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


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.


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.


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.


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.


Bacterial vaginosis (BV) is a polymicrobial condition in the female reproductive tract (FRT) that is known to increase the risk of sexually transmitted infection (STI) acquisition and reproductive sequelae. The BV rates among women vary widely globally; recent estimates suggest a 30% prevalence in the United States, whereas the rates in sub-Saharan Africa may exceed 50%. , Despite this high prevalence, no single causative agent of BV is known.

AJOG at a Glance

Why was this study conducted?

The current approaches for the management of bacterial vaginosis (BV) do not adequately assess the risk of treatment failure, which is demonstrated by the unacceptably high levels of relapse. This study sought to define the aspects of microbiota composition at the time of BV treatment initiation that predispose women to treatment failure.

Key findings

Although the levels of the most common BV-associated bacteria do not predict the risk of treatment failure, the accumulation of diverse, low-abundance taxa may predispose women toward treatment-resistant BV.

Shifts to Lactobacillus dominance improved the markers of mucosal tissue health, but these gains were only temporary among the women who experienced recurrence.

What does this add to what is known?

Our study provides evidence that a highly diversified vaginal ecosystem leads to BV treatment failure, which concerningly leads to increased diversity above the baseline levels. The failure of metronidazole therapy could entrench existing polymicrobial communities.

The most universal feature of BV is the loss of Lactobacillus dominance in the vaginal ecosystem, which is accompanied by an increase in the proportion of polymicrobial anaerobic taxa. The disruption of Lactobacillus dominance increases the pH levels and nutrient availability, enabling the outgrowth of bacterial vaginosis-associated bacteria (BVAB) such as Gardnerella vaginalis . , Persistent colonization by Gardnerella vaginalis is associated with the development of biofilms that support other BVAB, including Atopobium, Prevotella, Sneathia, and Shuttleworthia .

The large-scale availability of marker gene sequencing data has enabled the stratification of vaginal communities into discrete categories defined by their composition; the categories are frequently referred to as the cervicotype (CT) or community state types. Dominance by Lactobacillus crispatus (CT1) is considered as an optimal state and is linked to a marked decrease in STI susceptibility. CT2 is dominated by Lactobacillus iners and is frequently considered a transitional phenotype to CT3 and CT4, which are collectively referred to as “molecular BV.” , These communities, which are dominated by Gardnerella vaginalis or a highly diverse BVAB population (CT3 or CT4, respectively), strongly correlate with an increase of inflammatory factors, reproductive sequelae, and STI susceptibility.

Despite these known risks, the BV recurrence rates remain above 70% within 12 months of treatment. The current Centers for Disease Control and Prevention guidelines recommend metronidazole as a first line treatment. As a prodrug, metronidazole is reduced to its active state under anaerobic conditions—a mechanism that spares beneficial Lactobacillus . However, the complete removal of the BVAB is likely complicated by the presence of biofilms, which shield the BVAB by preventing metronidazole penetration.

Here, we build on an examination of women with symptomatic BV who enrolled in a clinical study (CONRAD 115, Identifier: NCT01347632 ). The initial analysis identified several BV-associated changes; these included increased inflammatory mediators and HIV target cells in the FRT and reduced antimicrobial activity of the cervicovaginal lavage (CVL). We hypothesized that 16S ribosomal RNA (rRNA) gene analysis would identify the keystone taxa or community structures that were present at diagnosis and contribute to BV recurrence or treatment failure; our primary object was to leverage these data toward the development of a molecular predictor of treatment failure.

Materials and Methods

Human and nonhuman experimentation: enrollment and sampling

The longitudinal, open-label study was approved by the Chesapeake Institutional Review Board (Pro #00006122) with a waiver of oversight from the Eastern Virginia Medical School, and it was registered in ( Identifier: NCT01347632 ). Thirty-three women seeking treatment for symptomatic vaginal discharge with or without atypical vaginal odor were enrolled on getting a Nugent scoring of ≥4 ( Supplemental Table 1 ); the Amsel criteria were not utilized in this study. All the participants were nonpregnant, free of reproductive tract infections, and were denied the use of antibiotics within the previous 14 days. On enrollment, vaginal swabs, cervicovaginal tissue biopsies, and CVL were collected. Following the collection of the biologic samples, all the women were prescribed 14 500-mg metronidazole tablets, with 1 tablet to be taken twice daily; patient adherence was self-reported. The biologic samples were collected again between 7 and 10 days following the conclusion of treatment (visit 2) and again between 28 and 32 days after treatment (visit 3). The biologic samples included vaginal pH testing, and the CVL was collected through administration of 10 cc of normal saline. Three full-thickness cervico-vaginal biopsies were obtained under topical anesthesia at the conclusion of CVL collection.

