Download Slide 1: Title slide

Microbiology
09/22/2008
Gram Negative Bacilli
Transcriber: Laura Rayne
1:15:04
Slide 1: Title slide
Slide 2
To bring a little bit of relevancy to this – a couple of years ago there was an E. coli outbreak in
our spinach. This was the E. coli 0157:H7 and it really kills people. There were 103 hospitalizations, 31
hemolytic uremic syndromes (and often these are kids) and they have kidney problems and often they
will need transplants later in life if they survive this. It was all over the US. Since 1995 there have been
19 outbreaks in leafy and green vegetables. In this outbreak the spinach came from California. The
cattle a mile away from the spinach fields had this E. coli in them and there were feral pigs that would go
and root around in the manure and then root around in the spinach and this is how the E. coli got to the
spinach. It turned out that this was more widespread than it should have been because the spinach
didn’t look good when it got to the packing house so they mixed it with good looking spinach. So it was
more widespread and took longer to find because of the way it was handled in the system.
Slide 3
September 14 through October2 there was Salmonella in restaurant tomatoes. This was in 21
states with 183 cases of illness and 22 hospitalizations. This was one of the more common Salmonella
strains, Salmonella enteric typhimurium. As you can see these are things we all eat: greens, spinach,
tomatoes. This year there was also an outbreak with tomatoes that I haven’t added to this. Tomatoes
were implicated but really it was probably peppers. They found farms in Mexico and a packing house in
Texas that had this right Salmonella on peppers, but it was really hard to trace.
Slide 4
This is another one that kind of hits home. In England they had an outbreak of Salmonella. It
was in the Cadbury chocolate around Easter time. The Salmonella strain was Montevideo, which isn’t
that important except that it is one that you can see easily because it is not that common. They had 65
cases of Salmonella Montevideo, which is way more than they would normally expect. Whoever was
overseeing these tests saw that the Salmonella in their ingredients was very low so they just ignored it.
It usually takes a lot of Salmonella to cause disease, 105 or so. But chocolate is one of the things that
allows Salmonella to get through the stomach because it is a real oily type thing. It goes through the
stomach, doesn’t see the acid, and the organism survives. So very low numbers of Salmonella in
chocolate or cheese or things like that put you at risk. In this case they went through and found it and
so they have fingerprinted it and can tell it’s the same one. This is important when you’re thinking
about what you’re eating. There have been several outbreaks with chocolate. If you get Salmonella you
get diarrhea and vomiting, you lose about 10 pounds and a week of your life, so it kind of counteracts
the chocolate.
Slide 5
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 2
We’re going to discuss the common characteristics of Enterobacteriaceae. It is important
because if someone has an infection you are able to group them if they are Enterobacteriaceae vs. other
organisms. So these are all gram negative rods with nice big round ends and flat parallel sides. These all
pretty much look the same in the lab so when you see a gram stain that’s an Enterobacteriaceae you
really can’t tell much from it. Colony morphology on sheep blood agar usually doesn’t help much either.
They are shiny, dull gray colonies usually, fairly large (couple of mm). Some of them are hemolytic,
which isn’t as important in gram negatives as it is in gram positives for identification but it is something
to note. Some are mucoid and really slimy. Sometimes in the incubator some of the colonies will drop
from the agar side of the plate to the lid side of the plate and you’ll get this slime of organisms. Some of
them swarm. This is the case if you had one colony on the plate, it will swim all over the whole plate.
That makes it very difficult to isolate anything else. Some of them are pigmented but most of them
aren’t.
Slide 6
This is a sputum gram stain. You can see the gram negative rods here. Some of them are
longer, some are shorter. This is exactly what Enterobacteriaceae would look like on a gram stain of
sputum from Enterobacteriaceae pneumonia; except this isn’t Enterobacteriaceae it is Pseudomonas,
which is much more resistant than Enterobacteriaceae for the most part. This is to bring home the point
that looking at a gram stain doesn’t tell you which Enterobacteriaceae. It doesn’t even tell you that it is
Enterobacteriaceae; it is just a gram negative rod.
Slide 7
This is a picture I took of an E.coli plate. You can see the colonies; they are pretty nondescript.
This is a real fresh plate. You can see it is a motile organism kind of spreading out. This isn’t swarming
but it is spreading a little bit. It also has a nice characteristic odor. These are nice colonies of an
Enterobacteriaceae – this is Escherichia coli (E. coli) but it can be a lot of other things.
