BACTE COMPILED

HIGH YIELD CLINICAL BACTERIOLOGY REVIEWER

BACTERIA BASICS

Prokaryotic, both RNA and DNA, binary fission, 0.4-2 µm. Smallest: Mycoplasma. Largest: Bacillus.

Exotoxins — protein, systemic, heat unstable, from gram positives (except Listeria); also Vibrio and E. coli. Released by living cells.

Endotoxins — polysaccharide (LPS), local, heat stable, gram negatives only. Released on cell death. Detected by Limulus lysate test (+) = clumping.

BACTERIAL STRUCTURES

Spore locations: C. tetani = terminal; C. botulinum = subterminal; B. anthracis = central

Flagella motility types: Gliding = Capnocytophaga; Darting = Campylobacter; Tumbling = Listeria; Twitching = Kingella; Shooting star = V. cholerae; Corkscrew = Spirochetes

OXYGEN REQUIREMENTS

• Obligate aerobes: Brucella, Francisella, Mycobacterium, Pseudomonas, Neisseria

• Obligate anaerobes: Clostridium, Bacteroides, Veillonella, Actinomyces

• Facultative anaerobes: Enterobacteriaceae, Staphylococcus

• Microaerophilic: Campylobacter, Helicobacter, Streptococci

• Capnophilic (need CO2): Neisseria, Haemophilus, Brucella abortus

Blood bag contaminants at 4°C: P. fluorescens, Y. enterocolitica, Serratia liquefaciens (NOT Listeria — animal pathogen)

STAINING

Gram stain steps: Crystal violet → Gram's iodine (mordant) → Decolorizer (95% alcohol/acetone) → Safranin. Most critical step = decolorization.

Gram rules:

• All cocci = gram (+) EXCEPT Neisseria, Moraxella, Veillonella

• All bacilli = gram (-) EXCEPT Mycobacterium, Corynebacterium, Clostridium, Bacillus, Lactobacillus, Erysipelothrix, Listeria

• NOT gram stained: Rickettsiae, Chlamydia (intracellular); Mycoplasma, Ureaplasma (no cell wall); Spirochetes (can't resolve by brightfield)

Acid-Fast Stain:

AFB = red against blue background. Smear size: 2x3 cm. Report negative after 300 fields.

BIOSAFETY LEVELS

BSC Class I — open front, least effective. Class II — laminar flow, for BSL 2-3. Class III — completely closed, for BSL-4. Class IIB = exhaust air outside building.

STERILIZATION & DISINFECTION

Autoclave: 121°C, 15 psi, 15-30 min. Indicator = B. stearothermophilus. Does NOT kill prions.

Dry heat oven: 160-180°C, 1-2 hours. Indicator = B. subtilis var. niger. For glassware.

Ionizing radiation (cold sterilization): gamma rays. Indicator = B. pumilus. For disposables.

Ethylene oxide: chemical sterilant for heat-sensitive equipment. Indicator = B. subtilis var. globigii.

Glutaraldehyde 2%: sporicidal in 3-10 hours. "Cold sterilization" along with peracetic acid.

Best disinfectant for blood spillage: 10% sodium hypochlorite (household bleach), 1:10 dilution. Substitute = vinegar.

Pasteurization: 63°C/30 min (VAT); 72°C/15 sec (HTST/Flash); 140°C/3 sec (UHT). Removes pathogens only, not spores.

70% ethanol > 95% ethanol as disinfectant. Alcohol = non-sporicidal. Best antiseptic = iodophor (iodine + detergent).

SPECIMEN COLLECTION & TRANSPORT

Transport media:

• Cary Blair = stool pathogens

• Stuart's = viral

• Amies = clinical swabs

• Transgrow/JEMBEC = Neisseria

Storage temperatures:

• CSF = 37°C (priority #1 specimen)

• Urine, stool, sputum, viral = 4°C

• Serum for serology = -20°C

• Long-term tissue = -70°C

• Anaerobic specimens = NEVER refrigerate; room temperature

Anticoagulants: SPS 0.025% = blood culture. EDTA = viral PCR. NOT used in micro = EDTA and citrate. Urine preservative = boric acid.

Blood culture: dilution 1:10; adult = 10 mL (5 aerobic + 5 anaerobic); children = 1-5 mL. Hold 5-7 days routinely; Brucella = 3-4 weeks; Leptospira = 8 weeks.

