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Educational Workshop Educational Workshop EW04: Practical approach to diagnose mixed anaerobic infections in "real time“ Arranged with the ESCMID Study Group for Anaerobic Infections (ESGAI) Convenor: Elisabeth Nagy (Szeged, HU) Faculty: Diane .M. Citron (Culver City, US) Ulrik Stenz Justesen (Odense, DK) Georg Conrads (Aachen, DE) Elisabeth Nagy (Szeged, HU) Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Classical methods for identification of anaerobes from mixed infections: do we still need them? Diane M. Citron R.M. Alden Research Lab Culver City, California Survey of USA Hospital Labs (2006-2007) 150 hospital laboratories (200-1000 beds); 85% responded to questionnaire 85% processed anaerobic cultures 15% sent to reference lab 100% used selective and differential media for isolation (in addition to blood agar) 30% use identification disks 66% use preformed enzyme kits 30% use other biochemical tests 4% GLC 0% used molecular methods Goldstein EJ et al, 2008, Anaerobe 14:68-72 Practical considerations Few commercial molecular kits available for routine anaerobic specimens except in specialized areas (C. difficile toxin) Molecular methods mostly limited to in-house preparations in large labs or research institutions 1 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Incidence of anaerobes in clinical specimens B. fragilis group 35% B. fragilis (40-50%) B. thetaiotaomicron (20%) Other Bf group (20-40%) Prevotella-Porphyromonas etc. 15-20% Fusobacterium 5-10% Anaerobic cocci 25-30% Clostridium 8-15% Non-sporeforming GPR 5-10% What classical methods are being used? Selective and differential agar media to culture anaerobes Rapid tests such as catalase, spot indole, urease, nitrate Identification kits – RapID ANA II, Rapid ID 32a and others Tube biochemicals GLC for short and long chain fatty acids Selective and differential media Bacteroides bile-esculin- gentamicin agar (BBE) Rapid presumptive ID of B. fragilis group, Alistipes, and Bilophila. Some strains of Fusobacterium mortiferum and F. varium may grow. (bile resistant) Enterococci highly resistant to gentamicin will grow 2 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Selective and differential media Laked blood with kanamycin and vancomycin brucella agar (LKV) Inhibits gram-positives and enterics Grows Bacteroides, Prevotella, Alistipes some fusobacteria(and Van-R Clost. innocuum!) Enhances pigmentation of Prev. melaninogenica group Growth on Selective and differential media EYA-Fusobacterium necrophorum (FEA) PEA Porphyromonas LKV-BBE B. fragilis Prevotella BBE-Bilophila species CCFA- Clostridium difficile F. nucleatum Identification disks: kanamycin 1000 ug vancomycin 5 ug colistin 10 ug P. intermedia B. fragilis 3 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Spot indole test p-DMACA reagent Colonies can also be rubbed onto filter paper moistened with reagent. Blue color is + Rapid urease test (disks, tablets) preformed enzyme; incubate aerobically, read 1-2h neg pos Grouping of gram-negative rods Bile Kana Van Col Cat Nit B.fragilis grp R R R R + - Prevotella S R R V - - Porphyromonas S R S R - - Fusobacteria V S R S - - Bilophila R S R S + + Desulfovibrio VSRR-+ + C. ureolyticus grp. V S R S -+ + 4 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Bacteroides fragilis group bile resistant (BBE plate) resistant to kana, vanco, colistin disks most clinical isolates are catalase pos comprise 1/3 of clinical isolates most virulent (capsule) most antibiotic resistant Parabacteroides distasonis, merdae, goldsteinii, johnsonii, gordonii Prevotella species Isolated from oral and pelvic infections, abdominal and soft tissue Growth inhibited on BBE (but may turn agar black from hydrolysis of esculin if colonies plated directly) Resistant to kana, vanco, variable colistin Catalase and indole usually negative Fusobacterium spp species bile NO3 ind lipase esc cells nucleatum S - + - - r,pt necrophorum SR -++-r,pleo naviforme S - + - - boat gonidiaform. S - + - - gonidia mortiferum R - - - v pleo varium R - +- -+ -pleo 5 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Fusobacterium nucleatum S to kana, colistin disks; R to vanco indole positive; lipase negative slender rods with pointed ends several different colony types; subspecies Isolated from all types of infections and all areas of the body polymicrobial infections or single isolate Fusobacterium necrophorum S to kana and colistin disks ; R to vanco indole and lipase