Microbiology Quiz: Campylobacter and Vibrio spp.

Non-Enteric Gastrointestinal Pathogens

• Vibrio spp. are oxidase positive, except V. metschnikovii
• Vibrio spp. produce gas from glucose, except V. fumissii, which causes voluminous "rice water" diarrhea.
• Vibrio spp. are best cultured in alkaline peptone broth (pH 8.4)
• Thiosulfate citrate bile salts (TCBS) agar is the best medium for isolating Vibrio.
• Vibrio spp. are gram-negative, comma-shaped, curved, or straight bacilli.
• Vibrio spp. are facultative anaerobes.
• Vibrio spp. are catalase negative and oxidase-positive, reducing nitrate to nitrite except V. metschnikovii.

Vibrio spp. Other Reactions of V. cholerae

• V. cholerae: MR is negative.
• V. cholerae: VP is positive/variable.
• V. cholerae: Citrate is positive.
• V. cholerae: Urease is negative.
• V. cholerae: TSI shows A/A, gas is negative, and H2S is negative.
• String test (emulsify organism in 0.5% sodium deoxycholate on a slide)
• Cholera red reaction (add concentrated sulphuric acid to 24hrs peptone water culture with V. cholerae results in positive pink color)

Vibrio spp. Salt Concentration (0.5%)

• Nutrient agar or sheep blood agar (SBA) are commonly used laboratory media.
• Colonies on SBA or chocolate agar (CHOC) appear smooth, opaque, and iridescent with a greenish hue (medium to large colonies).
• Cefsulodin-Irgasan-Novobiocin (CIN) agar is a selective differential medium for Vibrio spp.
• MAC agar is a selective differential medium.

Differentiation of Pathogenic Vibrio Species

• Table detailing the differentiation based on TCBS, Lact. Ferm., Cholera Red React., String Test, and Kanagawa Pheno for different pathogenic Vibrio species.

Summary for Vibrio spp.

• Summarized traits specific to different Vibrio species: positive oxidase with different sugars, lysines, decarboxylase, salt concentrations, color on TCBS, etc

Indole for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Indole.

Glucose for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Glucose.

Sucrose for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Sucrose.

Lysine Decarboxylase for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Lysine Decarboxylase.

Arginine Dihydrolase for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Arginine Dihydrolase.

Ornithine Decarboxylase for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative Ornithine Decarboxylase.

Growth in 0% NaCl for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative growth in 0% NaCl.

Growth in 6% NaCl for Vibrio spp.

• Table of Vibrio spp. categorized by positive and negative growth in 6% NaCl.
• Colony color and characteristics on TCBS agar.

Aeromonas spp. Key Characteristics

• Gram-negative, straight bacilli
• Facultative anaerobe
• Fermentative
• Indole positive
• Oxidase positive
• Motile
• Resistant to vibriostatic agent O/129 (2,4 diamino-6,7-diisopropylpteridine)
• Non-inositol fermenter
• A. hydrophila is the most common human isolate

Aeromonas spp. Selective Culture Media

• MAC Agar, ferment lactose (A. caviae), unusual universal susceptibility to ampicillin.
• CIN Medium, pink-centered colonies from fermentation of mannitol, uneven clear apron resembling Yersinia enterocolitica.
• Alkaline peptone water.

Aeromonas spp. Presumptive Identification

• Oxidase test (positive)
• Spot indole test on suspicious colonies (especially beta-hemolytic colonies)
• Ability to grow in the presence of NaCl.

Campylobacter spp. Key Characteristics

• Gram-negative, comma, curved, S-shaped, seagull wing-shaped bacilli
• Microaerophilic (5% O2)
• Capnophilic
• Motile (one polar flagellum)
• Oxidase-positive
• Non-fermentative
• Sodium Hippurate Hydrolysis-positive
• Urease-negative
• Exhibit characteristic motility (hanging drop preparations/phase contrast microscopy).

Campylobacter spp. (Campylobacter jejuni and Campylobacter coli)

• Causes enteritis (esp. children)
• Leading cause of acute diarrhea worldwide.
• Sometimes responsible for bacteremia or endocarditis
• Culture: Incubate in 5% O2, 10% CO2, and nitrogen atmosphere for 48 hours.
• Isolation media: "Campy-thio" or "Campy-blood" agar.
• Use antibiotics in the media to make it selective.

Campylobacter spp. Biochemical reactions of C. jejuni

• Non-fermentative
• Oxidase (+), Catalase (+), Nitrate (+), H2S (-)
• Grows in 1% glycine
• Hippurate (+). C. coli is negative.
• C. jejuni and other enteric Campylobacters
• Moist, runny look; spreading, usually non hemolytic, some round and others flat.
• C. fetus subsp. fetus
• Produces smooth, convex, translucent colonies.
• C. mucosalis and C. hyointestinalis
• Can produce a dirty yellow pigment.

