Metronidazole response profiles of Gardnerella species are congruent with phylogenetic and comparative genomic analyses.

Abstract

Background: Bacterial vaginosis (BV) affects 20-50% of reproductive-age female patients annually, arising when opportunistic pathogens outcompete healthy vaginal flora. Many patients fail to resolve symptoms with a course of metronidazole, the current first-line treatment for BV. Our study was designed to identify genomic variation associated with metronidazole resistance among strains of Gardnerella vaginalis spp. (GV), a genus of biogenic-amine-producing bacteria closely associated with BV pathogenesis, for the development of a companion molecular diagnostic.

Methods: Whole-genome sequencing and comparative genomic metrics, including average nucleotide identity and GC content, were performed on a diverse set of 129 GV genomes to generate data for detailed taxonomic analyses. Pangenomic analyses were employed to construct a phylogenetic tree and cluster highly related strains within genospecies. G. vaginalis spp. clinical isolates within our collection were subjected to plate-based minimum inhibitory concentration (MIC) testing of metronidazole (n = 60) and clindamycin (n = 63). DECIPHER and MAFFT were used to identify genospecies-specific primers associated with antibiotic-resistance phenotypes. PCR-based analyses with these primers were used to confirm their specificity for the relevant genospecies.

Results: Eleven distinct genospecies based on standard ANI criteria were identified among the GV strains in our collection. Metronidazole MIC testing revealed six genospecies within a closely related phylogenetic clade contained only highly metronidazole-resistant strains (MIC ≥ 32 µg/mL) and suggested at least two mechanisms of metronidazole resistance within the eleven GV genospecies. All strains within the six highly metronidazole-resistant genospecies displayed susceptibility to clinically relevant clindamycin concentrations (MIC ≤ 2 µg/mL). A PCR-based molecular diagnostic assay was developed to distinguish between members of the metronidazole-resistant and mixed-response genospecies, which should be useful for determining the clade membership of various GV strains and could assist in the selection of appropriate antibiotic therapies for BV cases.

Conclusions: This study provides comparative genomic and phylogenetic evidence for eleven distinct genospecies within the genus Gardnerella vaginalis spp., and identifies genospecies-specific responses to metronidazole, the first-line treatment for BV. A companion molecular diagnostic assay was developed that is capable of identifying essentially all highly metronidazole-resistant strains that phylogenetically cluster together within the GV genospecies, which is informative for antibiotic treatment options.