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Research Article | DOI: https://doi.org/10.31579/2688-7517/208
1 Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, 11884 Nasr, Cairo, Egypt.
2 Department of Food Chemistry and Metabolism, National Nutrition Institute, 16 Elkaser Alainy St. Cairo, Egypt.
*Corresponding Author: Khalifa Ali, Department of Botany and Microbiology, Faculty of Science (Boys), Al-Azhar University, 11884 Nasr, Cairo, Egypt.
Citation: Khalifa Ali, Mohammed G. Barghoth, Said E. Desouky, Hanaa H. Elsayed, Gleisy Kelly N. Gonçalves, (2024), Highly Efficiently Inactivation of Microbial Pathogensusing Advanced Ozone Generator Unit as An Eco- Friendly Promising Strategy, J. Pharmaceutics and Pharmacology Research, 7(9); DOI:10.31579/2688-7517/208
Copyright: © 2024, Khalifa Ali. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 17 May 2024 | Accepted: 04 June 2024 | Published: 02 September 2024
Keywords: ozone treatment; microbial elimination; s. aureus; b. subtilis; b.spizizenii; e. coli; p.aeroginosa; c. albicans
Searching for an alternative disinfection and sanitization strategy to control and prevent the contamination and diseases caused by microbial pathogens represents one of the critical challenges for all world governments. So that the antimicrobial efficiency of ozone gas as a terminal disinfectant was estimated at a relatively small level (1.2 mg/l/h) using a unit that was generated as a local unit assembled at the faculty of science against six reference strains including Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichia coli (ATCC 8739), Pseudomonas aeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) for 10, 20, 30 and 40 minutes under laboratory conditions. After 10 min of ozone treatment, the log reduction of cell viability was 97.15%, 59.25%, 24.20%, 24.09%, 14.50 %, 13.47%, and 0.46% for P. aeroginosa, strains combination, E. coli, C. albicans, B. spizizenii, B. subtilis, and S. aureus, respectively. The twenty-minute exposure to ozone resulted in a reduction in microbial viability percent 98.17%, 82.88%, 69.63%, 62.79%, 49.43%, 29.57%, and 28.08%, for P. aeroginosa, strains combination, B. subtilis, and E. coli, C. albicans, S. aureus, and B. spizizenii, respectively. The efficacy of ozone for P. aeroginosa, E. coli, and strains combination increased by more than 98% after 30 min of ozone treatment followed by 90.41%, 86.76%, 52.63%, and 36.64% for B. subtilis, C. albicans, B. spizizenii, and S. aureus, respectively. The maximum ozone efficacy reached 100% for all reference stains except B. spizizenii (62.10%) after 40 min of ozone treatment making this strategy a candidate tool recommended for the management and control of the pathogenic microorganisms.
Effective sanitation and cleaning of surgical materialsand wastes as well as food manufacturing equipment are major challenges in the medical and industrial fields. Due to the inappropriate cleaning method, the contamination by pathogenic microorganisms increased dramatically during the last decades raisingsubstantial public healthconcerns Silva et al., (2021).Disinfection used in the medical and industrial fields has typically been accomplished using heat, such as hot wateror steam, or traditional chemicals, such as chlorinethat are completely dissolved in water and easy to use at low cost. Unfortunately, not only are there growing environmental implications about the presenceof chemical by-products such as halo-organics created when liquidchemicals are employedas disinfectants but there are also developing public health problemssuch as respiratory tract and skin irritation (Alvaro et al., 2009; Selma et al., 2008; Shen et al., 2012).
The kind of disinfecting agent for using is determined by the nature of microorganisms to be destroyed as well as the quality of the cleaningequipment. If the sanitizing agent is ineffective, the microorganisms multiply and accumulate again on the equipment surfaces (Dosti et al., 2005). Among the new disinfectant strategies, ozone (O3) appearsto be a viable methodfor preventing microbiological contamination by spoilageor microbial pathogens. Ozone, in fact,has a significant antimicrobial activity and is considered an environmentally beneficial technology because of its low environmental impact (Bigi et al., 2021).
