Recurrent bacterial vaginosis following metronidazole treatment is associated with microbiota richness at diagnosis

Abstract

Background: Bacterial vaginosis-a condition defined by a shift from Lactobacillus dominance to a polymicrobial, anaerobic bacterial community-increases the risk of acquiring sexually transmitted infections and other complications of the female reproductive tract. Antibiotic treatment frequently fails to return the microbiome to an optimal Lactobacillus-dominated state. No criteria currently exist to identify the patients likely to experience treatment failure.

Objective: We sought to identify the pretreatment community signatures associated with treatment failure through 16S ribosomal RNA gene analysis.

Study design: Twenty-eight women who were enrolled in an oral metronidazole treatment trial of bacterial vaginosis were studied. Cervicovaginal lavage samples were collected before metronidazole treatment and at 7 and 30 days posttreatment. Cervicovaginal lavage DNA was amplified and sequenced using a paired-end, V4 region 2×150 MiSeq run.

Results: Of the 28 women, 25% failed to clear bacterial vaginosis; 35.7% demonstrated a transient clearance, shifting to community-type 2 (Lactobacillus iners dominant) at visit 2 only; 7.1% demonstrated a delayed clearance, reaching community-type 2 at the final visit only; and 32.1% of patients experienced sustained bacterial vaginosis clearance. Examination of the community composition and structure demonstrated that both the richness and the evenness were significantly lower for the women who experienced sustained clearance, whereas the women who failed to clear bacterial vaginosis possessed the highest median levels of richness, evenness, and diversity pretreatment. Soluble immune factors in the lower reproductive tract improved significantly following a shift from community-type 4 to a Lactobacillus-dominant microbiome, with the samples categorized as community-type 2 possessing significantly higher levels of secretory leukocyte protease inhibitor, growth-regulated alpha protein, and macrophage inflammatory protein-3 and significantly lower levels of intercellular adhesion molecule-1. Although the shifts to Lactobacillus dominance improved the markers of mucosal tissue health, these gains were only temporary among the women who experienced recurrence.

Conclusion: Assemblies of highly diverse microbiota are associated with the enhanced resilience of bacterial vaginosis to standard metronidazole treatment. These communities may be foundational to treatment resistance or simply an indication of a well-established community made possible by canonical biofilm-forming taxa. Future studies must target the transcriptional activity of these communities under the pressure of antibiotic treatment to resolve the mechanisms of their resistance.

Trial registration: ClinicalTrials.gov NCT01347632.

Keywords: antibiotics; bacterial vaginosis recurrence; biofilms; molecular bacterial vaginosis; mucosal immunity; vaginal microbiome.

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Disclosures: The authors report no conflict of interest.

Copyright © 2021. Published by Elsevier Inc.

Figures

Figure 1 –

Baseline and post-treatment vaginal microbiota profiles of women diagnosed with Nugent-BV. Relative abundance taxonomic plots of cervicovaginal lavage assessed by 16S rRNA gene profiling; women were sampled at baseline upon BV diagnosis (A), 7 – 10 days after conclusion of 10 day course of metronidazole (B), and again at 1 month post-treatment (C). A histogram demonstrates the majority of genera considered BV-associated were universally prevalent at baseline, including Prevotella, Sneathia, Gardnerella,Lactobacillus, Dialister, and Peptonophilus; Shuttleworthia and Atopobium were present in 27 of 28 analyzed samples. Several rare taxa were also highly prevalent (D). Box-and-whisker plots define median and interquartile ranges; all BV-associated bacteria deteceted in this study were significantly reduced at visit 2 following metronidazole treatment, though mean levels roseagain at visit 3. Lactobacillus was significantly increased at visits 2 and 3 (E).

Figure 2 –

Alpha and Beta Diversity assessment. Box-and-whisker plots define median and interquartile ranges for measures of alpha diversity such as richness, evenness, and the inverse Simpson index, which were significantly decreased one week post-treatment; only evenness remained significantly reduced at visit 3 (A). A principal coordinate analysis of weighted UniFrac distances with samples (dots) colored here by visit: visit one (red), two (green), and three (blue); shaded ellipses represent 95% confidence intervals for cervicotype distribution. The plot illustrates samples clustered at visit 1 prior to treatment, while samples were considerably more varied at visits 2 and 3, reflecting divergent responses to metronidazole therapy (B).

Figure 3 –

Immune profiles segregate by cervicotype. Principal coordinate analysis based on weighted UniFrac distances demonstrated clustering of samples by cervictoype. Samples (dots) are colored here by cerivcotype: CT2 in red; CT3 in green, and CT4 in blue. CT1 was not observed in this study. Shaded ellipses represent 95% confidence intervals for cervicotype distribution (A). Assessment of sample pH based on assigned cervicotype shows significant elevations in pH for women with cervicotype 3 or 4 (B). Several soluble factors analyzed from cervicovaginal lavage showed signficant differences between CTs 2, 3, and 4; additionally, CD4+ cells in the lamina propria were significantly elevated for women in CT4 (C). Vaginal immune profiles were generated through measurement of select soluble factors present in CVL. Samples were then compared based on the aggregated measurements through Bray-Curtis dissimilarity and visualized through principal coordinate analysis, with the plot split into visits 1, 2, and 3. 16S rRNA gene data was visualized in the same plot through procrustes rotation. By visit 2, women who transititioned to Lactobacillus dominance possessed a statistically distinct immune profile (p.adjusted = 0.003 by permanova), and this distinction remained significant at visit 3 one-month post-treatment (p.adjusted = 0.003 by permanova). The emergence of a distinct immune profile for women with Lactobacillus dominance is paralleled by the emergence of distinct bacterial community compositions associated with a shift to CT2 (p.adjusted = 0.006 at visit 2 and visit 3 by permanova) (D).

Figure 4 –

Post-treatment shifts to Lactobacillus-dominance vary in timing and duration. Only a portion of women experiened significant increases in Lactobacillus levels following treatment; of these women who were cleared of SeqBV at visit 2, only a portion remained clear at visit 3 (A). Relative abundance levels of several BV-associated bacteria seen here were inversely related to gains in Lactobacillus; levels of these bacteria were at or above baseline level for many women at the final visit (B). Women who sustained SeqBV clearance following treatment had significantly lower levels of observed taxa and measured evenness prior to treatment initiation (C). SeqBV clearance patterns were not predicated by immune profile at baseline (D). Smoothed plot of select soluble factors over time that were shown to differ significantly by CT (E).