APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2008, p. 4898–4909 Vol. 74, No. 150099-2240/08/$08.00�0 doi:10.1128/AEM.02884-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Diversity of Human Vaginal Bacterial Communities and Associationswith Clinically Defined Bacterial Vaginosis�†

Brian B. Oakley,1‡ Tina L. Fiedler,1 Jeanne M. Marrazzo,2 and David N. Fredricks1*Program in Infectious Diseases, D3-100, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle,

Washington 98109-1024,1 and Harborview Medical Center, 325 Ninth Avenue,Mailstop 359931, Seattle, Washington 981042

Received 20 December 2007/Accepted 12 May 2008

Bacterial vaginosis (BV) is a common syndrome associated with numerous adverse health outcomes inwomen. Despite its medical importance, the etiology and microbial ecology of BV remain poorly understood. Weused broad-range PCR to census the community structure of the healthy and BV-affected vaginal microbialecosystems and synthesized current publicly available bacterial 16S rRNA gene sequence data from thisenvironment. The community of vaginal bacteria detected in subjects with BV was much more taxon rich anddiverse than in subjects without BV. At a 97% sequence similarity cutoff, the number of operational taxonomicunits (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 hiddendiversity for many vaginal bacteria that are currently poorly represented in GenBank. Our sequencing effortsyielded many novel phylotypes (123 of our sequences represented 38 OTUs not previously found in the vaginalecosystem), including several novel BV-associated OTUs, such as those belonging to the Prevotella speciescomplex, which remain severely underrepresented in the current NCBI database. Community composition washighly variable among subjects at a fine taxonomic scale, but at the phylum level, Actinobacteria and Bacte-roidetes were strongly associated with BV. Our data describe a previously unrecognized extent of bacterialdiversity in the vaginal ecosystem. The human vagina hosts many bacteria that are only distantly related toknown species, and subjects with BV harbor particularly taxon-rich and diverse bacterial communities.

A growing body of evidence suggests that human health isfundamentally affected by the composition and function of thevast human microbial ecosystem. Microbial cells in and on ourbodies are approximately 10 times more numerous than ourown cells and contain, in aggregate, about 100 times moregenes, leading to the suggestion that humans and our microbialsymbionts be considered superorganisms (12). There is in-creasing evidence that microbial communities strongly influ-ence human health and quality of life. For example, in thegastrointestinal tract, the taxonomic composition of the micro-bial community may affect the propensity to develop obesity(30). In the vagina, bacterial vaginosis (BV) is marked bydramatic shifts in the types and relative proportions of a di-verse community of bacteria as the vaginal ecosystem changesfrom a healthy to a diseased state (7, 9).

BV is strongly associated with several adverse health out-comes, including preterm labor and delivery (13, 14), pelvicinflammatory disease (29), and the acquisition and transmis-sion of sexually transmitted diseases, such as human immuno-deficiency virus (23–25). BV is the most common cause ofvaginal discharge among women of reproductive age and re-

sults in millions of health care visits annually in the UnitedStates (26). Because BV is fundamentally a result of changes tothe vaginal microbial community, full understanding and suc-cessful treatment will require knowledge of how the healthycommunity is altered in its taxonomic composition, communitystructure, and, ultimately, function.

Historically, a search for single etiologic agents in BV andthe constraints of traditional cultivation techniques limited ap-preciation of the full extent of vaginal microbial diversity in thissyndrome. BV has long been recognized as a complex condi-tion, but until recently, lactobacilli found in the healthy vaginawere thought to be replaced by a relatively small suite of taxatypically including Gardnerella vaginalis, Prevotella spp., Por-phyromonas spp., Mobiluncus spp., and Mycoplasma hominis(7, 27). The advent of molecular tools, such as broad-rangebacterial 16S rRNA gene PCR, has enabled community assess-ments independent of cultivation-based techniques and hasgreatly expanded our knowledge of the phylogenetic range andtaxonomic diversity of bacteria found in the vagina. For exam-ple, intensive sequencing has revealed extensive diversity, notonly within the Lactobacillus species complex (15), but alsowithin other taxa most closely related to Atopobium, Clostrid-ium, Leptotrichia, Prevotella, Peptostreptococcus, and Pepto-niphilus, species not previously thought to be common in thehuman vagina (3, 9, 15, 33, 34).

