Professional Documents
Culture Documents
Heat Fixing: After air drying a slide, applying gentle heat helps the bacteria to adhere to the slide.
Simple Stain (Methylene Blue): Stains all cells and can illuminate morphology.
GRAM STAIN:
o PRIMARY STAIN: Crystal Violet stains both Gram+ and Gram- and makes all bacterial cells appear purple.
Both Gram+ and Gram- will stain with this dye.
o CHELATING AGENT: Add Gram's Iodine which will adhere only to cell walls that have a lot of peptidoglycan in
them (i.e. Gram+)
o DECOLORIZATION: Add Acetone very briefly to wash away the Crystal Violet dye.
Gram-: The purple dye will be washed away.
Gram+: The purple dye will remain adherent.
o COUNTERSTAIN: Then add Safranin which will stain cells pink.
GRAM-POSITIVE: Will appear purple (from Crystal Violet) -- Safranin doesn't stain it.
GRAM-NEGATIVE: Will appear pink, as it takes up the Safranin.
o FALSE GRAM-NEGATIVES: Dead Gram+ cells will appear as though they are Gram-. In antibiotic-treated
specimens, a certain portion of such cells is expected.
ACID-FAST STAIN: Stains Mycobacteria such as Mycobacterium Tuberculosis.
o LIPID cell wall gives them the property of weak initial staining, plus strong retention of initial dye (like Gram+)
once stained.
o PROCESS:
Carbol-Fuscin dye is used initially, with heat, for 20 to 30 minutes (long time)
Acid-Alcohol is then used as a decolorizing agent.
If the organism resist the decolorization, it is considered acid-fast.
FLUORESCENT STAIN:
o It has the advantage that you can scan at low power until you find something that fluoresces, then you can hone in
on it.
IMMUNODIAGNOSTICS
o Hemagglutination Inhibition: Certain organisms can agglutinate red blood cells. If you have antibodies to impede
that agglutination, then you have a positive test result (antibody is present).
o Direct Fluorescent Antibody: Fluoro tagged antibody checks for presence of antigen directly.
o Indirect Fluorescent Antibody: First allow antigen-antibody reaction, then use a second Fluoro tagged antibody
(anti-antibody) to identify presence of the first antibody.
o Complement Fixation: Usually avoided; expensive.
o ACUTE and CONVALESCENT TITERS: Take an antibody titer at initial infection (acute), and again four to five
weeks later. You would expect a four-fold increase in titer if the suspected organism is indeed the infecting
organism.
There is usually a baseline level of antibody present with or without infection, because of cross-reactivity
with other antigens. Thus you use acute:convalescent titers as a means of comparison.
For some organisms, there are also absolute antibody levels that are diagnostic.
o Radio-Immunoassay (RISA, ELISA): Take the patient's direct specimen and try to detect residual evidence of the
organism's presence. Used when recovery of bacteria itself is not possible.
Molecular Probes
Polymerase Chain-Reaction (PCR): Can also be used for direct detection but it's expensive.
CULTURE:
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PROCESS: Take an inoculating stick, flame it, dip it into the bacteria (tissue sample), and then streak across the
plate. Then flame it again, and streak it again.
SUSCEPTIBILITY TESTS:
o KIRBY-BAUER METHOD of Antibiotic Susceptibility Testing: Testing is done using the Disk-Diffusion
Method
PROCEDURE:
Grow confluent bacterial colony on plate
Add antibiotic disc in center.
Measure the Zone of Inhibition (diameter) to determine bacterial susceptibility.
RESULTS: The larger the zone of inhibition, the more susceptible are the bacteria.
Resistant: No zone of inhibition is found.
Intermediate: Intermediate zone of inhibition.
Susceptible: Larger zone of inhibition.
An interpretive table or computer, along with known concentrations of antibiotic, must be used to
interpret results.
o E-TEST: ANAEROBE Susceptibility Test. Strict Anaerobes must be cultures under special conditions.
Instead of a disk, the antibiotics are contained in capillary disks.
o SPIRAL GRADIENT ENDPOINT TEST: Another susceptibility test
Line several isolates (can be from different patients) up on a single plate in wheel-spoke fashion.
Growth inhibition at each line can then be observed and interpreted by computer.
o SERUM BACTERICIDAL TEST: Susceptibility test for patients that are extremely sick. Take serum sample and
test amount of antibody actually in patient's serum. Then plate that antibody out onto culture and verify that it
actually does kill the bugs at its peak and trough concentrations in the patient's serum.
A 1:8 dilution of serum inhibiting growth should be indicative that antibiotics are effective enough to kill
bacteria in vivo.
MINIMUM INHIBITORY CONCENTRATION (MIC): The minimum amount of antibiotic necessary to inhibit bacterial
growth to the point that no bacteria are visible with the naked eye.
o This doesn't necessarily mean that no bacteria are there at all. Healthy person should be able to handle this bacterialevel with normal host-defenses.
o PROCEDURE:
Take test-tubes containing varying concentrations of antibiotic, plus a control tube with no antibiotic.
Add growth media and 107 bacteria to each tube.
The most dilute tube in which no turbidity is seen is the MIC -- the minimum antibiotic concentration that
inhibits bacterial growth.
o MINIMUM BACTERICIDAL CONCENTRATION (MBC): Amount of antibiotic necessary to kill 99.9% of all
organisms. This value must be used when treating immunocompromised patients.
PROCEDURE: To determine MBC, we must take all of our clear tubes from the MIC and culture them
onto plates. Then, the plate on which the fewest number of colonies grows is termed the MBC.
o Antibiotic Tolerance occurs when an isolate has a serial difference of five tubes between its MIC and MBC.
Normally, the difference is only one or two tubes. This translates to a 32-fold difference in concentration.
CHOCOLATE AGAR: Heat-lysed red-blood cells in agar.
o
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Streptococcus Pneumoniae
Causes of NEONATAL (less than 3 months old) MENINGITIS and SEPSIS: In order
1.
2.
Group-B Strep
Escherichia Coli
E. Coli
Proteus Vulgaris / Mirabilis:
o 10-15% of hospital acquired UTI's.
Pseudomonas Aeruginosa
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STAPHYLOCOCCI
STAPHYLOCOCCUS AUREUS: Gram(+) cocci in pairs or clusters.
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Exotoxins:
HEMOLYSINS:
alpha-Hemolysin: Leads to beta-Hemolysis, or complete hemolysis of WBC's and RBC's
Kills WBC's as well as RBC's
Transposon mediated
Causes pore formation in blood cell membrane.
beta-Hemolysin: Only damages membranes at a cold temperature, 4C; unique to Staph Aureus.
At 37 it alone is not sufficient to produce hemolysis.
gamma-Hemolysin: not much known
delta-Hemolysin: Detergent, or surfactant-like action.
Kills WBC's as well as RBC's.
Panton-Valentine Leukocidin: Causes WBC lysis and divided into two components.
SLOW COMPONENT binds to GM1 ganglioside in PMN membrane.
