Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants

Objectives: Human clinical isolates of Bacteroides spp. originating from patients in the UK were investigated for the presence of metronidazole resistance determinants (nim genes) and their presence was related to the MIC of metronidazole for the isolates. Methods: Isolates were screened for susceptibility to a metronidazole disc and had their MIC determined by the Etest method. They were investigated for the presence of nim genes by PCR. An experiment to determine the effect of prolonged exposure to metronidazole was applied to nim-positive isolates with MICs below the therapeutic breakpoint. Results: Fifty of 206 isolates (24%) were found to possess nim genes and these had MICs of metronidazole ranging from 1.5 to >256 mg/L with 24 (11.6%) above the therapeutic breakpoint of 16 mg/L. The remaining 26 nim-gene-positive isolates had MICs that were still below the therapeutic breakpoint, ranging from 1.5 to 6.0 mg/L. nim genes were not found in 156 (76%) isolates, and all but seven of these were susceptible to a 5 mug disc of metronidazole. Ten members of the group for which the MICs were below the therapeutic level were found to have slow-growing sub-populations with metronidazole MICs ranging from 8.0 to >256 mg/L that became evident after prolonged exposure to metronidazole in vitro. This resistance selection process was sometimes reversible after passage in the absence of metronidazole; however, seven of the 10 slow-growing mutants converted to stable high-level resistance (MIC >256 mg/L). Conclusions: Although the presence of nim genes per se does not always equate to therapeutic resistance, and other metronidazole resistance mechanisms may exist, this study has shown that prolonged exposure of nim-gene-carrying Bacteroides spp. to metronidazole can select for therapeutic resistance.