Although there is agreement in the literature that no singleagent likely causes BV, there is no consensus about what con-stitutes a pathogenic bacterial community in this syndrome.Comprehensive cultivation-independent comparisons of thevaginal bacterial communities from subjects with and withoutclinically defined BV have been rare. Without such fundamen-

* Corresponding author. Mailing address: Program in InfectiousDiseases, D3-100, Fred Hutchinson Cancer Research Center, 1100Fairview Avenue N, Seattle, WA 98109-1024. Phone: (206) 667-6706.Fax: (206) 667-4411. E-mail: dfredric@fhcrc.org.

‡ Present address: Microbiology Research Group, Department ofBiological Sciences, University of Warwick, Coventry CV4 7AL,United Kingdom.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 16 May 2008.

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tal data, our understanding of the pathogenesis of BV is con-strained. In one study that compared broad-range bacterialcommunity composition among patients with objectively de-fined BV status, subjects with BV had nearly four times asmany taxa as subjects without BV, including uncultivated Clos-tridia-like bacteria, which were shown to be highly specific forBV (9).

In this paper, we extend this previous work to present asystematic phylogenetic analysis of vaginal bacterial commu-nity composition from subjects with defined BV status as de-termined by commonly accepted clinical criteria. We also syn-thesize current public 16S rRNA gene sequence data frombacteria found in the vaginal ecosystem (with inclusion of stud-ies where BV status was not assessed) and place these data ina phylogenetic context. Our goal was to provide a comprehen-sive picture of our current knowledge of the community struc-ture of the vaginal bacterial ecosystem.

MATERIALS AND METHODS

Sample collection and clinical assessment of BV status. Vaginal swabs werecollected from subjects attending either the Public Health-Seattle and KingCounty Sexually Transmitted Diseases Clinic or the Harborview Medical CenterWomen’s Research Clinic and immediately frozen for later analysis, as previ-ously described (9). BV status was assessed using Amsel clinical criteria (1) forall subjects and confirmed using Gram stain criteria (Nugent [18] scores) forsubjects attending the Women’s Research Clinic. In this paper, we analyze datacollected from 34 vaginal samples, including 21 collected from subjects withclinically defined BV and 13 samples collected from subjects without BV. Thisanalysis includes data on six vaginal samples not previously reported (9) andprovides a more exhaustive phylogenetic analysis based on the presence ofhigh-quality sequence data (detailed below) representative of the two patientpopulations.

Written informed consent was obtained from all participants in the study,

which was also approved by the Institutional Review Boards at the Fred Hutchin-son Cancer Research Center and the University of Washington.

Molecular methods. DNA was obtained from swabs using the UltraClean SoilDNA extraction kit (MoBio, Carlsbad, CA), chosen after testing guanidinium-based lysis protocols with and without bead beating. Out of the 969 sequencesgenerated, 3 were detected only in the protocol without bead beating and 7 weredetected only in the bead-beating protocol. Accordingly, we did not find thatbead beating substantially affected our results. For broad-range PCR, the prim-ers 338F (5�-ACTCCTRCGGGAGGCAGCAG-3�) and 1407R (5�-GACGGGCGGTGTGTRCA-3�) were used with a thermal-cycling protocol of denaturationat 95°C, followed by 21 to 25 cycles of 95°C for 30 s, 55°C for 30s, and 72°C for90s, with a final extension at 72°C for 7 min.

Successfully amplified products were cloned using the Topo blunt cloning kit(Invitrogen, Carlsbad, CA), and PCR-amplified inserts were sequenced usingvector primers and BigDye version 3 (Applied Biosystems, Foster City, CA).Single reads were generated for 736 sequences, with an average length of 811base pairs. Two reads were obtained for 233 sequences, resulting in about 1-kbsequence lengths.

Data analysis. Raw sequence data were edited, and contigs were assembled foreach clone using Sequencher (Gene Codes, Ann Arbor, MI). The 969 sequencesobtained from our sequencing efforts and all publicly available sequences fromvaginal bacteria (554 sequences, listed below) were then submitted to the Green-Genes 16S rRNA gene database (5) for alignment using the NAST algorithm (4)and taxonomic classification of each sequence. All sequences were checked forchimeric anomalies using the Mallard program (2).