FAST COMPONENT then uses phosphatidylcholine as a receptor to gain entry.
EXFOLIATIN: Cleaves the stratum granulosum and causes desquamation in Scalded Skin Syndrome.
Protein a: chromosomal.
Protein b: plasmid-mediated.
ENTEROTOXINS A, B, C1, C2, D: Affects the GI-tract in S. AURES food poisoning.
They are heat stable -- heating (or reheating) food does not denature the toxin.
Toxic-Shock Syndrome (TSS) Toxin: It is a superantigen.
o Cell-Associated:
Protein A: Binds the Fc portion of IgG, all four subclasses.
It is antiphagocytic, anticomplement, antigenic, and mitogenic for T-Cells.
It activates cell-mediated immunity and Type IV hypersensitivity response.
Peptidoglycan: Confers hardiness and activates Alternative Complement pathway.
Teichoic Acids: They act as a phage receptor.
Ribitol Phosphate (Polysaccharide A) results in a lot of antigen-antibody complex formation -----> immediate type hypersensitivity.
Surface Polysaccharides: Adhesins, antiphagocytic.
Vaccine / Prevention: Wash your hands!
Treatment: Antibiotics do not work!
o
STAPHYLOCOCCI SAPROPHYTICUS:
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ZOONOTIC INFECTIONS
All REPORTABLE diseases
BACILLUS ANTHRACIS: Gram(+) large rods with square ends.
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BACILLUS CEREUS:
Epidemiology / At Risk:
o Food-Poisoning = refried beans and rice
o Bacteremia = OPPORTUNISTIC. Nosocomial, immunocompromised patients.
Manifestations:
o Food-Poisoning in refried rice and beans is self-limiting to about 24 hrs.
Early on (1-6 hrs): Nausea and vomiting
Later (24 hrs): Profuse diarrhea
o Bacteremia (contaminated catheters) in immunocompromised leads to meningitis, endocarditis, death.
Identification:
o B. Cereus is penicillin-resistant. They will form chains of rods rather than string of pearls in penicillin suspension.
o gamma-PHAGE does not lyse it.
Virulence:
o Enterotoxins:
Emetic Toxin: Heat stable, basis for early symptoms.
Diarrheal Toxin: Heat labile, basis for late onset.
Stimulates host cell adenyl cyclase ------> cAMP
o Chromosomal beta-Lactamase and Cephalosporinase
BACILLUS SUBTILIS:
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STREPTOCOCCI
GRAM (+) COCCI, LACTOSE (+)
STREPTOCOCCUS PYOGENES (GROUP A): Gram (+) Cocci in pairs or chains of 4 to 8.
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Virulence:
o Cell-Associated:
Cell-Wall Carbohydrate: Not an effective human antigen
Peptidoglycan: Induces inflammation
Lipotechoic Acid: Adheres to buccal mucosa, allowing colonization. Adhesin.
M-Protein: Fibers extending from surface.
Antigenically diverse (85 types) and immunogenic, to which we make protective antibodies.
This means we could get 85 strep-throats in our lifetime.
Antiphagocytic:
Clumps PMN's and white cells.
Interferes with alternate complement by binding Factor H
Interferes with classical complement by inhibiting C3B deposition onto cells.
M-Associated Protein (MAP): Antigenic, can cause cross-reaction with myocardial sarcolemma.
T-Antigen: Associated with Glomerulonephritis.
G-Protein: Binds Fc portion of IgG.
Streptococcal Chemotactic Factor: Inactivates C5a. Shuts down inflammatory response in host.
Capsule: During early inflammation, it is made antiphagocytic due to hyaluronidase. We can't develop
antibody to the bugs until the capsule is lost.
Capsule accounts for the smooth texture in colony.
o Extracellular: Release right outside of cellular environment.
DNAase: Four Types
Type B: Is antigenic and is diagnostic for glomerulonephritis. Most prevalent in skin infections.
Streptokinase: Works with TPA to digest clots. Can be immunogenic. Repeated therapies with
streptokinase as a blood-thinner can result in hypersensitivity.
Hyaluronidase: Degrades its own capsule, plus host connective tissue.
Proteinase: Used to digest its own M-Protein.
o Exotoxins
Streptolysin O: Oxygen labile and not expressed in vitro. It is hemolytic in vivo.
It lyses RBC's and WBC's under reduced oxygen tension. Damages platelets.
Antigenic: Basis for the ASO test.
Streptolysin S: Oxygen-stable. Hemolytic, but there is no lysis in vivo. Very small and not antigenic.
Erythrogenic Toxin: Three types. Causes the rash seen in Scarlet Fever.
Type-A also known as Superantigen Erythrogenic Toxin A (SEA), and causes Toxic-Shock Like
Syndrome in the elderly.
Host Immune Response:
o Antibody to M-Protein is protective.
o Antibody to Streptolysin O (ASO) is diagnostic for suppurative infection to Strep A, C, or G.
o Antibody to DNAse B is diagnostic for non-suppurative sequelae.
Vaccine / Prevention:
Treatment: Penicillin-G will work. Strep to date is not resistant.
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Virulence:
o Capsular Antigens: Type III is associated with neonatal meningitis.
o Can withstand bile in GI tract.
Vaccine / Prevention: Under development for mother.
Name-Derivation: feces.
Epidemiology / At Risk: Normal GI flora.
Manifestations: Multiple infections. Often a complication of cholecystitis.
o GI obstruction may lead to bacteremia and endocarditis, due to bacterial resistances.
Processing:
o Stain:
GRAM-VARIABLE -- both Gram (+) and Gram (-) found, alive.
o Culture: Use blood agar with 40% bile and 6.5% NaCl. All three types of hemolysis found in culture.
Identification: Compared to Strep Bovis (Group D)
o Grows in the presence of bile.
o Penicillin resistant.
Virulence:
o LIPOTECHOIC acid, very lipid rich, leads to gram-variable appearance.
Treatment: Penicillin resistant, strongly, due to altered Penicillin-binding proteins.
o Also have acquired vancomycin and gentamycin resistance.
GROUP G STREPTOCOCCI:
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o Culture:
Identification: ASO (+)
Virulence:
o Streptolysin O: Oxygen labile hemolysin.
o DNAse: Invasive.
o Streptokinase: Antigenically unique.
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VIRIDANS STREPTOCOCCUS:
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Bugs of Childhood
HAEMOPHILUS INFLUENZAE: Short Pleomorphic Gram (-) Rods.
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Name-Derivation:
o Haemophilus = blood-loving
o Influenzae = discovered as part of flu epidemics.
Epidemiology / At Risk: Infantile Meningitis.
Manifestations: There are typable (encapsulated) and non-typable strains
o TYPABLE: Haemophilus Type-B infections
Infantile Meningitis: #1 cause in kids older than 3 months.
Epiglottitis in kids 2 - 5, possibly with sepsis.
Cellulitis in kids.
