Numerous alternatives for the treatment of chronically infected wounds have been described in the literature. The real role of various agents, topically applied, remains undetermined [1–3].Topical application of antibiotics has shown limited efficacy in the management of infected wounds, without conclusive results [4, 5]. In the last several years, various in vitro and in vivo experimental and clinical studies have investigated the bactericidal effect of the topical application of ozone in different situations, including the management of infected wounds [6–8]. O₃ is a potent oxidant and an important disinfectant, acting on microorganisms by means of oxidation of their biological material [9]. It has been reported that O₃ can be employed as a bactericidal agent under various forms, such as ozonized saline solution [10], ozonized water [11], ozonized oil [7], ozone associated with other substances [12], and more frequently the gaseous O₃/O₂ mixture [13]. The topical use of O₃ has also been reported for environment decontamination in diverse situations: in agriculture for food decontamination [14], in odontology [15], and in clinical settings such as in the treatment of infected wounds [7]. Gaseous ozone has also been potentially considered for the disinfection of the hospital environment, which can be a source of microorganisms for patients [16]. However, a minimal effective antibacterial dose of gaseous ozone for topical application has not been clearly determined [17].

The objective of this study was to determine whether a low dose of O₃, in a gaseous O₃/O₂ mixture, applied to Petri dishes containing bacterial culture completely eliminates the growth of different bacterial strains. The strains chosen in this study were those that were important pathogens of nosocomial and community-acquired infections and those with known mechanisms of antimicrobial resistance.

This study was performed at the 3^rd Division of Clinical Surgery and Laboratory of Medical Investigation (LIM-54 and 62), of the Hospital das Clínicas of the University of São Paulo School of Medicine. The project was approved by the Ethics Committee of the Institution.

For bacterial culture, Petri dishes (90 mm internal diameter) were adapted with the addition of two special tips in order to allow continuous gas entry and exit (Figure 1). The study was done in two phases: a pilot test (1^st phase) to determine the dose of O₃/O₂ to be later employed in the evaluation of its bactericidal activity when applied to different bacterial strains (2^nd phase). In both phases, bacterial strains were inoculated on agar-blood in the adapted Petri dishes, at a concentration of 10⁵ bacteria/dish. Ozone gas was produced from medicinal oxygen (liquid oxygen with a degree of purity of 98%) by means of a medicinal O₃ generator provided by the Gas Department of the Aircraft Technology Institute (Instituto de Tecnologia da Aeronáutica – ITA, Brazilian Army, São José dos Campos, Brazil). This instrument was equipped with an oxygen flow controller (MKS – Type 1179ª) with 0.01 min precision, in which a controlled oxygen flow passes through a glass cylinder and is exposed to an electric discharge by a dielectric barrier with controlled potency and voltage (Figure 2). The electric discharge generates O₃ and its concentration can be measured. The final product is a gaseous O₃/O₂ mixture, in which the concentration is regulated by variation of oxygen flow and the voltage applied to the electrodes.

The eight bacterial strains employed in this study were selected to represent pathogenic bacteria commonly present in patients with severe nosocomial infections, with known resistance to antibiotics. Our results showed that the application of a low dose of gaseous ozone completely prevented the in vitro growth of all bacterial strains. On the other hand, in both control groups, bacterial growth occurred in all eight bacterial strains and treatment with 100% O₂ had no effect on bacterial proliferation, compared with the Baseline group.

In the 1960s, Scott et al., using topical application of ozonized saline, showed that approximately 2 × 10⁷ molecules of O₃ per bacterium provoked 50% death [10]. They attributed this to O₃ reacting with lipid double bonds, thus leading to bacterial wall lysis and bacterial cell content extravasation [10]. By entering the cell, O₃ promotes oxidation of nucleic and amino acids; and cell lysis depends on the extent of these reactions [1].

The culture medium employed in this study was agar-blood in Petri dishes. The use of agar for bacterial culture in Petri dishes is usual practice in microbiology, including the evaluation of bactericidal effects of different substances, such as ozone. An in vitro study aiming at decontamination with prolonged (4 h) application of gaseous O₃ (2 ppm), revealed a reduction of viability of various bacteria, such as E. coli, S. aureus, Serratia liquefaciens, and Listeria innocula, suggesting a disinfectant effect of O₃. The bacteria were cultured on agar in Petri dishes as well as in other culture media, and the author considered agar as the best culture media for measuring the efficacy of O₃ [14].

