on the center of a plate, the next day you will see that plate is coated completely with these bacteria. They are swarmers. Why are we concerned about them? Well they can produce the enzyme urease. Urease that breaks down uric acid, which is found in urine, and when they do this… so lets say these bacteria, they can find their way sometimes to the blood and get into the kidney, and when they grow there, they elevate the pH of urine flowing through the kidney, and you know the pH of urine is slightly acidic, and when it’s acidic, everything’s fine. But when these bacteria grow there and produce the enzyme urease, that breaks down uric acid, the pH goes up. The pH rises above what it’s supposed to be in urine in the kidney. As a result of that elevation in pH, you get kidney stones or renal stones. So there are certain salts like magnesium, calcium that with an elevated pH start to precipitate out of your urine and form these so called stones, that block the transmission of urine through the kidney. Very painful. Those are caused by bacteria, often. Dr. Tierno will talk about the Weil-Felix story later on, it’s a cross reaction story. Some components of the cell wall of this bacterium cross react with typhus protozoa. Typhus…I think it’s typhus. He’ll tell you the story of Weil-Felix and world war II story and how this one test called the Weil-Felix test actually saved the people in a village from being taken over by the nazi’s. But that’s for him. I’ll let him tell that. But refer back to proteus. It’s a component in the cell wall of proteus that cross reacts with this other microbe he’ll talk about. So kidney stones, renal stones, elevation of pH, urease breaking down uric acid, pH goes up.

[27] Other coliform – pneumonia[Boylan] This one you’re going to hear a lot more about over the next couple years, because we’re hearing more about it now and the danger it poses to us, all of us, worldwide. And it’s klebsiella pneumoniae, k. pneumoniae, shown under klebsiella pneumoniae. It can be a primary or secondary infection. People with impaired pulmonary function, pneumonias, and sepsis, especially. So the top part, that part, we always knew about – above the red. Could cause pneumonia and sepsis in alcoholics, chronic obstructive pulmonary disease, it comes with a fever and a cough. You get these lung infections, they cough up the sputum, very productive cough, looks like currant jelly sputum. So that’s a characteristic of this particular infection. Pneumonia. The bacteria reside in the gut, but every now and then fecal oral transmission, they can get sometimes in the lungs, especially in people who are immunocompromised and have those conditions shown there. And it’s luxuriant capsule it produces, it’s a very large capsule that is it’s main virulence factor. Attention…attention. New superbug strain recently emerged. Called klebsiella peumoniae NDM1-Kp. Ok, lets talk a little bit more about it first. It has a plasmid gene called NDM1. That gene codes for…oh I had it written down here…New Dehli…ok. You have the slides? You know it’s on the bottom, maybe underneath the slides, it shows something? It doesn’t?

[student] New Dehli Motila beta lactamase

[Boylan] New Dehli protenase…New Dehli beta lactamase. This bug is becoming resistant to almost every drug we have out there on the market. Phenomenally resistant. And it is spreading among (?) throughout the world, and a very interesting story, it may have even been first diagnosed here at Langone medical center maybe 8 or so years ago. This particular strain is very very virulent, deadly, and spreading, and resistant to many antibiotics. That combination of properties makes it very serious. We have to monitor it very carefully. So klebsiella pneumoniae, New Dehli Metalo-beta lactamase 1. Klebsiella pneumoniae. It’s a gene on a plasmid that is transmitted among not only this bacterium but other gram negative bacterium – other enterics as well. Spreading rapidly. Even the last resort drugs like vancomycin and others. I wanted you to be aware of this particular bacteria, klebsiella pneumonia, not just for what we have always known it’s able to do on the top of the slide, but also this new superbug. Some other bacteria are given the name super bug because they are resistant to one or two antibiotics. This one is pan resistant, even those we use. There used to be many drugs to treat infection caused by this bacterium, but now they are useless. So keep your eye on this one because it is spreading worldwide. And finally, one more.

[28] Enteric infections – treatments[Boylan] Ok, so treatments. Some antibiotics are shown here. But for these GI infections, cook all meat thoroughly, consume only pasteurized milk and juices, pasteurized thoroughly. Wash hands carefully and frequently. If it’s gastroenteritis, you want to replace fluids, electrolytes with Gatorade, pedialyte for kids. Often better than using some antibiotic because you want to replenish the fluids that are being lost to restore the health of the patient.

[29] Yersinia pestis – an enteric organism with a different type of infection[Boylan] And finally, this other one which I didn’t think should be included among these but is, it’s an enteric because of the biochemical and serological properties it has, is the bacterium that causes bubonic plague. Bubonic plague, dysentery, cholera, three of the major scouragers of mankind. I mentioned this briefly in the last conference we had on bioterrorism and bioweapons that we use, and this is one of the ones we’re concerned about being used as a bioweapon by terrorists because of the deadly disease it can cause – bubonic plague. The black death. May be responsible for the deaths of a third of the European population in the 13th century. There have been three really serious outbreaks, epidemics, not quite pandemics, but epidemics of the plague in Europe that wiped out a third to maybe more of the population. Terrible history. It’s called the black death because it causes hemorrhaging and the skin and all the blood vessels hemorrhage and die, they turn black. It comes from animals primarily. The carrier of the reservoir of this bacteria in the middle ages and even today was the rat, although there are some other animals like prairie dogs in the US can carry it and be a reservoir. (?) Urban plague and sylvatic plague. Sylvatic plague refers to the plague out there in the woods – sylvatic. Urban plague is when humans and cities begin to get the plague. And the fleas are of course the vectors for the bubonic plague. Pneumonic plague is when the plague proceeds. It’s called bubonic plague because these bacteria infect our lymph

nodes, and our lymph nodes begin to swell, particularly in the axillae and the groin area, and the swollen lymph nodes are called buboes. So it’s called bubonic plague because of these swollen lymph nodes. If you’re bitten by a flea in the leg, it’ll be your groin, and if you’re bitten by a flea in your arm it’ll be your axilla, your armpits. So that’s the essentials of it. And the protein capsule is anti-phagocytic. Pneumonic plague, once it gets to the lungs, you’re gonna die, and pneumonic plague, if you get exposed to someone who coughs up these bacteria, and it goes from their lungs to your lungs, yersinia pestis bacteria, it’s going to be fatal. This is the fatal form of the plague – pneumonic plague, when it gets to the lungs. And it happened in many cases, many people who died in the middle ages and more recently as well from this, were exposed to the bacteria in this way. They inhaled it from patient’s who had pneumonia and within hours they were gone. Believe it or not this is one of the enterics. It likes to grow in our gut too. Ok I guess that’s about it. If you’re not completely dead I’ll go on. That’s enough. We’ve covered everything.