key: cord-0868863-o0vqfpef authors: Biurrun, J.; Garcia, B.; Perez, A.; Kochan, G.; Escors, D.; Crespo, J.; Lasa, I.; Echarri, A. title: Evaluation of the disinfecting capacity of ozone in emergency vehicles. date: 2020-05-26 journal: nan DOI: 10.1101/2020.05.24.20111666 sha: 48ac978f84ed51fa0d942d3c8c02787c2cd05c1d doc_id: 868863 cord_uid: o0vqfpef Objective: As a consequence of the health crisis arising from the SARS-CoV-2 coronavirus pandemic, ozone treatments are being applied as disinfectant in emergency vehicles, without objective evidence on its efficacy. Here we evaluate the efficacy of ozone treatment over bacterial strains and virus-like particles. Method: A preparation of a lentiviral vector (lentivector) and dried cultures of two bacterial strains (gram + Staphylococcus aureus and gram - Salmonella enterica ser. Enteritidis) were placed inside an ambulance at two different locations. The interior of the vehicle was subjected to 10 min and 20 min treatments (3 and 6 times the recommended time by the manufacturer). Following the treatments, lentivector preparations were titrated, and viable bacteria (colony forming units, CFUs) counted and compared to pre-treatment titers and infectious CFUs of the same lysates and cultures. Results: None of the treatments significantly reduced either lentivector titer or the number of viable bacteria. Conclusions: At least in the analyzed conditions and for the microorganisms used in this study, it can be concluded that ozone treatment is not advisable for the disinfection of emergency vehicles. As a consequence of the pandemic caused by SARS-CoV-2 the disinfection of emergency vehicles has become a priority, as this is the main transport means of infected patients. Therefore, these vehicles can be a potential source for new infections of patients not related to SARS-CoV-2 infections. The different governmental organizations, emergency services and private transport companies of biomedical materials are putting in place several measures for disinfecting vehicles. Ozone cannons are currently the most used method for disinfecting the surfaces of vehicles, due to their rapid and straightforward application. Ozone is an oxidizing gas with a demonstrated disinfecting capacity in aqueous solutions, and it is widely used for disinfecting food and water (1-4). However, its efficacy to disinfect surfaces by nebulization has not been properly tested. Therefore, the Spanish Ministery of Health does not include ozone in the list of authorized virucides for disinfecting surfaces (https://www.mscbs.gob.es/profesionales/saludPublica/ccayes/alertasActual/nCov-China/documentos/Listado_virucidas.pdf). Here we have tested the efficacy of ozone disinfection using ozone cannons as currently applied in ambulances. To this end, we have studied its efficacy in reducing the viability of three model microorganisms: a lentivector and two bacterial pathogens In addition, these treatments have also been proposed to as broad spectrum disinfectants. Hence, our study has also tested the capacities of ozone treatments All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint over two model bacterial pathogens, Staphylococcus aureus y Salmonella enterica. These gram-positive and gram-negative strains are recomended by American (EPA) and European (EN) norms (7) (8) for the evaluation of the efficacy of disinfecting treatments over surfaces. The pSIN-GFP lentivector was prepared by the three-plasmid co-transfection method as previously described. This lentivector encodes the GFP gene under the control of the SFFV promoter (9) . Lentivector preparations were titrated in 293T cell cultures as (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint As a control, UV-C light was used as a disinfectant agent, which is demonstrated to be efficacious for disinfecting surfaces (13) (14) (15) (16) . Hence, open Petri dishes containing 1 ml of pSIN-GFP lentivector stock in DMEM as well as Petri dishes with desiccated bacterial cultures were located in the interior of a laminar flow cell culture cabinet and subjected to UV-C irradiation for 20 min (TUV 30W, 100W/cm 2 at 1 m). Lentivector titres and bacterial counts were performed as described above. During the test, ozone levels higher than 10 ppm were reached within the ambulance for both 10 and 20-min treatments (Figure 1 ). The humidity during the experiments ranged from 37 to 48% (Figure 1 ). Ozone treatments barely affected the lentivector titres ( Figure 2 and 3) . Independently of the time and location from the ozone cannon, the differences were always inferior to a logarithm (10 -1 ) (Figure 3 ). In contrast, the UV light treatment reduced the lentiector titre below the detection limit of the titration technique (< 5000 partículas/ml), reaching at least 4 logs reduction in lentivector titre (Figures 2 y 3) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint Importantly, no significant effects over the viability of bacterial samples were observed. The decrease in viable cells did not reach have a log for S. aureus MW2 and 1,4 logs for S. Enteritidis 3934. In contrast, UV irradiation reduced the number of viable cells below the detection limit of the titration technique (<10 Infectious colony-forming units /ml), reaching a decrease of at least 6 logs ( Figure 4 ). These assays were repeated independently twice at different days, with equivalent results. