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.

TiO 2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

“…Therefore, SOD inhibition of the O 2 · Ϫ produced by irradiated TiO 2 induced a half-diminution of lipid peroxidation. This is in agreement with our previous work, which showed that scavenging the O 2 · Ϫ superoxide radical by adding the SOD enzyme completely restored cell viability (31). Impact of TiO 2 photocatalysis on E. coli whole-cell proteome.…”

“…It was noteworthy that addition of SOD decreases by half the amount of lipid peroxidation products, confirming that O 2 · Ϫ is implicated in the lipid peroxidation process. However, we consider the possibility that other ROS, such as singlet oxygen Ϫ radicals as important active ROS in E. coli cell death, i.e., in the photocatalytic antimicrobial effect, as previously shown in G. Carré et al (31).…”

“…Bacterial suspensions were maintained for 30 min in the dark or under UV-A radiation without TiO 2 or with 0.1 or 0.4 g/liter of TiO 2 . The duration under UV-A and TiO 2 concentration were chosen to obtain sublethal stress according to our previous work (31). Indeed, we have previously shown by cytometry that no significant modification of membrane integrity was observed for those TiO 2 concentrations in the dark or under UV-A light.…”

Effect of ball-milling and Fe-/Al-doping on the structural aspect and visible light photocatalytic activity of TiO2 towards Escherichia coli bacteria abatement

“…The capillary cytometry technique was used to assess possible aggregation phenomena within the TiO 2 -E. coli bacteria system in suspension, since this can be considered as the first step of the photocatalytic process towards microbiological targets in water, by analogy with the adsorption step in the degradation of chemical pollutants [39][40][41]. Fig.…”

Cotton Terry Textiles with Photo- and Bio-Activity in a Model Study and Real Conditions

“…Metal/non-metal doping extends their spectral response to visible light [ 12 , 17 , 19 , 20 , 21 ]. Caschera et al showed that europium doped TiO 2 finished cotton textiles have photocatalytic activity in UV and visible light and were even more effective in visible light [ 22 ]. Zahid at al.…”