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Bacterial vaginosis (BV), the overgrowth of diverse anaerobic bacteria in the vagina, is the most common cause of vaginal symptoms worldwide. BV frequently recurs after antibiotic therapy, and the best probiotic treatments only result in transient changes from BV-associated states to “optimal” communities dominated by a single species of Lactobacillus. Therefore, additional treatment strategies are needed to durably alter vaginal microbiota composition for patients with BV. Vaginal microbiota transplantation (VMT), the transfer of vaginal fluid from a healthy person with an optimal vaginal microbiota to a recipient with BV, has been proposed as one such alternative. However, VMT carries potential risks, necessitating strict safety precautions. Here, we present an FDA-approved donor screening protocol and detailed methodology for donation collection, storage, screening, and analysis of VMT material. We find that Lactobacillus viability is maintained for over six months in donated material stored at − 80 °C without glycerol or other cryoprotectants. We further show that species-specific quantitative PCR for L. crispatus and L. iners can be used as a rapid initial screening strategy to identify potential donors with optimal vaginal microbiomes. Together, this work lays the foundation for designing safe, reproducible trials of VMT as a treatment for BV.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations.We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities.The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa. Video Abstract.

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

Background Cervicovaginal microbiota domination by Lactobacillus crispatus is associated with beneficial health outcomes, whereas L. iners dominance has more adverse associations. However bacterial vaginosis (BV) treatment with metronidazole (MTZ) typically leads to domination by L. iners rather than L. crispatus. L. iners differs from other lactobacilli by its inability to grow in MRS media. We hypothesized that exploring this growth difference would identify targets for selective L. iners inhibition. Methods Bacteria were grown anaerobically. Nutrient uptake and metabolism were assessed using UPLC-MS/MS and isotopically labeled substrates. Bacterial genome annotation employed Prodigal, Roary, and EggNOG. Competition experiments with mock mixed communities were analyzed by 16S rRNA gene sequencing. We confirmed result generalizability using a diverse collection of South African and North American strains and genomes. Results Supplementing MRS broth with L-cysteine (Cys) or L-cystine permitted robust L. iners growth, while L. crispatus grew without Cys supplementation. Despite their different growth requirements, neither species could synthesize Cys via canonical pathways. Adding the cystine uptake inhibitors S-methyl-L-cysteine (SMC, Fig 1) or seleno-DL-cystine (SDLC) blocked growth of L. iners but not other lactobacilli, suggesting L. iners lacks mechanisms other lactobacilli use to exploit complex exogenous Cys sources. Notably, cydABCD, an operon with Cys/glutathione transport and redox homeostasis activities, is absent from L. iners but present in non-iners Lactobacillus species. Consistent with possible roles for cydABCD in explaining the observed phenotypes, (1) L. iners failed to take up exogenous glutathione and (2) supplementing MRS with reducing agents permitted L. iners growth, which could be blocked by SMC or SDLC. In growth competitions testing L. iners and L. crispatus within mock BV-like communities, SMC plus MTZ outperformed MTZ alone in promoting L. crispatus dominance (Figs 2&3). Figure 1: S-methyl-L-cysteine (SMC) selectively blocks growth of L. iners but not other cervicovaginal Lactobacillus species in cysteine-supplemented MRS broth. Growth was measured by optical density and inhibition calculated relative to Cys-supplemented no-inhibitor control during exponential growth. Values displayed are median (+/- maximum/minimum) for 3 replicates from a single experiment. In all panels, representative data are shown from 1 of >=2 independent experiments for each bacterial strain and media condition. Results are representative of multiple strains for L. iners (n = 16), L. crispatus (n = 7), and L. jensenii (n = 2). Figure 2: Relative abundance of L. crispatus, L. iners, or various BV-associated bacteria in mock bacterial communities grown in rich, non-selective media with or without metronidazole (MTZ) and/or SMC. Relative abundance was determined by bacterial 16S rRNA gene sequencing. Data are shown for three representative mock communities with 5 replicates per media condition. Figure 3: Ratio of L. crispatus to other species in the mock bacterial communities depicted in Figure 2. Statistical significance determined via 1-way ANOVA of log10-transformed ratios with post-hoc Tukey test; selected pairwise comparisons are shown (***, p < 0.001). Conclusion L. iners has unique requirements for exogenous cysteine/cystine or a reduced environment for growth. Targeting cystine uptake to inhibit L. iners is a potential strategy for shifting cervicovaginal microbiota towards L. crispatus-dominant communities. Disclosures Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

Vaginal microbiota has been postulated as a key contributor to the disproportionately higher HIV acquisition risk in women. The commensal bacterial communities in the vaginal tract have been implicated in HIV pathogenesis, with strict anaerobes such as Gardnerella and Prevotella causing inflammation and increased frequency of HIV target cells. Young African women are up to six times more likely to be infected with HIV than their male counterparts. The underlying biological mechanisms for increased susceptibility to HIV infection are not fully known, particularly among pregnant women who are also at risk of transmitting the infection to their unborn babies. We characterized the vaginal microbiome of pregnant women receiving antenatal care. Using 16S rRNA sequencing, we analyzed the richness and abundances of the commensal bacterial communities within the female genital tract. Data was analyzed using qiime2 version 2018, Dada2 plugin, and Naive-Bayes classifier for Taxonomic assignment. We report that 19% (35/179) of pregnant women had a Lactobacillus-dominant vaginal microbiota profile. Our findings show that the main cervicotypes (“CTs”) were CT1 which was predominantly non-iners Lactobacillus (6%, 11/179), CT2 which was dominated by L. iners (13%, 24/179), CT3 that was Gardnerella dominant (49%, 87/179) and CT4, a mixed CT co-dominated by L. iners, Gardnerella and Atopobium. Cervical lavage of women with non-Lactobacillus CT had significantly higher levels of inflammatory cytokines IL-1beta, TNF-alpha, and chemokines IL-6 and IL-8. Highly diverse cervicotype (CT4) was associated with inflammation, a known catalyst of HIV acquisition and transmission, within pregnant women regardless of HIV sero-status.