Analysis of metronidazole susceptibility in different Clostridium difficile PCR ribotypes

Susceptibility to metronidazole was investigated in 81 Clostridium difficile strains, belonging to nine different PCR ribotypes, by three different laboratory methods. MICs for 81 C. difficile clinical isolates were determined by Etest, the agar dilution method (ADM) and the agar incorporation method (AIM). Twenty selected strains were also subjected to subinhibitory concentrations of metronidazole and the MIC heterogeneity was analysed in colonies from each strain that showed increased values before and after exposure to the antibiotic, using ADM and AIM. Overall, the MICs obtained by Etest were lower compared with those obtained by ADM and AIM, causing discrepancies in the categorization (as susceptible or having reduced susceptibility) of some strains. Reduced susceptibility to metronidazole was observed using both ADM and AIM, with higher MIC values by AIM in isolates belonging to PCR ribotypes 001 and 010. An increase in MICs after exposure to metronidazole was observed for strains belonging to these PCR ribotypes (by Etest and ADM, but not by AIM). In particular, MICs for colonies from strains belonging to either PCR ribotype 001 or 010 were less heterogeneous by AIM compared with by ADM, suggesting a better ability of AIM to detect strains with reduced susceptibility. These results suggest that the presence of C. difficile subpopulations with reduced susceptibility to metronidazole in the human intestine may be one of the factors responsible for reduced antibiotic efficacy in vivo. The possibility that higher MICs may have often gone unnoticed underlines the importance of choosing the best method for MIC determination and the necessity to monitor C. difficile susceptibility to metronidazole.