Pereira et al. reported that application of a gaseous O₃/O₂ mixture (0.4%/99.6%) for 1 h, at constant pressure and flow (11 mm Hg and 2 L/min, respectively) and controlled temperature, in plates containing 10⁴ CFU/mL of E. coli, S. aureus, and P. aeruginosa led to total inhibition of growth of these bacteria [18]. Compared to this study, the O₃ concentration in the gaseous O₃/O₂ mixture in the present study was 2.5 times greater, the duration of application was much shorter (1/12), and the gas flow was half (1 L/min). Furthermore, in the present study, potentially pathogenic bacteria with higher inoculums and known antimicrobial resistance were selected. Due to these differences, comparison of these studies is not viable. Other in vitro studies involving gaseous ozone have been performed but cannot be compared with our study as they involve Thichophyton spp. [19], mutans streptococci [20] and Listeria innocua [21].

The potential of our findings is interesting. The hospital environment has been increasing implicated in the transmission of resistant bacteria such as methicillin-resistant S. aureus and enterococci [22, 23]. Environmental cleaning and the application of hydrogen peroxide in the environment have recently deserved attention [24, 25] and ozone may have a similar use. In 1973, Broadwater et al. determined the minimum dose of O₃ dissolved in water (ozonized water) needed to eliminate the growth of three bacterial species when applied for 5 min. They observed that 0.12 mg/L of ozone was lethal for Bacillus cereus; and that 0.19 mg/L was lethal for Bacillus megaterium and E. coli [11]. Although O₃ dissolved in water was employed for surface decontamination, there was no clear definition of a minimum effective dose for its application. Likewise, there is no clear dose for O₃ in the form of an aerosol (O₃ dissolved in air) to be employed for surface decontamination of scientific instruments [26, 27]. Also focusing on environment decontamination, Li analyzed the resistance of various bacteria exposed to O₃ for surface disinfection, and pointed out the importance of the species (E. coli was more susceptible) and of the O₃ dose (concentration × time of exposure) on resistance to O₃ ²⁹. In the present study the dose of O₃ employed totally prevented the growth of all bacterial strains although Acinetobacter baumannii had a greater inoculum than the other bacterial strains. A concern involving the use of environmental O₃ for environmental disinfection is its toxicity, especially to the lungs [28] as the epithelial lining fluid has a relatively poor antioxidant capacity when compared with the blood. To enable the use of ozone in the hospital environment, exposure of patients and healthcare workers to inhalation would have to be avoided.

Another potential use for ozone is in the treatment of infected wounds. A clinical study reported that in the case of superficial wounds with antibiotic resistant sepsis following trauma and surgery, the application of O₃/O₂ resulted in wound healing and control of sepsis [8]. Sanchez et al. reported the efficient management of diabetic foot with gaseous O₃/O₂ application [29]. In a clinical prospective study [30], 61 patients with “diabetic foot” infections were randomized into two groups: topical gaseous O₃ application (80μ/mL maintained during 20min/session) + conventional (debridement + wound dressing) vs placebo (O₂ treatment). Although in the whole population the wound closure in the ozone group vs placebo (41% vs 33%) was not significant, it was observed that in the 34 patients who completed the study (16 of O₃ and 18 placebo) the wound closure was significantly higher in the O₃ group (81% vs 44%); and for patients with wounds ≤ 5cm² the total closure was higher in the O₃ group when compared with placebo (100% vs 50%; p=0.006). This suggested that O₃ was superior to conventional treatment. However it is difficult to draw conclusions from such a small study. A point of concern is the toxicity of ozone to the skin. The skin is protected against oxidative stress by a variety of antioxidants [31], but chronic exposure to O₃ can be deleterious to the skin, especially to the stratum corneum, leading to a cascade of effects in the deeper layers. Brief topical exposures of O₃, however, have been shown to be non-toxic [31].

Our study presents the following limitations: it is a preliminary evaluation and focuses on the in vitro effect of a minimal dose of ozone applied in Petri dishes containing bacteria seeded superficially on the Agar medium. It is not yet clear how well our findings may translate into clinical practice in which factors such as variable blood flow, with ischemia, necrotic tissue and high bacterial burdens may play an important role, especially in the diabetic foot.

In conclusion, the results of the present in vitro study showed that a dose of 20 μg of O₃/mL in a gaseous O₃/O₂ mixture (1% O₃/99% O₂₎, in a single topical application by nebulization for 5 min under atmospheric pressure, effectively inhibited the growth of all potentially pathogenic bacterial strains with known antimicrobial resistance.

This study did not receive external funding.