Diversity of human vaginal bacterial communities and associations with clinically defined bacterial vaginosis

Abstract

Bacterial vaginosis (BV) is a common syndrome associated with numerous adverse health outcomes in women. Despite its medical importance, the etiology and microbial ecology of BV remain poorly understood. We used broad-range PCR to census the community structure of the healthy and BV-affected vaginal microbial ecosystems and synthesized current publicly available bacterial 16S rRNA gene sequence data from this environment. The community of vaginal bacteria detected in subjects with BV was much more taxon rich and diverse than in subjects without BV. At a 97% sequence similarity cutoff, the number of operational taxonomic units (OTUs) per patient in 28 subjects with BV was nearly three times greater than in 13 subjects without BV: 14.8 +/- 0.7 versus 5.2 +/- 0.75 (mean +/- standard error). OTU-based analyses revealed previously hidden diversity for many vaginal bacteria that are currently poorly represented in GenBank. Our sequencing efforts yielded many novel phylotypes (123 of our sequences represented 38 OTUs not previously found in the vaginal ecosystem), including several novel BV-associated OTUs, such as those belonging to the Prevotella species complex, which remain severely underrepresented in the current NCBI database. Community composition was highly variable among subjects at a fine taxonomic scale, but at the phylum level, Actinobacteria and Bacteroidetes were strongly associated with BV. Our data describe a previously unrecognized extent of bacterial diversity in the vaginal ecosystem. The human vagina hosts many bacteria that are only distantly related to known species, and subjects with BV harbor particularly taxon-rich and diverse bacterial communities.

FIG. 1.

(A) Numbers of taxa per subject using four different taxon definitions. Taxon definitions of 99%, 97%, and 95% OTUs were assigned using the DOTUR package, and NCBI definitions were based on taxonomic classifications using the GreenGenes 16S rRNA database, as described in the text. The data represent 13 subjects without BV and 28 subjects with BV. The shaded boxes encompass the 25th to 75th percentiles of the data, the boldface lines indicate means, the lightface lines indicate median values, and the whiskers span the 5th to 95th percentiles. The asterisks indicate significant (P < 0.001) differences in mean values for subjects with BV versus subjects without BV, as determined by t tests. (B) Total number of taxa found across all subjects that are unique (e.g., found only in subjects with BV) and the number that are shared for each clinical state.

FIG. 2.

Proportions of subjects for which each of the 97 OTUs classified at a 97% sequence similarity were encountered. The list indicates genus-level identification of each OTU based on the NCBI taxonomy. Note that sequences designated Bifidobacterium by NCBI correspond to sequences classified as Gardnerella by the RDP. The accession number for each OTU is listed in Table S1 in the supplemental material.

FIG. 3.

Taxonomic affiliations and relative proportions of sequences summarized at the genus (A) and phylum (B) levels. The taxa in panel A represent the closest matches from the NCBI database, with the numbers in parentheses representing the mean percent match for each group of sequences for subjects with BV (829 sequences) and subjects without BV (140 sequences). Note that some sequences designated Bifidobacterium by NCBI correspond to sequences consistent with G. vaginalis; there is significant sequence heterogeneity within the G. vaginalis species complex. The y axes in both panels show the proportion of sequences from subjects with and without BV calculated separately. The numbers above the bars represent the number of OTUs within each group defined at a minimum sequence similarity of 97%; only numbers >1 are shown. Note the break in the axis for Lactobacillus-like sequences from subjects without BV, where lactobacilli comprise 86% of the sequences.

FIG. 4.

Phylogenetic relationships among representative taxa belonging to the phylum Bacteroidetes. The tree was reconstructed using the maximum-likelihood method in ARB with a 75% similarity column filter. Representative taxa were defined using a 97% OTU definition, as described in the text. Additional taxa represent a nonredundant list of nearest neighbors current as of April 2008. Taxa in red represent OTUs unique to our sequencing efforts, OTUs in green were common to our study and at least one other, and OTUs shown in blue were detected in other studies but not encountered in our sequencing. The numbers after each OTU indicate the number of sequences with unknown BV status, the number of sequences from subjects without BV, the number of sequences from subjects with BV, and the total number of sequences within each OTU. OTUs shown in boldface were found only in subjects with BV. Triangles at nodes represent bootstrap values from 100 resamplings of maximum-likelihood (ML) trees built in PHYLIP and neighbor-joining (NJ) trees, as shown in the legend and described in the text. str., strain.

FIG. 5.

Phylogenetic relationships among representative taxa belonging to the phylum Actinobacteria. The tree reconstruction methods, color scheme, and bootstrap representations are as described in the legend to Fig. 4.

FIG. 6.

Phylogenetic relationships among representative taxa belonging to the phylum Firmicutes. The tree reconstruction methods, color scheme, and bootstrap representations are as described in the legend to Fig. 4.