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.

Keywords: Antibiotic resistance; Bacterial vaginosis; Comparative genomics; Distributed genome hypothesis; Gardnerella vaginalis; Metronidazole resistance; Molecular diagnostic; Pangenome; Phylogenetics; Supragenome.

Fig. 1

Comparative genomic techniques suggest eleven distinct genospecies of Gardnerella vaginalis spp. A ANI approximates genome similarity using BLAST. Each of the 129 total strains of G. vaginalis are represented on each axis in the same order, where the diagonal represents comparison to self, and off-diagonal represents comparison to a different strain. The range of ANI from least to greatest is 79.0% (white) to 99.9% (dark blue). Cluster diagrams were built using single-linkage hierarchical clustering. B Violin plot of ANI values for 129 strains of G. vaginalis (median: 86 ± 0.07%) and 25 strains each of four well-characterized species: Escherichia coli (99.7 ± 1.0%), Staphylococcus aureus (99.8 ± 0.5%), Pseudomonas aeruginosa (98.8 ± 2.1%), and Bacillus cereus (95.0 ± 2.4%). Density of data points at a given percentage is indicated by the width of the plot. Median value is indicated by a black diamond. C Distance matrix of G. vaginalis strains calculated from 1-ANI values and hierarchically clustered using the average linkage method. A cutoff was drawn at 95% similarity (horizontal line) and the number of genospecies counted. Eleven genospecies were observed using this cutoff value. Comparison to genospecies seen in phylogeny is displayed in colored boxes and labeled with clade numbers from the phylogenetic tree in Fig. 2. See also Additional File 1: Table S1 – S4

Fig. 2

Concatenated core-genome maximum likelihood tree displays the presence of eleven genetically distinct genospecies. The pangenome for 129 strains of G. vaginalis was calculated and 431 core genes were aligned with PRANK to create a phylogenetic tree with RAxML. Eleven individual genospecies (GS) are highlighted by color and marked with the range of GC content percentages within each. Singleton strains (genospecies 3 and 9) are included with the nearest genospecies with 5 or more strains and marked with a different shaded box. Genospecies designated as individual species by Vaneechoutte et al. [35] and Sousa et al. [37] are indicated by colored rings and labeled with their proposed species names. See also Additional File 1: Table S1-S2

Fig. 3

Core genome percentage is more reflective of a single species after GV is separated into individual genospecies. The count of core genes (orange bar) of all 129 GV strains is less than half of the proportion of the independently calculated genospecies’ pangenomes. See also Additional file 2: Fig. S1

Fig. 4

GV genospecies display two major phenotypic responses to metronidazole, but analysis of genes does not uncover the mechanism of resistance. A Representative images of MIC assays. Each metronidazole MIC strip contains fifteen twofold dilution concentrations. An ellipse of inhibition forms at the minimum concentration of metronidazole required to stop GV growth. Left: Resistant strain with MIC > 256 µg/mL. Middle: Resistant strain with MIC of 64 µg/mL. Right: Susceptible strain with MIC of 8 µg/mL. B Concatenated core gene tree labeled with MIC data indicate the existence of genospecies correlated with antibiotic resistance. Results of MIC analyses were plotted against the tree. At least one strain from each genospecies reported a result except for genospecies 8. On the right, MIC data are shown for each tested strain. A color legend is available at the top left. Length of MIC bar corresponds with MIC value. Color corresponds to CLSI classification. C Alignment of syntenic regions identified in the metronidazole-resistant partner of three resistant/susceptible strain pairings within the mixed-response genospecies. Colored arrows represent each of the genes with one of five KEGG-annotated functions (see legend). Arrow direction corresponds with coding strand. See Additional file 1: Table S5

Fig. 5

Selected metronidazole-resistant strains are susceptible to clindamycin. Sixty-three strains from various genospecies were subjected to antibiotic susceptibility testing with clindamycin MIC strips. A bar plot is superimposed above the gradient of measurable MIC values, indicating the number of strains with each measured MIC. The clinical sensitivity breakpoint as determined by CLSI for clindamycin is marked with an arrow (2.0 µg/mL). A representative image is shown at right, demonstrating the ellipse of inhibition observed for a clindamycin-susceptible strain with an MIC of 0.125 μg/mL. See Additional file 1: Table S5

Fig. 6

aruH primers specifically amplify strains in metronidazole-resistant genospecies. A set of two forward and two reverse primers for aruH, encompassing the range of diversity seen in this gene among the various GV species, successfully discriminated between selected clinical isolates within the metronidazole-mixed response genospecies 1a, 2, 3, 4a, and 4b and the entirely metronidazole-resistant genospecies (5–11). Successful amplification coincided with membership in the highly metronidazole-resistant genospecies (5–11), with the exception of strain B648 (BS610), a resistant strain and the only member of genospecies 9. See also Table 2, Additional file 1: Table S6, and Additional file 2: Fig. S2