To classify sequences based on self-similarities rather than matches to anexternal database, sequences were grouped into operational taxonomic units(OTUs) with cutoffs of 99%, 97%, and 95% sequence similarity using theDOTUR software package implemented with the furthest-neighbor option, inwhich all of the sequences within an OTU are at least x% similar to all of theother sequences within the OTU (22). The commonly accepted phylogeneticspecies definition of 97% 16S sequence similarity (32) was used to define a coredata set of representative sequences that were used for phylogenetic analyses.

Sequences were incorporated into ARB (17) for phylogenetic analysis and treeconstruction. Sequences from a nonredundant list of three nearest-neighborisolates for each sequence were also added to the database. Alignments weremanually corrected based on the secondary structure of the 16S rRNA and tree

FIG. 1. (A) Numbers of taxa per subject using four different taxon definitions. Taxon definitions of 99%, 97%, and 95% OTUs were assignedusing the DOTUR package, and NCBI definitions were based on taxonomic classifications using the GreenGenes 16S rRNA database, as describedin the text. The data represent 13 subjects without BV and 28 subjects with BV. The shaded boxes encompass the 25th to 75th percentiles of thedata, the boldface lines indicate means, the lightface lines indicate median values, and the whiskers span the 5th to 95th percentiles. The asterisksindicate significant (P � 0.001) differences in mean values for subjects with BV versus subjects without BV, as determined by t tests. (B) Totalnumber 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 clinicalstate.

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construction performed using the maximum-likelihood method in ARB (FastdnaML)with a 75% similarity filter constructed for each phylum. Bootstrap values(100 resamplings) were generated for maximum-likelihood trees created inPHYLIP (8) using the DNAML algorithm and neighbor-joining trees createdin ARB and superimposed on nodes supported by all three methods.

Publicly available sequences were collected from previous surveys of vaginalbacterial diversity, which included 439 sequences from Hyman et al. (15)(AY958774 to AY959212), 7 unpublished sequences from Zozaya-Hinchliffe etal. (EF120360 to EF120366), 25 sequences from Verhelst et al. (31) (AJ585206to AJ619714), 15 unpublished sequences from Zhang et al. (DQ666091 toDQ666105), and 68 sequences from Zhou at al (33) (AY267541 and -2,AY269020 to -34, AY271931 to -53, AY283264 to -75, AY335493 to -504, andDQ987868 to -9). Because subject BV status was not available for most of thesestudies, the corresponding sequences were excluded from comparisons of bac-terial community structure in subjects with and without BV but were included inphylogenetic analyses of vaginal bacterial diversity.

Nucleotide sequence accession numbers. Sequences from the 97 OTUs gen-erated in this study were deposited in GenBank (AY738660, AY738684,AY738687, AY738691, AY738694, AY738697, AY738701 to -5, EF428974, andEU188937 to EU189021) and are listed in Table S1 in the supplemental material.

RESULTS

The structures of the bacterial communities were dramati-cally different in samples from women with and without BV.Increased taxonomic richness and diversity of BV-associatedflora relative to normal vaginal flora was evident in a variety ofanalyses. The mean number of taxa detected per patient wassignificantly higher (P � 0.001) in subjects with BV than insubjects without BV for all phylotype definitions (Fig. 1A).Similarly, diversity was higher in subjects with BV; the Shan-non diversity index was 1.4 to 4.1 times greater for subjects withBV than for subjects without BV, depending on the taxonomicdefinition (Table 1).

High interpatient variability of vaginal bacterial communitystructure was evident in several analyses, particularly for sub-jects with BV. First, the total number of phylotypes foundacross all subjects in each clinical group was dramaticallyhigher than the per-patient mean (Fig. 1). Second, many taxawere relatively rare; for subjects with BV, 47 of the 97 OTUsidentified were found in fewer than 25% of subjects, while onlya single taxon, most closely related to Bifidobacterium/Gard-nerella, was found in all subjects with BV (Fig. 2).

