Agar dilution anaerobic susceptibility studies using rifampin, vancomycin, metronidazole, and bacitracin individually and in combination were performed with Clostridium difficile. Fifty-five strains of C. difficile were studied. Eighty-five percent of strains tested (29 of 34) were synergistically inhibited by the combination of bacitracin and rifampin (fractional inhibitory concentration, .0.50).Antimicrobial therapy is indicated in patients with Clostridium difficile-associated disease who do not respond to appropriate nonspecific treatment or have severe progressive disease. Oral vancomycin and metronidazole are the recognized therapeutic agents (6,10,11). In addition, oral bacitracin has recently been investigated with variable success (3, 13). Despite optimal therapy, up to 20% of patients develop relapses of diarrhea. Prompt repopulation of the gut with "normal flora" is considered the ideal goal by many, and therapeutic instillation of normal flora organisms has been proposed by some investigators (12).The use of combination therapy with vancomycin and rifampin in patients with multiple relapses has shown some promise (1). In vitro studies of synergy of antibiotic combinations with C. difficile are lacking. We evaluated the in vitro activities of bacitracin, vancomycin, and metronidazole alone and in combination with rifampin against C. difficile. Rifampin is a very active antimicrobial agent against C. difficile, with excellent inhibitory activity (7).(This study was presented in abstract form at the 1990 annual meeting of the American Society for Microbiology, Anaheim, Calif.) C. difficile strains were isolated on CCFA agar from stool specimens of patients (2) Individual-drug MIC plates were run with each synergy test. Aerobic contamination, anaerobic growth control, and uninoculated sterile plates were included with each run, as were the control organisms. Determinations of all MICs for representative strains and combination testing were performed in duplicate. Antimicrobial interaction was quantified according to the fractional inhibitory concentration (FIC) and FIC index.The FIC index was interpreted as follows: c0.5, full synergy; 0.51 to 0.75, partial synergy; and 0.76 to 1.25, additivity (4, 8). Table 1 shows the MICs of each of the antimicrobial agents tested. All 55 strains tested were susceptible to rifampin at an MIC of 0.002 ,ug/ml or less. Almost 90% were inhibited at a concentration of 0.001 ,ug/ml. Vancomycin susceptibility data revealed all 47 strains to be susceptible at or below 1.6 ,ug/ml. Metronidazole MICs were from 0.8 to 3.2 ,ug/ml. All 34 strains tested were susceptible to bacitracin at or below 32 U/ml. Only 44% of our isolates were susceptible to bacitracin at an MIC of 16 U/ml.The results of synergy studies are shown in