Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria

Abstract

Background: Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV.

Methodology/principal findings: Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel's clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel's criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV.

Conclusions/significance: The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in Black women. Tools developed in this project can be used to study microbial ecology in diverse settings at high resolution.

Conflict of interest statement

Competing Interests: D. N. Fredricks and T. L. Fiedler have developed intellectual property related to the use of PCR for the diagnosis of BV. Refer to US patent 7625704 on use of PCR for diagnosis of BV. No products to report. David Fredricks is a PLoS ONE editorial board member. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Vaginal bacterial communities in all women.

A novel variant of hierarchical clustering was used to generate a clustering tree depicting the bacterial diversity in 220 women with and without BV (A). The scale bar represents KR distance, a generalization of UniFrac (see Methods). The colored bars represent bacterial taxa that were most abundant in each sample. Less abundant taxa are grouped in the “other” category. BV status by Amsel’s criteria (B) and Gram stain (C) is provided for all women in the 2 vertical bars. In the absence of BV, the vaginal microbiota was dominated with Lactobacillus crispatus or Lactobacillus iners. Women with BV had more diverse bacterial communities.

Figure 2. Association of vaginal bacterial communities with BV status, clinical features and race.

A dendrogram shows association of meta data with vaginal bacterial communities. Nugent score and Amsel’s clinical criteria for BV are expressed as negative (light) and positive (dark). Nugent scores are presented in 2 formats, one considering BV positive (7–10) vs. BV negative (0–6), and the other separating data on women with intermediate scores (4–6) indicated in orange vs. women without BV (0–3) in white). pH values range from 4 to 6. Numerical values are shaded from light (low) to dark (high). Whiff test is expressed as positive (dark) or negative (light) and abundance of clue cells is expressed as none, 20% (light to dark). Vaginal discharge is represented as normal (light) and abnormal (dark). The race band denotes white (light) and black (dark) women. Gray bars denote that data not available or do not fit the criteria outlined. Meta data are also available in Table S3.

Figure 3. Lactobacillus species profiles in relation to BV status (A) and pH (B).

Edge principal components analysis, a variant of classical principal components analysis facilitates the labeling of the two components. The algorithm was not informed by the Nugent score or taxonomic classifications and the axes were independently selected by the algorithm. The x-axis represents presence of Lactobacillus species and the y-axis represents abundance of the two key Lactobacillus species, Lactobacillus crispatus and Lactobacillus iners. BV status is indicated by Nugent score; 0–6: BV Negative, 7–10: BV positive.

Figure 4. Co-occurrence analysis of bacterial taxa.

Hierarchically clustered Pearson correlation coefficients between abundant bacterial taxa are displayed. Correlation values range from −0.51 (light) to 0.81 (dark). Diagonal elements have correlation of 1. Several sub-groups of bacteria associated with BV show strong positive associations. The coefficients are available in Table S7.

Figure 5. Association of race with vaginal microbiota.

A general linear model (see Methods for additional detail) was used to describe interactions between race and BV status as determinants for bacterial prevalence. On the x-axis, BV was assessed by Gram stain: 0–6, BV negative; 7–10, BV positive. On the y-axis counts were sequence reads in each group; values summarized in the figure are log-transformed average counts in each group. Values below each taxonomic identifier are the estimate of the strength of the interaction +/− one standard error; large positive values indicate a more positive (less negative) slope amongst Black women than White women. Eight of 28 bacterial taxa showing greatest interaction with race are displayed.

Figure 6. Bacterial taxa associated with Amsel’s clinical characterisitics.

A model representing all bacterial taxa associated with the four clinical variables used to diagnose BV. Leptotrichia amnionii and Eggerthella sp. were found to be associated with each of the four clinical criteria. Stars denote bacteria that were present in greater than 75% of women with BV. Taxa in bold denote those showing associations with Amsel’s criteria as a composite unit.