Bacteremia: Can either be serum-sensitive (serum + complement lyse the bugs) or serum-resistant (serum
+ complement can't lyses the bug).
Serum sensitivity is conferred by the LOS coat. The higher the molecular weight of the LOS, the
worse is the bacteremia.
High molecular weight LOS: Serum resistant.
Low molecular weight LOS: Serum sensitive.
o NON-TYPABLE:
Otitis Media
Bronchitis and pneumonia
Processing:
o Specimen: CSF from infants.
o Stain: Gram (-) rods, but the poles tend to remain purple. Thus distinguishing with Strep Group-B can be a problem.
o Culture: Both Smooth and rough colonies seen in culture.
ABSOLUTE GROWTH REQUIREMENTS: Both derived from RBC's.
Factor X: Hemin precursor.
Factor V: NAD, used for organism pyridine synthesis.
Chocolate Agar: Heat-lysed red blood cells.
Fildes's Agar: Enzymatically (rather than heat) lysed RBC's.
Satellite Growth: Put the bugs in blood agar with Staph Aureus, they will grow around the perimeter of the
Staph. This is because the Staph Aureus lyses the RBC's and provides the needed nutrients for the Hib to
grow around perimeter.
Identification:
o TYPABLE: Types a thru f, but Type b is the only important one. Other strands are not typable.
QUELLUNG REACTION: Can be done on CSF to identify Hib. Add methylene blue to visualize:
Positive test shows rod-shaped organisms with halo, due to swelling of the capsule.
Can use CIE, ELISA, or Latex Agglutination to look for residual antigens left in blood, CSF, or urine.
o NON-TYPABLE: Pleomorphic appearance on stain, still with intensity at the poles.
Virulence:
o Capsule: When present, it is the basis for typing.
Type-B is made out of poly-ribitol phosphate. We don't really make antibodies to it, thus it is more
virulent. We do make antibodies after repeated exposure. Presence of Ab is AGE-DEPENDENT:
Infants younger than 3 months are protected by maternal IgG.
Infants older than 3 months have lost maternal IgG immunity, thus they become susceptible to Hib
meningitis.
Immunity is re-acquired by age 10 due to cross-reactivity with Staph Aureus and E. Coli ribitol
moieties.
o Outer Membrane Proteins: Looking at them for a vaccine. Various ones are antiphagocytic and invasive.
P2, porin protein is most promising candidate.
o Lipo-oligosaccharide (LOS): Differs from LPS in that it has less sugar. Bacteremia does not result in the serious
endotoxic shock of E. COLI, for example.
The LOS coat inhibits movement of cilia in the airway and helps to establish infection.
Molecular weight of LOS determines serum-resistance in bacteremia.
o IgA Protease: Allows Hib to establish in upper airway.
o beta-Lactamase: Plasmid-mediated.
Host Immune Response: Antibodies are protective. IgG is the most protective.
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Vaccine / Prevention: DPT Vaccine contains the Hib Conjugate -- Diphtheria Toxoid plus Poly-Ribitol Phosphate. 100%
effective.
o Vaccine administered at 2 months, 4 months, and 6 months.
o PRP by itself can only be given to kids older than 2 years. PRP by itself is not as effective. The conjugate tends to
elicit more of an IgG response (rather than IgM), which is what we want.
Treatment: Treated based on antibiotic susceptibility test results.
o Penicillin-Resistant.
o Chloramphenicol-Resistant, via Chloramphenicol Acetyl-Transferase (which inactivates the drug).
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100 kDa: Interferes with PMN phagocytosis and respiratory burst; hinders chemotaxis; hemolysin; impairs
alveolar macrophage metabolism.
o Other Toxins:
Heat-Labile Toxin: Released upon lysis of the bug. Causes dermonecrosis (or surface necrosis) of ciliated
epithelial cells upon release. Remember that B. Pertussis is not invasive.
Heat-Stable Toxin: Endotoxin with Lipid-A, similar to LPS.
Also contains Lipid X which stimulates protective antibodies.
Tracheal Cytotoxin: A small peptidoglycan fragment. Ciliastatic.
Vaccine / Prevention: DPT vaccine, at 2, 4, and 6 months.
o Initial shots: DPT. Pertussis component is methiolate-killed whole organisms.
o Booster Shots: DTaP. At 15 months and preschool, we use an acellular vaccine for a booster of FHA + Pertussis
Toxoid.
o Side-Effect: Risk of 1/300,000+ of encephalopathy.
Treatment: Erythromycin.
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TOXOID
REPORT
36 hrs
120 hrs
36 hrs
120 hrs
Neg
Pos
Neg
Neg
erythema and
inflammation
found
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Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Pos
Pos
Neg
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GRAM-NEGATIVE PNEUMONIAS
KLEBSIELLA PNEUMONIAE: Gram (-) Rod
LEGIONELLA PNEUMOPHILA: Faintly staining Gram (-) rods. Part of its own family, Legionellaceae.
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MYCOBACTERIA
Facultative Intracellular Parasites
Catalase (+)
MYCOBACTERIUM TUBERCULOSIS, MYCOBACTERIUM BOVIS: Acid-Fast Rods
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MYCOBACTERIUM LEPRAE:
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Epidemiology / At Risk:
Manifestations: LEPROSY
o Tuberculoid Leprosy: Small, organized tuberculoid lesions (granulomas) on skin.
Hypopigmented lesions. Cannot be distinguished from contact dermatitis.
Skin, nerves.
Cutaneous anesthesia results from occupations of nerves.
o Lepromatous Leprosy: Unable to mount cellular response (anergic) to the bugs, resulting in much worse skin
lesions.
No granulomas are formed. "Lepra" cells can be seen.
Affects skin, nerves, eyes.
Processing:
o Specimen:
o Stain: Acid-Fast
o Culture: Has never successfully been grown up in culture!
Can use Armadillo or Mouse footpad models for live hosts.
Identification:
Virulence:
o Obligate Intracellular Parasite inside Macrophages. Can inhibit macrophage phagolysosome fusion.
o CD8+ Suppressor Cells prevent Granuloma formation in Lepromatous form.
o Phenolic glycolipid appears to be key antigen.
Host Immune Response:
Vaccine / Prevention:
o SKIN TEST: Antigen was sequestered from Armadillos, who are quite affected Leprosy.
Skin Test can be used only for prognosis. A good (> 10 mm) response indicates that patient can mount a
good response, so lepromatous form should result.
Treatment: Sulfa drugs.
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ENTEROBACTERIACEAE
YERSINIA ENTEROCOLITICA: Gram-Negative Rod
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Name-Derivation: Originally part of Campylobacter, then given its own genus. Helical = wavy.
Epidemiology / At Risk:
Manifestations:
o Type-B Gastritis: Inflammatory, pyogenic infection of antrum and goblet cells. pH remains normal.
o Type-A Gastritis: Autoimmune disease against parietal cell resulting in pernicious anemia. pH is high due to
damaged parietal cells.
