Approximately 75,000 individuals acquire healthcare-associated infections (HAIs) annually in U.S. acute care hospitals (Popovich et al., 2019; Halverson et al., 2022). Environmental contamination significantly contributes to the acquisition of HAIs, particularly in intensive care units (ICUs) (Huang et al., 2006; Drees et al., 2008; Dancer, 2014; Cohen et al., 2018) Many clinically relevant pathogens can survive on inanimate surfaces for extended periods (Porter et al., 2024), and the presence of a previously infected occupant significantly increases the risk of subsequent residents developing HAIs (Mitchell et al., 2023). Current disinfection protocols, which include manual and mechanical cleaning with disinfectants, germicides, and ultrasonic cleaners (Rutala and Weber, 2015; Sheppeard et al., 2022), face challenges due to the development of resistance in many clinical isolates, rendering decontamination both expensive and labor-intensive (Dancer, 2014; Lourbopoulos et al., 2021). Given the persistent issues of understaffing and the growing antibiotic resistance crisis, there is a critical need for new, hands-free disinfection methods (Dancer, 2014).

Ozone (O₃), a potent oxidant composed of three unstable oxygen atoms, is a naturally occurring, pungent gas found at 10–20 parts per billion (ppb) in the atmosphere and up to 100 ppb in polluted areas. Ozone demonstrates remarkable microbicidal activity by oxidizing bacterial lipids, viral envelopes and capsids, and fungal membranes (Allison et al., 2009; Brié et al., 2018; Xue et al., 2023). Additionally, it generates reactive oxygen species (ROS) that collapse cellular membranes. O₃ is frequently employed as an environmental decontaminant, particularly in its aqueous form in water treatment plants (Ding et al., 2019; Epelle et al., 2023).

Ozone has also been used therapeutically for skin and oral pathologies for decades (Kim et al., 2009; Liu et al., 2023). Despite these successes, the use of gaseous ozone as an environmental disinfectant has been limited due to its toxicity to humans at levels exceeding 70 ppb. The severity of these effects can be mitigated by using lower doses, shorter durations, and less frequent applications of ozone (Bette et al., 2022; Singh et al., 2023). While high doses of ozone can be harmful, it also offers advantages as a treatment therapy for various human diseases including cancer, bacterial infections, asthma, and viral diseases, due to its disinfectant properties and anti-inflammatory effects (Singh et al., 2023; Elvis and Ekta, 2011; Zanardi et al., 2016). Additionally, ozone has demonstrated effective antimicrobial properties in sustainable food production, wound healing in healthcare, and public environments by penetrating intracellular components of microorganisms and causing oxidative damage (Epelle et al., 2023; Agarwal et al., 2020; Moraes et al., 2021; Premjit et al., 2022; Roth et al., 2023; Neves et al., 2023).

In recent years, interest in low-dose ozone as a cheap and effective environmental disinfectant has grown, particularly in the agriculture and healthcare industries (Shen et al., 2021). Gaseous ozone is a Generally Recognized as Safe (GRAS) agent and is approved by the U.S. Food and Drug Administration (FDA) for use (Bobka, 1993). Several studies have reported conflicting evidence regarding the efficacy of low-dose gaseous ozone against pathogens, with effectiveness against some bacteria but ineffectiveness against COVID-19 (Fontes et al., 2012; Westover et al., 2022; Misawa et al., 2023). Additionally, several studies have assessed ozone’s capability in decontaminating food and bacterial surfaces (Xue et al., 2023; Coll Cárdenas et al., 2011; Cantalejo et al., 2016). Most of these studies utilized a limited number of targets and exposure conditions, making it difficult to generalize the efficacy of low-dose gaseous ozone as a disinfectant.

We aimed to characterize the impact of environmental conditions, including bacterial growth conditions, temperature, and humidity, on the microbicidal capacity of low-dose ozone on clinically relevant bacterial pathogens. We found that ozone was effective at killing both Gram-positive and Gram-negative species in a dose-dependent manner. Additionally, we found that gaseous ozone was equally effective at low and ambient temperatures and was relatively unaffected by humidity. These studies address the current gap in the literature and provide evidence for the use of low-dose gaseous ozone as a potential environmental disinfectant.

