Applied and Environmental Microbiology, January 2010, p. 400-405, Vol. 76, No. 2

Biosynthesis and Degradation of H₂O₂ by Vaginal Lactobacilli

Area de Microbiología, Instituto Universitario de Biotecnología, Universidad de Oviedo, Oviedo, Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, Spain

Received 10 July 2009/ Accepted 15 November 2009

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify theability 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 renderedthe 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. gasseristrains. 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 ofLactobacillus 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 itsantimicrobial effect. The production of the enzyme appears to be constitutive, the Fe³⁺ ions being a necessary cofactor of the reaction.