Helicobacter pylori

• Curved, flagellated, motile gram-negative rods
• "Triple positive": catalase (+), oxidase (+), urease (+)
• Diagnosis: Biopsy, Urea breath test, Stool antigen

Aerobic Gram-Positive Bacilli

• Corynebacterium

Corynebacterium

• Non-spore-forming, nonbranching, catalase-positive bacilli.
• Also called Kleb Coeffler's bacillus
• Microscopy: Highly pleomorphic gram-positive bacilli.
• Club-shaped swellings and beaded forms are common.
• Metachromatic areas (Babes-Ernst granules) stain intensely.
• Facultative anaerobes.
• Best growth at 37°C, but can multiply from 15°C to 40°C.
• Grows well on nutrient agar, serum/blood-containing media (ex. Coeffler Serum or Pai Agar).
• Cystine-tellurite blood agar (CTBA) - modification of Tinsdale medium, sheep red blood cells, bovine serum, cystine, potassium tellurite. Inhibits non-Coryneform bacteria.
• Characteristics of C. diphtheriae- non motile, nitrate reduction (+), urease (-), catalase (+), glucose & maltose fermented, sucrose not fermented.

Corynebacterium (continued)

• Culture from threat, nasopharynx, skin wounds
• Special culture media: Coeffler's or Tinsdale
• Black colonies on Tinsdale media
• Metachromatic (blue/red) granules on Coeffler's media
• Media to enhance pleomorphism and granule production
• Coeffler serum agar + poached egg colonies, Pai coagulated egg medium

Listeria monocytogenes

• Facultative intracellular Catalase positive
• Tumbling motility (at room temp) but usually nonmotile at 35-37°C
• Hippurate hydrolysis-positive
• Esculin-positive, salicin (+)
• CAMP positive (hemolysis looks like a shovel, not arrowhead)
• Grows from 0.5-45°C
• Cold enrichment may be used

Listeria monocytogenes (Culture)

• Sheep blood agar (detect beta-hemolysis, blue-green colonies)
• McBride Cisteria medium (sheep blood).
• Phenylethylalcohol agar (inhibits gram-negative organisms).
• Hold suspect cultures at 4°C up to 6 months, subculture weekly for 1 month, monthly thereafter.
• Umbrella-shaped pattern.

Erysipelothrix rhusiopathiae

• Slender, pleomorphic gram-positive rods that form filaments
• Alpha hemolytic (on blood agar)
• Decolorizes easily in gram stain, may appear gram-negative.
• Catalase, oxidase, indole negative
• Produces H2S, differentiating it from Listeria monocytogenes.
• Test tube brush growth.

Mycobacterium

• Obligate aerobes
• Slender, non-spore-forming, acid-fast, gram-positive rods.
• Do not gram stain well.
• Culture media (incubate in 5-10% CO2):
• -Cowenstein-Jensen (C-J) (green color and opaque) - Contains egg glycerol, promotes growth of more species (less drying), good for niacin test. Difficult to prepare, often used for cultures.
• -Middlebrook 7H10 or 7H11 agar (Clear agar)
• Good for antimicrobial susceptibility testing, fewer contaminants grow.
• Contains glycerol. Enhances growth of human strain of M. tuberculosis.

Mycobacterium (Identification)

• Pigment production, growth rate, biochemical reactions.
• Niacin test (best for M. tuberculosis) - enzymatic hydrolysis of Tween 80 releases oleic acid. The indicator (neutral red) changes color depending on the species.
• Runyon groups I and III positive in 5 days.
• M. tuberculosis positive in 5 days.

Mycobacterium (Nitrate reduction)

• M. tuberculosis is positive
• M. bovis is negative
• M. kansasii is a positive control.

Mycobacterium (Catalase reaction)

• Almost all Mycobacteria are catalase-positive.
• M. tuberculosis and M. bovis are catalase-negative after incubation at 69°C for 30 minutes.

Biochemical tests (NTM identification)

• Includes tests for Tween-80 hydrolysis, Niacin test, Nitrate reduction test, Tellurite reduction test, Urea hydrolysis test and Citrate utilization test for Mycobacteria other than tuberculosis.

