key: cord-0844951-eaphli9e
authors: Tuñón-Molina, Alberto; Martí, Miguel; Muramoto, Yukiko; Noda, Takeshi; Takayama, Kazuo; Serrano-Aroca, Ángel
title: Antimicrobial Face Shield: Next Generation of Facial Protective Equipment against SARS-CoV-2 and Multidrug-Resistant Bacteria
date: 2021-09-01
journal: Int J Mol Sci
DOI: 10.3390/ijms22179518
sha: 32f0258fc8a8acba6fcfff3a48d9e57b1cd3fb10
doc_id: 844951
cord_uid: eaphli9e

Transparent materials used for facial protection equipment provide protection against microbial infections caused by viruses and bacteria, including multidrug-resistant strains. However, transparent materials used for this type of application are made of materials that do not possess antimicrobial activity. They just avoid direct contact between the person and the biological agent. Therefore, healthy people can become infected through contact of the contaminated material surfaces and this equipment constitute an increasing source of infectious biological waste. Furthermore, infected people can transmit microbial infections easily because the protective equipment do not inactivate the microbial load generated while breathing, sneezing or coughing. In this regard, the goal of this work consisted of fabricating a transparent face shield with intrinsic antimicrobial activity that could provide extra-protection against infectious agents and reduce the generation of infectious waste. Thus, a single-use transparent antimicrobial face shield composed of polyethylene terephthalate and an antimicrobial coating of benzalkonium chloride has been developed for the next generation of facial protective equipment. The antimicrobial coating was analyzed by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This is the first facial transparent protective material capable of inactivating enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one minute of contact, and the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Bacterial infections contribute to severe pneumonia associated with the SARS-CoV-2 infection, and their resistance to antibiotics is increasing. Our extra protective broad-spectrum antimicrobial composite material could also be applied for the fabrication of other facial protective tools such as such as goggles, helmets, plastic masks and space separation screens used for counters or vehicles. This low-cost technology would be very useful to combat the current pandemic and protect health care workers from multidrug-resistant infections in developed and underdeveloped countries.

Even though the severe lockdowns carried out in many countries of the world, the coronavirus disease 2019 (COVID- 19) pandemic is still increasing the number of global deaths in most countries [1] [2] [3] . The causative agent of this disease is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is an enveloped positive-sense Figure 1 . AFM topography images (height, amplitude error and phase images) and phase 3D representation recorded in tapping mode of the Untreated plastic (U Plastic), plastic treated by dip coating with the ethanol-based solvent (S Plastic) and filter with the biofunctional benzalkonium chloride (BAK) coating (BAK Plastic) scanning a 10 μm × 10 μm area. These images show how the BAK coating was formed on the polyethylene terephthalate surface after the dip-coating treatment. The U Plastic shows some impurities on its surface which disappear after immersion in ethanol (S Plastic). Thus, Figure 1 . AFM topography images (height, amplitude error and phase images) and phase 3D representation recorded in tapping mode of the Untreated plastic (U Plastic), plastic treated by dip coating with the ethanol-based solvent (S Plastic) and filter with the biofunctional benzalkonium chloride (BAK) coating (BAK Plastic) scanning a 10 µm × 10 µm area. These images show how the BAK coating was formed on the polyethylene terephthalate surface after the dip-coating treatment. The U Plastic shows some impurities on its surface which disappear after immersion in ethanol (S Plastic). Thus, the clean S Plastic shows some surface imperfections (irregular stripes) which disappear after the BAK coating (BAK Plastic). The BAK plastic shows slightly higher zones observed as white zones in the phase image.