There were dramatic differences in the relative proportionsof major groups of bacteria between subjects with and withoutBV. Relative to subjects without BV, those with BV had muchhigher proportions of Actinobacteria (4.5 times) and Bacte-roidetes (6.7 times) and a much lower proportion of Firmicutes(0.45 times) (Fig. 3B). Several phylogenetic groups were foundonly in samples from subjects with BV; Fusobacteria, candidatedivision TM7, and Betaproteobacteria together accounted for9% of clones from subjects with BV but were not detected inclone libraries from subjects without BV (Fig. 3B).

TABLE 1. Richness and diversity values and best matches to taxondesignations in NCBI

Taxondefinition

Total no. of taxa Shannon diversity index

BV�

(n � 829)BV�

(n � 140)BV�

(n � 829)BV�

(n � 140)Ratio

(BV�/BV�)

99% OTU 85 (101) 39 (67) 1.54 1.12 1.497% OTU 61 (75) 25 (21) 1.37 0.93 1.595% OTU 49 (48) 17 (16) 1.33 0.81 1.6NCBI 36 9 1.20 0.29 4.1

a Richness and diversity values for taxon definitions of sequence similaritycutoffs of 99%, 97%, and 95% and best matches to taxon designations in NCBIfor all 16S rRNA gene sequences generated from subjects with (BV�) andwithout (BV�) BV. Richness values in parentheses represent average Chao1richness estimates calculated as described in the text.

FIG. 2. Proportions of subjects for which each of the 97 OTUs classified at a 97% sequence similarity were encountered. The list indicatesgenus-level identification of each OTU based on the NCBI taxonomy. Note that sequences designated Bifidobacterium by NCBI correspond tosequences classified as Gardnerella by the RDP. The accession number for each OTU is listed in Table S1 in the supplemental material.

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Subjects without BV had bacterial communities dominatedby Lactobacillus species, accounting for 86% of all sequencesfrom subjects without BV (Fig. 3). In contrast, subjects withBV did not possess a single dominant taxon but rather har-bored a diverse array of vaginal bacteria, many present at lowrelative abundance. Of phylotypes unique to subjects with BV,nine were singletons (encountered only once), and the fourmost common (sequences most closely related to Olsenella,Megasphaera, Streptobacillus, and Shuttleworthia) accounted for4%, 7%, 8%, and 9%, respectively, of all sequences retrievedfrom subjects with BV (Fig. 3).

For several taxonomic groups in particular, the true extent ofdiversity is not reflected in the current GenBank database. Forexample, even though about 20% of sequences from subjectswith BV were classified as Prevotella according to the bestmatch in GenBank, this group of sequences actually contained21 different taxa based on a species definition of 97% sequencesimilarity (Fig. 3A). Similarly, within the group of sequencesmost closely related to Lactobacillus in GenBank, there wereactually 15 different phylotypes (Fig. 3A). Estimates of taxo-nomic richness and diversity based on best matches to named

taxa may severely underestimate the true extent of bacterialdiversity in the vagina.

Taxonomic designations of sequences using two differentclassification schemes, NCBI and the Ribosomal DatabaseProject (RDP) (http://rdp.cme.msu.edu), were largely congru-ent (Tables 2, 3, and 4) but also demonstrated important dis-crepancies. For example, sequences classified as Bifidobacte-rium by NCBI belonged to Gardnerella in the RDP scheme(Tables 2 and 4). Similarly, sequences classified as Clostridiumby NCBI were called Acetivibrio by RDP (Table 2). In general,NCBI designations split sequences into more groups, while theRDP scheme lumped sequences together: the 60 sequencesclassified as Acetivibrio by RDP belonged to four differenttaxonomic groups in the NCBI scheme, and 47 sequences clas-sified as Coriobacteriaceae by RDP represented five taxonomicgroups according to NCBI (Table 2).

