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Rahmani Z, Sharafati Chaleshtori R, Abdoli Arani A, Taghizadeh M. Efficacy of Ozonized Water Spray for the Sanitization of Food Contact Surfaces Contaminated with E. Coli and S. Aureus as an Eco-Friendly Asepsis Approach. JNFS 2023; 8 (4) :587-596
URL: http://jnfs.ssu.ac.ir/article-1-589-en.html
URL: http://jnfs.ssu.ac.ir/article-1-589-en.html
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran.
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1 Department of Laser and Photonics, Faculty of Physics, University of Kashan, Kashan, Iran; 2 Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran.
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Efficacy of Ozonized Water Spray for the Sanitization of Food Contact Surfaces Contaminated with E. Coli and S. Aureus as an Eco-Friendly Asepsis Approach
Zeinab Rahmani; PhD1, Reza Sharafati Chaleshtori; PhD*2, Abbas Abdoli Arani; PhD1 &
Mohsen Taghizadeh; PhD2
Zeinab Rahmani; PhD1, Reza Sharafati Chaleshtori; PhD*2, Abbas Abdoli Arani; PhD1 &
Mohsen Taghizadeh; PhD2
1 Department of Laser and Photonics, Faculty of Physics, University of Kashan, Kashan, Iran; 2 Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran.
| ARTICLE INFO | ABSTRACT | |
| ORIGINAL ARTICLE | Background: Cross-contamination between food, equipment, and contact surfaces during food processing may occur with some pathogenic bacteria, such as E. coli and S. aureus. This study was conducted to investigate the bactericidal potential of ozonized water spray in food contact surfaces (plastic, glass, steel, and ceramic) for the inactivation of E. coli and S. aureus isolated from traditional yogurt. Methods: Two isolated bacteria were tested for antibiotic resistance and the strains were exposed to ozonized water with concentration of 4 mg/l for 0, 2, and 10 minutes and after drying on food contact surfaces (FCS). Results: E. coli and S. aureus were defined as multidrug-resistant. Ozonized water inhibited two bacteria growth on the FCS after two minutes. On plastic, glass, and ceramic surfaces, E. coli was more sensitive than S. aureus, while S. aureus on steel surface was more sensitive than E. coli. Conclusion: Based on the results, it is recommended to use the ozonized water spray at 4 mg/l concentration to disinfect surfaces in contact with food, especially glass and ceramic surfaces. Keywords: Ozonized water; Sanitization; Food contact surfaces; E. Coli; S. Aureus |
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| Article history: Received: 28 Feb 2022 Revised: 22 May 2022 Accepted: 22 May 2022 |
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| *Corresponding author: sharafati.reza@gmail.com Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran. Postal code: 87015981151 Tel: + 31 55540021 |
Introduction
According to reports of World Health Organization (WHO), about 600 million people are involved in food-borne diseases, and 420,000 die each year due to contaminated food. Various pathogenic microorganisms may contaminate food contact surfaces (FCS). Contaminated FCS can be responsible for such incidences (World Health Organization, 2019). Several factors cause contamination of FCS, including cross-contamination, workers’ hands and clothing, raw food, materials of packaging, lack of proper washing, and disinfection (Mohammed et al., 2018). Mohammed et al. reported that 13 (26%) FCS of restaurants were contaminated with E. coli (Mohammed et al., 2018). In a study, 63 S. aureus samples were isolated from food industry surfaces such as dairy, meat, and seafood (Gutiérrez et al., 2012).
Biofilms of S. aureus and E. coli are important causes of food poisoning and contamination, causing risk for consumers' health. Biofilms are a natural bacterial lifestyle; first, the cells attach to FCS, and then they start to reproduce and discharge a stable matrix of extracellular polymeric substances in which cells are involved (Yin et al., 2019). Therefore, they are a major problem in many sections of the food industry.
There are many hygienic practices related to preventing microbial contamination of FCS. The Food Safety Modernization Act (FSMA) was approved for a preventative food safety system in the USA. Sanitization or disinfection indicates the reduction of the number of microorganisms to a safe level. Various methods such as thermal, mechanical, chemical, physical, and biological methods have been classified for the disinfection of FCS (Torres Dominguez et al., 2019). Recent investigations have focused on the production of sanitizing solutions with less toxic effects. Jiménez-Pichardo et al. demonstrated that the aqueous solution of NaOH on stainless steel plates followed by rinsing with an aqueous chlorine compound could be recommended as an alternative sanitizing method (Jiménez-Pichardo et al., 2016). Also, the effect of ClO2 to inactivate food-borne pathogens on FCS indicated that surface hydrophobicity such as silicon, rubber, Teflon®, stainless steel, and PVC is a major factor compared to the inhibition of bacterial growth by ClO2 gas from the surface (Park and Kang, 2017).
One of the most promising anti-microorganism agents in the list of sanitizers is ozone gas, with a half-life of about 1 hour at room temperature. Gas of ozone can be used as an antimicrobial compound with a powerful oxidizer activity against viruses, bacteria, and protozoa. Nonetheless, the gaseous form of O3 can be toxic. Therefore, O3 can be dissolved in water to reduce its toxicity, and maintain its sanitizing effect (Santos et al., 2021). Previous studies have shown that aqueous O3 effectively reduced several pathogenic microorganisms, including viruses, bacteria, and fungi (Giuroiu et al., 2020, Nakamura et al., 2018). Due to the absence of harmful disinfectant residues after ozone processing by exposure to gaseous ozone or rinsing in ozonated water, its use in the fruit and vegetable industries has been strongly recommended (Botondi et al., 2021).
However, despite the economic and environmental benefits and the high effectiveness of ozone in disinfecting food products, it should be essential to note that this processing does not damage the nutrition facts, appearance, and shelf life of food products. One of the factors increasing food contaminations is the contamination of the FCS in preparing vegetables and meat products. Disinfection of these surfaces using chemicals such as alcohol and chlorine in addition to environmental disadvantages can lead to toxic compounds and detrimental effects on the quality and taste of food products.
