Anaerobes

Chapter 18: Anaerobes

Introduction to Anaerobes

  • Overview
    • Anaerobes are microorganisms that require a reduced oxygen environment for growth.
    • They are prevalent throughout various environments, especially within the human body.
    • Normal Flora (NF): Commonly found in the human mouth, gastrointestinal tract, colon, female genital tract, and skin.
    • In lower intestinal tract, anaerobes outnumber aerobes by a ratio of 1000:1.
    • They are important human pathogens and can cause devastating infections, particularly in cases of trauma, tissue necrosis, or vascular stasis.
    • Anaerobic infections are frequently polymicrobic, meaning they often involve multiple microbial species.

Types of Infections

  • Exogenous Infections

    • Originating from outside the body, typically associated with pathogens such as Clostridium botulinum and Clostridium tetani.

  • Endogenous Infections

    • More common; these infections arise from commensal organisms that are normally present within the body, introduced through trauma or surgical procedures.
    • Example: Bacteroides fragilis can cause abscesses after a crush injury.

Common Anaerobic Infections

  • Types of Anaerobic Infections
    • Appendicitis
    • Aspiration pneumonia
    • Abscesses: brain, intra-abdominal, liver, lung abscesses
    • Cellulitis
    • Dental infections
    • Gynecologic infections
    • Myonecrosis
    • Post-abortion sepsis
    • Pseudomembranous and ulcerative colitis
    • Wound infections following surgery or trauma

Frequently Isolated Anaerobes

  • Common Groups and Organisms
    • Gram-Negative Bacilli: Bacteroides fragilis group, Fusobacterium spp., Prevotella spp., Porphyromonas spp., Clostridium spp. (e.g. C. difficile).
    • Gram-Positive Bacilli: Propionibacterium, Cutibacterium.
    • Gram-Negative Cocci: Veillonella.
    • Gram-Positive Cocci: Peptostreptococcus.

Oxygen Tolerance

  • Obligate Anaerobes: Unable to use oxygen and inhibited by its presence.
  • Moderately obligate anaerobes: Tolerate low oxygen levels (2-8%), often found among human pathogens.
  • Strict Obligate Anaerobes: Inhibited by O2 concentrations of less than 0.5%.
  • Facultative Anaerobes: Can tolerate and grow in either presence or absence of oxygen.
  • Microaerophiles & Aerotolerant: Require low levels of oxygen (around 5% O2).
  • Protective Enzymes: Organisms may produce enzymes such as superoxide dismutase, catalase, and peroxidase that protect against toxic oxygen products like superoxide radicals and hydroxyl radicals.

Suitable Specimens for Anaerobic Cultures

  • Acceptable Specimens:

    • Fluid exudates from normally sterile sites: amniotic, pleural, peritoneal, synovial fluid.
    • Cerebrospinal fluid.
    • Suprapubic urine aspirate.
    • Wounds from bites (human or animal).
    • Suspicion of botulism from food samples.
    • Specimens containing sulfur granules from draining fistulas.
    • Diarrheal stool samples for Clostridiodes difficile toxin testing.

  • Non-Acceptable Specimens:

    • Nasal, gingival, pharyngeal, urethral, vaginal, cervical, and rectal swabs.
    • Voided or catheterized urine.
    • Swabs of surface skin ulcers, exposed wounds, or abscesses.
    • Expectorated sputum, feces (other than C. difficile), gastric contents, and intestinal contents.

Specimen Transport

  • Transport Requirements
    • Maintain anaerobic conditions during transport using gassed-out collection tubes or swab systems equipped with reducing agents.
    • PRAS (Pre-reduced Anaerobically Sterilized Media) may use cysteine as a reducing agent and incorporate a redox indicator.
    • Importance of swift transport and proper storage to avoid oxygen exposure.