In the entire data set of 1,523 publicly available sequences,we found a total of 195 OTUs using the bacterial speciesdefinition of 97% 16S rRNA gene sequence similarity, includ-ing 38 novel OTUs representing 123 sequences unique to thedata presented here. Of these, 31 OTUs (115 sequences) were

FIG. 3. Taxonomic affiliations and relative proportions of sequences summarized at the genus (A) and phylum (B) levels. The taxa in panel Arepresent the closest matches from the NCBI database, with the numbers in parentheses representing the mean percent match for each group ofsequences for subjects with BV (829 sequences) and subjects without BV (140 sequences). Note that some sequences designated Bifidobacteriumby 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 barsrepresent the number of OTUs within each group defined at a minimum sequence similarity of 97%; only numbers �1 are shown. Note the breakin the axis for Lactobacillus-like sequences from subjects without BV, where lactobacilli comprise 86% of the sequences.

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found only in subjects with BV. We encountered 59 OTUs inour sequencing set that were also reported elsewhere in thedata set of publicly available sequences, while 98 OTUs weredetected in other studies (all but 5 of these by Hyman et al.[15]) but not in our patient populations.

Six sequences deposited in GenBank (AY959158, AY958981,AY959016, AY959073, AY958933, and AY959044) were foundto be consistent with chimeric data (P � 0.001) and so wereexcluded from our analyses.

Phylogenetic analyses of BV-associated bacteria. The truediversity and novelty of BV-associated bacteria were apparentwhen sequences generated in this study were compared to pub-licly available sequences from previous surveys of vaginal bacte-rial diversity and their nearest cultivated and uncultivatedmatches.

Within the phylum Bacteroidetes, sequences in our data setwere most closely related to one of three genera: Prevotella,Bacteroides, and Porphyromonas (Fig. 4). Most (196/222) be-longed to the Prevotella group but were only distantly related totheir nearest relative and represented many novel sequencetypes not found in previous studies of vaginal bacteria. In thePrevotella species complex, we found 13 novel OTUs (Fig. 4).Two novel OTUs representing 24 sequences were most closelyrelated to Porphyromonas uenonis (Fig. 4) and were found onlyin subjects with BV.

Within the Actinobacteria, most sequences (276/302) be-longed to clades affiliated with Atopobium, Eggerthella, orGardnerella, and all of the shared OTUs across studies be-longed to one of these three groups (Fig. 5). Of these 306sequences, only eight representing two OTUs in the Gard-nerella group were from subjects confirmed not to have BV(Fig. 5). Our sequencing also revealed several novel OTUs inthis phylum: one related to Atopobium, two most similar toMobiluncus, and two Gardnerella-like OTUs (Fig. 5). Of the 17OTUs not found in our sequences, 13 were singletons repre-senting a wider phylogenetic range outside of the three mainBV-associated genera of the Actinobacteria, and all were frompatients with unknown BV status (Fig. 5).

Within the Firmicutes, the majority of sequences (313/387) be-longed to one of four main groups related to known taxa: Shuttle-worthia, Megasphaera/Veillonella, Dialister, and Clostridium (Fig.6). Of these, only a single sequence was from a subject confirmednot to have BV. We identified 11 novel OTUs representing 35sequences within the Firmicutes; of these, only three sequencesfrom two OTUs related to Clostridium perfringens and Ureaplasmawere from subjects without BV (Fig. 6).

Previously identified (9) BV-associated bacteria appear tobe truly novel taxa that are commonly encountered in subjectswith BV. Ninety-three sequences most closely related toShuttleworthia encompass those previously designated (9) asBV-associated bacterium 1 (BVAB-1) (Fig. 6). Three addi-tional OTUs closely related to BVAB-1 were also identified(Fig. 6). Sequence types designated BVAB-2 and BVAB-3 (9)were most closely related to Acetovibrio cellulolyticus and sev-eral Clostridium strains and together accounted for 47 se-quences (Fig. 6). Three additional OTUs (two of them novel)closely related to BVAB-2 represented an additional 27 se-quences (Fig. 6). All three of the BVAB phylotypes had lowsimilarities to their nearest known cultivated neighbors and

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together accounted for 14% of all sequences from subjectswith BV.

DISCUSSION

The microbial ecosystem resident on the human vaginalepithelium remains poorly understood in terms of census, mi-crobe-microbe interactions, and microbial interactions with thehost. In particular, details about the relationship betweennewly described fastidious BV-associated bacteria and themanifestations of BV are just starting to emerge. In this study,we compared vaginal bacterial community structures betweensubjects with and without clinically defined BV and synthesizedpublicly available data to better understand the diversity ofhealthy and diseased vaginal bacterial communities.