The present study investigates antibacterial activities of ozonized water spray on the sanitization of FCS contaminated with E. coli and S. aureus in the use of ozone-containing water in cleaning equipment surfaces for pre-operational and in-process sanitation.
Materials and Methods
Preparation of materials and bacteria: In this experimental study, four materials with many applications in the food industry were used, including plastic, metal, glass, and ceramic. These surfaces for each material were prepared with dimensions of 10 x 10 cm and sterilized in an autoclave for 15 minutes at 121 °C immediately before use.
For isolation and identification of S. aureus and E.coli, 10 g of traditional yogurt was added to 90 mL of nutrient broth (Merck Co., Darmstadt, Germany) and homogenized for one minute. From the homogenate, 0.1 ml of aliquots was spread over the surface of the Baird-Parker agar plate (Merck Co., Darmstadt, Germany) supplemented with 5% Egg Yolk Tellurite (Merck Co., Darmstadt, Germany) and the MacConkey as well as EMB agar (Merck Co., Darmstadt, Germany), respectively, in duplicate following incubation for 24-48 hr at 37 °C. Following incubation, S. aureus and E. coli suspected colonies (up to five colonies) were selected and confirmed using biochemical tests such as Gram stain, catalase, coagulase, DNase, oxidase, triple sugar iron agar, and IMViC tests (Chaleshtori et al., 2017, Rahimi, 2013).
After isolation of strains, antibiotic susceptibility testing was carried out according to the Clinical and Laboratory Standards Institute (CLSI) by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (MHA) (Merck Co., Darmstadt, Germany) (Clinical and laboratory standards institiute, 2014). A bacterial suspension with turbidity equivalent to 0.5 (McFarland standard) (1.5 × 108 CFU/mL) was prepared in sterile phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). The bacterial suspension was uniformly inoculated with a sterile swab stick on the surface of MHA, and then five antibiotic disks were placed for each plate and incubated at 35 °C for 24 hr. Inhibition zones on agar plates were measured after 24 hr, and the results were recorded according to interpretive criteria provided by CLSI. The isolates of bacteria were tested against a panel of seventeen antibiotic discs (HiMedia Laboratories Pvt. Ltd, Mumbai, India): ampicillin (10 µg), amoxicillin (30 µg), clindamycin (2 µg), erythromycin (15 µg), gentamycin (10 µg), oxacillin (1 µg), penicillin (10 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (10 µg), vancomycin (30 µg), nitrofurantoin (300 µg), methicillin (5 µg), norfloxacin (30 µg), kanamycin (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), Ceftazidime (30 µg), (10 µg) and cefoxitin (30 µg).
Preparation of ozonized water: In the present study, the ozone generator PLASOZI made by Plasma Fanavaran Kavir Company (Kashan, Iran) was used to produce ozonized water. This system uses cold plasma technology to produce ozone and other oxidants active species from the ambient air as feed gas by a dielectric barrier discharge (DBD) structure. DBD reactors operate at atmospheric pressure and are made of two electrodes, at least one coated with a dielectric. The reactor configuration can be flat or cylindrical depending on its application. A high voltage AC or pulse is applied to the electrodes, which results from instantaneous electron emission from the cathode. These electrons pass through two electrodes as a filament. When this group of electrons reaches the dielectric coating of the opposite electrode, it spreads on its surface and reduces the local electric field, thereby limiting electric current, and preventing uncontrolled discharge progression and arc generation. The electrons reach very high energies by accelerating in the strong electric field while passing through. By colliding with oxygen molecules, these energetic electrons can break down molecular bonds and turn them into oxygen atoms. The combination of atomic oxygen with oxygen molecules leads to the formation of ozone. In the air-cooled ozone generator PLASOZI, ambient air was drawn into the device by a pump. By applying pulse high voltage (15 kV and 20 kHz), an electrical discharge was performed, the air was ionized and passed from the gas phase to the plasma phase. The created plasma contained ozone as the most efficient active species. Ozone is only partially soluble in water. In order to efficiently transfer ozone to the solution, it was necessary to inject ozone into the water in the form of fine bubbles. A bubble diffuser was used to transfer ozone to water and create 4 mg/L ozonized water. The half-life of ozone in water is much shorter than in air. The solubility of ozone and its half-life in water is inversely proportional to water temperature. On the other hand, to apply the experiment results in the food industry and surface disinfection, it was decided to ozonize the treated municipal water at room temperature, and the resulting solution was tested as a bacterial substance. In the present study, the water temperature was 27 °C, and its pH was 3.56.
Contamination and sanitization of FCS with ozonized water: This study evaluated the effectiveness of spraying ozonized water (4 mg/l) against S. aureus and E. coli in three exposure times (2, 6, 10 minutes, and after drying). The positive control group was a spray of normal saline solution on FCS impregnated with S. aureus and E. coli. For contamination of FCS, 1 mL of each bacterial suspension with turbidity equivalent to 1.5 × 105 CFU/ml in sterile normal saline (0.9%) was spread on the FCS with a sterile swab at room temperature for 15 min. Then, each FCS sample was sprayed with 2 ml of the ozonized distilled water at an initial concentration of 4 mg/l. After 0, 2, 6, and 10 minutes, it was dried with a sterile swab thoroughly and then cultured on Baird-Parker and MacConkey agar. The plates were incubated at 37 °C for 48 h. All the results were expressed as the number of CFU/ m2 (César et al., 2012).
Data analysis: The obtained results based on CFU/m2 were converted into a log10 form and subjected to an analysis of variance (ANOVA) followed by the Tukey test to compare multiple means. P-value < 0.05 was considered to indicate a statistically significant difference. The data were analyzed using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA) and Excel software version 2013 (Microsoft, Redmond, WA, USA). Log10 reduction factor (RF) was also calculated using Equation 1 (Megahed et al., 2018):
10 RF = 10 Control - 10 Treated
Results
The resistance pattern of S. aureus and E. coli isolates to antibiotics tested in this study is shown in Table 1.
