Ozonized water – Background, General Use in Medicine and Preclinic Support

The industrial applications of ozone exploit its antiseptic properties making it very useful in the disinfection of water, both for its purification and for the treatment of sewage (Fig. 1). It is more effective than chlorine and also eliminates the viruses that are able to survive in presence of high concentrations of chlorine. In October 1893, in Holland (Ousbaden), the first system of water treatment with ozone was invented and realized and is currently extended to more than 3.000 municipalities, including some big cities, all over the world.¹

In 1902 the London practical dictionary of medicine contained some notes by J.H Clark who enthusiastically described the use of ozonized water (O3W), called Oxygenium, in the treatment of anemia, cancer, diabetes, flu, morphine poisoning and aphtha. The Swiss dentist E.A Fish (1899-1966) was the first medical doctor to exploit the benefits of ozone in local treatment. He treated his patients with ozone and O3W from 1932 obtaining good results in cases of gangrenous pulpitis with a simple injection of the gas. One of the patients he treated, was Dr. E. Payr (1871-1946) who immediately understood the usefulness of ozone and was very excited about its application in general surgery. In 1935, Dr. Payr published a work of 290 pages entitled Ozone Treatment in Surgery and presented it at the 59th Meeting of the German Surgical Society.

In terms of water purification, the use of ozone is also important in the treatment of the water used in medical environments. During the last decade nosocomial infections have become common because the resistance of pathogens to antibiotics has increased to a point where we no longer have an effective drug for some strains.

Recent studies suggest that environmental contamination plays a significant role in healthcare-associated infections and in the unrecognized transmission of nosocomial pathogens during outbreaks, as well as ongoing sporadic transmission. Several pathogens can persist in the environment for extended periods and serve as vehicles of transmission and dissemination in the hospital setting. Cross- transmission of these pathogens can occur via hands of healthcare workers, who become contaminated directly from patient contact or indirectly by touching contaminated environmental surfaces.

Less commonly, a patient could become colonized by direct contact with a contaminated environmental surface.² This is a complex problem, partly due to the extensive use of antibiotics in animal food and the improper use in patients. O3W, due to it high antimicrobial potential could eliminate the resistant bacteria³ and as consequence several thousand deaths could be avoided each year.⁴

In spite of a large use of chlorine, 4.5 billion people (6 out of 10 peoples in the world) do not have access to adequate water sanitation (Fig. 2).⁵ Unfortunately, chlorine has unsatisfactory organoleptic characteristics and it is being widely substituted by ozone all over the world.

Ozone is possibly an even more potent drinking water disinfectant able to inactivate several human pathogens, e.g. as many as 63 different bacteria (Salmonella, Shigella, Vibrio, Campylobacter jejuni, Yersinia enterocolitica, Legionella, Staphylococcus aureus,⁶ etc.), some 15 viruses (polio, echo, Coxsackie viruses, etc.), some 25 fungi and mould spores (Aspergillus, Penicillium, Trichoderma, etc.), several yeast varieties, and up to 13 fungal pathogens (Alternaria, Monilinia, Rhizopus, etc.).

More recently, due to contamination of groundwater with faecal material, the problem of disinfection has become more complex, since encysted protozoa, such as Giardia lamblia, Cryptosporidium parvum oocysts and helminth eggs (Ascaris suum and Ascaris lumbricoides), require a much longer time of contact with ozone than bacteria and viruses.⁴ Every year Cryptosporidium spp. causes outbreaks of sickness, which can be fatal for elderly and very ill patients (AIDS).⁷

We can define ozonized water as the water obtained after the ozonation (a process of infusing water with ozone) of water. During the ozonation of water there are no chemical reactions between ozone and pure water.

The only chemical reactions take place between ozone and organic, inorganic, or biological material present in the water. The solubility of ozone in water depends on different variables: pH, temperature, salts contents, and others. The half-life of ozone in water also depends on these variables.

The treatment of domestic well drinking water⁸ and waste waters⁹ is also an important application of ozone. Is estimated that today, over 1 100 water treatment plants around the world use ozone for disinfection. Ozone it has been used for water purification prior to chlorine and only its high cost has hampered its wider application.¹⁰

However, considering the mode of effect of the various oxidizing agents and the longer flow time through the biological activated carbon filters (as a result of the ozonation), ozone is – according to studies made by Grob and the Zurich Water Supply with ozonized Lake Zurich water – the most economical oxidation process with the best effect, i.e., without formation of toxic or carcinogenic substances.