Soluble factor analysis

The CVL secretory leukocyte protease inhibitor (SLPI) was measured by the enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) using a victor2 reader (Perkin Elmer Life Sciences, Boston, MA). Interleukin (IL)-1b, IL-6, IL-8, IL- 10, ICAM-1, and tumor necrosis factor alpha were measured in undiluted CVLs by a multiplex electrochemiluminescence assay (Meso Scale Discovery, Gaithersburg, MD). All the measurements were performed in duplicate.

DNA extraction

The bacteria from the CVL samples were pelleted by centrifugation and resuspended in 200 μl of lysis buffer (30 mM tris hydrochloride, 10 mM ethylenediaminetetraacetic acid, 200 mM sucrose, pH 8.2). The samples were heated at 65°C for 10 minutes before the addition of 100 mg/mL lysozyme solution to a final concentration of 10 mg/mL. The samples were then incubated for 1 hour at 37°C. 5% sodium dodecyl sulfate was then added to a final concentration of 1% w/v and incubation was done at 56°C for 10 minutes. DNA extractions were then performed using Qiagen’s DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).

16S ribosomal RNA sequencing

The extracted DNA was amplified using the Earth Microbiome Protocol for 16S Illumina sequencing utilizing 515F-806R primers that were originally described by Caporaso et al to target the V3-V4 region of the 16S small subunit rRNA. This broadly applicable and widely utilized primer set was chosen to maximize the comparability of this data set with the existing and future marker gene sequencing of the vaginal microbiome. The amplicon concentrations were normalized, pooled, and cleaned before KAPA quantification. The pooled library was sequenced using a 2×150 bp Illumina MiSeq run.

Metataxonomic analyses

16S rRNA amplicon sequencing reads were demultiplexed in Illumina Basespace. A demultiplexed read object was generated in qiime2-2018.2. Amplicon sequence variants were generated using the dada2 denoize-single with 0 trim-left and trunc at 145. The phylogeny was determined using the mafft-fastree option and the amplicon sequence variants were assigned taxonomy using the 99% Greengenes 515-806 classifier. The Qiime2 objects were exported for downstream analysis in R. All fastq files were deposited in the sequence read archive under ascension PRJNA691964.


Lactobacillus recovery varies following metronidazole treatment

Twenty-eight of the 33 women enrolled were confirmed to have molecular BV by 16S rRNA sequencing, whereas 5 patients found to have Lactobacillus -dominant microbiomes pretreatment were removed from further analyses. At diagnosis, the mean Lactobacillus abundance was 4.4% ( Figure 1 , A and E). The known BVAB, such as Shuttleworthia , Sneathia, Prevotella , and Gardnerella, were most likely to be the dominant genus in a given sample at baseline ( Supplemental Figure 1 , A and B). Several other highly prevalent bacterial genera frequently associated with BV diagnosis, including Megasphaera, Atopobium, Clostridium , Parvimonas , Dialister , Peptonophilus , and Gemella ( Figure 1 , D and E), were observed at mean abundances of <5.

Figure 1

Baseline and posttreatment vaginal microbiota profiles of women diagnosed with Nugent-BV

Relative abundance taxonomic plots of cervicovaginal lavage assessed by 16S ribosomal RNA gene profiling; women were sampled at baseline on BV diagnosis ( A ) 7–10 days after conclusion of 10-day course of metronidazole ( B ) and again at 1 month posttreatment ( C ) A histogram demonstrates most of the 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 detected in this study were significantly reduced at visit 2 following metronidazole treatment, though the mean levels rose again at visit 3. Lactobacillus was significantly increased at visits 2 and 3 ( E ).

