In a new report, researchers describe the identification of 11 distinct genomic species within the genus formerly known as Gardnerella vaginalis, with 6 of these species showing complete resistance to metronidazole, the first-line treatment for bacterial vaginosis.
Investigators from Drexel University and the University of Pittsburgh developed a polymerase chain reaction (PCR)-based molecular diagnostic test that can identify strains belonging to the highly metronidazole-resistant genomic species, potentially guiding more effective antibiotic selection for patients with bacterial vaginosis (BV). Their findings were published in Genome Medicine.
"Eleven distinct genospecies based on standard average nucleotide identity (ANI) criteria were identified among the Gardnerella vaginalis [GV] strains in our collection," the researchers reported. "Metronidazole minimum inhibitory concentration (MIC) testing revealed 6 genospecies within a closely related phylogenetic clade containing only highly metronidazole-resistant strains (MIC ≥ 32 µg/mL) and suggested at least two mechanisms of metronidazole resistance within the 11 GV genospecies."
BV affects 20% to 50% of reproductive-age female patients annually and is associated with an increased risk of post-cesarean endometritis, urinary tract infections, and HIV infection. Current BV treatment protocols recommend metronidazole as first-line therapy, but up to 30% of cases fail to resolve with standard treatment, and recurrence rates range from 50% to 70%.
Comprehensive Genomic Analysis
The research team, led by Katherine Innamorati, PhD, and Joshua Earl, PhD, both of Drexel University, conducted whole-genome sequencing and comparative genomic analyses on 129 diverse Gardnerella strains, including 57 newly sequenced clinical isolates and 72 previously characterized genomes.
Using ANI and phylogenetic tree construction from 431 core genes, the researchers confirmed that Gardnerella comprises multiple distinct species rather than a single species with variants. The median ANI value among all strains was 84.1%, well below the 95% threshold typically used to define a bacterial species.
Further evidence supporting the multispecies distinction came from pangenomic analysis. When analyzed as a single species, only 18% of genes were identified as core genes across all strains, significantly lower than the typical 39% median for bacterial species. However, when individual genospecies were analyzed separately, core genome percentages increased to 44% to 51%.
Antibiotic Susceptibility Testing
The researchers performed antibiotic susceptibility testing on 60 clinical isolates representing 10 of the 11 identified genospecies.
The testing revealed two distinct patterns of metronidazole response:
1) All strains tested in genospecies 7a/7b (G. swidsinskii and G. leopoldii), 9, 10, and 11 showed high-level resistance (MIC ≥ 128 μg/mL, with 95% of strains having MIC ≥256 μg/mL).
2) Genospecies 1 (amended G. vaginalis and genospecies 1b) and 4 (G. pickettii and G. piotii) showed variable responses, with some strains demonstrating susceptibility or intermediate resistance.
Importantly, all 63 tested strains showed high sensitivity to clindamycin (MIC range = 0.016–0.5 μg/mL, median = 0.125 μg/mL), regardless of their metronidazole resistance status.
Diagnostic Tool Development
The striking difference in metronidazole response profiles between genospecies led the researchers to develop a PCR-based diagnostic test. They identified the aruH gene as a marker which was reliably amplified in highly resistant genospecies (5, 6, 7a/b, 10, and 11) but not in the mixed-response genospecies (1a, 2, 3, 4a/b).
"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," the authors stated.
The aruH gene encodes an enzyme involved in arginine catabolism and biogenic amine production, contributing to the pathogenesis of BV by increasing vaginal pH.
Clinical Implications
This research potentially explains why approximately 30% of BV cases fail to respond to metronidazole treatment. The findings support at least two mechanisms of metronidazole resistance within the eleven GV genospecies.
The identified PCR assay could represent a significant advance in personalized BV treatment, allowing clinicians to determine whether a patient’s Gardnerella strain belongs to a highly resistant genospecies before antibiotic selection. As all tested strains remained susceptible to clindamycin, this alternative antibiotic could be prescribed for patients identified as harboring metronidazole-resistant strains.
Disclosures can be found in the study.