The 2D phase provides images whose contrast is produced by differences in the adhesion and viscoelastic properties of the sample surface [36] . Thus, the pictures of the topography and phase angle clearly indicated that a BAK coating was formed onto the PET surface (BAK Plastic) after the dip-coating treatment with the solvent containing BAK (Figure 1 ). It can be clearly observed that the untreated plastic (U Plastic) possesses some impurities on its surface which disappear after immersing the disk in ethanol 70% for 1 min. Thus, the surface imperfections produced by the plastic fabrication procedure can be clearly observed in the 2D phase image of the PET plastic treated by dip coating with the absolute ethanol/distilled water (70/30 v/v) solvent mixture (S Plastic). However, the AFM images of the BAK Plastic clearly show that a BAK coating was formed covering all the imperfections observed in the 2D phase image of the S Plastic sample. This coating presents slightly higher zones that are observed white zones in the phase images. These results are in good agreement with the FESEM images shown in the following Figure 2 . The FESEM micrographs show clearly how a microcoating of BAK (light grey phase) is formed onto the PET surface (dark grey phase) with a thickness of approximately 25 μm (see Figure 2 ). Furthermore, the energy-disperse X-ray spectroscopy (EDS) analysis shows a nitrogen content of 0.37% weight on the BAK coating in good agreement with the nitrogen atom present in the BAK compound [34] . However, the EDS analysis on the PET matrix (polymer without nitrogen atoms) does not show any nitrogen content as expected. The FESEM micrographs show clearly how a microcoating of BAK (light grey phase) is formed onto the PET surface (dark grey phase) with a thickness of approximately 25 µm (see Figure 2 ). Furthermore, the energy-disperse X-ray spectroscopy (EDS) analysis shows a nitrogen content of 0.37% weight on the BAK coating in good agreement with the nitrogen atom present in the BAK compound [34] . However, the EDS analysis on the PET matrix (polymer without nitrogen atoms) does not show any nitrogen content as expected. Figure 3 shows that there are no statistically significant differences of opacity (or transparency), calculated with Equation (1), of the PET disks before and after the treatments with solvent or the dip-coating treatment with BAK, which is essential to be used as transparent facial protective equipment (face shield screens, plastic masks, protective screens, protective glasses, etc.).

Scanning Electron Microscopy with energy-disperse X-ray spectroscopy (EDS) for elemental analysis: PET with 0.182 ± 0.034% w/w of biofunctional BAK coating (BAK Plastic) at two magnifications: (a) ×150 and (b) ×720, and (c) EDS elemental analysis of the coating and PET matrix.

The FESEM micrographs show clearly how a microcoating of BAK (light grey phase) is formed onto the PET surface (dark grey phase) with a thickness of approximately 25 µ m (see Figure 2) . Furthermore, the energy-disperse X-ray spectroscopy (EDS) analysis shows a nitrogen content of 0.37% weight on the BAK coating in good agreement with the nitrogen atom present in the BAK compound [34] . However, the EDS analysis on the PET matrix (polymer without nitrogen atoms) does not show any nitrogen content as expected. Figure 3 shows that there are no statistically significant differences of opacity (or transparency), calculated with Equation (1), of the PET disks before and after the treatments with solvent or the dip-coating treatment with BAK, which is essential to be used as transparent facial protective equipment (face shield screens, plastic masks, protective screens, protective glasses, etc.). Therefore, we can observe that the plastic with the biofunctional coating of BAK showed potent antibacterial activity against MRSA and MRSE with similar normalized antibacterial halos of 0.61 ± 0.03 and 0.57 ± 0.05, respectively.

The phage phi 6, which is an enveloped double-stranded RNA virus (group III of the Baltimore classification [5] ), was used as biosafe viral model of SARS-CoV-2 and other enveloped viruses such as influenza due to safety reasons.

The BAK Plastic showed potent antiviral activity against phage phi 6 (100% of viral inhibition, see Figure 5 ). Therefore, we can observe that the plastic with the biofunctional coating of BAK showed potent antibacterial activity against MRSA and MRSE with similar normalized antibacterial halos of 0.61 ± 0.03 and 0.57 ± 0.05, respectively.

The phage phi 6, which is an enveloped double-stranded RNA virus (group III of the Baltimore classification [5] ), was used as biosafe viral model of SARS-CoV-2 and other enveloped viruses such as influenza due to safety reasons.

The BAK Plastic showed potent antiviral activity against phage phi 6 (100% of viral inhibition, see Figure 5 ). Thus, no plaques were produced on the bacterial lawns after 1 min of contact between the BAK Plastic and the biosafe viral model. However, similar plaques to control can be observed on the bacterial lawns after 1 min of contact between the U Plastic or S Plastic and the biosafe viral model (see Figure 5 ). The phage titers of each type of sample were calculated and compared with the control (Table 1) . Table 1 . Reduction of infection titers in plaque-forming units per mL (PFU/mL) determined by the double-layer assay for the phage phi 6. Logarithm of plaque-forming units per mL (log (PFU/mL)) of the control, untreated PET (U Plastic), PET treated by dip coating with the ethanol-based solvent (S Plastic) and PET with the biofunctional BAK coating (BAK Plastic) at 1 min of viral contact.

