The microbial ecosystem resident on the human vaginal epithelium remains poorly understood in terms of census, microbe-microbe interactions, and microbial interactions with the host. In particular, details about the relationship between newly described fastidious BV-associated bacteria and the manifestations of BV are just starting to emerge. In this study, we compared vaginal bacterial community structures between subjects with and without clinically defined BV and synthesized publicly available data to better understand the diversity of healthy and diseased vaginal bacterial communities.
For patients afflicted with BV, there is a profound shift in the types of bacteria present in the vagina and their absolute and relative abundances. Data presented here clearly demonstrate that BV is associated with a dramatic increase in the taxonomic richness and diversity of the vaginal bacterial community. Our analyses highlight three main points.
First, the true extent of vaginal bacterial diversity may be severely underestimated by attributing taxonomic identities to sequences simply according to their best match to currently known taxa. Although the use of the NCBI taxonomic classification scheme gives a familiar nomenclature for sequences obtained from any environment, in the case of the vaginal ecosystem, it also clearly hides a significant amount of diversity. Multiple OTUs within sequence groups matching a single taxon as defined by NCBI and low similarity scores to these taxa demonstrate that we are just beginning to reveal the true extent of the diversity of the vaginal flora.
Vaginal bacteria are generally underrepresented in the NCBI database, but for some groups in particular, the true extent of diversity is hidden by their poor representation in GenBank. For example, sequences classified as Prevotella
actually contained 21 different OTUs using a 97% definition. Similarly, within sequences classified as Lactobacillus
, there were 15 different OTUs. The diversity contained within the Lactobacillus
species complex of the vagina has been noted previously by other researchers (15
), but the functional importance of this level of diversity remains unknown. Ecological theory suggests that each phylotype occupies its own niche in its environment and thus must be somehow unique, but the details of how BV-associated microbes partition niche space in the vaginal ecosystem and the correspondence between 16S rRNA-defined phylotypes and various functional capabilities remain relatively unexplored.
It is likely that additional sampling will reveal even greater richness and diversity in subjects with BV. When a taxon was defined using a 97% OTU definition, we observed a total of 61 taxa across all subjects with BV, but richness estimates of the true number of taxa present ranged from 63 to 111 (95% confidence interval), with an average estimate of 75 taxa (Table ). For subjects without BV, our sampling effort was more complete; we encountered 25 taxa with a 97% OTU definition, and estimates of the total ranged from 20 to 30 (95% confidence interval), with an average of 21 (Table ).
The loss of information caused by lumping multiple OTUs into a single taxon and the subsequent underestimation of the true richness and diversity of the vaginal ecosystem highlight the second important result of our analyses. The application of an OTU-based analysis to the full data set of our sequences and those from previous surveys of the vaginal ecosystem revealed many novel phylotypes associated with BV. We found 38 OTUs not previously encountered in the vaginal ecosystem, including 31 OTUs that were found only in subjects with BV. These OTUs were found across all of the major groups of BV-related taxa (Fig. , , and ), providing additional evidence that many more taxa are associated with BV than was previously thought.
Third, although the vaginal bacterial communities found in subjects with and without BV are distinctly different, the structures of these vaginal bacterial communities have high intersubject variability within each clinical group. For both groups, the total number of taxa encountered across all subjects was at least four times greater than the per-patient mean (Fig. ), and many taxa were relatively uncommon (Fig. ). This variability in community membership and structure has important implications for understanding the etiology of BV and for developing diagnostic tools. Why is the taxonomic composition of BV-associated bacteria so different for each patient? High interpatient variability has been observed previously for microbial communities in the vagina (3
) and other areas of the human body (6
). Stochastic differences in colonization could generate this pattern, but so could other factors, such as differences in host immune response, expression of ligands for bacterial attachment to epithelial cells, the chemical and physical environments of the host, or intra- and interspecific microbial competition.
Although no single bacterium can be identified as uniquely associated with BV, aggregating our data at higher taxonomic levels made it clear that several taxonomic groups do have a strong association. Common practice has generally been to describe sampled communities using the finest level of taxonomic discrimination possible, but aggregating data into higher taxonomic groups could be more informative and may avoid currently unresolved problems in classifying many BV-related bacteria at the genus or species level. For example, when treated at the genus level, taxa such as Eggerthella, Mobiluncus, and Slackia were relatively rare in subjects with BV, but when considered as a phylum, Actinobacteria accounted for 26% of all sequences from subjects with BV versus 6% from those without. Actinobacteria and Bacteroidetes in particular were much more common in subjects with BV than in those without BV (4.5 times and 6.7 times, respectively). The ability to identify a set of signature taxa common to BV could have important clinical and diagnostic implications.
Aggregating taxonomic data may also reduce problems associated with the current poorly resolved systematics of many BV-associated bacteria. For example, under the NCBI naming scheme, sequences classified as Gardnerella were relatively rare in subjects with BV, which contradicts generally held notions of a strong association between BV and Gardnerella. However, the 110 sequences classified as Bifidobacterium by NCBI were all classified as Gardnerella by the RDP (Table ). The discrepancies among different taxonomic classification schemes highlight the current state of systematics for many BV-associated taxa and emphasize the importance of OTU-based analyses to capture the true diversity within a pool of sequences.