Urine: clean-catch midstream. Significant UTI = >1.0 x 10⁵ CFU/mL. 1µL loop → colonies x 1,000. 10µL loop → colonies x 100. Major UTI cause = E. coli. UTI in young females = S. saprophyticus.

CSF: tube #2 (or #4 if available) for microbiology. Centrifuge, use sediment. Transport at room temperature.

Sputum: >25 PMNs, <10 SECs = acceptable specimen.

CULTURE MEDIA SUMMARY

GRAM POSITIVE COCCI

Staphylococci (catalase +, grape clusters, non-motile, 7.5% NaCl tolerant)

S. aureus: coagulase (+), DNAse (+), mannitol fermenter (+), beta hemolytic, PYR (-). Golden colonies on LSS. Normal flora of anterior nares.

• Virulence factors: coagulase (major), hyaluronidase (Duran-Reynal factor), beta-lactamase (penicillinase), TSST-1, exfoliatin, enterotoxins A&B, PVL

• Toxin diseases: food poisoning (enterotoxin A/B), TSS (TSST-1), scalded skin/Ritter's disease (exfoliatin)

• Non-toxin: boils, cellulitis, bullous impetigo, sty

Coagulase test: reagent = rabbit plasma with EDTA. Slide = bound coagulase (clumping factor). Tube = free coagulase (incubate 37°C x 4 hrs; if neg, RT x 20 hrs total).

MRSA: resistant via PBP2A (mecA gene). DOC = Vancomycin. Detect with: chromogenic agar (mauve-rose), cefoxitin disk, D-test, PCR (nasal swab = gold standard).

CoNS differentiation:

S. saprophyticus = #1 UTI in young women. S. epidermidis = prosthetic valve infection, stitch abscess.

Micrococci: tetrads, strict aerobe, modified oxidase (+) = blue, bacitracin-S, furazolidone-R, lysostaphin-R.

Streptococci (catalase -, chains, non-motile)

Medium of choice: Sheep's blood agar. Starting point = hemolytic pattern.

Hemolytic pattern:

• Alpha (partial/green): S. pneumoniae, Viridans

• Beta (complete/clear): Group A, B, C

• Gamma (none): Enterococci, Group D non-enterococci

Group A — S. pyogenes:

• Bacitracin-S, PYR (+), beta hemolytic

• Diseases: pharyngitis, necrotizing fasciitis, erysipelas, scarlet fever

• Sequelae: rheumatic fever, acute glomerulonephritis

• Virulence: M protein (major), streptolysin O (antigenic, O2-labile), streptolysin S (non-antigenic, O2-stable), erythrogenic toxin (scarlet fever rashes)

• Dick's test = susceptibility to scarlet fever. Schultz-Charlton = confirms rash is scarlet fever

Group B — S. agalactiae:

• CAMP (+), hippurate hydrolysis (+), bacitracin-R, SXT-R

• #1 cause of neonatal meningitis

• Transport: Todd-Hewitt broth / LIM broth

S. pneumoniae:

• Lance-shaped diplococci, alpha hemolytic

• Bile solubility (+), optochin-S, Neufeld Quellung (+), encapsulated

• Dome-shaped colonies → crater/nail-head colonies after 48 hrs

• #1 cause of adult bacterial meningitis (>29 yrs), lobar pneumonia (rusty sputum), otitis media

Viridans Streptococci:

• #1 throat flora. Causes SBE (S. sanguis), dental caries (S. mutans)

• Optochin-R, bile insoluble, alpha hemolytic

• NOT under Lancefield

Enterococci vs Group D Non-Enterococci:

S. bovis in blood = suspect colon cancer.

GRAM NEGATIVE COCCI

Neisseria (oxidase +, capnophilic, non-motile, cold-sensitive)

N. gonorrhoeae:

• Never normal flora; kidney/coffee bean-shaped intracellular diplococci in PMNs

• Ferments glucose ONLY

• Superoxol test (+) — 30% H2O2

• Major virulence factor = pili

• Causes gonorrhea, ophthalmia neonatorum (prevent with silver nitrate), PID

• Collect with Dacron/Rayon (NOT calcium alginate or cotton)

• Media: Thayer-Martin (VCN), Modified Thayer-Martin (VCN-T), Martin-Lewis (VCAT), NYC Media

N. meningitidis:

• Ferments glucose AND maltose

• Capsule = major virulence factor (Quellung +)

• Causes meningitis (5-29 yrs), meningococcemia, Waterhouse-Friderichsen syndrome

• Specimen for carrier state = nasopharyngeal swab

Sugar fermentation:

Moraxella catarrhalis: butyrate disk test (+) = blue; hockey puck colonies; DNAse (+); asaccharolytic; causes otitis media (with S. pneumoniae and H. influenzae).