Ozone is widely applied in several applications as a disinfectant including; treatment of food plant wastes, reuse of waste water, sterilization of food plant equipment’s, food surface hygiene and dairy industry (Graham, 1997; Panebianco et al., 2022). Ozone is the second strongest oxidizing agent after fluorine (Miller et al., 1978). Because of its high oxidation capability, ozone is particularly effective at killing microorganisms. Ozone exhibited biocidal effects on a wide range of species, including Gram- positive and Gram-negative bacteria, spores, and vegetative cells (Guzel-Seydim et al., 2004; Lezcanoetal., 1999). Ozone has also been shown to kill a variety of viruses, including the hepatitis A, influenza A, and others (Dosti et al., 2005).
For practicaluses of ozone, different generation techniques are available, with the electrically andphotocatalytic (UV) techniques being the most widespread for instance, in the electric corona discharge process, in which the oxygen molecules pass through an electrical field between two electrodes and split (Brodowska et al., 2018). Ozone can also be produced by ultraviolet radiation, which involves the passing of oxygen gas molecules through a high-energy short-wave UV light (Baggio et al., 2020).
Chemical, thermal,chemo-nuclear, and electrolytic processes are among the othermethods of ozone generating (Varga& Szigeti2016).
Based on the Environmental Protection Agency, ozone is more effective than chemicals in the disinfection process because it has a faster reaction time and does not allow bacteria to regrowth again. The residual ozone breaks down rapidly into oxygen and therefore does not pose a real threat (EPA, 1999). Also, no toxic by- productsare found during the ozone treatment process, in comparison to the generation of disinfection by-products (DBP) during using chemicals such as chlorination, which may be mutagenic and carcinogenic. Moreover, the resistances of some organisms to disinfectants, as well as the demandfor considerably increased doses for microbial inactivation, are two of the most critical problemsin disinfection (Gomes et al., 2019; Greene et al., 1993).
Thus, the purpose of this investigation is to evaluate the action of direct application of ozone gas generated through advanced units against several bacterial species and yeast actingas pathogenic and spoilage organismsincluding; S. aureus,
B. subtilis, B. spizizenii, E. coli, P. aeroginosa, and C. albicans. Also, to evaluatethe optimal time and the efficacyof ozone gas required to attenuate or control the growth of these pathogens.
Ozone was generated during this study based on plasma technology through a new design reactor called Dielectric Barrier Discharge plasma reactor (DBD plasma reactor). In this unit, plasma is produced over the dielectric barrier's surfaceand along the perimeter of the top electrode that is exposedto the surrounding air as represented in Figure. [1].
This device was designed and manufactured by Prof. Dr. Safwat Hasaballah at the Faculty of Science, Al-Azhar University. As follows, the two copper electrodes were placed around the outer plastic tube and the inner electrode of the dielectric material (composed of hydrocarbon/ceramic RO4350B™).The thickness of the outer Cu+2 wire
electrode is 0.2 mm and the length of the inner metal electrode is 60 cm. An alternating potentialdifference between the two electrodes is used to create DBD plasmaaround the outer Cu+2 wire electrode that is connected to the higher potential (0–10 kv, 50Hz) and exposed to air. Plasma does not form on the bottom side of the reactor because the inner electrode is grounded and covered by a layer of Kapton tape (Portugal et al., 2017; Choudhury et al., 2018).
Reference strainsthat were used during this study includedGram- positive bacteriasuchas Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 9372,and Bacillus spizizenii ATCC6633 as well as Gram-negative bacteria represented in Escherichia coli ATCC 8739, and Pseudomonas aeroginosa ATCC 9027 while unicellular yeast represented in Candida albicans ATCC 10231. Bacterial strains were seeded on nutrient agar plates and incubated for 24 h at 37 °C while C. albicans was cultured onSabouraud dextrose agar at 30 °C for 24 h.
Separate colony of each referencestrains was transferred from the agar medium to 300 mL of the Muller Hinton broth and allowed to multiply at 37°C for 24 hours. Cells cultured in Muller Hinton brothmedium were collected at room temperature using centrifugation at 3000 ×g for 10 min. The supernatantwas removed and the cells were harvested and re-suspended in the phosphate-buffered saline (PBS; 0.07M, pH 7.0) until the bacterial inoculum density was equal to 2 McFarland (2MF) approximately 6 x 108 cells/ml when measured spectrophotometrically at absorbance 620 nm (CLSI, 2018; Sowhini et al.,2020). TwoMcFarland standard was prepared by mixing 0.2 mL of
1.175 percent barium chloridedihydrate (BaCl2.2H2O) with 9.8 mL of 1 percent sulfuricacid (H2SO4) (Zapata& Ramirez-Arcos,2015).
