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Background Women’s increased risk of HIV acquisition during pregnancy and postpartum may be mediated by changes in vaginal microbiota and/or cytokines. Methods A cohort of 80 HIV-1-seronegative Kenyan women contributed 409 vaginal samples at six pregnancy timepoints: periconception, positive pregnancy test, first trimester, second trimester, third trimester, and postpartum. Concentrations of vaginal bacteria linked with HIV risk and Lactobacillus species were measured using quantitative polymerase chain reaction. Cytokines were measured by immunoassay. Results Using Tobit regression, later pregnancy timepoints were associated with lower concentrations of Sneathia spp. (p = 0.01), Eggerthella sp. Type 1 (p = 0.002) and Parvimonas sp. Type 2 (p = 0.02), and higher concentrations of L iners (p Conclusions Increases in proinflammatory cytokines, but not vaginal bacterial taxa linked with higher HIV risk, could provide an explanation for increased HIV susceptibility during pregnancy and postpartum.

Background Bacterial vaginosis (BV) treatment failures and recurrences are common. To identify features associated with treatment response, we compared vaginal microbiota and host ectocervical transcriptome before and after oral metronidazole therapy. Methods Women with BV (Bronx, New York and Thika, Kenya) received 7 days of oral metronidazole at enrollment (day 0) and underwent genital tract sampling of microbiome (16S ribosomal RNA gene sequencing), transcriptome (RNAseq), and immune mediator concentrations on day 0, 15, and 35. Results Bronx participants were more likely than Thika participants to clinically respond to metronidazole (19/20 vs 10/18, respectively, P = .0067) and by changes in microbiota composition and diversity. After dichotomizing the cohort into responders and nonresponders by change in α-diversity between day 35 and day 0, we identified that transcription differences associated with chemokine signaling (q = 0.002) and immune system process (q = 2.5 × 10–8) that differentiated responders from nonresponders were present at enrollment. Responders had significantly lower levels of CXCL9 in cervicovaginal lavage on day 0 (P < .007), and concentrations of CXCL9, CXCL10, and monocyte chemoattractant protein 1 increased significantly between day 0 and day 35 in responders vs nonresponders. Conclusions Response to metronidazole is characterized by significant changes in chemokines and related transcripts, suggesting that treatments that promote these pathways may prove beneficial.

Background The association between vaginal washing and HIV risk may be mediated by vaginal washing-associated changes in vaginal microbiota. Methods Data from a cohort of HIV-negative US and Kenyan women enrolled in the Preventing Vaginal Infections trial were analyzed. Vaginal fluid samples and vaginal washing data were collected every 2 months for 12 months. Bacterial relative abundances were measured by broad-range 16S rRNA gene polymerase chain reaction with next generation sequencing. Generalized estimating equations were used to evaluate the association between vaginal washing and i) the Shannon Diversity Index (SDI); and ii) mean change in percent bacterial relative abundances, with application of a 10% false discovery rate (FDR). Results Participants ( N = 111) contributed 93/630 (14.8%) vaginal washing visits. Mean SDI was 0.74 points higher (95% CI 0.35, 1.14; p < 0.001) at washing visits among US participants ( N = 26). Vaginal washing was not associated with SDI in Kenyan participants ( N = 85). There were no associations between vaginal washing and vaginal bacterial relative abundances after applying the FDR. Conclusions The discordant results in Kenyan versus US women suggests the link between vaginal washing and sub-optimal vaginal microbiota may be context specific. Vaginal microbial shifts may not fully explain the association between vaginal washing and HIV acquisition.

The Nugent score is the reference standard for bacterial vaginosis (BV) diagnosis but has not been validated in postmenopausal women. We compared relative abundances from 16S ribosomal RNA gene sequencing of vaginal microbiota with Nugent score in cohorts of premenopausal (n = 220) and postmenopausal (n = 144) women. In premenopausal women, 33 taxa were significantly correlated with Nugent score, including the classic BV-associated taxa Gardnerella, Atopobium, Sneathia, Megasphaera, and Prevotella. In postmenopausal women, 11 taxa were significantly associated with Nugent score, including Prevotella but no other BV-associated genera. High Nugent scores should not be used to infer BV in postmenopausal women.

Background Vaginal yeast is frequently found with Lactobacillus-dominant microbiota. The relationship between vaginal yeast and other bacteria has not been well characterized. Methods These analyses utilized data from the Preventing Vaginal Infections trial. Relative abundance of vaginal bacteria from 16S ribosomal ribonucleic acid gene amplicon sequencing and quantities of 10 vaginal bacteria using taxon-directed polymerase chain reaction assays were compared at visits with and without detection of yeast on microscopy, culture, or both. Results Higher relative abundances of Megasphaera species type 1 (risk ratio [RR], 0.70; 95% confidence interval [CI], 0.52–0.95), Megasphaera species type 2 (RR, 0.81; 95% CI, 0.67–0.98), and Mageeibacillus indolicus (RR, 0.46; 95% CI, 0.25–0.83) were associated with lower risk of detecting yeast. In contrast, higher relative abundances of Bifidobacterium bifidum, Aerococcus christensenii, Lactobacillus mucosae, Streptococcus equinus/infantarius/lutentiensis, Prevotella bivia, Dialister propionicifaciens, and Lactobacillus crispatus/helveticus were associated with yeast detection. Taxon-directed assays confirmed that increasing quantities of both Megasphaera species and M indolicus were associated with lower risk of detecting yeast, whereas increasing quantities of L crispatus were associated with higher risk of detecting yeast. Conclusions Despite an analysis that examined associations between multiple vaginal bacteria and the presence of yeast, only a small number of vaginal bacteria were strongly and significantly associated with the presence or absence of yeast.