Processing:
o Specimen: Must be taken by gastric endoscopy. No specimen is usually collected.
o Stain:
o Culture: Campy Agar
NORMAL TEMP 37, as compared to the Campylobacter
Identification: Microaerophilic
o UREASE (+)
o Catalase (+)
o UREA BREATH TEST: Give patient radiolabeled 14C urea, then monitor for the appearance of the 14C in their
breath or blood, indicating that it has been broken down. Quick and easy test.
Virulence:
o Flagellum: Number one property allowing it to invade gastric mucosa.
o Mucinase helps penetrate mucin layer.
o Fibrillar Hemagglutinin: Adhesin. It hooks to a glycero lipid in host cell membrane.
o Catalase: Can survive (not replicate) inside PMN's.
o Urease: Thought to create a basic microenvironment which damages intracellular junctions in stomach.
o Oxidase
Treatment: Three antibiotics, plus Bisthmus (Pepto-Bismol) as a coating against acid damage.
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Epidemiology / At Risk: Spores found in soil and are usually ingested. Can be opportunistic.
o Endogenous Infection: Spores that were otherwise silent become activated when normal flora is depressed by
antibiotics.
o Exogenous Infection: Nosocomial infection in the hospital.
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Manifestations:
Processing:
o Specimen: Fecal filtrate is taken for toxin assay.
o Stain:
o Culture: Egg-yolk agar with antibiotics.
Identification:
o Obligate anaerobe
o Serogroups A - G
o Can be identified through Gas Liquid Chromatography.
o Toxin Test: Toxin is present only in some of the strains. Must determine whether it is present.
Put feces specimen filtrate in two dishes. Add antitoxin to one of the dishes.
Positive Test: One dish remains toxic, while the other dish is neutralized.
Negative Test: Neither dish is toxic.
Low Specificity: The antitoxin can cross react with other toxins.
o ELISA can identify C. Difficile toxin.
Virulence:
o Exotoxin A: Protein that damages the intestinal mucosa. It attracts PMN's and causes them to degranulate, resulting
in more damage.
o Exotoxin B: AB-Toxin that disrupts the cytoskeleton of enterocytes.
Fragment-A gets inside with the help of a host-cell protease. Mechanism of damage unknown.
Treatment: Treatment has a high relapse rate.
o Discontinue broad-spectrum antibiotic therapy. Replace lost fluid and electrolytes.
o Do not interfere with diarrhea. Let it run its course.
o Vancomycin (some resistance has shown) or metronidazole is used when necessary.
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Epidemiology / At Risk:
o UTI's
o Gastroenteritis
o Wound Infections
o Pneumonia
o Meningitis in Infants
o Sepsis
Manifestations: GI manifestations depends on strain. Virulence factors included.
o Enteropathogenic E. COLI (EPEC): Cause Travelers's Diarrhea -- loose stools, plus mild GI complaints, such as
nausea, vomiting, or even tenesmus.
Has also been called Entero-Aggregative E. COLI (EAEC) because of their tendency to aggregate.
o Enterotoxigenic E. COLI (ETEC): Watery diarrhea, as opposed to loose stools. Diarrhea acts on the small
intestine.
Labile Toxin (LT): Heat labile AB-toxin kicks water out by up-regulating cAMP.
Fragment-B: Binds GM1 Ganglioside in small intestinal enterocytes.
Fragment-A: ADP-Ribose Transferase. Transfers ADP to a Gs Stimulatory subunit ------> cAMP.
LT is also a large molecule and a potent antigen.
LT-IIa and Iib: Antigenic variants of labile toxin.
Stable Toxin (ST): Heat stable AB-Toxin, prevents water from being reabsorbed in small intestine.
Fragment-A: Up regulated cGMP ------> inhibit reabsorption of Na, Cl, and water in brush border.
o Enteroinvasive E. COLI (EIEC): Causes dysentery in addition to the watery diarrhea. The new cytotoxin acts in
the colon, while watery diarrhea continues to occur from the small intestine.
Verotoxin: Phage-mediated Shiga-Like Toxin is cytotoxic to colonic enterocytes. It inactivates protein
synthesis at the 60s ribosome and kills the cell, resulting in hemorrhagic necrosis.
Invasin: Gene allows the E. COLI to live intracellularly inside colonic enterocytes.
o Entero hemolytic E. COLI (EHEC): Causes Hemolytic Uremic Syndrome (HUS). In addition to the dysentery, it
has a hemolysin and is tropic for transitional epithelial cells.
Symptoms:
Hemolytic Crisis
Thrombocytopenia
Disseminated Intravascular Coagulopathy (DIC)
Acute Renal Failure
Hemolysin: Plasmid-mediated factor that lyses red-blood cells. It is also a nephrotoxin.
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Processing:
o Specimen:
o Stain: Gram-Stain is not done on fecal specimens.
o Culture: Selective Medium (always used on Enterics), which inhibits Gram (+) and contains lactose, in order to
differentiate lactose-fermenting genera.
Identification: Lactose-Fermenting
o Motile
o Huge Antigenic Diversity: Flagellar H-Antigen is divided L, A and B subtypes.
With Neonatal sepsis and meningitis, lab will report a B-subtype as it is associated with prognosis.
The LAB subtypes indicate how easily the flagellar antigens come off with heat.
o Mannose-sensitive hemagglutination, because of F1 pilus antigen.
Virulence: Only cell-associated factors.
o Pili (F-Antigen): Fimbriae. 10 F Antigen types. Adhesin.
F1 Antigen is found in all E. Coli. (chromosomally coded). It is Mannose-Sensitive, i.e. does not
agglutinate RBC's in the presence of mannose (because it prefers to stick to the mannose).
Mannose sensitive is important to us as normal carriers of E. COLI. It helps E. COLI stick to
mucosal (vaginal, GI, buccal) surfaces and protect them.
F2 - F10 Antigens: They are all mannose-resistant.
One of them is the P-Antigen, associated with Pyelonephritis.
o Capsule (K-Antigen): Important in UTI's and Meningitis. Antiphagocytic, serum resistance.
o Outer Membrane Proteins: Protein-A confers serum resistance.
o Siderophore: Aerobactin
o LPS: Lipid-A is the specific component which is a superantigen and makes up endotoxin.
Vaccine / Prevention:
o The Core Polysaccharide, common to all strands, has been looked at. But whenever we target it, E. COLI respond by
making new surface (O-Antigen) polysaccharides.
Treatment: Run the risk of inducing endotoxic shock when treating bacteremia.
SHIGELLOSIS:
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Different Species:
o S. Sonnei: Found in U.S.
o S. Flexneri: Found in U.S.
o S. Boydii: Found in U.S.
o S. Dysenteriae: Found mostly abroad, and contains the extra Shiga Toxin.
Epidemiology / At Risk: Communicable. Very low infective dose of only 200 - 500 bugs.
Manifestations: Ulcerative colitis. Invasive disease.
o Children in U.S. with Shigellosis can experience bacteremia and CNS toxicity.
