Learning Outcomes

  • Corynebacterium

    • State the general characteristics of the genus Corynebacterium.
    • State the criteria for considering "diphtheroids" as potential pathogens.
    • Demonstrate awareness of the following emerging pathogens:
    • C. jeikeium (lipophilic, antibiotic resistance).
    • C. pseudodiphtheriticum (urease POS, respiratory infections).
    • C. urealyticum (urease rapid POS, lipophilic, UTIs).
    • Compare and contrast classic C. diphtheriae, C. ulcerans, and "diphtheroids" based on:
    • Appearance on Tinsdale, urease activity, nitrate reduction, clinical significance, virulence factors.
    • Describe selective media for toxigenic Corynebacterium (Tinsdale, CTBA):
    • Principle (selective and differential ingredients, appearance of organisms), advantages and disadvantages.
    • Briefly describe Loeffler’s serum slant and its use.
    • Explain the term “lysogenic conversion” in C. diphtheriae: how it occurs and its effects on virulence and disease.
    • Discuss specimens and plate selection for the recovery of C. diphtheriae (carriers and diagnosis of diphtheria).
    • Briefly describe the purpose and principle of the Elek plate test.
    • Describe the role that immunization status and low socioeconomic status play in maintaining a reservoir of C. diphtheriae.
    • Describe symptoms of classic (respiratory) diphtheria concerning diphtheria toxin, pseudomembrane formation, and its impact on the patient.
    • Describe treatment of classic (respiratory) diphtheria (contrast the use of antitoxin and antimicrobials).
    • Briefly describe cutaneous diphtheria: patient population, lesion, co-infection.
    • Discuss the prevention of diphtheria using DTaP.

  • Listeria

    • Briefly describe how food can be a source of infection.
    • Explain the implications of Listeria monocytogenes being a “facultative intracellular parasite.”
    • Describe the morphology of Listeria monocytogenes (BAP, smear).
    • Describe tumbling motility and umbrella motility testing for Listeria monocytogenes.
    • Compare and contrast Listeria monocytogenes with Streptococcus agalactiae based on:
    • Neonatal disease, appearance on BAP, Gram-stain morphology, catalase activity, motility, BEA, CAMP, hippurate.
    • Describe “cold enrichment.”
    • Briefly differentiate disease processes in:
    • Immunocompetent individuals.
    • Pregnant women.
    • Fetus/neonate (early-onset vs. late-onset).
    • Immunocompromised individuals.

  • Bacillus

    • State the general characteristics of the genus Bacillus.
    • Briefly describe endospores: function, appearance, relevance to culture and identification.
    • Outline the clinical significance of B. cereus (two foodborne forms; ocular).
    • Outline the clinical significance of B. anthracis (how the organism is acquired; disease forms).
    • Briefly describe safety considerations when working with suspected B. anthracis.
    • Differentiate between B. anthracis and B. cereus based on colonial morphology, hemolysis, and motility.

  • Catalase-negative gpb

    • For Lactobacillus, describe atmospheric requirements, macroscopic/microscopic morphology, and catalase result; list normal flora sites; describe “minimal ID.”
    • For Arcanobacterium, describe disease spectrum, macroscopic/microscopic morphology, catalase results, and CAMP reaction.
    • For Erysipelothrix, outline disease spectrum, macroscopic/microscopic morphology, catalase results, and TSI reactions.

Aerobic Gram-positive Bacilli

Macroscopic and Microscopic Morphology

  • Morphological investigations are crucial for identifying aerobic Gram-positive bacilli.
  • Regular rods: parallel sides, not curved; includes some spore-formers.
  • Irregular rods: non-parallel sides, often curved; can display coccus-to-rod variation and rudimentary branching.
  • Most species grow on BAP, showing a wide range of macroscopic appearances.

Corynebacterium

  • Part of the “coryneform group” of bacteria (from the Greek word "koryne" meaning club).
    • Only true Corynebacterium species show a club shape; others exhibit irregular morphologies.
    • The coryneforms include:
    • Rudimentary branching: e.g., Actinomyces and Arcanobacterium.
    • Coccus-to-rod variation: e.g., Brevibacterium.
    • Previously classified as staphylococci: e.g., Rothia mucilaginosa.

General Characteristics of Corynebacterium

  • Appearance: small, irregular, slightly-curving, possibly club-shaped.
  • Arrangement: sometimes in V, L, K, or palisade (“picket fence”) formations due to daughter cell attachment during division.
  • Oxygen requirement: most are facultative anaerobes (fermentative), others are obligate aerobes.
  • Lipophilic: require lipids for optimal growth.
  • Catalase: positive.
  • Non-spore-forming (asporogenous), non-motile, non-acid-fast.
  • Habitat: found in human and animal mucous membranes and skin, also in soil and plants.

Significant Species

  • Historically, Corynebacterium diphtheriae has been known for causing diphtheria.
    • Toxigenic strains of C. diphtheriae, C. ulcerans, and rarely, C. pseudotuberculosis are significant.
    • Other species (often termed “diphtheroids”) were previously viewed as commensals but are increasingly recognized as potential pathogens.

Conditions for Testing Corynebacterium

  • Corynebacterium spp. should be considered for further testing in the following scenarios:
    • Isolated from sterile body fluids.
    • Predominant organism in a quality specimen.
    • Significant colony count in urine cultures.
  • Strengthened significance noted if:
    • The same species isolated from multiple specimens.
    • Observable inflammatory response in immunocompetent patients (↑PMNs, particularly if intracellular).
    • Predominant in mixed cultures with low pathogenicity organisms.

