6: bacillus & clostridium

8/10/2019 6: Bacillus & Clostridium

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Transcribed by Erica Manion 11/14/2014

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Infectious Diseases Lecture 6 Gram-Positive Rods by Dr. Boylan

[1] [Title Slide]

[Dr. Boylan]Good morning. I have some bad news and some good news. The bad news is

that one of our other faculty usually gives this series of lectures on gram positive rods, Dr.

Saxena, but hes away, so I said ok, Ill do it this year. He said, ok, Ill send you my slides.So I can use them. So I said great, send me your slides, so he did. There were about 150

slides, so I spent the last two days whittling them down to about 40. So I guess thats the

good news, thats a little less youve got to study. But you know, really for every infection

we discuss, you could go through the whole thing about what the bacteria look like, what

kind of tests you do to identify them in the lab, incubation periods, symptoms, pathology,

immunology, yeah, but I think we know that those things do happen, we covered them

already in general. So I think when we get to these other bacteria its good to focus on what

is unique about them. Or at least, a little bit different. Or some combination of properties

they have that distinguish them from other bacteria were going to discuss. So that is what I

think is more important and you may agree with me sometime, maybe sometimes youll

think it is too much information, but I tried to whittle it down to the core identificationfactors for these bacteria. By the way, a couple of days ago we had the lecture on

Streptococcus, and I had two slides to go. Why dont you just look those over. Because the

last two slides, one was on the genus Enterococcus,which used to be a Streptococcusbut

they, a couple of unique properties it has, that particular gene will sort of put it in a new

genus, Enterococcus. And E. faecalisis one of the major species, and look at that slide, and

why do we even study E. faecalis? What is unique about that one? And Ill tell you one of

the things youll see is that it is becoming very resistant to antibiotics. There are these

strains of enterococci called VRE, very resistant enterococci. Meaning they are resistant to a

lot of antibiotics. But also there is another property we have to identify it in the lab that is

similar to a test we do to identify Staph aureusin the lab. So look at that test and figure out

which test is used to help identify Staph aureusand Enterococcusspecies as well, thats onefeature they have in common. And the next slide, the last slide was on viridans

streptococci, meaning, of course, streptococci that hemolyze red blood cells only partially,

so you get a greening hemolysis around the colonies that appear. Streptococcus mutans, the

culprit in caries is one of those, and Streptococcus sobrinus. And I think its next Tuesday

that Dr. Caufield will come and talk about mutans streptococciand the different, the

bacteria we think are the major ones in causing caries. He always likes a great turn out. If

you could come again on his lecture, scheduled for Tuesday, he will be thrilled. He gets

depressed when he walks in and only sees five or ten students. I know you have other

things to do but I appreciate it when you do come. So look that over, and we are going to

have a little bit more about the involvement of viridans streptococci with bacterial

endocarditis in our conference next week. I have some case histories I want to go throughabout Staph and Strep.And we will discuss at least one where these viridans streptococci

are involved with bacterial endocarditis. So look those over.

And now Ill go on to discuss the gram-positive rods. So the first series of lectures in

Infectious Diseases are talking about gram-positive bacteria, the Cocci,the Streptococci,and

the Staphlococci were just covered, now we have the gram-positive rods. So when you

think back when you are studying for the exam, the first exam, usually the point that

separates the gram-positive infections from the gram-negative which will come up next, is


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the lecture on tuberculosis. Mycobacterium tuberculosiswhich Dr. Tierno will present next

week as well. So if you never can remember which is positive or negative, gram-positive or

gram-negative, just think about if we discussed that before T.B. or after. Before, its gram-

positive, for the most part. And after, its going to be gram-negative infections.

So gram-positive rods, the genera here are Bacillusand Clostridium, Corynebacterium, and

Listeria. And the next, if we get to them, hopefully later today, are Actinomyces andNocardia.

So Bacillus and Clostridium. Gram-positive, spore-forming rods. Another name for rods is

bacilli, bacillus. Small b means all these rods are bacilli with a smallb. But theres only

one genus, Bacilluswith a capital B, italicized usually. So these are all bacilli, rods, Bacillus

and Clostridium,spore-forming gram-positive rods. And all other bacteria really do not

form spores. These are the only two spore formers. And you are going to see, one habit that

bacteriologists have gotten into over the years when they discuss some new bacterium,

they will say, and its a non-spore former. Well it is really redundant, or not necessary,

because other than these two, and as youll see on the next slide, they dont have to be

there. The Corynebacteriais not a spore former, its not a Clostridiumor a Bacillus. The bigdifference between those first two bacteria, the Bacillusand the Clostridium,is that Bacillus

is a strict aerobe and Clostridiumis a strict anaerobe. Some species of each of those two

cause some serious, even life-threatening, infections. Well briefly talk about them. Then

Corynebacterium, really only one infection to be concerned about, and that is Diptheria.