We first tested the microbicidal activity of gaseous ozone, selecting E. coli and S. typhimurium as representative Gram-negative strains, and GAS and MRSA as representative Gram-positive strains. Strains were exposed to gaseous ozone at 5 ppm for varying CT. We observed a 3 log₁₀-fold reduction in both E. coli and S. typhimurium after exposure compared to controls (Figures 1A,B). GAS and MRSA showed a 1–2 log₁₀-fold reduction after exposure to increasing concentrations of O₃ (Figures 1C,D), indicating that gaseous ozone kills in a dose-dependent manner. To assess whether repeated exposure enhances killing, we exposed strains to a single 360 CT dose or three doses of 200 CT with recovery periods between each dose. We found no significant difference in bacterial survival between single and multiple exposures, suggesting that a single potent dose is sufficient to reduce bacterial survival (Figures 1E–H).

Strains were exposed to gaseous ozone at 5 ppm for varying contact times (CT). We observed a three log₁₀-fold reduction in both E. coli and S. typhimurium after exposure compared to controls (Figures 1A,B). GAS and MRSA showed a 1–2 log₁₀-fold reduction after exposure to increasing concentrations of O₃ (Figures 1C,D), indicating that gaseous ozone kills in a dose-dependent manner. To assess whether repeated exposure enhances killing, we exposed strains to a single 360 CT dose or three doses of 200 CT with recovery periods between each dose. We found no significant difference in bacterial survival between single and multiple exposures, suggesting that a single potent dose is sufficient to reduce bacterial survival (Figures 1E–H).

Next, we tested the impact of environmental conditions on the bactericidal capacity of gaseous ozone by exposing bacteria grown in logarithmic and stationary phases to O₃. We did not find consistent differences in killing between growth phases of E. coli and MRSA, although stationary-phase MRSA was modestly, though not significantly, more resistant to killing (Figures 2A,B). Given the significant interest in applying ozone to refrigerated food products, we examined the impact of temperature on gaseous ozone efficacy by exposing E. coli and MRSA to gaseous O₃ at ambient temperature (~21–22°C) and at 4°C. Gaseous ozone was slightly more effective at killing MRSA and E. coli at colder temperatures, though the difference did not achieve statistical significance (Figures 2C–F). We also investigated the impact of humidity on ozone’s killing capacity by testing low humidity (<40%), ambient humidity (40–60%), and high humidity (>70%). Different humidity levels did not impact the ability of ozone to kill E. coli (Figure 2G). There was a modest, though not statistically significant, improvement in the bactericidal activity of gaseous O₃ against MRSA at higher humidity levels (Figure 2H). Collectively, these results indicate that gaseous ozone is effective against bacteria in both quiescent and active growth phases and that its killing capacity is relatively unaffected by low temperatures and varying humidity levels.

Lastly, we examined the ability of ozone to kill a panel of clinical isolates of important human pathogens. We exposed strains of A. baumannii, MRSA, E. coli, P. aeruginosa, GAS, GBS, E. faecium, and S. marcescens to a single dose of gaseous ozone and found significant bactericidal activity against all strains tested (Figure 3A). We further tested a broader panel of isolates relevant to both human health and the food industry. Consistent with previous findings, a single low-dose exposure of gaseous ozone effectively reduced microbial counts by at least 10-fold (Figure 3B). As a proof of principle, we conducted a pilot study where an ozone generator was placed in a fitness studio over a weekend. Six sites were sampled for microbial counts before and after exposure to gaseous ozone at 0.5 ppm for 1 h over three consecutive nights. All six sites showed reduced bacterial counts after O₃ exposure (Figure 3C). These findings collectively demonstrate the potential of gaseous ozone as a hands-free microbicide effective against MDR bacterial species.

The findings from our study demonstrate the robust bactericidal efficacy of low-dose gaseous ozone against a variety of MDR, clinically relevant pathogens, highlighting its potential as a hands-free environmental disinfectant. Our results showed that a single potent dose of ozone was sufficient to achieve significant reductions in bacterial counts. The lack of enhanced killing with repeated exposures suggests that ozone’s bactericidal effect is dose-dependent rather than frequency-dependent.

