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Microbiology
9/29/2008
Mycoplasmas & Fastidious Gram-negative Bacteria: Dr. Ken Waites
Transcriber: Kimberly Watkins
46:36
Slide 1
Today we are going to talk about the listed bacteria.
Slide 2
We will use the same format as before in terms of talking about the organisms and their
characteristics, the epidemiology of their diseases, how they produce diseases, and how we detect and
identify them.
Slide 3
The first one we are going to talk about is Haemophilus, which means “blood-loving.” It is a
gram-negative coccobacillus (seen on the gram stain). It is a facultative anaerobe and it is also nonhemolytic, which means that you have to provide it with things that it otherswise would get from the
lysis of RBCs. We say that this organism is fastidious because most Haemophilus will not grow on unsupplemented blood agar since they are not hemolytic (they cannot get to the things that they need
from RBCs). We use an enriched medium such as chocolate agar. Some of the strains are invasive and
some are colonizing, primarily in the respiratory tract. In the upper respiratory tract (URT), the nonencapsulated strains of H. influenzae and other species are very commonly found. The encapsulated
strains are the ones that tend to cause more severe disease.
Slide 4
These are organisms that primarily cause disease in the respiratory tract. They are transmitted
through aerosols. In many causes, it is actually activation of endogenous disease. A typical example is
someone with an underlying lung disease, such as chronic obstructive pulmonary disease (COPD), where
you may be colonized with the organisms in the respiratory tract, even the non-encapsulated strains
that are there. These are often times older people that have smoked that have impaired cilia activity,
impaired cough reflex, maybe have chronic bronchitis, and have a lot of excessive secretions in the
respiratory tract. These bacteria will then grow and proliferate (grow to very large numbers) and can
cause symptoms such as fever and productive cough, so you have an exacerbation of chronic bronchitis
that you see in that setting,
The major virulence factor for H. influenzae (prototype pathogen) is an anti-phagocytic capsule.
There are several different capsule serotypes (at least 6) named in letters a-f for H. influenzae and it’s
the serotype b that is the most significant pathogen. Like we have discussed with some of the other
bacteria that have multiple serotypes that are immunologically distinct and in this case one of them
tends to be more pathogenic than others. This was formerly an important cause of meningitis, epiglottis
and bacteremia in young children who did not develop an antibody to protect them and this was the
basis for the vaccine developed in the 1980s (all children get as part of their immunization). It is based
on the antigen for the serotype b.
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pg. 2
Since it gram-negative, it has endotoxin as part of the cell wall and is part of the pathogenesis. It
damages the respiratory epithelium which can lead to bacteremic spread of the organism. Unlike some
of the bacteria we have talked about before (staphylococci and streptococci), the Haemophilus do not
produce any exotoxins of consequence; they mediate disease primarily through their endotoxin.
They do have an IgA protease and about 30% of the Haemophilus strains will produce a betalactamase enzyme that will break down penicillin. So non beta lactamase stable penicillin such as
ampicillin will no longer work on those organisms.
Slide 5
If you are going to detect H. influenzae in the lab, you have to grow it on chocolate agar (RBCs
have been heated so that they lyse and will release the things that the Haemophilus needs)> it will not
grow on MacConkey agar. It is capnophillic (give it CO2). The Haemophilus species will vary on whether
that will require the X and V factors. We put them on a nutrient agar plate (which the organism would
otherwise not grow) and use filter paper strips that contain the X or V factor or both. In the lower right,
you can see the haze of growth of the Haemophilus on the agar plate only around the X and the V and
that is how you can tell that it is H. influenzae. Other species would require either X or V but not both.
H. influenzae requires X (hemin) and V (NAD, an enzyme co-factor for catabolizing reactions). Something
else that is seen sometimes in culture is if you put a S. aureus strain (strongly beta-hemolytic) on a blood
agar plate it will hemolyze the RBCs and that will release the X and V factor and the Haemophilus will
then grow around where the staph is growing. This is called satelliting (see satellite colonies in lower
left).