In a vaginal 16S ribosomal RNA gene quantitative PCR study of 17 pelvic inflammatory disease (PID) cases and 17 controls who tested positive for Chlamydia trachomatis, women who additionally tested positive for Atopobium vaginae, Sneathia spp., BVAB-TM7, Megasphaera spp., Eggerthella-like bacterium or Mobiluncus spp. were more likely to develop PID.

Key Points The presence of butyrogenic bacteria after the onset of acute GVHD associates with subsequent steroid-refractory GVHD or chronic GVHD. Butyrate inhibits human colonic stem cells from forming an intact epithelial monolayer.

ObjectivesRecent studies have identified vaginal bacterial taxa associated with increased HIV risk. A possible mechanism to explain these results is that individual taxa differentially promote cervicovaginal inflammation. This study aimed to explore relationships between concentrations of bacteria previously linked to HIV acquisition and vaginal concentrations of proinflammatory cytokines and chemokines.MethodsIn this cross-sectional analysis, concentrations of 17 bacterial taxa and four proinflammatory cytokines (interleukin (IL)-1β, IL-6, IL-10 and tumour necrosis factor alpha (TNFα)) and two proinflammatory chemokines (IL-8 and interferon gamma-induced protein 10) were measured in vaginal swabs collected from 80 HIV-uninfected women. Cytokine and chemokine concentrations were compared between women with bacterial concentrations above or below the lower limit of detection as determined by quantitative PCR for each taxon. Principal component analysis was used to create a summary score for closely correlated bacteria, and linear regression analysis was used to evaluate associations between this score and increasing concentrations of TNFα and IL-1β.ResultsDetection of Dialister micraerophilus (p=0.01), Eggerthella sp type 1 (p=0.05) or Mycoplasma hominis (p=0.03) was associated with higher TNFα concentrations, and detection of D. micraerophilus (p, Eggerthella sp type 1 (p=0.04), M. hominis (p=0.02) or Parvimonas sp type 2 (p=0.05) was associated with significantly higher IL-1β concentrations. Seven bacterial taxa (D. micraerophilus, Eggerthella sp type 1, Gemella asaccharolytica, Sneathia sp, Megasphaera sp, M. hominis and Parvimonas sp type 2) were found to be highly correlated by principal component analysis (eigenvalue 5.24, explaining 74.92% of variability). Linear regression analysis demonstrated associations between this principal component and concentrations of TNFα (β=0.55, 95% CI 0.01 to 1.08; p=0.048) and IL-1β (β=0.96, 95% CI 0.19 to 1.74; p=0.016).ConclusionsThis study provides evidence that several highly correlated vaginal bacterial taxa may influence vaginal cytokine and chemokine concentrations. These results suggest a mechanism where the presence of specific bacterial taxa could influence HIV susceptibility by increasing vaginal inflammation.

ObjectivesVaginal washing has been associated with reductions in cultivable Lactobacillus and an increased risk of both bacterial vaginosis (BV) and HIV infection. The effect of vaginal washing on the quantity of individual Lactobacillus species is not well characterised. This analysis tested the hypothesis that vaginal washing would be associated with a lower likelihood of Lactobacillus spp. detected by both culture and quantitative PCR (qPCR).MethodsWe conducted a cross-sectional study of 272 HIV-seronegative women enrolled in an open-cohort study in Mombasa, Kenya. Vaginal washing and sexual risk behaviours were assessed using face-to-face interviews. Vaginal Lactobacillus spp. were detected using cultivation and PCR methods, with L. crispatus, L. jensenii and L. iners concentrations measured using qPCR assays targeting the 16S rRNA gene. Poisson regression with robust SEs was used to assess associations between vaginal washing and Lactobacillus detection by culture and qPCR.ResultsEighty percent (n=217) of participants reported vaginal washing in the prior week. One-fifth (n=58) of participants had BV by Nugent score. In unadjusted analysis, vaginal washing was associated with a 45% decreased likelihood of Lactobacillus spp. detection by culture (prevalence ratio (PR): 0.55, 95% CI 0.37 to 0.82). Adjusting for age and condomless sex in the prior week did not change the magnitude of the association (adjusted PR (aPR): 0.56, 95% CI (0.37 to 0.85). Vaginal washing was associated with approximately a 40% reduction in L. crispatus detection (aPR: 0.57, 95% CI 0.36 to 0.92), but was not significantly associated with L. jensenii (aPR: 0.68, 95% CI 0.42 to 1.09) or L. iners detection (aPR: 1.03, 95% CI 0.92 to 1.15).ConclusionsVaginal washing in the prior week was associated with a significantly reduced likelihood of detecting cultivable Lactobacillus and L. crispatus by qPCR. Given associations between Lactobacillus detection and improved reproductive health outcomes, these results provide motivation for additional study of vaginal washing cessation interventions to improve vaginal health.

