Resistance of anaerobic bacteria Can we find any clinical impact ? Szeged ESCMID School 2005 L. Dubreuil Faculté de Pharmacie Université de Lille II, France Antibiotic resistance among anaerobes Intrinsic resistance all anaerobes aminoglycosides, quinolones, fosfomycin,trimethoprim,aztreonam species -dependant : metronidazole : Propionibacterium & Actinomyces rifampicin : F. necrophorum, F. mortiferum cephalosporins : C. difficile cefotetan & vancomycin : C. innocuum Bacteroides fragilis group : C1G, aminopenicillins Fusobacterium : macrolides Acquired resistance Antibiotic resistance among anaerobes Acquired resistance : ß-lactams : ß -lactamase Clostridia (butyricum, + RIC group) Prevotella, Fusobacterium B. fragilis gene + promotor cfiA carbapenemase, cfxA Hyperproduction of the chromosomal enzyme lack of porin & PBP modifications Antibiotic resistance among anaerobes Acquired resistance : MLSb erm genes clindamycin Metronidazole nim genes Fluroquinolones gyrase, topoisomerase mutations resistance to Moxifloxacin Chloramphenicol cat genes The situation in Europe Gram positive cocci susceptible penicillins > cephalosporins glycopeptides, linezolid, rifampicin acquired resistance clindamycin > 20% metronidazole < 5% variable fluoroquinolones The situation in Europe Actinomyces susceptible penicillins > cephalosporins glycopeptides, linezolid, rifampicin acquired resistance tetracycline macrolides (rare) variable fluoroquinolones The situation in Europe Propionibacterium susceptible penicillins > cephalosporins fluoroquinolones glycopeptides, linezolid, rifampicin acquired resistance tetracyclin macrolides & clindamycin increasing The situation in Europe C. perfringens susceptible penicillins > cephalosporins, cefoxitin metronidazole, glycopeptides, linezolid, rifampicin most fluoroquinolones acquired resistance tetracyclins macrolides & clindamycin rare One strain resistant to metronidazole Faris & Poxton 1999 The situation in Europe C. difficile susceptible penicillins, metronidazole, glycopeptides, fusidic acid, bacitracin acquired resistance tetracyclins, chloramphenicol macrolides & clindamycin > 60% emerging ? Vancomycin & metronidazole The situation in Europe Other clostridia (with exception), susceptible penicillins > cephalosporins glycopeptides linezolid, metronidazole acquired resistance tetracyclins macrolides & clindamycin cefoxitin & cefotetan chloramphenicol glycopettides :C. boltae,C. hathewayi (Van B2) The situation in Europe clostridia C. butyricum : penicillinase inhibited by clavulanic acid RIC group C. clostridioforme broad cephalosporinase low-level resistance to teicoplanin, daptomycin, ramoplanin C. ramosum broad cephalosporinase not inhibited by clavulanic acid low-level resistance to vancomycin, linezolid, daptomycin C. innocuum intrinsic resistance to cefoxitin and cefotetan low-level resistance to vancomycin & daptomycin And now ? Gram positive cocci Non-sporulated gram positive bacilli Clostridia No major clinical problems except for individual patients low rate of antibiotic diffusion (brain, bone,) be aware of RIC group glycopeptide resistance in clostridia. What about gram negative anaerobes? Prevotella 2/3 ß-lactamase production + acquired resistance clindamycin 5% metronidazole rare variable fluoroquinolones Porphyromonas Fusobacterium penicillinase < 10% Les mousquetaires