clinical isolates. Jiang et al. (2000) reported high rifaximin concentrations (4000 to 8000 mg g21) in stools 3 days after a single oral administration. Johnson et al. (2007) reported on the possible prevention of recurrence of CDI by administering rifaximin immediately after completion of the last course of vancomycin therapy of CDI. In Austria, rifaximin is widely used to treat CDI, and microbiological laboratories often receive requests for in vitro susceptibility testing of clinical C. difficile isolates against this substance. The aim of this study was to evaluate whether the disc diffusion method can be applied to test in vitro susceptibility of C. difficile to rifaximin. METHODS Micro-organisms. One hundred and eighty-four C. difficile isolates were obtained from the reference strain collection of the Austrian national C. difficile reference centre, including ATCC strain 9689, and 50 strains previously provided by Leeds University Hospital (UK), 3 strains from the Public Health Laboratory Maribor (Slovenia) and 24 strains from Leiden University Hospital (Netherlands). In addition, 898 non-duplicate clinical isolates were tested, cultured in 21 Austrian medical laboratories in 2009. All 1082 isolates were stored at 280 uC in cryobank tubes (Mast Diagnostics) until testing. Isolates were recultivated on Columbia blood agar plates (bioMe´rieux); all were tested for PCR ribotype (RT) (Indra et al., 2008), toxin A (A) (van den Berg et al., 2004), toxin B (B) (Kato et al., 1991, 1999), binary toxin (BT) (Stubbs et al., 2000) and tcdC deletion (Spigaglia & Mastrantonio, 2002). Tables 1 and 2 list the PCR ribotypes of these isolates. All 1082 isolates except one (PCR ribotype 010) produced at least toxin B; 149 of them (14 %) were A/B/BT positive and belonged to the following ribotypes: 027 (n599), 078 (n510), 176 (n59), 023 (n55), 080 (n53), 126 (n52), 419 (n52), 045 (n52), 411 (n52), 429 (n52), 250 (n51), 413 (n51), 018 (n51), 515 (n51), 344 (n51), 605 (n51), 606 (n51), 616 (n51), 654 (n51), 655 (n51), 656 (n51), 657 (n51), 658 (n51). If possible nomenclature was used according to that given by the John Brazier’s laboratory, due to the higher quality of resolution gained with capillary sequencer-based PCR-ribotyping (Indra et al., 2008) strains not assigned by John Brazier’s laboratory were given numbers starting with 400. Antimicrobial agents and susceptibility testing. The 184 isolates from the strain collection were tested by an in-house rifaximin disc (40 mg) diffusion test (Oxoid; custom-made product), by an in-house rifaximin broth microdilution test and by rifampicin Epsilon-test (Etest) (bioMe´rieux). The 898 clinical isolates from 2009 were tested only by disc diffusion test and rifampicin Etest (bioMe´rieux). Rifaximin discs were custom-made (Oxoid) by Gebro Pharma. The disc diffusion tests were performed on Brucella blood agar plates supplemented with 5 mg haemin l21 and 1 mg vitamin K l21 (Oxoid). The rifaximin broth microdilution test was performed as follows. Rifaximin was purchased as a powder from Alfa Wassermann. Sterile stock solutions were prepared according to the instructions of CLSI for testing of anaerobes (Clinical and Laboratory Standards Institute, 2007). In short, rifaximin was dissolved in methanol and then diluted in 0.9 % saline solution. Serial twofold dilutions in ATB S-medium containing menadione (vitamin K3) at 0.5 mg l21 and haemin at 15 mg l21 (bioMe´rieux) were prepared for rifaximin concentrations covering a range from 0.000125 to 1024 mg ml21; 96-well microdilu- tion plates were filled with ATB S-medium (bioMe´rieux) containing http://jmm.sgmjournals.org Rifaximin disc diffusion test for C. difficile Table 1. Distribution of ribotypes among 184 C. difficile isolates from the strain collection of the Austrian national C. difficile reference centre Ribotype No. of strains Percentage 027 053 001 014/0 005 056 078 239 408 002/2 012 017 020 029 031 043 046 404 503 510 Other* 26 16 7 6 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 92 14.13 8.70 3.80 3.26 1.63 1.63 1.63 1.63 1.63 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 1.09 50 *Represented by one isolate each: 002/0, 002/1, 003, 004, 006, 007, 009, 010, 014, 015, 016, 018, 019, 023, 025, 026, 033, 035, 036, 037, 039, 040, 042, 045, 047, 049, 050, 051, 052, 054, 055, 057, 058, 060, 062, 063, 064, 066, 067, 068, 070, 072, 075, 076, 077, 079, 080, 081, 083, 084, 085, 087, 094, 095, 106, 115, 117, 118, 122, 126, 131, 153, 169, 174, 201, 209, 212, 220, 411, 413, 434, 441, 444, 448, 466, 497, 504, 523, 524, 539, 542, 548, 622, 627, 633, 643, 649, 650, 651, 652, 653, 654. the respective antibiotic concentrations. The antibiotic stocks were freshly prepared on the day of testing. For inoculum preparation, test organisms were cultured for 48 h anaerobically on Columbia blood agar plates (bioMe´rieux) at 37 uC; bacteria were suspended in 0.9 % saline solution to yield McFarland 0.5 and diluted 1 : 10 into the medium, so that the final test concentration of bacteria was approximately 16106 c.f.u. ml21. Minimal inhibitory concentration (MIC) was defined as the lowest concentration at which no growth was observed after incubation for 48 h at 37 uC in an anaerobic atmosphere using anaerobic jars and GasPak (BD). Growth controls were performed by inoculation of antibiotic-free medium with an aliquot of the primary inoculum at a concentration of 105 c.f.u. per well; purity testing was performed by transferring an aliquot of 10 ml onto two Columbia blood agar plates with incubation under aerobic and anaerobic conditions, respectively. Rifampicin Etests (MIC range 0.002–32 mg ml21) were performed according to the manufacturer’s instructions (bioMe´rieux) using Brucella blood agar plates supplemented with haemin (5 mg l21) and vitamin K (1 mg l21) (Oxoid). In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ¢32 mg ml21 as criterion for reduced in vitro susceptibility, as suggested by O’Connor et al. (2008) and Jiang et al. (2010). Setting of zone 1207