The spray of aqueous O3 at a concentration of 4 ppm reduced S. aureus and E. coli within 2 minutes of exposure in plastic, glass, steel, and ceramic surfaces. The results showed that the exposure time of aqueous O3 had a significant effect on S. aureus and E. coli on all surfaces (P<0.05).
In plastic, glass, and ceramic surfaces, the concentration of aqueous O3 demonstrated a higher antibacterial activity against E. coli compared to S. aureus, while S. aureus was more susceptible on steel surface (Figure 1). In plastic surface, the mean log10 reduction in S. aureus counts and killing percentage increased after drying aqueous O3 (3.42 log CFU/m2 and 59.8%, respectively), compared to 2 minutes of exposure (0.95 logs CFU/m2 and 16.75%, respectively). Also, the mean log10 reduction in E. coli counts and killing percentage increased after drying aqueous O3 (5.34 log CFU/m2 and 100%, respectively), compared to 2 minutes exposure (1.35 log CFU/m2 and 25.29%, respectively) (Figure 2).
In steel surface, the mean log10 RF increased numerically at 2, 6, and 10 minutes for S. aureus (2.44, 2.84, and 5.62 log CFU/m2, respectively), compared to E. coli (1.32, 1.43, and 1.98 log CFU/m2, respectively). Aqueous O3 at concentration 4 ppm after 2 minutes exposure killed 100% E. coli on the glass surface, while S. aureus at 10 minutes exposure only killed 54.19% (Figure 2).
According to the results, the concentration of aqueous O3 is considered the most powerful antibacterial against S. aureus and E. coli on the ceramic surface. After 2 minutes of exposure, approximately 50% of the reduction in S. aureus and E. coli counts was observed, while killing percentage at 6 and 10 minutes for S. aureus and E. coli was 100%, respectively (Figure 2).
Biofilms of S. aureus and E. coli are important causes of food poisoning and contamination, causing risk for consumers' health. Biofilms are a natural bacterial lifestyle; first, the cells attach to FCS, and then they start to reproduce and discharge a stable matrix of extracellular polymeric substances in which cells are involved (Yin et al., 2019). Therefore, they are a major problem in many sections of the food industry.
There are many hygienic practices related to preventing microbial contamination of FCS. The Food Safety Modernization Act (FSMA) was approved for a preventative food safety system in the USA. Sanitization or disinfection indicates the reduction of the number of microorganisms to a safe level. Various methods such as thermal, mechanical, chemical, physical, and biological methods have been classified for the disinfection of FCS (Torres Dominguez et al., 2019). Recent investigations have focused on the production of sanitizing solutions with less toxic effects. Jiménez-Pichardo et al. demonstrated that the aqueous solution of NaOH on stainless steel plates followed by rinsing with an aqueous chlorine compound could be recommended as an alternative sanitizing method (Jiménez-Pichardo et al., 2016). Also, the effect of ClO2 to inactivate food-borne pathogens on FCS indicated that surface hydrophobicity such as silicon, rubber, Teflon®, stainless steel, and PVC is a major factor compared to the inhibition of bacterial growth by ClO2 gas from the surface (Park and Kang, 2017).
One of the most promising anti-microorganism agents in the list of sanitizers is ozone gas, with a half-life of about 1 hour at room temperature. Gas of ozone can be used as an antimicrobial compound with a powerful oxidizer activity against viruses, bacteria, and protozoa. Nonetheless, the gaseous form of O3 can be toxic. Therefore, O3 can be dissolved in water to reduce its toxicity, and maintain its sanitizing effect (Santos et al., 2021). Previous studies have shown that aqueous O3 effectively reduced several pathogenic microorganisms, including viruses, bacteria, and fungi (Giuroiu et al., 2020, Nakamura et al., 2018). Due to the absence of harmful disinfectant residues after ozone processing by exposure to gaseous ozone or rinsing in ozonated water, its use in the fruit and vegetable industries has been strongly recommended (Botondi et al., 2021).
However, despite the economic and environmental benefits and the high effectiveness of ozone in disinfecting food products, it should be essential to note that this processing does not damage the nutrition facts, appearance, and shelf life of food products. One of the factors increasing food contaminations is the contamination of the FCS in preparing vegetables and meat products. Disinfection of these surfaces using chemicals such as alcohol and chlorine in addition to environmental disadvantages can lead to toxic compounds and detrimental effects on the quality and taste of food products.
The present study investigates antibacterial activities of ozonized water spray on the sanitization of FCS contaminated with E. coli and S. aureus in the use of ozone-containing water in cleaning equipment surfaces for pre-operational and in-process sanitation.
Materials and Methods
Preparation of materials and bacteria: In this experimental study, four materials with many applications in the food industry were used, including plastic, metal, glass, and ceramic. These surfaces for each material were prepared with dimensions of 10 x 10 cm and sterilized in an autoclave for 15 minutes at 121 °C immediately before use.
For isolation and identification of S. aureus and E.coli, 10 g of traditional yogurt was added to 90 mL of nutrient broth (Merck Co., Darmstadt, Germany) and homogenized for one minute. From the homogenate, 0.1 ml of aliquots was spread over the surface of the Baird-Parker agar plate (Merck Co., Darmstadt, Germany) supplemented with 5% Egg Yolk Tellurite (Merck Co., Darmstadt, Germany) and the MacConkey as well as EMB agar (Merck Co., Darmstadt, Germany), respectively, in duplicate following incubation for 24-48 hr at 37 °C. Following incubation, S. aureus and E. coli suspected colonies (up to five colonies) were selected and confirmed using biochemical tests such as Gram stain, catalase, coagulase, DNase, oxidase, triple sugar iron agar, and IMViC tests (Chaleshtori et al., 2017, Rahimi, 2013).
