S. Huhulescu and others Fig. 2. Rifaximin disc (40 mg) diffusion testing performed on 898 clinical C. difficile isolates yielding inhibition zone diameters ranging from 6 mm (zero inhibition) to 78 mm. DISCUSSION All the rifamycins are semisynthetic derivatives of rifamycin B, a fermentation product of Amycolatopsis mediterranei, formerly named Streptomyces mediterranei. Rifamycin B exerts poor antimicrobial activity, but is easily produced and readily converted chemically into rifamycin S, from which most active derivatives are prepared (Parenti & Lancini, 2003). Rifaximin is a semi-synthetic derivative of rifamycin S formulated for oral administration. In the 1980s it was only available in Italy, but today it is marketed worldwide mainly for the treatment of gastrointestinal infections and the treatment of chronic hepatic encephalopathy (Corazza et al., 1988; Festi et al., 1992). Jiang et al. (2000) reported high rifaximin concentrations (4000 to 8000 mg g21) in stools 3 days after single oral administration. Although adequate antimicrobial drug concentrations should be achieved by the high dosage of the non- absorbable rifaximin, the constraints imposed by possible drug resistance (O’Connor et al., 2008) often impel clinicians to request in vitro susceptibility testing of C. difficile isolates from patients not responding to therapy. Our results on 11 arbitrarily chosen isolates from the group with reduced susceptibility to rifaximin (MIC .1024 mg ml21) showed MICs ranging from 4096 to 32 678 mg ml21, so that a breakpoint for resistance between .32 mg ml21 and 1024 mg ml21 can be considered for the future. We evaluated a rifaximin disc diffusion test and found it substantially equivalent to an in-house rifaximin broth microdilution test. The level of performance considered acceptable for US Food and Drug Administration (FDA) clearance in premarket notification of commercial anti- microbial susceptibility systems is (among others) .89.9 % categorical agreement (same susceptible, intermediate or resistant classification), ¡1.5 % very major errors (false susceptibility based on the number of resistant organisms) and ¡3 % major errors (false resistance based on the 1210 number of susceptible isolates) (Richter & Ferraro, 2007). Accepting an MIC ¢32 mg ml21 as criterion for reduced rifaximin susceptibility, a rifaximin inhibition zone of ,38 mm would represent a valid resistance breakpoint in the 40 mg disc diffusion test described here. We also found good correlation between the rifaximin and rifampicin susceptibility testing results. The number of discordant results (n58) concerning supposed resistance based on the rifampicin Etest and rifaximin disc diffusion test on C. difficile isolates could be seen as an argument to introduce the criterion ‘intermediate’ for rifampicin susceptibility testing. If we take the EUCAST breakpoints for staphylococci (S¡0.06/R.0.5 mg ml21) for the rifam- picin Etest, the correlation would increase to 99.45 % (five discordant results) (EUCAST, 2010). Our suggestions for preliminary breakpoints between susceptible, indeterm- inate and resistant are summarized in Table 4 for the Etest and the broth microdilution method to be validated in future studies. In our opinion no reliable intermediate breakpoint result can be given for the disc test; however, all isolates in question showed rpoB mutations yielding an amino acid change. Rifamycin resistance is commonly the result of a mutation that alters the b-subunit of RNA polymerase, reducing its binding affinity for rifamycins (Struelens, 2003). This study found rpoB mutations in all of the 62 strains showing reduced susceptibility with the rifaximin broth microdilu- tion test. Interestingly, only H502N and D492V mutations showed discordant results between the rifampicin Etest and the rifaximin broth microdilution test in the 184 strains analysed, indicating a potential connection between muta- tion in the rpoB region and the efficacy of the antibiotic used. Future studies would enable examination of this potential connection. Our molecular investigations confirmed mutations result- ing in amino acid substitutions in RpoB for all eight isolates with discordant results with reduced susceptibility to rifaximin and rifampicin. Interestingly, two strains showing seven or eight silent mutations also showed reduced susceptibility with the disc diffusion test, but not with any of the other methods tested. Since no other mutation could be found in the rpoB gene a mutation in another gene influencing the activity of rifaximin has to be Table 4. Suggested preliminary breakpoints Susceptible Intermediate Resistant ¢38 – ,38 *DD, disc diffusion. Rifaximin DD* Rifaximin MIC inhibition zone (mg ml”1) (mm) ¡0.25 0.5–16 ¢32 Rifampicin MIC (mg ml”1) ¡0.06 0.012–16 ¢32 Journal of Medical Microbiology 60