Slide 8
We’ll discuss the cell walls. What makes them gram positive? Gram positive bacteria have a
real thick peptidoglycan layer. The crystal violet and iodine form a crystal inside the cell and it can’t get
out through this large peptidoglycan layer, so it is not decolorized. The gram negative cell wall is
different because it has a small peptidoglycan layer. So when you put the acetone/ethanol decolorizer
on it, it removes the outer layer LPS (the endotoxin, the toxic part of gram negative) and then the crystal
violet/iodine crystals come out and you don’t have any color until you re-stain with safranin. So you get
a red color for gram negative, while the gram positives stay purple.
Slide 9
Skipped
Slide 10
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 3
So what do we have with LPS? This is a lipopolysaccharide, also called endotoxin, that is part of
the cell. It is not released until the bacterium dies. So as long as the bacteria are happy we don’t see
the LPS in a way that stimulates us to recognize it. When we see LPS it is an alarm – we recognize that
we are infected. We attack it with anything possible. We often overreact and cause more damage with
the immune response that the organism would have caused otherwise. If we didn’t have antibiotics and
treatment, this reaction would probably save us but it kills more people that the bacteria would in our
system.
When our cells see LPS, granulocytes increase their oxygen potential to make oxygen radicals to
kill organisms, and make adhesion molecules so they bind to cells. They become more active and seek
out what the source of this is. Macrophages produce TNF, interleukins produce oxygen radicals,
vascular cells or endothelial cells release cytokines IL-1 and IL-6 and adhesion molecules that also bind
granulocytes, and B cells and T cells then proliferate, preparing to make a directed immune response.
All this happens quickly and we get fever, hypotension, tachycardia, tachypnea, neutropenia because all
the granulocytes are bound to endothelial cells looking for a place to attack something and you wind up
with sepsis, multi-organ failure and often death.
Slide 11
One thing that was found a while back was mice that were endotoxin resistant. They lack a TLR4
so they don’t recognize LPS. You can inject a milligram of LPS in them and they’re fine, whereas 50 mg is
essentially a lethal dose in a normal mouse. We expected these to be resistant to Salmonella but they
were actually more susceptible. You inject LPS into these mice and they don’t realize they are infected
as quickly as mice that have the TLR for LPS and they die of Salmonella. If you inject LPS by itself then
mice that are normal overreact to this molecule that isn’t toxic at all by itself (because if you don’t have
the receptor and go through all the cytokine cascades they really aren’t sick). So it’s really our response
to the LPS that is dangerous but in a real infection it is a useful response. When we start treating with
antibiotics it gets worse because we are killing the organisms, releasing LPS. We have tried many
different ways to blunt that response but so far none of them have been very successful and septic
shock is still a real important cause of death from gram negative infections.
Slide 12
So now we’re going to talk a little bit about the antigenic structure of Enterobacteriaceae. How
this was found was you inject killed organisms into a rabbit, take that rabbit’s blood, mix them with
bacterial cells that you injected and then see if they will agglutinate (so see if they will make antibodies
that will cause the cells to clump). It turns out that we can identify bacteria that way, so we have
serotypes of bacteria. This has been very useful in that if we have an outbreak of Salmonella (there are
2400 different serotypes of Salmonella) we can go back and trace it by recognizing if it is one of the
more rare Salmonella.
The one antigen we use is the LPS antigen. It has a long polysaccharide chain on it that is really
different with different strains. This is the major typing antigen. There are about 28 or 30 of these
different O antigen polysaccharide chains in Salmonella. We give an O number. This is E. coli O157 –
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 4
this is the 157th O antigen that was discovered in E. coli. To break it down further, the flagella are the
motility organ in these bacteria. They are several times the length of the cell and they have a big motor
at the base. These are very variable and allow bacteria to escape the immune response. The flagella
antigen is the H antigen. By combining the flagella antigen and the O antigens then you can separate
the different species of bacteria into a lot of different strains. E.coli O157:H7 – H7 is the flagella antigen.
If you have a non-motile one, it will be O157 but it won’t have an H antigen and that makes it a little
more difficult to trace. There are a lot of other antigens but these are the ones we use to type it. There
is a capsular antigen that we use for Salmonella typhi and it is a K or a Vi antigen. The Vi antigen is the
virulence antigen. Klebsiella pneumoniae also uses a K antigen.
Slide 13
E.coli O157:H7. Another E.coli is O75:K100:H5. K100 is the capsule, H5 is the flagellar antigen.
Typhi so typhoid fever is the O9,12 and the Vi (virulence antigen) and then Hd antigen. These are real
complex but just understanding where these letters and numbers come from is the important part.