The pre – and/or intermediate oxidation with ozone surely will be the right way in the foreseeable future.¹¹ Chemical and pharmaceutical industry also use ozone in order to treat residual water, as a bleaching agent,¹² and other applications.

Food industry and agricultural industry also employed successfully O3W. For example: Ozonation of water used for rinsing prevented fruits from consecutive contamination by pesticide residues present in water after several rinsing cycles. It was proven that applying O3W as a part of post-harvest treatment of apples may be beneficial for the fruit quality.¹³

A food toxin “Patulin” can be present in pear juice and especially in apple juice, this toxin can be destroyed by the action of ozone.¹⁴ In addition, the effect of ozonized water in Xanthomonas oryzae pv. oryzae, a seed borne bacterium causing leaf blight disease in Paddy (Oryza sativa) was study. Washing the paddy seeds with O3W would help control the bacterial blight of rice, the most serious disease of rice in Asia.¹⁵

Moreover, washing tomatoes (Lycopersicon esculentum Mill) with O3W 1 mg/L by 15 min, achieve an adequate disinfection.¹⁶ However, in case of tomatoes slices it is recommended to wash with 0.4 mg/L O3W for 3 min only.¹⁷

Salmonella inactivation by O3W washing was also demonstrated in tomatoes, lettuces and green onions. Mild heat and low pH improved inactivation efficacy on tomatoes and lettuces, but not on green onions. Processors should consider adjustments that are most appropriate for their produce.¹⁸

Ozone facilitates the extraction of some heavy metals from soil using ethylenediaminetetraacetic acid (EDTA). EDTA forms strong, water-soluble coordination compounds with some heavy metals (Pb, Zn) thereby making it possible to dissolve them out from contaminated soil. If contaminated soil is pre-treated with ozone, the extraction efficacy of Pb, Am and Pu increases by 11.0–28.9%,¹⁹ 43.5%²⁰ and 50.7%²⁰ respectively.

In aquaculture, O3W can be used to facilitate organic breakdown. Ozone is also added to recirculating systems to reduce nitrite levels²¹ through conversion into nitrate. If nitrite levels in the water are high, nitrites will also accumulate in the blood and tissues of fish, where it interferes with oxygen transport and causes oxidation of the heme-group of haemoglobin from ferrous (Fe²⁺) to ferric (Fe³⁺), making haemoglobin unable to bind O₂.²² Despite these apparent positive effects, ozone use in recirculation systems has been linked to reducing the level of bioavailable iodine in salt water systems, resulting in iodine deficiency symptoms such as goitre and decreased growth in Senegalese sole (Solea senegalensis) larvae.²³

Ozonate seawater is used for surface disinfection of haddock and Atlantic halibut eggs against nodavirus. Nodavirus is a lethal and vertically transmitted virus which causes severe mortality in fish. Haddock eggs should not be treated with high ozone level as eggs so treated did not hatch and died after 3–4 days.²⁴

O3W is also used successfully in the disinfection of drinking water, high purity water for Pharmaceuticals industries, and swimming pools.^25,26 The use of ozonized seawater to reduce and eliminate bacterial pathogens in mariculture facilities and to extend shelf life of marine food products is demonstrated. After treatment, Vibrio counts and shrimp disease were eliminated, ozonized seawater decreased the time required for normal molting of shrimp and the total growth cycle was reduced by three days versus control water.²⁷

General applications in medicine

The main medical applications of O3W are: the washing of wounds, surgical areas, ulcers and to eliminate pus and clean necrotic areas, as a mouthwash for periodontitis, gingivitis, and for the washing of cavities during surgical operations. When swallowed, it can be used in the treatment of gastric ulcers, Helicobacter pylori,²⁸ Giardia lambliaor gastritis.