Processing:
o Specimen: Ulcer swab or feces.
Special transport medium required. The organic acid by-products of other normal flora are toxic to the
Shigella bugs.
o Stain:
o Culture: Selective Medium
Identification: Non-Lactose Fermenting
o Non-Motile, thus no H-Antigen.
Virulence:
o Surface Polysaccharide: Plasmid-mediated, adhesin and invasin.
o Outer Membrane Proteins: Invasin allows mucosal penetration by receptor-mediated endocytosis. Details uncertain.
o Hemolysin: Plasmid-mediated (only some strains have it). Disrupts phagolysosome formation and allows
intracellular replication in PMN's.
They can also actually infect neighboring cells, eventually leading to an ulcer.
o SHIGA TOXIN: Only present in the S. Dysenteriae strain, making it the most virulent.
In the small intestine, it blocks absorption of NaCl, glucose, and water.
In the colon, AB-Toxin.
Fragment-B binds a glycolipid on colonic enterocyte.
Fragment-A gets internalized and is then known as Fragment-A1, which is an N-Glycosidase. It
removes adenine from the 28s rRNA and irreversibly in activates protein synthesis at the 60s
ribosomal subunit.
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Some of the U.S. bugs produce a Shiga-Like toxin that produces similar effects -- not but Shiga toxin, and
details are not characterized.
Treatment: Shigellosis is a public health issue. Antibiotic treatment is required.
SALMONELLA TYPHI:
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COMPLICATIONS:
Intestinal perforations
Thrombophlebitis.
Cholecystitis and gallstones
Patchy necrosis caused by complement fixation and PMN inflammation.
DIC and focal abscesses.
Processing:
o Specimen:
Blood can be taken in early in infection.
Feces can be taken in late infection (when diarrhea starts)
o Stain: Not useful.
o Culture: Enteric Selective Culture containing lactose and bile.
Culture under aerobic conditions 1- 7 days.
Will show motile bugs with smooth colonies.
Identification:
o Non-Lactose fermenting
o Motile
o Facultative intracellular parasite.
Virulence:
o Capsule Antigen (Vi): Polysaccharide, antiphagocytic, serum resistance. This enhances survival inside monocytes.
Immunogenic and basis for partial-vaccine.
o Intracellular in Monocytes: Facultative intracellular parasites of monocytes. The capsule protects them from
destruction.
o Flagellar (H) Antigen: Biphasic antigenic expression.
o Outer Membrane Proteins (O-Antigens): Antiphagocytic inside monocytes. Enhances resistance to non-oxidative
cationic proteins (defensins) inside PMN's.
o Endotoxin.
Vaccine / Prevention:
o TAB Vaccine: For travelers, short-term temporary protection. Passive antibody to the Vi antigen, allowing for
phagocytosis and good complement response.
o Prevention: Chlorinate water, sewage disposal, cook your food.
Treatment: Carrier state may be prolonged by antibiotic treatment. Cholecystectomy may be needed.
o
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Virulence:
o Polar Flagellum: Provides motility and penetration through mucin.
o Pilus: Along with an accessory colonizing protein, this is an adhesin that allows adhesion to brush border.
o Mucinase: Aids invasiveness -- but not beyond the intestinal lumen.
o Cholera Toxin: Exotoxin causes diarrhea.
AB-Mechanism:
B-Fragment binds GM1-ganglioside of enterocyte.
A-Fragment enters the cell, and a disulfide bond is broken so that it cleaves into two parts.
A1 Fragment transfers ADP-Ribose to the Gs-subunit, making it permanently activated ------>
perpetually high cAMP, diarrhea.
Cholera toxin also prevents absorption of water in the brush-border. Double-duty diarrhea.
Vaccine / Prevention: Live attenuated vaccine currently provides short-term protection.
Treatment:
o Oral Rehydration Therapy: Glucose and water. IV replacement fluids are only used in most severe cases.
o Tetracycline may be used, but can also produce resistant strains. Use with caution.
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Epidemiology / At Risk:
o Associated with raw shellfish.
o High infective dose.
o People with underlying liver disease are at risk.
Manifestations:
o Self-limiting diarrhea + GI symptoms lasting 3 days.
o Wound infections in fisherman.
Processing:
o Specimen:
o Stain: Small gram (-) rods with single large flagellum.
o Culture: Salty (3% NaCl) selective medium.
No hemolysis in vitro -- only in vivo.
Identification:
o V. Parahaemolyticus grows in 8% NaCl whereas V. Cholera does not.
o Facultative Anaerobe.
Virulence:
o Hemolysins: they only act in vivo.
Treatment: Not usually treated.
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Epidemiology / At Risk:
o #1 organism responsible for UTI's.
o Organisms originate from endogenous flora.
Manifestations: UTI
o Dysuria, Frequency, Urgency
o Cystitis, Urethritis
o Pyelonephritis
Processing:
o Specimen: Mid-stream clean catch
Catheter
Suprapubic aspiration
o Stain: Gram-stain with one or more organisms per oil-immersion field is diagnostic.
o Culture: Enteric Selective Medium (lactose).
Identification:
o Lactose-Positive
o Typical E. COLI Profile: O126:B4:H15:F1,P+
All E. COLI have F1 (mannose-sensitive) pilus
P+ means the Pyelonephritis antigen is present.
B4 represents subtype of flagellar antigens
H15 is another flagellar antigen
Virulence:
o P-Antigen (Pili): Mannose-resistant F2-10 antigen that is associated with pyelonephritis.
o K-Antigen (Capsule): Associated with adherence to transitional epithelium.
o O-Antigen: Cell-wall polysaccharide.
Treatment: No treatment if asymptomatic
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Name-Derivation:
Epidemiology / At Risk: 3rd most common cause of UTI's.
Manifestations: Opportunistic
o UTI's
o Burn Infection: Leads to bacteremia and sepsis.
o Bacteremia: Immunocompromised
Endocarditis
o Pneumonia: Especially in Cystic Fibrosis patients.
Luminal infection is leads to buildup of mucus and pus.
micro-abscesses and necrosis can result.
o Otitis Externa: Swimmer's ear
o Eye infection: Conjunctivitis, keratitis, endophthalmitis.
Processing:
o Specimen: Take blood to test for bacteremia
Clean-catch urine for UTI
Sputum for CF patient
Surface swab of burn
o Stain: Direct microscopy not helpful.
o Culture: Blood agar shows smooth, beta-hemolytic, blue-green (due to pyocyanin) colonies.
Sputum colonies from CF patient will be mucoidy, because of the presence of Alginate which sticks to the
glycocalyx of epthelial cells.
Identification:
o Strict Aerobe, thus they are non-fermenting of all sugars.
o Oxidase (+) -- cytochrome oxidase; again, strict aerobes.
o Juicy-Fruit Smell
o Bug-Typing: Pyocins are used in hospitals to type-strains. These are proteins made by one strain of Pseudomonas
that is lethal to another strain of Pseudomonas. This property allows for mapping of infections by different strains.