Listeriais one species of that, that is a bacterium that can cause food poising and has the

unusual ability to grow at very low temperatures. It grows very well in a refrigerator. It

contaminates food and people get sick if they eat contaminated food. AndActinomyces and

Nocardia, Ill tell you more about them after we get through the first four.

[2] [Introduction]So, Bacillus,gram-positive, aerobic, spore-forming rod.

Clostridium, gram-positive/negative, strict anaerobic. Now I would say they are definitely

gram-positive. I got this slide from Dr. Saxena, I think I know what he means. Sometimes

when you stain bacteria that are gram-positive, when they are really old, theyve been on a

plate or theyve been in broth for a long time,they are starting to lose their strength and

ability. The wall becomes a little bit weaker, and so sometimes for older cultures of bacteria

that are gram-positive, they will stain gram-negative because they cant retain the positive

stain inside as well when they get old and weak, when the wall is weak, but they are really

gram-positive, the Clostridium.

Corynebacterium, gram-positive, aerobic, see that, non-spore-forming rod. Well, you dont

have to say that, we know its not going to form spores.

Listeria, gram-positive, facultative, this anaerobe.

[3] [Arrangements of bacillus (rod)]


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Bacillus, a rod, they can divide by binary fission. You can have pairs, you can have singles,

you can have a chain of bacillus, a chain of rods called streptobacillus, just like

streptococcus, a chain of cocci, bacilli form long chains, often as well, streptobacillus. And a

coccobacillus, I mean sometimes these bacilli, these rods, are tough to tell microscopically

whether they are rods or cocci, because they sort of round up when they are short. So you

hear the term coccobacillus sometimes meaning it is a rod that looks more round than theother nice, rectangular cells, the other bacteria. Then we see some arrangements here,

scanning EM and how they grow in culture and on a slide.

[4] [Bacillus species]

The two major species of Bacillusare Bacillus anthracis, the agent of anthrax, and Bacillus

cereus, which causes food poisoning.

[5] [Anthrax]

Anthrax, a little bit about the history about that, thats really an infection of animals,herbivores. Cattle and sheep for the most part. And it used to be found even in the first part

of the history of this country around the 1800s, cattle and sheep would have this particular

infection and there was no vaccine. People got very sick too when the spores of this

bacterium, Bacillus anthracis, were transmitted from animals to humans via direct contact

or animal products or inhalation of spores. Endospores, spores, same thing.

A serious problem is in developing countries, even today there are very few cases of

anthrax in the United States, because we do vaccinate the animals, the cattle and sheep.

Rarely seen here. A few cases were reported in 2001. Remember after 9/11 there was a

bioterrorist at work who mailed out envelopes to people in the media and publications, and

politicians as well, envelopes that were laden or coated with spores of anthrax bacteria,and they got sick, many of them did, and five of them died. So 2001 there were cases of

anthrax but that was because of some guy releasing, mailing spores of it, which were

inhaled by postal workers and people in the media, or the spores got into the breaks in the

skin of the postal workers, and they came down with another type of anthrax on the skin

that we will talk about.

[6] [Anthrax]

So the key, also about anthrax is that the spores are the infective form in all three types.

The spores. What does that mean? You know the spores are living cells that are not

growing, theyre not multiplying, they are dormant. But sooner or later those spores maycrack open like an egg shell and out comes the vegetative cell. And then that starts to

undergo binary fission, to two, four, eight, etc. So contrast a spore of a bacterium, Bacillus

anthracis, with the vegetative cells. The vegetative cells of these bacteria are not infectious.

