CLBACT - Aerobic Gram-positive Bacilli

Bacillus

• Spore-forming, aerobic or facultative anaerobic, rod-shaped bacteria isolated from the soil.
• Forms endospores only aerobically.
• Motile with peritrichous flagella, except B. anthracis and B. mycoides.
• Survives extreme environmental conditions due to its endospores.

Bacillus microscopy

Large, boxcar-shaped gram-positive rods with clear, unstained, central spores or "empty spaces".

Bacillus biochemical test

(+) Catalase; ferments glucose; hydrolyzes starch.

Clinically significant Bacillus

B. anthracis, B. cereus, B. thuringiensis and B. mycoides.

Bacillus anthracis

Anthrax bacillus — causative agent of anthrax.

B. anthracis

• Not part of the indigenous human microbiota.
• Grows aerobically or anaerobically.
• Not highly contagious.
• Non-motile; halophilic — withstands up to 7% NaCl.
• Biological weapon of mass destruction.

B. anthracis

Grows in a low-pH (<6.0) environment, produces lecithinase, and ferments glucose.

B. anthracis virulence factors

D-glutamic acid capsule and protein exotoxins.

B. anthracis microscopy

Gram-positive, large, encapsulated, square-ended rods; "bamboo fishing rod" appearance, with unstained central spores.

B. anthracis on BAP

Non-hemolytic colonies with a "Medusa head" appearance — swirling projections ("curly lock" or "comet tail"); "beaten egg white" appearance when an inoculating loop is used.

B. anthracis growth factor

Thiamine (B1).

String-of-pearls

B. anthracis on MHA with penicillin, to which it is susceptible.

Cutaneous anthrax

• Acquired through skin cuts and abrasions.
• Small papule at the site of spore inoculation 2–5 days after exposure.
• "Black eschar" — black, necrotic, painless central area that does not produce pus.

Pulmonary anthrax

• Woolsorter's disease / ragpicker's disease.
• Spores inhaled into the pulmonary parenchyma.
• Resembles an upper respiratory tract infection.
• Mild fever, fatigue, dyspnea.

Gastrointestinal anthrax

• Spores inoculated into a lesion on the intestinal mucosa following ingestion.
• Abdominal pain, nausea, anorexia, vomiting, bloody diarrhea.

B. anthracis specimens

Malignant pustule, sputum, blood; typically isolated from normally sterile sites — blood, lung tissue, CSF.

B. anthracis processing

In a biological safety cabinet with biosafety level 3 (weaponized anthrax).

B. anthracis old cultures

Gram-variable cells.

B. anthracis spore stains

Malachite green and McFadyean stain (as listed in the lecture; McFadyean is the capsule stain).

B. anthracis capsule stain

India ink (for blood or CSF specimens).

Fluorescent microscopy

Rapid presumptive diagnosis of B. anthracis.

B. anthracis culture media

BAP, CAP, EYA, PEA, polymyxin-lysozyme-EDTA-thallous acetate (PLET), bicarbonate agar, nutrient broth.

Heat or alcohol shock

Enrichment and selective technique applied before plating for Bacillus.

PEA

Recommended for identification of Bacillus in fecal specimens; with PLET, isolates Bacillus from contaminated specimens.

PLET

Polymyxin-lysozyme-EDTA-thallous acetate — isolating Bacillus species from contaminated specimens.

EYA

Determination of lecithinase production in B. anthracis.

Gelatin medium

Shows the "inverted pine-tree" appearance of B. anthracis.

Ascoli test

• Precipitin test.
• Detects thermostable anthrax antigens using rabbit antiserum.
• Sample: 2 g in 5 mL saline, 1/100 final concentration of acetic acid.
• (+) Precipitate band in less than 15 minutes.

Catalase test (Bacillus)

Differentiates Bacillus (catalase-positive) from Clostridium (catalase-negative).

Oxidase test (Bacillus)

Gives variable results.

Direct fluorescent antibody

Positive cell wall polysaccharide and capsule antigen of B. anthracis.

B. anthracis rule-out

• Large gram-positive bacillus.
• Ground glass, non-pigmented, gamma (no) hemolysis on BAP, no growth on MAC or EMB.
• Catalase positive, non-motile.
• Yes to all: not ruled out — do not attempt further identification; refer to reference laboratory.

Bacillus cereus

"Fried rice" bacillus.

B. cereus

• Food poisoning from contaminated cooked rice dishes (typical source) or other food.
• Most common Bacillus in opportunistic infections — eye and ear infections.
• Motile; resistant to penicillin.

B. cereus on BAP

Large, feathery colonies with spreading growth; frosted-glass appearance; β-hemolytic.

B. cereus biochemical test

Ferments salicin; (+) lecithinase.