For patients afflicted with BV, there is a profound shift inthe types of bacteria present in the vagina and their absoluteand relative abundances. Data presented here clearly demon-strate that BV is associated with a dramatic increase in thetaxonomic richness and diversity of the vaginal bacterial com-munity. Our analyses highlight three main points.

First, the true extent of vaginal bacterial diversity may be se-verely underestimated by attributing taxonomic identities to se-quences simply according to their best match to currently knowntaxa. Although the use of the NCBI taxonomic classificationscheme gives a familiar nomenclature for sequences obtainedfrom any environment, in the case of the vaginal ecosystem, it alsoclearly hides a significant amount of diversity. Multiple OTUswithin sequence groups matching a single taxon as defined byNCBI and low similarity scores to these taxa demonstrate that weare just beginning to reveal the true extent of the diversity of thevaginal flora.

Vaginal bacteria are generally underrepresented in theNCBI database, but for some groups in particular, the trueextent of diversity is hidden by their poor representation inGenBank. For example, sequences classified as Prevotella ac-tually contained 21 different OTUs using a 97% definition.Similarly, within sequences classified as Lactobacillus, therewere 15 different OTUs. The diversity contained within the

Lactobacillus species complex of the vagina has been notedpreviously by other researchers (15, 34), but the functionalimportance of this level of diversity remains unknown. Ecolog-ical theory suggests that each phylotype occupies its own nichein its environment and thus must be somehow unique, but thedetails of how BV-associated microbes partition niche space inthe vaginal ecosystem and the correspondence between 16SrRNA-defined phylotypes and various functional capabilitiesremain relatively unexplored.

It is likely that additional sampling will reveal even greaterrichness and diversity in subjects with BV. When a taxon wasdefined using a 97% OTU definition, we observed a total of 61taxa across all subjects with BV, but richness estimates of thetrue number of taxa present ranged from 63 to 111 (95%confidence interval), with an average estimate of 75 taxa (Ta-ble 1). For subjects without BV, our sampling effort was morecomplete; we encountered 25 taxa with a 97% OTU definition,and estimates of the total ranged from 20 to 30 (95% confi-dence interval), with an average of 21 (Table 1).

The loss of information caused by lumping multiple OTUsinto a single taxon and the subsequent underestimation of thetrue richness and diversity of the vaginal ecosystem highlightthe second important result of our analyses. The application ofan OTU-based analysis to the full data set of our sequencesand those from previous surveys of the vaginal ecosystem re-vealed many novel phylotypes associated with BV. We found38 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 ofBV-related taxa (Fig. 4, 5, and 6), providing additional evi-dence that many more taxa are associated with BV than waspreviously thought.

Third, although the vaginal bacterial communities found insubjects with and without BV are distinctly different, the struc-tures of these vaginal bacterial communities have high inter-subject variability within each clinical group. For both groups,the total number of taxa encountered across all subjects was atleast four times greater than the per-patient mean (Fig. 1), and

TABLE 4. Comparison of NCBI and RDP taxonomic designations for sequences obtained from subjects without BV (n � 140)

NCBI designation

No. of sequences for RDP designation:

TotalActinobacteria Bacteroidetes Firmicutes

Gardnerella Prevotella Bacteroidales Anaerococcus Clostridiaceae Dialister Lactobacillus Staphylococcus Ureaplasma

ActinobacteriaBifidobacterium 8 8

BacteroidetesBacteroides 1 1Prevotella 4 4

FirmicutesAnaerococcus 1 1Clostridium 1 1Dialister 1 1Lactobacillus 120 120Staphylococcus 2 2Ureaplasma 2 2

Total 8 4 1 1 1 1 120 2 2 140

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many taxa were relatively uncommon (Fig. 2). This variabilityin community membership and structure has important impli-cations for understanding the etiology of BV and for develop-ing diagnostic tools. Why is the taxonomic composition ofBV-associated bacteria so different for each patient? Highinterpatient variability has been observed previously for micro-bial communities in the vagina (3) and other areas of thehuman body (6, 11). Stochastic differences in colonizationcould generate this pattern, but so could other factors, such asdifferences in host immune response, expression of ligands forbacterial attachment to epithelial cells, the chemical and phys-

ical environments of the host, or intra- and interspecific mi-crobial competition.