Slide 6
H. influenzae is still an important cause of otitis media in children because this is usually not the
type b encapsulated strains that cause this. The type b encapsulated strains have largely been
eliminated as pathogens in children because of the H. influenzae vaccine but we still see this organism as
a cause of disease in adults (in the non-encapsulated strains primarily) such as sinusitis, epiglottis and
bacteremia. Epiglottis and bacteremia are not nearly as common as they used to be just because that
was usually the invasive encapsulated type b strains and at least in children and in young adults we do
not see these diseases very often. You can get laryngotracheobronchitis, meningitis (which is very rare
because it is almost always the encapsulated type b strains that have been wiped out by the vaccine),
and exacerbation of chronic bronchitis in COPD. It is also an important cause of community acquired
pneumonia and also causes cellulitis and conjunctivitis.
Slide 7
Because of the vaccine, we hardly ever see the invasive disease in young children. The vaccine
that is given now as part of the series to all infants is a polysaccharide based vaccine to the capsular
serotype b. Polysaccharides are poor immunogens in young children so it is conjugated to a protein
carrier to make it more immunogenic. Three to four doses are given in infancy. Since introduced over 90
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pg. 3
% of invasive disease has been eliminated. However, this does not have any impact on the non-typeable
(non-encapsulated) H. influenzae infections.
Slide 8
Some other Haemophilus species that are just for note. H. ducreyi is a cause of STD (not a
common cause of STD in the US) and it produces a chancroid (painful genital ulcer) which can be
mistaken for herpes virus infection. H. aegyptius (very similar to H. influenzae) is also a cause of bacterial
conjunctivitis.
Slide 9
Bordetella pertussis is the cause of pertussis or whooping cough. It is an encapsulated gramnegative coccobacillus (seen in gram stain) that is slow-growing, fastidious and aerobic. It does not do a
lot biochemically (does not utilize any sugars so it is not easily picked up in the lab). It generates
metabolic energy by oxidizing amino acids.
Slide 10
Whooping cough is a very contagious disease and before the vaccine it was a very common
disease encountered in pediatrics (could cause death especially in young infants). Generally there are 3
different stages after an incubation period of 5-21 days:
1. Catarrhal stage: cough and sneeze which gets worse and progresses to the second stage
2. Paroxysmal stage: you have a severe irritation of the airways in the URT because of the exotoxin
produced and it causes necrosis of the airways and stimulates the coughing. You get very rapid
coughing which makes it hard to breathe and then you take a very deep breath which sounds
like a whooping sound. The child can literally turn blue because they are not oxygenating well
due to the severe coughing.
3. Convalescent stage: after you get over it and your immunity develops can take several months
to get back to normal. You can cough for a long time with this organism.
You will typically see lymphocytosis in the blood and if you ever have whooping cough you get a lifelong
immunity (it simulates the immune system to make Abs against several of the toxins).
Slide 11
This is an old disease (known since the 16th century) and even though we vaccinate for it in the
in US we do not have complete eradication. The vaccine that has been used in the past and the
immunity wanes over time and then adults can get this disease as a result of their immunity waning,
especially if you get exposed to non-vaccinated individuals. Worldwide there have been 285,000 cases
(deaths) as of 2001. Also, in 2005 there were 25,000 cases in the US and it is increasing. There is no
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pg. 4
environmental or animal reservoir; it is person-to-person spread. Over half of the cases in the US are in
adolescents and young adults (vaccinated as infants but their vaccine immunity has waned). If adults
and adolescents are getting this disease, they can spread it to young children before they have been
adequately immunized.
Slide 12
Data from Alabama over the past few years shows that it is a reportable disease that is
increasing.
Slide 13
Why is it going up? Infants are being under-vaccinated (immigrants may not get their infants
immunized and also no health insurance) and they can acquire the disease and spread it to other
children as well as adults. The other reason is that physicians have been taught that this is a vaccine
preventable illness and that everybody gets vaccinated and so you do not see this and they do not think
about it when diagnosing patients. A person is very contagious until you get them out of circulation and
on the proper antibiotics. In addition, with the older vaccine it sort of wears out in early adulthood (do
not have enough Abs) and this is contributing to the reservoir.
