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BACTERIOLOGY
ASEPTIC TECHNIQUE: In handling bacteria specimens, do everything possible to assure that extraneous bacteria from you
do not contaminate the specimen. This is not sterile technique.
• 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:
1. Streptococcus Pneumoniae
Causes of NEONATAL (less than 3 months old) MENINGITIS and SEPSIS: In order
1. Group-B Strep
2. Escherichia Coli
1. E. Coli
2. Proteus Vulgaris / Mirabilis:
o 10-15% of hospital acquired UTI's.
3. Pseudomonas Aeruginosa
STAPHYLOCOCCI
STAPHYLOCOCCI SAPROPHYTICUS:
ZOONOTIC INFECTIONS
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:
STREPTOCOCCI
• 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:
VIRIDANS STREPTOCOCCUS:
Bugs of Childhood
• 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.
Bacteriology Page 18 of 50
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.
• 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).
TOXIN TOXOID
GRAM-NEGATIVE PNEUMONIAS
LEGIONELLA PNEUMOPHILA: Faintly staining Gram (-) rods. Part of its own family, Legionellaceae.
MYCOBACTERIA
Catalase (+)
MYCOBACTERIUM LEPRAE:
• 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.
Bacteriology Page 26 of 50
ENTEROBACTERIACEAE
• 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.
Bacteriology Page 28 of 50
CLOSTRIDIUM DIFFICILE: Gram (+) Rod, Spore-former. Not an Enterobacteriaceae.
• 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.
• 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.
• 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.
Bacteriology Page 29 of 50
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.
• 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:
• 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.
Bacteriology Page 30 of 50
• 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.
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:
Bacteriology Page 31 of 50
• Epidemiology / At Risk: Human to human transmission only.
o Large infective dose.
o Mortality Rate: 2-10%
o Relapse Rate: 20%
• Manifestations: Typhoid Fever
o Symptoms:
First Week: Constipation with step-wise fever and malaise.
Second week: Bacteremia. Sustained fever.
Rose Spot Rashes are discrete rashes on the trunk.
Diarrhea: Intestinal necrosis, severe bleeding, thrombophlebitis, cholecystitis, pneumonia, focal
abscesses.
Carrier State following resolution -- may last up to a year. Bugs localize to the bile ducts.
o PATHOGENESIS:
Organisms multiple in the lamina propria, lymphatics, and monocytes of the small intestine.
They then invade and disseminate to reticuloendothelial system, leading to sustained bacteremia.
At the liver, organisms re-invade the GI-Tract via the gallbladder.
o 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.
• 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.
Bacteriology Page 33 of 50
• 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
• 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.
Bacteriology Page 35 of 50
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.
SEXUALLY TRANSMITTED
• 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.
Bacteriology Page 36 of 50
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.
o 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.
• 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.
BACTEREMIAS
• 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!
Bacteriology Page 39 of 50
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.
• 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.
SPIROCHETEMIAS
ANAEROBES
• 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.
• 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.
Bacteriology Page 45 of 50
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.
• 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.
• 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.
Bacteriology Page 46 of 50
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.
Bacteriology Page 47 of 50
• 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.
Bacteriology Page 49 of 50
o Acute-Convalescent titer of 4X
• Treatment: Tetracycline.
MYCOPLASMA HOMINIS: Emerging bug, associated with post-abortal and post-partum fevers, and PID.