XTT assay for evaluating the effect of alcohols, hydrogen peroxide and benzalkonium chloride on biofilm formation of Staphylococcus epidermidis

To analyze the degree of biofilm formation on three ica-positives Staphylococcus epidermidis as a function of biocides, the medium was supplemented with increasing concentrations of isopropanol, ethanol, and methanol at 0, 1, 4, 6, 8, 10, 12 and 14% (v/v), hydrogen peroxide (0, 0.125, 0.25, 0.5, 1, 2, 3, 4 and 5% v/v) and benzalkonium chloride (0, 0.125, 0.25, 0.5, 1, 2, 3, 4 and 6 μg ml⁻¹). In biocide-free biofilms, the results showed that two strains (S. epidermidis CIP106510 and E24) were strongly biofilm positive displaying a high oxidative activity (1.254 and 0.855, respectively) in comparison with the non-adherent one (S22). In addition biofilm formation was induced with 1% alcohol (isopropanol and ethanol) supplementation. The three studied strains cultured in TSB supplemented with 2% methanol displayed a strong oxidative activity (P=0.008). Moreover wells with 0.125% hydrogen peroxide enhanced increasing oxidative activity of S. epidermidis CIP106510 and S22. A significant induction of biofilm was noted after treatment with 1 μg ml⁻¹ of benzalkonium chloride. This study suggests that some biocides currently used in hospitals are ineffective against nosocomial pathogens growing in biofilms when used at weak concentration and fail to control this reservoir for hospital-acquired infection.