Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates * Elisabeth Nagy a, * , Ulrik Stenz Justesen b , Zsuzsa Eitel a , Edit Urb  an a , on behalf of ESCMID Study Group on Anaerobic Infection a Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary b Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark article info Article history: Available online xxx Keywords: Bacteroides fragilis group Susceptibility testing Disc diffusion MIC determination Breakpoints abstract With the emergence of antibiotic resistance among Bacteroides fragilis group isolates the need of sus- ceptibility testing in routine laboratories is increasing. The aims of the present study were to evaluate the disk diffusion method for susceptibility testing in case of different clinical isolates of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic suscep- tibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to amoxicillin/clavulanic acid, cefoxitin, clindamycin, imipenem, metronidazole, moxifloxacin, piperacillin/tazobactam, tigecycline by agar dilu- tion method previously. The inhibition zones of the same antibiotics including meropenem disc were determined by the disc diffusion on Brucella blood agar supplemented with haemin and vitamin K1. Plates were incubated at 37  C in an anaerobic atmosphere for 24 h. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement between the inhibition zone di- ameters and the MICs for imipenem, metronidazole, moxifloxacin and tigecyclin. The inhibition zone diameters of meropenem also separated clearly the isolates, which can be considered wild-type isolates. In case of amoxicillin/clavulanic acid and piperacillin/tazobactam intermediate and susceptible isolates according to the MIC determination, overlap during the zone diameter determination. Isolates with an inhibition zone <23 mm for amoxicillin/clavulanic acid and <25 mm for piperacillin/tazobactam should be retested by a MIC determination method. The 10 mg clindamycin disc clearly separated the resistant and the susceptible population of B. fragilis group strains. In the case of cefoxitin only resistant popu- lation could be separated with an inhibition zone <17 mm, intermediate and susceptible isolates overlap. In conclusion, we suggest that disk diffusion can be an option for susceptibility testing of B. fragilis group isolates for most relevant antibiotics in routine laboratories. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Antimicrobial susceptibility testing of anaerobic bacteria is important because variable resistance to penicillins, cephalospo- rins and clindamycin undermines empiric antimicrobial choices. Furthermore, increasing resistance to carbapenems and beta- lactams in combination with beta-lactamase inhibitors are also registered [1,2]. The rate of metronidazole resistance or reduced susceptibility in Bacteroides spp. is generally low, around 1%, but some reference laboratories observed an increased number of metronidazole resistant strains (up to 7.5%) among the referred Bacteroides isolates at the end of the previous century [3]. This reported trend may be the result of a selection bias as these isolates were referred to the reference laboratory solely to confirm putative resistance to a 5 mg metronidazole disc used in the routine. To identify resistance among anaerobic bacteria including Bacteroides fragilis group isolates is important, as anaerobic bacteria are asso- ciated with life threatening infections with high morbidity and mortality, especially if inappropriate antimicrobials are adminis- tered [4]. Increasing number of cases is reported where multi- resistant Bacteroides strains are isolated [5e7]. * Part of this study was presented during the 23rd ECCMID in Barcelona 2014. * Corresponding author. Institute of Clinical Microbiology, University of Szeged, Semmelweis u. 6, H-6725 Szeged, Hungary. Tel.: þ36 62 541699; fax: þ36 62 545712. E-mail address: nagy.erzsebet@med.u-szeged.hu (E. Nagy). Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe http://dx.doi.org/10.1016/j.anaerobe.2014.10.008 1075-9964/© 2014 Elsevier Ltd. All rights reserved. Anaerobe xxx (2014) 1e7 Please cite this article in press as: Nagy E, et al., Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.008