And we will see why that is important later. So the three types of anthrax are inhalational,

thats very dangerous, where youinhale the spore-laden dust. Rare, but we have to be

concerned about bioweapons-if they are used as bioweapons. Also called woolsorters

disease, thats because this particular type of anthrax was common in the men and women


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who sheared sheep. The sheep were carrying the spores of this bacterium in their wool,

and they are shearing it, and theres often aerosol that is created with the spores as they

take the wool off, and they inhale them and they came down with pulmonary or

inhalational anthrax, called woolsorters disease because of that. Cutaneous anthrax is on

the skin, well talk about that. And a rare form we wont say much about is gastrointestinal

anthrax, from eating infected meat from these animals. Eating the meat that iscontaminated with these spores of anthrax bacterium.

[7] [Anthrax]

Clinical symptoms of inhalational anthrax. Well, like so many infections, this one also

begins with flu-like symptoms. So initially you dont the childs got a cold or maybe the

flu. Well, we wont beterribly concerned because you get over the flu in a matter of days

usually so you dont anticipate what is going to happen later on potentially with other

infections that begin with flu-like symptoms. Now a little fever, a dry cough, aches and

pains. And that is how this anthrax begins, inhalational especially. GI symptoms, diarrhea.

And then, this is characteristic of this pulmonary, or inhalational anthrax. The personseems to get better for about a day. You think, oh, you feel bad, flu-like symptoms, but then,

ok its starting to fade away. With anthrax, inhalational, all of a sudden the signs and

symptoms and disease comes back with a vengeance, really. And thats the danger, its

sudden onset of hyper acute illness with trouble breathing, turning blue, lack of oxygen,

high fever, disorientation, and it can lead to death. So pulmonary anthrax can be very

serious, as you see, and it is biphasic. I call it biphasic. In other words, the initial flu-like

symptoms, a period of seeming well-being, and then boom. The major symptoms you see

before you die or hopefully you can recover of course sometimes but it can lead to death.

[8] [B. anthracis virulence factors]Ok,this is what I think is an important slide.Why is this bacterium so dangerous? What

is unique about it, and why are we concerned that this particular bacterium and the spores

that it forms are potential bioweapons that a terrorist group may use, what is it? Well, you

look at the virulence factors of this bacterium. And two things it has are the capsule and

what is called the anthrax toxin, which I call a toxic trinity. It is said to be one toxin, but it

really has three different components to it, three exotoxins. So one toxin with three

different components to it, they all work together, they all have different functions but they

are referred to as the anthrax toxin.

The capsule, you know how important that is. Never underestimate forget that. Role:

protects the cells from phagocytosis. Bacteria survive longer in the body. It is encoded by aplasmid gene. Not a chromosomal gene, not a phage gene. A plasmid in the anthrax codes

for the particular capsule. And it is a unique capsule in that it isnt a carbohydrate. It is

made up of a single amino acid, a polymer of a single amino acid, D-glutamic acid. This is

the only capsule we know of that is not composed of a polysaccharide, its an amino acid.

So it is a unique capsule, and that contributes to its ability to protect the bacteria from

phagocytosis.


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The toxin itself, Im not going to go through the particular functions that the three different

toxins carry out, but together they also are encoded by a plasmid. So heres Bacillus

anthracisliving a nice leisurely life, and all of a sudden and without the plasmids that are

responsible for these virulence factors, they arent pathogenic. If these anthrax bacteria do

not have both these plasmids, and most of those in nature do not, they will not be

pathogenic. It is only because they come infected themselves with plasmids. Harbor theseplasmids. There is a little bit more about that on the next slide. But they are able to these

plasmids encode these virulence factors and its the capsule and the toxin that work

together. You have to have all of these together for anthrax to cause the problems that it

does. So capsule and toxin works together, Three toxins, they are exotoxins. Protective

antigen, lethal factor and edema factor. Remember those names.And the capsule and

toxin work together. Once again, we arent going to go through what the toxins do, but they

both must be present, they destroy the host tissues, in the lung especially, and then they

also inhibit the hosts immune response, both the toxins and the capsule [something

inaudible, maybe inhibit?] phagocytosis. So therefore they can survive, and they cause

pneumonia. Then the vegetative cells. We inhale the spores, they get to the lung, they

germinate there. The vegetative cells come out, and after a while they kill those cells andthey spread in our blood stream, and other organs are effected. But if a person has anthrax,

that means the vegetative cells, not the spores, are teaming in their blood, in their

circulatory system, in their body. You can still work with someone who has anthrax if you

want to without fear of contagion. Once again, they are sick because they have the

vegetative forms of this bacterium in their body, not the spores. The spores are the

infective form. If you want to if there ever was a bio attack, or somebody gets sick with

anthrax, you might need to come in contact with them, especially in any other countries

you may go to, you can work with them without fear of getting the infection. They no longer

have the spores.