positive; beta- hemolytic cells have rounded ends or may be very pleomorphic important pathogen in Lemmiere’s disease and postanginal sepsis in young people (deep vein thrombosis, metastatic thrombi) Throat cultures? Bacteroides ureolyticus-like grp Kana-S, Vanco-R, Col~S URE MOT CAT BILE CO2 NO2 B. ureolyticus +--+ --+ Camp. gracilis -----+ Camp. rectus -+---+ Bilo. wadsworthia +-+R -+ Sut. wadsworthen. ---R-+ Dial. pneumo. Col-R ------ Desulfovibrio Col-R -+ +/- -+ -R -+ Eiken. corrod. - - --++ ( Use disk test for bile resistance) 6 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Clostridium perfringens Box car shaped GPB Double zone of beta-hemolysis on BA Lecithinase positive Produces abundant gas in liquid media (blood culture bottles?) Clostridium innocuum Resistant to cefoxitin, other cephs, vancomycin (MIC= 8-16), some strains R to clindamycin, quinolones Grows on CCFA medium – resembles C. difficile but (PRO-neg) Disks- resistant to Ka, Va, Cl Misidentified in preformed enzyme kits ( often as C. subterminale) Gram variable rod, occ large terminal spores Isolated from IA infections, blood Clostridium ramosum Gram-variable long slender rod Chains in broth media Spores are rarely visible – terminal May be resistant to antimicrobials Identify using enzyme kits, biochemicals 7 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Clostridium clostridioforme group Typically stains Gram-negative Spores are rarely seen Sensitive to vanco and kana disks One of most frequently encountered clostridia ~10-30% produce beta-lactamase; generally more resistant to antimicrobials (clinda, moxifloxacin) Identify with enzyme kits or biochems New species: boltae, hathewayii, citroniae, aldenense Clostridium septicum Swarmer; subterminal spores; indole-neg Bacteremia associated with cancer of large bowel Portal of entry – ileocecum Myonecrosis, gas gangrene) Underlying conditions- (leukemia, lymphoma, diabetes Susceptible to usual antimicrobials Toxins not eliminated with abx treatment High mortality Gram-positive rods– non-sporeforming catalase nitrate indole Propionibacteria + +- +- Actinomyces -+ +- Eggerthella lenta + + - “Eubact. grp.” - -+ - Lactobacilli - - - Bifidobacteria - - - 8 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Propionibacterium acnes Frequent skin contaminant in blood, csf cultures Occasionally pathogenic (shunts, implants, post- op cultures from eye) Relatively slow-growing ID based on pos rxns for catalase, indole, nitrate Acne strains may be R to tetra and macrolides All strains R metronidazole Importance of basic tests – ID to group >75% of clinical isolates Gram stain – cell morph Growth in 20% bile Susceptibility to 1mg kana Susceptibility to 5 ug vanco Catalase Spot indole Nitrate Fluorescence Limitations of phenotypic identification tests Current and newer taxonomy is based on 16S rRNA and other gene sequences Identification kits have limited databases resulting in misidentifications of some organisms Expensive and time-consuming for the results obtained 9 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? What can classical methods still do? Provide a basic level of identification based on phenotypic appearance Provide isolates for further study Susceptibility to antimicrobials and to detect novel resistance mechanisms Sequencing to identify new species Molecular characterization of resistance mechanisms – limitations The cfiA gene is present in 3-5% of bacteroides fragilis, yet resistance is not expressed unless insertion sequences are present NimB genes may be present if Finegoldia magna, yet the strains remain susceptible to metronidazole Multiple mechanisms may be present in one strain – eg beta-lactamase + efflux pumps Emergence of novel mechanisms Diversity of Anaerobic Bacteremia: Gram-negative isolates Group, Genus 16S rRNA vs Conventional Genus Species Genus Species None Bacteroides (129) 129 124 127 102 26 Fusobacterium (15) 15 15 15 14 0 Parabacteroides (7) 7 7 7 5 2 Porphyromonas (1) 1 1 1 0 0 Prevotella (11) 11 11 10 8 4 Veillonella (4) 4 3 0 0 4 Other (4)* 4 3 0 0 4 *Alistipes, Bilophila, Campylobacter Simmon KE el al, J Clin Microbiol 2008,46:1596 10 Citron - Classical methods for identification of anaerobes from mixed infections: do we still need them? Diversity of Anaerobic Bacteremia: Gram-positive isolates Group, Genus 16S rRNA Conventional Genus Species Genus Species None Anaerococcus (7) 7 1 7 0 0 Clostridium (97) 97 93 93 71 13 Eggerthella (14) 14 14 9 7 5 Eubacterium
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