Download 1,2: Going to talk about anaerobic bacteria

Microbiology
09/17/08
Anaerobic Bacteria
Transcriber: Sara McGowan
48:54
1,2: Here are the relevant chapters in Murray. I strongly suggest you read these chapters, don’t just rely
on the lecture because this is an overview and there is other information in the book.
3: Basic difference between anaerobic bacteria and others that you’ve studied, which are mostly
facultative organisms that can grow with or without oxygen. There’s a whole spectrum and aerobic
bacteria are obligate. They must have oxygen to grow, e.g. Pseudomonas. The Microaerophilic require
oxygen in reduced quantities, e.g. Campylobacter that cause intestinal disease. They will not grow in
atmospheric oxygen. Capnophilic means they require CO2 in the atmosphere, e.g. Neisseria gonorrhea,
Hemphylius influenza; more fastidious organisms like Pneumococcus. The vast majority of bacteria in
human infections are the facultative. We are going to talk today about the spectrum in anaerobic
bacteria, the obligate anaerobes and aerotolerant. Keep in mind that there are varying conditions
required, not just in temperature but also in atmospheric conditions.
4. They live in certain places and they live there because of certain conditions. You have to have
reduced or no oxygen. The pH and oxidation-reduction potential are also important. The basis of either
being able to grow in oxygen or not are these enzymes: cytochrome systems, superoxide dismutase and
catalase are the fundamental mechanisms by which we, as well as bacteria, can detoxify the high-energy
radicals that are produced from oxygen metabolism. If they lack those, they kill the cell. Facultative
anaerobes have some of these, but not sufficient quantities to be totally able to grow in atmospheric
oxygen.
5. This is a schematic showing the same thing. If you have anaerobic bacteria, it lacks this detoxifying
pathway. You have high-energy radicals including hydrogen peroxide that are toxic to the cell. Down
here, it’s all turned into water or atmospheric oxygen. Facultative organisms or organisms that grow in
the presence of oxygen have those enzymes. They have those cytochrome systems; that’s how they
survive. Anaerobic bacteria do not have these, but there is a spectrum. Aerotolerant organisms have
some enzymes, but not the quantity that’s required.
6. Where do they live in people? We have a lot of anaerobes that we carry around everyday. The
majority of the microbial flora in your colon are anaerobes (over 90%). We usually only think about
pathogens or problems with that flora like with Shigella or Campylobacter. The facultative bacteria
cause intestinal disease or often extra-intestinal disease, but they are a small portion of the population.
Anaerobes live in a variety of places, based upon this condition (oxidation-reduction potential and
anatomic site), e.g. periodontal pockets, dental plaque, colon. The extreme locations are H electrode
and O electrode. They are not floating around in the body; they live in places like the colon, like the oral
cavity, but not on the surface like Streptococcus mutans. They live in pockets so they are protected
from oxygen and have these other conditions.
7. This is a way of putting them in different categories: gram-negative rods, gram-positive rods, gramnegative cocci and gram-positive cocci. There’s a commonality to a lot of them. The colon is mentioned
in all categories, so there’s a very mixed flora in the colon. There are others that are focused in the oral
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pg. 2
cavity. Some are specific to the vagina or skin. Propionibacteria is an aerotolerant anaerobe whereas
some of these others are strict anaerobes. The only ones found in soil are the Clostridium. Bacillus are
the spore forming aerobic rods and Clostridium are the spore forming anaerobic rods. The spore allows
them to exist in condition s that they would otherwise not grow in.
8. Where can there be problems with anaerobic bacteria? First, remember where they live. Areas
adjacent to those places are often subject to infection. If there’s reason for dissemination e.g. infection
from a human bite can be nasty, involving anaerobes. If someone has aspiration pneumonia and gets an
abscess in the lung, could get in the bloodstream and to the brain. There’s a lot going on in the mouth.
If you have surgical or other disruptions like appendicitis that ruptures, your peritoneal cavity can have
abscess formation. If they get loose from the spot where they normally live as part of our normal flora,
that’s when they can cause problems.
9. Some of the oral diseases like Vincent’s Angina is a good example s synergy. This is not caused by a
single bacterium. It’s hard to see in the picture, but there are three morphological types. Scrape the
mouth and do a gram stain; they are all gram negative. Treponema are very thin and corkscrew-like.
Fusobacterium are long and fusiform. The short fat rods are probably Prevotella/Bacteroides. There is
overgrowth of these organisms that are normally there, but because of poor oral hygiene have
multiplied and cause damage.
