In Vitro Evaluation of Ozone Activity on Recent Clinically Isolated Bacterial Strains

Abstract Full-Text HTML XML Download Download as PDF (Size:889KB) PP. 106-115
DOI: 10.4236/aim.2014.42015    3,180 Downloads   4,940 Views   Citations


This study aims to evaluate the cozone bactericidal activity in different suspension media (saline, broth and whole blood) at different exposure times. Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, ESBLpositive Escherichia coli, MDR Pseudomonas aeruginosa were suspended in different media. We used a bacterial concentration of 0.2 MF for all experiments, as this concentration is consistent with the results of septic shock blood experiments. We performed ozone insufflations in a “sealed environment”. The total number of insufflations for each experiment ranged from one to four. The gas concentration was maintained at 80 mcg/ml. We confirmed the bactericidal activity of ozone on saline for all the bacterial strains. Experiments in broth revealed no changes in the bacterial growth. Ozone is primarily bactericidal against E. coli and bacteriostatic on P. aeruginosa, S. aureus and E. faecalis on whole blood. This study confirms the bactericidal efficacy of topical ozone applications and supports the need for further evaluations of the therapeutic potential of major ozone autohemotherapy. The results in E. coli promote further investigations of ozone activity on other Enterobacteriaceae and its potential use in the treatment of urinary infections. In general, these results suggest that ozone-therapy might be an alternative therapy to overcome antibiotic resistance.

Cite this paper

P. Tordiglione, F. Morselli, I. Scarpa, G. Puggioni, C. Mancini, G. Rosa and A. Giordano, "In Vitro Evaluation of Ozone Activity on Recent Clinically Isolated Bacterial Strains," Advances in Microbiology, Vol. 4 No. 2, 2014, pp. 106-115. doi: 10.4236/aim.2014.42015.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Safe Practices for Food Processes, Chapter V, “Methods to Reduce/Eliminate Pathogens from Produce and FreshCut Produce,” US Food and Drug Administration.
[2] D. S. Boyce, O. J. Sproul and C. E. Buck, “The Effect of Bentonite Clay on Ozone Disinfection of Bacteria and Viruses in Water,” Water Research, Vol. 15, No. 6, 1981, pp. 759-767.
[3] W. H. Glaze, “Drinking Water Treatment with Ozone,” Environmental Science & Technology, Vol. 21, No. 3, 1987, pp. 224-230.
[4] A. Joss, H. Siegrist and T. A. Ternes, “Are We about to Upgrade Wastewater Treatment for Removing Organic Micropollutans?” Water Science and Technology, Vol. 57, No. 2, 2008, pp. 251-255.
[5] T. Glauner, P. Waldmann, F. H. Frimmel and C. Zwiener, “Swimming Pool Water-Fractionation and Genotoxicological Characterization of Organic Constituents,” Water Research, Vol. 39, No. 18, 2005, pp. 4494-4502.
[6] T. Masaoka, Y. Kubota, S. Namiuchi, T. Takubo, T. Ueda, H. Shibata, H. Nakamura, K. Yoshitake, H. Doi and T. Kamiki, “Ozone Decontamination of Bioclean Rooms,” Applied and Environmental Microbiology, Vol. 43, No. 3, 1982, pp. 509-513.
[7] A. Baysan, R. A. Whiley and E. Lynch, “Antimicrobial Effect of a Novel Ozone-Generating Device on Micro-Organisms Associated with Primary Root Carious Lesions in Vitro,” Caries Research, Vol. 34, No. 6, 2000, pp. 498-501.
[8] D. Bialoszewski, E. Bocian, B. Bukowska, M. Czajkowska, L. B. Sokó? and S. Tyski, “Antimicrobial Activity of Ozonated Water,” Medical Science Monitor, Vol. 16, No. 9, 2010, pp. 71-75.
[9] A. Castillo, P. Galindo Moreno, G. Avila, M. Valderrama, J. Liébana and P. Baca, “In Vitro Reduction of mutans streptococci by Means of Ozone Gas Application,” Quintessence International, Vol. 39, No. 10, 2008, pp. 827831.
[10] T. G. Fragell, W. Dietz, P. Lingstr?m, F. Steiniger and J. G. Norén, “Effect of Ozone Treatment on Different Cariogenic Microorganisms in Vitro,” Swedish Dental Journal, Vol. 32, No. 3, 2008, pp. 139-147.
[11] R. S. Hems, K. Gulabivala, Y. L. Ng, D. Ready and D. A. Spratt, “An in Vitro Evaluation of the Ability of Ozone to Kill a Strain of Enterococcus faecalis,” International Endodontic Journal, Vol. 38, No. 1, 2005, pp. 22-29.