Ozone createdby the plasma reactor was transferred to a beakercontaining sterile broth media and inoculated with 2 McFarland (6 x 108 CFU/ mL) of the reference test organisms. At pH 6–8 and room temperature 20–25 oC, 50 ml of bacterial suspension was exposed to 1.2 mg/l/h of ozone dosage for interval times ranging from 10 to 40 minutes. The experiments were conducted for each strain alone and in combination for all testorganisms with each other. After each test was measured, 180 µL of ozonated bacterial suspension was kept in sterile Eppendorf tubes until the end of the experiments César et al.,(2012).
The collected Eppendorf tubes were incubated for 30 min at 37 oC to make sure that the ozone was completely consumed before adding the indicator dye (resazurin). Then 20 µL of resazurinsolution was added to each Eppendorf tube and incubated for 4 h at 37oC. All experiments were conducted in triplicate for each test organism Morganet al., (2009).
Resazurin solution (7-Hydroxy-3H-phe-noxazin-3-one 10-oxide) was prepared by dissolving 337.5 mg of this dye in 50 mL of sterile distilled water and homogeneity obtained by mixing for 1 hour in a sterile vortex mixer. The preparation processes werecarried out in the dark, and the resazurin solutionwas maintained in a dark bottle to avoid light exposure (Teh et al., 2013). The color of resazurin dye was converted from blue topink or red fluorescent material (resorufin) by oxidoreductase within live bacterial cells that were used to determine the antibacterial impact of ozone. The change in resazurin color was measured at 570 nm and 600 nm (Pettit et al., 2005; Morgan et al., 2009).
Data analysiswas performed using ANOVA and the Tukey test after converting the CFU/ml countsto a logarithmic. A statistically significant difference was defined as a P value of less than 0.05. Bycomparing the groups that were sterilized with ozonated broth media for 10, 20, 30, and 40 minutes to the control group, the hundredpercent reductions in CFU/mL for each microorganism investigated was calculated (César et al., 2012). Two control groups were used during this study firstly negative group: contained 180 µL of sterile nutrient broth media and 20 µL indicator, without a test organism in which the color of dye remained blue even at the end of the experiment. Secondlypositive group: contained180 µL of inoculated nutrient broth media and a 20 µL indicator of 180 µL without exposure to ozone in which the color of the dye was changed to pink at the end of the experiment.
Managing and controlling microbialcontamination are represented as one of the main challenges in all life aspects, especially in the medicalfield. Maintaining a suitablehygienic environment is critical to prevent the dispersion and contamination formed by microbial pathogens. Also searching for advanced strategies to preventmicrobial contamination or pathogenic bacteriarepresents one of the largestchallenges facing all industrial and medical branches in the following upcoming years. Therefore, newly ozone designed unit was used to generate ozone gas with a low dosage of 1.2 mg/l/h in an aqueous solution.
The antimicrobial action of generated ozone was studied on six microbial reference strains including Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichia coli (ATCC 8739), Pseudomonas aeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) for 10, 20, 30 and 40 minutesas represented in Table 1. The initial microbialconcentration was adjustedat 2 MF approximately equal to 6 x 108 CFU/ ml or 8.76 ±0.02 log number of cells.
For E. coli (ATCC 8739), there are statistically significant differences observed after ozone exposurefor 10 min up to 40 min if comparedwith the controlgroup. The obtained results showed that the viability of E. coli cells gradually decreased after 20 min until disappeared of bacterial cells, this indicated that the activityof ozone is based actuallyon the exposure time duration.A significant effect
of ozone at a concentration of 2 mg/l/h was used to remove E. coli cells in biofilms on lettucesurfaces as reportedby Ölmez & Temur (2010). Moreover, the effective inactivation of E. coli O157:H7 by ozonated water dose 35 and 45 mg L-1 for 0, 5, 15, and 25 min was studied by Souza et al. (2019). Also, Mohammadet al. (2019) demonstrated that the mean log reductions of Shiga toxin-producing E. coli when treated with 5 mg/l/h of aqueous ozone after 10, 15, and 20 min, were found to be 1.5 ± 0.4, 1.6 ± 0.4, and 2.1 ± 0.5, respectively.
