Antibacterial Effect of Ozone on Pseudomonas aeruginosa growth from ocular isolate in vitro cultures

Abstract

Purpose : Ozone therapy is well known for its antimicrobial and anti-inflammatory activity that stem from its strong oxidative properties. The aim of this study was to investigate the in vitro antibacterial effect of different ozone exposures to known microbial loads of Pseudomonas aeruginosa.

Methods : The Pseudomonas aeruginosa strain was cultivated in a chocolate agar culture medium for 24 hours and posteriorly used for bacterial suspension, which was performed at a 0.5 concentration in the McFarland nephelometer. With this concentration, posterior serial dilutions were performed at 10^-1, 10^-2, 10^-3, and 100 µL of each dilution was inoculated into three Mueller-Hinton plates each. This process was repeated for the three studied groups: the no-ozone control group, the 1-hour ozone exposure group, and the 2-hour ozone exposure group. The 1- and 2-hour ozone exposure plates were placed in an incubator at 37°C that was connected to an ozone generator (Generador de Ozono Gl2186, America Water Treatment, CDMX, Mexico), where bacteria were susceptible to ozone at a concentration of 400 mg/hr for 1 and 2 hours, with a total exposure of 800 mg and 1200 mg, respectively. Posteriorly, they were repositioned to an incubator with no exposure to ozone at 37°C, along with the control group plates that were not exposed to ozone. All plates were finally incubated for a total 48 hours, with inspection for bacterial count every 24 hours. The results were recorded for analysis.

Results : We observed a significant reduction in Pseudomonas aeruginosa growth according to the ozone exposure time, with the least amount of growth detected in the 2-hour ozone exposure group, specifically in the 10^-3 dilution, where microbial counts decreased 51% compared to the control group. Pseudomonas aeruginosa cultures also showed increased production of pyoverdine and pyocyanin pigments with prolonged exposure to ozone, which are involved in bacterial virulence and survival against oxidative stress and harsh environmental conditions.

Conclusions : This study demonstrates the antibacterial activity of ozone in reducing known microbial loads of Pseudomonas aeruginosa in vitro cultures, with increased effectivity according to the length of exposure. Further research is needed to establish the effectivity of different ozone time exposures and microbial loads, as well as the role of ozone as an adjunct therapy for in vivo models.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.