Phi 6 at 1 min (PFU/mL) Control 4.36 × 10 6 ± 2.92 × 10 5 U Plastic 4.38 × 10 6 ± 1.98 × 10 5 S Plastic 4.23 × 10 6 ± 1.36 × 10 6 BAK Plastic 0.00 ± 0.00 Table 1 shows that the titers obtained by contacting the phages with the U or S Plastic are similar to the control. However, the BAK plastic displayed a strong phage inactivation. The results achieved with SARS-CoV-2 after 1 min of contact with the U Plastic, the S Plastic and the BAK Plastic containing the biofunctional coating are shown in Figure 6 . Thus, no plaques were produced on the bacterial lawns after 1 min of contact between the BAK Plastic and the biosafe viral model. However, similar plaques to control can be observed on the bacterial lawns after 1 min of contact between the U Plastic or S Plastic and the biosafe viral model (see Figure 5 ). The phage titers of each type of sample were calculated and compared with the control (Table 1) . Table 1 . Reduction of infection titers in plaque-forming units per mL (PFU/mL) determined by the double-layer assay for the phage phi 6. Logarithm of plaque-forming units per mL (log (PFU/mL)) of the control, untreated PET (U Plastic), PET treated by dip coating with the ethanol-based solvent (S Plastic) and PET with the biofunctional BAK coating (BAK Plastic) at 1 min of viral contact. Table 1 shows that the titers obtained by contacting the phages with the U or S Plastic are similar to the control. However, the BAK plastic displayed a strong phage inactivation. The results achieved with SARS-CoV-2 after 1 min of contact with the U Plastic, the S Plastic and the BAK Plastic containing the biofunctional coating are shown in Figure 6 .

These results clearly demonstrate that the BAK Plastic is very effective against SARS-CoV-2 even after 1 min of contact. This is also in good agreement with the antiviral results of the biosafe viral model used in this study (see Figure 5 and Table 1 ). However, since BAK is highly water-soluble and therefore could come out when the PET sheet with the BAK coating is in contact with water, the antiviral and antibacterial tests were performed again after washing with distilled water to analyze the antimicrobial durability of the BAK coating to water. The results of these experiments (results not shown) showed that the antimicrobial BAK coating dissolves really fast in distilled water and loses its antimicrobial activity as expected. However, the developed antimicrobial face shield is presented here as a single-use face protective equipment for the current and future microbial menaces. These results clearly demonstrate that the BAK Plastic is very effective against SARS-CoV-2 even after 1 min of contact. This is also in good agreement with the antiviral results of the biosafe viral model used in this study (see Figure 5 and Table 1 ). However, since BAK is highly water-soluble and therefore could come out when the PET sheet with the BAK coating is in contact with water, the antiviral and antibacterial tests were performed again after washing with distilled water to analyze the antimicrobial durability of the BAK coating to water. The results of these experiments (results not shown) showed that the antimicrobial BAK coating dissolves really fast in distilled water and loses its antimicrobial activity as expected. However, the developed antimicrobial face shield is presented here as a single-use face protective equipment for the current and future microbial menaces.

These advanced face shields can provide superior protection to virologists working with highly infective pathogens in high-level biosafety labs, surgeons and healthcare workers in general. Furthermore, the proposed PET plastic is a recyclable material that can be reutilized [32] and thus contribute to decrease the increasing amount of this type of waste generated in the current pandemic. Furthermore, the antimicrobial coating can be produced easily as many times as necessary as a reusability characteristic of this technology. The BAK coating can be performed onto the outside side only for people not infected or also on the inside side if the protective equipment is going to be used for infected patients. It could also be performed on other types of transparent synthetic polymers such as polycarbonate, polymethyl methacrylate, poly (2-hydroxyethyl methacrylate), on nontransparent materials or on biodegradable polymers that would provide a solution to the need for bio-based protective tools for environmental reasons [37] . This next generation equipment will significantly reduce the increasing generation of infectious biological waste. These new technologies could revolutionize the face protective tool industry because other face protective equipment could be developed applying the same low-cost technology providing high antimicrobial activity (see Figure 7 ). These advanced face shields can provide superior protection to virologists working with highly infective pathogens in high-level biosafety labs, surgeons and healthcare workers in general. Furthermore, the proposed PET plastic is a recyclable material that can be reutilized [32] and thus contribute to decrease the increasing amount of this type of waste generated in the current pandemic. Furthermore, the antimicrobial coating can be produced easily as many times as necessary as a reusability characteristic of this technology. The BAK coating can be performed onto the outside side only for people not infected or also on the inside side if the protective equipment is going to be used for infected patients. It could also be performed on other types of transparent synthetic polymers such as polycarbonate, polymethyl methacrylate, poly (2-hydroxyethyl methacrylate), on non-transparent materials or on biodegradable polymers that would provide a solution to the need for bio-based protective tools for environmental reasons [37] . This next generation equipment will significantly reduce the increasing generation of infectious biological waste. These new technologies could revolutionize the face protective tool industry because other face protective equipment could be developed applying the same low-cost technology providing high antimicrobial activity (see Figure 7) .