The same results were obtained for Pseudomonas aeroginosa (ATCC 9027) except that the log- transformed CFU/ml was rapidly full down during the first 10 min of ozone exposure from log 8.76±0.02 to 0.25±0.02 and these results indicated that the highest effectiveness of ozone on P. aeroginosa occurred at short exposure time [Table 1]. These results are full consistence with data reported by Dosti et al. (2005) they found that the largest log reduction of P. fragi (ATCC 4973), P. putida (ATCC 795), and P. fluorescens (ATCC 948) was produced after the 10 min of ozonation process. Our observation is greater than the results observed by Marino et al. (2018) who studied the effectiveness of ozonated water instatic and dynamic conditions to demonstrate the sensitivity of P. fluorescens to ozone treatments. After 60 minutesof treatment, the authors found that the gaseous ozone at 20 ppm reducedthe number of P. fluorescens by 5.51 log CFU/cm2 (Marino et al., 2018).
On the other hand, no statistically significant differences were observed by
S. aureus (ATCC 6538) after 10 min of exposure to gaseous ozone this may be due to the bacterial resistance or the duration of ozone dose not being enough to reduce the number of bacterial cells. The higher reduction of S. aureus cells was obtained after 30 min of ozone exposureand this confirmedthat the reduction of bacterial cell numbers was dependent on the ozone exposure duration and this indicated that the greater log reduction was obtained after 30 min of treatment than 10 and 20 min [Table 1].
| Table 1 Effect of ozone on reference strains suspended in broth culture at different experimental times. | ||||||
| Reference Strains | Groups | Mean ± SD. | Minimum | Median | Maximum | (p) |
| E. coli (ATCC 8739) | Control | 8.76a ±0.02 | 8.74 | 8.76 | 8.78 | (<0> |
| 10 min | 6.64b ±0.09 | 6.53 | 6.67 | 6.71 | ||
| 20 min | 3.26c ±0.75 | 2.40 | 3.59 | 3.78 | ||
| 30 min | 0.06d ±0.01 | 0.05 | 0.06 | 0.07 | ||
| 40 min | 0.0d ±0.01 | 0.0 | 0.0 | 0.01 | ||
| S. aureus (ATCC 6538) | Control | 8.76a ±0.02 | 8.74 | 8.76 | 8.78 | (<0> |
| 10 min | 8.72a ±0.01 | 8.71 | 8.72 | 8.73 | ||
| 20 min | 6.17b ±0.10 | 6.07 | 6.17 | 6.27 | ||
| 30 min | 5.55c ±0.04 | 5.52 | 5.53 | 5.60 | ||
| 40 min | 0.03d ±0.01 | 0.02 | 0.03 | 0.04 | ||
| P. aeroginosa (ATCC 9027) | Control | 8.76a ±0.02 | 8.74 | 8.76 | 8.78 | (<0> |
| 10 min | 0.25b ±0.02 | 0.23 | 0.25 | 0.27 | ||
| 20 min | 0.16c ±0.03 | 0.13 | 0.17 | 0.19 | ||
| 30 min | 0.03d ±0.03 | 0.01 | 0.02 | 0.07 | ||
| 40 min | 0.0d ±0.0 | 0.0 | 0.0 | 0.0 | ||
| C. albicans (ATCC 10231) | Control | 8.76a ±0.01 | 8.75 | 8.76 | 8.77 | (<0> |
| 10 min | 6.65b ±0.03 | 6.63 | 6.65 | 6.68 | ||
| 20 min | 4.43c ±0.02 | 4.41 | 4.43 | 4.45 | ||
| 30 min | 1.16d ±0.05 | 1.11 | 1.15 | 1.21 | ||
| 40 min | 0.21e ±0.02 | 0.19 | 0.20 | 0.23 | ||
| B. spizizenii (ATCC 6633) | Control | 8.76a ±0.01 | 8.75 | 8.76 | 8.77 | (<0> |
| 10 min | 7.49b ±0.01 | 7.48 | 7.49 | 7.50 | ||
| 20 min | 6.30c ±0.01 | 6.29 | 6.30 | 6.31 | ||
| 30 min | 4.15d ±0.03 | 4.12 | 4.15 | 4.17 | ||
| 40 min | 3.32e ±0.42 | 3.01 | 3.14 | 3.80 | ||
| B. subtilis (ATCC 9372) | Control | 8.76a ±0.02 | 8.74 | 8.75 | 8.78 | (<0> |
