LAB 13 – ANAEROBIC BACTERIOLOGY Flashcards
What factors contribute to O2 sensitivity of anaerobes? How do they do this?
- Direct toxic effects of oxygen
- Toxic products of oxygen metabolism
- Strict anaerobes lack superoxide dismutase (SOD), which converts superoxide anion (O2-) to H2O2
- Some anaerobes lack catalase, which neutralizes H2O2 - Oxidation-reduction potential (Eh) must be low
- Oxidized organic constituents of media form in media stored in O2:
- C. perfringens can grow on BAP stored for weeks
- C. haemolyticum cannot grow if media exposed for 1-2 hrs
What clues may signal that a patient has an anaerobic infection?
- foul odor
- location of infection in proximity to mucosal surface
- infections secondary to human or animal bite
- gas
- previous therapy with aminoglycoside antibiotics (gentamicin, tobramycin, amikacin)
- black discoloration of blood-containing exudates (may fluoresce red)
- sulfur granules in discharges (Actinomyces)
- failure to grow, aerobically, organisms seen on Gram stain of original exudate
- growth anaerobically
- characteristic colonies
How long should primary anaerobic plates be incubated for?
at least 2 days/ 48 hours
Why are transport systems used for anaerobic specimens? Name one.
designed to avoid exposure of specimen to oxygen (Cary-Blair transport)
What media is used to isolate Clostridium difficile? What type of media is it?
Cycloserine-Cefoxitin-egg yolk-Fructose agar (CCFA):
- selective for flat, gray to white rhizoid colonies (flt gry/ wh rhz)
- yellowish under regular light
- yellow-green fluorescence under UV
BAP* or PEA: flt gry/ wh rhz
PEA - Phenyl Ethyl Alcohol agar
Why are reducing agents used for anaerobic media? What is the most commonly used reducing agent?
- Reducing agents prevent formation of oxidized constituents in media
Eg. cysteine
What are 2 indicators used to detect anaerobiosis?
- Methylene blue
- colorless when reduced
- blue when oxidized - Resazurin
- colorless when reduced
- pink when oxidized
Describe EYA agar and the 2 things it detects.
Egg-Yolk Agar detects enzymes produced by Clostridium sp.:
1. Lipase
2. Lecithinase
What are 2 conditions caused by Clostridium difficile? What causes them?
- Antibiotic-associated diarrhea
- Pseudomembrane colitis
- due to Toxins A and B
What are some diseases/conditions that Clostridium species cause?
C. botulinum = Botulism
C. tetani = Tetanus
C. perfringens = Food poisoning, gas gangrene
C. difficile = Antibiotic-associated diarrhea and pseudomembrane colitis (toxins A and B)
How would you work up a Gram positive bacilli, sporulating anaerobe?
- suspected Clostridium sp.
1. Aerotolerance test
2. Colony morph
3. Gram stain
4. EYA lipase + lecithinase
5. Reverse CAMP
6. GLC
Why do we do aerotolerance?
To confirm that a suspect isolate is actually an anaerobe
Describe the Nagler inhibition test.
- neutralization of C. perfringens alpha toxin (lecithinase) by an antitoxin
- 3-4 drops antitoxin is spread on half of EYA
- C. perfringens is inoculated heavily on both halves
Result: C. perfringens will grow on both halves, however lecithin will be precipitated (wide zone of opacity) only on the half without antitoxin - alpha toxin (lecithinase) acitivty is neutralized on the other half
What is the identification scheme for an anaerobic Gram negative bacilli?
- Aerotolerance test
2.Colony morph - Gram stain
- Antibiotic susceptibility
- Biochemical tests
- GLC
B. fragilis group ID
- g-b, pleo with capsule
- shy gy
- most frequently isolated anaerobes
- found in mixed infections of abdominal cavity
- Bile esculin agar (+)
- penicillin (B-lactamase), kanamycin, vancomycin, and colistin resistant
Pigmented Prevotella sp. ID
- bile esculin (-)
- saccharolytic, black colonies incubated for >7 days on KVLB
- kanamycin and vancomycin resistant
- young colonies = red fluorescence under UV
KVLB = Kanamycin-Vancomnycin-Laked-Blood agar
Non-pigmented Prevotella sp.
- bile esculin (-)
- may fluoresce pink or orange under UV
Fusobacterium ID
- g-b; long, slender, tapered ends
- wh op β/α-hem on BAP*
- vancomycin resistant
- kanamycin and colistin susceptible
- GLC end-product: butyric acid
What are the Kanamycin Vancomycin Colistin (KVC) results for the following organisms?
a. Bacteroides fragilis
b. Prevotella melaninogenica
c. Fusobacterium sp.
a. Bacteroides fragilis = KVC resistant
b. Prevotella melaninogenica = Kanamycin and vancomycin resistant
c. Fusobacterium sp. = vancomycin (R), 1000 µg Kanamycin (S) and Colistin (S)
NOTE: melanin = pigmented Prevotella sp.
What are the results for the following tests for Clostridium perfringens?
a. Colonial morphology
b. Gram
c. EYA
d. Reverse camp
a. Colony morp = rzdzh on BAP*
b. Gram = g+b, boxcars, no spores
c. EYA = white zone of opacity; lecithinase (+)
d. Reverse camp = arrowhead (+) on BHI
BHI = brain-heart infusion agar
GLC end products of Genus Mobiluncus
succinic acid
GLC end products of Propionibacterium
propionic and acetic acids
GLC end products of Bifidobacterium
acetic and lactic acids
GLC end products of Eubacterium
acetic and butyric acids
GLC end products of Fusobacterium
butyric acid
GLC end products of Veilonella
acetic and propionic acids
GLC end products of Actinomyces
acetic, lactic, succinic acids
GLC end products of Lactobacillus
lactic acid
What media is used to show the pigmented colonies of Prevotella? What is the pigment?
Kanamycin-Vancomycin-Laked-Blood agar:
- black pigmented due to protohemin