The antimicrobial mechanism of action of BAK against both bacterial and enveloped viruses is attributed to its positively charged nitrogen atoms that can eradicate the bacterial surface or disrupt the phospholipid bilayer membrane, the glycoproteinaceous envelope, and the spike glycoproteins of viruses such as phi6, SARS-CoV-2 and IFV [38, 39] . BAK is a Food and Drug Administration-approved product for a broad-range of disinfecting applications such as additives in soaps and hand sanitizers [40] [41] [42] . We have demonstrated here that these transparent PET-based composites possess high antiviral and antibacterial activity to reduce the spread of COVID-19 and methicillin-resistant bacteria. This extraprotective composite material has been developed by a low-cost method of dip coating that let BAK to physically adsorbed [43] onto the surface of a commercial PET plastic commonly used for the fabrication of face shields and other protective equipment providing high antimicrobial activity. Nonetheless, further research is necessary to overcome the possible shortfalls on the applicability of this coating technology to other protective tools and to other types of transparent materials. The effect of packing, storage and transportation on the antimicrobial properties of the BAK coating need also to be studied before applying this technology at large-scale. The antimicrobial mechanism of action of BAK against both bacterial and enveloped viruses is attributed to its positively charged nitrogen atoms that can eradicate the bacterial surface or disrupt the phospholipid bilayer membrane, the glycoproteinaceous envelope, and the spike glycoproteins of viruses such as phi6, SARS-CoV-2 and IFV [38, 39] . BAK is a Food and Drug Administration-approved product for a broad-range of disinfecting applications such as additives in soaps and hand sanitizers [40] [41] [42] . We have demonstrated here that these transparent PET-based composites possess high antiviral and antibacterial activity to reduce the spread of COVID-19 and methicillin-resistant bacteria. This extra-protective composite material has been developed by a low-cost method of dip coating that let BAK to physically adsorbed [43] onto the surface of a commercial PET plastic commonly used for the fabrication of face shields and other protective equipment providing high antimicrobial activity. Nonetheless, further research is necessary to overcome the possible shortfalls on the applicability of this coating technology to other protective tools and to other types of transparent materials. The effect of packing, storage and transportation on the antimicrobial properties of the BAK coating need also to be studied before applying this technology at large-scale.

Sheets of PET with a thickness of 0.3167 ± 0.0408 mm used for the fabrication of commercial face shields were purchased from Plasticos Villamarchante S.L [44] (Valencia, Figure 7 . Applications of the coating technology of transparent polyethylene terephthalate (PET) with an antimicrobial coating of benzalkonium chloride (BAK) for the next generation of facial protective equipment: face shields, plastic masks, helmets, goggles, helmets and space separation screens.

Sheets of PET with a thickness of 0.3167 ± 0.0408 mm used for the fabrication of commercial face shields were purchased from Plasticos Villamarchante S.L [44] (Valencia, Spain). Six disk specimens (n = 6) of approximately 10 mm in diameter of these transparent PET sheets (BAK Plastic) were treated with 70% ethyl alcohol with 0.1% w/w BAK (Montplet, Barcelona, Spain) by the dip-coating method [45] for 1 min at 25 • C to achieve a dry BAK content of 0.182 ± 0.034% w/w determined gravimetrically. Six more PET disks (n = 6) were subjected to the same dip-coating treatment but using only an absolute ethanol/distilled water solution (70/30% v/v) without BAK for 1 min at 25 • C (S Plastic). Untreated PET disks (U Plastic) disks (n = 6) were produced as reference material. The disks were subsequently dried at 60 • C for 48 h and sterilized under UV radiation for 1 h per disk side. The BAK used in this study was previously characterized by nuclear magnetic resonance on a BRUKER AVIIIHD 800 MHz (Bruker BioSpin AG, Fälladen, Switzerland) equipped with a 5 mm cryogenic CP-TCI [34] .