Ozone’s microbial inactivation stems from its ability to induce microorganisms such as bacteria, fungi, and mold, to generate reactive oxygen species (ROS), which then attack their cell membranes, particularly the polyunsaturated fatty acids, leading to lipid peroxidation and microbial inactivation (Brié et al., 2018; Xue et al., 2023; Epelle et al., 2023; Elvis and Ekta, 2011; Agarwal et al., 2020; Moraes et al., 2021; Premjit et al., 2022; Roth et al., 2023; Neves et al., 2023; Pagès et al., 2020; Rangel et al., 2021). This effect is more evident in humidified ozone applications, where spore swelling occurs more severely, enhancing the passage of ROS and resulting in the collapse or rupture of the cell membrane (Epelle et al., 2023; Agarwal et al., 2020; Moraes et al., 2021; Premjit et al., 2022; Roth et al., 2023; Neves et al., 2023; Bhilwadikar et al., 2019; Tizaoui et al., 2022). When ozone is used against viruses, it damages their lipid envelopes and protein capsids, rendering them unable to infect hosts (Allison et al., 2009). This damage extends to the genome and RNA, impairing the virus’s ability to reproduce. Furthermore, the reaction with ROS can produce secondary reactive species, which can intensify the inactivation process (Farooq and Tizaoui, 2023).

A critical aspect of our study was evaluating the impact of environmental conditions on ozone’s efficacy. Previous studies have found mixed effects of microclimate on O₃ (Pironti et al., 2021; Blanco et al., 2021; Grignani et al., 2020; Hudson et al., 2009). Our findings indicate that gaseous ozone maintains its bactericidal properties across various temperatures and humidity levels. Specifically, ozone was slightly more effective at colder temperatures (~4°C) compared to ambient temperature (~21–22°C), and higher humidity levels modestly improved killing, particularly for MRSA, though these differences were not statistically significant. Our findings are consistent with those of De Caro et al., which showed mild impacts of humidity and temperature on the bactericidal activity of O₃ against E. coli (Pironti et al., 2021). Furthermore, we observed a slight but nonsignificant increase in microbicidal efficacy at higher humidity levels compared to low humidity, aligning with previous reports demonstrating maximal antiviral O₃ activity against COVID-19 at high humidity (Hudson et al., 2009). Collectively, these results suggest that ozone can be an effective disinfectant in diverse environmental settings, including refrigerated environments. This is particularly relevant to the food industry, which continually combats mold and bacterial growth in walk-in refrigerators and refrigerated trucks where food is stored and transported. Regular low-dose gaseous ozone application to these spaces could reduce microbial contamination, prevent food spoilage, and result in cost savings.

The effectiveness of ozone against a broad range of clinical isolates, including A. baumannii, P. aeruginosa, and S. marcescens, underscores its potential application in healthcare settings to combat healthcare-associated infections (HAIs). The growing threat of MDR infections is difficult to overstate, with projections estimating 10 million deaths annually by 2050 if infection trends continue unchecked (O’Neil, 2014). Of the 22 new antibiotics approved between 2012 and 2022, only two were considered first-in-class, or novel, antibiotics (García-Castro et al., 2023), and five are no longer available (McKenna, 2020). Despite standardized cleaning protocols, a previously room occupancy by infected patients remains a major risk factor for HAIs due to the ability of MDR pathogens to leverage virulence factors that facilitate their persistence in highly inhospitable healthcare environments (Mitchell et al., 2023). Given the dwindling supply of antibiotics and limitations of current disinfection methods, ozone offers a promising alternative that could be integrated into infection control protocols in a cost-effective manner.

While gaseous O₃ cannot completely replace manual cleaning, we envision its use in conjunction with current treatments to reduce the risk of HAIs and eliminate environmental reservoirs of nosocomial pathogens. O₃ could be particularly useful in disinfecting hard-to-reach spaces or areas where manual cleaning is impractical. As a proof of concept, we conducted a pilot study in which an ozone generator was placed in a fitness studio. Three one-hour exposures to 0.5 ppm gaseous O₃ on successive evenings reduced microbial counts by 2–3 log₁₀-fold across multiple surfaces. These findings support the practical application of ozone and suggest it could be implemented in clinics, schools, and other high-risk settings.

While our study provides compelling evidence for the use of low-dose gaseous ozone as an environmental disinfectant, it also highlights areas for future research. Long-term studies assessing the safety and efficacy of continuous ozone use in occupied spaces are necessary to establish implementation guidelines. Additionally, exploring the synergistic effects of ozone with other disinfection methods could further extend its utility in various settings.