Slide 14
How do you get the disease? For successful bacteria to cause disease, they have to have a way
to attach to the host. Pertussis has a number of different structures that allows it to attach and affiliate
with the epithelium of the URT. You have the filamentous hemagglutinin (FHA) and the pertussis toxin
(PTx), which are part of the attachment moieties. The toxin not only helps attach but also damages the
cells. Once the bacteria get in, they have to get the host immune system blocked in some way so it does
not get rid of them. One of things that the pertussis does is it impairs chemotaxis of WBCs and then the
exotoxins damage the respiratory epithelium and it can help it spread throughout the body. The main
disease that you get is in the respiratory tract.
Slide 15
There are a lot of different toxins. I do not know that you have to learn the details of these
toxins but the main things to remember are 1) there are several of them 2) this is an exotoxin-mediated
disease 3) toxins not only facilitate the attachment but also the local damage to the cell over the
respiratory tract and stimulate the host immune system and 4) are the basis for the vaccine. These
toxins actually inhibit the leukocyte migration and phagocytosis, cause necrosis of the respiratory tract
epithelium, stimulate the release of interleukins (why you get the fever: IL-1 is a mediator of fever) and
also the endotoxin (LPS). So the bug hits you with two different things: all the exotoxins and (since it is
gram-negative) the endotoxin. The endotoxin activates the alternate complement pathway and
stimulates cytokine release, which accounts for some of the systemic effects that can spread beyond
the respiratory tract.
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pg. 5
Slide 16
If you encounter someone that you suspect has pertussis, the diagnostic test of choice is first to
collect a nasopharyngeal swab (all the way in the posterior nasopharynx because that is where it is
replicating). You have to have a special media, such as the Bordet-Gengou or Regan-Lowe that is
enriched with horse blood and charcoal. You incubate it in a moist environment and then identify by
immunoflourescence or agglutination. This will not be done in a community hospital lab because it is not
seen often (would be sent to reference lab). You can measure antibody titers but is retrospective. PCR is
recommended to augment the sensitivity of culture (enhance the diagnosis). A direct fluorescent
antigen test (DFA) is done on NP secretions but like so many DNA based tests you have to have a very
high organism load to detect (since it is uncommon this test is not very good).
Slide 17
The old vaccine contained whole heat-killed organisms that presumably had all the antigens for
the toxins that you would make Abs against. People had a lot of side effects to the vaccine because you
were injecting whole killed bacteria and your body was reacting to a lot of things that were not really
the way the organisms were mediating disease. In 1996, a new acellular vaccine was developed called
the DaPT (D: diphtheria, aP: acellular pertussis, T: tetanus) that is given in 5 doses (beginning at 2
months up to the time of school age). The acellular vaccine was developed to contain primarily just the
toxins that cause disease (help prevent the attachment of the organism and the introduction of disease,
the cell damage). Because of the concern of the resurgence of pertussis and the waning immunity, there
is a new vaccine for adults and adolescents (ages 11-64) as a single dose. This is separate from the
tetanus toxoid vaccine. The Td protects against tetanus and diphtheria but not against pertussis and is
recommended every ten years.
Slide 18
Legionella is a very fastidious (why it was not identified until the 70s), catalase-negative and
facultative intracellular organism. It does not gram stain very well (very pale because it does not take up
the saffarin well). It is non-fermentative (so you will not see sugar fermentations). There are over 30
different species and multiple serogroups.
Slide 19
Came to attention with the convention in 1976 of WWII veterans; they knew that there was
something else causing the pneumonia; developed a medium to grow the organism in vitro. The
organism was infected through air conditioning. It lives in water systems, cooling systems, and A/C. It is
a somewhat opportunistic organism; tends to cause severe disease in people with underlying lung
disease. We normally think of pneumonia as being contagious from person to person. Legionella is
usually not spread from person to person; it is spread from an environmental source. The organism is
inhaled, complement deposits on the bacteria and then the bacteria bind macrophages. Since it is
facultative intracellular it lives in the macrophage and it is not killed because it prevents the phagosome
and the lysosome from fusing. It lives and multiplies in the phagosome and produces enzymes. The cell
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pg. 6
dies and the bacteria are released. When the cells die in the respiratory tract, it stimulates inflammation
and also coughing. You can get a purulent pneumonia with abscesses because of the intense stimulation
of the host immune response and bacteria growing in the cells. Fairly high mortality if it is not treated.
