The implausible "in vivo" role of hydrogen peroxide as an antimicrobial factor produced by vaginal microbiota.

In the cervicovaginal environment, the production of hydrogen peroxide (H2O2) by vaginal Lactobacillus spp. is often mentioned as a critical factor to the in vivo vaginal microbiota antimicrobial properties. We present several lines of evidence that support the implausibility of H2O2 as an "in vivo" contributor to the cervicovaginal milieu antimicrobial properties. An alternative explanation is proposed, supported by previous reports ascribing protective and antimicrobial properties to other factors produced by Lactobacillus spp. capable of generating H2O2. Under this proposal, lactic acid rather than H2O2 plays an important role in the antimicrobial properties of protective vaginal Lactobacillus spp. We hope this commentary will help future research focus on more plausible mechanisms by which vaginal Lactobacillus spp. exert their antimicrobial and beneficial properties, and which have in vivo and translational relevance.

Vaginal pH and microbicidal lactic acid when lactobacilli dominate the microbiota.

Lactic acid at sufficiently acidic pH is a potent microbicide, and lactic acid produced by vaginal lactobacilli may help protect against reproductive tract infections. However, previous observations likely underestimated healthy vaginal acidity and total lactate concentration since they failed to exclude women without a lactobacillus-dominated vaginal microbiota, and also did not account for the high carbon dioxide, low oxygen environment of the vagina. Fifty-six women with low (0-3) Nugent scores (indicating a lactobacillus-dominated vaginal microbiota) and no symptoms of reproductive tract disease or infection, provided a total of 64 cervicovaginal fluid samples using a collection method that avoided the need for sample dilution and rigorously minimized aerobic exposure. The pH of samples was measured by microelectrode immediately after collection and under a physiological vaginal concentration of CO2. Commercial enzymatic assays of total lactate and total acetate concentrations were validated for use in CVF, and compared to the more usual HPLC method. The average pH of the CVF samples was 3.5 ± 0.3 (mean ± SD), range 2.8-4.2, and the average total lactate was 1.0% ± 0.2% w/v; this is a five-fold higher average hydrogen ion concentration (lower pH) and a fivefold higher total lactate concentration than in the prior literature. The microbicidal form of lactic acid (protonated lactic acid) was therefore eleven-fold more concentrated, and a markedly more potent microbicide, than indicated by prior research. This suggests that when lactobacilli dominate the vaginal microbiota, women have significantly more lactic acid-mediated protection against infections than currently believed. Our results invite further evaluations of the prophylactic and therapeutic actions of vaginal lactic acid, whether provided in situ by endogenous lactobacilli, by probiotic lactobacilli, or by products that reinforce vaginal lactic acid.

Evaluation of microbicide gel adherence monitoring methods.

BACKGROUND: An objective and accurate method that measures adherence to vaginal microbicide gel regimens during clinical trials could provide more accurate estimates of microbicide efficacy, aid in targeting adherence promotion resources, and enable objective assessment of adherence promotion strategies. METHODS: We evaluated 4 methods to assess whether or not gel applicators had been vaginally inserted. At the study site, 50 women inserted hydroxyethylcellulose universal placebo gel through a polypropylene vaginal applicator and handled, but did not insert a second "sham-inserted" applicator. Applicators were discarded into a container capped with a medical event monitor system (MEMS) that recorded the time and date of opening. Fifteen additional participants did likewise at 2 study site visits, and administered gel on 6 intervening days at home. Applicators were scored as inserted, or not, by direct inspection under ambient light, ultraviolet (UV) light, staining with Alcian blue, and microscopic detection of vaginal cells stained with iodine. RESULTS: Mean sensitivity/specificity of 2 readings each by 3 test readers for UV, Alcian blue, ambient light, and iodine methods were 84/83, 79/83, 76/63, and 65/80%, respectively. Sensitivity of all methods was significantly higher in applicators inserted after one or more prior insertions of gel, with the highest sensitivity (95%) obtained with UV. MEMS caps accurately recorded applicator disposal time. CONCLUSIONS: The modest accuracy of all 4 methods for applicator insertions without prior gel applications may limit their accuracy in monitoring coital regimens. However, for daily dosing regimens, MEMS monitoring and UV inspection should provide a rapid, reliable, and quantitative assessment of adherence.

In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide.

BACKGROUND: Hydrogen peroxide (H2O2) produced by vaginal lactobacilli is generally believed to protect against bacteria associated with bacterial vaginosis (BV), and strains of lactobacilli that can produce H2O2 are being developed as vaginal probiotics. However, evidence that led to this belief was based in part on non-physiological conditions, antioxidant-free aerobic conditions selected to maximize both production and microbicidal activity of H2O2. Here we used conditions more like those in vivo to compare the effects of physiologically plausible concentrations of H2O2 and lactic acid on a broad range of BV-associated bacteria and vaginal lactobacilli. METHODS: Anaerobic cultures of seventeen species of BV-associated bacteria and four species of vaginal lactobacilli were exposed to H2O2, lactic acid, or acetic acid at pH 7.0 and pH 4.5. After two hours, the remaining viable bacteria were enumerated by growth on agar media plates. The effect of vaginal fluid (VF) on the microbicidal activities of H2O2 and lactic acid was also measured. RESULTS: Physiological concentrations of H2O2 (< 100 µM) failed to inactivate any of the BV-associated bacteria tested, even in the presence of human myeloperoxidase (MPO) that increases the microbicidal activity of H2O2. At 10 mM, H2O2 inactivated all four species of vaginal lactobacilli but only one of seventeen species of BV-associated bacteria. Moreover, the addition of just 1% vaginal fluid (VF) blocked the microbicidal activity of 1 M H2O2. In contrast, lactic acid at physiological concentrations (55-111 mM) and pH (4.5) inactivated all the BV-associated bacteria tested, and had no detectable effect on the vaginal lactobacilli. Also, the addition of 10% VF did not block the microbicidal activity of lactic acid. CONCLUSIONS: Under optimal, anaerobic growth conditions, physiological concentrations of lactic acid inactivated BV-associated bacteria without affecting vaginal lactobacilli, whereas physiological concentrations of H2O2 produced no detectable inactivation of either BV-associated bacteria or vaginal lactobacilli. Moreover, at very high concentrations, H2O2 was more toxic to vaginal lactobacilli than to BV-associated bacteria. On the basis of these in vitro observations, we conclude that lactic acid, not H2O2, is likely to suppress BV-associated bacteria in vivo.

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli.

BACKGROUND: H2O2 produced by vaginal lactobacilli is believed to protect against infection, and H2O2-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H2O2-blocking activity. METHODS: We measured the H2O2 concentration of CVF and the H2O2-blocking activity of CVF and semen using fluorescence and in vitro bacterial-exposure experiments. RESULTS: The mean H2O2 measured in fully aerobic CVF was 23 +/- 5 microM; however, 50 microM H2O2 in salt solution showed no in vitro inactivation of HSV-2, Neisseria gonorrhoeae, Hemophilus ducreyii, or any of six BV-associated bacteria. CVF reduced 1 mM added H2O2 to an undetectable level, while semen reduced 10 mM added H2O2 to undetectable. Moreover, the addition of just 1% CVF supernatant abolished in vitro pathogen-inactivation by H2O2-producing lactobacilli. CONCLUSIONS: Given the H2O2-blocking activity of CVF and semen, it is implausible that H2O2-production by vaginal lactobacilli is a significant mechanism of protection in vivo.