GRAM POSITIVE SPORE-FORMING BACILLI

Bacillus

B. anthracis: non-motile, gamma hemolytic, box-car morphology, bamboo fishing rod. Capsule = D-glutamate. Medusa head colonies on BAP. String of pearl on penicillin MHA. Beaten egg white consistency when lifted. Lecithinase (+). Causes anthrax (cutaneous = most common; pulmonary/woolsorter's = most severe). Selective media = PLET. Ascoli test = serologic precipitin test.

B. cereus: motile, beta hemolytic (on BAP), causes food poisoning (fried rice bacillus). Diarrheal type (diarrhea) and emetic type (vomiting). Growth at 45°C (+). Penicillin-R.

B. stearothermophilus: biological indicator of autoclave. Agent of flat sour spoilage.

Clostridium (strict anaerobes, catalase -)

C. perfringens: encapsulated, NON-motile, double/target hemolysis, reverse CAMP (+), gas production, Nagler's reaction (+). Causes gas gangrene/myonecrosis. Stormy fermentation of milk.

C. tetani: terminal swollen spores (drumstick/tennis racquet), non-saccharolytic, tetanospasmin → spastic paralysis → risus sardonicus. Iron-loving.

C. botulinum: subterminal spores, botulinum toxin → blocks ACh → flaccid paralysis. Floppy baby syndrome. Alpha hemolytic on BAP.

C. difficile: pseudomembranous colitis, antibiotic-associated diarrhea. Media = CCFA. Horse stable odor. Yellow fluorescence on BAP. Toxin A (enterotoxin) + Toxin B (cytotoxin).

GRAM POSITIVE NON-SPORE FORMING BACILLI

Listeria monocytogenes: tumbling motility at room temp (presumptive), CAMP (+) with Rhodococcus equi (block hemolysis), cold enrichment, inverted Christmas tree in SIM, narrow beta hemolysis on BAP, VP (+). Causes meningitis in elderly/immunocompromised, neonates, food poisoning, stillbirth.

Corynebacterium diphtheriae: club-shaped, Chinese letter/palisade arrangement, Babes Ernst granules. CTBA = gray-black colonies. Modified Elek test (+) = in vitro toxigenicity (lines of precipitation). Diphtheria toxin → pseudomembrane → bull's neck. Shick's test = susceptibility to diphtheria.

Erysipelothrix rhusiopathiae: H2S (+) on TSI, non-motile, catalase (-), bottle-brush growth on gelatin. Causes erysipeloid (occupational hazard for butchers).

Nocardia: partially acid-fast, strict aerobe, sulfur granules, aerial hyphae on tap water agar. N. asteroides = pulmonary in immunocompromised. N. brasiliensis = skin infection.

GRAM POSITIVE BACILLI — ACID FAST

Mycobacterium tuberculosis

Aerobic, non-motile, slow grower (6-10 weeks on LJ). Cauliflower-like growth; rough colonies with cording. Virulence = cord factor and sulfatides. Beaded appearance (Much granules).

Key tests:

• Niacin test (+) = yellow (MTB +)

• Nitrate reduction (+) = red initial, no color change with zinc = confirmed (+)

• Heat-stable catalase (-) = MTB, M. bovis, M. leprae

• Pyrazinamidase (+) = MTB

• TCH susceptibility = M. bovis susceptible; MTB resistant

Mantoux test: intradermal PPD injection. Tuberculin syringe. Von Pirquet = scratch test.

M. bovis: TCH-S, niacin (-), causes intestinal TB in man. Water droplet colonies.

M. leprae: non-culturable; maintained in mouse foot pads or armadillo. Cigar packet arrangement. Lepromin test. Tuberculoid (few bacteria) vs Lepromatous (many bacteria).

Runyon's Classification (MOTT)

ENTEROBACTERIACEAE

All are: glucose fermenters, catalase (+), oxidase (-), reduce nitrates. Non-motile: Klebsiella, Shigella, Yersinia (at 37°C).

