Tina L. Fiedler

Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria

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.

Targeted PCR for Detection of Vaginal Bacteria Associated with Bacterial Vaginosis

ABSTRACT Several novel bacterial species have been detected in subjects with bacterial vaginosis (BV) by using broad-range PCR assays, but this approach is insensitive for detecting minority species. We developed a series of taxon-directed 16S rRNA gene PCR assays for more sensitive detection of key vaginal bacteria. We sought to determine the prevalence of each species in the vagina, its association with BV, and the utility of PCR for the microbiological diagnosis of BV. Targeted PCR assays were developed for 17 vaginal bacterial species and applied to 264 vaginal-fluid samples from 81 subjects with and 183 subjects without BV. The results were compared to those of two widely accepted methods for diagnosing BV, the use of clinical findings (Amsel criteria) and the interpretation of vaginal-fluid Gram stains (Nugent criteria). Leptotrichia/Sneathia, Atopobium vaginae, an Eggerthella-like bacterium, Megasphaera species, and three novel bacteria in the order Clostridiales are among the bacterial species significantly associated with BV. PCR detection of either a Megasphaera species or one of the Clostridiales bacteria yielded a sensitivity of 99% and a specificity of 89% for diagnosis of BV compared to the Amsel clinical criteria and a sensitivity of 95.9% and a specificity of 93.7% compared to the Nugent criteria (Gram stain). PCR detection of one or more fastidious bacterial species is a more reliable indicator of BV than detection of bacteria, such as Gardnerella vaginalis, previously linked to BV, highlighting the potential of PCR for the diagnosis of BV.

Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis

Background Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV). Methodology/Principal Findings Twenty-two women assessed for BV using Amsels criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p<0.001). Women with BV had a variety of fastidious bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated bacteria after treatment followed by subsequent increases at relapse. Conclusions/Significance The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of fastidious bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in bacterial concentrations, but these bacteria later reemerge, suggesting that antibiotic resistance in these bacteria is not an important factor mediating BV recurrence.

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.

Randomized, Placebo-Controlled Phase 2 Trial of a Lactobacillus crispatus Probiotic Given Intravaginally for Prevention of Recurrent Urinary Tract Infection

BACKGROUND Urinary tract infections (UTIs) are common among women and frequently recur. Depletion of vaginal lactobacilli is associated with UTI risk, which suggests that repletion may be beneficial. We conducted a double-blind placebo-controlled trial of a Lactobacillus crispatus intravaginal suppository probiotic (Lactin-V; Osel) for prevention of recurrent UTI in premenopausal women. METHODS One hundred young women with a history of recurrent UTI received antimicrobials for acute UTI and then were randomized to receive either Lactin-V or placebo daily for 5 d, then once weekly for 10 weeks. Participants were followed up at 1 week and 10 weeks after intervention and for UTIs; urine samples for culture and vaginal swabs for real-time quantitative 16S ribosomal RNA gene polymerase chain reaction for L. crispatus were collected. RESULTS Recurrent UTI occurred in 7/48 15% of women receiving Lactin-V compared with 13/48 27% of women receiving placebo (relative risk [RR], .5; 95% confidence interval, .2-1.2). High-level vaginal colonization with L. crispatus (≥10(6) 16S RNA gene copies per swab) throughout follow-up was associated with a significant reduction in recurrent UTI only for Lactin-V (RR for Lactin-V, .07; RR for placebo, 1.1; P < .01). CONCLUSIONS Lactin-V after treatment for cystitis is associated with a reduction in recurrent UTI. Larger efficacy trials of this novel preventive method for recurrent UTI are warranted. CLINICAL TRIALS REGISTRATION. NCT00305227.