Another application of O3W is in dentistry. Those applications are supported by pre-clinical studies. One study examined the effect of O3W on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with O3W (4 mg/L) for 10 s. When the experimental dental plaque was exposed to O3W, the number of viable S. mutans remarkably decreased. O3W strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to O3W in vitro, almost no viable bacterial cells were detected. These results suggest that O3W should be useful in reducing the infections caused by oral microorganisms in dental plaque.²⁹

One study examined the effect of O3W against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with O3W, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, O3W had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). The study also compared the cytotoxicity against L-929 mouse fibroblasts between O3W and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with O3W, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl.³⁰

O3W was found effective in reducing the bacterial load in both the 24 h plaque and stimulated whole saliva samples, but it did not eliminate it completely. In addition, there was no statistically significant effect of the O3W rinse on the salivary flow rate, salivary calcium and protein concentration. As consequence O3W rinse can be an effective adjunct to tooth brushing and flossing to maintain plaque and salivary bacterial load.³¹In biofilms containing Actinomyces naeslundii, Streptococcus mutans and Lactobacillus paracasei O3W had a level of antimicrobial activity equivalent to chlorhexidine.³²

One study compared the antimicrobial efficacy of O3W and minocycline against mixed microbial cultures after their initial adherence to tooth surfaces. Teeth were incubated in saliva suspension containing reference strains of Streptococcus mitis, Fusobacterium nucleatum, Actinomyces naeslundii and Candida albicans. The teeth were immersed either in O3W or in minocycline solution. Sterile saline served as control. Adherent microorganisms were resuspended, serially diluted, cultured, and statistically analyzed. In comparison to the control group, O3W showed significant reduction of all four strains, whereas minocycline was effective only on two strains tested.³³

Different in vitro assessments demonstrate the antibacterial activity of O3W against microorganisms associated with illnesses such as abscesses, bacteremia, endocarditis and osteomyelitis, which were resistant to multiple drugs.³⁴ Significant reduction of the microorganisms Staphylococcus aureus,^35-37 Pseudomonas aeruginosa,^36,37 Enterococcus faecalis,³⁶ Candida albicans,³⁸ was also found. However, a study shown that the irrigation of infected human root canals with O3W, 2.5% NaOCl, 2% chlorhexidine and the application of gaseous ozone for 20 min was not sufficient to inactivate E. faecalis.³⁹ Moreover, a study comparing the effect of O3W, O3W with ultrasonication, NaOCl, and chlorhexidine in human primary root canals contaminated by E. faecalis, provided evidence of its antibacterial effect in vitro conditions, but NaOCl showed the best effects.⁴⁰

Water disinfection of dental unit waterlines (DUWL) using ozone was also study with excellent results. When ozone disinfection methods were compared to hydrogen peroxide/silver ion, best results were obtained using ozone in particular against Pseudomonas aeruginosa.⁴¹ In addition, a study of Acanthamoeba strains, isolated from DUWL shown high levels of disinfection in vitro by the application of ozone as disinfectant.⁴²

One study analysed the bactericidal and fungicidal activity of O3W in multidrug-resistant bacterial strains. The scope was the application of O3W during intraoperative ozone treatment for tissue protection against infection with pathogenic bacteria. O3W was useful to kill almost all cells of the bacterial and yeast strains tested after 30 s (60 clinical bacterial isolates were analysed). Effective action against Aspergillus brasiliensis spores required a longer time than those required in the case of bacterial cells or vegetative cells of yeast.⁴³

The effect of O3W in remineralizing artificially created initial enamel caries was also investigated using laser fluorescence and polarized light microscopy in an in situ study. Results shown that O3W can be used to remineralize incipient carious lesions, and it enhances the remineralizing potential of nano-hydroxyapatite thereby preventing the tooth from entering into the repetitive restorative cycle.⁴⁴

Because of the antiseptic properties of O3W, its efficacy in the treatment of wounds has been studied. An in vivo study in diabetic rats, showed that wounds irrigated with O3W revealed diffuse haemorrhage and increased blood vessel neoformation. There was no statistically significant difference in bone trabeculae neoformation. After seven and 14 days, the number of osteoclasts was higher in O3W groups than in those treated with pure water.⁴⁵ The use of O3W are also extended to keratomycosis⁴⁶ and contact dermatitis.⁴⁷

The effect of ozonated saline solution (O3SS) in was also testet in vitro⁴⁸and in vivo⁴⁹against deferments strains of Mycobacterium tuberculosis. The O3SS was found to produce bactericidal and bacteriostatic effects, including multiresistant strains. In addition, it was investigated the anti-biofilm potential of O3SS against biofilms of Staphylococcus aureus, a microorganism commonly implicated in wound infections. The results shown a 99 % reduction in bacterial numbers within 15 min of exposure. These results suggesting that O3SS may be suitable as an adjuvant therapy to treat patients as an instillation fluid for wound irrigation and sterilization.⁵⁰

