fall mico week 4

primary anaerobic plates

under 48 hrs killed by oxygen, grow best during log phase, NG at 48 hrs keep for 5-7 days or 14 is slow grower suspected

indicator of anaerobes

foul odar, type of media growing org, numerous colony types, morph

preliminary ID of anaerobes

descripe colony morph, subcultures to purity plates, gram stain and cell morph, observe growth on media, check aerotolerance

aerotolerance

subculture colony to BAP (ambient air), CHOC (increased CO2), ANA BAP (anaerobic condition)

BAP will grow

obligate aerobes and facultative anaerobes

CHOC will grow

Obligate aerobe, capnophilic aerobe, and facultative anaerobe

ANA BAP will grow

facultative anaerobe and obligate anaerobe

most commonly isolated anaerobe

bacteroides fragilis

normal skin flora and common contaiminants in wound and blood cultures

Peptostreptococcus and Propionobacterium species/Cutibacterium acnes

actinomyces

GPR, nuggets- pus, branching, beaded.banded, thin, filamentous, ANA BAP- white opaque molar tooth like A. israelli

bacteroides fragilis

GNR, safety pin (pleomorphic), ANA BAP, BBE, KV Agar, disks (K, V, C, bile resistant)

bacteroides fragilis ANA BAP

white gray, circular, entire, convex, large, translucent-semiopaque, gamma

bacteroides fragilis BBE

black-dark gray, esculin hydrolysis, pitts with B. ureolyticus

clostridium botulinum

GPR, straight, subterminal spores, tennis racket, appear GNR, ANA BAP- gray white circular-irregular Beta

C. diff

GPR, chains up to six, subterminal rare spores, can be gram variable, ANA BAP- large white circular matte-glossy, convex, opaque, gamma, horse stable odor, fluoresces yellow green (chartreuse)

C. perfingens

GVR, blunt ends, seldom spores- large central-subterminal oval swell cell, double beta, large boxcar shape, nonmotile, ANA BAP, EYA- lecithinase pos

C. perfingens ANA BAP

gray yellow, circular, glossy, dome, entire, translucent, double beta zone, NG on KV, inhibited by vancomycin

C. sordellii

GPR, straight, free oval or subterminal spores, single or pairs, ANA BAP- circular-irregular, mottled internal structure, scalloped or lobate margin, variable hemolysis

C. tetani

GPR, GNR after 24 hr incub, spores oval and terminal or subterminal drumstick tennis racket, ANA BAP- gray, matte, irregular-rhizoid margin, translucent, flat, narrow beta zone, can swarm

cutibacterium (propionibacterium) spp

GPR, pleomorphic, anaerobic diphtheroid, club shape to palisade, ANA BAP- small white gray become large yellow tan, P. avidum beta

most commonly isolated non spore forming GPR

cutibacterium (propionibacterium) spp

fuscobacterium necrophorum

GNR, round tapered ends, can filament, ANA BAP- fluoresces chartreuse, green agar with air, some Beta

Fusobacterium necleatum

GNR, sharply pointed or tapered ends, ANA BAP- bread crumb (white), speckled or smooth (gray-white), green agar with air, fluoresces chartreuse (yellow-green), gamma

porphyromonas spp

GNR, tiny, ANA BAP- black due to heme, shiny, round, fluoresces brick red

peprostreptococcus anaerobius

GPCB, large, chains, ANA BAP- medium, gray white, opaque, sweet fetid odor, larger than most anaerobic coci

prevotella sp

GNR, pairs or short chains, ANA BAP- white circular convex shiny gamma fluoresces brick red, LKV black

veillonella spp

GNDC, clusters pairs short chains, large, ANA BAP- small, transparent, gray-white, smooth, opaque, greasy, red fades fluorescence with UV (360 nm)

PEA

anaBAP overgrown with swarming orgs, discard after 48-72 hrs

BBE

slect colonies over 1 mm, perform spot catalase

LKV

select each colony type, check for fluorescence or pig

postive plate broth

incubate 7 days, use only as backup

neg plate broth

subculture and gram stain at 7 days or when turbid, subculture and gram stain only if primary plates are neg and broth is turbid

ana BAP PEA BBE LKV selected colonies

subculture to anaBAP for isolation with potency disks, subculture to EYA, RBA if needed, subculture to CAP for aerotolerance incub in CO2, prepare gram stain

