R505K+I548M). In 181 (98.4 %) of the 184 strains tested, the use of the rifampicin Etest yielded results (reduced suscep- tibility or non-resistant) in accordance with those obtained by the rifaximin broth microdilution test. Discordant results (rifaximin vs rifampicin) were found for three strains. Strain 2347/655 (showing an H502N rpoB mutation) and 3153/PCR ribotype 002/0 (showing a D492V rpoB mutation) had a rifaximin broth microdilution MIC of ¢1024 mg ml21 and a rifampicin Etest MIC of 0.064 mg ml21 and 1 mg ml21, respectively. Strain Lee047/PCR ribotype 047 had a rifaximin broth microdilution MIC of 64 mg ml21 and a rifampicin Etest MIC of 0.25 mg ml21. Molecular analyses of these strains revealed a mutation in the rpoB gene giving an H502N amino acid substitution (Table 3). Using an inhibition zone of ,38 mm as breakpoint for reduced susceptibility, the use of rifaximin disc diffusion yielded results correlating with those received by the use of rifaximin broth microdilution in 180 (97.8 %) of the 184 strains tested. Strain 2203/PCR ribotype 053 and 3018/PCR ribotype 018 had rifaximin inhibition zones of 32 mm and rifaximin broth microdilution MICs of 1 mg ml21 and showed an H502N or S550Y rpoB gene mutation, respectively. Strain 3109/PCR ribotype 002/0 had a rifaximin inhibition zone of 30 mm and a rifaximin broth microdilution MIC of 16 mg ml21 and showed a number of silent mutations (T501T, L506L, G510G, G512G, F521F, E541E, K556K) in the rpoB gene. Strain 3141/PCR ribotype 539 had a rifaximin inhibition zone of 16 mm and a rifaximin broth microdilution MIC of 4 mg ml21, and also showed a number of silent mutations (S475S, F481F, D492D, T501T, A508A, G510G, T539T, K556K) (Table 3). The 58 strains with reduced susceptibility to rifampicin and rifaximin showed rpoB gene amino acid substitutions as follows: R505K (n546), H502N+R505K (n55), H502Y Rifaximin disc diffusion test for C. difficile (n52) and one each of H502L, S550F, H502N+A555A, L487F+H502Y and R505K+I548M. Of the 117 rifampicin- and rifaximin-susceptible strains with MICs of ¡0.002 mg ml21, 98 (84 %) showed no point mutations in rpoB; 19 showed only silent amino acid sub- stitutions as follows: T501T+L506L+G510G+G512G+ F521F+E541E+K556K (n511), A555A (n56), L500L (n52). Correlation of rifampicin MICs obtained by broth dilution with rifaximin disc diameters was tested by Kendall’s tau-b correlation coefficient: 20.42 (P,0.001 for the hypothesis that both parameters were independent). Results for clinical isolates Rifaximin disc (40 mg) diffusion testing performed on 898 clinical C. difficile isolates yielded inhibition zone dia- meters ranging from 6 mm (n567) to 78 mm (n52) (Fig. 2). Using an inhibition zone ,38 mm as breakpoint for reduced susceptibility a total of 68 strains with reduced susceptibility were identified. Rifampicin Etests performed on the 898 isolates revealed that 67 isolates had MICs of ¢32 mg ml21 for rifampicin and that 819 had MICs of ¡0.002 mg ml21; 12 isolates (1.3 %) exhibited rifampicin MICs between these extremes. By rifaximin disc diffusion test, the 67 strains with reduced rifampicin susceptibility exhibited no inhibition zone. No discordant results were observed among these 67 isolates with reduced susceptibility using an MIC of ¢32 mg ml21 as breakpoint for reduced rifampicin susceptibility and a ,38 mm inhibition zone as breakpoint for reduced rifaxi- min susceptibility. Isolate 2663/PCR ribotype 053 had a rifampicin Etest MIC of 0.064 mg ml21 and a rifaximin inhibition zone of 36 mm; molecular analyses revealed a D492N mutation in the rpoB gene (Table 3). Table 3. Amino acid substitutions detected by rpoB sequence analysis in C. difficile strains with discordant results Isolate ID no. 2203 2347 2663 3018 3109 3141 3153 Lee047 rpoB mutation* H502N H502N D492N S550Y T501T, L506L, G510G, G512G, F521F, E541E, K556K S475S, F481F, D492D, T501T, A508A, G510G, T539T, K556K D492V H502N *Resulting amino acid substitution shown. DDD, disc diffusion. http://jmm.sgmjournals.org Rifampicin MIC (mg ml”1) Rifaximin MIC (mg ml”1) Rifaximin DDD inhibition zone (mm) 0.5 0.064 0.064 0.016 0.125 4 1 0.25 1 ¢1024 1 1 16 4 ¢1024 64 32 34 36 32 30 16 26 30 1209