Atomic force microscopy (AFM) was performed with a Bruker MultiMode 8 scanning probe microscope (SPM, Karlsruhe, Germany) operating in tapping mode in air and with the NanoScope V Controller and NanoScope 8.15 software version (Bruker, Karlsruhe, Germany). An antimony (n) doped silicon cantilever from Bruker was used with a scan rate of 0.500 Hz. The phase signal was set to zero at the resonance frequency of the tip. The tapping frequency was 5-10% lower than the resonance frequency. The drive amplitude and amplitude setpoints were 308.5 and 644.8 mV, respectively, and the aspect ratio was 1.00.

A Zeiss Ultra 55 field emission scanning electron microscope (FESEM, Jena, Germany) was operated at an accelerating voltage of 10 kV to observe the biofunctional coating morphology of the treated PET surface at a magnification of ×150 and ×720. The plastic samples were prepared to be conductive by platinum coating with a sputter coating unit. This FESEM microscope is equipped with energy-disperse X-ray spectroscopy (EDS) for elemental ratio estimation at 2.00 kV.

The opacity of the synthesized films was evaluated according to the spectrophotometric method utilized by Park and Zhao [46] . Thus, rectangular specimens (4 mm × 50 mm) of the dry films were directly placed in a spectrophotometer cell to measure the absorbance at 600 nm with a UV/VIS Nanocolor UV0245 spectrophotometer (Macherey-Nagel, Düren, Germany). An empty cell was utilized as a reference. After that, the opacity (O) of the films can be determined with Equation (1), in which Abs600 is the absorbance value at 600 nm and x is film thickness in mm.

The measurements were performed with three specimens of each material and were reported as absorbance divided by film thickness (mean ± standard deviation).

The phage phi 6 host is Pseudomonas syringae (DSM 21482). This Gram-negative bacterium was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell cultures GmbH (Braunschweig, Germany). P. syringae was cultured in solid tryptic soy agar (TSA, Liofilchem, Teramo, Italy). After that, the microorganism was cultured in liquid tryptic soy broth (TSB, Liofilchem, Roseto degli Abruzzi, Italy) incubated in an orbital shaker (CERTOMAT IS, Sartorius Stedim Biotech, Göttingen, Germany) at 25 • C and 120 rpm.

The specifications provided by the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH were followed to propagate the phage phi 6 (DSM 21518) by using the double agar layer technique with top and bottom agar, which allows to perform lysis in bacterial host cultures [47] .

50 µL of TSB with phages was placed onto each material disk at a titer of about 1 × 10 6 plaque-forming units per mL (PFU/mL) and allowed to incubate for 1 min. Each material disk was placed in a falcon tube with 10 mL TSB, and subsequently sonicated for 5 min and vortexed for 1 min at room temperature (24 ± 1 • C).

Phage titration was performed by serial dilutions of each falcon sample. 100 µL of each phage dilution was mixed with 100 µL of the host strain at OD 600 nm = 0.5.

The infective activity of the phage phi 6 was measured based on the double-layer method [48] . Thus, 4 mL of top agar (TSB + 0.75% bacteriological agar, Scharlau) and 5 mM CaCl 2 were added to the mixture containing phages and bacteria, which was then poured on TSA plates. Incubation of the plates was performed for 24-48 h in a refrigerated oven at 25 • C. Phage titers of each sample were calculated in PFU/mL and compared with a control consisting of 50 µL of phage added directly to the bacterial culture without being in contact with any type of disk and without sonication or vortexing.

The antiviral activity of the material disks was estimated at 1 min of contact with the biosafe virus model in log reductions of titers. It was ensured that the residual disinfectants present in the titrated samples did not interfere with the titration process. It was also checked that sonication and vortexing did not affect the infectious activity of the phage phi 6. The antiviral assays were carried out three times during two different days (n = 6) to ensure reproducible results.