[9] [Genetics of B. anthracis]

Genetics here we see, that is a B. anthraciscell. The capsule, and you can see here are the

two plasmids. Whenever you see a small p like that it means plasmid. So plasmid with

X02 (pX02) is the one that codes for the capsule, the D-glutamic acid capsule. And pX01

has three different genes in it, they code for the trinity, the anthrax toxin. Protective,

edema factor, and lethal factor. So without those plasmids, if you could cure the anthrax

of those toxins, and there are ways to do that in the lab, it is no longer pathogenic.

[10] [Capsule of B. anthracis]

The capsule is just shown here in the stain, the gram stain. Gram-positive clusters of themand heres the capsule in different highlight here (points to green area in bottom image).

And sometimes you can actually see spores, endospores being formed inside these bacteria.

You have to look closely but youll see, even in the ones on the upper left there, a clear zone

around them. Maybe its even a negative stain as well. That looks more like a gram stain

but you will see if you look closely there, you can see it better up there than on my screen,

the capsule. Clear zone around them.


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[11] [Cutaneous anthrax development of an anthrax eschar]

Cutaneous anthrax, where the spores enter a cut or lesion in the skin. They get - the spores

once they get in under your skin when you scratch your skin perhaps, or just introduce the

spores into a little scratch or break you dont even see, then the spores germinate and they

cause cutaneous anthrax, which leads to the development of a lesion called an eschar. An

eschar a black lesion that is shown primarily here on the bottom, a hand of thatindividual. A black lesion. So thats what happens when the spores of anthrax make their

way into a cut, cutaneous anthrax, the skin anthrax. And actually this is how it got its name,

Bacillus anthracis, Anthracismeans coal, black coal. So it got its name of the species from

the cutaneous anthrax that it can cause when it gets under the skin. A black lesion.

Anthracis. Coal-like.

[12] [Anthrax]

Cutaneous, characteristic papule, eschar develops, history of exposure to animal or their

products. Clinical symptoms, well, you see the lesion developing. You can isolate the

bacterium from blood or even some times cerebral spinal fluid. So it can be invasive once itleaves that area with the eschar, it gets into the blood, it can be serious and life threatening

as well.

[13] [Anthrax]

Control animals, control animals that die suddenly should be handled cautiously, livestock

should be vaccinated. Individuals at high risk should be vaccinated and wear protective

clothing. So, there are vaccines for anthrax, but we dont all get the vaccine for anthrax

because it is so rare in this country. There is a vaccine, and maybe it is given to army

personnel, it is given to animals of course and it is given to people who work in areas of the

world where anthrax is still a problem. And in certain parts of the world it is a problem.And so you have to vaccinate the people who work with these animals and protect them in

that way.

[14] [Bioterrorism]

In 9/11, late September of that year, early October, there were a couple of mailings of

envelopes by some, bioterrorist really, someone who had worked in our biological

weapons program during World War II and afterwards. We had a biological warfare

program, as did many other countries, and we tried all different types of microbes and

bacteria, and viruses, and bacterial toxins to see which ones might be best used as

bioweapons to help defeat our enemy. To spread them, have them be airborne, and then beinhaled and in the food or water.

That program was thankfully stopped in the 60s. But a lot of countries who were

concerned with bioterrorism and wanted to develop their own programs thought about

using anthrax. Because anthrax spores are pretty easy to prepare. You just grow a

bacterium in a lab and as they get older, they begin to form spores and it is easy to isolate

spores. Relatively easy. Purify spores, dry them, and aerosolize them and distribute them


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over quite large areas. And so that is essentially what happened after 9/11. Here is Tom

Brokaw, he was one of the people in the media who was sent these envelopes with spores

in them from anthrax bacteria. Luckily someone intercepted it. But people did get sick, and

I think five people did die from inhalational anthrax, and thirteen or sixteen, something like

that did develop cutaneous anthrax they all survived. But people died and there was a lot

of panic because it wasnt just in one area. There were mailings to New York, andWashington, D.C., and some to Florida, and one to Connecticut. I remember even here at

NYU at Langone Medical Center, our friend Dr. Tierno, who is going to give a lecture next

week, he was the chief microbiologist there. So they were going to him, What should we

do?? What can we do to protect ourselves? What antibiotic is the best one?? So there was a

lot of fear, because people didnt really know what to do. And really, for anthrax, Penicillin

is good. There are a whole variety, they used to use Cipro at that time. They thought