10. Not quite as acute is Adult Periodontitis. The same process is going on, but it’s not as acute. Usually
not caused by that same group of organisms. What you see is progressive receding of the gums because
of all of the toxins being produced by the bacteria that are hiding in crypts. No matter how good the
teeth are, eventually the gums are gone and they can’t hold their roots into the bone.
11. Much more serious are things like brain abscess. When you eat, if you have poor dentition and your
teeth are loose or you get your teeth cleaned, with all of that activity you get showering of oral bacteria.
That puts you at risk if, for example, you have a defective heart valve, you could get endocarditis. If it’s
chronic, it may get to the brain and cause an abscess. In aspiration peneumonia, the classic example is
an alcoholic who passes out and they regurgitate stomach acid and contents and they aspirate. They get
oral flora and foreign body material from the stomach and acid. All of that destroys tissue and once you
get destroyed tissue, anaerobes are happy because they have a place to hide from oxygen. They will
cause abscesses and bacteremias as well.
Back to slide 10. This is a gram stain of an aspiration of an anaerobic abscess. These are neutrophils
around the white cells. There are gram-positive cocci and rods and there are gram-negative rods. Like
in Vincent’s Angina, this is characteristic of anaerobic infections: abscess formation and mixed flora.
Often if you have a Staphylococcus infection of the arm it’s one organism, S. aureus. This is different,
most of theses abscesses have multiple organisms in it and can include facultative organisms they don’t
have to be strict anaerobes.
12. In people with poor peripheral circulation, like diabetics, just like other bacteria it can get into sores
that do not heal well and then cause tissue damage. This is polymicrobic cellulitis. It’s not the most
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pg. 3
common cause of this sort of thing, but it can be involved. It depends on a number of things like
personal hygiene.
13. We group them by gram stain and morphology and how closely they are related. This group is three
genera: Bacteroides, Prevotella and Porphyromonas. They all fit these criteria: no spores, pleomorphic,
normal flora of respiratory and GI tract as well as the female genital tract. They normally don’t cause
problems. The important groups are the Bacteroides fragilis and the Prevotella melaninogenica, which
are more commonly involved in infections. Above the diaphragm you think of P. melaninogenica and
below the diaphragm you think B. fragilis, so it ‘s colon associated or oral associated. There are some
other niches, like the female genital tract has particular species of Prevotella.
Again, abscess formation, mixed flora sometimes also mixed with facultative organisms are
characteristic of anaerobic infections.
14. This is not a mixed flora; this is necrotizing fasciitis caused by Bacteroides fragilis. This is the same
sort of flesh-eating bacteria like Streptococci. It’s the same idea: fasciitis is fasciitis. It’s not as toxic as
the streptococci, but it can cause this. This is an example of introducing bacteria that are normally part
of our flora into a place they don’t belong and you get infection and disease.
15. This is the bad outcome of that. You can’t leave dead tissue; you get devitalized tissue in this sort of
condition. You have to remove it, so this guy will have additional surgery to get rid of black tissue, which
is dead.
16. Fusobacterium is the long fusiform rods. Fusiform means pointed. They are pointed at the ends.
They are pleomorphic, virtually all of these are pleomorphic. This is distributed in the upper respiratory
and GI tracts. Pleomorphic means the same organism can have multiple shapes.
17. Already talked about pulmonary abscess. This is lower lung abscess, probably a result of aspiration
like the circumstances already described.
18. Fusobacteria are pointed. There’s nothing unique about them when they grow on plates, except
that they would not have grown unless you collected them properly and they did anaerobic cultures in
the lab.
19. Of the gram-positive bacteria, there are some of the more classic diseases. Actinomyces are a group
of higher order bacteria that are strict anaerobes; they cannot be exposed to oxygen at all. If they are
exposed to oxygen, more than likely they will not live. This is the most common species, A. israelii. They
are very slow growing. Most of the bacteria we deal with grow a colony in 18-24 hours on a plate. It
may take 3-4 days for Actinomyces to multiply enough to make colonies. What that means is that if you
ever suspect this is a part of an infection, you have to let the lab know to hold these longer and not to
look at them early in their incubation. Taking them out might kill them if they haven’t grown enough.
They create draining sinuses with sulphur granules. If you were to aspirate this organism, it makes an
abscess in your lung and it can come out and causes sinus in the chest walls. Out of that drains material
and pus, including the organisms that are in small colonies like little sand grains. They are the organism
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microcolonies covered in host material. The original ones seen were yellow, so they were called sulphur
granules. They can be yellow, white, or different colors, but that’s a characteristic. This is mostly
associated with oral and respiratory. The copper IUD’s can get colonized and have to be removed, but
the major problems are in the oral and respiratory tract.