Virulence:
o Cell-Associated
Pigments:
Pyocyanin: It is an effective antibiotic against Staph Aureus, thus it overtakes Staph in CF lung
infections.
Pyoverdin:
Helps to acquire iron.
Ciliostatic.
Flagella: Adhesin in the urinary tract.
Pili: Adhesin to skin and upper respiratory tract.
o Toxins
Exotoxin A: AB-Toxin acts similar to Diphtheria toxin. Especially toxic to liver cells (as compared to
Diphtheria which likes heart, nerve, kidneys).
Fragment-A: Transfers ADP to Elongation-Factor 2 (EL-2) ------> inhibit protein synthesis.
Exotoxin S: General AB-cytotoxin that targets a lot of cells. ADP-ribosyl transferase that interrupts protein
synthesis.
beta-Hemolysin: Consists of Phospholipase-C and Glycolipid, which act synergistically to lyse RBC's.
o Enzymes
Alkaline Protease: Ruins PMN interaction with antibodies, thus preventing opsonisation and making a
vaccine difficult.
Alginate: In CF patients, this sticts to the glycocalyx and is toxic to PMN's, resulting in the mucoidy
colony.
Elastase: Responsible for the toxic effects of the bug.
Attacks elastin in vessels and lung.
Damages cornea
Causes pinpoint hemorrhages in skin
Impairs PMN function.
Vaccine / Prevention: Under develpoment, but not much progress.
Treatment: Highly drug-resistant. Big problem.
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SEXUALLY TRANSMITTED
NEISSERIA GONORRHEA (GONOCOCCUS): Gram-negative reniform Diplococci
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Name-Derivation:
Epidemiology / At Risk:
o Sexual Contact
o Fomite (inanimate object) may be means of transmission in prepubescent female, but it is highly unlikely. We must
investigate for sexual abuse.
Manifestations:
o Gonorrhea
Male: Painful urethritis. Purulent discharge and dysuria.
Female: Vaginitis; 80% are asymptomatic.
UTI symptoms
Vaginal discharge
Vaginitis progresses to PID, leading to sterility
Common to have coinfection with Chlamydia (one predisposes to the other).
Anorectal: From anal sex; rectal discharge and bleeding; usually asymptomatic.
Pharyngitis: From oral sex; usually asymptomatic.
o Bacteremia: Can result from failed, incomplete, or inadequate treatment.
Fever, rash, skin lesions. Rash has no bugs in it.
Septic Arthritis: The leading cause of arthritis in people 20-30 years of age!
o Neonatal Conjunctivitis: Rapidly invasive. Destroys cornea and leads to blindness.
By law, NaNO3 or Tetracycline eyedrops are given prophylactically to neonates at birth.
Processing:
o Specimen: Urethral exudate preferred, or swab.
o Stain: Only useful on urerthral exudate. Sensitive for males but not females.
Stain will show lots of variants, but they're from the same isolate. Variation reflects presence or absence of
pili.
o Culture:
Thayer-Martin Medium is Chocolate Agar, with iron and antibiotics added to supress Gram (-)'s, Gram
(+)'s, and Candida. It is usually inoculated at the bedside, because the bugs die very easily.
Vancomycin gets rid of Gram (+)
Colistin gets rid of other Gram (-)'s
Nystatin gets rid of Candida.
Then subculture to Chocolate Agar to speciate (glucose and maltose fermentation)
Identification:
o Neisseria: Catalase(+), Oxidase(+) is key factor.
o Glucose-fermenting
o Non-maltose fermenting (differ from N. Meningiditis).
Virulence:
o Cell-Surface:
Pili
Porin Protein:
Protein I (PI): Complexes with PIII to form the Porin protein.
Structure:
Antigenically diverse: N and C termini are within membrane, and central loop
sticks out which is highly antigenically diverse.
High Molecular Weight: Associated with disseminated disease and increased
serum resistance.
Low Molecular Weight: Associated with localized (UG) disease and decreased
serum resistance.
Action: It triggers phagocytosis.
Protein III (PIII): Complexes with PI to form the Porin protein.
Binds IgG and blocks killing mediated by IgG (serum resistance).
Protein II (PII): Adhesin; causes autoagglutination and adherence to epithelium.
H8: Immunogenic antigen. Antibody and complement will bind to this and lyse the bug.
Penicillin-Binding Protein (PBP):
Peptidoglycan:
Lipooligosaccharide (LOS): Not endotoxin. It damages fallopian tube mucosa and is ciliostatic.
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Enzymes:
IgA Protease: Degrades IgA antibodies; two types
beta-Lactamase
Intracellular survival: Catalase, Superoxide Dismutase, Peroxidase
Host Immune Response: Antibodies are effective, but not protective because of antigenic diversity.
Vaccine / Prevention: Tetracycline or NaNO3 eyedrops given to babies prophylactically.
Treatment: Completely Penicillin-resistant, due to both beta-Lactamase and altered PBP's.
o
Epidemiology / At Risk:
Manifestations: Chancroid. Looks like a Syphillus chancre, but it isn't.
o Painful
o Purulent Exudate
o Non-indurated
o Usually found on genitalia, but can be on lip.
o Suppurative lymph nodes found; may develop abscess.
Processing:
o Specimen: Purulent exudate
o Stain: Gram-negative rods. If you see bugs on the gram stain, then you have established that it is not Syphillus.
o Culture: Chocolate agar, but it is very difficult to grow.
Identification:
o It stains on the gram-stain.
Virulence:
o Pili: Adhesin
o beta-Lactamase
Treatment: Penicillin-resistant. Use erythromycin or bactram.
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BACTEREMIAS
Only cause disease when in bloodstream
NEISSERIA MENINGIDITIS (MENINGEOCOCCUS): Gram-negative diplococcus
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Name-Derivation:
Epidemiology / At Risk: Communicable bia inhaled droplets.
o #2 cause of infantile meningitis.
o Transient normal flora of throat.
Manifestations:
o Meningeococcemia: Bugs in the bloodstream leads to a rash with lots of bugs in it.
Gram negative DIC and shock are possible complications.
o Meningitis: Vomiting and fever may be the only signs in infants. No treatment leads to death.
Processing:
o Specimen: CSF, rash exudate, blood, synovial fluid.
o Stain:
Mostly extracellular gram-negative cocci. Because of their thick capsule they are not phagocytosed.
Lots of PMN's.
o Culture: Smooth colonies. Capsule.
Thayer-Martin (chocolate agar with antibiotics)
Sterile specimens (CSF, blood) will can be grown on chocolate agar alone.
CO2
Subculture for glucose / maltose fermentation tests.
Identification:
o Catalase(+), Oxidase(+): Neisseria
o Glucose-fermenting, Maltose-fermenting (Meningidits)
o Serotypes: Based on the capsule antigen.
A,B,C,D,29E: Originally known to be virulent
W135,X,Y,Z: Orginally thought to be harmless; now it's known otherwise.
