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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.