Gram Stain Short, coccoid, or pyriform. Starch & Glycogen + NO3 + Hemolysis - * **C. diphtheriae biotype intermedius:** Small (0.5 mm), sometime pinpoint, flat, and gray. “FROG’S EGGS

Gram-Positive Bacilli

Unit 1: Non-Spore-Forming Gram-Positive Bacilli

  • Includes genera: Corynebacterium, Listeria, Erysipelothrix, and Lactobacillus.
  • Some are common isolates but considered contaminants (Corynebacterium, Lactobacillus).
  • Others are rare but cause significant disease (Listeria, Erysipelothrix, Corynebacterium diphtheriae).
Corynebacterium diphtheriae

  • Common Name: Klebs-Loeffler's bacillus

  • First observed by Edwin Klebs in 1883, cultivated by Friedrich Löffler in 1884.

  • General Characteristics:

    • Gram-positive bacilli
    • Pleomorphic, club-shaped appearance due to irregular swellings at one end. Term "diphtheroid" is used for this morphology.
    • Arranged in pairs at angles (X, Y, V, L configuration or "Chinese letters") or parallel to each other ("palisades").
    • Metachromatic granules (volutin or Babe-Ernst granules) irregularly distributed in cytoplasm, giving a beaded appearance.
    • Nonmotile
    • Non-spore-forming
    • Aerobic/Facultative anaerobic
    • Catalase (+)
    • Oxidase (+)

  • Habitat and Transmission:

    • Habitat: Human nasopharynx
    • Transmission: Direct contact with respiratory secretions or exudates from skin lesions.

  • Pathogenesis and Clinical Manifestations:

    • Causes diphtheria, an acute disease caused by toxigenic strains.
    • Incubation: 2-5 days (range 1-10 days).
    • Classified by anatomic site:
      • Respiratory Diphtheria:
        • Most common sites: pharynx and tonsils.
        • Gradual onset of pharyngitis; early symptoms include sore throat, low-grade fever, and dyspnea.
        • Localized Manifestations:
          • Pseudomembrane Formation: Gray-white patches composed of fibrin, necrotic host cells, and bacteria over the pharynx, tonsils, uvula, and palate. Firmly adherent; forcible removal causes bleeding. Extension into larynx or nasal area may cause respiratory obstruction, coma, and death.
          • Bull Neck: In severe cases, marked edema of submandibular areas and anterior neck with lymphadenopathy.
        • Systemic Manifestations:
          • Myocarditis, polyneuritis, nephritis, and thrombocytopenia.
            • Paralysis of eye muscles, limbs, and diaphragm can occur after the fifth week. Diaphragmatic paralysis can lead to secondary pneumonia and respiratory failure.
            • Heart damage leads to heart failure, the most common cause of mortality.
          • Death occurs in 5-10%.
      • Cutaneous Diphtheria (Wound Diphtheria):
        • Infected skin lesions lacking characteristic appearance; a membrane forms on the infected wound that fails to heal.
        • May be associated with non-toxigenic strains.
        • Less frequently results in systemic complications.

  • Virulence Factors:

    • Diphtheria Exotoxin (Diphtherotoxin):
      • Produced by lysogenized strains infected by β-prophages carrying the tox gene. All strains produce the same antigenic type of toxin.
      • Absorption of toxins causes local tissue destruction and damage to the peripheral nervous system, heart, and other organs.
      • Composed of two polypeptide fragments:
        • Fragment A: Interacts with cytoplasm factors to stop protein synthesis by inhibiting polypeptide chain elongation, causing necrotizing and neurotoxic effects.
        • Fragment B: Binds to and facilitates entry of toxin into cytoplasm of heart and nervous system cells via receptor-mediated endocytosis.