Changes in Vaginal Bacterial Concentrations with Intravaginal Metronidazole Therapy for Bacterial Vaginosis as Assessed by Quantitative PCR

Several fastidious bacteria have been associated with bacterial vaginosis (BV) using broad-range bacterial PCR methods such as consensus sequence 16S rRNA gene PCR, but their role in BV remains poorly defined. We describe changes in vaginal bacterial concentrations following metronidazole therapy for BV. Vaginal swabs were collected from women with BV diagnosed using Amsel clinical criteria, and vaginal fluid was assessed by Gram stain to generate Nugent scores. Follow-up swabs were collected 1 month after a 5-day course of vaginal 0.75% metronidazole gel and analyzed for 24 subjects with cured BV and 24 subjects with persistent BV. Changes in bacterial concentrations were measured using eight bacterium-specific 16S rRNA gene quantitative PCR assays. DNA from several fastidious BV-associated bacteria (BVAB) were present at high concentrations in the vagina prior to treatment. Successful antibiotic therapy resulted in 3- to 4-log reductions in median bacterial loads of BVAB1 (P = 0.02), BVAB2 (P = 0.0004), BVAB3 (P = 0.03), a Megasphaera-like bacterium (P < 0.0001), Atopobium species (P < 0.0001), Leptotrichia/Sneathia species (P = 0.0002), and Gardnerella vaginalis (P < 0.0001). Median posttreatment bacterial levels did not change significantly in subjects with persistent BV except for a decline in levels of BVAB3. The presence or absence of BV is reflected by vaginal concentrations of BV-associated bacteria such as BVAB1, BVAB2, Leptotrichia/Sneathia species, Atopobium species, Gardnerella vaginalis, and a Megasphaera-like bacterium, suggesting that these bacteria play an important role in BV pathogenesis and may be suitable markers of disease and treatment response.

Relationship of Specific Vaginal Bacteria and Bacterial Vaginosis Treatment Failure in Women Who Have Sex with Women