Slide14
So the physiology or what makes it Enterobacteriaceae. They are all facultative anaerobes so
they can ferment glucose. They can grow anaerobically by fermenting glucose or other sugars but they
can also grow aerobically. They would rather grow aerobically but if the oxygen runs out then they will
go ahead and grow anaerobically fairly effectively.
They are oxidase negative. This is the test that we use in the lab commonly to split up
organisms. A lot of the non-Enterobacteriaceae gram negative rods are oxidase positive and
Enterobacteriaceae are all oxidase negative. They are nitrate positive so they will reduce nitrate to
nitrite. This is used in urine cultures especially. If you have a urine dipstick, you can predict whether
you have a gram negative rod causing the urinary tract infection (UTI) if you have nitrates in the urine,
so that is something that is done often and really detects Enterobacteriaceae well. It doesn’t detect
many of the gram positives so it is a good test to detect a day earlier than the culture. They are all
catalase positive. This isn’t used much on gram negatives because a lot of the gram negatives are
catalase positive. If you put hydrogen peroxide on you get bubbling with oxygen release. If they are
mobile, they are mobile by peritrichous flagella, meaning they have flagella all the way around the cell
and not just a single flagellum on one end. (Pseudomonas just has a flagellum on one end.)
Slide 15
The oxidation fermentation test. This is something you will see in the lab and is a nice, easy
thing to ask questions about. These are agar tubes with soft agar that have glucose in them. We are
seeing if the organism can utilize glucose with and without oxygen. We put this paraffin plug in here
that inhibits oxygen from getting into the media and so in this case you can see that glucose is
fermented both with and without oxygen. We have produced acid and the phenol red indicator turns
yellow and we just stabbed it with the bacteria. We produced gas also in this case. There is a good
chance that this is E.coli, which produces a lot of gas from glucose, blows the paraffin plug out, and
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 5
produces acid both aerobically and anaerobically. An aerobic organism can ferment glucose with oxygen
but can’t without. An example would be Pseudomonas, which is an oxidative organism but not
fermentative. Then we have some that are asaccharolytic and don’t really use glucose for anything and
they don’t produce acid. These are less well known but can cause some pretty severe disease. Some of
them are hospital (nosocomial transmitted), resistant bugs.
Slide 16
How do we identify organisms? This is a microscan walkaway. We can put 100 plates in here a
day so it will identify 100 bacterial cultures per day. It incubates them and then reads them and spits
out a computer readout the next day and tells us what it is.
Slide 17
This is an example of what the plate looks like. It is a 96 well plate. Up here are the
biochemicals. Glucose – you can look to see if glucose is positive or not. Also other chemicals – lysine,
urease. It is the same tests we would run to identify organisms. It is just automated. The machine looks
for color changes and is able to tell if it is positive or not. The bottom half of the plate is antibiotic
susceptibility. There is gentamicin, tobramycin, ampicillin etc. with different levels of antibiotic in each
well. This is how essentially all of the gram negatives and many of the gram positives are read.
Slide 18
I’ve just mentioned most of the way we identify bacteria is biochemical reactions. You set it up on the
plate based on the gram stain – so we still do a gram stain. From the fermentation we get
decarboxylation of amino acids and deaminization of amino acids so we get different pH changes.
Release of ammonia will increase the pH instead of decreasing it so we have detectors for that.
Another way of doing it that really hasn’t been done clinically is genetic relatedness. If you
sequence the 16S ribosomal RNA genes you can identify a lot of organisms. Sometimes we send off
organisms that we can’t identify in the lab for sequencing identification. DNA hybridization is another
method, but sequencing is easier and faster. It is not something we do very often.
Slide 19
Enterobacteriaceae host interactions. These are really our normal flora. E. coli is in everyone’s
gut and we get along with it well, but there are a few bad players in E. coli and other things that cause
trouble and compromise our immune system, and we wind up with trouble from our normal flora. They
are normally beneficial nutritionally. We get vitamins, such as vitamin K, from our normal flora. Some
people that eat a real high carbohydrate diet can get protein and fixed nitrogen from Klebsiella from
some of the Enterobacteriaceae.
They also are important for competition. If someone takes antibiotics and kills a lot of their
normal flora, and then they eat something that has real low numbers of Salmonella in it which wouldn’t
have caused disease before the antibiotics now can cause disease. Also, this is used in chickens.
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 6
Salmonella is normal flora in chickens. If you can infect them with flora without Salmonella early then
they won’t be infected with Salmonella the rest of their lives. It is harmless to them but it causes
trouble to our system. 50-75% of chicken on the shelf has Salmonella and/or Campylobacter, which
cause GI problems in us. They take the cecum from an uninfected chicken, mash it up and feed it to
newly hatched chicks and that will prevent them from being colonized the rest of their life. That is the
same with us. If we get the right flora, we can be protected from a lot of things.
