Microbiology Quiz on Corynebacterium & Gardnerella

Aerobic Gram Positive Bacilli

• Spore formers include Bacillus
• Non-spore formers include Corynebacterium, Arcanobacterium, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Rothia
• Branching non-spore formers include Actinomycetes, Nocardia
• Some cause significant disease
• Most are contaminants or commensals

Non-Spore Forming, Non-Branching Catalase Positive Bacilli

• Corynebacterium
  • More than 60 species, 40 clinically significant
  • Common microbiota of skin and mucous membranes
  • Cell walls contain m-DAP
  • All are catalase positive, and non-motile
  • Divided into lipophilic and non-lipophilic
  • Lipophilic types are fastidious and grow slowly (at least 48 hours) on standard media
  • Gram stain shows slightly curved, club-shaped bacilli
  • Coryneform-like isolates require 16s rRNA sequencing to identify species. Species include: C. bovis, C. ulcerans, C. xerosis, C. jeikeium, C. pseudodiphtheriticum, C. pseudotuberculosis
• Virulence factors of Corynebacterium diphtheriae:
  • Diphtheria toxin produced by lysogenic β-phage strains carrying tox gene
  • Non-toxigenic strains can be converted to toxigenic by phage infection
  • Only toxin-producing strains cause diphtheria
  • Two fragments (A and B linked by disulfide bridge) form the toxin
    • Fragment A is cytotoxic
    • Fragment B binds to receptors on eukaryotic cells causing high potency and lethality (130ng/kg body weight)
    • Acts by blocking protein synthesis
    • Excreted by the bacterial cell, non-toxic until exposed to trypsin

Clinical Significance: Respiratory diphtheria

• Humans are the only natural host
• Carried in upper respiratory tract (URT and spreads via droplet and hand to mouth contact)
• Incubation period of 2-5 days
• Illness starts gradually, marked by low-grade fever, malaise, mild sore throat
• Common site of infection: tonsils or pharynx
• Rapid multiplication occurs on epithelial cells
• Necrotic cells and exudate form a pseudomembrane
• Toxin causes demyelinating peripheral neuritis which may cause paralysis following the acute illness

Clinical Significance: Cutaneous diphtheria

• Prevalent in the tropics
• Toxin is absorbed systematically
• Marked by non-healing ulcers with a dirty gray membrane
• Treatment via horse-derived anti-toxin (P or E drug of choice)

Lab DX

• Microscopy - highly pleomorphic gram-positive bacilli in palisades or V and L forms, club-shaped swellings and beaded forms common, metachromatic areas stain intensely (Babes-Ernst granules).
• Culture - grows best in aerobic conditions at 37ºC, requires 8 essential amino acids, may have a small zone of β-hemolysis, use CTBA, Loeffler's serum, or Pai agars, catalase-positive, non-motile, C. diphtheriae is urease negative, ferments glucose and maltose with acid but no gas, reduces nitrates.
• Toxigenicity tests

Listeria monocytogenes

• Comprises 6 species, only L. monocytogenes and L. ivanovii are pathogenic
• Found in environment (soil, water, vegetation, animal products like raw milk, cheese, poultry, and processed meats)
• Isolated from crustaceans, flies and ticks
• Serious infections primarily in neonates, pregnant women, elderly, and immunocompromised hosts
• Virulence factors: Listeriolysin O (hemolysin), Catalase superoxide dismutase, Phospholipase C, Surface protein p60
• Clinical infections:
  • Listeriosis in pregnant women is most common during third trimester, responsible for spontaneous abortion and stillbirth
  • Flu-like illness (fever, headache, myalgia) is often self-limited due to elimination during childbirth
  • Disease in newborns can be fatal (up to 50%). 2 forms exist: early onset (intra-uterine/shortly after birth, sepsis is often the outcome) and late onset (occurs several days to weeks after birth, meningitis is often the manifestation)
  • Immunocompromised patients can develop CNS infections and endocarditis from contaminated food ingestion.
• Lab DX:
  • Microscopy - gram-positive coccobacillus, short chains, palisades
  • Culture - grows well on SBA, chocolate agar, nutrient agar, BHIB, Thio broth; small, round, smooth, translucent colonies; surrounded by a narrow zone of β-hemolysis; growth over a wide range (0.5-45°C). Optimum: 30-35°C
  • Cold enrichment is often necessary
  • Catalase-positive, motility in wet smear (umbrella pattern at room temperature). Other tests: Hippurate hydrolysis (positive), BE (positive), CAMP (positive), block-type hemolysis on CAMP, acid from glucose (positive), VP, MR (positive).