BV , bacterial vaginosis.

Gustin et al. Microbiota correlates of bacterial vaginosis treatment failure. Am J Obstet Gynecol 2022.

At visit 2 that was one week after treatment, the Lactobacillus mean relative abundance reached 70.1%, whereas several genera that were dominant at baseline decreased significantly ( Figure 1 , B and E). At visit 3 that was approximately one month after treatment, the mean Lactobacillus abundance was down to 24.7% across all samples, whereas Sneathia , Prevotella , and Shuttleworthia increased on average ( Figure 1 , C and E). The mean values were found to obscure divergent treatment outcomes; these are discussed in detail below.

The alpha diversity measurements of richness and evenness assess the number of observed taxa per sample and the degree of parity among the taxa, respectively, whereas inverse Simpson suggests the complexity of a sample by estimating the likelihood that 2 randomly chosen reads from a sample would identify the same taxa. In the context of BV treatment, positive therapeutic responses include reducing the observed diversity and increasing dominance by Lactobacillus . Although all the measurements of diversity were significantly decreased at the level of the cohort for visit 2, a number of individuals actually experienced increases in richness and diversity ( Figure 2 , A). Further analysis revealed that a subset of women who did develop Lactobacillus iners dominance by visit 2 failed to retain these communities at visit 3, indicating BV recurrence. The variable response to treatment can be measured by beta diversity, which estimates the degree of difference between 2 communities; these differences can be visualized using dimensionality reduction techniques such as principal coordinates analysis (PCoA), where an increasing distance between any 2 samples represents decreasing similarity. In the present study, although the patients clustered together at baseline, the community composition and structure varied widely following treatment ( Figure 2 , B). It should be noted that the patients were not found to cluster by age, body mass index, cycle day, contraception method, or self-reported race ( Supplemental Figure 6 , A–E).

Figure 2

Alpha and beta diversity assessment

Box-and-whisker plots define the median and interquartile ranges for measures of alpha diversity such as richness, evenness, and the inverse Simpson index, which were significantly decreased 1 week posttreatment; 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 1 (red), 2 (green), and 3 (blue); shaded ellipses represent 95% confidence intervals for cervicotype distribution. The plot illustrates samples clustered at visit 1 before treatment whereas the samples were considerably more varied at visits 2 and 3, reflecting divergent responses to metronidazole therapy ( B ).

Gustin et al. Microbiota correlates of bacterial vaginosis treatment failure. Am J Obstet Gynecol 2022.

Soluble immune factors in the female reproductive tract respond to microbiota shifts

Given the diverse patterns of change in the microbial structure following treatment for BV, we cervicotyped the patients at each visit. We elected to use the classification strategy described by Anahtar et al, which utilizes 4 CTs assigned according to the dominant organism, with CT1 dominated by Lactobacillus crispatus , CT2 by Lactobacillus iners , CT3 by Gardnerella vaginalis , and CT4 by any of a number of diverse anaerobes associated with BV. We first validated the distinct composition of the CTs through weighted UniFrac assessment ( Figure 3 , A), finding significant differences between each CT pairwise (2v3: adjusted P (adj .P )=.003; 2v4: adj. P =.003; 3v4: adj. P =.027); CT1 was not observed in the current study. We next assessed the vaginal pH levels across these groupings and found that the women supporting CT2 had a significantly lower pH at the time of sampling than the women in either CT3 or CT4 ( Figure 3 , B) ( P =.001; P =3.9 × 10 –12 ). In addition to the differences in the vaginal pH, we identified significantly higher levels of SLPI, growth-regulated alpha protein, and macrophage inflammatory protein-3 in the CVL from women in CT2 than those in CT4 ( Figure 3 , C); these women also had significantly lower levels of ICAM-1 ( P= 2 × 10 –4 ) and a trend toward lower IL-1B ( P =.067). CD4+ cells from the lamina propria, as assessed by microscopy, were significantly higher in the women categorized as CT4 ( P =.034), which is in agreement with recent reports. ,

Apr 16, 2022 | Posted by in GYNECOLOGY | Comments Off on Recurrent bacterial vaginosis following metronidazole treatment is associated with microbiota richness at diagnosis
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