anaerobes

Describe the different atmosphere requirements of the organisms in this group

Obligate anaerobe = do not grow in the presence of oxygen
Aerotolerant organisms = grow best in anaerobic conditions but can grow in up to 5% oxygen
Facultative anaerobes = can grow in both aerobic and anaerobic conditions
Specimen processing
- anaerobic cultures should only be ordered when a site is truly anaerobic
  - ACCEPTABLE: biopsies, body fluids, aspirates, tissue
  - NOT ACCEPTABLE: sputum, swabs of superficial wounds, vaginal swabs, urine
- plates are kept in anaerobic jars or bags until they are read either on the bench or in an anaerobic box

Identify the special types of media used to cultivate anaerobes

Culture media
- Brucella blood agar
  - non-selective, grows most anaerobic organisms and facultative anaerobes
  - has vitamin K and hemin for pigment production
  - growth nutrients added
    - tryptophan to facilitate production of indole
  - detects hemolytic patterns
  - good for special potency discs and nitrate discs
  - addition of PEA prevents proteus from swarming
- Bi Plate - LKV and BBE
  - LVK is selective and differential
    - antibiotics inhibit most facultative gram positive and gram-negative organisms
    - vitamin K for pigmented Prevotella species
  - BBE is selective and differential and has gentamich which inhibits most facultative anaerobes
    - Bile inhibits bile sensitive organisms and allows bile resistant ones to grow
    - Esculin detects esculin hydrolysis
    - Hemin growth factor good for catalase testing of colonies
- Egg-Yolk agar
  - Detects lecithinase production
    - lecithinase splits lecithin (found in egg yolk) to insoluble diglycerides resulting in an
    - positive reaction: opaque halo surrounding a positive colony
  - Detects lipase production
    - free fats present in egg yolk are broken down by lipase to produce glycerol and fatty acids
    - positive reaction: “oil on water” layer that covers the colony and may extend beyond or as a zone of opacity directly beneath the colony
  - Detects proteolysis
    - produces a clearing of the medium around the colony
- Thioglycolate broth
- Cycloserine cefoxitin fructose agar (CCFA)
- Chopped meat glucose or carbohydrate broth media
Methods for identification
- Gram stain
- media reactions
- catalase, indole
- antibiotic discs
- nitrate and bile discs

Recognize the resistance patterns seen in anaerobes and how they are used for identification

Clostridium perfringens
- Vancomycin: sensitive
- Colisten: resistant
Clostridium tetani
- Vancomycin: sensitive
- Colisten: resistant
Clostridium botulinum
- Vancomycin: sensitive
- Colisten resistant
Actinomyces
- Vancomycin: sensitive
- Colistin: resistant
Cutibacterium acnes
- Vancomycin: sensitive
- Colistin: resistant
Peptostreptococcus
- Colistin: resistant
Bacteroides fragilis
- Vancomycin: resistant
- Colistin: resistant
- Bile: resistant
Prevotella
- Vancomycin: resistant
- Kanamycin: resistant
- Bile: sensitive
Porphyromonas
- Vancomycin: sensitive
- Kanamycin: resistant
- Colistin: resistant
- Bile: sensitive
Fusobacterium necrophorum
- Colistin: sensitive
- Kanamycin: sensitive