The action mechanism of O3SS is complex and involved mediators such as hydroxynonenal (HNE) and H₂O₂ and the activation of the Nrf2 pathways. A study involved different non-toxic doses of O3SS ranging from 2 μM to 300 μM, in an in vitro wound scratch model by the use of human keratinocytes supports that hypothesis. The results showed that O3SS is able to improve in vitro wound healing by stimulating cell proliferation.⁵¹

Disinfectant effect of ozonized water. Preclinical support

The disinfectant effect of O₃W is a direct consequence of the presence of dissolved ozone in water. Different variables such as: pH, ion content, water temperature, exposure time, and ozone concentration may impact in the germicide effect. For example, a concentration of ozone of 0.1 mg/L yields a low impact in oral cavity bacterial control,³¹ however using 8 mg/L yielded good results.³⁶ Although the inhibitory and lethal effects of ozone on pathogenic organisms has been observed since the latter part of the 19^th century, the mechanisms for these actions have not yet been satisfactorily elucidated. Ozone is a strong germicide needing only a few micrograms per litter for measurable action. At low concentrations, ozone rapidly inactivates, virus, coliform bacteria, Staphylococcus aureus, Aeromonas hydrophilia, and many other microorganims.⁵²

Virus inactivation by ozone

Viruses are parasites at the genetic level, separated into families based on their structure, type of nucleic genome and mode of replication. Many virions contain a phospholipid envelope with glycoprotein spikes encasing the nucleocapsid which contains nucleic acids (DNA or RNA), and structural proteins (including enzymes).

Lipid-containing viruses are sensitive to treatment with ether, assorted organic solvents, and ozone, indicating that disruption or loss of lipids results in impaired or destroyed infectivity. Viruses containing lipid envelopes include the Herpes viridae a large family grouping the Simplex, Varicella-Zoster, Cytomegalovirus and Epstein-Barr viruses; the Paramyxoviridae (mumps, measles); the Orthonyxoviridae (influenza); the Rhabdoviridae (rabies); and the Retroviridae (HIV). The HIV virus has an outer envelope made of a double layer of lipids penetrated by proteins of several types encasing two molecules of RNA.⁵³

Many of the above viruses have complex, sometimes baffling life cycles and replicative strategies with progressions from host cell attachment of the virus particle, to penetration, uncoating of the viral envelope, synthesis of molecular components, and release of new generations of virions to the surrounding medium, most often through cell lysis. Many chronic viruses have eclipse phases alternating with phases of viremia, when waves of viral particles flood the bloodstream.

Viruses differ in their susceptibility to destruction by ozone. The resistance of polio virus type 2 was 40 times that of coxsackie AS,^54,55 and in an experiment using a continuous flow mixed reactor under controlled laboratory conditions, relative resistance in descending order was found to be: polio virus type 2, echovirus type 1, polio virus type 1, coxsackie virus type B5, echovirus type 5, coxsackie virus type A9. In pure water, at maximal solubility of ozone and room temperature, Echovirus type 29 is inactivated in one minute, polio virus type 1 in two, type 3 in three and type 2 in seven minutes.⁵⁵ The mechanism of enteroviral inactivation by ozone was investigated with poliovirus 1 (Mahoney) as the model virus. Ozone was observed to alter two of the four polypeptide chains present in the viral protein coat of poliovirus 1. However, the alteration of the protein coat did not significantly impair virus adsorption or alter the integrity of the virus particle. Damage to the viral RNA after exposure to ozone was demonstrated by velocity sedimentation analysis. It was concluded that the damage to the viral nucleic acid is the major cause of poliovirus 1 inactivation by ozone.⁵⁴ The inactivation rate of enteroviruses⁵⁶ is more rapid than for E. coli, takes place in relatively small concentrations of ozone, and is influenced by pH, temperature, and the presence of ambient organic compounds. On the basis of the measured second-order ozone inactivation rate constants, typical ozone exposures applied in water and wastewater treatment, are considering as a highly effective disinfectant for virus control.⁵⁷