After isolation of strains, antibiotic susceptibility testing was carried out according to the Clinical and Laboratory Standards Institute (CLSI) by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (MHA) (Merck Co., Darmstadt, Germany) (Clinical and laboratory standards institiute, 2014). A bacterial suspension with turbidity equivalent to 0.5 (McFarland standard) (1.5 × 108 CFU/mL) was prepared in sterile phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). The bacterial suspension was uniformly inoculated with a sterile swab stick on the surface of MHA, and then five antibiotic disks were placed for each plate and incubated at 35 °C for 24 hr. Inhibition zones on agar plates were measured after 24 hr, and the results were recorded according to interpretive criteria provided by CLSI. The isolates of bacteria were tested against a panel of seventeen antibiotic discs (HiMedia Laboratories Pvt. Ltd, Mumbai, India): ampicillin (10 µg), amoxicillin (30 µg), clindamycin (2 µg), erythromycin (15 µg), gentamycin (10 µg), oxacillin (1 µg), penicillin (10 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (10 µg), vancomycin (30 µg), nitrofurantoin (300 µg), methicillin (5 µg), norfloxacin (30 µg), kanamycin (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), Ceftazidime (30 µg), (10 µg) and cefoxitin (30 µg).
Preparation of ozonized water: In the present study, the ozone generator PLASOZI made by Plasma Fanavaran Kavir Company (Kashan, Iran) was used to produce ozonized water. This system uses cold plasma technology to produce ozone and other oxidants active species from the ambient air as feed gas by a dielectric barrier discharge (DBD) structure. DBD reactors operate at atmospheric pressure and are made of two electrodes, at least one coated with a dielectric. The reactor configuration can be flat or cylindrical depending on its application. A high voltage AC or pulse is applied to the electrodes, which results from instantaneous electron emission from the cathode. These electrons pass through two electrodes as a filament. When this group of electrons reaches the dielectric coating of the opposite electrode, it spreads on its surface and reduces the local electric field, thereby limiting electric current, and preventing uncontrolled discharge progression and arc generation. The electrons reach very high energies by accelerating in the strong electric field while passing through. By colliding with oxygen molecules, these energetic electrons can break down molecular bonds and turn them into oxygen atoms. The combination of atomic oxygen with oxygen molecules leads to the formation of ozone. In the air-cooled ozone generator PLASOZI, ambient air was drawn into the device by a pump. By applying pulse high voltage (15 kV and 20 kHz), an electrical discharge was performed, the air was ionized and passed from the gas phase to the plasma phase. The created plasma contained ozone as the most efficient active species. Ozone is only partially soluble in water. In order to efficiently transfer ozone to the solution, it was necessary to inject ozone into the water in the form of fine bubbles. A bubble diffuser was used to transfer ozone to water and create 4 mg/L ozonized water. The half-life of ozone in water is much shorter than in air. The solubility of ozone and its half-life in water is inversely proportional to water temperature. On the other hand, to apply the experiment results in the food industry and surface disinfection, it was decided to ozonize the treated municipal water at room temperature, and the resulting solution was tested as a bacterial substance. In the present study, the water temperature was 27 °C, and its pH was 3.56.
Contamination and sanitization of FCS with ozonized water: This study evaluated the effectiveness of spraying ozonized water (4 mg/l) against S. aureus and E. coli in three exposure times (2, 6, 10 minutes, and after drying). The positive control group was a spray of normal saline solution on FCS impregnated with S. aureus and E. coli. For contamination of FCS, 1 mL of each bacterial suspension with turbidity equivalent to 1.5 × 105 CFU/ml in sterile normal saline (0.9%) was spread on the FCS with a sterile swab at room temperature for 15 min. Then, each FCS sample was sprayed with 2 ml of the ozonized distilled water at an initial concentration of 4 mg/l. After 0, 2, 6, and 10 minutes, it was dried with a sterile swab thoroughly and then cultured on Baird-Parker and MacConkey agar. The plates were incubated at 37 °C for 48 h. All the results were expressed as the number of CFU/ m2 (César et al., 2012).
Data analysis: The obtained results based on CFU/m2 were converted into a log10 form and subjected to an analysis of variance (ANOVA) followed by the Tukey test to compare multiple means. P-value < 0.05 was considered to indicate a statistically significant difference. The data were analyzed using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA) and Excel software version 2013 (Microsoft, Redmond, WA, USA). Log10 reduction factor (RF) was also calculated using Equation 1 (Megahed et al., 2018):
Results
The resistance pattern of S. aureus and E. coli isolates to antibiotics tested in this study is shown in Table 1.
The spray of aqueous O3 at a concentration of 4 ppm reduced S. aureus and E. coli within 2 minutes of exposure in plastic, glass, steel, and ceramic surfaces. The results showed that the exposure time of aqueous O3 had a significant effect on S. aureus and E. coli on all surfaces (P<0.05).
In plastic, glass, and ceramic surfaces, the concentration of aqueous O3 demonstrated a higher antibacterial activity against E. coli compared to S. aureus, while S. aureus was more susceptible on steel surface (Figure 1). In plastic surface, the mean log10 reduction in S. aureus counts and killing percentage increased after drying aqueous O3 (3.42 log CFU/m2 and 59.8%, respectively), compared to 2 minutes of exposure (0.95 logs CFU/m2 and 16.75%, respectively). Also, the mean log10 reduction in E. coli counts and killing percentage increased after drying aqueous O3 (5.34 log CFU/m2 and 100%, respectively), compared to 2 minutes exposure (1.35 log CFU/m2 and 25.29%, respectively) (Figure 2).
In steel surface, the mean log10 RF increased numerically at 2, 6, and 10 minutes for S. aureus (2.44, 2.84, and 5.62 log CFU/m2, respectively), compared to E. coli (1.32, 1.43, and 1.98 log CFU/m2, respectively). Aqueous O3 at concentration 4 ppm after 2 minutes exposure killed 100% E. coli on the glass surface, while S. aureus at 10 minutes exposure only killed 54.19% (Figure 2).
