Abstract

BackgroundHydrogen 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.MethodsAnaerobic 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.ResultsPhysiological 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.ConclusionsUnder 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.

Highlights

  • 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

  • The results presented here report the effects of a twohour exposure to H2O2, lactic acid, or acetic acid, with or without the addition of vaginal fluid (VF)

  • Microbicidal activity of hydrogen peroxide A two-hour anaerobic exposure to 100 mM H2O2 with 50 mU/mL MPO at pH 7 reduced the viability of all four vaginal lactobacilli species and all seventeen BVassociated bacterial species to undetectable levels

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Summary

Introduction

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. 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. VF has sufficient anti-oxidant activity to block the microbicidal activity of H2O2 even when H2O2 is supplied at concentrations much higher than lactobacilli are capable of producing. We believe these findings make protection by H2O2 implausible in vivo. Given the broad spectrum of BV-associated bacteria and the diverse reproductive tract infections that occur more frequently in women with BV, we chose to compare the microbicidal activities of the most robust broad-spectrum antimicrobials that lactobacilli are known to produce: H2O2 and lactic acid. To clarify whether cytosolic acidification is the primary anti-microbial action of lactic acid, we observed the effects of acetic acid, which is elevated during episodes of BV [20,21], and which, by being smaller and more lipid soluble, can acidify cytosol more rapidly than lactic acid [22]

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