Type-B is the most virulent because it is not immunogenic -- we cannot make protective antibodies against
it.
It is made out of alpha-2,8-n-acetylneuraminic acid. It is rapidly degraded and thus not
immunogenic.
o Quellung Reaction: Add antiserum to look for Type-B. Swelling shows positive test.
o CIE, Latex Agglutination: On CSF or urine. Two specific antibody tests to look for capsular antigens.
Virulence:
o Cell-Associated:
Capsule: Thick polysaccahride determining the serogroup. Antiphagocytic.
Type-B is not immunogenic.
Outer Membrane Proteins: Another basis for serotyping.
They are being used to search for a conjugate vaccine.
They have homology with the Outer Membrane Proteins (I thru III) of N.Gonorrhea.
LPS: Induces the Schwartzman Reaction. Causes the rash and can lead to necrosis.
Pili: Adhesins in upper respiratory tract.
o Enzymes:
IgA Protease: Two types, cleaves bonds at hinge region of IgA1
Oxidase
Needs Iron: Iron is taken up by an energy-dependent (not siderophore) mechanism.
Host Immune Response: Serological
o Antibody is essential to phagocytosis of this bug.
o Problem: cross-reacting antigens (E.Coli, E.Fecaelis) stimulate IgA, which blocks IgM, hindering serum immunity.
Not good.
o Group-B can be phagocytosed but remains serum resistant.
Vaccine / Prevention: Given to military recruits. Contains A, C, Y, W135. Naturally it doesn't contain B because Type-B is
not immunogenic!
o Vaccine can only be given to people older than two.
Treatment: Prophylactic antibiotic treatment for kids younger than 6 years old, with documented contact with an index case.
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Species
B. Melitensis goat
B. Abortus cattle
B. Suis pig
B. Canis dog
Epidemiology / At Risk: Zoonotic. Cattle ranchers and people who handle cattle.
Manifestations: Undulating Fever, Bang's Disease
o Infecting organisms go into bloodstream and then are taken up by fixed RES macrophages, where they reside
happily.
o Organisms are occassionally released from fixed macrophages and go back into the bloodstream. Fever waxes and
wanes according to the presence of organisms in the bloodsteam.
o Symptoms: Fever, chills, sweats, myalgia, weakness, recurring at 10-day intervals.
o Chronic disease causes microabscesses, granulomas, and caseation in the spleen and liver.
This results from Type-IV cell mediated response to the bugs.
Processing:
o Specimen: Blood, bone marrow
o Stain: Not useful
o Culture: Brucella Agar = Selective Agar containing Erythritol, which the bugs subsist on.
Erythritol is found in reproductive tract of cattle, hence these bugs cause abortion in cattle. Humans don't
have erythritol so no abortion happens.
Identification:
o Facultative Intracellular Parasite of macrophages
o Catalase (+)
o Oxidase (+)
o Gas-liquid chromatography can be used to identify the species.0
Virulence:
o Catalase
o 5'-GMP, Adenine: They inhibit the release of peroxidase by PMN's, thus hindering the halide pathway of killing.
o Facultative Intracellular Parasite of macrophages
o LPS: Responsible for fever.
Host Immune Response:
o IgM persists throughout infection. IgG waxes and wanes with fever.
o IgG blocks IgM's bactericidal action in the serum.
o Acute:Convalescent titer of 1:160.
Vaccine / Prevention: Cattle are vaccinated in Kansas. Live attenuated B.Abortus.
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Virulence:
o Listerolysin O: Oxygen labile, beta-Hemolytic, like Streptolysin-O
Cytotoxic to RBC's, WBC's, PMN's (at low pH intracellularly), and myocardium
Disrupts phagolysosome.
o Catalase
o Facultative Intracellular Parasite of macrophages: actin polymerizes to allow the bugs to travel from cell to cell, like
Shigella.
o Cell Wall Lipid: Induces granulocytic response in humans. Induces an IgM response.
In humans, hemolytic due to cross-reaction with blood-group antigen. It's not gram-negative, but this leads
to a hemolytic crisis.
Host Immune Response:
o Monocytic response in animals. Granulocytic with left-shift (standard) response in humans.
o Cross-reaction with Staph and Strep leads to high diagnostic titer of 1:200.
o Protective Cell-mediated immunity is induced.
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SPIROCHETEMIAS
LEPTOSPIRA INTERROGANS: Spirochete
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ANAEROBES
BACTEROIDES FRAGILES: Gram-Negative Pleomorphic Rod
Name-Derivation:
Epidemiology / At Risk: #1 bacteria of normal GI flora. 1010-1011 bacteria / gram
o Can survive on skin and mucosa.
o At Risk: Immunocompromised
Manifestations: Bugs enter through a compromise in GI-tract. Initial infection is polymicrobic, but as other bugs use up the
oxygen, Bacteroides can grow.
o Pelvic Inflammatory Disease
o Intra-abdominal abscess; peritonitis
o Sustained Bacteremia: Not endotoxic, and not transient, but in between.
Processing:
o Specimen: Exudate. Will be polymicrobic early on.
Must use anaerobic transport medium.
o Stain: Gram-negative rods contain intracytoplasmic vacuoles.
o Culture: Blood agar with antibiotics.
Identification:
o Strict (Aerotolerant) Anaerobe
o Direct-FA on capsule to verify its presence. It is not readily visible in culture.
o Gas-Liquid Chromatography of metabolic byproducts. Anaerobes are difficult to culture.
Virulence:
o Isobutyric and Succinic Acid: Biochemical byproducts are toxic to Salmonella and Shigella. Thus our normal flora
are protective against Salmonella.
o Capsule: Allows adhesin to peritoneum.
Antichemotactic and antiphagocytic for PMN's.
Aids to inhibit intracellular killing once the bugs are phagocytosed.
o Superoxide Dismutase: makes it aerotolerant
o Catalase: induced by hemin, thus produced in the blood.
o Enzymes: Hyaluronidase, DNAse, Heparinase contribute to invasiveness.
o LPS: Contains Lipid-A, but it is not as toxic as E. Coli endotoxin. Only mild endotoxic effect.
o Enterotoxin: Diarrhea. This toxin only carried by a few strains.
o beta-Lactamase
Treatment: Abscesses must be surgically drained. Metronidazole.
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Double-zone of hemolysis: beta-hemolysis on inside, plus hazy zone of partial hemolysis on outside.
Identification:
o Strict (Aerotolerant) Anaerobe
o SEROTYPING: Based on ability to produce four different exotoxins: alpha, beta, ,
There are types A-E
Type-A is most prevelant in U.S.
o Gas-Liquid Chromatography for speciation.
Virulence:
o Very fast (10 minute) generation time.
o Exotoxins: At least 12 are produced. Some main ones
Alpha Toxin: Lecithinase. Hydrolyzes lecithin and sphingomyelin, to lyse mitochondria, RBC's, and
WBC's.
Responsible for the larger zone of incomplete hemolysis.
