On the use of capillary cytometry for assessing the bactericidal effect of TiO2. Identification and involvement of reactive oxygen species

Abstract

The photocatalytic antimicrobial properties of TiO₂ were studied on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa bacterial strains taken as model strains for pathogenic species mainly implied in nosocomial infections. Capillary cytometry, coupled to a double-staining method for visualizing membrane integrity as a cell viability indicator, was highlighted as a rapid, easy-to-use, and automated numeration technique for quantitative and reproducible determination of cellular viability and thus, was able to give an accurate evaluation of the bactericidal effect of UV-A photocatalysis. Cytometry also enabled the study of TiO₂–bacteria interactions and aggregation in the dark as well as TiO₂ cytotoxicity. Compared with the traditional agar plate cultivation method, a significatively weaker reduction in cell viability was recorded by cytometry whatever the bacteria, TiO₂ concentration, and duration of the photocatalytic treatment. The mismatch between both numeration methods was attributed to: (i) the presence of mixed bacteria–TiO₂ aggregates that could interfere with bacteria measurement on plates, (ii) prolonged contact of the bacteria with TiO₂ during incubation, which could cause additional cytotoxic damage to the bacterial wall, and (iii) the counting of viable but non-culturable bacteria as live bacteria in cytometry, whereas they cannot grow on solid media. A more pronounced difference was observed for P. aeruginosa and S. aureus bacteria, for which 2.9 and 1.9 log₁₀ survival reduction overestimations were measured by plate counting, respectively. Using chemiluminescence, full restoration of cell viability by controlled addition of the O₂˙⁻ scavenger superoxide dismutase enzyme suggests that O₂˙⁻ acts, in our conditions, as the main reactive oxygen species responsible for the photocatalytic attack towards the targeted bacteria.