| 10 min | 7.58b ±0.02 | 7.56 | 7.59 | 7.60 | ||
| 20 min | 2.66c ±0.05 | 2.62 | 2.64 | 2.71 | ||
| 30 min | 0.84d ±0.05 | 0.79 | 0.84 | 0.89 | ||
| 40 min | 0.0e ±0.0 | 0.0 | 0.0 | 0.0 | ||
| Combination of all referencestrains | Control | 8.76a ±0.02 | 8.74 | 8.75 | 8.78 | (<0> |
| 10 min | 3.57b ±0.05 | 3.51 | 3.59 | 3.60 | ||
| 20 min | 1.50c ±0.01 | 1.49 | 1.50 | 1.51 | ||
| 30 min | 0.11d ±0.02 | 0.09 | 0.11 | 0.13 | ||
| 40 min | 0.0e ±0.0 | 0.0 | 0.0 | 0.0 | ||
| SD: Standarddeviation, means in the same column with small commonletters are not significant at p ≤ 0.05 (i.e., Means with Different letters are significant) | ||||||
S. aureus was highly sensitive to ozonation treatments under dynamic conditions, according to Marino et al. (2018), while contact with gaseous ozone at high doses (20 ppm) resulted in a lowering of 4.72 Log CFU/cm2 of S. aureus biofilm.
Genus Bacillus is one of the most significant spore-forming bacteria, with considerable variety and adaptation to many environmental conditions, as well as the capacity to attach and survive on a variety of surfaces and the ability to producesome biofilm forms(Shemesh & Ostrov, 2020).They are easilycapable of formingresistant spores that can surviveafter the conventional cleaning methods that increased the additional disinfectant compounds and raised the cost during the lastfew yearsto manage the microbial contamination formed by spoilage or pathogenic microorganisms (Panebianco etal., 2022).
The bactericidal action of gaseous ozone on Bacillus subtilis (ATCC 9372) and Bacillus spizizenii (ATCC6633) demonstrated that lowering reductionin log- transformed CFU/ml 7.58 ±0.02 and 7.49 ±0.01, respectively after the first 10 min of ozone exposure comparing in Contrast to the control group 8.76 ±0.02. Both strains showed some resistance toward ozone gas for the first 30 min of contact with ozone followed by a quick fall down in the bacterial cell numbers and this was revealed primarily by B. subtilis thenB. spizizenii and this may be due to the presenceof sporulation character of Bacillus genus and hence it required for prolongation of ozone gas exposure also the results indicated that the 30 min of ozone treatment is effective sanitization method to destroy these pathogens
All of the treatments after 10 min of Bacillus exposure differ substantially from the controls. The presence of spores may decrease ozone's efficiency. Ozone penetration is likely prevented by the multilayer structure of spores' outer coat. Bacillus cereus and Bacillus megaterium spores were found to be 10–15 times more resistant to ozone than their vegetative cells as determined by Broadwater et al. (1973). Ozone did not considerably reduce the number of Bacillus subtilis spores, according to the data recorded by Herbold et al. (1989). The same results were obtained by Dosti et al. (2005) who noted that the response of B. licheniformis, was found after 10-min of ozonation treatment. Also, the effect of ozonated water on B. cereus biofilms was evaluated by Babu et al. (2016) on dairy processing membranes and they found that theaverage reduction in treat membranesis 1.0 log CFU/cm2.
