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

Laerte Marlon Conceição Dos Santos,Leticia De Alencar Pereira Rodrigues,Arlene Souza Lessa,Carlos Nascimento,Marcelo Gerhardt,Cássio Santana Meira,Bruna Aparecida Souza Machado,Luis Alberto Breda Mascarenhas,Paulo Roberto Freitas Neves,Gabriela Monteiro Lobato,Eduardo Santos Da Silva,Greta Almeida Fernandes Moreira,Fabricia Oliveira Oliveira

doi:10.3390/biology10060525

Abstract

Simple SummaryControlling 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.O3 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 O3 was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O3 (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, O3 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.

Highlights

Ozone (O3 ) is a gas that has a natural configuration of three oxygen atoms
The concentration of O3 in water decays depending on the physicochemical state of the water
The decrease in pH and temperature was clearly positive in terms of increasing the initial concentration of O3 in the water as well as decreasing the percentage of reduction

Summary

Introduction

Ozone (O3 ) is a gas that has a natural configuration of three oxygen atoms. It is considered an elemental form of oxygen that occurs naturally in the Earth’s atmosphere, protecting the Earth from harmful solar ultraviolet radiation [1]. O3 can be produced artificially by the use of electricity generators [2,3], in which both air and oxygen can be used for gas formation. Its formation occurs from oxygen (O2 ) atoms that are supplied by the separation of the O2 molecules through the application of electrical discharges [2]. The free atom of O2 quickly combines with an available molecule of O2 (O + O2 > O3 ) forming O3 [2,4].

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