In view of the above considerations, what part can ozone play as an antiviral agent? In one study,⁵⁴ polio virus 1 was exposed to 0.21 mg/L of ozone at pH 7.2. After 30 seconds 99 % of the viruses were inactivated (lost their ability to replicate within host cells), but appeared to maintain their structural integrity. Analysis of viral components showed damage to polypeptide chains and envelope proteins, which could result in attachment capability compromise, and breakage of the single-stranded RNA into two parts, producing replicating dysfunction at its root level. Other researchers⁵⁸ in similar experiments concluded that in ozonation, it is the viral capsid which sustains damage. It is to be noted however, that the polioviridae (Picornavirus family) contain four structural proteins encapsulating a single RNA strand and are devoid of lipids (Fig. 3).

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/L at pH 7 and 5 °C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log₁₀ within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods.⁵⁹

The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by ozone was compared at 4 °C by using single-particle virus stocks. Although the human strain was clearly more sensitive, both virus types were rapidly inactivated by ozone concentrations of 0.25 mg/L or greater at all pH levels tested. Comparison of the virucidal activity of ozone with that of chlorine in identical experiments indicated little significant difference in rotavirus-inactivating efficiencies when the disinfectants were used at concentrations of 0.25 mg/L or greater.⁶⁰

There is evidence of inactivation of some others virus by ozone as: Hepatitis A visus,⁶¹ papillomavirus (HPV),⁶² adenovirus, norovirus, sapovirus, parechovirus, hepatitis E virus, astrovirus, pecovirus, picobirnavirus, parvovirus, gokushovirus,⁶³ coxsackievirus B5 (CVF, CVEnv1, and CVEnv2), human adenovirus (HAdV), echovirus, bacteriophages (MS2, Qβ, T4, and Φ174),⁵⁷ bacteriophage lambda⁶⁴ human H5N1/H1N1 influenza viruses,⁶⁵ herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV),⁶⁶ and others.

Bactericidal effects of ozone

The most cited explanation for ozone’s bactericidal effects centres on disruption of envelope integrity through peroxidation of phospholipids and lipoproteins. There is evidence for interaction with proteins as well,⁶⁷ enzymes⁶⁸ and DNA^68,69 (Fig. 4).

The cell envelope of Gram-negative microorganisms such as E. coli is a complex multiplayer system composed of an inner cytoplasmic membrane made of phospholipids and proteins invaginating into the cytoplasm, a peptidoglycan layer, and an outer membrane of polymers such as polysaccharides. Gram positive cells have a less complex, three-layer envelope with a thick peptidoglycan middle layer.

In one study⁶⁹ exploring the effect of ozone on E. coli, evidence was found for ozone’s penetration of the cell membrane, reacting with cytoplasmic substances and converting the closed circular plasmid DNA to open circular DNA, which would presumably lessen the efficiency of bacterial proliferation. It is notable that higher organisms have enzymatic mechanisms to restabilize disrupted DNA and RNA, which could provide a partial explanation for why, in clinical treatment with ozone at doses prescribed, ozone appears to be toxic to infecting organisms and not to the patient.⁷⁰

Vibrio parahaemolyticus is a bacterium in the same family as those that cause cholera. It lives in brackish saltwater and causes gastrointestinal illness in humans. Vibrio parahaemolyticus contamination, causes serious foodborne illness, and it has become a global health problem. A study, shown that under low aqueous ozone concentrations (less than 0.125 mg/L), the bacterial cell membranes remained intact, and the ozone was detoxified by intracellular antioxidant enzymes (e.g., superoxide dismutase and catalase). Under high aqueous ozone concentrations (more than 1 mg/L), cell membranes were damaged by the degree of peripheral electro negativity at the cell surface and the concentration of lactate dehydrogenase (LDH) released into the extracellular space, and the ultra-structures of the cells were confirmed by transmission electron microscopy. O₃W penetrated the cells through leaking membranes, inactivated the enzymes, inhibited almost all the genes, and degraded the genetic materials of gDNA and total RNA, which eventually lead to cell death.⁶⁸

