Selection, phenotyping and identification of acid and hydrogen peroxide producing bacteria from vaginal samples of Canadian and East African women.

The common but poorly understood condition known as bacterial vaginosis (BV) increases vulnerability to HIV infection and is associated with the absence of H(2)O(2)-producing Lactobacillus. Vaginal lactic acid bacteria (LAB) produce anti-HIV factors such as organic acids and hydrogen peroxide (H(2)O(2)), and may bind and inactivate HIV particles during scavenging of mannose. These factors define potential criteria for initial selection of candidate probiotics to block heterosexual transmission of HIV. Therefore, the primary goal of this study was to characterize acid production on mannose and H(2)O(2) production in vaginal isolates from Canadian adolescents (192 isolates, 16 individuals) and commercial sex workers in Nairobi, Kenya (576 isolates, 96 individuals). Selection of isolates from H(2)O(2)-detecting media suggested an idiosyncratic individual-level profile and extensive phenotypic diversity, including the identification of a subset of "double-strong" acid- and H(2)O(2)-producers with phenotypes similar to well-characterized probiotic strains. Molecular fingerprinting of all isolates by capillary electrophoresis of 16S-23S rRNA interspacer amplicons was coupled with chaperonin-60 universal target (cpn60 UT) sequencing in a subset, tentatively identifying 96% of isolates although only 19% were sequenced. Most isolates belonged to Lactobacillus, Streptococcus, Bifidobacterium or Gardnerella, with a total of 37 species in 15 genera, as well as 5 potentially novel organisms, identified in this study. This sensitivity was likely enhanced by phenotype-based selection on two chromogenic media formulations. Identification of double-strong isolates may provide a rational basis for selection and further characterization of vaginal probiotics, with potential application as part of HIV prevention initiatives in western Canada and East Africa.

Bacterial vaginosis, HIV serostatus and T-cell subset distribution in a cohort of East African commercial sex workers: retrospective analysis.

OBJECTIVE: Although bacterial vaginosis is a known correlate of HIV infection, no previous studies have investigated whether women defined as HIV-exposed seronegative (HESN) are less likely to have bacterial vaginosis. Little is known about the effects of bacterial vaginosis on systemic immune activation associated with HIV+ serostatus. DESIGN: Cohort-based retrospective analysis of bacterial vaginosis in relation to HESN status, HIV+ serostatus and peripheral T-helper cells, with cross-sectional analysis of bacterial vaginosis in relation to peripheral T-regulatory cells (Tregs). METHODS: Bacterial vaginosis diagnosis by Gram stain and determination of systemic CD4(+) and CD8(+) T-helper cell frequency by flow cytometry for 3504 vaginal samples from 988 commercial sex workers over 4 years. Treg phenotyping by FoxP3 staining and multiparameter flow cytometry in peripheral blood of 97 women at a single time-point. RESULTS: No differences in bacterial vaginosis diagnosis were observed between HESN and other HIV-negative (HIV-N) controls; however, HIV+ women were more likely to be diagnosed with bacterial vaginosis compared to all HIV-negative women (HESN/HIV-N combined). HIV+ women with bacterial vaginosis had significantly higher CD4(+)/CD8(+) T-helper cell counts and a lower CD4/CD8 ratio, as well as fewer Tregs as a proportion of total T-helper cells, compared to bacterial vaginosis-negative women. The number of bacterial vaginosis diagnoses in this cohort has decreased significantly over time. CONCLUSION: Bacterial vaginosis is associated with HIV serostatus and shifts in distribution of T-cell subsets. A concomitant reduction in bacterial vaginosis and HIV infections over time suggests that the elucidation of bacterial vaginosis-HIV interactions will be critical to further understanding of HIV pathogenesis and prevention in this high-risk group.

Molecular definition of vaginal microbiota in East African commercial sex workers.

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.