According to the results, the concentration of aqueous O3 is considered the most powerful antibacterial against S. aureus and E. coli on the ceramic surface. After 2 minutes of exposure, approximately 50% of the reduction in S. aureus and E. coli counts was observed, while killing percentage at 6 and 10 minutes for S. aureus and E. coli was 100%, respectively (Figure 2).
| Figure 1. Bacterial counts (Log CFU/m²) of S. aureus and E. coli after spray of aqueous O3 (4 ppm) on plastic, glass, steel, and ceramic surfaces for 2, 4, 10 minutes and after drying. Different capital letters within a time point differ significantly (P < 0.05) |
| Figure 2. Mean ± SD Log10 reduction in bacterial counts of S. aureus and E. coli after spray of aqueous O3 (4 ppm) on plastic, glass, steel and ceramic surfaces for 2, 4, 10 minutes and after drying. The killing percentage is demonstrated above on columns. Different capital letters within a time point differ significantly (P < 0.05). |
| Table 1. Antimicrobial resistance of S. aureus and E. coli isolated from traditional yogurt in Kashan, Iran. | ||
| Antimicrobials | Isolates | |
| S. aureus | E.coli | |
| Ampicillin (10 µg) | Resistant | Resistant |
| Amoxicillin (30 µg) | Resistant | Resistant |
| Clindamycin (2 µg) | Susceptible | - |
| Erythromycin (15 µg) | Susceptible | - |
| Methicillin (5 µg) | Resistant | - |
| Gentamycin (10 µg) | Resistant | Resistant |
| Oxacillin (1 µg) | Resistant | - |
| Penicillin (10 µg) | Resistant | - |
| Tetracycline (30 µg) | Resistant | Resistant |
| Trimethoprim/Sulfamethoxazole (10 µg) | Susceptible | Resistant |
| Vancomycin (30 µg) | Resistant | - |
| Nitrofurantoin (300 µg) | Resistant | Resistant |
| Norfloxacin (30 µg) | Resistant | Resistant |
| Kanamycin(30 µg) | Resistant | Resistant |
| Ceftriaxone (30 µg) | Resistant | Resistant |
| Cefoxitin (30 µg) | Resistant | - |
| Chloramphenicol (30 µg) | Susceptible | Susceptible |
| Ceftazidime (30 µg) | - | Resistant |
| Ciprofloxacin (10 µg) | Susceptible | Susceptible |
Discussion
The study results indicated that spray of aqueous O3 at a concentration of 4 mg/l is sufficient to reduce 60 to 100% of multidrug resistance food-borne pathogens (MRFBP) to a safe level on FCS after drying. Also, the finding showed that 2 and 6 minutes exposure of aqueous O3 is not an adequate means of controlling MRFBP under heavy load on different surfaces. Moreover, on smooth surfaces, an aqueous O3 had a significant impact on RF. In line with the present study, Megahed et al. reported that a 2 min exposure to aqueous ozone at concentrations of 4 and 9 mg/l reduces manure-based pathogens to a safe level on smooth and moderately rough surfaces, respectively (Megahed et al., 2018).
High oxidative capacity of ozonized water in higher concentrations increases the redox potential in bacteria and decreases the bacterial load on surfaces (de Paula Nascente et al., 2021). Initially, the ozone involves considerable changes in fluidity and permeability of the bacterial membrane. As a result, cell death occurs due to leaching of ions and intracellular molecules out of bacteria (Kahrilas et al., 2015). A study showed that DNA damage of bacteria by oxidative stress increased by increasing exposure time, concentration, and continuous exposure to O3 (Devatkal et al., 2016).
Pomposiello and Demple, demonstrated that E. coli via protective mechanisms including SoxR and OxyR causes activation of different genes such as soxS and sod in E. coli cells and reduces the oxidative stress of O3 through DNA repair or removal of the radicals (Pomposiello and Demple, 2001). However, these protective mechanisms cannot hold up for long at doses of 4 mg/l or greater of O3 (Aydogan and Gurol, 2006, Megahed et al., 2018).
Marino et al. reported that the antibacterial activities of O3 against Gram-positive or Gram-negative group of bacteria had no significant correlation. They showed that S. aureus and L. monocytogenes were more resistant than P. fluorescens (Marino et al., 2018), which might be due to cellular defense systems against oxidative stress. At the same time, P. fluorescens with an external membrane (as a cellular cover against other antibacterial agents) has not shown a defense (Clauditz et al., 2006, Marino et al., 2018).
A previous study demonstrated that growth inhibition of multidrug-resistant Salmonella Heidelberg strains occurred in the presence of aqueous O3 and the lack of organic material after 1 min, with changes in metabolic profile and bacterial DNA concentration (de Paula Nascente et al., 2021, Kumar et al., 2016). Studies have shown antiviral activities (coronaviruses such as SARS-CoV-1 and 2) of O3 with interaction with the envelopes of the lipid layer (Kampf et al., 2020, Kumar et al., 2016, Tizaoui, 2020).
One important factor related to the increase of sensitivity of bacterial biofilm to aqueous O3 is the dynamic condition compared to static conditions at the time of its use (Sarron et al., 2021). It is known that almost under dynamic conditions the amount of ozone in water remains constant; therefore, in a study, the biofilm of S. aureus was more sensitive than attached cells (bacterial reduction after 2 min exposure at least 3 Log of biofilm viability) (Marino et al., 2018).
Smooth surfaces such as glass, ceramic, and steel enhance the mass transfer of O3, and consequently, an efficient diffusion and penetration of ozone among MRFBP cause increased damage to bacteria and cell death. In addition, the physical removal of ozone on smooth surfaces is lower than rough surfaces due to a decrease of surface reactivity and irreversible consumption of ozone on smooth surfaces when O3 strikes the surface (Nicholas et al., 2013, Wang and Morrison, 2010).
In contrast with a previous study (Megahed et al., 2018), a considerable impact of exposure time on RF was identified, especially on the glass, steel, and ceramic, except for plastic. Another study showed that O3 could completely kill klebsiella michiganensis and p. aeruginosa during the first 4 minutes of exposure (Saha et al., 2014). The present study findings can be hopeful and provide a practical method to use ozonized water as a complementary way in the disinfection of the contact surfaces in the food industry to reduce E. coli and S. aureus environmental contamination. Performing tests in a controlled environment was the main limitation of this study. Therefore, future studies are recommended in the various food industries.