Non-Spore Forming, Non-Branching Catalase Negative Bacilli

• Erysipelothrix rhusiopathiae

  • Gram-positive, non-spore-forming, pleomorphic rods (can produce filaments)
  • Distributed in nature, particularly affecting swine, turkey, sheep.
  • humans are affected by occupational cuts & scratches (fish handlers, animal products)
  • Clinical infections include acute erysipeloid (self-limiting localized infection, usually on hands/fingers, painful swelling, heals 3-4 weeks), Endocarditis (less common).
  • Diagnostics
    • Colony morphology (CO2 required, grows on blood or chocolate agars, colonies may appear gray or translucent, pinpoint, alpha hemolysis or non-hemolytic)
    • Microscopy (pleomorphic, gram-positive thin rods, may form long filaments form long filaments or short rods, arranged singly, in short chains, or in V shape decolorizes easily so it may appear gram-variable).
    • Identification (catalase, nitrate, urease negative, non-motile, produce H2S on TSI, VP negative, does not hydrolyze esculin, growth in semi-solid motility media, two colony types – pin point non-hemolytic, glistening colonies, or larger rough colonies with matte, curled, irregular edges)

• Other important genera - Arcanobacterium, (3 medically important species,A. haemolyticum, A. pyogenes, A. bernardiae). Actinomycetes, and Nocardia.

Gardnerella vaginalis

• Member of the normal flora of the female genital tract
• Associated with bacterial vaginosis
• Marked by foul odor, vaginal pH >4.5
• Diagnostics:
  • Wet prep – look for clue cells (large epithelials with various bacterial types on edges)
  • Gram stain – small, thin gram-variable rods.
  • Culture – growth on BAP, CA; no growth on MAC; beta-hemolytic in human blood bilayer tween "V" agar - requires a CO2 environment. Catalase negative

Mycobacterium

• Aerobic bacilli, non-spore forming. Nonmotile, Cell walls rich in lipids, Acid fast bacilli, Very slow growing
• Diseases associated include Tuberculosis, Leprosy (M.leprae), Non Tuberculosis Mycobacteria.
• Classifications: Various types of mycobacteria are associated with human diseases
  • Tuberculois, Leprosy, Non Tuberculosis Mycobacteria (Nocardia)
• Lab Diagnosis for Mycobacterium:
  • Microscopy, Gram stain, culture, Oxidative type metabolism, no specific growth factors for media or growth conditions needed, growth after 3 to 6 days.

Bacillus

• Aerobic, Catalase positive, Not fastidious
• Important Bacillus include B. anthracis, B. cereus.
• B. anthracis
  • large bacilli (3-5 μm), single or paired in clinical isolates, polypeptide capsule and exotoxins, highly resistant central spores.
  • Symptoms - Cutaneous (malignant pustule, incubation 2-3 days erythematous papule, increasing necrotic/later ruptures to form a painless black eschar), Gastrointestinal (contaminated meat), Pulmonary
  • Diagnosis: Specimen aspiration or swab from cutaneous lesion, blood culture, sputum investigation, gram stain and culture, Identification via isolate identification
  • Treatment: Penicillin / tetracycline / chloramphenicol, Erythromycin or Clindamycin
  • Prevention: Vaccination of animal herds, proper disposal of carcasses, active immunization using attenuated bacilli.
  • B. cereus:
    • Large, motile, saprophytic bacillus - heat resistant spores, airborne and dust-borne contaminants, forms heat-stable (emetic syndrome) and heat-labile toxins (diarrhoeal disease) – multiplies readily in cooked foods (rice, potato, meat).
    • Lab diagnosis - demonstration of large number of bacilli in food.
    • Symptoms - Emetic (incubation <6 hours, severe vomiting, lasts 8-10 hours), Diarrhoeal (incubation >6 hours, diarrhoea, lasts 20-36 hours)

Note: Additional details and specifics for each topic and species are available within the provided text.