Immunological stimulation – people or animals that are grown germ-free don’t develop a good
immune system. The normal flora stimulate the immune system.
Enterobacteriaceae also cause opportunistic infections. These aren’t that common in the whole
population but are very common in people who are sick.
You can have infections in otherwise healthy individuals. These are with things like Salmonella
and Shigella that really cause GI infections any time they are in a person. Rarely do people carry
Salmonella and Shigella. They can also cause invasive systemic diseases. Something we don’t normally
think about in this country is typhoid fever, but it has killed a lot of people in the past and in the rest of
the world. It causes an invasive disease where the bugs are in the blood, liver, and spleen and causes a
fairly high rate of death – 10-15% of untreated people die.
Slide 20
The most common Enterobacteriaceae is E.coli. It is normal flora of mammals and birds. It is
used for the coliform count in water. If you checking a well or a water source for how much fecal matter
is in that water you look at the E. coli count. We are really more worried about human E. coli but we
really don’t have a way to test that so we look for all fecal contamination of water. It doesn’t survive
very long in the water so it is a good measure of recent fecal contamination. All drinking water is tested.
It is the most common Enterobacteriaceae in the gut. It is lactose positive. This is something we
use in the lab. We use a MacConkey plate, which has lactose in it, and if the organism will ferment
lactose we get this nice pink color.
Slide 21
Here you can see E.coli that is lactose positive fermented the lactose and around the colonies
has formed a bright pink color showing it has decreased the pH. This is the E. coli that has the mutation
in the lac operon. In the wild this is very rare – only about 1% of E. coli are lactose negative. That makes
this a very good method of identifying E. coli. In the lab we consider E. coli a normal inhabitant of the
host. When we have a stool we put it on this MacConkey media and then don’t even pay any attention
to the lactose positive ones. Both Salmonella and Shigella are lactose negative so we pick those and
look for Salmonella and Shigella. “It seems I always make a question about lactose”
Slide 22
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 7
E.coli is real common for causing urinary tract infections. A lot of the Enterobacteriaceae cause
UTIs. Since E. coli is the most common one in the GI tract, which is where most of these come from, it is
the most common cause of UTIs. One thing that predisposes people to having UTIs is the female
anatomy – a very short urethra makes it very easy for the organisms to get through the urethra into the
bladder and there they multiply and cause cystitis. Intercourse exacerbates this process and you wind
up with honeymoon cystitis. Pregnancy and childbirth are also hard on the bladder and cause increased
UTIs. Males catch it at the other end when they get older. They get prostatic hypertrophy and can’t
empty the bladder well. That causes UTIs. Anytime you catheterize or do any other mechanical
manipulations of the urinary tract, you can introduce organisms and facilitate growth in the bladder,
causing cystitis. Any reason that causes a failure to empty the bladder will predispose to UTIs.
Slide 23
Other opportunistic infections caused by E.coli. Peritonitis from a ruptured gut. Gunshot
wounds to the abdomen often cause peritonitis and you often isolate E. coli as well as all the other gram
negatives and strict anaerobes, which are more common in the gut than Enterobacteriaceae.
Septicemia secondary to UTI and pneumonia. The organisms get in the blood and grow and cause
septicemia which is often lethal. Wounds often get infected with fecal organisms, especially in the
lower half of the body.
Slide 24
E.coli diarrhea. This is something we really don’t diagnose much in this country or essentially
anywhere in the world, except for E. coli O157. An estimated 4% of diarrhea in the US is caused by nonO157 E. coli but we really don’t have a good way to look for it in the lab so we just kind of ignore those.
Most people get over them and become immune as kids and then do better later in life, so it is fairly
uncommon. When we go to other countries we run into this fairly often so we get traveler’s diarrhea.
30-40% of visitors to Mexico will drink water or eat food that is bad and get traveler’s diarrhea. Most of
our lettuce comes from Mexico in the winter so we are really at risk for having things like this
transmitted here on our vegetables. That is something you need to think of but there is not much we
can do about it.
Slide 25
When you look at the E.coli that causes diarrhea there is enteropathogenic E. coli that causes
infantile diarrhea, enteroaggregative E. coli that causes traveler’s diarrhea, enterotoxigenic E. coli that is
the most common cause of traveler’s diarrhea. You don’t really need to remember all these terms.