Identify characteristics and morphology in each organism

Gram positive spore-forming bacilli
- Clostridium perfringes
  - boxcar shaped, double zone of beta hemolysis
  - reverse CAMP, catalase negative
  - produces several endotoxins that destroy and disrupt host cells
  - causes gangrene, necrotizing fasciitis, food poisoning, necrotizing enteritis
- Clostridium tetani
  - subterminal spores
  - indole positive, catalase negative
  - produces TeNT neurotoxin that disrupts impulses to muscles
  - causes tetanus (lockjaw)
- Clostridium botulinum
  - subterminal spores
  - catalase negative
  - produces BoNT, a very potent neurotoxin that causes “rag doll” paralysis of essential muscle groups
  - causes botulism
- Clostridium difficile
  - produces toxin A (enterotoxin) and toxin B (cytotoxin), only strains that produce at least one of these toxins can cause infections
  - infection cause by decrease in normal gut flora (usually due to antibiotic usage)
  - can progress into pseudomembranous colitis (surface of inflamed bowel becomes coated in necrotic debris, WBCs, and fibrin)
  - Can do a rapid test kid for toxin and/or antigen
- Clostridium sordellii
  - gram-positive rods, subterminal spores, can swarm on brucella agar
  - causes skin and soft tissue infections
- Clostridium septicum
  - stains poorly as it gets older (start to look gram-negative)
  - gram-positive rods, subterminal spores, swell cells, swarms, beta hemolytic, irregular rhizoid margins, “medusa head”
  - causes bacteremia and trauma-associated gangrene
Gram-positive non-spore forming bacilli
- Actinomyces
  - filamentous branching rods, “molar tooth” colonies
  - indole negative, nitrate positive
  - causes periodontal disease and soft tissue infections
- Bifidobacterium
  - gram-positive diphtheroid, pointed or curved rods, branching, can have forked ends, usually “dog bones” or “Y” shaped
  - usually found in mixed pelvic or abdominal infections
- Cutibacterium acnes (Propionibacterium acnes)
  - yellow, raised colonies, gram positive rods, can look diphtheroid-like
  - indole positive, nitrate positive, catalase positive
  - opportunistic pathogen, common blood culture contaminant
- Eubacterium
  - gram-positive, pleomorphic rods/coccobacilli, can have bending, filaments, and branching
  - causes oral infections, sinusitis, tonsillitis, and other various mixed infections
- Lactobacillus
  - gram-positive pleomorphic rods/coccobacilli
  - some species in this group are aerobic but some can also be anaerobic
  - often a contaminant (usually vaginal flora)
  - can cause sepsis in immunocompromised patients (rare)
Gram- positive cocci
- Peptostreptococcus
  - indole positive, catalase positive, nitrate negative
  - P. asaccharolyticus
    - GPC pairs, tetrads, or irregular clusters
  - P. anaerobius
    - GP coccobacilli chains
    - “sweet rotting” odor
Gram-negative bacilli
- Bacteroides fragilis
  - indole negative, nitrate negative, catalase positive
  - black on BBE agar
  - most common cause of anaerobic infections
- Biophilia
  - pale staining, delicate gram-negative rods
  - On BBE agar, after 3-5 days, colonies can have a black center that may disappear when exposed to oxygen
  - cause intra-abdominal infections and abscesses
- Prevotella
  - pigmented and non-pigmented varieties
  - catalase negative, indole negative
  - normal flora of the oral cavity
  - pigmented Prevotella will have brick red fluorescence and be black on LKV agar
- Porphyromonas
  - P. gingivalis
    - gram-negative coccobacilli
    - dark brown/black pigmented colonies, fluoresces brick red
    - indole positive, nitrate negative, catalase negative
    - causes periodontal diseases and gingivitis
- Fusobacterium necrophorum
  - most virulent species in this group
  - gram-negative, pale staining, slender spindle shaped rods, pointed ends, can be filamentous, fluoresce chartreuse
  - indole positive, nitrate negative, catalase negative
  - Fusobacterium nucleatum
    - gram-negative, pale staining, slender spindle shaped rods, pointed ends, bread-crumb like colony, chartreuse fluoresce
    - causes head/neck and pleuro-pulmonary infections
- Fusobacterium mortiferum
  - gram-negative, pale staining, filamentous rods with swollen areas, “fried egg” colony
  - causes various infections, oral, bacteremia, etc.
  - biochemically the same as others but indole negative
- Mobiluncus
  - normal vaginal flora but can cause bacterial vaginosis
  - gram-variable, small, thin curved rods
  - very small colonies
- Veillonella
  - rarely significant but can be found in mixed infections
  - tiny gram-negative diplococci in clusters, pairs, and chains