20. Dacryocystitis is not a serious condition. This is A. israelii and the sulphur granules are blocking the
tear ducts and they can’t function. Prior to the (availability of) haemophilus influenza B vaccine a child
may look like this, so it would require a differential diagnosis between dacryocyctitis and haemophilus
infection. It’s important when you’re dealing with people to know their immunization history. If the
child had not been immunized you would want to look into that. The treatment is to get those things
(bacteria?) out of the tear duct.
21. The classic condition of Actinomycosis: Lumpy Jaw. This guy is asymmetric; normal face on one side
and greatly swollen on the left side.
22. Not the same patient, but the same process. Looking down at the root of the crown and looking at
the x-ray, you see the roots and the bone are being eaten away. An abscess forms down there and is
quite painful. People will come in to have it looked at and if you leave it alone they make these draining
sinuses. This will come out the side of the jaw. If it’s opened up and put some gauze on it, the gauze
soaks up the fluid and you can capture the sulphur granules and diagnose without having to go into the
abscess inside, but not everyone waits this long. This is the classic description of Actinomyces israelli
infection.
23. These are the sulphur granules, about the size of a grain of sand. The picture is black and white, so
they may or may not be colorless. They are important to look at and crush into a gram stain. In the
gram stain they are filamentous branching rods. There are fungal infections that also cause draining
sinuses, generally not in the oral cavity, but elsewhere and they make sulphur granule-type material.
The treatment for a fungal versus bacterial infection is quite different. It’s important that the lab looks
to be sure they have a bacteria or fungus.
24. The closer view shows that in culture they look long and branched, kind of unusual for bacteria, but
the size is 1-2 microns in diameter. That is bacterial size. Fungi are roughly 10 times larger, so size can
be important when you look at things with similar morphology. You may see in the future that
staphylococcus is a certain size and yeast is about 10-15 times bigger, but they both are round, so you
can be fooled. When it’s grown (this is an old culture) it makes what’s called a “molar tooth colony.”
The colony looks like a molar.
25. There are less important, nuisance anaerobes and they fall in the gram-positive bacilli category.
They can be part of the mixed flora but the others we’ve discussed, the gram-negatives, are more
important in the pathogenesis. This can be a nemesis in the lab because they are normal skin flora. It’s
aerotolerant and it grows down in the hair follicles. It’s a problem because we get blood cultures in the
lab and if someone doesn’t decontaminate the area properly, there are two things they can put in there.
There’s staphylococcus epidermis, which most of us have on our skin and propionobacteium; it causes
confusion. Staph can cause true infection, for example, of artificial heart valves or artificial joints. So
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pg. 5
you don’t know did it come from the skin or from somewhere else? Propionobacteria are easier, except
they show up about day 4-5 of the incubation. Clinician is told they have a gram-positive rod in the
blood culture and they have to do something about it until they get a final answer because there are
other gram-positive rods that can cause a problem. We try to stress to the medicine people and nurses
… it depends on who collects the blood. Phlebotomists specialize in it, so you get lower contamination.
Nurses are OK and less often the clinician does it, but whoever does can save the lab trouble by doing it
right.
Lactobacilli are good because they help protect the vagina from bacterial vaginosis; they keep it acidic.
They are there in large numbers, that’s healthy. The acidic pH protects from other organisms. There are
some other gram-positives, so there’s a variety. Actinomyces are the most important.
26. Classic diseases you should know about. These are mostly toxin-mediated diseases, with one
exception and the only ones that form spores. You’ve all been immunized against the tetanus toxin. This
is the result of in vivo toxin production, so this is an infection. You step on a nail, get a splinter, some
kind of gross contamination of a wound and the organisms spores in the environment are now in your
tissue and they grow and produce this toxin called tetanospasm. Tetanospasm blocks the inhibitory
neurotransmitters, the gamma-aminobutyric acid and glycine. That causes the inability to relax the
muscle, so you get constant firing of muscles. This is lockjaw. It’s called “sardonic smile,” because the
muscles are clamped and you can’t undo them.
The other end of the spectrum is botulism, aka food poisoning. It’s mostly pre-formed toxin that we
ingest. This organism lives in the soil and it can come from poor handling of foods, improper canning.
Bacterial spores are harder to kill than cells, so there has to be higher temperature and pressure and it
takes a period of time to kill them all off. If canning isn’t done properly and all the spores aren’t killed
then the organism grows in the anaerobic environment. This toxin is extremely toxic; it does not take
much of it. This toxin blocks Ach and you don’t get firing of the muscles and you get flaccid paralysis.