These are just all the things that we don’t look for. It is fairly mild disease, lasts a few days and is really
not something we need to worry too much about. The one we do look for is enterohemorrhagic E. coli,
E. coli O157. We look for this routinely. Enteroinvasive E. coli almost looks like Shigella. It is really
closely related to Shigella and should really maybe be the same species. It causes bacillary dysentery so
we can find it if it is Shigella but if it is not Shigella then we don’t find it.
Slide 26
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 8
Now we go to Klebsiella pneumoniae. This is another Enterobacteriaceae. It is lactose positive
so it is not a stool pathogen essentially. It is non-motile so these don’t have flagella and don’t have an H
antigen number in their name. They have a large capsule and these have been typed. There are 77
different serotypes of Klebsiella pneumonia based on capsule. They also produce a β-lactamase so that
is important because all Klebsiella are resistant then to ampicillin and carbenicillin, but are still sensitive
to the cephalosporins (unless other cephalosporinases are present).
Slide 27
This is what Klebsiella looks like on a MacConkey plate. The organisms are pink so they
fermented lactose and produced acid. You can see how slimy these are. They are much more different
than the E. coli, which are pretty flat. Sometimes they look like bubble gum and drip.
Slide 28
Klebsiella pneumoniae gets its name from causing pneumonia. Here you can see a gram stain of
sputum. These are PMNs in the sputum showing inflammation and these organisms in the cells. Around
these cells you can see a clearing and that’s the capsule. This is an Enterobacteriaceae, a short gram
negative rod and you can see a capsule.
Slide 29
Predisposing factors to Enterobacteriaceae pneumonia: hospitalization (so nosocomial - often
people on respirators get nosocomial infections. Enterobacteriaceae is probably one of the better ones
to get because it is probably more treatable than some of the other gram negatives), increased age
(people don’t clear their lungs as well when they get older), aspiration of oral secretions so alcoholism
(people pass out and often aspirate vomitous stomach contents and you get a mixed growth in
abscesses in the lungs), diabetes and chronic bronchopulmonary pulmonary disease are all things that
prevent us from clearing our lungs well and we wind up with more pneumonia.
Slide 30
Klebsiella pneumoniae disease pneumonia: 25-50% mortality. These are mostly people who are
already compromised some way. If they get Klebsiella pneumoniae pneumonia it really is bad. It is thick
non-purulent bloody sputum, and necrosis and abscess formation are common and they can destroy a
lot of lung tissue that way. Often then it will progress to septicemia, meaning an infection in the
bloodstream. You have circulating organisms growing in the bloodstream and it is often fatal. Klebsiella
pneumoniae also causes UTIs, but not as commonly as E. coli. It can also cause meningitis. When it gets
in the blood stream it can get to the meninges through spinal fluid and often it is fatal.
Slide 31
Enterobacter cloacae and E. aerogenes: these are other Enterobacteriaceae that are lactose
positive and non-motile. UTIs are commonly caused by Enterobacters. These have cephalosporinase –
ampC so they are ampicillin and cephalothin resistant. Some are still susceptible to the some of the
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 9
third generation cephalosporins but all are resistant to ampicillin and cephalothin and can be more
resistant if they get plasmids etc. These are less common than the other two, though.
Slide 32
One way we measure antibiotic susceptibility is we put a disk that has antibiotic on it on the
agar plate and then you look for the zone of inhibition after growing it overnight. The plate is first
streaked with a fairly low concentration of organism all over the plate, then you drop the disks on,
incubate it overnight, and look to see what was inhibited. The diffusion of the drugs is very different.
Each drug has a different zone of inhibition that determines resistance or susceptibility so you have to
look at them. You can pretty well tell that it is resistant to these two drugs and they are ampicillin and
cephalothin (top left on plate).
Slide 33
Proteus mirabilis and P. vulgaris. The defining test for these is urease positivity. If it is urease
positive it will probably be a Proteus. These are highly motile and they will swarm all over the whole
plate. They are also hydrogen sulfide (H2S) positive. These produce H2S if we put them on the right
medium. I think you will look at an API strip that has H2S indicator on it that will turn black. Proteus can
cause UTIs, wound infections, pneumonia, septicemia and it is not an uncommon cause. These are all
tetracycline resistant as well as ampicillin and cephalosporin resistant. The organism has a real
characteristic smell. If you think that it might be Proteus don’t put your nose too close because it smells
real bad.
Slide 34
This is the urease test. If urease is there it will break the urea into ammonia and increase the pH
and you get a color change in phenol red. It will turn bright red. It is the normal test that is important
for Proteus.