Comparison Chart of Toxigenic Corynebacterium & Diphtheroids

  • Corynebacterium diphtheriae

    • Normal Flora: Found in asymptomatic carriers (nasopharynx & skin lesions), never normal flora.
    • Tinsdale Appearance: Black colonies with brown halo.
    • Urease: Negative.
    • Clinical Significance: Toxigenic strains cause diphtheria; non-toxigenic strains associated with endocarditis, foreign body infections, pharyngitis.

  • Corynebacterium ulcerans

    • Habitual flora among healthy hosts on skin and mucous membranes.
    • Tinsdale Appearance: Similar to C. diphtheriae but may lack halo.
    • Urease: Positive.
    • Clinical Significance: Exudative pharyngitis, skin ulcers.

  • Corynebacterium pseudotuberculosis

    • Common in sheep, not normal flora in humans.
    • Tinsdale Appearance: Black colonies, no halo; minor clinical significance.

Brief Notes on Species Within Corynebacterium

  • Various species are associated with specific pathologies:
    • Wound infections: C. diphtheriae (cutaneous diphtheria), C. ulcerans, C. jeikeium.
    • Respiratory infections: C. diphtheriae, C. pseudodiphtheriticum, C. pseudotuberculosis.
    • Urinary tract infections: C. urealyticum.

Detailed Overview of Notable Corynebacterium Species

  • Corynebacterium amycolatum
    • Common skin flora; associated with serious infections (e.g., UTI).
    • Resistant to various antibiotics; tiny colonies.
  • Corynebacterium jeikeium
    • Resistant to most antibiotics; susceptible to vancomycin.
  • Corynebacterium urealyticum
    • Lipophilic; rapidly positive urease (within 15 minutes).
  • Corynebacterium striatum
    • Present in normal skin flora; associated with nosocomial infections.

Corynebacterium in the Laboratory

Growth Requirements

  • Generally non-fastidious; grow on routine media but slowly (16-18 hr).
  • Some species require lipids (lipophilic) and may need 3 days incubation on routine media.
  • Plates should be incubated at 35-37°C in ambient air or CO2.

Media for Corynebacterium

  • BAP (Blood Agar Plate):
    • Supports growth of lipophilic and non-lipophilic species.
    • Typical colony appearance: NH, sm, wh, op, dry/waxy/crumbly.
  • TINS (Tinsdale Agar):
    • Selective and differential; produces black colonies with or without a brown halo based on tellurite reduction.
  • CTBA (Cystine-Tellurite Blood Agar):
    • More inhibitory; can produce black colonies without halos (depends on strain).

Identification and Testing

  • Loeffler’s Serum Slant: Utilized for enhanced recovery & identifying metachromatic granules in C. diphtheriae.
  • Specimens for C. diphtheriae:
    • Collect throat/nasopharyngeal swabs, skin/wound swabs; culture on specialized agars (TINS, BAP, Loeffler’s).
    • Important processes include precise media use and careful monitoring of colony characteristics.

Mechanisms of Pathogenicity

  • Toxigenic Strains:
    • Produce diphtheria toxin via infection with a lysogenic tox+ corynephage.
    • Exotoxin inhibits protein synthesis in eukaryotic cells, causing severe disease.
  • Non-toxigenic Strains:
    • Cause less severe disease; include virulence factors like neuraminidase, sialidase, and adhesion factors.

Specimen Collection for Diphtheria Investigation

  • When investigating diphtheria, specific specimen collections are vital:
    • Throat swab from inflamed area & nasopharyngeal swabs for carrier status.
    • Skin/wound swabs for cutaneous diphtheria.

Toxigenicity Testing

  • Guinea Pig Toxicity Test:
    • Determines potential toxin production; control guinea pig survives with antitoxin.
  • Elek Plate Test:
    • Confirmatory test for toxin production using antitoxin-impregnated filter paper to assess precipitate lines.

Clinical Significance of C. diphtheriae

  • Main cause of respiratory diphtheria and cutaneous infections.
  • Presents as an epidemic disease in areas with low immunization coverage; asymptomatic carriers contribute significantly to its transmission.

Symptoms and Treatment for Diphtheria

  • Respiratory Diphtheria: Symptoms include fever, sore throat, pseudomembrane formation (risk for airway obstruction).
  • Treatment:
    • Clinical diagnosis via symptoms, lab confirmation expected post-Antitoxin administration; antibiotics (penicillin or erythromycin) reduce bacterial load but not toxin binding.

Listeria

  • Found commonly in soil, plants, animals; significant as a foodborne pathogen, especially in the context of immunocompromised populations.
  • Listeria monocytogenes:
    • A small, regular Gram-positive bacilli, exhibiting tumbling motility and an ability to survive and reproduce at refrigeration temperatures.

Clinical Significance of Listeria

  • Primarily affects:
    • Immunocompetent (asymptomatic).
    • Pregnant women (risk for fetal transmission).
    • Newborns: high-risk for severe infection outcomes.

Testing for Listeria

  • Media: BAP, BEA for identification, key tests include motility observation in broth (tumbling, 'umbrella' motility).

Bacillus

  • A diverse genus commonly found in the environment, often presenting as large Gram-positive bacilli.
  • Endospores: Dormant forms that enable survival in adverse conditions.
  • Bacillus cereus and Bacillus anthracis represent clinically significant species, each associated with distinctive disease processes and types of harmful effects.

Treatment for Bacillus Infections

  • B. cereus usually treated with vancomycin or ciprofloxacin, while anthrax requires prolonged protocols and post-exposure prophylaxis in at-risk populations.

Conclusion

  • Understanding various Gram-positive bacilli, particularly their morphology, pathogenic mechanisms, and clinical implications, is crucial for effective diagnosis and treatment in clinical settings.
  • Comprehensive testing using specialized media, toxin testing, and specimen handling is vital to accurately identify and manage infections caused by these microorganisms.