key: cord-1026676-pxe7kv33 authors: Martí, Miguel; Tuñón-Molina, Alberto; Aachmann, Finn Lillelund; Muramoto, Yukiko; Noda, Takeshi; Takayama, Kazuo; Serrano-Aroca, Ángel title: Protective face mask filter capable of inactivating SARS-CoV-2, and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis date: 2020-11-24 journal: bioRxiv DOI: 10.1101/2020.11.24.396028 sha: 238760d6f5dd055368c3a82d4135dc3109aec955 doc_id: 1026676 cord_uid: pxe7kv33 Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating neither SARS-CoV-2 nor multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2 mediated pneumonia disease complex and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a novel protective non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating SARS-CoV-2 in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many health care workers and researchers working in this urgent and challenging field. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, Hubei province, China, in December 2019 1 . The rapid spread of this pathogen, which has caused the current COVID-19 pandemic, is putting at high risk the health and economy of the most developed and underdeveloped countries. According to the World Health Organisation (WHO), the current COVID-19 outbreak has 59,342,857 global cases and 1,399,373 global deaths in more than 200 countries (data of 24 th November 2020) 2 . SARS-CoV-2 is the third coronavirus causing severe pneumonia 3, 4 , an infection of the lungs caused usually by bacteria and viruses 5, 6 . The death risk of viral pneumonias can increase when co-infection can be caused by viruses in the setting of community-acquired bacterial pneumonia such as the lethal Streptococcus pneumoniae [7] [8] [9] [10] , with additional symptoms of bacterial pneumonia 11 . New pathogens, such as SARS-CoV-2, that can coexist with a broad range of other types of clinically relevant bacteria, including multidrug-resistant strains, constitutes a real life-threatening to humans during the approaching cold season. In addition, antibiotic resistance in bacterial pneumonia treatment is a wide-spread problem that is increasing at an alarming rate 12, 13 . The SARS-CoV-2 pathogen is stable from hours to days in aerosols and surfaces of different chemical nature such as copper, cardboard, plastic, aluminium, or stainless steel surfaces, demonstrating that infections can be easily transmitted through the air via the microdroplets or direct contact after touching contaminated surfaces [14] [15] [16] [17] [18] . This coronavirus can spread faster than its two ancestors SARS-CoV and MERS-CoV 19 through coughing, sneezing, touching or breathing 20 , and more broadly through asymptomatic carriers [21] [22] [23] onto the surface of a commercial non-woven fabric filter, which are commonly used in the production of face masks in the present pandemic, could produce antiviral filters that can inhibit the infection capacity of SARS-CoV-2. Non-woven filters are lightweight, flexible, resilient, provides good bacteria filtration and air permeability, and are costeffective materials for masks, has a lower manufacturing cost, are hygienic and clean as they are for single use 38 . Face masks have been accepted as effective protective tools by blocking the pass of viral and bacterial particles 39 . However, if the filters that contain the face masks are made by composite materials with antimicrobial activity, the protection of these tools could increase even more. Furthermore, due to the previously reported antibacterial activity of BAK against Gram-positive bacteria 40 , we also expect that the developed BAK filter will be able to inhibit the bacterial grow of two clinically relevant multidrug resistant bacteria: methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). In addition to the current COVID-19 pandemic, antibiotic resistance is another increasing challenge of the present century. According to the World Health Organization (WHO), antibiotic resistance will be one of the leading causes of death over other important diseases such as cancer by the year 2050 41 . In fact, MRSE is a nosocomial pathogen that is spreading globally and is often cause of catheterassociated disease, especially among low birth weight prematures 42, 43 . MRSA is causing global health problems specially in medical instruments and catheters because S. aureus is a human pathogen that can easily develop resistance to antibiotics 44, 45 . Finally, in this study, we attempted to develop