Anaerobes: Microscopy and Identification

  • Gram Stain and Morphological Identification:
    • Typical appearances of anaerobes on gram stain may include:
    • Bacteroides, Porphyromonas, Prevotella: pale, pleomorphic gram-negative coccobacilli with bipolar staining.
    • Fusobacterium species: long thin filamentous gram-negative bacilli with tapered ends.
    • Actinomyces species: show sulfur granules, branching gram-positive bacilli.
    • Clostridia: large gram-positive bacilli, e.g., C. perfringens has a distinct boxcar arrangement.

Endospores in Anaerobes

  • Definition of Endospore: Dormant, tough non-reproductive structures formed by some bacteria under environmental stress, aiding in survival.
  • Endospore Characteristics:
    • Presence, location, and shape can indicate species, as seen in Clostridium. Examples include:
    • Clostridium tetani: swollen round terminal spores.
    • Clostridium botulinum: oval swollen spores, which begin to germinate in optimal conditions similar to seeds.

Anaerobic Media and Cultivation Techniques

  • Growth Conditions:

    • Anaerobic media supplemented with hemin, blood, yeast, vitamin K, and sources of sodium bicarbonate (as CO2 source).
    • Use of reducing agents like cysteine or thioglycollate which absorb oxygen, plus holding media anaerobically 8–16 hours before inoculation.

  • Types of Media:

    • Anaerobic Blood Agar (AnBAP): Enriched, nonselective media for primary isolation of anaerobes.
    • Selective Media:
    • PEA: Phenylethyl alcohol agar that inhibits facultative anaerobes.
    • KV: Kanamycin–Vancomycin media to isolate gram-negative obligate anaerobes.
    • BBE: Bacteroides bile esculin agar promoting growth in 20% bile.
    • CCFA: Cycloserine-cefoxitin fructose agar, selective for Clostridiodes difficile.

Incubation of Anaerobes

  • Incubation Methods:

    • Anaerobic Jar: For small to medium lab volumes, employing gas generator envelopes to remove oxygen.
    • Anaerobic Chamber/Glove Box: Used for larger volumes where all processes are contained in a self-contained anaerobic system.

  • Temperature and Duration: Recommended incubation at 35°C to 37°C for a minimum of 48 hours.

    • Additional examination of growth may extend for 2-5 days.

Identification of Anaerobes

  • Methods of Identification:

    • Use of Gram stain, biochemical tests, and gross examination for presumptive ID.
    • Characteristic odors, colonial morphology, and pigmentation are noted for certain species, like A. israelii (molar tooth appearance).
    • Further tests to include catalase activity, nitrate reduction, and susceptibility to sodium polyanethol sulfonate (SPS).

  • Biochemical Identification:

    • Important tests like lipase, protease, and motility assessed through wet mounts or egg yolk agar.

  • Utilization of Advanced Systems:

    • Enzyme-based systems and gas liquid chromatography for metabolic end product measurement. Methods using MALDI-TOF MS and 16s rDNA sequencing for high-level identification.

Specific Anaerobic Organisms

  • Bacteroides Genus:

    • Predominantly Bacteroides fragilis among over 20 species.
    • Known for virulence factors including a capsule for abscess formation and various enzymes impacting immune response and tissue integrity.

  • Clostridium species:

    • Exhibit a range of clinical conditions including tetanus (Clostridium tetani) characterized by immobilization results from tetanospasmin toxin.
    • Clostridium botulinum: Responsible for botulism, with variations like foodborne, infant, and wound types.
    • Gas Gangrene: Infections from Clostridium perfringens, often arising from trauma leading to myonecrosis.

  • Clostridiodes difficile: Significance in antibiotic-associated diarrhea and associated infections due to resistance and virulence.

Gram-Positive Non-Spore-Forming Bacilli

  • Actinomyces israelii: Associated with actinomycosis, characterized by yellow “sulfur” granules in exudates, known for polymicrobial infections.

Gram-Negative Cocci

  • Veillonella: Typically part of the normal flora but can be implicated in rare infections. Treatment typically involves penicillins and cephalosporins.

Summary of Susceptibility Testing

  • Challenges exist in testing due to slow growth and variable reproducibility. New antibiotic introductions and resistance patterns warrant specific susceptibility testing in clinically significant cases.