Slide 35
Now we’ll go to food borne illnesses. It is the other Enterobacteriaceae like Salmonella and
Shigella that we haven’t discussed. I will also talk about other organisms that cause disease that aren’t
Enterobacteriaceae.
Slide 36
The normal adult has 9 liters of fluid that enters the duodenum. I don’t know about you but I
don’t take in that much, so a lot of that is saliva that is being recirculated all the time. So if you have
disease of the GI tract, you wind up with a large volume diarrhea. If the disease is in the lower part of
the GI tract, you wind up with a fairly small volume diarrhea. So something like Shigella that causes
disease in the colon has really low volume, bloody-mucus diarrhea called dysentery. The volume can
give you an idea of where it is in the GI tract and gives you an idea of what the organism might be.
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 10
Slide 37
Osmotic diarrhea is caused by an increased intra-luminal osmotic pressure. If you don’t absorb
any of your intake then you have a lot of salts and other molecules in the gut and fluid is actually drawn
into the gut. Some causes of this are Giardia and lactose intolerance. If you can’t digest lactose, it gets
into the lower gut and E. coli and other gram negative organisms produce acid from that lactose and this
acid then causes an osmotic diarrhea. If you stop taking things in by mouth, then the diarrhea stops.
Slide 38
Secretory diarrhea is a malfunction of the absorption mechanisms and increased Cl- secretion.
This is often caused by toxins from bacteria. The way we regulate this is by having cAMP in the gut but
the organisms come and stimulate more cAMP. This causes a huge secretion. In this case, if you stop
taking things in orally, you will still secrete so you can become very dehydrated and die quickly.
Slide 39
Bacillary dysentery is an infection of the colon that is caused by bacterial invasion of the
mucosa, so this is a bloody-mucus diarrhea. Frequent small volume stools, often blood and mucus, and
severe abdominal cramps.
Slide 40
Now we’ll look at the causes of food borne problems. The incidence of food borne illness in the
US is estimated to be 76 million cases annually. Out of the 76 million or so there are really only 80,000
hospitalizations, so rarely are people hospitalized from food borne illnesses. 5000 deaths is a fairly small
number, but it is a significant number.
Slide 41
Your risk of food borne disease per meal for selected animal foods. If you eat seafood that is
cooked it is 1 in 5 million, which is good. But if you like oysters or something that are uncooked it is
about 1 in 250, so if you eat them every day for a year you probably will escape but maybe not.
Slide 42
What causes food borne illness? These are studies put out by the CDC. They are looking at all
known outbreaks of food borne illness. This is where people ate the same thing, got sick, and then they
go back and examine it. The most important thing here is the unknown etiology. Studies are done on
these outbreaks and still 68% or so are not identified.
Slide 43
If we look at what caused the food borne outbreaks, Salmonella is the most common.
Campylobacter is as common as Salmonella, but in this case it wasn’t detected causing outbreaks as
much. This is partially because Campylobacter doesn’t cause as severe a disease. You get over it pretty
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 11
quickly so less people are diagnosed with Campylobacter. E. coli, often these are E. coli O157 because
these cause more severe disease and often deaths, especially of children. Clostridium and Staph. aureus
are gram positives. Many other things cause bacterial food borne illness more rarely.
Slide 44
Vibrio cholerae is spectacular. We rarely see it in this country, although it is in our coastal
waters. So the oysters from Texas, Louisiana, and a little bit in Alabama have Vibrio cholerae but it is not
an epidemic Vibrio cholerae. It causes a 1-5 day incubation period and a profuse watery diarrhea. This
is a secretory diarrhea. You can secrete 20 liters of stool per day, but you can’t do that for very long so
this is a deadly disease. If you take fluid orally you can actually treat it without any antibiotics. This was
shown very well in Peru a number of years ago when they had newly arrived cholera there. They had
very few deaths because the health minister did a good job convincing people to take oral rehydration
fluid, which is just a little bit of sugar and salt and water.
Cholera is spread by contaminated water. It takes 10 million organisms to cause disease in fairly
normal people. The reason is that the stomach acid kills most of them and just a few will get through,
depending on what you are eating at the time. The cholera toxin has a B subunit that allows it to bind to
our cells. The A subunit then goes in and irreversibly activates the Adenyl Cyclase (AC) so they turn on
all the cAMP production they can in the gut and then we secrete huge amounts.
Slide 45
This is a cholera cot that I saw in Zambia. They put the person here and they have a hole in the
center over a 5 gallon bucket underneath, so you can measure how much output they had and you can
go back and put that much back in orally.
