Download Mycoplasmas and Fastidious Gram Neg Bacteria

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I.
Mycoplasmas and Fastidious Gram-negative Bacteria [S1]
a. Continuing with miscellaneous bacteria.
II.
Objectives [S2]
a. Will be working with Haemophilus in the lab tomorrow.
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III. Haemophilus [S3]
a. Look at the origin of the name
i. Heme means blood, philus means loving.
ii. Blood-loving bacteria
b. Gram-negative coccobacilli
c. Sometimes so small you can’t tell what shape they are.
d. Facultative anaerobes
e. Non-hemolytic
f. They are fastidious because they can’t grow on regular blood agar.
g. Find many in upper respiratory tract.
h. We all have them in our throat.
i. There are also invasive strains.
j. We primarily have non-encapsulated strains that cause normal flora and encapsulated invasive ones cause
disease.
IV. Haemophilus: Pathogenesis [S4]
a. When we talk about the pathogenis of haemophilus, we are mainly talking about haemophilus influenza.
b. A lot of people get Haemophilus influenza, a gram-negative bacteria, and viral influenza mixed up.
i. Very different things.
ii. Haemophilus influenza can cause pneumonia and viral influenza can cause pneumonia.
iii. Same nomenclature used for two different types of diseases- make sure you don’t get them confused.
c. Uses same name for two diff types of diseases.
d. Haemophilus influenzae- main pathogenic feature is its capsule.
e. Type b encapsulated type is the most important.
i. There’s a, b, c, d, e, f types – 6 types total
f. Has endotoxin because its gram negative.
i. Allows it to damage respiratory epithelium and spread.
g. Does not make exotoxins.
h. Does have IgA protease analogous to Neisseria meningitidis.
i. Beta lactamase allows it to destroy beta lactam antibiotics.
i. That’s a virulence factor.
ii. Means you can’t treat it with plain ampicillin or penicillin.
V. Detection: H. influenzae [S5]
a. Can grow it on chocolate agar.
b. Looks like neisseria.
c. Oxidase positive.
d. Requires CO2.
e. Can be distinguished from other haemophilus species that require both the x and v factor
f. X factor is hemin, which is part of hemoglobin.
g. V is nicotine adenine dinucleotide, which is a cofactor for number of biochemical reactions that haemophilus
has to do.
h. You can take nutrient agar that doesn’t contain these things and put a filter paper disc and then start doing
the V factor on there. If you have both factors together, haemophilus will grow there and no where else on
the plate. This was an old way laboratories were able to tell which species of haemophilus you had. Some
species require the X or the V, but H. influenzae requires both.
i. Sometimes you can have a mixed infection
j. S. aureus is very beta hemolytic.
i. It will open the red cells and will release x and v factor.
ii. Sometimes you get haemophilus around the beta hemolytic staph.
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k. Small colonies of haemophilus.
l. Staph makes the big creamy, yellow colonies.
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VI. Diseases: H. influenzae [S6]
a. Causes disease primarily in respiratory tract.
b. Ear infections- otitis media in kids.
c. Sinusitis
d. Bacteremia
e. Epiglottitis- no longer common with H.influenzae because it’s an invasive disease and we vaccinate kids
routinely in infancy. It has dramatically reduced invasive haemophilus disease like meningitis.
f. Years ago this was a common cause of meningitis in children up to age 5 years when they had an immature
immune system and did not have protective antibodies.
g. Haemophilus vaccines have started in early 1980s and since then meningitis and epiglottitis due to this
organism have been wiped out. There are very few cases of it anymore.
h. The nonencapsulated and non-type B strains can still produce disease, although less likely to produce
invasive diseases.
i. We sometimes see cellulitis (the girl’s swollen face in picture)
j. H. influenzae or sometimes H. aegyptius can cause conjunctivitis (red swollen eye).
k. Laryngotracheobronchitis
l. One of the most common places we still see H influenzae is COPD in older people.
i. Typical person that has been a smoker, chronic bronchitis, coughs a lot, produces a lot of sputum. Most of
the time they go along okay. But then the low numbers of haemophilus in respiratory tract all of a sudden
start growing. They produce a lot of pure yellow sputum and start running a fever. You culture the sputum,
and there will be a lot of H.influenzae.
ii. Most common place we see it now is pneumonia and exacerbations of obstructive bronchitis in middle age
to older people with history of smoking and it cause significant disease there.