BACKGROUND Bacterial vaginosis (BV) is associated with an increased risk of high-risk human papillomavirus (hrHPV), whereas Lactobacillus-dominated vaginal microbiotas are associated with reduced burden of hrHPV. Few epidemiologic studies have prospectively investigated the relationships between vaginal bacteria and hrHPV, particularly among women from countries in Africa. METHODS We conducted a prospective cohort study nested within the Preventing Vaginal Infections trial to evaluate associations between vaginal bacteria and hrHPV incidence and persistence. Sexually active, HIV-seronegative women aged 18 to 45 years who had a vaginal infection at screening were eligible to enroll. Analyses were restricted to participants enrolled in Kenya and randomized to placebo. At enrollment and months 2, 4, 6, 8, 10, and 12, hrHPV testing, quantitative polymerase chain reaction (measuring taxon quantity per swab), and 16S rRNA gene amplicon sequencing of the vaginal microbiota were performed. Generalized estimating equations multinomial logistic regression models were fit to evaluate associations between vaginal bacteria and incident and persistent hrHPV. RESULTS Eighty-four participants were included in this analysis. Higher concentrations of Lactobacillus crispatus were inversely associated with persistent hrHPV detection. Specifically, 1 tertile higher L. crispatus concentration was associated with 50% reduced odds of persistent hrHPV detection (odds ratio, 0.50; 95% confidence interval, 0.29-0.85). CONCLUSIONS This study is consistent with reports that vaginal L. crispatus is associated with reduced susceptibility to hrHPV persistence. Evidence from in vitro studies provides insight into potential mechanisms by which L. crispatus may mediate hrHPV risk. Future studies should further explore in vivo mechanisms that may drive this relationship and opportunities for intervention.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species., Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency—defined by a dominant negative stop codon polymorphism, rs5744168—is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro. We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

Background Bacterial vaginosis (BV) is a common cause of vaginal discharge and associated with vaginal acquisition of BV-associated bacteria (BVAB). Methods We used quantitative polymerase chain reaction assays to determine whether presence or concentrations of BVAB in the mouth, anus, vagina, or labia before BV predict risk of incident BV in 72 women who have sex with men. Results Baseline vaginal and extra-vaginal colonization with Gardnerella spp, Megasphaera spp, Sneathia spp, BVAB-2, Dialister sp type 2, and other BVAB was more common among subjects with incident BV. Conclusions Prior colonization with BVAB is a consistent risk for BV.

Summary Background Disruptions of vaginal microbiota might increase women's susceptibility to HIV infection. Advances in molecular microbiology have enabled detailed examination of associations between vaginal bacteria and HIV acquisition. Therefore, this study aimed to evaluate the association between the concentrations of specific vaginal bacteria and increased risk of HIV acquisition in African women. Methods We did a nested case-control study of participants from eastern and southern Africa. Data from five cohorts of African women (female sex workers, pregnant and post-partum women, and women in serodiscordant relationships) were used to form a nested case-control analysis between women who acquired HIV infection versus those who remained seronegative. Deep sequence analysis of broad-range 16S rRNA gene PCR products was applied to a subset of 55 cases and 55 controls. From these data, 20 taxa were selected for bacterium-specific real-time PCR assays, which were examined in the full cohort as a four-category exposure (undetectable, first tertile, second tertile, and third tertile of concentrations). Conditional logistic regression was used to generate odds ratios (ORs) and 95% CIs. Regression models were stratified by cohort, and adjusted ORs (aORs) were generated from a multivariable model controlling for confounding variables. The Shannon Diversity Index was used to measure bacterial diversity. The primary analyses were the associations between bacterial concentrations and risk of HIV acquisition. Findings Between November, 2004, and August, 2014, we identified 87 women who acquired HIV infection (cases) and 262 controls who did not acquire HIV infection. Vaginal bacterial community diversity was higher in women who acquired HIV infection (median 1·3, IQR 0·4–2·3) than in seronegative controls (0·7, 0·1–1·5; p=0·03). Seven of the 20 taxa showed significant concentration-dependent associations with increased odds of HIV acquisition: Parvimonas species type 1 (first tertile aOR 1·67, 95% CI 0·61–4·57; second tertile 3·01, 1·13–7·99; third tertile 4·64, 1·73–12·46; p=0·005) and type 2 (first tertile 3·52, 1·63–7·61; second tertile 0·85, 0·36–2·02; third tertile 2·18, 1·01–4·72; p=0·004), Gemella asaccharolytica (first tertile 2·09, 1·01–4·36; second tertile 2·02, 0·98–4·17; third tertile 3·03, 1·46–6·30; p=0·010), Mycoplasma hominis (first tertile 1·46, 0·69–3·11; second tertile 1·40, 0·66–2·98; third tertile 2·76, 1·36–5·63; p=0·048), Leptotrichia/Sneathia (first tertile 2·04, 1·02–4·10; second tertile 1·45, 0·70–3·00; third tertile 2·59, 1·26–5·34; p=0·046), Eggerthella species type 1 (first tertile 1·79, 0·88–3·64; second tertile 2·62, 1·31–5·22; third tertile 1·53, 0·72–3·28; p=0·041), and vaginal Megasphaera species (first tertile 3·15, 1·45–6·81; second tertile 1·43, 0·65–3·14; third tertile 1·32, 0·57–3·05; p=0·038). Interpretation Differences in the vaginal microbial diversity and concentrations of key bacteria were associated with greater risk of HIV acquisition in women. Defining vaginal bacterial taxa associated with HIV risk could point to mechanisms that influence HIV susceptibility and provide important targets for future prevention research. Funding National Institute of Child Health and Human Development.