anaérobies des infections pulmonaires et O.R.L QuickTime™ et un décompresseur TIFF (non compressé) sont requis pour visualiser cette image. Prevotella : MIC 50/90% ß-lactamase - ß-lactamase + Amoxicillin 0,12 0,25 8 >64 Amox +clavu 0,06 0,06 0,06 2 Cefalotin 0,25 1 16 >64 Cefuroxime 0,12 1 8 >64 Cefixime 0,25 1 16 >64 Cefpodoxime 0,12 0,5 4 >64 Ceftriaxone 0,12 0,5 4 >64 Prevotella : MIC distribution Nb of strains 25 Amoxycillin 20 15 10 5 0 MIC in mg/l Prevotella : distribution des CMI Nb of strains 12 Cephalotin 10 8 6 4 2 0 ß-lac + ß -lac - MIC in mg/l Prevotella : distribution des CMI Nb of strains 14 Cefuroxime 12 10 8 6 4 2 0 ß-lac + 0.03 0.06 ß lac - 0.12 0.25 0.5 1 2 4 8 16 32 64 128 >128 MIC in mg/l Prevotella : distribution des CMI Nb of strains 14 12 Cefpodoxime 10 8 6 4 2 0 ß-lac + ß -lac - MIC in mg/l Antibiotic susceptibility in Porphyromonas Antibiotic MIC range MIC 50% MIC90% Amoxicillin 0,03-0,5 0,5 0,5 Co-amoxiclav 0,03-0,25 0,25 0,25 Cefotaxime 0,06-1 1 1 Clindamycin 0,03-0,25 0,06 0,25 Metronidazole 0,03-0,25 0,125 0,25 Roxithromycin 0,25 Pristinamycin 0,25 FQ susceptibility of Prevotella & Fusobacterium Antibiotic Prevotella Fusobacterium MIC 50% 90% 50% 90% Ciprofloxacin 1 4 2 4 Ofloxacin 2 8 1 4 Levofloxacin 2 4 1 2 Moxifloxacin 0,12 0,5 0,12 1 Gatifloxacin 0,25 2 0,5 4 Trovafloxacin 1 2 0,25 0.5 Anaerobic lung infections. High rate of penicillin failures associated with penicillin-resistant Prevotella melaninogenica 10/47 resistant strains 5 patients received benzyl-penicillin 2 M every 4 hours all failed Failure penicillin 8/18 vs 1/19 clindamycin Gudiol Arch inter Med 1990 150 :2525 Role of ß-lactamases from gram neg anaerobes Nord-Brook ß-lactamases excreted by Prevotella or Fusobacterium caused clinical failures when patients were treated by penicillins otitis - sinusitis-dental abcess-lung abcess-recurrent tonsillitis Anaerobic infections Bacteroides fragilis group + Clostridium : intra-abdominal gyneco-obstetrics, biliary infections Perforated appendicitis Decubitus ulcers Last but not least the B. fragilis group Chromosomal cep A ß-lactamase AMX + C1G+ C3G+C4G + overproduction TIC + PIP + lack of porin AMC, TTC, PTZ ? carbapenemase All ß-lactams MLSb Clindamycin (20-40%) nim genes Nitro-imidazole AMX TIC PIP CTX IMP MTZ CFT CFX CTT ERY CLN AMC TCC PTZ TE CM Resistance to the combinations of ß- lactams and ß-lactamase inhibitors IS 1224 upstream the cep A gene increases the production of the chromosomal cephalosporinase Rogers et al.J Bacteriol 1994;176:4376-4384 High level production of the cephalosporinase alone or associated with modifications in the permeability barrier (defect of porin) Phenotypes of resistance among imipenem- susceptible strains of the B. fragilis group Antibiotic wild hyperproduction of ß-lactamase or/and lack type of porin or silent carbapenemase % (72) (19) (6) (2) (1) Ticarcillin S R R R R Coamoxiclav S S S/I I/R R Ticar + clavu S S S S I/R Pipera+tazo S S S S I Cefotetan S/I I/R R R R Figure 1 : Antibiotic resistance rates among the B. fragilis group (256 strains) in 2003 30 27,5 28 Antibiotic resistance rates (%) 25 20 15 10 6,2 4,3 1,9 1,9 5 0 0,8 0 Metronidazole Imipenem Ticar+ clavu Pipéra +tazo Co-amoxclav Cefoxitin Ticarcillin Clindamycin Activity of fluoroquinolones against the B. fragilis group (130 strains) 60 50 40 30 CIP 20 LEV 10 MOX 