The incidence of Clostridium difficile infection is increasing, with reports implicating fluoroquinolone use. A three-stage chemostat gut model was used to study the effects of three fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) on the gut microbiota and two epidemic C. difficile strains, strains of PCR ribotypes 027 and 001, in separate experiments. C. difficile total viable counts, spore counts, and cytotoxin titers were determined. The emergence of C. difficile isolates with reduced antibiotic susceptibility was monitored with fluoroquinolone-containing medium, and molecular analysis of the quinolone resistance-determining region was performed. C. difficile spores were quiescent in the absence of fluoroquinolones. Instillation of each fluoroquinolone led to C. difficile spore germination and high-level cytotoxin production. High-level toxin production occurred after detectable spore germination in all experiments except those with C. difficile PCR ribotype 027 and moxifloxacin, in which marked cytotoxin production preceded detectable germination, which coincided with isolate recovery on fluoroquinolone-containing medium. Three C. difficile PCR ribotype 027 isolates and one C. difficile PCR ribotype 001 isolate from fluoroquinolone-containing medium exhibited elevated MICs (80 to > or =180 mg/liter) and possessed mutations in gyrA or gyrB. These in vitro results suggest that all fluoroquinolones have the propensity to induce C. difficile infection, regardless of their antianaerobe activities. Resistant mutants were seen only following moxifloxacin exposure.