Anaerobes Flashcards
require atmosphere of 15-21% oxygen as found in air or CO2 incubator
i.e., Mycobacteria
Obligate aerobe
require oxygen content lower than ambient air (<5%)
i.e., Campylobacter, Neisseria, Helicobacter
Microaerophilic
organisms that multiply equally in the presence or absence of air
i.e., Enterobacteriaceae
Facultative anaerobe
require increased CO2 (5-10%)
i.e., Haemophilus, Neisseria
Capnophile
can survive some oxygen exposure, but will not be able to preform metabolic processes unless in an anaerobic environment
i.e., Bacteroides
Aerotolerant anaerobe
requires strict anaerobic environment and are killed almost immediately in the presence of oxygen
i.e., Clostridium
Obligate anaerobe
Exogenous anaerobes
- exist outside of the body
- infection develops at site of entry
- i.e., Clostridium, Fusobacterium
Endogenous anaerobes
- exist inside the body
- source of infection in/throughout body
- usually polymicrobial infections
- i.e., Bacteroides, Peptostreptococcus, Actinomyces
Skin anaerobes
Propionibacterium, Peptostreptococcus
Respiratory tract anaerobes
Prevotella, Porphyromonas, Fusobacterium
Gastrointestinal tract anaerobes
Bacteroides, Bifidobacterium, Clostridium, Eubacterium
Genitourinary tract anaerobes
Fusobacterium, Prevotella, Bacteroides, Lactobacillus
Predisposition to Anaerobic infections
- trauma to protective barriers (bowel, genital tract)
- vascular stasis (no oxygen to site)
- bite wounds, oral surgery, oral flora into deep lungs, burns with soil contamination
Anaerobic bacteria (characteristics)
- close proximity to mucosal surface
- produces a foul odor
- large quantity of gas
- black color or brick-red fluorescence
- distinct morphological characteristics (pleomorphic, fusiform)
Gram-positive, spore-forming, anaerobic, bacilli
Clostridium
- boxcar-shaped gram-positive bacilli
- double zone of hemolysis on Brucella blood agar or blood agar
- lecithinase +
C. perfringens
- heavily swarming
- terminal spores
C. tetani
- heavily swarming
- subterminal spores
C. septicum
Clostridium Clinical Infections
- mostly exogenous (wounds, ingest toxin)
- endogenous is C. difficile
- tetanus, gas gangrene, botulism, food poisoning
C. perfringens food poisoning
- 3rd most common after Salmonella and Shigella
- Type A and Type C
C. perfringens Type A food poisoning
- ingestion of enterotoxin
- causes diarrhea and cramps
- usually self-resolving
C. perfringens Type C food poisoning
- causes Enteritis necroticans
- acute onset of severe abdominal pain and bloody diarrhea, vomiting
- necrotic inflammation of small intestines
- fatal without treatment
- causes Botulism
- Botulism toxins A-G
- Toxins A, B, E associated with human disease
- causes flaccid paralysis
- caused by ingestion of toxin, ingestion of spores (infants), or wound botulism
- 2 hours to 8 days
C. botulinum
infant botulism
- ingestion of spores in contaminated honey
- colonize colon and produce toxins
Tetanus
- caused by C. tetani
- tetanospasmin neurotoxin (inhibits neurotransmitters)
- symptoms begin after 7 days (long incubation)
- muscular rigidity of jaw, neck and lumbar
- vaccine available (DPT)
Myonecrosis (gas gangrene)
- most commonly caused by C. perfringens
- contaminated wounds from trauma or surgery
- alpha toxin causes tissue necrosis and deeper invasion
- tissue death that leads to amputation
- treat with antibiotics, surgical debridement and hyperbaric oxygen (to kill anaerobes)
Bacteremia
- C. perfringens is most common
- C. septicum is marker for GI malignancy
- C. bifermentans and C. tertium are secondary
Antibiotic-associated diarrhea
- C. difficile
- causes pseudomembranous colitis (bloody diarrhea, necrosis)
- antibiotics destroy flora, but not Clostridium so increased toxin production (Toxin A and B)
- nosocomial infection
- test for toxin, not culture
Gram-positive non-spore-forming anaerobic bacilli