0 GAR 0.06 0.25 1 4 16 64 GAR MOX LEV CIP - B. fragilis group Are antibiotic resistance rates increasing ? Antibiotic resistance rates (%) 35 MTR 30 25 AMC 20 CLN 15 10 5 0 1996 1998 2000 2003 1996 1998 2000 2003 MTR 0,4 1,9 4,7 4,3 AMC 4 3,8 5,6 4,3 CLN 20 29 32 28 Metronidazole resistance nim on mobilisable plasmids nim A B.vulgatus nim C B. thetaiotaomicron nim D B. fragilis nim B chromosomal B. fragilis, P. acnes, A. odontolyticus, C. bifermentans nim A in Prevotella bivia nim G B. fragilis B. fragilis S or Intermediate to metronidazole B. fragilis metronidazole-R Metronidazole resistance Gal & Brazier, JAC 2004,54:109-116 15/ 206 isolates were found to possess nim genes and these had MICs of MTZ from 1.5 to >256 mg/L with 11.6% above the therapeutic breakpoint of 16 mg/L Disc diffusion 28-30 mm test on FAA with a 5 µg MTZ disc. 70% showed reduced zone of susceptibility or no zone 30% with no reduced zone MIC 1.5 to 4 mg/L No zone for 7 strains without nim gene. M 17 22 23 29 31 39 ND Results (2) E c o Ø Bacteroides fragilis group R (PCR-RFLP) V Strain N° 17: nimB 22: nimE 23: nimD 29: nimD F 31: nimB o 39: nimA k Strain N° 40: nimA (sequencing) I Ø Veillonella sp. Strain N° 18 : nimE (sequencing) H i n f I Results (3) MTZ /R MTZ /R MTZ /I 2 copies of nimA gene in the genome of: - MTZ Resistant strain (MIC = 64 mg/L) - MTZ intermediate strain (MIC = 16 mg/L) nimA SfiI nimA EcoRI nimA EcoRI Clinical significance of intermediate or resistant metronidazole clostridia ? Elsaghier , Brazier et al. JAC 2003 Failure of treatment due to Bacteroides fragilis with reduced susceptibility to metronidazole. Pelvic collections grew mixed coliforms susceptible to cefuroxime Patient treated by Cefuroxime +metronidazole +gentamicin. Pyrexia after four days of Tt. WBC 16000/mm3 B. fragilis MTR-R cultured from blood cultures (disk diffusion) MIC by E test 6 mg/L. Metronidazole resistance in France French resistant strains MTZ-R Prevotella buccae et 3 souches de P. loescheii CMI = 16 mg/L B. fragilis MIC = 64 mg/L H. Marchandin et al. Anaerobes 2002;8:137 Veillonella nim E in a metronidazole-susceptible Veillonella sp. strain MIC = 4 mg/L H. Marchandin, Dubreuil, AAC 2004 48:3207 Bacteremia caused by Prevotella sp. with reduced susceptibility to metronidazole Mory et al.ECC Paris december 2004 78 old patient with sepsis Cefotaxime (3g)+ ofloxacin (400mg) E. coli S + Streptococcus anginosus S Patient pyrexial new blood cultures Teicoplanin 400mg and metronidazole 500mg tid added Day 7 Prevotella AMX, CTX, OFL-R, MTR S but colonies appearing later in the inhibition zone MIC 64 mg/L. Day 16 persistance of fever Piperacillin + tazobactam 12g + Cipro 800 mg. Apyrexia in 48h Metronidazole resistance B. fragilis & B. ovatus from appendicetomy MIC >32 mg/l : failure in Koweit Rotimi et al. Clin Microbiol Infect 1999 ; 5:166-169 B. fragilis MIC =256 in New Delhi with clinical failure Chauldry, Emerging Infect Dis 2001,7,485-486 B. fragilis MIC >32 mg/L in Seattle Shapiro et al. J Clin Microbiol 2004 ;42:4127-4129 B. fragilis in Hungary (Nagy et al.), Poland (Wojcik) Prevotella loescheii MIC 12 mg/L subdural empyema in Cardiff Sandoe JAC 2001;47:366-367. Metronidazole resistance C. difficile MIC =16 mg/L in Hong Kong, Madrid and Cardiff Wong et al.Diagn Microbiol Infect Dis 1999 ;34:1-6 Pelaez AAC 2002,46:1647-1650 Brazier et al.