We characterized clinically occurring and novel mutations in the ␤ subunit of RNA polymerase in Clostridium difficile (CdRpoB), conferring rifamycin (including rifaximin) resistance. The Arg 505 Lys substitution did not impose an in vitro fitness cost, which may be one reason for its dominance among rifamycin-resistant clinical isolates. These observations were supported through the structural modeling of CdRpoB. In general, most mutations lacked in vitro fitness costs, suggesting that rifamycin resistance may in some cases persist in the clinic. The nonabsorbed rifamycin antibiotic rifaximin has been considered an adjunctive therapy to reduce the recurrence of Clostridium difficile infection following vancomycin treatment (1, 2). Rifaximin, which is approved for the treatment of traveler's diarrhea, inhibits DNA transcription by selectively binding to the ␤ subunit of RNA polymerase (RpoB). Substitutions in the rifamycin resistance-determining region (RRDR) of RpoB confer resistance to rifamycins, including rifaximin, in clinical isolates of C. difficile (3,4). An arginine to lysine substitution at position 505 (i.e., Arg 505 Lys) in C. difficile (CdRpoB) is the most common mutation among rifamycin-resistant clinical isolates (3, 5, 6). Other mutations in clinical isolates also occur at His 502 , Ser 488 , and Ser 550 . However, it is unknown whether fitness costs influence the spectra of rifamycin resistance alleles among C. difficile isolates.Fitness cost is a leading factor that affects the clinical prevalence of specific resistance alleles (7,8). In the present study, we characterized clinically occurring and novel rifamycin resistance mutations in terms of their impacts on the growth and competitive fitness of C. difficile and by in silico structural modeling of the CdRpoB (Fig. 1).The rifamycin-susceptible C. difficile strains were CD43 and CD1679 (both epidemic ribotype 027) and were kindly provided by Scott Curry at the University of Pittsburgh. They were cultivated in brain heart infusion tryptone yeast (BHITY) broth or agar at 37°C in a Whitley A35 anaerobic workstation (Don Whitley Scientific). The MIC of rifaximin was defined as the lowest concentration of drug that prevented growth on BHITY agar (9). Spontaneous mutants were recovered by plating aliquots of overnight cultures onto selective agars containing rifaximin at 4ϫ the MIC. Mutations were identified in an ϳ200-bp PCR amplicon containing the RRDR (3). The competitive fitness (W) of rifaximin-resistant mutants was determined by pairwise competition between the wild-type parents and their respective derivative mutants (7,8). Briefly, aliquots from overnight cultures of wild-type and mutant bacteria were coinoculated in BHITY broth at a 10:1 ratio (ca. 10 4 :10 3 CFU/ml) and grown for 24 h. The numbers of mutant and wild-type bacteria at the start and at the end of the experiments were determined by plating onto selective agar containing 4ϫ the rifaximin MIC and on nonselective BHITY agar (7,8). W was calculated from ln[N R (24)/N R (0)]/ln[N S...