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

Abstract

Gardnerella spp., a hallmark of bacterial vaginosis, can form biofilm and it has been suggested that failure of antibiotic treatment of bacterial vaginosis and recurrent vaginosis are linked to its ability to form biofilm. Here, we tested the hypothesis that biofilm formation provides protection from the effects of metronidazole. We performed a broth microdilution assay to measure the minimum inhibitory concentration (MIC) of metronidazole for thirty-five Gardnerella isolates in two different growth media: one medium in which Gardnerella spp. grow primarily as biofilm and the other medium in which Gardnerella spp. grow primarily in planktonic form. The MIC of Gardnerella isolates observed in the two conditions were highly correlated (R2= 0.69, p <0.001) and 27/35 isolates had no difference in MIC between the two growth modes. When established biofilms were treated with metronidazole, live Gardnerella could be recovered following treatment in most cases (7/9 isolates tested). Metabolic activity of established biofilms of thirty-one isolates with and without metronidazole treatment was measured using a resazurin assay. Most (27/31) isolates showed reduced metabolic activity following treatment with μ128 g/ml of metronidazole relative to untreated controls. The amount of biofilm produced by Gardnerella isolates was not enhanced by sub-inhibitory concentrations of metronidazole and scanning electron microscopy revealed no architectural differences between treated and untreated biofilms. Our results suggest that Gardnerella spp. growing in established biofilms reduce metabolic activity as a mechanism of protection from the bactericidal effects of metronidazole.