Conclusion
E. coli and S. aureus were defined as multidrug-resistant. Ozonized water inhibited two bacteria growth on the FCS after two minutes. On plastic, glass, and ceramic surfaces, E. coli was more sensitive than S. aureus, while S. aureus on steel surface was more sensitive than E. coli. The results demonstrated that spray with a concentration of 4 mg/L of aqueous O3 had the most antibacterial power against S. aureus and E. coli on the ceramic surface. After 6 and 10 minutes of exposure, the killing percentage for S. aureus and E. coli was 100%, respectively.
Acknowledgments
The authors are grateful to the Research Deputy and the personnel of the Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran (grant number 98000780).
Authors’ contributions
Rahmani Z and Sharafati Chaleshtori R designed the research; Rahmani Z, Sharafati Chaleshtori R, Abdoli Arani A, and Taghizadeh M managed the whole project and contributed in all steps; Rahmani Z, Sharafati Chaleshtori R, and Abdoli Arani A wrote the draft of the manuscript and its finalization. All the authors read and approved the final manuscript.
Conflict of interest
The authors declare that there is no conflict of interest.
References
Aydogan A & Gurol MD 2006. Application of gaseous ozone for inactivation of Bacillus subtilis spores. Journal of the air & waste management association. 56 (2): 179-185.
Botondi R, Barone M & Grasso C 2021. A review into the effectiveness of ozone technology for improving the safety and preserving the quality of fresh-cut fruits and vegetables. Foods. 10 (4): 748.
César J, Sumita TC, Junqueira JC, Jorge AOC & Do Rego MA 2012. Antimicrobial effects of ozonated water on the sanitization of dental instruments contaminated with E. coli, S. aureus, C. albicans, or the spores of B. atrophaeus. Journal of infection and public health. 5 (4): 269-274.
Chaleshtori FS, Arani NM, Aghadavod E, Naseri A & Chaleshtori RS 2017. Molecular characterization of Escherichia coli recovered from traditional milk products in Kashan, Iran. Veterinary world. 10 (10): 1264.
Clauditz A, Resch A, Wieland K-P, Peschel A & Götz F 2006. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infection and immunity. 74 (8): 4950-4953.
Clinical and laboratory standards institiute
2014. Performance Standards for Antimicrobial Susceptibility Testing; Twenty - Fourth Informational Supplement, http://microbiolab-bg.com/ wp-content/uploads/2015/05/CLSI-2014. pdf.
de Paula Nascente E, et al. 2021. Inactivation of Multidrug-Resistant Salmonella Heidelberg Isolated from Pigs by Ozonated Water under Different Conditions. Ozone: science & engineering. 44 (4): 363-367.
Devatkal SK, Jaiswal P, Kaur A & Juneja V 2016. Inactivation of Bacillus cereus and Salmonella enterica serovar Typhimurium by aqueous ozone: modeling and UV-VIS spectroscopic analysis. Ozone: Science & Engineering. 38 (2): 124-132.
Giuroiu CL, et al. 2020. The combination of diode laser and ozonated water in the treatment of complicated pulp gangrene. Applied sciences. 10 (12): 4203.
Gutiérrez D, et al. 2012. Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces. Applied and environmental microbiology. 78 (24): 8547-8554.
Jiménez-Pichardo R, et al. 2016. Evaluation of electrolyzed water as cleaning and disinfection agent on stainless steel as a model surface in the dairy industry. Food control. 60: 320-328.
Kahrilas GA, Blotevogel J, Stewart PS & Borch T 2015. Biocides in hydraulic fracturing fluids: a critical review of their usage, mobility, degradation, and toxicity. Environmental science & technology. 49 (1): 16-32.
Kampf G, Todt D, Pfaender S & Steinmann E 2020. Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents. Journal of hospital infection. 104 (3): 246-251.
Kumar GD, Williams RC, Sumner SS & Eifert JD 2016. Effect of ozone and ultraviolet light on Listeria monocytogenes populations in fresh and spent chill brines. Food control. 59: 172-177.
Marino M, Maifreni M, Baggio A & Innocente N 2018. Inactivation of foodborne bacteria biofilms by aqueous and gaseous ozone. Frontiers in microbiology. 9: 2024.
Megahed A, Aldridge B & Lowe J 2018. The microbial killing capacity of aqueous and gaseous ozone on different surfaces contaminated with dairy cattle manure. PLoS One. 13 (5): e0196555.
Mohammed SS, Ayansina A, Mohammed S, Oyewole OA & Shaba A 2018. Evaluation of food contact surfaces in selected restaurants of kaduna state university for the presence of escherichia coli and Staphylococcus aureus. Science world journal. 13 (3): 45-49.
Nakamura K, et al. 2018. Evaluation of ozonated water using ASTM E1174 for standardized testing of handwash formulations for healthcare personnel. Journal of hospital infection. 100 (2): 211-213.
Nicholas R, Dunton P, Tatham A & Fielding L 2013. The effect of ozone and open air factor on surface-attached and biofilm environmental L isteria monocytogenes. Journal of applied microbiology. 115 (2): 555-564.
Park S-H & Kang D-H 2017. Influence of surface properties of produce and food contact surfaces on the efficacy of chlorine dioxide gas for the inactivation of foodborne pathogens. Food control. 81: 88-95.
Pomposiello PJ & Demple B 2001. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends in biotechnology. 19 (3): 109-114.
Rahimi E 2013. Enterotoxigenicity of Staphylococcus aureus isolated from traditional and commercial dairy products marketed in Iran. Brazilian journal of microbiology. 44: 393-399.
Saha R, Saha N, Atwain A & Donofrio RS 2014. Evaluation of disinfection efficacy of ozone and chlorinated disinfectant against the biofilm of Klebsiella michiganensis and Pseudomonas aeruginosa. Annals of microbiology. 64 (4): 1607-1613.
Santos LMCd, et al. 2021. Ozonized water in microbial control: analysis of the stability, in vitro biocidal potential, and cytotoxicity. Biology. 10 (6): 525.
Sarron E, Gadonna-Widehem P & Aussenac T 2021. Ozone treatments for preserving fresh vegetables quality: A critical review. Foods. 10 (3): 605.