VII. Prevention: H. influenzae [S7]
a. Type b is also known as Hib vaccine reduces number of invasive cases.
b. Four doses of this given in children, so hardly see this again.
c. Specific for type b encapsulated organisms, so there is no impact.
VIII. Other Haemophilus sp [S8]
a. H. ducreyi is related to H. influenzae that causes chancroid, which one of the lesser common sexually
transmitted diseases
b. H. aegyptius like H. influenzae can cause conjunctivitis.
i. Organism is very similar to H. influenzae.
IX. Bordetella pertussis [S9]
a. Organism that is the cause of whooping cough.
b. Upsurge of Bordetella pertussis in California recently
i. People that get vaccination in infancy, the immunity wains over several years.
ii. Some kids not protected because don’t get vaccinated (some people don’t believe in vaccination), and this
is one disease in which there is a big increase because of that.
iii. Very successful pathogen.
c. Encapsulated, gram-negative coccobacillus, slow-growing, strict aerobe
d. Does not ferment sugars, does oxidize amino acids.
e. Don’t encounter it in laboratory.
i. We send a request to reference public health library because it is sufficiently uncommon because of
vaccination.
X. B. pertussis: Whooping Cough [S10]
a. Produces whooping cough.
b. Very contagious infection
c. 5-21 day incubation
d. First stage is catarrhal stage where you cough or sneeze for a week or two.
e. Paroxysmal stage- several weeks
f. Convalescent stage- white cell count goes up.
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g. Once you get over it, you’re immune to it. But if you take the vaccine, you won’t have it.
h. Website listed for you on slide to listen to whooping cough.
i. In prevaccine days, you would go to the doctor’s office and just listen to the whoopers.
j. The whoop is the deep breath they take after coughing so much.
k. Bacteria destroy the respiratory mucosa.
l. It’s an upper respiratory infection.
m. It is growing back there and producing toxins that are killing the respiratory epithelial cells and producing
irritation.
n. It’s in the upper throat and is producing all this inflammation back there and a terrible cough.
XI. Pertussis Epidemiology [S11]
a. Disease has been known for number of years.
b. Well-described even when they didn’t know what bacteria was in the 16th century.
c. Vaccine-preventable disease.
d. 285,000 cases worldwide.
e. 2007- at least 10,000
f. There’s probably more cases than reported. Can be treated without a diagnosis ever having been made.
g. No environmental or animal reservoir.
h. Spread from person to person by droplets.
i. Adolescents and adults are more than half the cases because get the vaccine when we are babies and now
we’re older.
j. Antibodies level taper off so older people sometimes spread the disease to younger children.
k. The older vaccine is not as good as the new vaccine that has been out.
l. Some people come across the border from Mexico.
i. If they’re illegal, they don’t go to doctor.
ii. So they don’t always get kids vaccinated and these are the people that are susceptible to it and can spread
it because they’re not part of the public health system.
XII. Pertussis in Alabama [S12]
a. Since 2001, the cases are going up.
b. They are going up just about every year.
c. In California, there are now hundreds of cases every year identified.
i. Their population is bigger than Alabama’s, and they probably have a bigger problem with undocumented
immigrants.
XIII. Reasons Pertussis is Increasing [S13]
a. Under-vaccination.
b. If not treated properly, can further spread disease.
XIV.
Pertussis Pathogenesis [S14]
a. For bacteria to cause disease, they have to attach to something in body so they can invade and multiply.
b. Pertussis attaches to respiratory epithelium by a lot of different adhesions.
c. It has a filamentous hemagglutinin (FHA) and a pertussis toxin (PTx) which is also an attachment (inaudible)
that the bacteria produces there.
i. These are structural components of bacteria.
d. In addition to making toxins, it causes impaired neutrophil chemotaxis
i. It helps it avoid the human immune system.
e. You get local tissue damage because of the toxins that occur there.