To examine associations between the composition of the vaginal microbiota and genitourinary menopausal symptoms, serum estrogen, and vaginal glycogen.For this cross-sectional study, 88 women aged 40 to 62 years, enrolled in a hot flash treatment trial, provided vaginal swabs and a blood sample at enrollment. Bacterial communities were characterized using 16S rRNA PCR and deep sequencing targeting the V3-V4 region. Quantities of Lactobacillus crispatus and Lactobacillus iners were measured using qPCR. Self-reported genitourinary symptoms included: presence and severity of individual symptoms and identification of most bothersome symptom. Glycogen was measured fluorometrically in swab eluate. Serum estradiol (E2) and estrone (E1) were measured by liquid chromatography/mass spectrometry. Associations between bacteria, symptoms, glycogen, and serum estrogens were tested by linear regression or Wilcoxon signed-rank test, adjusted for multiple comparisons. Comparisons between groups used Kruskall-Wallis or Fisher's exact test.Of the 88 women, 33 (38%) had a majority of Lactobacillus species, whereas 58 (66%) had any Lactobacillus detected. Over half (53%) reported at least one vulvovaginal symptom (most commonly dryness), but symptoms were not associated with the presence of Lactobacillus species. Women with Lactobacillus-dominant communities had higher unconjugated serum estrone, but no difference in vaginal glycogen levels, compared with those with non-Lactobacillus-dominant communities. Higher serum E2 and E1 were not associated with either higher vaginal glycogen or detection of individual genera.Presence of Lactobacillus-dominant vaginal microbiota was not associated with fewer vulvovaginal symptoms. Serum estrone was higher in women with Lactobacillus dominance, but vaginal-free glycogen was not associated with composition of the vaginal microbiota.

Low alpha diversity in stool microbiota at neutrophil recovery associates with increased severe graft-versus-host disease (GVHD), greater abundance of oral Actinobacteria or Firmicutes, and decreased abundance of intestinal Lachnospiraceae in stool microbiota at neutrophil recovery associated with increased severe GVHD., Background Graft-versus-host disease (GVHD) is common after allogeneic hematopoietic cell transplantation (HCT). Risk for death from GVHD has been associated with low bacterial diversity in the stool microbiota early after transplant; however, the specific species associated with GVHD risk remain poorly defined. Methods We prospectively collected serial weekly stool samples from 66 patients who underwent HCT, starting pre-transplantation and continuing weekly until 100 days post-transplant, a total of 694 observations in HCT recipients. We used 16S rRNA gene polymerase chain reaction with degenerate primers, followed by high-throughput sequencing to assess the relative abundance of sequence reads from bacterial taxa in stool samples over time. Results The gut microbiota was highly dynamic in HCT recipients, with loss and appearance of taxa common on short time scales. As in prior studies, GVHD was associated with lower alpha diversity of the stool microbiota. At neutrophil recovery post-HCT, the presence of oral Actinobacteria and oral Firmicutes in stool was positively correlated with subsequent GVHD; Lachnospiraceae were negatively correlated. A gradient of bacterial species (difference of the sum of the relative abundance of positive correlates minus the sum of the relative abundance of negative correlates) was most predictive (receiver operator characteristic area under the curve of 0.83) of subsequent severe acute GVHD. Conclusions The stool microbiota around the time of neutrophil recovery post-HCT is predictive of subsequent development of severe acute GVHD in this study.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera . UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6–94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA–DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera . We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