Slide 20
To culture Legionella we can use a medium with buffered charcoal yeast extract and cysteine
(why it looks black). It may take several days for them to grow. A tracheal aspirate or a lung biopsy tends
to be more reliable than sputum to get the organism out. It is grown very rarely in the hospital lab. If it
grows on the agar, it is sent off to be identified by agglutination or immunofluorescence.
Slide 21
There are other ways to detect Legionella. You can stain lung specimens with silver or also a
direct florescent antibody (it is not a good test unless you have lots of organisms). A test that is
commonly used is to test for Legionella antigen that has spilled over into the urine. The infection can
become systemic and after several days you will start excreting the antigen in the urine. Culture is the
only method that will pick up all the species. Antibody paired serum can be measured for Legionella
pneumophila but will not pick up some of the other species. PCR is also being looked at as a way to pick
up Legionella.
Slide 22
If you take all the community acquired pneumonias (CAP), maybe 5-10% is due to Legionella.
The unique things about Legionella as a cause of CAP is point source outbreaks. You may have a lot of
people that come down with this at one time if they have been exposed to the same source but it is not
generally contagious from person to person. It is used associated with water. In addition, you can get
vague non-specific complaints such as diarrhea and kidney problems because this organism doe spread
throughout the body and can cause damage. The main people that tend to get this are older men with
obstructive lung disease and transplant recipients that are immunosuppressed (not really seen in young
children or otherwise healthy individuals). Because of the unique epidemiology, it is more common in
the summer.
Slide 23
There is not a vaccine. Cell mediated immunity seems to be more important than Ab mediated
immunity and is why transplant recipients that have had a depressed cellular immunity because of
suppression tend to get it. The way to prevent it is to monitor the sources of contamination and you
clean them or eliminate them.
Slide 24
Moraxella has been through several name changes. It is a gram-negative coccus and it will grow
on regular blood agar (not very fastidious). Many people especially children may carry it in the URT and
it may occasionally cause diseases such as bronchitis, CAP, sinusitis and ear infections. They may be seen
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pg. 7
sometimes outside of the respiratory tract. An important virulence factor is that most strains contain a
beta lactamase so you cannot use ampicillin.
Slide 25
Mycoplasmas are unique among the prokaryotes because they lack a cell wall.
Slide 26
This is his cartoon to demonstrate the relative sizes. Streptococcus is about one micron and
Mycoplasma may be about 1/10 of a micron. You cannot classify it as a rod or coccus because it is not
surrounded by a rigid cell wall; they have a tri-laminar sterol containing membrane that allows them to
be very pleomorphic (change shape very easily). None of the antibiotics that work on cell walls are going
to affect them.
Slide 27
Mycoplasma are the smallest free living (that you can cultivate in vitro) organisms. They are very
fastidious; you cannot grow them on regular blood agar (need special media). They do grow on agar
medium if you provide them with a cholesterol source such as serum which they need to synthesize
their cell membranes. They are the smallest in terms of both their size as well as their genome. It
produces about 687 genes from a genome of about 800,000 bp. It is about a quarter of the size in both
cellular dimension and in genome size. The limited biosynthetic capabilities mean that you have to
provide them with everything if you want to grow them. Since they are small and lack a cell wall, they
are susceptible to environmental conditions. They will dry out immediately and the reason why you do
not see them in reservoirs. You do not find them free in nature; they go from host to host. There are
about 150 species but there is only about 17 species that infect humans. They evolved from grampositives through gene deletion. They are primarily extracellular pathogens that colonize mucosal
surfaces. Sometime they will live inside host cells.