Tizaoui C 2020. Ozone: a potential oxidant for COVID-19 virus (SARS-CoV-2). Ozone: science & engineering. 42 (5): 378-385.
Torres Dominguez E, Nguyen PH, Hunt HK & Mustapha A 2019. Antimicrobial coatings for food contact surfaces: Legal framework, mechanical properties, and potential applications. Comprehensive reviews in food science and food safety. 18 (6): 1825-1858.
Wang H & Morrison G 2010. Ozone surface reactions in five homes: surface reaction probabilities, aldehyde yields, and trends. Indoor air. 20 (3): 224-234.
World Health Organization 2019. Food safety. Key Facts. Retrieved from, https://www.who.int/ news-room/fact-sheets/detail/food-safety.
Yin W, Wang Y, Liu L & He J 2019. Biofilms: the microbial “protective clothing” in extreme environments. International journal of molecular sciences. 20 (14): 3423.
The study results indicated that spray of aqueous O3 at a concentration of 4 mg/l is sufficient to reduce 60 to 100% of multidrug resistance food-borne pathogens (MRFBP) to a safe level on FCS after drying. Also, the finding showed that 2 and 6 minutes exposure of aqueous O3 is not an adequate means of controlling MRFBP under heavy load on different surfaces. Moreover, on smooth surfaces, an aqueous O3 had a significant impact on RF. In line with the present study, Megahed et al. reported that a 2 min exposure to aqueous ozone at concentrations of 4 and 9 mg/l reduces manure-based pathogens to a safe level on smooth and moderately rough surfaces, respectively (Megahed et al., 2018).
High oxidative capacity of ozonized water in higher concentrations increases the redox potential in bacteria and decreases the bacterial load on surfaces (de Paula Nascente et al., 2021). Initially, the ozone involves considerable changes in fluidity and permeability of the bacterial membrane. As a result, cell death occurs due to leaching of ions and intracellular molecules out of bacteria (Kahrilas et al., 2015). A study showed that DNA damage of bacteria by oxidative stress increased by increasing exposure time, concentration, and continuous exposure to O3 (Devatkal et al., 2016).
Pomposiello and Demple, demonstrated that E. coli via protective mechanisms including SoxR and OxyR causes activation of different genes such as soxS and sod in E. coli cells and reduces the oxidative stress of O3 through DNA repair or removal of the radicals (Pomposiello and Demple, 2001). However, these protective mechanisms cannot hold up for long at doses of 4 mg/l or greater of O3 (Aydogan and Gurol, 2006, Megahed et al., 2018).
Marino et al. reported that the antibacterial activities of O3 against Gram-positive or Gram-negative group of bacteria had no significant correlation. They showed that S. aureus and L. monocytogenes were more resistant than P. fluorescens (Marino et al., 2018), which might be due to cellular defense systems against oxidative stress. At the same time, P. fluorescens with an external membrane (as a cellular cover against other antibacterial agents) has not shown a defense (Clauditz et al., 2006, Marino et al., 2018).
A previous study demonstrated that growth inhibition of multidrug-resistant Salmonella Heidelberg strains occurred in the presence of aqueous O3 and the lack of organic material after 1 min, with changes in metabolic profile and bacterial DNA concentration (de Paula Nascente et al., 2021, Kumar et al., 2016). Studies have shown antiviral activities (coronaviruses such as SARS-CoV-1 and 2) of O3 with interaction with the envelopes of the lipid layer (Kampf et al., 2020, Kumar et al., 2016, Tizaoui, 2020).
One important factor related to the increase of sensitivity of bacterial biofilm to aqueous O3 is the dynamic condition compared to static conditions at the time of its use (Sarron et al., 2021). It is known that almost under dynamic conditions the amount of ozone in water remains constant; therefore, in a study, the biofilm of S. aureus was more sensitive than attached cells (bacterial reduction after 2 min exposure at least 3 Log of biofilm viability) (Marino et al., 2018).
Smooth surfaces such as glass, ceramic, and steel enhance the mass transfer of O3, and consequently, an efficient diffusion and penetration of ozone among MRFBP cause increased damage to bacteria and cell death. In addition, the physical removal of ozone on smooth surfaces is lower than rough surfaces due to a decrease of surface reactivity and irreversible consumption of ozone on smooth surfaces when O3 strikes the surface (Nicholas et al., 2013, Wang and Morrison, 2010).
In contrast with a previous study (Megahed et al., 2018), a considerable impact of exposure time on RF was identified, especially on the glass, steel, and ceramic, except for plastic. Another study showed that O3 could completely kill klebsiella michiganensis and p. aeruginosa during the first 4 minutes of exposure (Saha et al., 2014). The present study findings can be hopeful and provide a practical method to use ozonized water as a complementary way in the disinfection of the contact surfaces in the food industry to reduce E. coli and S. aureus environmental contamination. Performing tests in a controlled environment was the main limitation of this study. Therefore, future studies are recommended in the various food industries.
Conclusion
E. coli and S. aureus were defined as multidrug-resistant. Ozonized water inhibited two bacteria growth on the FCS after two minutes. On plastic, glass, and ceramic surfaces, E. coli was more sensitive than S. aureus, while S. aureus on steel surface was more sensitive than E. coli. The results demonstrated that spray with a concentration of 4 mg/L of aqueous O3 had the most antibacterial power against S. aureus and E. coli on the ceramic surface. After 6 and 10 minutes of exposure, the killing percentage for S. aureus and E. coli was 100%, respectively.
Acknowledgments
The authors are grateful to the Research Deputy and the personnel of the Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran (grant number 98000780).
Authors’ contributions
Rahmani Z and Sharafati Chaleshtori R designed the research; Rahmani Z, Sharafati Chaleshtori R, Abdoli Arani A, and Taghizadeh M managed the whole project and contributed in all steps; Rahmani Z, Sharafati Chaleshtori R, and Abdoli Arani A wrote the draft of the manuscript and its finalization. All the authors read and approved the final manuscript.
Conflict of interest
The authors declare that there is no conflict of interest.
References
Aydogan A & Gurol MD 2006. Application of gaseous ozone for inactivation of Bacillus subtilis spores. Journal of the air & waste management association. 56 (2): 179-185.