CAP scant or NG after 24-48 hr incub

ANA BAP for Id and susceptability testing

CAP good growth after 24-48 hr incub

aerobic org

potency disks

used if cannot determine true gram stain rxn, antibiotic disk in 1st quadrant of ANA BAP

gram neg potency disks

V- kanamycin 1mg and colistin 10ug, R- vancomycin 5 ug, nitrate, bile

gram pos potency disks

S Vancomycin, V Kanamycin, R Colistin

GPC potency disks

nitrate, sodium polyanethol sulfonate SPS (Peptostreptococcus anaerobius)

GPR potency disks

nitrate

B. fragilis group potency disks

R Vancomycin, R Kanamycin, R Colistin

B. ureolyticus group potency disks

R Vancomycin, S Kanamycin, S Colistin

Fusobacterium spp potency disks

R Vancomycin, S Kanamycin, S Colistin

porphyromonas spp potency disks

S Vancomycin, R Kanamycin, R Colistin

Veillonella spp potency disks

R Vancomycin, S Kanamycin, S Colistin

indole and catalase test

24-48 hr pure culture, ANA BAP or EYA containing sufficient tryptophan, nitrate disk plate gives false neg

indole pos

Clostridium sordellii, C. tetani, Propionibacterium/Cutibacterium, Fusobacterium nucleatum, Porphyromonas, Bacillus sp (not anaerobe!)

catalase pos

Propionibacterium/Cutibacterium and Bacillus sp

fluorescence

over 72 hr pure cultire or tested directly to body site, any media containing blood, 365 UV light

P. biva/disiens fluorescence

light organge-pink coral

c. ramosum fluorescence

red

eggerthella lenta fluorescence

red or none

lipase test

24-48 hr pure culture from ANA BAP to EYA (fats broken down by lipase enzyme producing glycerol, FA, and iridescent layer), F. necrophorum

Lecithinase

24-48 hr pure culture from ANA BAP to EYA, splits, lecithin into water soluble diglycerides causing off white halo, C. perfringens

presumptive ID

growth on media, colony morph, rapid tests

definitive ID

commercial ID kits (same day as PC-SC plate 4 hr incub id most sig anaerobes), biochem kits (24-48 hr, id saccharolytic anaerobes), MALDI-TOF (under 1 hr most anaerobes),

susceptibility testing

done on anaerobes from brain abcess, endocarditis, prosthetic device/vascular graft infec, joint infec, osteomyelitis, bacteremia

wound culture growth on plates in ambiant CO2

SBA and CHOC- e coil s aureus, MAC- e coli, PEA/CNA- s aureus

wound culture growth in anaerobic incub BRU/BA

e coli, b. fragilis, pigmented prevotella, s aureus, peptostreptococcus anaerobius

wound culture growth in anaerobic incub PEA/CNA

b. fragilis, pigmented prevotella, s aureus, peptostreptococcus anaerobius

would culture growth in anaerobic incub BBE

b. fragilis

wound culture growth in anaerobic incub KYLB

b. fragilis, pigmented prevotella

susceptibility testing limitations

lack reproducibility, failure of some anaerobes to grow, lack comparability, cost, complex

primary choice for antimicrobial susceptilibity testing on GN anaerobes and B. fragilis group

Beta-bactam combination agent (ampicillin-sulbactam, amoxacillin-clavulanate, piperacillin-tazobactam)

supplemental choices for antimicrobial susceptilibity testing on GN anaerobes and B. fragilis group, and GP anaerobes

Clindamycin, carbpenem (ertapenem, imipenem, meropenem), metronidazole, ceft…, cefoxitin, moxifloxacin

primary choice for antimicrobial susceptilibity testing on GP anaerobes

ampicillin, penicillin, Beta-lactam combination agent (ampicillin-sulbactam, amoxacillin-clavulanate, piperacillin-tazobactam)