Staphylococcus aureus (S. aureus) belongs to the normal flora of the skin, mucosa and nasopharynx of several animal species, including man, but it is also associated to illnesses such as abscesses, bacteremia, endocarditis and osteomyelitis, and can show resistance to multiple drugs. O₃W reduces this bacterium.^35,36,50Treatment of Staphylococcus aureus infections, particularly that of methicillin resistant Staphylococcus aureus (MRSA), is a challenge in clinical practice. A study shown that 100 % S. aureus and 100 % MRSA were eliminated by O₃W (1.5 mg/L) in 1 min.⁶

Other bacteria that shown sensibility to ozone treatment are: Mycobacterium smegmatis,⁷¹ Mycobacterium tuberculosis,⁴⁸ Enterococcus faecalis,^36,40,72 Legionella pneumophila, Streptococcus faecalis, Streptococcus mutans,²⁹ Pseudomonas aeruginosa,^36,37,73 Helicobacter pylori,⁷⁴ Salmonella typhimurium,⁷⁵ Lactobacillus paracasei,³² Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans,⁷⁶ Actinomyces naeslundii, Streptococcus mitis and Fusobacterium nucleatum.⁶⁴

Fungicidal effects of ozone

Ozone possesses fungicidal effects, through poorly understood mechanisms. In one study, Candida utilis cell growth inhibition with ozone was greatly dependent on phases of their growth, budding cells exhibiting the most sensitivity to its presence.⁷⁷ In addition, with an increase in the ozone dose, neutral lipids (sterols) and nitrogen-containing phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) were modified to a greater extent.⁷⁸ The bacterial action of ozone consists mainly in damaging the surface cell structures.⁷⁹

Interestingly, in another study,⁸⁰ different doses of ozone were shown either to stimulate (1-2 x 10⁸ molecules O₃/cell) or inhibit (1 x 10⁹ molecules O₃/cell) respiration and reproduction of C. utilis yeast. The observed changes were preserved for several hours after treatment with ozone. Possible role of structural rearrangements of cell membranes in the ozone-induced effects has been involved. Moreover, low doses of ozone stimulated the growth and development of Monilia fructagen and Phytophtora infestans, while higher doses were inhibitory.⁸¹

The cell wall of fungi is multilayered and composed of approximately 80 % carbohydrates and 20 % of proteins and glycoproteins. The presence of many disulfide bonds making this a possible site for oxidative inactivation by ozone (Fig. 5). Ozone has the capacity to diffuse through the fungal wall, enters into its cytoplasm and disrupting vital cellular functions.⁸² The inhibitory effect of ozone on spore germination, spore production and biomass production in two Aspergillus species was also examined.⁸³

Dermatophytes are classified in three genera, Epidermophyton, Microsporum and Trichophyton. They have the capacity to invade keratinized tissue to produce a cutaneous infection known as dermatophytoses. Ozone is effective in killing keratinophilic dermatophyte fungi Trichophyton rubrum and Trichophyton mentagrophytes; two organisms commonly isolated as the etiological agent in onychomycosis and tinea pedis patients.⁸⁴ In addition, other study demonstrated that Microsporum gypseum and Microsporum canis were the most susceptible, while Trichophyton interdigitale and Trichophyton mentagrophytes were relatively resistant.⁸²

A study shown that Trichophyton rubrum, Trichophyton mentagrophytes and Candida albicans were shown to survive at least 123 days in chlorinated swimming-pool water of 28-30 °C, at least 18 days in O₃W at 34-35 °C, and at least 25 days in pipe water at room temperature of 23- 25 degrees.⁸⁵

C. albicans is an emerging multidrug-resistant fungal pathogen representing an important source of invasive disease in humans and generating high healthcare costs worldwide. This fungus is frequently found in different anatomical sites of healthy persons and could induce systemic and superficial infections under optimal environmental conditions. Invasive candidiasis, is an important nosocomial infection with high morbidity and mortality rates in hospitalized children. It represents a major source of prolonged infections in intensive care unit, particularly in immunosuppressed or elderly patients.⁸⁶ Ozone was highly effective in killing C. albicans,^38,64,87 but It seems that ozone therapy induces resistance to amphotericin B.⁸⁷ However, disinfection protocol combining chlorhexidine and ozone may act in synergic, promoting complete elimination of C. albicans.⁸⁸ Moreover, the combination of O₃W and ultrasonication had a strong effect on the viability of C. albicans adhering to the acrylic resin plates.³⁸