Ciprofloxacin, Cipro was the best one to use. But really there is a variety of antibiotics that

can be used to treat anthrax. Thats another good thing to know about these things. Yeah,

people who have it are not contagious and there are a lot of antibiotics that can be used to

kill the bacterium, the vegetative bacterium in the body of the people who have anthrax. So

they used spores, the CDC said anthrax spores are a potential bioweapon. Weve alreadyseen they are very effective in efficacy just by the incidents after 9/11. People got sick,

people that inhaled them, and people got cutaneous anthrax. Easy to make, easy to

disseminate, and a high lethality for an infectious disease. The mailings and the [inhaled?]

and cutaneous.

[15] [B. cereus]

Ok, thats it for anthrax. Once again, the good thing is youre not, a person with anthrax is

not contagious and there are a lot of antibiotics that can be used to treat anthrax. Penicillin,

Cipro, and others as well.

Another species of Bacillusis cereus. B. cereus. I always like the B. cereus. Theres another

one calledB. subtleus we wont talk about. Lets be serious about B. cereus [hahaha].And

gram-positive, aerobic. See, why say that, they are all that. It is very easy to grow in the lab.

Nonfastidious. Even anthrax is easy to grow actually. Very simple medium. Well talk about

some bacterium later that are very fastidious, meaning they need a very rich, nutrient

medium with a lot of vitamins, and salts, and sugars, and other things perhaps to give them

a little boost to grow. But these grow very easily.

[16] [B. cereus]

Ok, so here is one of Dr. Saxenas slides. Pathogenesis of it. Causes food poisoning. A lot ofdifferent enzymes and toxins. And Im concerned a little bit about it. Well see that it causes

food poisoning, but primarily when it effects rice. Rice is one of the main foods we eat that

happens to be contaminated with the spores of B. cereus. And right now, as you may know,

a lot of us moved from, a month or so ago, from the VA, where our offices were, to a

building in Curry Hill, you know Curry Hill? Where all the Indian restaurants are. Curry in a

Hurry? So were near, and boy I go out in that area, it is a great area if you like to eat. And a

few of us over there in our new offices like to eat a lot. We go out to these Indian


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restaurants and we get the buffet and we bring it back or we eat in the restaurants. And the

Asian restaurants I dont know why, I always think, oh rice, why do you have to be

concerned about B. cereusfoodborne infection, but if you boil, you know, if food is handled

properly youre not going to worry about it. But youll see every now and then an outbreak

of food poisoning caused by B. cereus.Usually it is because the rice was contaminated and

the rice was not heated properly. A whole bunch of toxins, these bacteria form thatcontribute to the food poisoning. And you want to look at the enterotoxins, these are the

toxins that bacteria produce that work in the gut. They are exotoxins released by the

bacteria, they get to our intestine, they get to our gut, and thats where they cause diarrhea

and problems, and food poisoning.

[17] [B. cereus]

Rice. Enteritis. Theres two the thing about the B. cereus,lets look at those two toxins

once again. Enterotoxins. A rapid onset, emetic form, HS-enterotoxin. Nausea and vomiting.

So the two toxins we have to be concerned about. There is a slower onset diarrheal form,

HL enterotoxin. So different strains of B. cereus[have at least? 28:52 into podcast] one orthe other of these two toxins. And some, once again, the symptoms may appear in a matter

of hours, especially with the rapid onset. Like Staph aureus, remember Staph aureus food

poisoning, 3-5 hours for people to get very, very sick, because the pre-formed toxin that

they ingest from the food the Staph aureusgrew in. Very short incubation period, similar to

the Staph aureustoxin. And the another one works a bit slower, the HL-enterotoxin. So a

combination is an unusual feature, a combination of enterotoxin. Or one or the other. One

strain may make you sick very soon, and the other, it may take a while for them to cause

the symptoms of food poisoning.

[18] [B. cereusinfections]Control, good hygiene, adequate cooking, avoidance of recontamination of cooked food,

proper storage, food poisoning.

[19] [BacillusOne more thing]

One more thing about Bacillus, that genus. Since they are spores, they can form spores -

spores are the hardest types of cells in the world to kill, destroy. We went through that in

the course on microbiology The fact that spores are living, vital cells, but they are dormant,

and they are heat resistant, and resistant to chemicals because of the make-up of the spore.