Slide 28
Mycoplasma has a specialized attachment structure that is composed of the P1 protein and
several other accessory proteins. When inhaled into the lungs, it seeks out the respiratory epithelium
and it takes this thing and attaches vigorously. This is not only an attachment protein but it is also
immunogenic and serology (measurement of Abs) is a common way to diagnosis it. The Abs you make
against M. pneumoniae are directed against this structure. The bacteria then stimulate peroxide
formation (cytotoxicity) and there is some evidence now that the organism produces an exotoxin that
damages the host respiratory cells. You can get ciliostasis and damage to cilia because of the hemolysin
produced. It affects the cell metabolism and ultimately the cells will die. It also stimulates inflammation
and cytokine release. When the P1 protein attaches, it stimulates the inflammatory response and
damages the cells. The first thing you get is interstial type pneumonia (most of the time you get
bronchitis). This is why you cough (dry hacking) because million of these bugs are attached. They also
vary their surface antigens so you can get this disease over and over. It can produce multi-system effects
Microbiology: Mycoplasmas & Fastidious Gram-negative Bacteria
Kimberly Watkins
pg. 8
because it stimulates a lot of autoantibodies (antigens stimulate Ab production that cross reacts with
antigens in other parts of the body) and you can get autoimmune phenomena, Guillian-Barre type
paralysis, meningitis, encephalitis, autoimmune hemolytic anemia. Many times when you get these
extra pulmonary things is because of the autoantibodies.
Slide 29
You can culture this organism but it takes about 3 weeks and you have to use special media. You
can identify the colonies by PCR (colonies are small; need microscope). Serology has been used in the
past especially in children you could mount an IgM response. Everyone has probably had “walking”
pneumonia; you feel tired, cough, low grade fever, and think you may have sinusitis (it is not real severe
like you get with Pneumococcus) and you may gradually get this over a period of several days. Without
antibiotics, it may take several weeks to get over it. It is rarely diagnosed because culture takes too long
and looking for Abs is generally retrospective (would like to see a rise and fall). PCR is becoming more
commonly used especially in children where you have a severe disease.
Slide 30
It causes diseases in all ages. A tracheobronchitis is the most common manifestation. The
atypical interstitial “walking” pneumonia occurs about 30% of the time. Overall, about 20-50% of all CAP
may be Mycoplasma. It is very similar to the other pneumonias. Extrapulmonary disease can occur
through direct invasion as well as through autoimmunity. If one person in the household gets it, it can
affect everyone else. There have been well described outbreaks in closed populations. It may play a role
in bronchial asthma. It colonizes the respiratory tract and triggers wheezing and it can certainly make
asthma worse but some suggestion that it may trigger asthma if those you do not have it. Re-infection is
common but there is no protective immunity. We are now seeing antibiotic resistant in this organism.
Slide 31
M. hominis, M. genitalium and the Ureaplasma species are important genital tract pathogens.
They can be grown at least M. hominis and Ureaplasma can. M. genitalium is actually the smallest freeliving organism. It is so fastidious and slow-growing that you cannot really culture it but you can detect
by PCR.
Slide 32
There are a lot of different diseases that they can cause. It has been problematic in learning
about what disease these organisms may cause because they are very common .Over half of sexually
active adults may have these organisms in their lower urogenital tract.
Slide 33
This table shows the diseases that have been shown to be associated. Non-gonnococcal, nonchlamydia urethrits that you can get as a STD can be caused by Ureaplasma as well as M. genitalium.
Also evidence that they can cause urinary calculi (similar to Proteus that splits urea, raises the pH of the
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pg. 9
urine and causes kidney stones). Infection of the kidney: pyleonephritis, certainly M. hominis can cause
this. Cervicitis caused by M. genitalium. Pelvic inflammatory disease (infection of the uterus and the
tubes) caused by both M. hominis and M. genitalium. Ureaplasmas have been implicated in recent years
as causing preterm labor (infection can cause women to go into labor before term because the bacteria
can cause an intrauterine infection, stimulate inflammatory cytokines in the amniotic fluid, stimulate
prostaglandin release and stimulate the onset of labor contractions). Because they stimulate the
inflammation they can infect the placenta and cause amnionitis and also enter the fetus’s lungs and
cause lung disease. They can also cause sepsis and meningitis in the newborn. This is a concern as well
as the propensity for chronic lung disease because of a low grade inflammation. People with impaired
Ab production such as congenital hypogammaglobulinemia and others are particularly prone to getting
arthritis and other systemic infections with these organisms.
Slide 34
You can culture them except for M. genitalium. Serology is not good because so many people
have the organisms in them and everybody has Ab. PCR is available for detecting M. genitalium (not
available commercially).
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pg. 10