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint diameter), with a single-stranded RNA genome packaged by the nucleoprotein. In turn, the viral nucleocapsid in enclosed within a lipid membrane in which the S, M and E proteins are incorporated. Due to the same chemical composition of their genomes (single-stranded RNA), we could hypothesize that both organisms as similarly susceptible to UV light and other oxidazing agents. Guidelines of the Environmental Protection Agency (EPA, United States of America) (7) and the European Guidelines (EN-14885) reccomend a decrease superior than 3 logs and 5-6 logs for viruses and bacteria, respectively, for a disinfecting treatment to be considered efficacious by nebulization in hospitals. None of the two ozone treatments significantly reduced the lentivector titer (Figures 2 and 3) , or S. aureus MW2 and S. Enteritidis 3934 bacterial counts ( Figure 4) . Therefore, these treatments cannot be considered effective for disinfecting contaminated surfaces in ambulances, in the tested conditions. Interestingly, the location of the samples from the ozone cannon did not influence the results, without significant reduction in lentivector treatments or bacterial counts (Figures 3 y 4) . The evidence from our study shows that gaseous ozone treatments currently applied in emergency vehicles do not significantly affect virus or bacteria viability. It has been previously shown virucide activities of gaseous ozone in surfaces at concentrations higher than 20-25 ppm in the presence of relative humidity higher than 90% (17) . In our present study, we have quantified ozone concentrations higher than10 ppm, although we cannot discard that superior concentrations may have been reached as the upper detection limit of the ozometer is 10 ppm. Relative humidity at the time of the experiments ranged from 37% to 48%, which may have influenced the efficacy of the treatment. Nevertheless, it has to be remarked that these humidity levels correspond to those normally found within the vehicle, without using humidifyiers. The ozone is rapidly decomposed into highly oxidative OH radicals in the presence of water, while it is more stable in air. This difference in stability could explain the differences in efficacy found in water compared to nebulization. Although we cannot discard that prolonged treatments with higher ozone concentrations may show some disinfecting capacities, our data indicates that the current treatments in emergency vehicles are insufficient. Therefore, we cannot recommend their use for this end. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.24.20111666 doi: medRxiv preprint Chapter Thirty-Three -Ozone Disinfection. Second Edition. Microbiology of Waterborne Diseases: Microbiological Aspects and Risks Ozone disinfection of chlorine-resistant bacteria in drinking water Application of ozone for enhancing the microbiological safety and quality of foods: a review Ozone Contribution in Food Industry in Japan The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability Lentiviral Vectors in Gene Therapy: Their Current Status and Future Potential Product Performance Test Guidelines TECHNICAL GUIDANCE DOCUMENT IN SUPPORT OF THE DIRECTIVE 98/8/EC CONCERNING THE PLACING OF BIOCIDAL PRODUCTS ON THE MARKET GUIDANCE ON DATA REQUIREMENTS FOR ACTIVE SUBSTANCES AND BIOCIDAL PRODUCTS Targeting dendritic cell signaling to regulate the response to immunization Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes Genome and virulence determinants of high virulence community-acquired MRSA Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose Room Decontamination with UV Radiation No touch" technologies for environmental decontamination. Current Opinion in Infectious Diseases Role of Ultraviolet Disinfection in the Prevention of Surgical Site Infections Understanding ultraviolet light surface decontamination in hospital rooms: A primer Development of a Practical Method for Using Ozone Gas as a Virus Decontaminating Agent. Ozone: Science & Engineering No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted We would like to acknowledge the use of the test ambulance from Escuela Sanitaria Técnico Profesional de Navarra (ESTNA), and to Protección Civil de Navarra for the use of the ozone cannon.