TSI interpretation:

IMViC:

• E. coli = + + - -

• Klebsiella = - - + +

• Enterobacter = - - + +

• S. typhi = - + - -

E. coli: #1 UTI, #2 neonatal meningitis, primary marker of fecal contamination. Greenish metallic sheen on EMB.

EHEC O157:H7: sorbitol MacConkey (-), MUG test (-), verotoxin → hemorrhagic colitis/HUS. Associated with undercooked hamburger.

Klebsiella pneumoniae: Friedlander's bacillus, non-motile, encapsulated (mucoid), currant jelly sputum, ESBL producer. String test (+).

Proteus: swarming motility on BAP, burnt chocolate odor on MAC. OX2 and OX19 antigens (P. vulgaris), OXK (P. mirabilis) = used in Weil-Felix test for Rickettsia.

PPM group (Proteus-Providencia-Morganella): all are rapid urease producers and deaminase (+), LIA = R/A.

Salmonella typhi: K/A, H2S(+), LDC(+), gas(-), citrate(-). IMViC = -+--. Vi antigen. Long-term site = gall bladder.

Shigella: non-motile, no gas, no H2S, invasive. S. dysenteriae = most severe (endotoxin + neurotoxin, catalase -). S. sonnei = late lactose fermenter, ODC(+).

Yersinia pestis: plague bacillus, non-motile, bipolar (safety pin) staining with Wayson stain. Flea vector (Xenopsylla cheopis). Stalactite growth in broth. Hammered copper colonies on BAP.

Yersinia enterocolitica: late lactose fermenter, cold enrichment, bull's eye on CIN, pseudoappendicitis syndrome. Ornithine and urease (+).

BIOCHEMICAL TEST POSITIVE RESULTS (MUST MEMORIZE)

GRAM NEGATIVE NON-ENTERIC PATHOGENS

Vibrio

Halophilic (8-10% NaCl), oxidase (+), TCBS media. V. cholerae = rice-water stool; string test (+); shooting star motility; sucrose fermenter (yellow on TCBS). Alkaline peptone water = enrichment broth.

Pseudomonas aeruginosa

#1 ICU isolate. K/K on TSI, strict aerobe, oxidase (+). Pyocyanin (blue) + pyoverdin (green). Corn tortilla odor. Blue pus in burn patients. Cystic fibrosis pneumonia. Cetrimide agar = selective. Acetamide utilization (+).

Campylobacter

S-shaped, wings of seagulls, darting motility, microaerophilic (5% O2, 10% CO2), 42°C optimal. C. jejuni = hippurate (+). Antecedent cause of Guillain-Barré.

Helicobacter pylori

Formerly Campylobacter coli. Strong urease (+). Causes gastritis, peptic ulcer, gastric carcinoma. Urea breath test = non-invasive. Warthin-Starry stain. Gold standard for infection = endoscopy + biopsy culture.

Haemophilus

Requires X (hemin) and V (NAD) factors. H. influenzae = both X and V, mousy odor, dew drop colonies, satellitism. Major virulence = capsule (type b). Cause of meningitis in <5 years old. H. ducreyi = soft chancre (STD), school of fish appearance, X factor only. H. aegypticus = pink eye/Koch-Weeks bacillus.

FASTIDIOUS GRAM NEGATIVE BACILLI

Brucella: undulating/Malta fever. Non-motile, strict aerobe, intracellular. Castaneda's media. B. abortus = cattle, capnophilic, H2S(+). B. melitensis = goats/sheep, most virulent. B. canis = dogs, not a bioterrorism agent. Serum agglutination significant titer = 1:160.

Bordetella pertussis: whooping cough. Non-motile. Mercury droplet colonies. Bordet-Gengou media. Best specimen = nasopharyngeal swab at catarrhal phase. Response = lymphocytosis. PCR = most sensitive.

Francisella tularensis: tularemia (rabbit fever). BSL-3. Faint bipolar staining. Media = BCGA. Forshay test = susceptibility test.

Legionella pneumophila: BCYE media. Bronchial washings specimen. Serotype 1 = Legionnaire's disease. Asaccharolytic. Requires L-cysteine.

Gardnerella vaginalis: bacterial vaginosis. Clue cells (Pap smear). Whiff test (+) = fishy amine odor with KOH. Beta hemolytic on HBT. Human blood bilayer tween media.

HACEK organisms: cause subacute bacterial endocarditis — Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella.

ANAEROBES

Clostridium difficile: CCFA media, horse stable odor, yellow fluorescence on BAP, Toxin A + B, pseudomembranous colitis.