Mycobacterium tuberculosis

• Rough, wrinkled, dry, elevated, buff-colored ("cauliflower-like") at 35-37°C
• Slow growth: 2-3 weeks or longer
• Innoculate in 2 C-J slants, wrap in aluminum foil.
• Incubate at 35°C until visible growth occurs in unshielded tube, expose covered tube to light for 1 hour, cover again, and reincubate overnight.
• Niacin positive/yellow
• Tween 80 hydrolyzed in 10-20 days (pink color)
• Nitrate positive
• Catalase at 68C: negative
• No growth in MacConkey Medium
• Arylsulfatase negative

Mycobacterium ulcerans

• Associated with skin lesions in tropical and subtropical areas (Buruli ulcers in Africa)
• Requires several weeks (6-9) of incubation at 32°C for growth to become visible.
• Negative for niacin, nitrate reduction, Tween 80.

Mycobacterium leprae

• Hansen's bacillus
• Causes Hansen's disease (leprosy)
• Acid-fast rod in nasal mucosa of affected patients.
• Smears of tissue juice - acid-fast stain for lepra cells (macrophages containing acid-fast bacilli).
• Culture: Grows very slowly, not cultured on artificial media or human tissue. Culture can be performed on footpad of mice.
• Lepromin test - skin test for leprosy using a sterile extract from lepromatous nodules (24-48 hrs = early/Fernandez; 3-4 weeks = late/Mitsuda reaction)

Nocardia

• Thin, branching and beaded modified acid-fast positive rods.
• Key characteristics: obligate aerobe, partially acid-fast, delicate mycelium, catalase (+), urease (+).
• Culture: growth on any media without antibiotics.
• Complete biochemical tests needed for identification.

Anaerobes

• Found throughout human bodies as part of normal microbiota.
• Frequently cause polymicrobial infections.
• Anaerobic culture methods: Enriched agar, selective media:
  • KV (kanamycin-vancomycin) blood agar (anaerobic gram (-) rods).
  • KVL (kanamycin-vancomycin-laked) blood agar (esp. Bacteroides melaninogenicus).
  • Neomycin-vancomycin blood agar (for Fusobacteria, Veilonella).
  • Neomycin blood agar (for clostridia and anaerobic gram-positive cocci).
  • Naegler agar (with egg yolk, neomycin) (for clostridia).
  • Chopped meat glucose broth, supplemented thioglycollate broth. Boil 10 min and cool rapidly to drive off oxygen prior to inoculation.

Commonly Isolated Anaerobes

• Clostridium (anaerobic, gram-positive, spore-forming, motile rods; mostly peritrichous flagella)
• May produce lecithinase and lipase, ferment sugars, digest proteins.
• Table displaying motility, lecithinase, lipase, glucose and lactose results for different Clostridium species. C. perfringens, C. botulinum, C. tetani, and C. difficile.

Clostridium tetani

• Terminal spores ('lollipop').
• Positive for gelatin hydrolysis, glucose fermentation.
• Produces neurotoxin (tetanospasmin) causes tetanus (spastic paralysis).

Clostridium botulinum

• Causes botulism (floppy baby syndrome in infants)

Clostridium perfringens

• Causes gas gangrene (clostridial myonecrosis).
• Alpha toxin destroys muscle tissue, causes hemolysis.
• Phospholipase (lecithinase) degrades phospholipids in cell membranes.
• Double zone of hemolysis around colonies on blood agar.

Clostridium difficile

• Causes pseudomembranous colitis in patients undergoing prolonged antibiotic therapy.
• Cycloserine-cefoxitin-fructose agar (CCFA) used to recover C. difficile from stool cultures.
• Naegler's test - inhibition by anti-perfringens antitoxin of the precipitate around growth on egg yolk agar.

Antimicrobial Susceptibility Testing (Kirby-Bauer Disk Diffusion Test)

• Principle: Inverse linear relationship between zone diameter inhibited growth and antibiotic MIC.
• Testing parameters:
  • Use Mueller Hinton Agar in a 150x15mm plate.
  • pH: 7.2-7.4
  • Agar depth constant at 4mm (some sources 4-6mm).
  • Inoculum density: equivalent to 0.5 McFarland standard (1.5 x 10⁸ CFU/mL) using 0.5 mL of 1.175% BaCl₂ and 99.5 mL of 1% H₂SO₄
  • Too heavy inoculum = smaller zones, too light = larger zones
• Incubate plate at 35 ± 2°C for 18 hours.
  • <16 hours = ↑ zone, false susceptible

  • 18 hours = ↓ zone, false resistant

• Measure zone of inhibition using ruler, caliper, or template (on the opposite of blood agar plate).
• Resistant, intermediate, or susceptible based on zone inhibition in millimeters.
• QC strains of E. coli, S. aureus, P. aeruginosa, Enteroccocus faecalis for 20-30 days for frequent testing. Reduce frequency to weekly if results are acceptable.

Test your knowledge on microbiological characteristics of Campylobacter and Vibrio species. This quiz covers aspects such as growth conditions, biochemical reactions, and disease associations. It is a great resource for students and professionals in microbiology or related fields.