It has the ability to interact with and invade host cells, and then to live within these cells (Ly & Casanova, 2007). We report that the two Lactobacillus strains display killing activity against G. vaginalis, UPEC and S. typhimurium by substances present in the cell-free culture supernatants (CFCSs). Moreover, our results show that the main metabolic product of Lactobacillus, lactic acid, displays
no killing activity at the concentration present in Lactobacillus cultures, whereas hydrogen peroxide dose-dependently killed these pathogens. We also provide evidence that at the concentration present in Lactobacillus cultures, lactic acid considerably enhances the killing activity of hydrogen peroxide. The prototype UPEC strain CFT073 (Mobley et al., 1990) and S. typhimurium SL1344 Selleck DAPT (Finlay & Falkow, 1990) were used. Bacteria were Transmembrane Transporters modulator cultured in Luria–Bertani (LB) agar (Difco Laboratories, Detroit, MI) and incubated at 37 °C for 24 h. Gardnerella vaginalis DSM 4944 was grown on Gardnerella agar plates purchased from BioMerieux (Lyon, France), as described previously (Atassi et al., 2006a, b). Bacteria were suspended in pH 7.0 buffered sodium chloride-peptone solution at about 106 CFU mL−1. Five hundred microliters of the prepared suspension was spread on the agar plate. The inoculated plates were dried under a sterile laminar airflow. The agar plates were then
incubated under anaerobic conditions in a sealed anaerobic jar (Becton Dickinson) at 37 °C for up to 36 h. Before being used, G. vaginalis was subcultured in brain–heart infusion supplemented with yeast
extract (1%), maltose (0.1%), glucose (0.1%) and horse serum (10%) under anaerobic conditions in a sealed anaerobic jar at 37 °C for up to 36 h. For each experiment, bacteria were subcultured for the exponential phase in appropriate media. Lactobacillus johnsonii strain NCC533 was from the Nestec Research Center PAK6 at Vers-chez-les-Blanc (Switzerland). The L. gasseri KS120.1 strain isolated from the vaginal flora of a healthy woman (Department of Obstetrics and Gynecology, Zurich University Hospital, Switzerland) was from Medinova (Zurich, Switzerland) (Atassi et al., 2006a, b). All the Lactobacillus strains were grown in De Man, Rogosa, Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for 24 h at 37 °C. The Lactobacillus culture was adjusted to pH 4.5 by adding HCl or NaOH to ensure standardized conditions. Cultures of the Lactobacillus strains (24 h) were centrifuged at 10 000 g for 30 min at 4 °C. Bacteria were collected and washed three times with sterile phosphate-buffered saline (Coconnier et al., 1997, 2000). Supernatants of the centrifuged cultures were collected and passed through a sterile 0.22-μm filter unit Millex GS (Millipore, Molsheim, France).
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