It's an exotoxin so it can go awayfrom the cell.
Beta Toxin: Cytotoxin, responsible for necrotizing enteritis.
Theta Toxin: Oxygen-labile hemolysin.
Responsible for smaller zone of beta-hemolysis.
Toxic to heart muscle, leading to myocarditis as a cardiac complication (from toxemia)
o Invasiveness: Due to extracellular enzymes; proteases, collegenases, DNAses.
o Enterotoxin: Only released upon sporulation and lysis of a vegetative cell.
Thus it is not a true exotoxin (only released upon lysis)
Causes watery diarrhea, no dysentery.
Vaccine / Prevention: Clean and debride wounds for prevention.
Treatment: Antibiotics, intermittent hyperbaric oxygen treatment.
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Epidemiology / At Risk: Spores are uniquitous in the soil and introduced through puncture wounds.
Manifestations: Tetanus. A microgram of toxin is sufficient to kill.
o Spore Germination:
Once spores introduced, they can lie dormant for years if wound remains well aerated.
As aeration is reduced (such as with age, for example), spores germinate and release exotoxin.
Exotoxin is taken up by nerve cells ------> retrograde transport to post-synaptic dendrites, where the
exotoxin exerts its effect.
o Generalized Tetanus: Lockjaw. Toxin may ascend to brain and affect respiratory center. ANS involvement and
generalized spasms. Death by respiratory arrest. Symptoms:
Dysphagia
Drooling
ANS involvement: sweating, hyperthermia, cardiac arrhythmias.
o Localized Tetanus: Localized spasm in area of infection. It may spread to generalized tetanus.
o Cephalic Tetanus: Primary infection in head.
Processing:
o Specimen: Only a few organisms in the lesions. Diagnosis is clinical -- not by microbiology.
o Culture: Swarming Growth in culture. Thin surrounding film and faint beta-hemolysis.
Amplified in cooked hamburger broth. Selected for by heat.
Identification:
o Opportunstic Strict Anaerobe
o Genus is identified by colony morphology.
o Somatic Antigen O: Fortunately there is only one serotype of Antigen-O, so that a vaccine is easy to make and
effective.
Virulence:
o Tetanospasmin: Heat labile, potent, antigenic exotoxin.
AB-Toxin cleaved in light (A) and heavy (B) fragments by an endogenous protease.
Fragment-B binds to ganglioside receptor of a nerve cell.
Fragment-A is taken up by endocytosis. Acidifcation of the vesicle causes Fragment-A to be
release into cytoplasm.
Effect: Fragment-A moves up axon retrograde to the post-synpatic receptor. It blocks release of GABA
and Glycerine at the post-synaptic terminal.
This results in unregulated excitatory post-synaptic potentials on nerve terminal, leading to spastic
paralysis of skeletal muscle and respiratory arrest.
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Vaccine / Prevention:
o VACCINE: Given against the O-Antigen in cell wall.
Alum-precipitated toxoid (more immunogenic but can cause hypersensitivity): given to infants and
children, and never-before immunized adults.
Children given at age 2, 4, 6, and 15-18 months.
Ammonium-precipiated toxoid: Given as a booster shot every ten years thereafter.
People are usually over-vaccinated for this bug.
o ANTITOXIN: Given prophylactically if someone is potentially exposed (has a puncture wound) and has not been
vaccinated within 5 years.
Tetanus toxin itself is not inherently immunogenic.
If a documented vaccine has been administered in last 5 years, no antitoxin is necessary.
Treatment: To someone who has never been vaccinated, the antitoxin is given in one arm, and a vaccine is given in the other
arm, simultaneously. The two will not intermix if given in separate arms.
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Epidemiology / At Risk: Spores are in soils and sediment. Found in foods preserved at home at room temperature; old canned
foods or self-canned foods.
Manifestations:
o Food-Born Botulism: Usually found in alkaline foods, consumed without heating.
Initial Symptoms: Weakness, diziness, constipation.
Toxin is then absorbed from small intestine into blood and nerve cells.
Terminal Symptoms: Blurred vision, dry mouth, peripheral and respiratory flaccid paralysis. Death.
o Infant Botulism: Infant at risk up to 1 year of age. After 1 year, GI ingestion of spores is not a problem.
Cause: Ingestion of spores, usually in unpasteurized honey.
Organisms replicate in GI tract and release toxin. Toxin is not readily absorbed through mucosa, but it can
be absorbed if the problem is neglected.
Presents as failure to thrive, but can progress to flaccid paralysis.
o Wound Botulism: Rare, similar clinical presentation as food botulism.
Processing:
o Specimen: Blood, gastric contents, or food. Feces for children.
Serology: Do an assay for the toxin in the blood.
o Stain: On infant feces, will find gram-positive rods.
o Culture: Blood agar. Heat to boiling for 10 minutes to induce sporulation.
Smooth colonies, beta-hemolysis.
Identification:
o ELISA can be used on culture for detection of toxin.
o Oppotunstic Strict Anaerobe
o Serotypes:
8 serologic types of toxin: A, B, C1, C2, D, E, F, G. All of them act the same.
Only types A, B, E affect humans.
Virulence:
o Botulin Exotoxin: The most potent toxin known to man, i.e. lowest amount required to kill.
Phage-mediated, heat labile toxin. Antigenic. The toxin is released upon cell lysis.
MECHANISM: AB-Toxin. Fragment-A is translocated up neuron and blocks release of Acetylcholine -----> Flaccid Paralysis.
Enzyme resistance: Acid stable. Our bodies actually break down the toxin into fragments that are more
toxic.
Host Immune Response: No persistent antibody to the toxin, therefore no lifelong immunity.
o Again, the toxin is not inherently immunogenic.
Vaccine / Prevention: Alum-precipitated toxoid, only for lab workers.
o PREVENTION: Head food, don't give unpasteurized honey to babies.
o Antitoxin is available for types A, B, and E.
Treatment:
o Ventilatory Support
o Administer antitoxin.
o Gastric Lavage to wash out any unabsorbed toxin.
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Epidemiology / At Risk: Respiratory droplets inhaled. Found in pet birds (parrots, parakeets), poultry birds. People talking to
their birds up close, etc.
Manifestations: Psitaccosis. An interstitial pneumonia.
o Headache (can be severe), and dry, non-productive, hacking cough.
o Sequelae: severe headache, encephalopathy, possible convulsions, coma, and death.
Processing:
o Specimen: Lung biopsy. Can take sputum late in disease. Blood.
o Stain:
o Culture:
Identification:
o Iodine-negative: Does not stain brown with iodine, as the inclusion bodies do not contain glycogen.
o Serology: Indirect FA. Test for patient antibodies to the bugs, by using labelled anti-antibodies.
o Acute-Convalescent titer of 4X
Treatment: Tetracycline.
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MYCOPLASMA HOMINIS: Emerging bug, associated with post-abortal and post-partum fevers, and PID.
UREAPLASMA UREALYTICA: Tiny bug.
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