Synergistic potential of aqueous ozone: Sublethal bacterial damage and enhanced antibiotic susceptibility

Abstract

Combat-related injuries are frequently complicated by infections caused by multidrug‑resistant microorganisms, posing significant challenges to treatment. To improve clinical outcomes in such cases, alternative adjunctive therapeutic strategies are required. This study aimed to assess the antimicrobial potential of electrolytically generated aqueous ozone, focusing on its ability to induce sublethal damage in bacteria and enhance their susceptibility to antibiotics. A total of 357 multidrug-resistant clinical isolates were obtained from wound exudates and blood samples of 284 wounded soldiers. The most frequently isolated pathogens included Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and coagulase-negative Staphylococcus spp. Ozonated water was generated at concentrations ranging from 3.6 to 11.2 mg/L and subsequently standardised to approximately 4 mg/L for bacterial treatment. Selected isolates of S. aureus and Escherichia coli were exposed to aqueous ozone for 10 and 15 minutes. Sublethal damage was assessed by comparing bacterial growth on selective and non‑selective media, revealing up to 89.2% sublethally injured S. aureus cells and up to 98.6% injured E. coli cells after 15 minutes of exposure. Initial estimations of the minimum inhibitory concentration using the Vitek-2 system were distorted by the combined effect of ozone and free chlorine, which forms during the electrolysis of saline solution. This prompted a shift to the Kirby-Bauer disc diffusion method. The results consistently demonstrated increased antibiotic susceptibility in treated isolates, as evidenced by larger inhibition zone diameters and a reduced number of antibiotics to which the isolates remained resistant. Electrolytically generated aqueous ozone effectively compromises bacterial integrity, enhancing their susceptibility to antibiotics. It represents a promising adjunctive strategy for managing multidrug-resistant infections, particularly in resource-limited settings or during wartime.