BackgroundWhile bacterial vaginosis has been associated with an increased risk of Trichomonas vaginalis (TV) acquisition, it is unknown whether other characteristics of the vaginal microbiota, including the presence of key bacterial species, influence a woman’s risk of TV acquisition.MethodsThe vaginal microbiota before 25 unique episodes of TV infection involving 18 women was compared to that of 50 controls who remained uninfected. TV was detected by transcription-mediated amplification. Vaginal microbiota were quantified using broad-range polymerase chain reaction analysis and taxon-specific quantitative PCR of the 16S ribosomal RNA gene.ResultsTV acquisition was significantly associated with the presence of Prevotella amnii (risk ratio [RR], 2.21; 95% confidence interval [CI], 1.12–4.38; P = .02) and Sneathia sanguinegens (RR, 2.58; 95% CI, 1.00–6.62; P = .049). When adjusted for menstrual phase, the association between P. amnii and TV acquisition remained similar (adjusted RR, 2.11; 95% CI, 1.03–4.33; P = .04), but the association between S. sanguinegens and TV acquisition was attenuated (adjusted RR, 2.31; 95% CI, .86–6.23; P = .10).ConclusionsKey vaginal bacterial species may contribute to the susceptibility to TV acquisition. Understanding how these bacterial species increase a woman’s risk of TV acquisition could help to guide the development of novel strategies to reduce women’s risk of TV infection.

BACKGROUND Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with

BACKGROUND Depot medroxyprogesterone acetate (DMPA) is associated with HIV acquisition. We studied changes in vaginal microbiota and inflammatory milieu after DMPA initiation. METHODS In a cohort of HIV-negative Kenyan women, we collected monthly vaginal swabs over 1 year before and after DMPA. Using quantitative polymerase chain reaction, we compared quantities of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus iners, Gardnerella vaginalis, and total bacterial load (16S ribosomal RNA gene levels). Six vaginal immune mediators were measured with enzyme-linked immunosorbent assay. Trends in the detection and quantity of bacteria were estimated by logistic and linear mixed-effects regression. RESULTS From 2010 to 2012, 15 HIV-seronegative women initiated DMPA, contributing 85 visits (median, 6 visits/woman; range, 3-8 visits/woman). The median time of DMPA-exposed follow-up was 8.4 months (range, 1.5-11.6 months). Seven women (46%) had bacterial vaginosis within 70 days before DMPA start. L. iners was detected in 13 women (87%) before DMPA start, but other lactobacilli were rarely detected. Gardnerella vaginalis decreased by 0.21 log10 copies per swab per month after DMPA exposure (P = 0.01). Total bacterial load decreased by 0.08 log10 copies per swab per month of DMPA (P = 0.02). Sustained decreases in interleukin (IL)-6 (P = 0.03), IL-8 (P = 0.04), and IL-1 receptor antagonist (P < 0.001) were also noted. Nine women (60%) had L. crispatus detected post-DMPA, which significantly correlated with reduced IL-6 (P < 0.01) and IL-8 (P = 0.02). CONCLUSIONS Initiation of DMPA led to sustained shifts in vaginal bacterial concentrations and levels of inflammatory mediators. Further studies are warranted to outline components of the vaginal microbiota influenced by DMPA use and impact on HIV susceptibility.

OBJECTIVES: Vaginal washing has been associated with reductions in cultivable Lactobacillus and an increased risk of both bacterial vaginosis (BV) and HIV infection. The effect of vaginal washing on the quantity of individual Lactobacillus species is not well characterized. This analysis tested the hypothesis that vaginal washing would be associated with a lower likelihood of Lactobacillus spp. detected by both culture and quantitative PCR (qPCR). METHODS: We conducted a cross-sectional study of 272 HIV-seronegative women enrolled in an open-cohort study in Mombasa, Kenya. Vaginal washing and sexual risk behaviours were assessed using face-to-face interviews. Vaginal Lactobacillus spp. were detected using cultivation and PCR methods, with L. crispatus, L. jensenii, and L. iners concentrations measured using qPCR assays targeting the 16S rRNA gene. Poisson regression with robust standard errors was used to assess associations between vaginal washing and Lactobacillus detection by culture and qPCR. RESULTS: Eighty percent (n=217) of participants reported vaginal washing in the prior week. One-fifth (n=58) of participants had BV by Nugent score. In unadjusted analysis, vaginal washing was associated with a 45% decreased likelihood of Lactobacillus spp. detection by culture (prevalence ratio (PR): 0.55, 95%CI 0.37–0.82). Adjusting for age and condomless sex in the prior week did not change the magnitude of the association (adjusted PR (aPR): 0.56, 95%CI (0.37, 0.85). Vaginal washing was associated with approximately a 40% reduction in L. crispatus detection (aPR: 0.57, 95%CI 0.36–0.92), but was not significantly associated with L. jensenii (aPR: 0.68, 95%CI 0.42–1.09) or L. iners detection (aPR: 1.03, 95%CI 0.92–1.15). CONCLUSIONS: Vaginal washing in the prior week was associated with a significantly reduced likelihood of detecting cultivable Lactobacillus and L. crispatus by qPCR. Given associations between Lactobacillus detection and improved reproductive health outcomes, these results provide motivation for additional study of vaginal washing cessation interventions to improve vaginal health.