Context Bacterial vaginosis, a condition that is particularly common among women who have sex with women, is an overgrowth of anaerobic bacteria and depletion of normally occurring lactobacilli. Bacterial vaginosis persists or recurs in 11% to 29% of women at 1 month after treatment. Contribution This study identified that the presence of specific vaginal bacteria at baseline (Clostridia BVAB1, BVAB2, or BVAB3; Peptoniphilus lacrimalis; or Megasphaera phylotype 2) and lower adherence to treatment were the only factors associated with persistence of bacterial vaginosis 1 month after treatment among women who have sex with women. Caution The results might not apply to women who have sex only with men. The Editors Bacterial vaginosis is characterized by depletion of hydrogen peroxideproducing lactobacilli that characterize normal vaginal flora and profound overgrowth of anaerobic bacteria (1). Bacterial vaginosis is the most prevalent vaginal infection in reproductive-age women, affecting 8% to 29%, and is the most common cause of vaginal symptoms prompting medical care (2). Of 3739 women enrolled during 2001 to 2004 in a nationally representative sample of the U.S. civilian noninstitutionalized population, almost 1 in 3 (29.2% [95% CI, 27.2% to 31.3%]) had bacterial vaginosis, as determined by Gram stain of vaginal fluid (3, 4). Bacterial vaginosis has been consistently associated with adverse outcomes related to the upper genital tract and with increased risk for HIV acquisition (57). Treatment of bacterial vaginosis targets the abundance of anaerobes that define the condition. With oral metronidazole used for 7 days or vaginal metronidazole for 5 days, symptoms improve in 83% to 87% of women by 2 to 3 weeks (8, 9). The improvement rate is similar for women who use vaginal clindamycin regimensboth antibiotics are recommended (10)and the restoration rate of vaginal lactobacilli at 30 days is similar (11, 12). Although short-term treatment response is acceptable, bacterial vaginosis persists or recurs in 11% to 29% of women at 1 month (8, 13, 14) and long-term recurrence rates exceed 70% (1517). Few studies have reported factors associated with bacterial vaginosis recurrence after successful treatment, such as black race, older age, higher Nugent score at enrollment (17), history of bacterial vaginosis, regular sex partner during study, female sex partner, and hormonal contraception (15). Even fewer studies have assessed risks associated with bacterial vaginosis persistence at 1 month after treatment. Available data suggest that condom use in the period immediately after treatment might support maintenance of normal vaginal flora and thus increase cure rates (16, 18). For unknown reasons, women who have sex with women have a high prevalence of bacterial vaginosis (25% to 52%) (3, 19). We hypothesized that this population might provide a unique opportunity to study the response to treatment of bacterial vaginosis because the potentially confounding exposure of unprotected vaginal intercourse was not likely to be common. In a cohort study of vaginal flora in this population, we assessed the incidence of bacterial vaginosis persisting at 1 month after treatment with vaginal metronidazole. In addition to measuring the contribution of recognized risk factors for bacterial vaginosis, including race, sexual behaviors, and douching, we assessed the contribution of specific species of bacterial vaginosisassociated bacteria (BVAB) present at initiation of bacterial vaginosis treatment. These bacteria include fastidious anaerobes detected by species-specific polymerase chain reaction (PCR) assays, some of which have not yet been cultivated and include 3 recently identified bacteria in the Clostridiales order (BVAB1, BVAB2, and BVAB3) that are highly specific (>97%) for bacterial vaginosis (20). Methods Participants and Clinical Definitions The study population comprised women age 16 to 30 years who reported sex with at least 1 woman in the previous year and who responded to recruitment through advertisements, media, and community referral between October 2004 and December 2006. Participants completed an extensive computer-assisted self-interview on demographic characteristics and medical, reproductive, and sexual history and underwent a standardized examination, including collection of vaginal fluid for Gram stain, saline microscopy, pH measurement, potassium hydroxide evaluation, and culture of Trichomonas vaginalis. We asked all participants to return for 4 quarterly visits or at any time if genital symptoms developed. To obtain specimens for bacterium-specific PCR assays, we brushed a polyurethane foam swab (Catch-All, Epicentre Biotechnologies, Madison, Wisconsin) against the lateral vaginal wall, resheathed it, and froze it immediately in a 80 C freezer until DNA extraction. We diagnosed bacterial vaginosis if 3 of 4 clinical (Amsel) criteria (vaginal pH >4.5, clue cells on saline microscopy >20% of epithelial cells, amine odor on addition of potassium hydroxide, or homogeneous vaginal discharge) were present (21) and Gram stain of vaginal fluid confirmed abnormal flora (Nugent score >3) (4). We treated women who had bacterial vaginosis with vaginal metronidazole gel (37.5 mg nightly for 5 days) and asked them to return in 1 month for test of cure, when we repeated examination and collected vaginal fluid for Gram stain in all women. Initially, we collected vaginal fluid samples for bacteria-specific PCR assays at test-of-cure visits for all women whose vaginal pH was greater than 4.0 and thus had suspected bacterial vaginosis or trichomoniasis. After approximately one half of the study participants were enrolled, we collected these samples routinely in all participants at the test-of-cure visit. For the analysis, we used the first visit at which a woman was found to have bacterial vaginosis (whether at the initial enrollment visit, at a later quarterly routine visit, or at a visit self-initiated for vaginal symptoms). We used test-of-cure visits after a womans first bacterial vaginosispositive visit that were completed before 31 March 2007 to examine incidence of persistent bacterial vaginosis and abnormal vaginal flora. We defined persistent bacterial vaginosis by Amsel criteria. We confirmed persistent bacterial vaginosis by Nugent score of vaginal fluid greater than 6 at the 1-month follow-up visit and confirmed abnormal vaginal flora by Nugent score greater than 3. We obtained written informed consent from all participants. Conduct of the study adhered to standard guidelines for research involving human participants and was approved by the University of Washington and Fred Hutchinson Cancer Center Human Subjects Review Committees. Microbiological Analysis For DNA extraction, we placed vaginal swabs for bacterial PCR assay in 15-mL conical vials with 2 mL of saline and vortex-mixed them for 1 minute to dislodge cells. We centrifuged the solution at 14000 rpm for 10 minutes and resuspended the pellet in 100 L of supernatant. We extracted DNA from the pellet by using the Ultra Clean Soil DNA Kit (MoBio, Carlsbad, California) according to the manufacturers instructions. We eluted DNA from silica columns in a 150-L volume buffer. We performed sham digests by using a swab without human contact with each round of DNA extraction (every 10 to 25 samples) to control for possible contamination from kit reagents or collection swabs. We developed bacterium-specific PCR assays to detect species-specific regions of the 16S rRNA gene. We aligned 16S rDNA sequences from vaginal bacteria detected by broad-range 16S rDNA PCR assays (22). We designed primers to target highly variable regions of the bacterial 16S rRNA gene that seem to be unique for each species. We developed PCR assays for 17 bacterial species that were commonly detected in vaginal samples (23). Each 50-L PCR reaction contained 1PCR Buffer II, 2 mmol of magnesium chloride, 0.8 mmol of deoxyribonucleotide triphosphate mix, and 1 U of AmpliTaq Gold DNA Polymerase (all from Applied Biosystems, Foster City, California), as well as 0.2 mol each of forward and reverse primer and 1 L of template DNA. The PCR conditions included a premelt at 95C for 10 minutes, then 40 to 45 cycles of 95C for 30 seconds (melt), 53C to 62C for 30 seconds (annealing), and 72C for 30 seconds (extension), followed by a final extension at 72C for 7 minutes. We visualized PCR products after electrophoresis in 2% agarose gels and stained them with ethidium bromide. We optimized bacterial PCR assays so that each could detect as few as 100 molecules of cloned 16S rDNA per reaction, although most assays could detect 1 to 10 molecules and thus had even lower detection thresholds. We sequenced every PCR assay with a visible band of the expected size on gel electrophoresis (BigDye, version 3; Applied Biosystems) to confirm that the PCR product had at least 99% similarity with the expected bacterial target, thereby assuring bacterial specificity. We considered PCR reactions to be negative if they did not have visible bands on gel electrophoresis or confirmed sequence homology. We ran no-template PCR controls (consisting of master mix, primers, and water) and sham digest controls (template consisting of water subjected to DNA extraction) with each PCR assay to monitor for contamination. We subjected each participants extracted DNA to a human -globin PCR assay to assure that amplifiable DNA was successfully extracted from the sample and to monitor for PCR inhibitors (24). The -globin PCR protocol used is the same as that listed for bacterial PCR, with the exception that the following primers were used: GH2O-5-GAAGAGCCAAGGACAGGTAC-3 and PCO4-5-CAACTTCATCCACGTTCACC-3. In addition, we performed an internal amplification control quantitative PCR assay by using DNA from each vaginal sample to detect more subtle PCR inhibitors by monitoring the amplification of an exogenously added template (jellyfish aequorin

