Optimization of media for detection of hydrogen peroxide production by Lactobacillus species

A healthy vaginal ecosystem has been shown to be protective against the acquisition of human immunodeficiency virus and gonorrhea, and women who are colonized with H2O2-producing lactobacilli are more likely to maintain a normal vaginal flora than women with lactobacilli that do not produce H2O2. The purpose of this study was to formulate a testing medium that better supports the growth and detection of H2O2 by a broader range of lactobacilli than a published, widely used agar formulation (TMB). The new medium (TMB-Plus) consists of brucella agar base, 3,3',5,5'-tetramethylbenzidine, horseradish peroxidase, starch, vitamin K, hemin, magnesium sulfate, manganese sulfate, and horse serum. To validate the new formula, 256 vaginal isolates and ATCC strains were inoculated onto TMB-Plus and, for comparison, onto TMB. Growth was enhanced for 69% of the isolates on TMB-Plus, and 48% had enhanced color production. The percentage of H2O2-positive isolates increased from 71% on TMB to 79% on TMB-Plus. Formulations using Rogosa or MRS agar base in combination with peroxidase and a chromogen did not support the growth of all of the strains of Lactobacillus, and fewer H2O2-producing strains were detected on these formulations than on TMB-Plus. This new medium better supports the growth of a wider range of Lactobacillus strains isolated from the vagina and enhances the color production of H2O2-producing strains.