It’s the opposite of tetanus. This is a problem as it progresses because the diaphragm is a muscle and it
quits firing. That’s why most of the treatment is support and can include ventilators to maintain the
patient until they recover.
An exception to it being pre-formed is infant botulism. Infants don’t have a GI flora established yet,
they’re not born with one, and so over time they pick up bacteria that colonize the GI tract and that
protects against introduction of different organisms. If during that period of time, they’re exposed to
Clostridium botulinum and the classic way this happens is by giving them honey. Honey is
environmental and it can have spores from the organism and you get production in the GI tract. This
still occurs occasionally and it’s the opposite of the usual ingestion of pre-formed toxin from food.
Although toxins are involved, gas gangrene is a very active infection. In the Civil War, people got
wounded in the trenches and they lost their arms or legs due to this infection. There are a number of
toxins, but the most important one in gas gangrene is Phospholipase C, also called alpha-toxin or lethal
toxin. It takes your tissue and makes hydrogen gas and CO2 out of it. That’s why it has gas in the tissue.
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Gangrene means dead tissue and gas is in the dead tissue, so you can poke it and it feels like packing
material. It has about 12 possible toxins that do lots of things. Basically, it chews up your tissue.
It also has milder conditions. Enterotoxin can be ingested. This is mostly in gravy that’s sat around at
the wrong temperature. You’d rather have this than gas gangrene. Septicum can be in blood cultures
and may indicate a malignancy, like GI malignancy. Most commonly seen is the antibiotic-associated
diarrhea in hospitalized patients and sometimes outpatients. It’s a result of broad-spectrum antibiotics
killing off a lot of the flora of the GI tract. Many of the anaerobes are knocked out. C. difficile is resistant
to many antibiotics, so it overgrows. When you get overgrowth of something, even if it’s normally
there, you can have problems. You can get pseudomembranous colitis, which is a more serious form.
It’s either diarrhea or colitis.
27. This is tetanus. This is the sardonic smile. He can’t open his mouth if he tried. The infant infection
usually comes from contamination of the umbilical cord. It wouldn’t happen in the hospital; it would
happen if the child were born outside. He’s being supported by the foot and the head because the
muscles have clenched so tightly. The wound doesn’t have to look like this; it can be a festered splinter
on your finger. It used to be that you’d get a tetanus booster shot, but the immunogen, the tetanus
toxoid, started giving people really high antibody titers and they started reacting to the immunizations.
They got really big reactions at the site. Now you get a booster every ten years unless you have a
traumatic incident, like a car accident where you get thrown into the dirt and you have lots of cuts.
28. The organism grows like a film; no distinct colonies. These are lollipop-like looking things. The
lollipop head is the spore. You can tell where the spore is on Clostridium with a gram stain. If it’s at the
end terminal and it’s bigger than the rest of the cell, it tells us one thing. Sometimes it’s internal and not
any wider than the cell and that belongs to another group.
29. Gas gangrene. This is the gas. You can see the blebs. You have to surgically remove the dead
tissue. That’s usually the outcome of a surgical procedure in the abdomen. Sometimes they nick your
colon in an emergent procedure. Sometimes it can’t be explained.
30. Clostridium perfringens looks like gram-positive large boxcars. If you do a special stain, the empty
spots are the spores. Unlike C. tetani that had it on the end, it’s hard to visualize the spores in c.
perfringens. The organism is growing anaerobically, has this morphology and does a few other things…
31. …Like this. (He doesn’t have a good picture of double zone hemolysis, so take his word for this)
This is beta-hemolysis. The toxins cause lysis of the red cells in the media. It destroys the membranes,
so it clears, it doesn’t look red. It has other toxins that partially destroy the red cells, so that’s what they
mean by a double zone. You can’t see it here. This is a manifestation of the multiple toxins that it can
produce.
You can also test it for phospholipase, the alpha-toxin, to show that it’s a toxigenic organism. You have
media that has lecithin in it and if you enzyme, one’s a positive and one’s the unknown and on one side
of the plate is antibody to the toxin and the other side not. What you see is the destruction of the
lecithin, turning it opaque, in the absence of antibody and (not turning opaque, i.e. no enzyme activity)
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in the presence of antibody shows the specificity of the reaction, so you can prove that it’s a toxigenic C.
perfringens. It’s called the Nagler Test.
32. C. difficile colitis still occurs. It was the original discovery of this whole issue because it’s a severe
expression. Antibiotic associated diarrhea that would lead to it was discovered subsequently. This is a
colon and it’s bad.