But especially as you can recall, the core, the central part of the spore, they call the core,

and there is no water there, it is dehydrated. And as also you may remember, hasdipicolinic acid. This acid is built up to very high levels, about 15% of the material in the

core is dipicolinic acid. So that, plus the absence of water makes them very resistant to

heat. Cells are usually resistant, sensitive to heat, why? Because when you boil them, the

cytosol gets hot of course, and the other components, protein congeals in our cells.. But

without water, that wont happen. Even when you boil spores, or heat them at high

temperatures, youre not going to destroy them. We are going to talk later on about the

autoclave. Oh, well talk now about it.


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So you know, in this building, we have to in the clinics and the stuff you use and the

instruments you use have to be sterilized. That means they have to kill all bacteria, even

spores! In order to do that, you take these necessary components down to the, its inthe

basement of the Weissman building. They have all these autoclaves there. Autoclaves.

Steam heat. These autoclaves. Its like a pressure cooker you might have used in yourkitchen, the same general purpose. Get some steam, bring up the temperature inside the

temperature over boiling. 100 degrees is boiling. Thats not going to kill all spores.

You have to elevate the temperature to 121 degrees centigrade, 21 degrees higher than

boiling, to actually make sure you kill spores. You cant do that ifyou boil, boil, boil, it is

going to stay at 100 degrees. You have to use an autoclave, or steam heat in enclosed space.

So as the pressure goes up, the temperature goes up to 121. So not only that, but you have

to make sure these autoclaves are functioning properly. What if you put all your stuff in

there to be sterilized, it has to be used when it is sterile, you put it in the autoclave, but

what if the autoclave is not functioning properly? What if there is a breakdown in one

component or another, and that happens a lot. The autoclave did not work. It went throughthe whole cycle and you think it worked, but it didnt necessarily kill all the spores. So the

point being, you have to have a very strict test, a strict test, to make sure the autoclaves are

working properly. How do you do that? You make sure they kill even the most heat

resistant cells on the face of the earth, spores. So every time we use the autoclave over

there, or maybe not every time, at least once a day, they put a little packet in there, in

glassine, and inside of that packet are spores of Bacillus. Two different species. Spores. And

then you take, you run it through the cycle, somebody picks up that glassine envelope, goes

up to the lab, opens it up aseptically, pours out the spores and puts it in broth and they

should not grow. But if they do that, they take the glassine up, put the spores up to the lab,

put them into broth and they come back the next day and there is growth, they know those

spores were not killed by the autoclave and you have to shut that autoclave down until it isrepaired. So, spores.

Ok, so what do they use, what kind of spores from bacteria? Bacillus. Bacillus

stearothermophilusand Bacillus subtilis. Spores of these two. We just buy them from a

manufacturer, you get a box, there may be a hundred different glassine envelopes in there

with the spores inside. Now youre thinking, ok. So the biological indicator for validation

of sterilization. By moist heat is autoclave, and by dry heat in the oven. So you can also

sterilize things, not liquids, in an oven if it reaches a high temperature for a long period of

time. So we use those envelopes too, with the spores, to determine that sterilization

occurred in dry heat ovens as well. So why use two species of Bacillus, wouldnt one be

enough, one spore former? Well they think yeah, maybe it would be, but we want thestrictest possible test to be done. And it turns out, I dont remember which is which, but

one of these two, Bacillus (also known as Geo.They changed the genus name) but Bacillus

stearothermophilusand Bacillus subtilis, spores of each of them.

Why do they use two? One is a little more resistant to moist heat, slightly more, and one is

a little bit more resistant to dry heat. So maybe it is just a degree or two but it is going to be

a stricter, more valid, stringent test if we have both types of spores together. And so that is


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why they use both types. It is a biologic indicator. There are also color indicators, there are

all types of different tests that can be used to see if autoclaves are working properly,

sterilizing the way they should be, but this is the best one. Biologically are the spores

killed? If the autoclave is running properly, if the dry heat oven maintains a high

temperature, it will kill spores when they run them through a cycle in the autoclave or the

dry heat oven.