  • Prevention and Control

    • Vaccination with diphtheria toxoid induces protective antitoxin antibodies. Toxin is converted to toxoid by treatment with formalin.
    • Given as part of DPT vaccine (diphtheria, pertussis, tetanus). Three vaccinations starting at 6-8 weeks of age, followed by boosters at 15 months and school age.
    • Multi-antigen vaccines (with Hep B, Hib, or IPV) are increasingly used.
    • Suspected respiratory diphtheria cases should promptly receive diphtheria antitoxin (produced in horses) without waiting for lab confirmation. Neutralizes circulating toxin but not toxin already fixed to tissues.
    • Diphtheria infections are also managed with antibiotics. Patients are usually non-contagious 48 hours after starting antibiotics. Antibiotic therapy should be used with antitoxin therapy.

  • Schick Test:

    • Determines susceptibility (lack of antitoxins) or immunity (presence of circulating antitoxins).
    • Determines hypersensitivity to diphtheria toxin or other proteins.
    • Procedure: Injection of 0.1 mL diphtheria toxin on test arm (TA) and 0.1 mL diphtheria toxoid (vaccine preparation or heat-inactivated toxin) on control arm (CA).
    • The injection site (TA and CA) is inspected daily up to the 6th day for erythema, induration or necrosis which consists of a positive reaction.
    • Interpretation:
      • Positive Reaction: TA - reaction persists until 6th day; CA - no reaction. Susceptible; NOT hypersensitive
      • Negative Reaction: TA - no reaction; CA - no reaction. Immune; Not hypersensitive
      • Combined Reaction: TA - reaction persists until 6th day; CA - reaction peaks at 48 hours and subsides by day 5. Susceptible and hypersensitive
      • Pseudo-reaction: TA - reaction subsides by day 5; CA - reaction subsides by day 5. Immune and hypersensitive

  • Laboratory Diagnosis:

    • Specimens: Swabs from oropharynx (beneath pseudomembrane), nasopharynx, or cutaneous lesions.
    • Microscopy:
      • Gram staining: Pleomorphic gram-positive rods in angular arrangements ("Chinese letters" or palisades), swollen ends producing a club shape.
      • Loeffler's alkaline methylene blue (LAMB) or Albert's staining: Pleomorphic beaded rods, reddish-purple metachromatic granules.
    • Cultural Method:
      1. 5% sheep BAM, Columbia CNA agar: Screen and rule out group A β-hemolytic streptococci.
      2. Media containing cystine and potassium tellurite: selective and differential for Corynebacterium species.
      • Tinsdale agar (TIN):
        • High concentration of potassium tellurite inhibits most upper respiratory tract normal flora (other than Corynebacterium species) and majority of gram-negative bacteria; organisms capable of growing on the medium are differentiated based on the tellurite reductase activity, resulting in the reduction to tellurium, thus will grow as black colonies.
        • Bovine serum and horse serum provide essential growth factors.
        • Sodium thiosulfate provides sulfur for H2SH_2S production; and cystine detects cystinase activity producing brown halos around the colonies.
      • Cystine-tellurite blood agar (CTBA): Heart infusion agar supplemented with 5% rabbit blood, tellurite, and L-cystine. Selective and differential medium for Corynebacterium species similar to TIN.
      1. Loeffler's serum medium: Contains eggs and beef serum. Stimulates growth of club-shaped cells and production of metachromatic granules. "Poached egg" colonies with no characteristic differential features.
      2. Pai medium: Contains coagulated egg in distilled water and glycerin.
    • Inoculation and Incubation: Specimens inoculated to blood agar and a selective medium (CTBA or TIN). Incubate in ambient air or 5-10% CO2CO_2 for 24-48 hours.
    • Colonial Characterization:
      • Four colony types (biotypes): gravis, mitis, intermedius, and belfanti.
      • On BAM: Colonies range from small, gray, and translucent (biotype intermedius) to medium, white, and opaque (biotypes mitis, belfanti, and gravis); C. diphtheriae biotype mitis may be β-hemolytic.
      • On CTBA: Colonies appear black or gray.
      • On TIN: All four biotypes grow as black colonies surrounded by dark brown halos.
        Gram Stain and growth characteristics of Corynebacterium diphtheriae Biotypes
    • C. diphtheriae biotype gravis: Large (2-4 mm), flatter, dark gray with radial striations and irregular edges. “DAISY HEAD