33. The normal flora being put somewhere they don’t belong and having toxins are the major things
these organisms have. They have synergy and mix with enteric bacteria that are facultative. They help
anaerobes because they use oxygen preferentially. They get 32 ATP aerobically and 2 ATP anaerobically,
so they use up the oxygen and will shift to the fermentative pathway and the anaerobes can now grow.
They can produce enzymes that breakdown antibiotics. Beta-lactamase works on, for example,
penicillin, so penicillin would not be effective against several Bacteroides. Whenever you see a capsule
it means it can evade phagocytisis, so it’s a virulence factor. This is true for many bacteria. B. fragilis is
anti-phagocytic. Toxins are important in a lot of anaerobes where they are the primary reason for the
pathogenesis of the disease.
34. What do you need to do and what does the lab need to do? Clinical signs that are hallmarks are foul
smelling discharge, proximity to the mucosal surface cause that’s where they live, gas in tissue and
abscess formation. Why does it stink? Anaerobic metabolism results in short chain fatty acid
production. 2,3,4 chain maybe up to 5 or 6 ad they stink. Gram stain can be helpful because it’s mixed.
It’s unusual for there to be a single morphological type in an anaerobic abscess.
35. This is a bigger view. These are all neutrophils, gram-positive roods and cocci, gram-negative rods
and cocci. This means you have a mixed infection. It doesn’t have to be all anaerobes; there can be
some facultative organisms. The likelihood of the lab getting every single organism and letting you
know all of the susceptibilities and how to treat it is low, so you have to think broadly when you see this
kind of infection.
36. Cultures are entirely dependent on sample collection and transport. If they are exposed to oxygen,
they won’t grow. Report: no anaerobes isolated. There are special collection methods, like aspirating a
closed abscess and putting the material in a transport vial that is filled with nitrogen or has been
evacuated. That’s fine for multiple cultures. Swab use is discouraged. Some require special conditions
like vitamin K. That’s important because you have to ask for anaerobes to be cultured. At UAB, you
have to indicate that you think that’s part of the culture. We can take advantage of the resistance to
aminoglycosides, e.g. gentamicin, by anaerobes. In high doses they work well against E. coli, many of
the enterobacteria, those facultative organisms that will overgrow anaerobes. We can put that in the
media and suppress those and look for anaerobes. We incubate them and work them up in oxygen
reduced or absent environment.
37. Some labs use these containers. The hydrogen generators have a metal catalyst that, in the
presence of hydrogen and oxygen, turns into water. This is for campylobacter. This can be put in a
regular incubator.
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38. Another way, which was used in the past, is a large anaerobic chamber where they set the plates up
and incubate them and work on them. That’s ideal, but a better system is to connect the portable
chambers to gas lines. With the chamber, it may take 5-10 minutes to go anaerobic and the portable
chambers takes 5-10 seconds to flush with nitrogen and add the right mix of gases and it tells you how
much oxygen is left. The catalyst gets rid of residual oxygen. That’s more effective for transport after
they’ve been worked on. It’s simpler and less expensive.
39. We can identify a few of these organisms by a few spot tests. B. fragilis is an example of
pleomorphic. They are longer, shorter, etc, that’s pleomorphic. The ability to grow in the presence of
aminoglycosides and to, in this case, turn this media black, which is an indicator of a certain enzyme.
That, with all the other info, would indicate that it’s B. fragilis.
40. Closed abscesses cannot be cured by antibiotics; you have to drain them. There are mixed
infections, so you have to have broad coverage. These are some of the more common anaerobic drugs.
This changes over time. You’ve got to watch out for penicillin resistance. We talked about
aminoglycosides. Sometimes there are antitoxins available. He’s not sure if there’s antitoxin for
botulism or tetanus, doesn’t think so.
41. Susceptibility tests are not routinely done, that is, test anaerobic bacteria for what antibiotic would
be most likely to work because they are predictable. If it were done it could look like this Etest. It’s a
diffusion of antibiotic over time and growth of the organisms. You can see an endpoint where it’s
isolated and see that, for example, with benzylpenicillin it only takes 0.32 micrograms per mL to kill the
organism on the plate. They gather anaerobes overtime to do an anobiogram, showing a profile of B.
fragilis in our institution or fusobacterium. It can be a good guide to use.
42. Know the gaseous requirements; who belongs where on the list. Know the different things or
special things you have to do for anaerobes if you’re collecting a specimen and getting it to the lab.
Epidemiology means where do they live, how do they get to causing infection, is it environmental, is it
endogenous, etc. Know the classic clinical syndromes associated with anaerobes: the clostridial
infection, actinomyces infection and others like C. difficile.