OBJECTIVE: Identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen and serum estrogen. METHODS: Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs. venlafaxine vs. placebo) who reported GSM and provided vaginal swabs at 0, 4 and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3–V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using chi-square, Wilcoxon signed-rank test, or Hotelling’s t test. RESULTS: Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study, and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared to placebo (54%; p = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, p = 0.08). Vaginal glycogen, serum estradiol and estrone increased significantly in women whose MBS improved. CONCLUSION: Improvement in GSM was more common in women receiving oral estradiol or venlafaxine and those with Lactobacillus-dominant vaginal microbiota at enrollment but differences were not statistically significant. Larger trials are need to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.

Background Cervicitis is an inflammatory condition of the cervix associated with upper genital tract infection and reproductive complications. Although cervicitis can be caused by several known pathogens, the etiology frequently remains obscure. Here we investigate vaginal bacteria associated with bacterial vaginosis as potential causes of cervicitis. Methods Associations between vaginal bacteria and cervicitis were assessed in a retrospective case-control study of women attending a Seattle sexually transmitted disease clinic. Individual bacterial species were detected using 2 molecular methods: quantitative polymerase chain reaction (qPCR) and broad-range 16S rRNA gene PCR with pyrosequencing. The primary finding from this initial study was evaluated using qPCR in a second cohort of Kenyan women. Results The presence of Mageeibacillus indolicus, formerly BVAB3, in the cervix was associated with cervicitis, whereas the presence of Lactobacillus jensenii was inversely associated. Quantities of these bacteria did not differ between cervicitis cases and controls, although in a model inclusive of presence and abundance, M. indolicus remained significantly associated with cervicitis after adjustment for other cervicitis-causing pathogens. M. indolicus was not associated with cervicitis in our study of Kenyan women, possibly due to differences in the clinical definition of cervicitis. Conclusions Colonization of the endocervix with M. indolicus may contribute to the clinical manifestations of cervicitis, but further study is needed to determine whether this finding is repeatable and applicable to diverse groups of women. Colonization of the cervix with L. jensenii could be a marker of health, perhaps reducing inflammation or inhibiting pathogenic infection.

Introduction We tested the hypothesis that vaginal microbiota influence women’s susceptibility to T. vaginalis (TV) acquisition. Methods We conducted a nested case-control study of 25 episodes of TV infection using vaginal samples collected 30–60 days prior to infection in 18 HIV-negative women and 50 incidence-density matched controls. Broad-range 16S rRNA gene PCR was used to measure total vaginal bacterial load. Deep sequencing was applied to 18 first episodes of TV infection and 36 matched controls to measure bacterial diversity (Shannon index) and species richness (Chao-1 index). Bacterium-specific quantitative PCR was performed for Lactobacillus crispatus , Prevotella amnii , Sneathia sanguinegens , Dialister species Type 2, Bacterial Vaginosis Associated Bacterium 1 (BVAB-1), and Mageeibacillus indolicus for all TV infection cases and controls. The associations between Shannon and Chao-1 indices and TV acquisition were evaluated using logistic regression. Generalised estimating equations with logit link were used to evaluate associations between TV acquisition, detection of bacterial species, and total vaginal bacterial load. Results There was no association between total vaginal bacterial load, species diversity, or richness, and likelihood of TV acquisition. Detection of S. sanguinegens (odds ratio [OR] 4.00, 95% CI 1.19–13.50) and P. amnii (OR 3.45, 95% CI 1.29–9.24) were both associated with TV acquisition. M. indolicus was also associated with TV acquisition (OR 2.47, 95% CI 0.88–6.93), although not significantly. Compared to women with none of these species, women with all three bacterial species had substantially higher likelihood of TV acquisition (none-reference category; one-OR 1.57, 95% CI 0.21–11.86; two-OR 4.50, 95% CI 0.93–21.76; three-OR 5.50, 95% CI 1.15–26.40). There was no association with the other three bacterial species. Conclusion The presence of three bacterial species commonly associated with BV may increase susceptibility to TV infection. Elimination of these bacteria could be explored as an approach to decrease women’s risk of trichomoniasis.

Background Steroid refractory acute graft- vs. -host-disease (GVHD) after hematopoietic cell transplantation (HCT) is highly morbid with limited treatment options. Murine studies show protection from GVHD with butyrate exposure but direct exposure of stem/progenitor cells to butyrate inhibits colonic stem cell proliferation. Methods Stool samples were collected weekly in a cohort of HCT recipients (n = 210) undergoing allogeneic transplant, and underwent 16S rRNA sequencing to determine the number and relative abundance of butyrogens. Dissociated primary human colonoid cell aggregates (200,000 per well) were plated onto collagen IV-coated transwells (0.4 µm pore size, 0.33 cm2, PET) in stem cell medium for 24 hours. From 24 hours onwards, the basal-lateral chamber was switched to differentiation medium; the apical chamber was Hanks Buffered Salt Solution (HBSS), HBSS with 10 mM butyrate sodium salt early (24 hours onwards) or late (72hours onwards). Trans-epithelial electrical resistance was measured daily. Results Retrospective chart review identified 27 recipients who developed acute GVHD of the gut, stratified to be either steroid refractory GVHD (failed to respond to 2 mg/kg of methylprednisolone) or responsive. The presence of butyrogens in the gut microbiome after the onset of severe acute GHVD of the gut associated with increased risk of steroid refractory GVHD (Figure 1; P < 0.05). Direct exposure of human colonic stem/progenitor cells to butyrate inhibits the development of trans-epithelial electrical resistance; exposure after differentiation had no inhibition of barrier formation (Figure 2; P < 0.05 by T-test). Conclusion Butyrogens may help prevent the development of acute GVHD of the gut, but once severe GVHD has developed may inhibit recovery due to the loss of crypt architecture exposing colonic stem cells to microbe-produced butyrate with impaired differentiation and cell replacement. Disclosures All Authors: No reported Disclosures.