The antimicrobial nature of ozone on Candida albicans (ATCC 10231) showed a statistically significant difference relative to the controlgroup after 10 min of exposure (6.65±0.03) and the largest reduction of Candida cells obtained after passing 20 min of ozone exposure. The effect of ozonated solution against the combination of all reference strains showed the reduction of microbial quantitiesfrom 8.76± 0.02 to 3.57±0.05 during the first ten minutes. The complete microbial reduction obtained after 30 min of exposure indicated the higher effectiveness of ozone against the collection of several strains of microbial pathogens.
The efficacy of ozone againstthe reference strains based on the mean logarithmic reduction concerning the control group after 10 min of exposure to 1.2 mg/l/h of ozone dose was found to be 97.15%, 59.25%, 24.20%, 24.09%, 14.50 %, 13.47%, and 0.46% for P. aeroginosa, strains combination, E. coli, C. albicans, B. spizizenii, B. subtilis, and S. aureus, respectively and this indicated that the largest reduction percent was observed by P. aeroginosa while the smallest was observed by S. aureus[Fig. 2].
Figure 2: the efficacy of ozone treatment against the reference strains after 10 min of exposure.
Whereas the highest reductionpercent achieve after 20 min of ozone treatment was 98.17%, 82.88%, 69.63%, 62.79%, for P. aeroginosa, strains combination, B. subtilis, and E. coli, respectively, and the smallest was found at 49.43%,29.57%, and 28.08%,for C. albicans, S. aureus,and B. spizizenii, respectively [Figure. 3].
Figure. 3 the efficacy of ozone treatment against the reference strains after 20 min of exposure.
After passing 30 min of ozone treatment the efficacy of ozone toward reference strainsincreased by more than 99% for P. aeroginosa and E. coli andmore than 98% for strainscombination followed by 90.41%, 86.76%,52.63%, and 36.64% for B. subtilis, C. albicans, B. spizizenii, and S. aureus, respectively [Figure. 4].
Figure 4: the efficacy of ozone treatment against the reference strains after 30 min of exposure.
Furthermore, the efficacy of ozone reachedthe maximum reductionyield (100%) for all reference stainsexcept B. spizizenii (62.10%) after exposure to ozone for 40 min [Figure. 5].
As a result, the net log reduction in the cell viability of the reference strains was found to be largest after 30min for P. aeroginosa and E. coli and 40 min for the other strains.
Recently ozone has an expansiveantimicrobial range, and each microorganism speciesis responsive to the gas in a different manner.Control of fungi,Gram-negative and Gram-positive bacteria, and viruses have all been investigated using the ozonationprocess (Alwi & Ali 2014; Khadreet al., 2001). Microorganisms are inactivated by ozone throughthe rupture of the cellularmembrane and subsequent dispersion of internal cytoplasmic components due to the higher oxidative potential of thisgas. Ozone interacted with the cellular component such as unsaturated fatty acids, nucleic acids, cell wall constituent’s enzymes, and other proteins (Sanchez et al., 2016). Based on Manousaridis et al.'s observation, the antimicrobial effect of ozone in aqueous media is attributed to their dissociation into molecular ozone, superoxide, and hydroxyl, as well as hydroperoxyl radicals (Manousaridis et al., 2005). However, in the presenceof moisture, the harmful effectsof ozone are hypothesized to be enhanceddue to the increase of these radicals’production
(Kowalski et al., 1998).
Our findings indicate that Gram-negative bacteria are more susceptible to gaseous ozone than Gram-positive bacteria similar to the observation reported by Moore et al. (2000) who concluded that the Gram-negative bacteria were significantly moresensitive than the Gram-positive bacteria than the yeast strain after being exposed to
Throughout this study, comparatively small concentrations of gaseous ozone exhibited good antimicrobial capabilities when applied to pathogenic microorganisms. Exposure of six reference strains included Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichiacoli (ATCC 8739), Pseudomonasaeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) to ozone gas at the level of 1.2 mg/l/h for 40 min resulted in at least 62 to 100% reductionof the viability of the test organisms. The higher efficiency of the ozone strategy used in this study appeared well among the combination of the strains with 98% and 100% reduction of cell viability after 30 and 40 min, respectively. Gaseous ozone could be utilized as a successful disinfectant agent in different fields, according to the findings of this study, if applied after regular cleaning. Although it is recommended ozone as an effective tool used for traditional sanitation approaches replacement of chemical disinfectants.
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