- Actinomyces, Bifidobacterium and Propionibacterium
- Mobiluncus
- Lactobacillus
Actinomyces, Bifidobacterium and Propionibacterium
- cause Actinomycosis (sulfur granules)
- P. acnes is frequent blood culture contaminant
- normal skin flora
Actinomycosis
chronic granulomatous disease characterized by sinus tracts and fistulae that erupt to the surface and drain pus that contains “sulfur granules”
- bacterial vaginosis
- overgrowth when Lactobacillus decreases
Mobiluncus
- highly pleomorphic bacilli
- contaminant
- bacteremia and endocarditis in ICP
Lactobacillus
Anaerobic gram-negative bacilli
- Bacteroides fragilis
- Prevotella
- Porphyromonas
- Fusobacterium
- # 1 normal flora in colon
- most commonly isolated anaerobe from blood cultures
- # 1 agent in peritonitis and intra-abdominal abscess
- grows on KVLB and BBE
- resistant to vanco, kanamycin and colistin disks
Bacteroides fragilis
- P. melaninogneica is #1 normal flora of gingiva
- also found in vagina
- peridontal disease, sinusitis, pneumonia, brain abscesses
Prevotella
- slow grower
- normal flora of oropharynx and urogenital tract
- head and neck infections
Porphyromonas
- commensal bacteria in gingiva
- normal in GI, GU and URT
- mixed infections (abscesses, peritoneal infections, diabetic foot ulcers)
- Lemierre disease (F. necrophorum)
Fusobacterium (necrophorum and nucleatum)
- long, thin rods with pointed ends
- breadcrumb, speckled or smooth colonies
- greening of agar with air exposure
- fluoresces chartreuse
- nonhemolytic, indole +, lipase -
F. nucleatum
- slightly pleomorophic with rounded ends
- circulate colonies
- greening with air exposure
- beta-hemolytic, indole +, lipase +
- fluoresces chartreuse
F. necrophorum
Anaerobic cocci
- Peptostreptococcus, Anaerococcus, Finegoldia, Parvimonas, Peptoniphilus (gram-positive)
- Veillonella (gram-negative)
Anaerobic cocci clinical infections
- brain abscesses, meningitis, aspiration pneumonia, lung abscess, gingivitis
- polymicrobial infections
Specimen collection
- acceptable specimens must be collected
- tissue biopsy or needle aspiration is best
- must be actual site of infection (not surface)
- rapid processing (because of oxygen toxicity)
Aspirates
- needle and syringe
- inject specimen into prereduced anaerobically sterilized media (PRAS)
- vortex and plate to isolation media
Anaerobic infections
MOST ARE POLYMICROBIAL
Swabs
not recommended for specimen collection
Blood
must collect anaerobic and aerobic blood culture bottles
Anaerobic chambers
- glove boxes, anaerobic jars, biobags
- oxygen-free environment
Direct examination of anaerobic specimen
- rules in or out pathogens
- establishes polymicrobial or monomicrobial
- presumptive identification
- aids in selection of culture media
- determines specimen quality
Gram-staining anaerobes
use methanol instead of heat as a fixative
Anaerobic chambers
- 5-10% Hydrogen gas
- 5-10% CO2 gas
- 80-90% Nitrogen gas
- Palladium-coated alumina pellets to remove all oxygen
- desiccant to absorb excess water
- indicator (turns when O2 present)
Anaerobic cultures
- foul odor
- characteristic morphology on anaerobic agars
- unique morphology on gram stain
- good growth on Bacteroides bile-esculin agar (BBE)
- double zone of hemolysis
- brick-red fluorescence on KVLB
Kanamycin vancomycin laked blood (KVLB)
- anaerobes have brick-red fluorescence on agar
- Porphyromonas and Prevotella turn it black
Processing suspected anaerobes
- Observe growth and morphology on selective media
- Observe gram-stain and cell morphology
- aerotolerance test
- inoculate pure culture plate and add disks
determines whether an organism is a strict anaerobe or a facultative anaerobe
Aerotolerance test
Aerotolerance test (plates)
- chocolate agar plate incubated in CO2 incubator
- anaerobic blood agar plate incubated anaerobically
Brick-red fluorescent anaerobes
Prophyromonas and Prevotella