B. cereus virulence factors

Enterotoxins (heat-stable and heat-labile), cerelysin, phospholipase C, pyogenic toxin.

B. cereus best specimen

Suspected food (≥10⁵ cells/gram).

Diarrheal type

• B. cereus food poisoning from meat, poultry, vegetables.
• Incubation 8–16 hours (long).
• Abdominal pain and watery diarrhea without fever.
• Heat-labile enterotoxin.

Emetic type

• B. cereus food poisoning from improperly stored cooked rice; B. cereus type 1.
• Incubation 1–6 hours (short).
• Abdominal cramps and profuse vomiting.
• Heat-stable enterotoxin.

Anthracis vs cereus

Motility: B. anthracis non-motile; B. cereus motile.

Anthracis vs cereus

Capsule: B. anthracis encapsulated; B. cereus non-encapsulated.

Anthracis vs cereus

Hemolysis: B. anthracis non-hemolytic; B. cereus β-hemolytic.

Anthracis vs cereus

Growth on PEA: B. anthracis V; B. cereus +.

Anthracis vs cereus

Growth at 45 °C: B. anthracis zero to slight; B. cereus rapid.

Anthracis vs cereus

Penicillin: B. anthracis sensitive; B. cereus resistant.

Anthracis vs cereus

String-of-pearl: B. anthracis +; B. cereus −.

Anthracis vs cereus

Salicin fermentation and gelatin hydrolysis: B. anthracis −; B. cereus +. Lecithinase: both +.

Bacillus subtilis

Hay bacillus.

B. subtilis

• Most commonly encountered laboratory contaminant.
• Halophilic; tolerates up to 7% NaCl.
• Source of the antibiotic bacitracin.
• Eye infection among prohibited drug users.

B. subtilis culture

BAP: large, flat, dull colonies with a ground-glass appearance; may be β-hemolytic; may be pigmented (pink, yellow, orange, brown). Ferments mannitol, xylose, arabinose.

Bacillus pumilus

Biological indicator in sterilization methods. BAP: large, moist, blister-like colonies; can be β-hemolytic.

Bacillus thuringiensis

Insect pathogen; produces parasporal crystals used as a pesticide.

Corynebacterium

• Most species are indigenous microbiota on skin and mucous membranes of humans and animals.
• Found in freshwater, saltwater, plants, humans.
• Non-motile, non-encapsulated, non-spore-forming, highly pleomorphic rods.

Corynebacterium fermentation

Glucose and maltose fermenters except C. urealyticum and C. pseudodiphtheriticum.

Corynebacterium microscopy

Slightly curved gram-positive rods with unparallel sides and slightly wider ends — "club-like" shape.

Corynebacterium on BAP

Small zone of β-hemolysis; some strains non-hemolytic.

Corynebacterium biochemical test

(+) Catalase. (The lecture also prints oxidase +; corynebacteria are oxidase-negative.)

Lipophilic corynebacteria

Fastidious; require 48 hours of incubation before growth is seen; lipids added to the medium enhance growth.

Non-lipophilic corynebacteria

Fermentative or oxidative metabolism. C. diphtheriae, C. pseudotuberculosis, C. pseudodiphtheriticum, C. striatum, C. ulcerans, C. xerosis.

Human-infection corynebacteria

C. diphtheriae, C. jeikeium, C. pseudotuberculosis, C. pseudodiphtheriticum, C. ulcerans, C. urealyticum.

Corynebacterium diphtheriae

Diphtheria bacillus or Klebs-Löffler bacillus.

C. diphtheriae

• Non-lipophilic facultative anaerobe; grows best aerobically.
• Not indigenous respiratory microbiota; inhabits the nasopharynx only in a carrier state.
• Inhalation of respiratory droplets or contact with infected cutaneous lesions (hand-to-mouth).

C. diphtheriae

• Rarely enters the bloodstream.
• Readily killed by heat and most disinfectants.
• Glucose and maltose fermenter.
• Virulence factor: diphtheria toxin.

C. diphtheriae preferred medium

Enriched medium with serum, cystine, and potassium tellurite.

C. diphtheriae microscopy

Rounded ends and "club-shaped swellings"; highly pleomorphic cells in pairs forming X, V, Y and L formations — Chinese letters.

C. diphtheriae biochemical test

(−) Urease; (+) nitrate reduction.

C. diphtheriae best specimens

Nasopharyngeal and throat swabs.

Intermediate biotype

Very small, flat, dry, grayish-black colonies; non-hemolytic.

Mitis biotype

Small, black, convex colonies with a "fried egg" appearance; hemolytic.

Gravis biotype

Large, flat, dark gray colonies with a "daisy-head" appearance; non-hemolytic.