[20] [Clostridium]

The other major gram-positive, spore-forming anaerobes are Clostridium. Many of them are

harmless saprophytes. In other words, they live out on vegetation and dead trees,

vegetation, saprophytes, live in the soil, and they are opportunistic. They can cause

infections in us if we are immunocompromised or when we inhale them, or we will see

other ways we get exposed to the spores of the Clostridiumspecies. They can survive

outside, spore formers, rapid growth Easy to grow. And they cause disease primarily

through the production of numerous toxins. So we are going to look at a couple of species

of Clostiridiumand see what infections they cause and pay attention to the toxins that theyform that help them cause these diseases.

[21] [Clostridium]

Here is an overview of them. The three, Clostridium tetani,causes tetanus. Clostridium

botulinumcauses botulism, botulism toxin. And Clostridium perfringenscauses gas

gangrene, myonecrosis, meaning these are bacteria that produce toxins that actually

destroy our muscle. These bacterium, the third one there, myonecrosis, death of our muscle

cells. They actually can break down the proteins in muscle and ferment the proteins. These

are very destructive infections, destroy the they ferment proteins, not carbohydrates. So

you can imagine how much damage that will do to the muscle, and will kill a lot of cells.And I guess when Dr. Saxena put this a less life-threatening disease, pseudomembranous

colitis. PC. Caused by Clostridium difficile. Youve heard of that one, Clostridium difficile?

Everybody say C. difficile. And say one more thing. C. diff.Okay, because it is also known as

C. diff, and it is becoming more and more prevalent, especially in hospital environments,

causing pseudomembranous colitis.

[22] [Clostridium tetani]

Tetanus, physiology. Small, gram-positive, motile, spore-forming, well, no kidding. Round,

terminal spores, extremely sensitive to oxygen. They are anaerobes, remember. And Id

like to point one other feature of some of these spore-forming bacteria. When they formspores.. Some of them form spores only at one end or one pole, and they look like tennis

rackets. And often, a trained microbiologist can look at a cell like this, and say Oh I know

that is Clostridium tetani. Because the spore is formed at the pole. Other Clostridiumform

spores in the middle of the cell. And some cause bulging of the cell. So these are cells that

are forming their spores, they are creating spores, and they have not yet released them.

Maybe up on the top left there, that might be a free spore. But you can see the spores, the

endospores are being formed. And often the position of the spore in the cell will help. Is it


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at the pole, tennis racket like? Is it in the center? Is there a big bulge or other characteristics

of the cells in which the spores are being formed that can help you identify the species, in

this case of Clostridium.

[23] [Pathogenesis C. tetani]So tetanus. Transmission from soil. You always think of this as being transmitted by

stepping on a rusty nail. Well yeah, it could be. You know, because these spores, where are

they found? They are found in the soil all over the world really. And so if they are there, and

if they are present on the rusty nail you step on, or any nail you step on, anything that

perforates your foot, and spores are there, well, that is how they enter your body. Through

a perforation or wound. The main virulence factors, the toxin tetanospasmin. A heat-labile

neurotoxin blocks release of neurotransmitter glycine, causing muscle spasms and

convulsions or lock-jaw. So think of that man there, that is a characteristic, historical

picture of someone with tetanus. Muscle spasms. Convulsions. Lock-jaw, their jaw is

locked. Theres hyperactive muscle contractions. So instead of having contractions and

having it being released at intervals between contractions, they continue all the time, all thetime. Because of the action of this tetanospasmin. Release of the neurotransmitter glycine,

causing muscle spasms and convulsions, lock-jaw, a fixed smile, seizures, as a result of this

toxin produced by these bacteria.

[24] [Clostridium perfringens]

Oh thats all I have on that one. Ok, Clostridiumperfringens. Large rectangular spores, point

being once again that the type of spores produced by these bacteria can vary and can help

in diagnosis.

[25] [Pathogenesis C. botulinum]

I think I have some more on that but I guess these slides are [in a different order] (? 41:00

minutes into podcast). Well lets talk now about Clostridium botulinum. And in contrast to

what we just saw with tetanus, look at the example of someone here who has botulism. The

floppy baby. Instead of being like this, it is paralysis. It is no activity at all. Floppy baby. So

there is something different. The way this toxin works is just the opposite from the toxin

that causes tetanus. Tetanus is spasms, this one paralysis. Floppy. Virulence factors,

exotoxin, heat-labile once again. It blocks the releases of the neurotransmitter

acetylcholine, causing at the neuromuscular junction, it blocks the release of this so as a

result there is no real transmission of nerve impulse to muscle. So muscle function

deteriorates in the body. More on that and how it works in that last line there.