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. Bacterial vaginosis (BV) is the most prevalent vaginal infection in reproductive-age women, and has been consistently associated with adverse outcomes related to upper genital tract complications and with increased risk of human immunodeficiency virus acquisition [1–3]. Of 3739 women enrolled during 2001–2004 in a nationally representative sample of the US civilian

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.

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.

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.

Background. Bacterial vaginosis (BV) is a common polymicrobial disease associated with numerous negative reproductive health outcomes, including an increased risk of human immunodeficiency virus acquisition. BV is treatable with antibiotics, but relapse is common. A more detailed understanding of bacterial dynamics during antibiotic therapy for BV could identify conditions that favor establishment, maintenance, and eradication of BV-associated bacterial species, thereby improving treatment outcomes. Methods. We used mathematical models to analyze daily quantitative measurements of 11 key bacterial species during metronidazole treatment for 15 cases of BV. Results. We identified complete reorganization of vaginal bacterial composition within a day of initiating therapy. Although baseline bacterial levels predicted a longer time to clearance, all anaerobic species were eliminated rapidly within a median of 3 days. However, reemergence of BV-associated species was common following treatment cessation. Gardnerella vaginalis, a facultative anaerobe, was cleared more slowly than anaerobic BV-associated species, and levels of G. vaginalis often rebounded during treatment. We observed gradual Lactobacillus species growth, indicating that untargeted microbes fill the transient vacuum formed during treatment. Conclusions. Under antibiotic pressure, the human microbiome can undergo rapid shifts on a scale of hours. When treatment is stopped, BV-associated bacteria quickly reemerge, suggesting a possible role for intermittent prophylactic treatment.

BACKGROUND Bacterial vaginosis affects millions of women and is associated with several serious health conditions. The cause of bacterial vaginosis remains poorly understood despite numerous studies based on cultures. Bacteria in microbial communities can be identified without cultivation by characterizing their ribosomal DNA (rDNA) sequences. METHODS We identified bacteria in samples of vaginal fluid with a combination of broad-range polymerase-chain-reaction (PCR) amplification of 16S rDNA with clone analysis, bacterium-specific PCR assay of 16S rDNA, and fluorescence in situ hybridization (FISH) performed directly on vaginal fluid from 27 subjects with bacterial vaginosis and 46 without the condition. Twenty-one subjects were studied with the use of broad-range PCR of 16S rDNA, and 73 subjects were studied with the use of bacterium-specific PCR. RESULTS Women without bacterial vaginosis had 1 to 6 vaginal bacterial species (phylotypes) in each sample (mean, 3.3), as detected by broad-range PCR of 16S rDNA, and lactobacillus species were the predominant bacteria noted (83 to 100 percent of clones). Women with bacterial vaginosis had greater bacterial diversity (P

Urethritis is the most common male reproductive tract syndrome and is caused primarily by Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT). However, up to 50% of cases have neither GC nor CT detected1 and are referred to simply as nonchlamydial nongonococcal urethritis (NGU). Clinically defined as symptoms of urethral irritation (dysuria, pruritis) or visible urethral discharge and 5 or more polymorphonuclear leukocytes per high-power field (PMNs/HPF) in urethral exudates, nonchlamydial NGU has been associated with a number of known pathogens; Mycoplasma genitalium (MG) in 15% to 25%, Trichomonas vaginalis (TV) in 5% to 15%, and, less commonly, herpes simplex virus (HSV) and adenovirus in 2% to 4%.1 The differentiated Ureaplasma urealyticum (UU) has been associated with urethritis in some2–4 but not all studies.4,5 However, in a recent case-control study, 45% of NGU cases had no known pathogen,4 even after testing for CT, MG, TV, and UU. Although noninfectious causes of male urethritis exist, most cases are likely caused by sexually transmitted pathogens. Therefore, bacteria involved in female reproductive tract disease are reasonable candidates for involvement in NGU. Bacterial vaginosis (BV) is the most prevalent genital tract syndrome in women, affecting 29% of reproductive-aged women,6 and has been associated with increased risk for perinatal complications,7 pelvic inflammatory disease,8 and HIV acquisition9 and transmission.10 Although there is debate as to whether BV can be sexually transmitted, the protective effect of condoms11 and the concordance of infection in lesbian couples12 support this concept. The syndrome is characterized by a shift in the vaginal microbiota from a predominance of lactobacilli to a predominance of anaerobic bacteria13 and does not seem to be caused by a single organism, but rather represents a change in the vaginal microbiota from one with several predominant Lactobacillus species to one marked by increased species richness and diversity.14–16 Several recently identified bacteria have been highly predictive of and specific for BV, including Leptotrichia/Sneathia spp., Atopobium spp., Megasphaera spp. and BVAB-1, BVAB-2, and BVAB-3 (all Clostridiales order bacteria)17 Most of these organisms, with the exception of BVAB-1, were also more common in women with than in those without cervicitis in a pilot study we conducted (unpublished data), suggesting that their presence in men may be associated with similar inflammation in the male urethra. To assess the role of these newly described bacteria in male urethritis, we conducted a case-control study of men with and without idiopathic NGU. All men were tested for 5 recently identified bacteria associated with BV in women, and we estimated the risk of urethritis associated with detection of each bacterial species.