Slide 46
Enterotoxigenic E. coli causes a watery diarrhea, a secretory diarrhea. It has a labile toxin, which
is the same toxin essentially as cholera so it has the same effect but it doesn’t cause as severe a
secretory diarrhea as cholera and most people survive very well. They also have a stable toxin, which is
heat resistant. You can cook it and it is still active. It has a little bit different mechanism but essentially
the same result, the secretory diarrhea. Some toxigenic E. coli have one, some have the other one.
Again, this is one we can’t really identify in the lab.
Slide 47
Bacterial food poisoning is caused by invasive causes of food poisoning. So Salmonella and
Shigella are two of the top ones we look for, as well as Campylobacter. Campylobacter is not an
Enterobacteriaceae but it is a gram negative rod that causes diarrhea. E. coli can invade and be one of
the invasive ones. Yersinia enterocolitica, E. coli O157 and Lysteria are things that invade and cause
more systemic disease.
Slide 48
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 12
There are 2400 named serotypes identified by using the O and H antigens. In the past this was
used for epidemiology. If we had an outbreak, we could try to trace it by which species it was. It is not
used as much anymore, but we still type.
Enteric fever is typhoid fever. This is uncommon in the US. It causes systemic disease by getting
into our bone marrow, liver, and spleen and is often fatal. The enterocolitis is when you lose 10 days
and 10 pounds and is very uncomfortable. Bacteremia, which is fairly uncommon, is a complication of
the enterocolitis and it gets in the blood and causes a pretty severe disease.
They are lactose negative. When we look at a stool culture we only look for lactose negative.
Most Salmonellas are H2S positive. This is the important thing you will see in the lab. It will be black on
XLD media.
Slide 49
Salmonella enterocolitis. There are a couple million cases per year in the US and a couple
thousand deaths. Common serotypes are Salmonella Typhimurium, Salmonella Enteritidis, and
Salmonella Newport. Typhimurium is in a lot of our animals like cattle and chickens. Salmonella
Enteritidis is really only in chickens. This is the scare where eggs had Salmonella in the yolk of the eggs.
The poultry industry has decreased this a little bit.
With Salmonella you have vomiting and profuse diarrhea for 8-48 hours and it resolves in 10
days or so. Sources: it can live in water for a fair amount of time, meat, dairy products, spinach,
tomatoes, and the outside of watermelon and cantaloupe etc. Sprouts also. Often the Salmonella is in
the alfalfa seeds and they grow during sprouting time.
Slide 50
This is what Salmonella looks like on an XLD plate. This is something we use in the lab. We will
go through a stool culture and pick out the one black colony and see if it is Salmonella.
Slide 51
PulseNet is the CDC’s fairly new weapon against food borne outbreaks. This is a system where
all state public health systems collect all Salmonella, Shigella, and other food borne organisms and
fingerprint them. So we cut their genome and look at the bands we get from this and then we can
identify each group. That is how all these food borne outbreaks now are traced to make sure we are
looking at the right organism.
Slide 52
This is just an example. The 2006-07 peanut butter outbreak. There was an increase in serotype
Tennessee when they PulseNeted this using the O and H antigen. In November and December there
were 26 patients in multiple states that all had the same fingerprint. They looked at what they had
eaten and 48% had eaten turkey (not too surprising in November and December) and 85% had eaten
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 13
peanut butter. Later they looked at 65 patients in February and 81% of the patients had eaten peanut
butter vs. 65% of the controls. Then when they looked at the brand of peanut butter it turned out to be
Peter Pan with 67% of the patients and only 13% of the controls. It turned out that this Salmonella was
resistant to heat treating. Right after this the plant ceased production and they found it in unopened
jars. They had heated it to 70 degrees but the Salmonella actually survived in that.
Slide 53
This just shows the numbers. When they shut the plant down it went away real quickly.
Slide 54
Typhoid fever is caused by Salmonella Typhi. Humans are the only host. There are 200-500
cases/year in the US. I have only known of 2 in my 17 years. Both were from India. Mortality is fairly
low in the US, but is about 10% or so in the third world.
Slide 55
Pathogenesis. You eat this, it goes through the gut and gets into the mesenteric lymph nodes. It
causes belly pain at that time, gets into the blood, goes to all of the organs, and then survives in
macrophages and grows in the liver, spleen, and bone marrow. You end up with secondary bacteremia
and often people die at that time, a couple weeks into the disease.