[12] K. C. Huth, M. Quirling, S. Maier, K. Kamereck, M. Alkhayer, E. Paschos, U. Welsch, T. Miethke, K. Brand and R. Hickel, “Effectiveness of Ozone against Endodontopathogenic Microorganisms in a Root Canal Biofilm Model,” International Endodontic Journal, Vol. 42, No. 1, pp. 3-13.
[13] G. M. Knight, J. M. McIntyre and P. S. Zilm, “The Inability of Streptococcus mutans and Lactobacillus acidophilus to Form a Biofilm in Vitro on Dentine Pretreated with Ozone,” Australian Dental Journal, Vol. 53, No. 4, 2008, pp. 349-353.
[14] I. R. Komanapalli and B. H. S. Lau, “Ozone-Induced Damage of Escherichia coli K-12,” Applied Microbiology and Biotechnology, Vol. 46, No. 5-6, 1996, pp. 610-614.
[15] P. Müller, B. Guggenheim and P. R. Schmidlin, “Efficacy of Gasiform Ozone and Photodynamic Therapy on a Multispecies Oral Biofilm in Vitro,” European Journal of Oral Sciences, Vol. 115, No. 1, 2007, pp. 77-80.
[16] M. Nagayoshi, T. Fukuizumi, C. Kitamura, J. Yano, M. Terashita and T. Nishihara, “Efficacy of Ozone on Survival and Permeability of Oral Microorganisms,” Oral Microbiology and Immunology, Vol. 19, No. 4, 2004, pp. 240-246.
[17] M. Oizumi, T. Suzuki, M. Uchida, J. Furuya and Y. Okamoto, “In Vitro Testing of a Denture Cleaning Method Using Ozone,” Journal of Medical and Dental Sciences, Vol. 45, No. 2, 1998, pp. 135-139.
[18] R. Stoll, L. Venne, A. J. Momeni, R. Mutters and V. Stachniss, “The Disinfecting Effect of Ozonized Oxygen in an Infected Root Canal: An in Vitro Study,” Quintessence International, Vol. 39, No. 3, 2008, pp. 231-236.
[19] B. Thanomsub, V. Anupunpisit, S. Chanphetch, T. Watcharachaipong, R. Poonkhum and C. Srisukonth, “Effects of Ozone Treatment on Cell Growth and Ultrastructural Changes in Bacteria,” The Journal of General and Applied Microbiology, Vol. 48, No. 4, 2002, pp. 193-199.
[20] D. E. Cortezzo, K. Koziol Dube, B. Setlow and P. Setlow, “Treatment with Oxidizing Agents Damages the Inner Membrane of Spores of Bacillus subtilis and Sensitizes Spores to Subsequent Stress,” Journal of Applied Microbiology, Vol. 97, No. 4, 2004, pp. 838-852.
[21] M. Bette, R. M. Nusing, R. Mutters, Z. B. Zamora, S. Menendez and S. Schulz, “Efficiency of Tazobactam/Piperacillin in Lethal Peritonitis Is Enhanced after Preconditioning of Rats with O3/O2-Pneumoperitoneum,” Shock, Vol. 25, No. 1, 2006, pp. 23-29.
[22] R. Polgnano, P. L. Vannucchi and R. Degli Innocenti, “Efficacia Antisettica Dell’Acqua Ozonizzata su Germi Isolati da Lesioni Croniche Della Cute,” Proceedings International Medical Ozone Society (IMOS) Congress, Ossigeno Ozono Terapia: Dall’Empirismo alla Metodologia Scientifica, Siena, 2000, pp. 49-50.
[23] Z. Z. Rodriguez, D. Guanche, R. G. álvarez, F. H. Rosales, Y. Alonso and S. Schulz, “Preconditioning with Ozone/ Oxygen Mixture Induces Reversion of Some Indicators of Oxidative Stress and Prevents Organic Damage in Rats with Fecal Peritonitis,” Inflammation Research, Vol. 58, No. 7, 2009, pp. 371-375.
[24] S. Schulz, Z. Z. Rodriguez, R. Mutters, S. Menendez and M. Bette, “Repetitive Pneumoperitoneum with Ozonized Oxigen as a Preventive in Lethal Polymicrobial Sepsis in Rats,” European Surgical Research, Vol. 35, No. 1, 2003, pp. 26-34.
[25] H. Steinhart, S. Schulz and R. Mutter, “Evaluation of Ozonated Oxygen in an Experimental Animal Model of Osteomyelitis as a Further Treatment Option for SkullBase Osteomyelitis,” European Archives of Oto-RhinoLaryngology, Vol. 256, No. 3, 1999, pp. 153-157.
