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 19th 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 DNA68,69 (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