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

Abstract

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.

Figure 1

Microbicidal activity of hydrogen peroxide (H₂O₂) with 50 mU/mL human myeloperoxidase (MPO) at pH 7, against four species of vaginal lactobacilli (solid lines) and seventeen species of bacteria associated with bacterial vaginosis (BV) (broken lines). The vertical dashed line indicates the concentration of H₂O₂ measured in vaginal fluid (VF) from women with a lactobacilli-dominated microbiota (~ 23 μM).

Figure 2

The blocking effect of bacterially-depleted VF on the microbicidal activity of 1 M H₂O₂ with 50 mU/mL MPO, which is otherwise sufficient to inactivate completely four species of lactobacilli (solid lines) and seven species of BV-associated bacteria (broken lines).

Figure 3

Microbicidal activity of lactic acid at pH 4.5 against four species of vaginal lactobacilli (solid lines) and seventeen species of BV-associated bacteria (broken lines). The vertical dashed line indicates the mean concentration of lactic acid measured in VF from women with a lactobacilli-dominated vaginal microbiota (93 mM).

Figure 4

Microbicidal activity of acetic acid at pH 4.5 against four species of vaginal lactobacilli (solid lines) and seventeen species of BV-associated bacteria (broken lines).

Figure 5

The effect of bacterially-depleted VF on the microbicidal activity of 100 mM lactic acid at pH 4.5, which is otherwise sufficient to inactivate completely seven species of BV-associated bacteria (broken lines), but not four species of lactobacilli (solid lines).