The polymicrobial nature of BV clearly raises the possibility of interactions among vaginal microbes, including syntrophies, which might contribute to the pathogenesis of BV (see reference 20
for a review). Ammonia transfer from Prevotella bivia
to G. vaginalis
has been demonstrated (19
), but much remains to be learned about interspecific interactions of vaginal microbes and their contribution to the etiology of BV. Subjects with BV have volatile amines in vaginal fluid (the basis for the “whiff test” used in the clinical diagnosis of BV) with elevated levels of trimethylamine, putrescine, cadaverine, and tyramine, but the microbes responsible for generating these metabolic products are not clearly identified. An interesting recent paper has shown the importance of direct competition between lactobacilli and G. vaginalis
, apparently independently of pH and H2
). Clearly, much work remains to be done to tease apart the microbial interactions, metabolic processes, and host factors that lead to BV; it is our hope that increased knowledge about the composition and structure of BV-associated bacterial communities will facilitate these studies.
The data presented here reflect different sample sizes for subjects with and without BV. However, initial screening of samples using restriction fragment length polymorphisms indicated that the extreme differences in taxonomic richness between the two patient populations justified the different sample sizes in order to achieve similar sampling depths. It is axiomatic that representative sampling of any community requires that the sampling effort be tied to the structure of the community; depauperate communities naturally require less sampling effort than taxon-rich and diverse communities to achieve equivalent sampling saturation. In the case of subjects without BV, fewer sequences were obtained due to many fewer restriction fragment length polymorphism patterns because of the dominance by Lactobacillus. Richness estimators (Table ) indicated subjects without BV were adequately sampled, and additional diversity will be discovered in subjects with BV. Future work utilizing techniques such as pyrosequencing could provide greatly improved coverage of BV-associated genetic diversity but with more limited phylogenetic resolution due to limited sequence lengths.
Although the use of cloning to identify bacteria from complex samples has been criticized for potentially biased amplification (28
), the clone library approach has also compared favorably to PCR-independent methods, such as fluorescence in situ hybridization, in its estimation of the relative proportions of various taxonomic groups (16
). The partial 16S rRNA gene sequences obtained in our study (~800 to 1,000 bp) may contribute to some of the discrepancies in taxonomic assignments between the NCBI and RDP classifications. Full-length sequences (~1.5 kb) would likely improve phylogenetic resolution for some taxa, but in this study, we opted for a relatively high-throughput approach to compare a large number of sequences from subjects with and without BV.
Comparison to previous results.
In our phylogenetic analysis, it is apparent that novel OTUs found in our study generally belong to different phylogenetic clades than OTUs not encountered in our study. This is perhaps most striking for the Bacteroidetes
(Fig. ), for which we found 13 OTUs that had not been discovered in previous studies of vaginal bacteria. Similarly, for the Actinobacteria
(Fig. ), we found only two OTUs representing five sequences that did not group with either Atopobium
, or Gardnerella
, while Hyman et al. found 15 OTUs outside of this main group. This pattern could reflect different study populations, the inherent variability of BV, PCR primer bias, or different sampling intensities across studies. Most (82/98) of the OTUs not found by us have two or fewer members represented, and all but six of these were found only by Hyman et al. (15
), suggesting that their intensive sequencing effort did find uncommon members of the community. However, it is also possible that these sequences could originate from low-level PCR contaminants, which are more likely to become evident with more intensive sampling of clone libraries (10
polymerase, used for PCR, is known to be contaminated with low levels of bacterial 16S rRNA genes. Demographic differences among patient populations have also been demonstrated (34
) and may explain these differences.
The structures of the vaginal bacterial communities differ dramatically between subjects with and without BV. BV is associated with increased taxonomic richness and diversity. At a species or genus level, the composition of the vaginal bacterial community has high interpatient variability, yet at higher taxonomic levels, several bacterial groups are strongly associated with BV, most notably Actinobacteria and Bacteroidetes. Our data describe a previously unrecognized extent of diversity in the vaginal ecosystem in general and of BV-associated bacteria in particular. The true extent of diversity within several key taxonomic groups is grossly underrepresented in the current NCBI database. The most prominent of these are Prevotella-like sequences, commonly found in subjects with BV, and Lactobacillus-like sequences, common in subjects without BV.
Using Web-based tools freely available to the research community, our analysis provides a comprehensive census of vaginal bacterial communities and their association with BV. It is our hope that the data presented here will stimulate the formulation of new hypotheses about the metabolic functions, syntrophic interactions, and niche partitioning of bacteria colonizing the vaginal ecosystem. Continuing investigations of BV will almost certainly reveal complex syntrophies, cell-to-cell signaling, and bacterial-host interactions that will shed light on how consortia of bacteria interact to form pathogenic communities in the human host.