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p

Background The presence of hydrogen peroxide (H2O2) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H2O2-producers. Methods Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR. Results Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45). Conclusions Among HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.

Objective: Evaluate predictors of vaginal colonization with lactobacilli after treatment for bacterial vaginosis (BV).Methods. Vaginal fluid specimens from women with BV underwent qPCR forLactobacillus crispatus,L. jensenii, andL. inerspre- and posttreatment.Results. Few women with BV were colonized withL. crispatus(4/44, 9%) orL. jensenii(1/44, 2%), though all hadL. iners.One month posttreatment 12/44 (27%) hadL. crispatus, 12/44 (27%)L. jensenii, and 43/44 (98%)L. iners.Presence ofL. jenseniiposttreatment was associated with cure (Risk Ratio (RR) 1.67; 95% CI 1.09–2.56);L. crispatusshowed a similar trend (RR 1.41; 95% CI 0.89–2.24,P=0.14). Receptive oral sex was associated with 2.2-log10lower concentration ofL. crispatus(95% CI −4.38, −.02), and digital-vaginal sex with 2.6-log10lower concentration (95% CI −4.87, −.33).Conclusion. One month after BV treatment, few women established colonization withL. crispatusorL. jensenii. Few behaviors were associated with colonization.

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 Amsel’s 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.

BACKGROUND: Bacterial vaginosis frequently persists after treatment. The role of newly defined bacterial vaginosis-associated bacteria (BVAB), which have a specificity for this condition of 97% or greater, has not been assessed. OBJECTIVE: To define risks for bacterial vaginosis persistence, including pretreatment detection of specific vaginal bacteria, among women reporting sex with women. DESIGN: Observational cohort study. SETTING: University-based research clinic. PATIENTS: 335 women age 16 to 29 years reporting sex with at least 1 woman in the past year. Participants were recruited through advertisements and provider referral. INTERVENTION: Bacterial vaginosis was treated with intravaginal metronidazole gel (0.75%), 37.5 mg nightly for 5 nights. MEASUREMENTS: Species-specific 16S recombinant DNA polymerase chain reaction assays targeting 17 bacterial species were applied to vaginal fluid obtained at baseline. Test of cure by clinical criteria and Gram stain analysis and repeated polymerase chain reaction assays of vaginal fluid were performed 1 month after treatment, and interim behaviors were assessed by using computer-assisted self-interview. RESULTS: Of 335 women, 24% of whom also reported sex with men within 3 months before enrollment, 131 (39%) had bacterial vaginosis. In the 120 (92%) women who returned for follow-up, the incidence of persistent bacterial vaginosis was 26% and was statistically significantly higher in women with baseline detection of 3 Clostridiales bacteria, designated as BVAB1 (risk ratio, 2.0 [95% CI, 1.1 to 4.0]), BVAB2 (risk ratio, 8.7 [CI, 2.5 to infinity]), or BVAB3 (risk ratio, 3.1 [CI, 1.7 to 5.8]); Peptoniphilus lacrimalis (risk ratio, 3.5 [CI, 1.6 to 15.5]); and Megasphaera phylotype 2 (risk ratio, 3.4 [CI, 1.4 to 5.5]). Persistence was lower with treatment adherence (risk ratio, 0.4 [0.2 to 0.9]). Detection of these bacteria at the test-of-cure visit was associated with persistence, whereas posttreatment sexual activity was not. LIMITATIONS: Findings may not be generalizable to women who have sex only with men, or to women whose bacterial vaginosis is treated with oral antibiotics. The study may be too small and may involve a population that is too highly selected to draw definitive conclusions about associations of persistent infection with posttreatment sexual behaviors. CONCLUSION: Persistent bacterial vaginosis is associated with several bacteria in the Clostridiales order, Megasphaera phylotype 2, and P. lacrimalis, suggesting that vaginal microbiology at diagnosis may determine risk for antibiotic failure.

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.

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.