XV. Pertussis Toxins [S15]
a. Don’t have to memorize all pertussis toxins but be familiar with the concept of what they do.
b. Read slide
c. Because it’s gram negative, it has LPS endotoxin.
d. All these things together give you whooping cough.
e. Good thing:
i. Of all these that are part of pertussis organism, a lot are immunogenic so they are good candidates for
developing vaccines.
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XVI.
B. pertussis: Detection [S16]
a. You can do a nasopharyngeal swab and send it to a reference laboratory
i. They will culture it on Bordet-Gengou or Regan-Lowe enriched charcoal media
b. Grow it for about a week in a humidified, moist environment.
c. if you grow the organisms on the selected media, you react them with immunofluroescent-stained antibodies
to see if they react so that you know if you have B. pertussis.
d. Will not grow on conventional media in hospital laboratory.
e. Can also measure antibodies doing convalescent serum.
f. PCR nucleic acid amplification testing is recommended in addition to culture because it’s more sensitive.
g. They still recommend doing cultures so that you can do an antibiotic susceptibility test on it.
h. There are direct fluorescent antibodies (DFA) on nasopharynx secretions that are sometimes done but they
have low sensitivity because have to have large number of organisms there to be positive.
i. These are not routinely done in hospitals because of complicated procedures and because disease is not
common.
i. Usually done in state public health reference laboratories.
XVII.
Pertussis Prevention [S17]
a. The old vaccines were the acellular vaccines
i. Whole pertussis organisms have been heat killed so all antigens sticking out and immune system makes
antibodies to them.
ii. Because they were whole cell organisms, you were exposed to all other things in there that don’t
necessarily protect you against pertussis disease.
iii. So there are a lot of side effects of vaccine, and vaccine sort of wears out after a few years.
b. 1996- introduced cellular vaccine for infants (DaPT)- contains antigens for all of the different toxins we
described here.
i. Protect against attachment of organism and the exotoxins.
c. If you take vaccine, you have antibodies against the things on the organism that allow it to attach to the
respiratory epithelium and against the toxin that causes disease.
d. If it became attached and you neutralize its toxins, it can’t do very much.
e. Given in a series of 5 doses over the first 6 years of life and gives good protection against pertussis.
f. 2005- New vaccine developed for adults and adolescents- to get better protection so that you will not catch it
yourself and spread it to children
i. Recommended for ages 11-64 as a single dose
g. Tdap vaccine- diphtheria and tetanus.
i. Td is tetanus and ap is acellular pertussis
ii. Td does not protect against pertussis.
h. Pertussis is an old disease that is coming back.
XVIII.
Legionella pneumophila [S18]
a. Gram-negative, fastidious organism.
b. Bacillus
c. Remained undetected many years.
d. Doesn’t gram stain well, doesn’t grow well on conventional media we have.
e. Not known until 1976.
i. Convention of World War II veterans of the American legion.
ii. It was hot in July in Pennsylvania, and air conditioners running high. Took place at Stratford hotel.
iii. Many men in their 50s and 60s, World War II generation, came down with respiratory illness of unknown
etiology and got pneumonia (many were smokers and had underlying lung disease and COPD).
iv. They were sick and put in the hospital.
v. CDC called in to see what was wrong. Everyone was getting sick from this one place.
1. They tracked source of infection to the air conditioner and water that circulates from the humidifier to
run the air condition.
2. They took samples from this air conditioner and they looked at several different things and found
charcoal medium that grew the organism.
3. The organism had never grown before, and they named it Legionella pneumophila because of the
American legion veterans that caught the illness.
4. Called it pneumophila because it liked the lung and caused pneumonia.
5. Since then, we have found over 30 different species of Legionella.
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a. They are generally named after city they were identified in.
b. There is a Legionella birminghamiensis (patient it was identified in worked in Volker Hall).
XIX.
