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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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    Clinically relevant Gram- positive anaerobic cocci (GPAC), new names and their antibiotic susceptibility © by author ESCMID Online Lecture Library Linda Veloo Contents Introduction Gram-positive anaerobic cocci Peptostreptococcus sp. (formerly) New species and taxonomy Identification : - Phenotypic - Vitek ANC© bycard author - Fluorescent in situ hybridisation (FISH) - MALDI-TOF MS ESCMID Online Lecture Library Antibiotic resistance Virulence F. magna Introduction Gram-positive anaerobic cocci (GPAC): Peptococcus P.niger Seldomly isolated from clinical material Ruminococcus Faecel microbiota Seldomly ©isolated by fromauthor clinical material Insufficient culture methods? ESCMIDCoprococcus Online Lecture Library Isolated from faecal samples Gram positive anaerobic cocci, D.A. Murdoch Clinical Microbiology reviews, 1998 Introduction Atopobium Faecal microbiota and clinical material Sarcina Soil, abdominal samples, faecal samples from vegetarians and patients with gastro-intestinal abnormalities Unique cell© morphology: by author Groups of 8 cells which might produce spores ESCMID Online Lecture Library Peptostreptococcus The genus Peptostreptococcus contained 13 species. Clinically most relevant (literature): P. anaerobius P. asaccharolyticus P. magnus P. micros © by author ESCMID Online Lecture Library New species, changed taxonomy (in chronological order) In 1997 3 new species were added to the genus Peptostreptococcus P. harei P. ivorii P. octavius Clinical relevance unknown© by author ESCMID Online Lecture Library Description of Three New Species of the Genus Peptostreptococcus from Human Clinical Specimens: P. harei sp. nov., P. ivorii sp. nov., and P. octavius sp. nov., D. A. Murdoch et al., Int. J. Syst. Bact. 1997 New taxonomy Reclassification of Peptostreptococcus magnus (Prevot 1933) Holdeman and Moore 1972 as Finegoldia magna comb. nov. and Peptostreptococcus micros (Prevot 1933) Smith 1957 as Micromonas micros comb.
  • Streptococcus Agalactiae, Arcanobacterium Haemolyticum과 Finegoldia Magna에 의한 괴사성 근막염 1예

    Streptococcus Agalactiae, Arcanobacterium Haemolyticum과 Finegoldia Magna에 의한 괴사성 근막염 1예

    대한진단검사의학회지 제 28권제3호 2008 � 증례∙임상미생물학 � Korean J Lab Med 2008;28:191-5 DOI 10.3343/kjlm.2008.28.3.191 개에게 물린 당뇨환자의 Streptococcus agalactiae, Arcanobacterium haemolyticum과 Finegoldia magna에 의한 괴사성 근막염 1예 이성실1∙노경호2∙김창기1∙용동은1,3∙최준용 4∙이진우∙이경원1,3∙정윤섭1 연세대학교 의과대학 진단검사의학교실 세균내성연구소1, BK21 의과학사업단 3, 내과학교실4, 정형외과학교실5, 고려대학교 의과대학 진단검사의학교실2 A Case of Necrotizing Fasciitis Due to Streptococcus agalactiae, Arcanobacterium haemolyticum, and Finegoldia magna in a Dog-bitten Patient with Diabetes Sungsil Lee, M.D.1, Kyoung Ho Roh, M.D.2, Chang Ki Kim, M.D.1, Dongeun Yong, M.D.1,3, Jun Yong Choi, M.D.4, Jin Woo Lee, M.D.5, Kyungwon Lee, M.D.1,3, and Yunsop Chong, Ph.D.1 Departments of Laboratory Medicine and Research Institute of Bacterial Resistance1; BK21 Project for Medical Sciences3, Internal Medicine4, and Orthopedic Surgery5, Yonsei University College of Medicine, Seoul; Department of Laboratory Medicine2, Korea University College of Medicine, Seoul, Korea We report a case of necrotizing fasciitis involving Streptococcus agalactiae, Arcanobacterium haemolyticum, and Finegoldia magna in a 36-yr-old female diabetic patient, which started after a minor dog bite to the toe of the patient. This case suggested that a trivial infection after a minor dog bite in an immunocompromised patient such as diabetes patient could result in a significant compli- cation such as necrotizing fasciitis. The life-threatening infection was cured by timely above-the-knee amputation, as well as penicillin G and clindamycin therapy. (Korean J Lab Med 2008;28:191-5) Key Words : Necrotizing fasciitis, Streptococcus agalactiae, Arcanobacterium haemolyticum, Finegoldia magna, Diabetic patient INTRODUCTION tions[1-3].