Risks for acquisition of bacterial vaginosis among women who report sex with women: a cohort study.

Background Bacterial vaginosis (BV) is common in women who have sex with women. While cross-sectional data support a role for sexual transmission, risks for incident BV have not been prospectively studied in this group. Methodology/Principal Findings We studied risks for BV acquisition in a prospective cohort study of women (age 16–35 years) who reported sex with other women (≥1 partner, prior year). Women were followed for one year with examinations at quarterly visits and for genital symptoms at any time. Species-specific 16S rRNA gene PCRs for BV-associated bacteria (BVAB) were applied to vaginal fluid obtained at enrollment. Sexual behaviors were ascertained by computer-assisted interview. Of 335 participants, 239 had no BV at baseline; 199 were seen in follow-up (median follow-up 355 days, 4.0 visits/subject). Forty women experienced ≥1 BV episode. Risks for incident BV were presentation ≤14 days since onset of menses (hazard ratio (HR) 2.3 (95% CI, 1.2–4.7), report of new sex partner with BV history (HR 3.63 (1.1–11.9)), change in vaginal discharge (HR 2.6 (1.3–5.2)) and detection of any of several BVAB in vaginal fluid at enrollment, including BVAB1 (HR 6.3 (1.4–28.1)), BVAB2 (HR 18.2 (6.4–51.8)), BVAB3 (HR 12.6 (2.7–58.4)), G. vaginalis (HR 3.9 (1.5–10.4)), Atopobium vaginae (HR 4.2 (1.9–9.3)), Leptotrichia spp (9.3 (3.0–24.4)), and Megasphaera-1 (HR 11.5 (5.0–26.6)). Detection of Lactobacillus crispatus at enrollment conferred reduced risk for subsequent BV (HR 0.18 (0.08–0.4)). Detailed analysis of behavioral data suggested a direct dose-response relationship with increasing number of episodes of receptive oral-vulvovaginal sex (HR 1.02 (95% CI, 1.00–1.04). Conclusions/Significance Vaginal detection of several BVAB in BV-negative women predicted subsequent BV, suggesting that changes in vaginal microbiota precede BV by weeks or months. BV acquisition was associated with report of new partner with BV; associations with sexual practices – specifically, receptive oral sex – require further investigation.