Pathogenesis of Legionellosis [S19]
a. Legionella is different from other organisms that cause pneumonia.
b. It caused a point-source outbreak from that air conditioner.
c. Different from other causes of community-acquired pneumonia we talked about (like pneumococcus,
mycoplasma) because it does not spread from person to person.
d. It is inhaled from environment
e. Complement deposit on bacteria
f. Bacteria bind macrophages by the C3 receptor
g. Bacteria are taken up by macrophages, and inside the macrophages, they prevent phagolysozome fusion
(like the Chlamydia did), and in doing so they multiply inside macrophages. They are protected from the
immune response.
i. They live inside macrophages.
h. They produce enzymes, kill the cell, cell dies, and bacteria are released.
i. Then you get the inflammation and consolidation because you’re stimulating white cells to come to the area.
j. Respiratory cells die from bacteria being released.
k. This process gives you pneumonia.
l. It’s a dead end from there unless someone gets exposed to air or the water.
m. Legionella likes to live in hot water.
i. Identified from hot tubs, hospitals where it got into water supply
n. Legionella is not that common in this part of the country.
XX. Legionella Culture [S20]
a. We can grow it on buffered charcoal agar or on respiratory specimens.
b. We grow it rarely and send it to a public health laboratory to identify it using immunofluroescence.
XXI.
Legionella Detection [S21]
a. If you have lung biopsy, you can do a silver stain and stain for bacteria.
b. Apply direct fluorescent antibody to respiratory secretions- not so good because requires many organisms,
more than you can produce from a clinical specimen
c. Urine antigen test- we use this now
i. The organism becomes bacteremic and spreads throughout body.
ii. If it’s positive, it’s quicker than culture, but it takes a few days to get there so not much better
d. Serology done occasionally but you need acute convalescent serum to measure antibody response- takes
several days after someone gets sick to measure again and compare first and second antibody
e. PCR is becoming popular but there is no commercial PCR available so most labs still can’t do it.
f. Most laboratories do culture for legionella and the urine antigen.
i. Problem is there is more than 30 different species and not all of these tests (like the urine antigen and
serology) will pick up all of them.
ii. Culture will pick up all of them. So we still do culturing in hospital.
XXII.
Legionnaires Disease [S22]
a. 5-10% of community-acquired pneumonias. 10-20,000 cases a year.
b. We don’t see much of it in this part of the country.
c. Associated with point source outbreaks.
i. Several people getting affected at one time.
d. Reportable disease to see what people have been exposed to.
e. Can also get GI and renal manifestations.
i. Most people that have this are older people or transplant patients.
f. Unlike other things that cause respiratory disease, it is worse in summer because caused by air conditioning
systems.
XXIII.
Prevention of Legionellosis [S23]
a. No vaccine
b. Good cellular immune system is important in helping you get sick from it.
c. Identify source and eliminate them.
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d. UAB- Have to submit water four times a year to make sure we don’t have Legionella in there and we are not
exposing people to it.
XXIV.
Moraxella catarrhalis [S24]
a. It’s an organism that is carried in respiratory tract.
b. Very similar to Neisseria, gram negative coccus, grows fine on blood agar, not as fastidious as Neisseria.
c. Can cause all kinds of respiratory trouble: bronchitis, CAP, sinusitis, otitis.
d. Most strains produce beta lactamase.
i. Can’t treat it ampicillin with anymore.
e. Not an important organism, see it occasionally.
XXV.
MYCOPLASMA PNEUMONIAE [S25]
XXVI.
Diagram [S26]
a. Everyone has probably had mycoplasmas at one time or another.
b. Mycoplasmas are unique microorganisms.
c. Do not get mycoplasmas mixed up with mycobacteria (tuberculosis).
i. These are two very different organisms.
d. Mycoplasmas are the smallest free-living bacteria.
e. Diagram shows relationship mycoplasmas have with other types of microbes.
f. Big eukaryotic cell. Streptococcus about 1 micron.
g. Pointed out parts of diagram.
h. Mycoplasma is much smaller than bacteria.
i. They are smallest bacteria you could put on agar plate and make it grow.
j. Since they are so small, you have to do a whole bunch of things to make them grow.
k. Unlike Chlamydia, which are obligative cellular organisms, these are free-living.