[26] Z. B. Zamora, A. B. Orlay, Y. L′opez, R. Delgado, R. González, S. Menéndez, F. Hernández and S. Schulz, “Effects of Ozone Oxidative Preconditioning on TNF-α Release and Antioxidant-Prooxidant Intracellular Balance in Mice during Endotoxic Shock,” Mediators of Inflammation, Vol. 2005, No. 1, 2005, pp. 16-22.
[27] S. Burgassi, I. Zanardi, V. Travagli, E. Montomoli and V. Bocci, “How Much Ozone Bactericidal Activity Is Compromised by Plasma Components?” Journal of Applied Microbiology, Vol. 106, No. 5, 2009, pp. 1715-1721.
[28] S. K. Fridkin, J. C. Hageman, M. Morrison, L. T. Sanza, K. Como Sabetti, J. A. Jernigan, K. Harriman, L. H. Harrison for the Active Bacterial Core Surveillance Program of the Emerging Infection Program Network, “MethicillinResistant Staphylococcus aureus Disease in Three Communities,” New England Journal of Medicine, Vol. 352, No. 14, 2005, pp. 1436-1444.
[29] R. M. Klevens, M. A. Morrison, J. Nadle, S. Petit, K. Gershman, S. Ray, L. H. Harrison and R. Lynfield, “Active Bacterial Core Surveillance (ABCs) MRSA Investigators, “Invasive Methicillin-Resistant Staphylococcus aureus Infections in the United States,” JAMA, Vol. 298, No. 15, 2007, pp. 1763-1771.
[30] G. J. Moran, A. Krishnadasan, R. J. Gorwitz, G. E. Fosheim, L. K. McDougal, R. B. Carey, D. A. Talan and Emergency ID Net Study Group, “Methicillin-Resistant S. aureus Infections among Patients in the Emergency Department,” New England Journal of Medicine, Vol. 355, No. 7, 2006, pp. 666-674.
[31] K. J. Popovich, R. A. Weinstein and B. Hota, “Are Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Replacing Traditional Nosocomial MRSA Strains?” Clinical Infectious Diseases, Vol. 46, No. 6, 2008, pp. 787-794.
[32] D. A. Robinson, A. M. Kearns, A. Holmes, D. Morrison, H. Grundmann, G. Edwards, F. G. O’Brien and F. C. Tenover, “Re-Emergence of Early Pandemic Staphylococcus aureus as a Community-Acquired Meticillin-Resistant Clone,” Lancet, Vol. 365, No. 9466, 2005, pp. 1256-1258.
[33] J. Vallés and R. Ferrer, “Bloodstream Infections in the ICU,” Infectious Disease of North America, Vol. 23, No. 3, 2009, pp. 557-569.
[34] V. Bocci and L. Paulesu, “Studies on the Biological Effects of Ozone: 1. Induction of Interferon Gamma on Human Leucocytes,” Haematologica, Vol. 75, No. 6, 1990, pp. 510-515.
[35] V. Bocci, “Ozone as Bioregulator: Pharmacology and Toxicology of Ozone Therapy Today,” Journal of Biological Regulators & Homeostatic Agents, Vol. 10, No. 2-3, 1996, pp. 31-53.
[36] V. Bocci, “Can Ozone Therapy Be Performed If the Biochemistry of the Process Cannot Be Controlled?” Archives of Medical Research, Vol. 38, No. 5, 2007, pp. 584-585.
[37] N. Di Paolo, V. Bocci, G. Marosi, E. Borrelli, A. Bravi, A. Bruci, C. Aldinucci and L. Capotondo, “Extracorporeal Blood Oxygenation and Ozonization in Man. Preliminary Report,” The International Journal of Artificial Organs, Vol. 23, No. 2, 2000, pp. 131-141.
[38] V. Bocci and L. Paulesu, “Studies on the Biological Effects of Ozone 5: Evaluation of Immunological Parameters and Tolerability in Normal Volunteers Receiving Ambulatory Autohaemotherapy,” Biotherapy, Vol. 7, No. 2, 1994, pp. 83-90.
[39] I. R. Komanapalli and B. H. S. Lau, “Inactivation of Bacteriophage k, Escherichia coli and Candida albicans by Ozone,” Applied Microbiology and Biotechnology, Vol. 49, No. 6, 1998, pp. 766-769.
[40] I. R. Komanapalli, J. B. Mudd and B. H. Lau, “Effect of Ozone on Metabolic Activities of Escherichia coli K-12,” Toxicology Letters, Vol. 90, No. 1, 1997, pp. 61-66.
[41] G. Valacchi, V. Fortino and V. Bocci, “The Dual Action of Ozone on the Skin,” British Journal of Dermatology, Vol. 153, No. 6, 2005, pp. 1096-1100.
