Antibiotic susceptibility testing : When ?

1. - single anaerobic organism isolated from a sample - anaerobes isolated from blood cultures -when severe infections occur : osteomyelitis, brain abcess, endocarditis, prosthetics, vascular grafts, bacteremiae, recurrent or refractory infections... - failure of empiric treatment - when asked by the clinician Thornsberry & the NCCLS Rev. Inf. Dis. 1979,139:83 Antibiotic susceptibility testing : When ? Présenté le 29 Janvier 2009 par le Pr Luc Dubreuil aux CMI à la faculté COCHIN

2. The disk diffusion method Not recommanded by the NCCLS results are related to many parameters : type of medium inoculum type of anaerobic atmosphere potency of disk, incubation time lack of correlation with reference method

3. Disk diffusion test difficult to apply for slow growing organisms May indicate : B. fragilis High resistance level to clindamycin (MIC >128 mg/l) carbapenemase production metronidazole resistance (to be confirm by E test) Clostridia, Prevotella and Fusobacterium ß-lactamase production (to be confirm by E test : MIC ≥ 0.38 mg/l) MIC in agr dilution ≥0.5 mg/l Peptostreptococcus High resistance level to clindamycin

4. The agar dilution procedure M11A6 inoculum McFarland 0.5 (1.5x108 organisms/ml) in brucella or clear broth + supplements: hemin 5µg/ml, NaHCO3 20µg/ml, Vit K1 10µgml,+ 5 % laked (lysed sheep blood) medium brucella blood agar + hemin +laked sheep blood no more than one month in the refrigerator Steers apparatus 1x105 CFU per spot of inoculation incubation 35-37°C in anaerobiosis Control strains : B. fragilis ATCC 25285, B. thetaiotaomicron ATCC 29741, E. lenta ATCC 43055

5. Brucella agar medium ! Brucella agar BBL or Difco 43g +1ml Vit K1+50ml blood + hemin or other supplements Tryptone Meat Dextrose NaCl Na bisulfite BBL 10g 10g 1g 5g 0,1g Oxoid 10g 5g 10g 5g none BBL for anaerobes + 2g yeast extract,10mg hemin, 10mg vit K1

6. Susceptibility testing in liquid medium macrodilution Inoculum effet +++ Contamination may occurs Lack of culture for 15% of anaerobic bacteria type of medium Degradation of antibiocs in solution imipenem and thioglycolate cystein chlorhydrate and piperacillin macrolides and reductors Growth without antibiotic must be adequate and optimal supplements are needed : formate,fumarate, arginin

7. Anaerobes & E-test in routine inoculation McFarland 1 in brucella broth (or Schaedler) agar medium Brucella+blood 5% + vitK1 + supplements drying 10mn incubation in anaerobiosis as soon as possible reading after 24 or 48h of incubation 48h if clindamycin

8. E test and anerobes reading at 24h 64% of strains including 77% of B. fragilis group 48h 95% 100% Agreement with agar reference method (+1log ) 85% Major discrepancies 1.7%, very major discrepancies 1.7% very major errors 16.6% (24h) 2.5% B. fragilis (48h) 37.5% 0 % Clostridia tendancy to lower MIC if Mc farland 0.5 E test 100µl on a 150mm Petri dish = 6,000 CFU/mm2 Steers replicator 2mm2 / spot = 40,000CFU/mm2

9. Anaerobes & E test Mc Farland 0.5, incubation 48h Author +1log + 2log discrepancies Citron 87 98 2 Wust 91 98 1 Smoczinski 82 93 4 Appelbaum 81 99 Croco 85 98

10. Resistance to Metronidazole false resistance Anaerobiosis is recquired to reduced metronidazole (-350 to - 450mV) bad anaerobiosis : jars open frequently, catalyst poisoned number of Petri dish inside the jar indicator of anaerobiosis in chambers resazurine >>> methylene blue gaz composition security (5% H2) lack of purity or true resistant strain check resistance by E test

11. - colonies from Columbia blood agar are picked and introduced into suspension medium (Mc farland 3) - 200µl are introduced into 7ml of antibiotic medium S -135µl delivered with an automatic pipette into each well - When adequate growth occurs read - susceptible no growth, intermediate growth only at low concentration, resistance growthin both wells of the pair Dubreuil et coll. J Clin Microbiol. 1999,37:1824 ATB ANA device

12. West Wilkins medium biotrypticase 10g biogelytone 10g yeast extract 5g dextrose 3g arginin HCl 5g sodium pyruvate 5g NaHCO3 1g cystein HCl 0.5g Tween 80% (solution 5%) 5 ml hemin (solution 50mg/100ml) 10ml vitamin K3(solution 5mg/100ml) 10ml QSP distillated water 1l