XXVII. Mycoplasma [S27]
a. Will grow without human cells.
b. Only a few species cause disease in humans.
c. Mycoplasma pneumoniae is the best known.
d. Have about 800,000 base pairs of 687 genes.
e. Regular E.coli will have four or five times the size of this.
f. They are about one-fourth the size of bacteria and have very few genes.
g. They have had gene deletions through evolution, so a lot of things they have lost.
h. Unique thing that separates them from all other prokaryotic bacteria:
i. They don’t have a cell wall
ii. No peptidoglycan
iii. They have lost the genes to make it.
i. Can’t say they are a coccus or rod because don’t have a rigid cell wall. Can be any shape they want to be.
j. Cilia in picture. Snake-like things in picture attach to organelle (the business end of the organism). Allows it to
attach to respiratory epithelium and cause disease.
k. Are essentially a naked gram positive bacteria
l. Probably evolved from gram positive but don’t have cell wall.
m. Mainly attach to mucosal surfaces of respiratory or urogentical tract.
n. Some may go inside cells to cause chronic disease.
i. Now being investigated.
XXVIII. Pathogenesis [S28]
a. You inhale respiratory secretions and they attach.
i. P1 and other proteins
ii. Immunogenic - you make antibodies against them when you have a mycoplasma there.
b. When mycoplasma attaches to respiratory epithelium, it will produce peroxides, damage cilia, stimulate white
cells to come there, stimulate production of cytokines
i. Can cause bronchospasm and wheezing, asthmatic-type manifestations
c. Like the Neisseria, it does antigenic variation- so you get no protective immunity because every time it comes
back it comes back in different costume.
i. Immune system does not recognize it, so it starts all over again and causes disease.
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d. Kids, adults, anyone can get it.
e. Autoimmune actions occur- that’s why some people get hemolytic anemia because antibodies cross-react
with red blood cells.
i. Get a lot of pulmonary manifestations because of autoimmune effect.
f. Oxidative damage because of superoxide radicals inhibit host catalase.
g. Now we know there is a toxin that is similar to the pertussis toxin, called the MP CARDS toxin. It causes
ciliostasis and vacuolation in the mucosa of the epithelium and is very similar structurally to some of the
pertussis toxins.
XXIX.
Mycoplasma Detection [S29]
a. Colonies growing on enriched agar. SP 4 + agar
b. Have to provide mycoplasmas with sterol.
c. They have a complicated membrane that requires sterol.
d. Slow-growing organism.
e. This takes a week or two or three weeks sometimes to grow.
f. Hydrolyzes glucose, and these days we can use PCR to detect mycoplasma.
g. Many years people did antibody titers for it, would take three weeks when you have to come back.
h. Now we can do PCR and tell them what they have today or tomorrow.
i. Antibody detection is useful in some cases.
i. Children are often making IgM.
ii. If you have an IgM that is positive, that may be the answer.
iii. Sometimes the IgM can hang around for a long time and serology is problematic
j. We are moving more to PCR because nothing else works too well.
XXX.
M. pneumoniae Disease [S30]
a. Most common disease is tracheobronchitis.
b. Mycoplasma is one of the most common things that causes coughs.
c. Kills respiratory cells, damage cilia, get cough.
d. Any age is affected: common in young children and teenagers.
e. By the time you are an adult and have had it several times, it is more of an aggravation than something that
makes you real sick.
f. Called walking pneumonia.
i. Not sick enough to go to the hospital, and you sort of drag around and cough.
ii. Somewhat similar to viral infection but difference is this will respond to antibiotic and virus won’t.
g. About one in three people that get mycoplasma get pneumonia.
h. May cause of up to 50% of all pneumonias.
i. Since it’s fairly mild, they might not even go to the doctor.
j. Some cases are more severe, and it is similar to chlamydia pneumonia.
k. Well known to cause outbreaks in boarding schools, military camps.
l. Don’t have vaccine because of antigenic variation.
m. There is some suggestion that this organism has a role in asthma.
n. Mycoplasmas is much more common in children with asthma than children without asthma.
o. If you give children with asthma antibiotics, their lungs function better.
p. Mycoplasmas are known to produce wheezing.
q. You can reproduce the symptoms of asthma in animals by putting the organisms in there.
r. It is very possible that this organism has a very important role in the pathogenesis of asthma.
s. If you don’t have any genetic tendencies or underlying problems of asthma, you can get it from mycoplasma
just by itself.
