Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity

SIMPLE SUMMARY: Controlling microbial infections, especially nosocomial infection, is a task that continues to be a burden in many societies. The current pandemic has brought new concerns on this matter, mainly on how to better control the dissemination of microbial agents in the environment/surfaces and among humans. Therefore, the search for alternative methods and agents for disinfection is warranted. The aim of our study was to analyze the disinfecting potential of ozonized water in different in vitro tests. By performing microbiological and cell lineage in vitro assays, the biocidal effect of ozonized water was confirmed. Upon a short incubation time, bacterial strains and a yeast were killed by ozonized water, whereas there was no cytotoxicity in the mammalian cell line. These findings lead to the conclusion this agent can be safely tested in technologies for water spray disinfection devices. ABSTRACT: O(3) dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O(3) was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O(3) (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O(3) remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.