Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure

Abstract

Background: Metronidazole is the first-line treatment for bacterial vaginosis, but cure rates are suboptimal and recurrence rates high.

Objectives: To evaluate the impact of a standard course of oral metronidazole treatment (500 mg twice per day for 7 days) on the vaginal microbiota of Rwandan bacterial vaginosis patients using microscopy and 16S rRNA gene sequencing, and to evaluate correlates of treatment failure.

Study design: HIV-negative, nonpregnant women aged 18-45 years with bacterial vaginosis and/or Trichomonas vaginalis (N=68) were interviewed and sampled before and after metronidazole treatment. They were also screened, and treated if applicable, for other urogenital infections. The vaginal microbiota was assessed by Gram stain Nugent scoring, Illumina 16S rRNA HiSeq sequencing (relative abundances), and BactQuant 16S gene quantitative polymerase chain reaction (estimated concentrations). Only women with a pretreatment Nugent score of 7-10 and a valid posttreatment Nugent score (N=55) were included in metronidazole treatment failure analyses, with treatment failure defined as a posttreatment Nugent score of 4-10.

Results: The bacterial vaginosis cure rate by Nugent scoring was 54.5%. The mean total vaginal bacterial concentration declined from 6.59 to 5.85 log₁₀/μL (P<.001), which was mostly due to a reduction in mean bacterial vaginosis-associated anaerobes concentration (all bacterial vaginosis-associated anaerobe taxa combined) from 6.23 to 4.55 log₁₀/μL (P<.001). However, only 16.4% of women had a bacterial vaginosis anaerobes concentration reduction of more than 50%, and only 3 women had complete eradication. The mean concentration of lactobacilli (all species combined) increased from 4.98 to 5.56 log₁₀/μL (P=.017), with L. iners being the most common species pre- and posttreatment. The mean concentration of pathobionts (defined as Proteobacteria, streptococci, staphylococci, enterococci, and a few others) did not change significantly: from 1.92 log₁₀/μL pretreatment to 2.01 log₁₀/μL posttreatment (P=.939). Pretreatment pathobionts concentration, and having a pretreatment vaginal microbiota type containing more than 50% Gardnerella vaginalis (compared with less than 50%), were associated with increased likelihood of treatment failure, but the latter did not reach statistical significance (P=.044 and P=.084, respectively).

Conclusions: Metronidazole alone may not cure women with high G. vaginalis relative abundance, potentially due to biofilm presence, and women with high pathobionts concentration. These women may benefit from additional biofilm-disrupting and/or pathobiont-targeting treatments.

Trial registration: ClinicalTrials.gov NCT02459665.

Keywords: 16S rRNA gene sequencing; Lactobacilli; anaerobes; antibiotics; bacterial vaginosis; biofilm; metronidazole; trichomoniasis; vaginal dysbiosis; vaginal microbiota.

Figure 1

Heatmaps of 16S rRNA gene sequencing data pre- and posttreatment A–B, Heatmaps at the pretreatment (A) and the posttreatment visit (B) depicting the 20 amplicon sequence variants with the greatest mean relative abundance on the y-axis and samples (N=67 at each visit) on the x-axis. The dendrogram above the heatmap depicts vaginal microbiota clusters based on Euclidean distance. The bar below the dendrogram depicts Nugent score categories (see legend; black means no score available). BVAB1, bacterial vaginosis-associated bacterium type 1. Verwijs et al. Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure. Am J Obstet Gynecol 2020.

Figure 2

Nugent scores, mean bacterial group relative abundances, and vaginal microbiota types pre- and posttreatment A–C, Changes in vaginal microbiota characteristics before and after metronidazole treatment. A, Nugent scores, B, mean bacterial group relative abundances, and C, vaginal microbiota types. D, Three-dimensional non-metric multidimensional scaling plot based on rarefied relative abundances of samples before and after metronidazole treatment. The figure shows that samples cluster together by visit (and hence, treatment status). BV, bacterial vaginosis; BV_GV, polybacterial Gardnerella vaginalis-containing; BV_noGV, polybacterial but low G. vaginalis; GV, G. vaginalis-dominated; IQR, interquartile range; LA, lactobacilli and anaerobes; Li, Lactobacillus iners-dominated; Lo, other lactobacilli-dominated; PB, pathobionts-containing; Post-tx, posttreatment visit; Pre-tx, pretreatment visit. Verwijs et al. Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure. Am J Obstet Gynecol 2020.

Figure 3

Individual bacterial group concentrations pre- and posttreatment A-D, Changes in total bacterial concentrations and bacterial group concentrations before (n=66) and after metronidazole treatment (n=63). A, Total bacterial concentration, B, total lactobacilli, C, total BV-anaerobes, and D, total pathobionts (boxplot not shown because of high proportion of zero values). See Table 2 for mean concentrations and 95% confidence intervals, and statistical significance. E-H, Change in concentrations between pre- and posttreatment, expressed as a percentage for every individual participant with valid quantitative polymerase chain reaction results at both visits (n=61), for total bacterial concentration (E), total Lactobacillus (F), total BV-anaerobes (G), and total pathobionts (H). In some women, concentration went from zero to non-zero; these increases were set at 100% or the greatest increase observed among the other participants, whichever was greatest. BV, bacterial vaginosis; conc, concentration; Post-tx, posttreatment visit; Pre-tx, pretreatment visit. Verwijs et al. Impact of oral metronidazole treatment on the vaginal microbiota and correlates of treatment failure. Am J Obstet Gynecol 2020.