Biosynthesis and Degradation of H 2 O 2 by Vaginal Lactobacilli

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H₂O₂, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H₂O₂ concentrations. Generation of H₂O₂ was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H₂O₂ only occurred upon agitation of the cultures, but the H₂O₂-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H₂O₂ production, while Cu²⁺ inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe³⁺, hemin, and hemoglobin did not accumulate H₂O₂. Fe³⁺ activated an extracellular peroxidase that destroyed the H₂O₂ being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe³⁺ ions being a necessary cofactor of the reaction.