Anaerobes

Page 1: Chapter 16

Anaerobes

Page 2: Overview

  • Anaerobes

    • Prevalent throughout various environments.

    • Normal flora in:

      • Human mouth

      • Gastrointestinal tract

      • Colon

      • Female genital tract

      • Skin.

    • Outnumber aerobic bacteria by 1000:1 in the lower intestinal tract.

    • Considered important human pathogens.

    • Can cause devastating infections that are often polymicrobic (involving multiple types of microorganisms).

Page 3: Infections

  • Exogenous Infections

    • Originates from external sources.

    • Examples: Clostridium botulinum and C. tetani.

  • Endogenous Infections

    • More commonly sourced from within the body.

    • Involves commensal organisms displaced due to:

      • Surgery or trauma.

    • Example: Bacteroides fragilis leading to abdominal abscess.

Page 4: Oxygen Tolerance Classification

  • Obligate Aerobes: Require oxygen for growth and metabolism (referred to as aerobes).

  • Strict/Obligate Anaerobes: Cannot tolerate oxygen; will die upon exposure (commonly called anaerobes).

  • Facultative Anaerobes: Can use oxygen for metabolism but can grow in anaerobic conditions.

  • Aerotolerant Anaerobes: Can survive and grow in the presence of oxygen but do not utilize it.

  • Microaerophiles: Require oxygen but only in low concentrations.

Page 5: Specimen Collection Guidelines

  • Preferable Specimens

    • Abscesses from the liver, lung, brain, and abdomen.

    • Necrotic tissue from poor vascular perfusion, trauma, or malignancy.

    • Wound infections close to mucous membranes.

    • Exudates from deep wounds or abscesses, especially with gas, necrosis, or foul odor.

    • Gram-stained samples indicating anaerobic bacteria.

    • Specific fluids: cerebrospinal fluid, serous fluid from sterile sites, suprapubic urine, transtracheal aspirate, etc.

  • Specimens to Avoid or Reject

    • Nasal, pharyngeal, vaginal, cervical, or rectal swabs.

    • Voided or catheterized urine.

    • Swabs of surface skin ulcers and abscesses.

    • Sputum samples, gastric contents, and feces (unless for Clostridium difficile).

Page 6: Specimen Transport

  • Maintain specimens in an anaerobic state during transport to the laboratory.

  • Use of:

    • Gassed out collection tubes to maintain anaerobic conditions.

    • PRAS (Pre-reduced Anaerobically Sterilized media) for culturing under anaerobic conditions.

Page 7: Infections

  • Common Types:

    • Abscesses (brain, lung, liver, intra-abdominal).

    • Dental infections.

    • Aspiration pneumonia.

    • Peritonitis and appendicitis.

    • Post-operative or trauma wound infections.

    • Gynecologic conditions including post-abortal sepsis.

    • Cellulitis and myonecrosis.

Page 8: Gram Stain Characteristics

  • Bacteroides, Porphyromonas, Prevotella

    • Pale, pleomorphic, gram-negative coccobacilli exhibiting bipolar staining.

  • Fusobacterium species

    • Long, thin gram-negative bacilli (GNB) with tapered ends, arranged end to end.

  • Actinomyces species

    • Characterized by branching gram-positive bacilli.

  • Clostridia

    • Large gram-positive bacilli; C. perfringens shows boxcar arrangement.

Page 9: Spores

  • Classification by presence, location, and shape:

    • Clostridium

      • Types of spore locations:

        • Terminal spores (e.g., C. tetani: round and swollen).

        • Central and subterminal spores (e.g., C. botulinum: oval and swollen).

Page 10

  • Spore Types

    • Terminal, central, and subterminal spores definitions and characteristics.

Page 11: Anaerobic Media

  • Cultivation requirements:

    • Supplement with hemin, blood, vitamin K, and Na bicarbonate for CO2.

    • Include reducing agents like cysteine and thioglycollate to lower redox potential and create an anaerobic environment.

    • Held anaerobically for 8-16 hours before inoculation.

Page 12: Anaerobic Media Continued

  • PEA: Phenylethyl alcohol agar, inhibits facultative anaerobes to isolate gram-positive and gram-negative obligate anaerobes.

  • KV: Kanamycin-Vancomycin to inhibit gram-positive anaerobes and facultative gram-negative anaerobes, isolating gram-negative obligate anaerobes like Bacteroides and Fusobacterium.

Page 13: Anaerobic Media Continued

  • PVLB: Paromomycin-Vancomycin Laked Blood Agar, isolates gram-negative obligate anaerobes.

  • Thioglycollate: Enriched broth with hemin and vitamin K, used for plating media.

Page 14: Anaerobic Media for Specific Pathogens

  • BBE: Bacteroides Bile Esculin for growth and esculin hydrolysis of Bacteroides fragilis.

  • CCFA: Cycloserine-Cefoxitin Fructose Agar for selective isolation of Clostridium difficile, incubated at 35-37°C for at least 48 hours.

Page 15: Incubation Techniques

  • Anaerobic Jar: Uses generator envelopes that release H2 and CO2, removing O2.

