Journal of Medical Microbiology (2011), 60, 1206–1212 DOI 10.1099/jmm.0.028571-0 Correspondence Alexander Indra Alexander.Indra@ages.at Received 1 December 2010 Accepted 1 February 2011 Rifaximin disc diffusion test for in vitro susceptibility testing of Clostridium difficile Steliana Huhulescu,1 Ulrich Sagel,1 Anita Fiedler,1 Verena Pecavar,1 Marion Blaschitz,1 Guenther Wewalka,1 Franz Allerberger2 and Alexander Indra2 1Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology, Hygiene, Waehringerstrasse 25a, A-1096 Vienna, Austria 2Paracelsus Medical University, Institute for Medical Microbiology, Hygiene and Infectious Diseases, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these,184 isolates from a strain collection were tested by an in-house rifaximin disc (40 mg) diffusion test, by an in- house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ¢32 mg ml”1 as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n51) to ¢1024 mg ml”1 (n561); 62 isolates showed a reduced susceptibility (MIC ¢32 mg ml”1). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of .1024 mg ml”1, rifaximin end-point MICs were determined by broth dilution: 4096 mg ml”1 (n52), 8192 mg ml”1 (n56), 16 384 mg ml”1 (n52) and 32 678 mg ml”1 (n51). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ¡0.002 (n5117) to ¢32 mg ml”1 (n559). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ¢32 mg ml”1. There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ¢32 mg ml”1 as breakpoint for reduced rifampicin susceptibility and a ,38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin – despite its water- insolubility – may be a suitable candidate for disc diffusion testing. INTRODUCTION Clostridium difficile is a spore-forming Gram-positive anaerobic bacterium and a major cause of nosocomial and community-acquired diarrhoea (Wistro¨m et al., 2001). Metronidazole and oral vancomycin are the main anti- biotics used to treat C. difficile infection (CDI). In 2005, in Austria, oral rifaximin was licensed for ‘treatment of Abbreviation: CDI, Clostridium difficile infection. 1206 gastrointestinal diseases caused or partially caused by rifaximin-susceptible bacteria, e.g. gastrointestinal infections, pseudomembranous colitis due to C. difficile, hepatic encephalopathy, small bowel bacterial overgrowth and diverticulitis’ (Willerroider, 2009). However, to our know- ledge, no guidelines have yet been published to test in vitro susceptibility of C. difficile to rifaximin. Two study groups previously used ¢32 mg l21 (O’Connor et al., 2008; Jiang et al., 2010) as breakpoint for resistance using agar dilution testing, a method not widely employed in routine testing of 028571 G 2011 SGM Printed in Great Britain