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.