  • Anaerobic Chamber/Glove Box: Contains a self-sufficient anaerobic system (85% N2, 10% H2, 5% CO2).

  • Gas-generating pouches: Disposable anaerobic bags used for transport.

Page 16: Identification Techniques

  • Identification methods include:

    • Gram stain analysis

    • Gross examination focusing on odors from volatile and non-volatile fatty acids.

    • Colonial morphology, pigment production, fluorescence and susceptibility to sodium polyanethol sulfonate (SPS).

Page 17: Biochemical Tests for Identification

  • Tests for identification

    • Include catalase test, nitrate reduction, indole production, growth in 20% bile, lipase production, lecithanase, and antibiotic susceptibility.

    • Gas-liquid chromatography (GLC) used for identifying anaerobic bacteria through fatty acid analysis.

Page 18: Gram Positive Spore Forming Bacilli - Clostridium

  • Characteristics:

    • Gram-positive, spore formers, form spores anaerobically, catalase negative, true exotoxin producers (neurotoxins).

    • Example neurotoxins:

      • Tetanospasmin

      • Botulism toxin

    • Can be found in soil, water, and as normal inhabitants of some animals.

Page 19: Clostridium tetani

  • Tetanus Infection

    • Introduced through puncture wounds, animal bites, or burns.

    • Tetanospasmin acts as a powerful neurotoxin causing:

      • Convulsive contractions of voluntary muscles.

      • Symptoms include lockjaw and characteristic back arching.

Page 20: Clostridium tetani - Prevention and Treatment

  • Prevention: DTaP Immunization, Td booster, tetanus toxoid.

  • Treatment: 50% mortality untreated; includes neutralizing antibodies (antitoxin), antibiotics, and supportive therapy.

Page 21: Clostridium tetani - Diagnosis

  • Diagnosis based on clinical symptoms, patient history, identification of:

    • Gram positive bacilli (GPB)

    • Round, swollen, terminal spores resembling a tennis racket.

    • Gelatinase positive, non-fermentative for sugars and rarely recovered except during autopsy.

Page 22: Clostridium botulinum

  • Botulism Infection

    • Caused by food with preformed toxin (often from home-canned foods) and from wounds or infants ingesting spores.

    • Resulting botulism toxin binds to nerve fiber synapses causing flaccid paralysis, especially affecting thoracic and diaphragm muscles.

    • Seven toxin types: A, B, C, D, E, F, G with A, B, E being the most common.

Page 23: Clostridium botulinum - Diagnosis

  • Diagnosis: Clinical signs, patient history.

  • Treatment: Antitoxin administration to bind botulism toxin.

  • Identification:

    • Lipase positive, ferments glucose, and oval, subterminal spores.

Page 24: Clostridium perfringens

  • Infections Caused By C. perfringens

    • Myonecrosis (gas gangrene).

    • Food poisoning from processed foods.

    • Post-abortion sepsis and intra-abdominal infections.

    • Associated with antibiotic-related diarrhea and pleuro-pulmonary infections.

Page 25: Clostridium perfringens - Toxins

  • Toxins produced include:

    • Alpha toxin (lecithanase), hemolysins, cardiotoxin, collagenase, fibrinolysin, DNAse, RNAse, and enterotoxin with various proteolytic enzymes contributing to pathogenicity.

Page 26: Clostridium perfringens - Identification

  • Identification features:

    • Large GPB arranged end-to-end.

    • Double zone of hemolysis on blood agar due to theta and alpha toxins.

Page 27: Clostridium perfringens - Identification Techniques

  • Identification Tests:

    • Lipase positive on egg yolk agar; demonstrates lecithanase production.

    • Reverse CAMP positive indicating arrowhead in hemolysis when tested with Group B Streptococcus.

    • Saccharolytic, ferments glucose, lactose, maltose, fructose.

Page 28: Clostridium difficile

  • Characteristics:

    • Gram-positive, non-spore forming bacilli.

    • Normal flora of the bowel with rare opportunistic infections.

    • Found in soil, water, intestinal tracts of animals, and in the stools of 5% healthy adults.

    • Causes antibiotic-associated pseudomembranous colitis and health care-associated infections in broad-spectrum antibiotic treated patients.

Page 29: Clostridium difficile - Identification

  • Gold standard: tissue culture.

  • Common identification through testing for toxin A (enterotoxin) or toxin B (cytotoxin) via immunoassay or tissue culture.

  • Isolation media: CCFA (yellow, umbunate colonies) and CDSA.

  • Biochemical reactions that are gelatinase positive and ferment glucose/fructose.

Page 30: Gram Positive Non-Spore Forming Bacilli

  • Normal flora of:

    • Oral cavity, bowel, vaginal tract, urinary tract.

    • Rare cases lead to opportunistic infections, such as actinomycosis caused by Actinomyces israelii presenting yellow “sulfur” granules, and Bifidobacterium expressing rare pulmonary, dental, or wound infections.

Page 31: Anaerobic Gram Positive Cocci

  • Normal flora located in:

    • Bowel, female genital tract, oral cavity, skin, respiratory tract.

  • Related infections include abscesses in the liver, brain, female genital tract, and abdominal cavity.