The SARS-CoV-2 strain (SARS-CoV-2/Hu/DP/Kng/19-027) was provided to us by Dr. Tomohiko Takasaki and Dr. Jun-Ichi Sakuragi from the Kanagawa Prefectural Institute of Public Health. SARS-CoV-2 was plaque-purified, propagated in Vero cells and stored at −80 • C. 50 µL of a virus suspension in phosphate-buffered saline (PBS) was placed onto each material disk at a titer of 1.3 × 10 5 median tissue culture infectious dose (TCID50) per disk, and then incubated for 1 min of contact at ambient temperature. After that, 1 mL PBS was added to each disk, and then vortexed for 5 min. After that, each tube was vortexed for 5 min at ambient temperature. Viral titers were determined by the TCID50 assay performed in a Biosafety Level 3 laboratory at Kyoto University. Thus, TMPRSS2/Vero cells [49] (JCRB1818, JCRB Cell Bank), cultured with the minimum essential media (MEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, were seeded into 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA). Serial dilutions of 10-fold (from 10 −1 to 10 −8 ) were performed in the culture medium. These dilutions were placed onto the TMPRSS2/Vero cells in triplicate and incubated at 37 • C for 96 h. Cytopathic effect was evaluated under a microscope and TCID50/mL was calculated using the Reed-Muench method [50] .

The antibacterial activity was studied by the agar disk diffusion tests [51, 52] . Thus, lawns of methicillin-resistant Staphylococcus aureus (MRSA), COL [53] and methicillinresistant Staphylococcus epidermidis (MRSE), RP62A [54] , in a concentration of approximately 1.5 × 10 8 colony forming units per mL (CFU/mL) in tryptic soy broth, were cultivated on trypticase soy agar plates. The lawns of bacteria were incubated aerobically at 37 • C for 24 h with the sterilized disks placed upon them. The antibacterial disks showed an inhibition zone (or halo) that can be normalized using Equation (1) [51] .

The term nw halo represents the normalized width of the antibacterial inhibition zone, d iz is the inhibition zone diameter and d indicates the material disk diameter. The material disk diameter was measured by image software analysis (Image J, Wayne Rasband (NIH), USA). The antibacterial tests were performed three times during two different days (n = 6) to ensure reproducible results.

The antiviral and antibacterial tests were performed again after washing 1 cm disks of the PET/BAK composite material (BAK Plastic) by immersion in 100 mL of distilled water during 1 min at 24 ± 1 • C to analyze the antimicrobial durability of the BAK coating to water.

The statistical analyses were performed by ANOVA followed by Tukey's post hoc test (* p > 0.05, *** p > 0.001) using the GraphPad Prism software (GraphPad Prism 6, GraphPad Software Inc., San Diego, CA, USA).

A single-use antimicrobial face shield has been developed as the next generation of face protective equipment capable of inactivating enveloped viruses such as phage phi 6 and SARS-CoV-2 after 1 min of contact and multidrug-resistant bacteria such as MRSA and MRSE. This antimicrobial composite material was fabricated by a low-cost procedure consisting of dip-coating of polyethylene terephthalate with benzalkonium chloride. The formation of the antimicrobial coating was demonstrated by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This composite material avoids viral and bacterial inhalation and entry into the body through the respiratory tract or by splashing (in a surgical operation for example), providing an extra biosafety due to its capacity of inactivating the infectious microorganisms as soon as they are in contact with the protective element. Furthermore, this antimicrobial material is recyclable, and it reduces the generation of infectious biological waste. This antimicrobial material can be used for the fabrication of other face protective equipment such as goggles, helmets, plastic masks and space separation counter or vehicles screens and thus are very promising for the current and future microbial menaces. Nonetheless, further research is needed to contribute significantly on the up-scaling of this technology.

Facial protection element against risks of exposure to infectious biological agents (Utility model). U202130782. 15 April 2021. 

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The authors would like to express their gratitude to the Fundación Universidad Católica de Valencia San Vicente Mártir, the Ministerio de Ciencia e Innovación and the Japan Agency for Medical Research and Development for their financial support. We would like to thank Yoshio Koyanagi and Kazuya Shimura (Kyoto University) for setup and operation of the BSL-3 laboratory.

The authors declare no conflict of interest. Int. J. Mol. Sci. 2021, 22, 9518