[26] [Botulism is caused by an exotoxin in improperly canned foods]

Botulism is most often found in this country in children, in infants who are being fed baby

food, canned baby food that has not been properly been sterilized. And so you have, and

again if there are a few spores from the environment, or from other people, or from soil

that just happen to find a way into this food that is being canned for in preparation for


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babies to eat. So spores are in there. And that is a fantastic anaerobic environment inside

canned food. And these are anaerobes, so then they find themselves in this milieu, inside of

a can with no oxygen, and it is food for them as well, so they begin to grow. They germinate,

they grow, and they produce these toxins and so when the baby eats this food they get sick.

Adults have a little bit better ability to fight off this toxin produced by the bacteria than do

children. Another common food they find in this country that causes botulism is honey,especially when given to children. So thats something else you have to be worried about I

guess. Honey. This is the most potent toxin that we know about. Anybody in the world

knows about. Any chemical in the world, tetanospasmin, other toxins, exotoxins,

endotoxins, chemicals that you can think of, this is the most potent toxin in the world. In

nature. It is a nerve toxin. One pint, if you could purify one pint of Botulinum toxin could

destroy the whole world. The CDC considers this toxin to be one of the candidates for

bioweapon use, and it is also category A. The CDC, as far as, after 9/11 and the release of

those anthrax items, look at all the potential bioweapons that could be out there, that could

be used by a terrorist. And the most dangerous ones and the most likely to be used and

maybe the easiest to prepare they put in category A. Well talk about those. And there are

others, yeah, like T.B. and Staph aureusin Category B, and some others in Category C. So farweve seen two today that the CDC puts in category A. Anthrax spores, dangerous, easily

made, easily disseminated, inhalational anthrax. Heres another one. Botulinum, not the

bacteria itself, but the toxin. The toxin. The toxin, the terrorists may purify the toxin, and

release it, maybe in the air conditioning system, or you know, not outside in the world, it

becomes too dilute probably, but it could be used to contaminate small areas where a lot of

people may come together for a meeting or lecture, stuff like that, and hurt them. So the

toxin also is a Category A bioweapon.

[27] [ClostridiumDiagnosis]

Diagnosis, if you are infected by the bacterium, or you inhale the toxin, it causes paralysis,which you saw with the floppy baby. Botulinum, floppy baby. And it causes a new type of

paralysis called descending paralysis, meaning that the initial symptoms of this disease

begin in the face and descend. Usually people who have this Botulinum type of disease,

infection, first of all have a minor headache. They have trouble seeing, double vision, weak,

dizzy, blurred vision, dry mouth, and its descending. And then after a couple hours, they

have trouble breathing. Muscle contraction, muscle activity, once again, is being destroyed.

And with the muscles that help them breathe are losing their ability to do that, and so they

begin to have problems breathing and eventually they will suffocate. They will die if it goes

on too long. Abdominal pain, no fever. Thats another odd feature of this particular

infection. The patient experiences this descending paralysis, not thinking right, not seeing,

trouble breathing and swallowing, then paralysis.. And at the same time they are mentallyalert, and they recognize this is happening to them and it makes them very, very afraid.

What can I do? And what you have to do, guess what the best treatment would be, if a

person was in that condition. How could you best treat them? An antibiotic? Too late for

that. Give them antitoxin. Give them antitoxin you have in the lab. The toxin is causing

these symptoms, descending paralysis is the toxin. Give them, inject them with antitoxin,

antibodies formed against the toxin, to neutralize the toxin, eliminating its ability to be

toxic. And then the patient will be okay. And even, that is what is done whenever they can


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identify it early, and the patient will get better, but it still takes quite a while, maybe

months before they are back to normal. But people have survived it.

[28] [Botox]

Botox, well stop with this one. Botox is Botulinum toxin. What does it do? Remember in thenerve, muscle, it relaxes the muscle in a way. Botox is

used actually for a lot of different conditions today, not just for getting rid of wrinkles in the

skin. Cosmetic purposes, facial wrinkles, but there is a whole list of very good therapeutic

uses for Botox. And that reminds me, its about time, I think its about time for my Botox

injection, let me check my calendar here [rummages around in his bag, cant find his phone]

I dont seem to have my calendar.. Oh, heres another way I can tell [whips out a mirror],

Im good for five more years!!!!So, okay, lets take a break and then Ill go on to the next

batch.

6: bacillus & clostridium

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