Although no single bacterium can be identified as uniquelyassociated with BV, aggregating our data at higher taxonomiclevels made it clear that several taxonomic groups do have astrong association. Common practice has generally been todescribe sampled communities using the finest level of taxo-nomic discrimination possible, but aggregating data into highertaxonomic groups could be more informative and may avoidcurrently unresolved problems in classifying many BV-relatedbacteria at the genus or species level. For example, when

FIG. 4. Phylogenetic relationships among representative taxa belonging to the phylum Bacteroidetes. The tree was reconstructed using themaximum-likelihood method in ARB with a 75% similarity column filter. Representative taxa were defined using a 97% OTU definition, asdescribed in the text. Additional taxa represent a nonredundant list of nearest neighbors current as of April 2008. Taxa in red represent OTUsunique 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 otherstudies but not encountered in our sequencing. The numbers after each OTU indicate the number of sequences with unknown BV status, thenumber of sequences from subjects without BV, the number of sequences from subjects with BV, and the total number of sequences within eachOTU. OTUs shown in boldface were found only in subjects with BV. Triangles at nodes represent bootstrap values from 100 resamplings ofmaximum-likelihood (ML) trees built in PHYLIP and neighbor-joining (NJ) trees, as shown in the legend and described in the text. str., strain.

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treated at the genus level, taxa such as Eggerthella, Mobiluncus,and Slackia were relatively rare in subjects with BV, but whenconsidered as a phylum, Actinobacteria accounted for 26% ofall sequences from subjects with BV versus 6% from thosewithout. Actinobacteria and Bacteroidetes in particular were

much more common in subjects with BV than in those withoutBV (4.5 times and 6.7 times, respectively). The ability to iden-tify a set of signature taxa common to BV could have impor-tant clinical and diagnostic implications.

Aggregating taxonomic data may also reduce problems as-

FIG. 5. Phylogenetic relationships among representative taxa belonging to the phylum Actinobacteria. The tree reconstruction methods, colorscheme, and bootstrap representations are as described in the legend to Fig. 4.

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FIG. 6. Phylogenetic relationships among representative taxa belonging to the phylum Firmicutes. The tree reconstruction methods, colorscheme, and bootstrap representations are as described in the legend to Fig. 4.

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sociated with the current poorly resolved systematics of manyBV-associated bacteria. For example, under the NCBI namingscheme, sequences classified as Gardnerella were relatively rarein subjects with BV, which contradicts generally held notions ofa strong association between BV and Gardnerella. However,the 110 sequences classified as Bifidobacterium by NCBI wereall classified as Gardnerella by the RDP (Table 2). The discrep-ancies among different taxonomic classification schemes high-light the current state of systematics for many BV-associatedtaxa and emphasize the importance of OTU-based analyses tocapture the true diversity within a pool of sequences.

The polymicrobial nature of BV clearly raises the possibilityof interactions among vaginal microbes, including syntrophies,which might contribute to the pathogenesis of BV (see refer-ence 20 for a review). Ammonia transfer from Prevotella biviato G. vaginalis has been demonstrated (19), but much remainsto be learned about interspecific interactions of vaginal mi-crobes and their contribution to the etiology of BV. Subjectswith BV have volatile amines in vaginal fluid (the basis for the“whiff test” used in the clinical diagnosis of BV) with elevatedlevels of trimethylamine, putrescine, cadaverine, and tyramine,but the microbes responsible for generating these metabolicproducts are not clearly identified. An interesting recent paperhas shown the importance of direct competition between lac-tobacilli and G. vaginalis, apparently independently of pH andH2O2 production (21). Clearly, much work remains to be doneto tease apart the microbial interactions, metabolic processes,and host factors that lead to BV; it is our hope that increasedknowledge about the composition and structure of BV-associ-ated bacterial communities will facilitate these studies.