Botondi R, Barone M & Grasso C 2021. A review into the effectiveness of ozone technology for improving the safety and preserving the quality of fresh-cut fruits and vegetables. Foods. 10 (4): 748.
César J, Sumita TC, Junqueira JC, Jorge AOC & Do Rego MA 2012. Antimicrobial effects of ozonated water on the sanitization of dental instruments contaminated with E. coli, S. aureus, C. albicans, or the spores of B. atrophaeus. Journal of infection and public health. 5 (4): 269-274.
Chaleshtori FS, Arani NM, Aghadavod E, Naseri A & Chaleshtori RS 2017. Molecular characterization of Escherichia coli recovered from traditional milk products in Kashan, Iran. Veterinary world. 10 (10): 1264.
Clauditz A, Resch A, Wieland K-P, Peschel A & Götz F 2006. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infection and immunity. 74 (8): 4950-4953.
Clinical and laboratory standards institiute
2014. Performance Standards for Antimicrobial Susceptibility Testing; Twenty - Fourth Informational Supplement, http://microbiolab-bg.com/ wp-content/uploads/2015/05/CLSI-2014. pdf.
de Paula Nascente E, et al. 2021. Inactivation of Multidrug-Resistant Salmonella Heidelberg Isolated from Pigs by Ozonated Water under Different Conditions. Ozone: science & engineering. 44 (4): 363-367.
Devatkal SK, Jaiswal P, Kaur A & Juneja V 2016. Inactivation of Bacillus cereus and Salmonella enterica serovar Typhimurium by aqueous ozone: modeling and UV-VIS spectroscopic analysis. Ozone: Science & Engineering. 38 (2): 124-132.
Giuroiu CL, et al. 2020. The combination of diode laser and ozonated water in the treatment of complicated pulp gangrene. Applied sciences. 10 (12): 4203.
Gutiérrez D, et al. 2012. Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces. Applied and environmental microbiology. 78 (24): 8547-8554.
Jiménez-Pichardo R, et al. 2016. Evaluation of electrolyzed water as cleaning and disinfection agent on stainless steel as a model surface in the dairy industry. Food control. 60: 320-328.
Kahrilas GA, Blotevogel J, Stewart PS & Borch T 2015. Biocides in hydraulic fracturing fluids: a critical review of their usage, mobility, degradation, and toxicity. Environmental science & technology. 49 (1): 16-32.
Kampf G, Todt D, Pfaender S & Steinmann E 2020. Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents. Journal of hospital infection. 104 (3): 246-251.
Kumar GD, Williams RC, Sumner SS & Eifert JD 2016. Effect of ozone and ultraviolet light on Listeria monocytogenes populations in fresh and spent chill brines. Food control. 59: 172-177.
Marino M, Maifreni M, Baggio A & Innocente N 2018. Inactivation of foodborne bacteria biofilms by aqueous and gaseous ozone. Frontiers in microbiology. 9: 2024.
Megahed A, Aldridge B & Lowe J 2018. The microbial killing capacity of aqueous and gaseous ozone on different surfaces contaminated with dairy cattle manure. PLoS One. 13 (5): e0196555.
Mohammed SS, Ayansina A, Mohammed S, Oyewole OA & Shaba A 2018. Evaluation of food contact surfaces in selected restaurants of kaduna state university for the presence of escherichia coli and Staphylococcus aureus. Science world journal. 13 (3): 45-49.
Nakamura K, et al. 2018. Evaluation of ozonated water using ASTM E1174 for standardized testing of handwash formulations for healthcare personnel. Journal of hospital infection. 100 (2): 211-213.
Nicholas R, Dunton P, Tatham A & Fielding L 2013. The effect of ozone and open air factor on surface-attached and biofilm environmental L isteria monocytogenes. Journal of applied microbiology. 115 (2): 555-564.
Park S-H & Kang D-H 2017. Influence of surface properties of produce and food contact surfaces on the efficacy of chlorine dioxide gas for the inactivation of foodborne pathogens. Food control. 81: 88-95.
Pomposiello PJ & Demple B 2001. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends in biotechnology. 19 (3): 109-114.
Rahimi E 2013. Enterotoxigenicity of Staphylococcus aureus isolated from traditional and commercial dairy products marketed in Iran. Brazilian journal of microbiology. 44: 393-399.
Saha R, Saha N, Atwain A & Donofrio RS 2014. Evaluation of disinfection efficacy of ozone and chlorinated disinfectant against the biofilm of Klebsiella michiganensis and Pseudomonas aeruginosa. Annals of microbiology. 64 (4): 1607-1613.
Santos LMCd, et al. 2021. Ozonized water in microbial control: analysis of the stability, in vitro biocidal potential, and cytotoxicity. Biology. 10 (6): 525.
Sarron E, Gadonna-Widehem P & Aussenac T 2021. Ozone treatments for preserving fresh vegetables quality: A critical review. Foods. 10 (3): 605.
Tizaoui C 2020. Ozone: a potential oxidant for COVID-19 virus (SARS-CoV-2). Ozone: science & engineering. 42 (5): 378-385.
Torres Dominguez E, Nguyen PH, Hunt HK & Mustapha A 2019. Antimicrobial coatings for food contact surfaces: Legal framework, mechanical properties, and potential applications. Comprehensive reviews in food science and food safety. 18 (6): 1825-1858.
Wang H & Morrison G 2010. Ozone surface reactions in five homes: surface reaction probabilities, aldehyde yields, and trends. Indoor air. 20 (3): 224-234.
World Health Organization 2019. Food safety. Key Facts. Retrieved from, https://www.who.int/ news-room/fact-sheets/detail/food-safety.
Yin W, Wang Y, Liu L & He J 2019. Biofilms: the microbial “protective clothing” in extreme environments. International journal of molecular sciences. 20 (14): 3423.
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Received: 2022/02/28 | Published: 2023/11/20 | ePublished: 2023/11/20
Received: 2022/02/28 | Published: 2023/11/20 | ePublished: 2023/11/20
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