Reduced metabolic activity in established Gardnerella spp. biofilms contributes to protection from the bactericidal effects of metronidazole

Gardnerella spp. can form biofilm and it has been suggested that failure of antibiotic treatment of bacterial vaginosis and recurrent vaginosis are linked to this property, however no specific mechanisms have been identified. Here, we performed broth microdilution assays to measure the minimum inhibitory concentration (MIC) of metronidazole for thirty-five Gardnerella isolates in one medium in which Gardnerella spp. grow primarily as biofilm and another in which growth is primarily planktonic. The MIC of isolates in the two conditions were highly correlated (R2= 0.69, p <0.001). No significant reduction in viable cell count of 7/9 isolates was observed when established biofilms were exposed to metronidazole at levels double the MIC. Subsequent measurements of metabolic activity in established biofilms formed by a larger collection of 31 isolates showed reduced metabolic activity following treatment with 128 g/ml of metronidazole relative to untreated controls in 27/31 cases. The amount of biofilm produced by Gardnerella isolates was not enhanced by metronidazole exposure, even at sub-MIC levels. Our results suggest that reduced metabolic activity in Gardnerella spp. growing in established biofilms may be a mechanism of protection from the bactericidal effects of metronidazole.