Limitations. The data presented here reflect different sam-ple sizes for subjects with and without BV. However, initialscreening of samples using restriction fragment length poly-morphisms indicated that the extreme differences in taxonomicrichness between the two patient populations justified the dif-ferent sample sizes in order to achieve similar sampling depths.It is axiomatic that representative sampling of any communityrequires that the sampling effort be tied to the structure of thecommunity; depauperate communities naturally require lesssampling effort than taxon-rich and diverse communities toachieve equivalent sampling saturation. In the case of subjectswithout BV, fewer sequences were obtained due to many fewerrestriction fragment length polymorphism patterns because ofthe dominance by Lactobacillus. Richness estimators (Table 1)indicated subjects without BV were adequately sampled, andadditional diversity will be discovered in subjects with BV.Future work utilizing techniques such as pyrosequencing couldprovide greatly improved coverage of BV-associated geneticdiversity but with more limited phylogenetic resolution due tolimited sequence lengths.

Although the use of cloning to identify bacteria from com-plex samples has been criticized for potentially biased ampli-fication (28), the clone library approach has also comparedfavorably to PCR-independent methods, such as fluorescencein situ hybridization, in its estimation of the relative propor-tions of various taxonomic groups (16). The partial 16S rRNAgene sequences obtained in our study (�800 to 1,000 bp) maycontribute to some of the discrepancies in taxonomic assign-ments between the NCBI and RDP classifications. Full-lengthsequences (�1.5 kb) would likely improve phylogenetic reso-

lution for some taxa, but in this study, we opted for a relativelyhigh-throughput approach to compare a large number of se-quences from subjects with and without BV.

Comparison to previous results. In our phylogenetic analy-sis, it is apparent that novel OTUs found in our study generallybelong to different phylogenetic clades than OTUs not encoun-tered in our study. This is perhaps most striking for the Bac-teroidetes (Fig. 4), for which we found 13 OTUs that had notbeen discovered in previous studies of vaginal bacteria. Simi-larly, for the Actinobacteria (Fig. 5), we found only two OTUsrepresenting five sequences that did not group with eitherAtopobium, Eggerthella, or Gardnerella/Bifidobacterium, whileHyman et al. found 15 OTUs outside of this main group. Thispattern could reflect different study populations, the inherentvariability of BV, PCR primer bias, or different sampling in-tensities across studies. Most (82/98) of the OTUs not found byus have two or fewer members represented, and all but six ofthese were found only by Hyman et al. (15), suggesting thattheir intensive sequencing effort did find uncommon membersof the community. However, it is also possible that these se-quences could originate from low-level PCR contaminants,which are more likely to become evident with more intensivesampling of clone libraries (10). Taq polymerase, used forPCR, is known to be contaminated with low levels of bacterial16S rRNA genes. Demographic differences among patientpopulations have also been demonstrated (34) and may explainthese differences.

Conclusions. The structures of the vaginal bacterial commu-nities differ dramatically between subjects with and withoutBV. BV is associated with increased taxonomic richness anddiversity. At a species or genus level, the composition of thevaginal bacterial community has high interpatient variability,yet at higher taxonomic levels, several bacterial groups arestrongly associated with BV, most notably Actinobacteria andBacteroidetes. Our data describe a previously unrecognized ex-tent of diversity in the vaginal ecosystem in general and ofBV-associated bacteria in particular. The true extent of diver-sity within several key taxonomic groups is grossly underrep-resented in the current NCBI database. The most prominent ofthese are Prevotella-like sequences, commonly found in sub-jects with BV, and Lactobacillus-like sequences, common insubjects without BV.

Using Web-based tools freely available to the research com-munity, our analysis provides a comprehensive census of vag-inal bacterial communities and their association with BV. It isour hope that the data presented here will stimulate the for-mulation of new hypotheses about the metabolic functions,syntrophic interactions, and niche partitioning of bacteria col-onizing the vaginal ecosystem. Continuing investigations of BVwill almost certainly reveal complex syntrophies, cell-to-cellsignaling, and bacterial-host interactions that will shed light onhow consortia of bacteria interact to form pathogenic commu-nities in the human host.

ACKNOWLEDGMENTS

This work was supported by the National Institute of Allergy andInfectious Diseases with grants to D.N.F. (RO1 AI061628) and toJ.M.M. (AI052228).

We thank Andrew Millard for assistance with Perl scripting.

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