[42] V. Bocci, E. Luzzi, F. Corradeschi, L. Paulesu, R. Rossi, E. Cardaioli and P. Di Simplicio, “Studies on the Biological Effects of Ozone 4: Cytokine Production and Glutathione Levels in Human Erythrocytes,” Journal of Biological Regulators and Homeostatic Agents, Vol. 7, No. 4, 1993, pp. 133-138.
[43] V. Bocci, E. Luzzi, F. Corradeschi and S. Silvestri, “Studies of the Biological Effects of Ozone 6: Production of Transforming Growth Factor b1 by Human Blood after Ozone Treatment,” Journal of Biological Regulators and Homeostatic Agents, Vol. 8, No. 4, 1994, pp. 108-112.
[44] W. Doroszkiewicz, I. Sikorska and S. Jankowski, “Studies on the Influence of Ozone on Complement-Mediated Killing of Bacteria,” FEMS Immunology & Medical Microbiology, Vol. 9, No. 4, 1994, pp. 281-285.
[45] L. Paulesu, E. Luzzi and V. Bocci, “Studies on the Biological Effects of Ozone 2: Induction of Tumor Necrosis Factor on Human Leucocytes,” Lymphokine and Cytokine Research, Vol. 10, No. 5, 1991, pp. 409-412.
[46] E. Riva Sanseverino, “Aspetti Immunologici Dell’Ozonoterapia,” Rivista Italiana di Omotossicologia, pp. 19-24.
[47] P. D. Thomson, G. O. Till and D. J. Smith, “Modulation of IgM Antibody Formation by Lipid Peroxidation Products from Burn Plasma,” JAMA Surgery, Vol. 126, No. 8, 1991, pp. 973-976.
[48] V. Bocci, “A Reasonable Approach for the Treatment of HIV Infection in the Early Phase with Ozone Therapy (Autohaemotherapy). How Inflammatory Cytokines May Have a Therapeutic Role,” Mediators of Inflammation, Vol. 3, No. 5, 1994, pp. 315-321.
[49] M. E. Bégin, “Fatty Acids, Lipid Peroxidation and Diseases,” Proceedings of the Nutrition Society, Vol. 49, No. 2, 1990, pp. 261-267.
[50] V. Bocci, G. Valacchi, F. Corradeschi, C. Aldinucci, S. Silvestri, E. Paccagnini and R. Gerli, “Studies on the Biological Effects of Ozone 7: Generation of Reactive Oxygen Species (ROS) after Exposure of Human Blood to Ozone,” Journal of Biological Regulators and Homeostatic Agents, Vol. 12, No. 3, 1998, pp. 67-75.
[51] R. D. Buckley, J. D. Hackney, K. Clark and C. Posin, “Ozone and Human Blood,” Archives of Environmental Health, Vol. 30, No. 1, 1975, pp. 40-43.
[52] B. D. Goldstein and O. J. Balchum, “Effect of Ozone on Lipid Peroxidation in the Red Blood Cell,” Experimental Biology and Medicine, Vol. 126, No. 2, 1967, pp. 356-358.
[53] L. Coppola and G. Verrazzo, “Oxygen-Ozone Therapy and Haemorheological Parameters in Peripheral Chronic Arterial Disease,” Trombosi e Aterosclerosi, Vol. 3, 1992, pp. 83-89.
[54] C. Di Filippo, M. Luongo, R. Marfella, F. Ferraraccio, B. Lettieri, A. Capuano, F. Rossi and M. D’Amico, “Oxygen/Ozone Protects the Heart from Acute Myocardial Infarction through Local Increase of eNOS Activity and Endothelial Progenitor Cells Recruitment,” Naunyn-Schmiedeberg’s Archives of Pharmacology, Vol. 382, No. 3, 2010, pp. 287-291.
[55] B. A. Freeman and J. B. Mud, “Reaction of Ozone with Sulphydryls of Human Erythrocytes,” Archives of Biochemistry and Biophysics, Vol. 208, No. 1, 1981, pp. 212-220.
[56] A. Van Der Vliet, C. A. O’Neil, J. P. Eiserich and C. E. Cross, “Oxydative Damage to Extracellular Fluids by Ozone and Possible Protective Effects of Thiols,” Archives of Biochemistry and Biophysics, Vol. 321, No. 1, 1995, pp. 43-50.
[57] P. Wentworth, J. E. McDunn, A. D. Wentworth, C. Takeuchi, J. Nieva, T. Jones and C. Bautista, “Evidence for Antibody-Catalyzed Ozone Formation in Bacterial Killing and Inflammation,” Science, Vol. 298, No. 5601, 2002, pp. 2195-2199.

comments powered by Disqus

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.