Chartreuse fluorescent anaerobes
Fusobacterium nucleatum, Fusobacterium necrophorum and Clostridium difficile
red fluorescent anaerobe
Veillonella
Catalase test
separates Bacillus (+) and Clostridium (-)
Anaerobe tests
catalase test
spot indole test
rapid urease test
rapid motility test
Spot indole test
- dilated cardiomyopathy with ataxia (DCMA) reagent
- blue/green is positive
- different from regular indole test
Anaerobe special potency disks
Vancomycin disk, Kanamycin disk, Colistin disk
Vancomycin disk
- gram-positive bacillus and Porphyromonas are susceptible
- Fusobacterium are resistant
Kanamycin disk
- Bacteroides (not fragilis), Bilophila, Fusobacterium and Veillonella are susceptible
- Porphyromonas, B. fragilis are resistant
Colistin disk
- Bacteroides (not fragilis), Bilophila, Fusobacterium, Veillonella and Prevotella are susceptible
- Porphyromonas, B. fragilis are resistant
Anaerobic antimicrobial disks
Bacteroides fragilis is resistant to all 3 (Vanco, Kanamycin and Colistin)
Nitrate disk
organisms that have ability to reduce nitrate
Sodium polyanethol sulfonate (SPS) disk
- Peptostreptococcus anaerobius is susceptible
- Peptoniphilus asaccharolyticus is resistant
Bile disk
- for organisms that can grow in 20% bile
- B. fragilis
Egg Yolk Agar (EYA)
- Lecithinase cleaves lecithin found in egg yolk and releases insoluble fat
- forms opaque zone around colony
- C. perfringens (lecithinase +)
Lipase
- hydrolyzes triglycerides and diglycerides to fatty acids and glycerol
- iridescent, multicolored sheen
- F. necrophorum (+)
Clostridium (identification)
- double zone of hemolysis
- Cycloserine-cefoxitin-fructose agar (CCFA)
- lecithinase and lipase reactions
- spot indole, rapid urease, gelatin hydrolysis
- selective and differential for C. difficile
- has yellow “ground glass” colonies
Cycloserine-cefoxitin-fructose agar (CCFA)
Bile-tolerant gram-negative bacilli
Bacteroides fragilis
Bile-sensitive pigmented gram-negative bacilli
Porphyromonas and Prevotella
Bile-sensitive nonpigmented gram-negative bacilli
Bacteroides ureolyticus, Campylobacter, Bilophila wadsworthia
B. fragilis (special media)
KVLB and BBE biplate
Pigmented Gram-negative bacilli
Bacteroides, Prevotella, Porphyromonas
- produces protoporphyrin
- brown-black color shift of macroscopic colonies over 2-3 weeks
- brick-red fluorescence
Prevotella
- brick-red fluorescence
- no growth on media containing vancomycin
Porphyromonas
- isolated following tooth extraction
- nonpigmented
- pits the agar
- urease +
B. ureolyticus
Prevotella (body sites)
vagina and oral cavities
- gram-negative
- long, thin, tapered rods
- may be pleomorphic
Fusobacterium
- classic fusiform shape
- indole +, lipase -
- butyric acid from PYG media
F. nucleatum
Other Fusobacterium
F. mortiferum, F. russi
- many clinical specimens contain these
- vagina and abscesses
- tetrads, chains or clusters
- P. anaerobius, P. asaccharolyticus
Anaerobic gram-positive cocci
- gram-positive cocci
- SPS sensitive
- indole -
- acid production in PYG
Peptostreptococcus anaerobius
- gram-positive cocci
- SPS resistant
- indole +
Peptostreptococcus asaccharolyticus
Antimicrobial susceptibility testing is performed in
brain abscesses endocarditis prosthetic device infection vascular graft infection joint infections osteomyelitis bacteremia
Highly virulent/commonly resistant anaerobes
B. fragilis Prevotella Clostridium Fusobacterium B. wadsworthia Sutterella wadsworthensis
Treatment of anaerobic diseases
- destroy reduced environment (debridement, draining)
- hyperbaric oxygen environment
- antimicrobials, antitoxins
Definitive ID tests
- biochemical systems
- gas-liquid chromatography
- ribosomal sequencing
Presumptive ID tests
- disks
- media
- morphology
- pitting of agar
- urease +
Bacteroides ureolyticus
Actinomyces
- cause Actinomycosis (sulfur granules)
- molar tooth colonies
