C. difficile forms biofilms in vitro Biofilm formation by C. difficile was first reported by Donelli and colleagues where they identified the role of polymicrobial biofilms in clogging of biliary stents using confocal and field emission scanning electron microscopy 4. Mutants deficient in stress-related proteins including the SOS response regulator, LexA, the RNA chaperone, Hfq, and the heat stress-associated chaperone, DnaK, have been associated with increased biofilm formation 8–10. In a recent global gene expression analysis of microfermentor biofilms, several genes controlled by the SinR-like regulators CD2214 and CD2215, including pilA1, were differentially expressed in biofilms, although pilA1 appeared to contribute to biofilm/aggregate formation only in c-di-GMP overexpressing strains 12. ...C. difficile forms complex biofilms in vitro which involves multiple regulatory pathways and several virulence-associated proteins. Paradoxically, subinhibitory concentrations of metronidazole and vancomycin induced biofilm formation and seemingly reduced antibiotic susceptibility 5,18. ...it is possible that low levels of antibiotics could induce C. difficile biofilm production, thus promoting persistence and recurrence of infection.