Bacteroides fragilis: most common anaerobic isolate. BBE media = black colonies. Bacteremia.

Fusobacterium nucleatum: toothpick shape (tapered ends), rancid butter odor (butyric acid), predominant in oral cavity.

Actinomyces israelii: molar tooth colonies, lumpy jaw (actinomycosis), sulfur granules, anaerobic.

Anaerobic indicators: Resazurin (pink) or Methylene blue (blue) in air → colorless in anaerobic conditions.

Unacceptable anaerobic specimens: expectorated sputum, feces, voided urine, swabs, bronchial washings, gastric juice.

SPIROCHETES

T. pallidum: syphilis. Stages: primary (hard/Hunterian chancre) → secondary (condyloma latum) → latent → tertiary (gummas). Non-culturable in vitro; maintained in rabbit testicular chancre. Darkfield microscopy to visualize.

Leptospira interrogans: leptospirosis/Weil's disease. Fletcher's/EMJH media. Hook-shaped ends. Blood/CSF in week 1; urine in week 2. Confirmatory = Microscopic Agglutination Test.

Borrelia burgdorferi: Lyme disease. Ixodes tick (deer tick). Bull's eye rash (ECM). Kelly's media. Best diagnostic = serology (EIA + Western blot).

INTRACELLULAR ORGANISMS

Rickettsiae

Gram negative, obligate intracellular, arthropod-borne. Giemsa = blue; Machiavello = red; Jimenez = reddish-black. Weil-Felix test uses Proteus OX antigens.

• R. rickettsii = Rocky Mountain spotted fever (wood/dog tick)

• R. prowazekii = epidemic typhus (body louse)

• R. typhi = murine/endemic typhus (rat flea)

• R. tsutsugamushi = scrub typhus (chigger/mite)

• Coxiella burnetii = Q fever (inhalation of aerosols)

• Bartonella henselae = cat scratch disease

• Ehrlichia chaffeensis = monocytic ehrlichiosis (lone star tick); clusters = morulae

Chlamydia

Obligate intracellular. Two forms: elementary body (infectious) → reticulate body (reproductive) → elementary body.

• C. trachomatis = trachoma, LGV, NGU; sensitive to sulfonamides; glycogen inclusions (Halberstaedter-Prowazek); McCoy cells = gold standard

• C. psittaci = psittacosis/parrot fever (aerosol from birds)

• C. pneumoniae = walking pneumonia; pear-shaped elementary body; sulfonamide-R

Mycoplasma

No cell wall; smallest free living organism; penicillin-R. Fried egg colonies. Dienes stain.

• M. pneumoniae = primary atypical/walking pneumonia (EATON agent)

• M. hominis = post-abortal/post-partum fever, PID

• U. urealyticum = NGU, crosses placenta, urease (+)

MENINGITIS BY AGE

SUSCEPTIBILITY TESTING

Kirby-Bauer: MHA, 4mm depth, pH 7.2-7.4, 6mm filter paper disks, McFarland 0.5 standard (= 1.5 x 10⁸ CFU/mL), incubate 35-37°C for 16-18 hrs, measure ZOI at underside.

MIC: lowest concentration that inhibits growth (no turbidity in tube dilution).

MBC/MLC: lowest concentration that kills organism.

D-test: detects inducible clindamycin resistance in S. aureus. Uses erythromycin 15µg + clindamycin 2µg. (+) = D-shaped zone = clindamycin resistant.

E-test: quantitative, MIC on a stick. For fastidious organisms and anaerobes. (+) = ellipse of inhibition.

False sensitive causes: light inoculum, thin/dry agar, acid pH (tetracycline, methicillin), alkaline pH (aminoglycosides).

False resistant causes: heavy inoculum, thick agar, too much moisture, prolonged incubation.

QUALITY CONTROL FREQUENCY

HIGH-YIELD MNEMONICS & ASSOCIATIONS

• PPM = Rapid urease + deaminase (+): Proteus, Providencia, Morganella

• Oxidase (+) GNB: Pseudomonas, Neisseria, Vibrio, Campylobacter, Helicobacter, Aeromonas, Plesiomonas, Moraxella

• Non-motile Enterics: Klebsiella, Shigella, Yersinia (at 37°C)

• Late lactose fermenters: Citrobacter, Serratia, S. arizonae, S. sonnei, Y. enterocolitica

• ESBL producers: K. pneumoniae and E. coli

• Antibiotic resistant enterics: Citrobacter, Enterobacter, Serratia