XXXI.
Other Mycoplasmas [S31]
a. Mycoplasma hominis and Mycoplasma genitalium - Urogenital mycoplasmas
b. Mycoplasma genitalium- important cause of non-chlamydial and non-gonococcal urethritis and cervicitis.
c. Mycoplasma hominis- opportunistic
i. Cause UTIs, nephritis, abscesses
ii. Can cause infection in newborn infants well because many people carry these in urogenital tract and can
be transmitted to babies.
d. Ureaplasma species are different from the mycoplasma
i. These are all cell wall-free organisms
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ii. Unique in that they metabolize urea, can produce kidney stones
iii. Most common organism that causes inflammation in placentas.
iv. Can cause premature birth and delivery because they can infect women in utero and cross fetal
membranes.
v. Can be inhaled by baby and cause pneumonitis.
vi. Linked to development of chronic lung disease in infancy because cause inflammation in lungs and
requires baby to have oxygen for long periods of time which leads to oxygen toxicity.
e. Mycoplasma genitalium- urethritis and cerviticis
XXXII. Diseases due to Genital Mycoplasmas [S32]
a. Talked about those
XXXIII. Diseases due to Genital Mycoplasmas [S33]
a. All diseases that have been associated with genital mycoplasmas.
b. Ureaplasma and M. hominis can be cultured within couple days.
c. Genitalium- PCR only way you can identify them because it’s slow-growing
XXXIV. Detection of Genital Mycoplasmas and Ureaplasmas [S34]
a. Went through that already
XXXV. Brucella [S35]
a. Small, gram negative coccobacillus
b. Looks like haemophilus.
c. It’s a zoonotic infection.
d. B. abortus causes abortion in cattle
e. B. melitensis- most common cause of human infection
i. Can be acquired if you’re around animals.
f. Read slide
g. Don’t see much brucellosis in US because cows are vaccinated for it and we have good handling of meat.
h. We don’t encounter it very much.
i. Intracellular, grows in macrophages
j. Fastidious, slow-growing
XXXVI. Brucellosis [S36]
a. Much more common in other parts of world.
b. Much more common in other parts of world (direct contact of consumption of contaminated foods, or direct
inhalation)
c. Undulant fevers- fevers of unknown origin
XXXVII. Brucellosis [S37]
a. Can identify by culture of bone marrow or blood.
b. Takes several days in laboratory.
c. Serology- looking for antibody response.
d. Agent of bioterrorism- very easy to concoct in laboratories.
i. Can do it in a barn.
ii. Scrape some off infected animal, put it in culture, and get large numbers of it.
e. Because it can be aerosolized, when we suspect patient of brucellosis we have to be careful because we
don’t want to infect laboratory with it.
f. Not allowed to use it for teaching purposes because can get in wrong hands.
XXXVIII. Francisella tularensis [S38]
a. Agent of tularemia, or rabbit fever
b. (no pepSmall, gram-negative coccobacillus
c. Intracellular pathogen
d. Common in many animals
e. Most common place you see it is in Midwest of US (Arkansas or Missouri).
f. Few cases a year.
g. Can be transmitted by ticks, or direct contact with rabbits.
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h. Can get it through inhalation or cutaneous exposure.
XXXIX. Tularemia [S39]
a. Person’s finger with tularemia.
b. If you suspect you have it, make sure you are careful of it in laboratory.
c. Can be transmitted to laboratory workers through aerosols or inoculation or injury.
d. Refer culture to public health laboratory.
e. Serology – four-fold rise in titer is commonly used to diagnose infection
f. There is vaccine but not used much because it’s uncommon.
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Scribe: FARAH BUTT
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