Slide 56
The typhoid vaccine is actually a live vaccine that people take. You are eating 1011 organisms for
3 days 2 times, which is amazing. I did this and didn’t have any symptoms at all. It is somewhat
protective. The whole killed vaccine has a lot of LPS so when you inject this in your arm you get a
localized reaction and inflammation. That is important to get the vaccine to work well but it is
unpleasant and you have a sore arm.
Slide 57
Campylobacter is a non-Enterobacteriaceae and again it causes food poisoning as much as
Salmonella so it is very common. It is a gram negative rod with a 1-7 day incubation. It causes fever,
bloody diarrhea, abdominal pain. It is more common/less severe than Salmonella. It is carried by a lot
of our food animals like cattle and chickens. About 70% of the chickens in the grocery store have
Campylobacter on them.
Slide 58
Enteroinvasive E. coli causes bacillary dysentery. It is very close to Shigella.
Slide 59
Enterohemorrhagic E. coli is E. coli O157 for the most part. The diarrhea often isn’t that severe
but it does invade the colon and often causes a bloody diarrhea and can be a problem. The most
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 14
common outbreak strain is O157:H7. Don’t worry about it being sorbitol negative for this class. At least
100 different strains have been shown to have the toxin that causes hemolytic uremic syndrome but
they aren’t as virulent as O157. So now we look for the toxin instead of looking for O157 but O157 is
still much more important than the rest.
Slide 60
It has this Shiga-like toxin, which is really specific for the ribosomal RNA of humans. It goes in
and takes out a single adenosine on the 28S ribosomal RNA and makes our cells not able to produce
protein, which kills a lot of endothelial cells especially. The blood surfaces then become sticky because
the sick cells get fibrin strands and small clots in the circulatory system cause hemolytic uremic
syndrome. Renal glomeruli, especially in infants, have a lot of receptors so they have a lot of kidney
problems from this. Acute renal failure, hemolytic anemia, thrombocytopenia mortality.
Slide 61
Shigella is the other one we worry about. It is lactose negative, which is very important. It is
closely related to E. coli. There are 4 species of Shigella. It is non-motile whereas E. colis are mostly
motile. There is no gas formed from glucose is another way we see it.
Slide 62
Bacillary dysentery. Usually in oral inoculation, after 1-3 days you have fairly high numbers of
organisms in the small bowel and have high volume watery diarrhea during that time, then they invade
the colon and you have dysentery. So small volume bloody-mucoid stools. It resolves in 2-7 days. It is
really fairly uncommon in the US except in kids. Children often have the less virulent one of the 4
strains.
Slide 63
Shigella epidemiology. Humans are the only hosts so we can’t blame cows or pigs or chickens.
200 organisms are the infectious dose. 108 CFU/gram in infected stool (CFU=colony forming unit) so
huge numbers in stool. The 4 Fs of Shigella transmission: feces, fingers, food, flies and water. This is
really a fecal-oral organism. It doesn’t survive really well in water. Fecal-oral is where daycares come in.
Kids often get it in daycares. It survives in food. Flies probably aren’t very efficient. If you want to
spread the organism through flies you hang the fly zapper over the picnic table. Anything the fly has on
it at the time it hits the zapper spreads in about a 6 feet radius. Modern sanitary facilities and hand
washing are good for controlling Shigella. 15% of pediatric diarrhea in the US is Shigella – “that seems
pretty high.”
Slide 64
These are the names of the organisms. Shigella dysentery is almost never seen in the US.
Nearly everything we see is Shigella sonnei, which is really not very pathogenic and really is only found
in children.
Microbiology: Gram Negative Bacilli
Laura Rayne
pg. 15
Slide 65
Yersinia enterocolitica is another organism that is Enterobacteriaceae again. Mucosal ulcerations
in the terminal ileum. It was found because it caused appendicitis symptoms in children and so they had
a rash of removing normal appendices. This is something we need to worry about. If someone has
symptoms like an appendicitis, it might not be and antibiotics would cure it. Abdominal diarrhea, pain, 3
weeks, confused with appendicitis (he just read these off like this). It is in pigs a lot so it is a fecal normal
flora of pigs. Around the holidays in the US, the CDC has released bulletins that young black kids less
than a year old are really at risk for Yersinia enterocolitica caused by eating chitlins.
Slide 66
Another Enterobacteriaceae that is not a gut bug is Yersinia pestis. This is the cause of bubonic
and pneumonic plague. It is contracted from the bite of a flea so fleas and rodents like prairie dogs and
rats. If we get bitten, the organism can be passed to the blood and cause severe disease. 25 million
people were killed in 14th century Europe. This organism has just now caused an outbreak in South
Dakota so it is in the prairie dogs there. So far there are no human cases. Plague is also in Colorado.