Biozone Manufacturing

Ozone effect on multi drug resistanT pathogens and clostridioides difficile spores


( Extract from https://www.nature.com/articles/s41598-022-18428-w.pdf)

Ozone produced by a DBD plasma device showed the ability of effective decontamination for MDROs and C. difficile spores, which is known as the main cause of healthcare-associated infections. Also, in our study, considering that environmental contamination of MDROs and C. difficile spores could be a source of healthcare-associated infection, the sterilisation effect of ozone was successful on materials that are mainly used in hospital facilities. After artificially contaminating materials, such as stainless steel, cloth, glass, plastic and wood with MDROs and C. difficile spores, a decontamination test was conducted with the DBD plasma device. As a result, although there was a difference in the decontamination effect depending on the materials, it showed ozone had significant ability for decontamination.

High-touch objects in hospital rooms warrant routine low-level disinfection. The standard decontamination of such objects is manual cleaning with a liquid disinfectant, such as a quaternary ammonium compound. Even if strict adherence to the recommended disinfectant application is carried out, MDROs are difficult to remove by conventional environmental cleaning, which is typically by manual cleaning. Therefore, new technologies, such as no-touch methods, are desirable. As a result, there has been an interest in gaseous disinfectants including hydrogen peroxide and ozone10. The advantage of gaseous disinfectants can reach places and objects inaccessible to conventional manual methods. Hydrogen peroxide has recently been used in healthcare settings; however, hydrogen peroxide is toxic in itself and should be dealt with according to strict handling procedures. Plasma sterilisation with hydrogen peroxide requires a relatively long purging time before the next sterilisation cycle. By comparison, ozone can be applied as a broad-spectrum antimicrobial that is effective against bacteria and viruses that can resist other disinfectants.  Moreover, ozone can be produced inexpensively with ambient air and does not require any additional toxic chemicals that may leave detrimental footprints on the environment, as it eventually decomposes into oxygen. Nevertheless, the reasons why ozone has not been widely used for disinfectant are as follows. Ozone is toxic to human health, thereby limiting its concentration below 0.07 ppm, averaged over an 8-h period so that ozone sterilisers have been developed and commercialised mainly for cleaning waste air. There is also the possibility of inhalation of the gas and an unpleasant odour being present after decontamination.  Ozone has not been actively used in healthcare settings yet. However, ozone can be used safely using sterilisation chambers and proper ventilation procedures, and the use of a catalytic converter can significantly speed up its removal. In this study, we demonstrated that a plasma ozone steriliser can be used for sterilisation of healthcare settings. We developed a device that had high sterilisation power, is easy to handle and had a rapid turnaround time for in-patient accommodations. In addition, we developed a sterilisation device with a simple structure that does not incur additional costs using ambient air. To date, there is no sufficient information on minimal ozone requirements for MDRO inactivation. The device used in our study had a simple configuration and short running-time, which is expected to be useful for sterilising equipment frequently.

Ozone’s mechanism of action for sterilisation has not been fully understood. Some studies have suggested that ozone destroys bacterial cell membranes, causing intracellular leakage and eventually cell lysis. Ozone may disrupt cellular enzyme activity by reacting with thiol groups, and it may modify purine and pyrimidine bases in nucleic acids. In this study, the morphologies of VRE, CRAB and C. difficile spores before and after ozone treatment were visualised and revealed not only the size had been shrunk but also the surfaces were significantly roughened, indicating damage or corrosion of the outermost membrane and inside materials due to the strong oxidising power of gaseous ozone. Such damage leads to cell inactivation depending on the severity of the cell alterations.

C. difficile spores are known to be difficult to eliminate from hospital environments. The spores have long-term persistence in areas where they are shed.  Also, in this study, although the maximum log10 reduction in counts on agar plates was 2.73 when ozone was used at 500 ppm for 15 min, the sterilisation effect of ozone on various materials for C. difficile spore decreased. Therefore, different strategies could be considered to reduce C. difficile contamination in healthcare settings. It may also be useful to adjust the exposure time and intensity of ozone treatment with application only in C.difficile isolation rooms. And, we should remembered that ozone decontamination method cannot totally replace routine manual cleaning using disinfectant and antimicrobial polices also can be very effective for controlling C. difficile5. The effectiveness of ozone as a steriliser varied among different types of MDROs in this study. Effectiveness might depend on several factors, such as growth stage, the cell envelope and the efficiency of repair mechanisms. The reasons for differences in the effectiveness of ozone sterilisation on the surface of each material may be relate to the formation of a biofilm. Previous studies have shown that A. baumanni and E. faecium confer increased environmental tolerance when existing as a biofilm. Nevertheless, this study showed that ozone has a significant sterilisation effect on MDROs and C. difficile spores.

The limitation of our study is that we evaluated the ozone antiseptic effect after re-recultivation. It might result in overestimation of the number of survived bacterial cells.

Although this study was conducted to evaluate the effectiveness of ozone as a steriliser in a hospital environment, it is difficult to generalise our results to all hospital settings. Therefore, more studies are needed to examine the applicability and compatibility of this DBD ozone steriliser in actual hospital settings.


The ozone generated by a DBD plasma reactor can provide a simple and valuable decontamination tool for MDROs and C. difficile. Ozone treatment may therefore be regarded as a valid alternate means of hospital environment disinfection.