a protective face mask filter capable of inactivating SARS-CoV-2, and MRSA and MRSE. Disks specimens of approximately 10 mm in diameter were prepared with a non-woven spunlace fabric filter from NV EVOLUTIA (commercial filters used for face masks) by dry cutting with a cylindrical punch. Face mask filter (BAK Filter) disks (n=6) were produced by the dip coating method 37 Nuclear magnetic resonance (NMR) was applied for the characterization of the benzalkonium chloride used in the biofunctional coating of the commercial nonwoven filters. Prior to NMR sample preparation, the ethanol/water solvent was evaporated from commercial Montplet 70% ethyl alcohol with benzalkonium chloride (99.9/0.1% w/w) at 25°C. After that, the sample of benzalkonium chloride was prepared by dissolving 10 mg in 550 µL D2O (D, 99.9 %) (Sigma-Aldrich, Norway) and transferred to 5mm LabScape Stream NMR tube. The NMR experiments were recorded on a BRUKER AVIIIHD 800 MHz (Bruker BioSpin AG, Fälladen, Switzerland) equipped with 5mm with cryogenic CP-TCI. All NMR recording was performed at 25°C or 37°C. For the characterization of benzalkonium chloride the following spectra were recorded: 1D proton, 2D double quantum filtered correlation spectroscopy (DQF-COSY) and 2D 13 C heteronuclear single quantum coherence (HSQC) with multiplicity editing. TMS was used as chemical shift reference for proton and carbon chemical shifts. The spectra were recorded, processed and analysed using TopSpin 3.7 software (Bruker BioSpin). A Zeiss Ultra 55 field emission scanning electron microscope (FESEM, Zeiss Ultra 55 Model) was operated at an accelerating voltage of 3 kV to observe the porous morphology of the treated and untreated non-woven face mask filters at a magnification of x100 and x1000. The filter samples were prepared to be conductive by platinum coating with a sputter coating unit. Pseudomonas syringae (DSM 21482) from the Leibniz Institute DSMZ-German collection of microorganisms and cell cultures GmbH (Braunschweig, Germany), was cultured in solid Tryptic Soy Agar (TSA, Liofilchem) and subsequently in Liquid Tryptic Soy Broth (TSB, Liofilchem). Liquid incubation was carried out at 25ºC and 120rpm. Phage phi 6 (DSM 21518) propagation was carried out according to the specifications provided by the Leibniz Institute DSMZ-German collection of microorganisms and cell cultures GmbH (Braunschweig, Germany). The SARS-CoV-2 strain used in this study (SARS-CoV-2/Hu/DP/Kng/19-027) was kindly gifted from Dr. Tomohiko Takasaki Dilutions were placed onto the TMPRSS2/Vero cells in triplicate and incubated at 37°C for 96 hours. Cytopathic effect was evaluated under a microscope. TCID50/mL was calculated using the Reed-Muench method. The agar disk diffusion tests were performed to analyse the antibacterial activity of the (1) where nwhalo indicates the normalised width of the antimicrobial inhibition zone, diz is the inhibition zone diameter and d referrers to the sample disk diameter. These diameters were measured by image software analysis (Image J). The tests were performed six times in different days to ensure reproducibility. The statistical analyses were performed by ANOVA followed by Tukey's posthoc test ( ⁎ p > 0.05, ⁎⁎⁎ p > 0.001) on GraphPad Prism 6 software. The benzalkonium chloride used in this study for the treatment of the unwoven filters analysed by NMR is shown in Figure 1 . The porous morphology images of the commercial and treated non-woven face mask filter are shown in Figure 2 . Phage phi 6 is a double-stranded RNA virus with three-part, segmented, totalling The phage titers of each type of face mask filter sample were calculated and compared with the control (see Figure 4 ). The results achieved with the TCID50/mL method about the reduction of infectious titers of SARS-CoV-2 after 1 minute of contact with the control, the U Filters, the S Filters and the BAK Filters containing the biofunctional coating are shown in Figure 5 . These results clearly demonstrate that the BAK Filter is very effective against SARS-CoV-2 even after 1 minute of contact. This is also in good agreement with the antiviral results of the biosafe viral model used in this study (see Figures 3 and 4) . The antibacterial results of the treated filters against MRSA and MRSE multidrugresistant bacteria are shown in Figure 6 . Non-woven benzalkonium chloride-containing filters capable of inactivating SARS- pandemic. Director-General's opening remarks at the media briefing on COVID-19 -11 