Diphtheria toxin

• Heat-labile polypeptide.
• Produced by strains with a lysogenic β-phage carrying the TOX gene.
• Tissue necrosis and exudate (pseudomembrane) over the tonsils, larynx and pharynx.

Diphtheria toxin production

Favors alkaline pH (7.8 to 8.0) and an aerobic environment; released when the iron in the medium is consumed. (The lecture also says "sufficient" iron favors production — iron represses the toxin.)

Respiratory diphtheria

• Systemic toxin and a false membrane (pseudomembrane) on the throat mucous membrane; may cause respiratory obstruction.
• Low-grade fever, thick mucopurulent nasal discharge, cough, bull neck.

Diphtheria control measure

Immunization (DPT vaccine).

Diphtheria antitoxin

Administered to neutralize unabsorbed exotoxin in the patient's tissues.

Cutaneous diphtheria

• Veldt sore or Barcoo rot.
• Slow-healing ulcers and membrane formation.
• Toxin absorbed systemically but less severe.

Corynebacterium urealyticum

• One of the most frequently isolated and clinically significant species.
• Urinary pathogen, strict aerobe, lipophilic.
• Does not ferment glucose or maltose.
• V-shaped forms and palisades.

C. urealyticum culture

BAP: pinpoint, white, smooth, non-hemolytic colonies. Rapid urease producer.

Corynebacterium pseudodiphtheriticum

Hofmann's bacillus.

C. pseudodiphtheriticum

• Indigenous microbiota of the human nasopharynx.
• Respiratory infections, UTI and cutaneous wound infections in the immunocompromised (e.g., AIDS).
• Parallel rows or palisades, without the pleomorphism of other corynebacteria.
• (+) Urease and nitrate.

Corynebacterium jeikeium

• Skin microbiota of inguinal, axillary and rectal sites.
• Obligate aerobe; multi-antibiotic-resistant (β-lactams).
• Isolated from immunocompromised individuals.
• Common cause of diphtheroid prosthetic valve endocarditis in adults.

C. jeikeium

Pleomorphic, club-shaped, V-shaped forms. BAP: colonies appear large with 1% Tween 80. (−) Urease and nitrate reduction.

Corynebacterium ulcerans

• Acquired through animal contact or unpasteurized dairy products.
• Isolated from skin ulcers and exudative pharyngitis.
• Diphtheria-like sore throats — many isolates produce a diphtheria-like toxin.

C. ulcerans culture

BAP: narrow zone of β-hemolysis. CTBA: surrounding brown halo. Löffler's serum agar: growth. (+) Urease and gelatinase; (−) nitrate reduction.

Corynebacterium pseudotuberculosis

• Animal pathogen; acquired by direct contact with infected animals.
• Dermonecrotic toxin kills various cell types.
• Can also produce diphtheria toxin.

C. pseudotuberculosis culture

CTBA: black colonies with a brown halo. BAP: small, yellowish-white. (+) Urease; (−) gelatinase.

Corynebacterium specimens

Nasopharyngeal and throat swabs (best for C. diphtheriae), urine, blood, wound discharge.

Calcium alginate swab

Preferred for collection of Corynebacterium specimens.

Corynebacterium Gram stain

Highly pleomorphic, gram-positive, short or slightly curved rods; arrangements of C. diphtheriae are due to incomplete fission.

Methylene blue stain

Corynebacterium cells exhibit a beaded formation.

Neisser's stain

Stains metachromatic granules; composed mainly of methylene blue or crystal violet.

Albert's stain

Stains metachromatic granules; components malachite green and toluidine blue.

Corynebacterium culture media

BAP, CAP, CTBA, Tinsdale agar, Löffler's serum agar.

Löffler slant

Primary inoculation of throat swabs, overnight incubation, then subculture to CTBA improves recovery of C. diphtheriae.

CTBA and Tinsdale incubation

At least 48 hours at 35 °C. Multiplication of corynebacteria occurs within 15 °C to 40 °C.

Cystine tellurite blood agar

• CTBA — preferred medium for isolation and identification.
• Modification of Tinsdale agar; selective and differential.
• Sheep blood, bovine serum, cystine, potassium tellurite.
• (+) Black or brown colonies after 48 hours.

CTBA brown halo

Black or brown colonies with a brown halo: C. diphtheriae, C. ulcerans, C. pseudotuberculosis.

Tinsdale agar

• Sheep blood, cystine, potassium tellurite, sodium thiosulfate.
• (+) Black colonies surrounded by a brown halo.
• All biotypes of C. diphtheriae have brown haloes.

Pai's slant

Pai's slant or Löffler's serum agar — microscopic morphology and metachromatic granules of C. diphtheriae; colonies have a "poached-egg" appearance.