Metabolic Signatures of Bacterial Vaginosis

ABSTRACT Bacterial vaginosis (BV) is characterized by shifts in the vaginal microbiota from Lactobacillus dominant to a microbiota with diverse anaerobic bacteria. Few studies have linked specific metabolites with bacteria found in the human vagina. Here, we report dramatic differences in metabolite compositions and concentrations associated with BV using a global metabolomics approach. We further validated important metabolites using samples from a second cohort of women and a different platform to measure metabolites. In the primary study, we compared metabolite profiles in cervicovaginal lavage fluid from 40 women with BV and 20 women without BV. Vaginal bacterial representation was determined using broad-range PCR with pyrosequencing and concentrations of bacteria by quantitative PCR. We detected 279 named biochemicals; levels of 62% of metabolites were significantly different in women with BV. Unsupervised clustering of metabolites separated women with and without BV. Women with BV have metabolite profiles marked by lower concentrations of amino acids and dipeptides, concomitant with higher levels of amino acid catabolites and polyamines. Higher levels of the signaling eicosanoid 12-hydroxyeicosatetraenoic acid (12-HETE), a biomarker for inflammation, were noted in BV. Lactobacillus crispatus and Lactobacillus jensenii exhibited similar metabolite correlation patterns, which were distinct from correlation patterns exhibited by BV-associated bacteria. Several metabolites were significantly associated with clinical signs and symptoms (Amsel criteria) used to diagnose BV, and no metabolite was associated with all four clinical criteria. BV has strong metabolic signatures across multiple metabolic pathways, and these signatures are associated with the presence and concentrations of particular bacteria. IMPORTANCE Bacterial vaginosis (BV) is a common but highly enigmatic condition that is associated with adverse outcomes for women and their neonates. Small molecule metabolites in the vagina may influence host physiology, affect microbial community composition, and impact risk of adverse health outcomes, but few studies have comprehensively studied the metabolomics profile of BV. Here, we used mass spectrometry to link specific metabolites with particular bacteria detected in the human vagina by PCR. BV was associated with strong metabolic signatures across multiple pathways affecting amino acid, carbohydrate, and lipid metabolism, highlighting the profound metabolic changes in BV. These signatures were associated with the presence and concentrations of particular vaginal bacteria, including some bacteria yet to be cultivated, thereby providing clues as to the microbial origin of many metabolites. Insights from this study provide opportunities for developing new diagnostic markers of BV and novel approaches for treatment or prevention of BV. Bacterial vaginosis (BV) is a common but highly enigmatic condition that is associated with adverse outcomes for women and their neonates. Small molecule metabolites in the vagina may influence host physiology, affect microbial community composition, and impact risk of adverse health outcomes, but few studies have comprehensively studied the metabolomics profile of BV. Here, we used mass spectrometry to link specific metabolites with particular bacteria detected in the human vagina by PCR. BV was associated with strong metabolic signatures across multiple pathways affecting amino acid, carbohydrate, and lipid metabolism, highlighting the profound metabolic changes in BV. These signatures were associated with the presence and concentrations of particular vaginal bacteria, including some bacteria yet to be cultivated, thereby providing clues as to the microbial origin of many metabolites. Insights from this study provide opportunities for developing new diagnostic markers of BV and novel approaches for treatment or prevention of BV.

Extravaginal Reservoirs of Vaginal Bacteria as Risk Factors for Incident Bacterial Vaginosis

BACKGROUND Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence. METHODS Risks for BV acquisition over the course of 1 year were defined. DNA in vaginal, anal, and oral swab samples from enrollment was subjected to quantitative polymerase chain reaction assays targeting 16S ribosomal RNA genes of Gardnerella vaginalis, Lactobacillus crispatus, BVAB1, BVAB2, BVAB3, Megasphaera spp., Lactobacillus jensenii, and Leptotrichia/Sneathia spp. A case-control approach analyzed BVAB detection at enrollment for case patients (BV acquisition) versus controls (none). RESULTS Of 239 women enrolled without BV, 199 were seen in follow-up, and 40 experienced BV; 15 had all samples for analysis. Detection of G. vaginalis in oral cavity or anal samples and Leptotrichia/Sneathia spp. in anal samples was more common at enrollment among case patients, who also had higher concentrations of these bacteria and Megasphaera relative to 30 controls at each site. In contrast, L. crispatus was detected more frequently in anal samples among controls. CONCLUSIONS Women who acquire BV are more likely have previous colonization of extravaginal reservoirs with some BVAB, and less likely to have L. crispatus, suggesting that BVAB may be acquired vaginally from extravaginal reservoirs.