COVID-19) Pandemic. World Health Organization Hosts and Sources of Endemic Human Coronaviruses Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study Previous lung diseases and lung cancer risk: A systematic review and meta-analysis Severe community-acquired pneumonia due to Pseudomonas aeruginosa coinfection in an influenza A(H1N1)pdm09 patient Recommendations and guidelines for the treatment of pneumonia in Taiwan Community-acquired adenoviral and pneumococcal pneumonia complicated by pulmonary aspergillosis in an immunocompetent adult Influence of Older Age and Other Risk Factors on Pneumonia Hospitalization in Switzerland in the Pneumococcal Vaccine Era Complications of Viral Influenza Mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance community-acquired pneumonia treatment guidelines: There is a need for a change toward more parsimonious antibiotic use Turbulent Gas Clouds and Respiratory Pathogen Emissions Potential Implications for Reducing Transmission of COVID-19 Evidences of SARS-CoV-2 virus air transmission indoors using several untouched surfaces: A pilot study Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1 Air, Surface Environmental, and Personal Protective Equipment Contamination by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from a Symptomatic Patient Stability of SARS-CoV-2 in different environmental conditions COVID-19: A promising cure for the global panic A Review of Coronavirus Disease-2019 (COVID-19) Presumed Asymptomatic Carrier Transmission of COVID-19 Inference of person-to-person transmission of COVID-19 reveals hidden super-spreading events during the early outbreak phase Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals SARS-CoV-2 is transmitted via contact and via the air between ferrets Airborne transmission of SARS-CoV-2: The world should face the reality The effect of climate on the spread of the COVID-19 pandemic: A review of findings, and statistical and modelling techniques A spatio-temporal analysis for exploring the effect of temperature on COVID-19 early evolution in Spain Prolonged presence of SARS-CoV-2 viral RNA in faecal samples Expression of animal virus genomes Different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus Are Quaternary Ammonium Compounds, the Workhorse Disinfectants, Effective against Severe Acute Respiratory Syndrome-Coronavirus-2? Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 by WHO-Recommended Hand Rub Formulations and Alcohols Virucidal Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome Coronavirus 2 Nanodroplet-Benzalkonium Chloride Formulation Demonstrates In Vitro and Ex-Vivo Broad-Spectrum Antiviral Activity Against SARS-CoV-2 and other Enveloped Viruses Broad-Spectrum Antiviral Activity of Nanodroplet-BZK Key Words Antiviral, Nanoemulsion Evaluation of benzalkonium chloride chemoneurolytic proximal gastric vagotomy Facile preparation of durable and robust superhydrophobic textiles by dip coating in nanocomposite solution of organosilanes The need for fully bio-based facemasks to counter coronavirus outbreaks: A perspective Respiratory virus shedding in exhaled breath and efficacy of face masks Antibacterial activity of preservativefree topical anesthetic drops in current use in ophthalmology departments Clinical Pharmacology of Anti-Infective Drugs Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis Methicillin-Resistant Staphylococcus aureus : Molecular Characterization, Evolution, and Epidemiology Staphylococcus aureus and Staphylococcus epidermidis Virulence Strains as Causative Agents of Persistent Infections in Breast Implants Enumeration of bacteriophages by double agar overlay plaque assay Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells Antimicrobial Characterization of Advanced Materials for Bioengineering Applications Development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial property Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain Nosocomial septicemia due to multiply antibioticresistant Staphylococcus epidermidis Increased Use of Quaternary Ammonium Compounds during the SARS-CoV-2 Pandemic and Beyond: Consideration of Environmental Implications Comparison of the virucidal effects of disinfectant agents against equine influenza a virus Quaternary ammonium biocides: Efficacy in application Inorganic micro-and nanostructured implants for tissue engineering The authors would like to express their gratitude to the Fundación Universidad Católica The authors declare no conflict of interest.