13. E -test vs ATB ANA bioMérieux E test* ATB ANA** % McFarland 1,48h CA-SFM NCCLS Agreement 95.1 94.3 94.4 Minor discrepancies 3.8 4.8 3.8 Major errors 0.5 0.3 0.7 Verymajor errors 0.6 0.6 0.6 Preference for CA-SFM metronidazole,amoxicillin and Gram+ NCCLS amoxicillin and gram negative *Pierrard **Dubreuil

14. Methodology- Conclusion no standard medium for all anaerobes need for supplements reading problems tetrazolium chloride, automats hazes clear endpoints couples from hell clostridia and clindamycin B; fragilis and cefoxitin, cefotetan I like intermediate susceptibility or I don’t know ?

15. What the experience tall us The reference agar dilution method leads to good correlation with clinical outcome even this correlation is difficult to assess : polymicrobial infection, surgery, necrotic tissues with poor vascularization. Inocula for anaerobes : ten fold higher comparatively to aerobes Blood : does not affect MIC. No standard medium convenient for all anaerobes Major problem assessment of breakpoints Clustering effect for some couples antibiotic-anaerobic species

16. ESGARAB Implications of EUCASTfor Testing of Anaerobes Arne C. Rodloff

17. The EUCAST Anaerobe Subcommittee The EUCAST Anaerobe Subcommittee is a joint project between EUCAST and ESGARAB: The subcommittee is chaired by Arne Rodloff (EUCAST and ESGARAB) and include Luc Dubreuil (France) and Elizabeth Nagy (Hungary). The committee has conducts its work via email. The remit of the subcommittee agreed with ESGARAB is to:

18. The EUCAST Anaerobe Subcommittee • determine whether separate columns are needed in breakpoint tables for Gram-positive and Gram-negative anaerobes. • determine which agents should be given anaerobe breakpoints. • collect MIC distributions for anaerobes. • consider whether a standardised method of susceptibility testing is required. • suggest appropriate quality control strains for susceptibility testing.

19. RD  Species-related breakpoints (S</R>) Non-species related breakpointsLS</R> Gram-negative, anaerobesK Gram-positive, anaerobes Benzylpenicillin 0.25/0.5 0.25/0.5 0.25/2 Ampicillin1 0.5/2 4/8 2/8 Ampicillin/sulbactam2 4/8 4/8 2/8 Amoxicillin 0.5/2 4/8 2/8 Amoxicillin/clavulanate2 4/8 4/8 2/8 Piperacillin 16/16 8/16 4/16 Piperacillin/tazobactam2 8/16 8/16 4/16 Ticarcillin 16/16 8/16 8/16 Ticarcillin/clavulanate2 8/16 8/16 8/16 Penicillins Susceptibility of Gram-negative anaerobes to ampicillin, amoxicillin and piperacillin without betalactamase inhibitors can be inferred from their susceptibility to benzylpenicillin

20. Species-related breakpoints (S</R>) Non-species related breakpoints1S</R> Gram-negative anaerobes Ertapenem 1/18 0.5/1 Imipenem 2/8 2/8 Meropenem 2/8 2/8 Carbapenems 8. The ertapenem S/I breakpoint for Gramnegative anaerobes was moved from 0.5 to 1.0 to avoid dividing the wild type MIC distributions

21. Species-related breakpoints (S</R>) Non-species related breakpoints1S</R> Gram-negative anaerobes Gram-positiveanaerobes Metronidazole 4/4 4/4 -- Chloramphenicol 8/8 8/8 8/8 Linezolid -- -- -- -- -- „Other“ - Antimicrobials

22. Tigecycline For anaerobic bacteria there is clinical evidence of activity in mixed intra-abdominal infections, but no correlation between MIC values, Pk/Pd data and clinical outcome. Therefore no breakpoint for susceptibility testing is given.

23. 11.4 Pepto-strepto-coccus spp., Bacteroides spp. Clinda-mycin If resistant to erythromycin but susceptible to clindamycin, report resistant to clindamycin. Resistance to macrolides in Peptostreptococcus spp.and Bacteroides spp. is generally due to the production of a ribosomal erm methylase conferring the MLSB phenotype. In the case of inducible MLSB resistance, resistance to clindamycin is poorly expressed in vitro and this antibiotic should not be considered as active. Table 11: Interpretive rules for macrolides, lincosamides and streptogramins