Virucidal efficacy of antimicrobial surface coatings against the enveloped bacteriophage Φ6

Abstract

Aims:

Antimicrobial coatings, for use in combination with routine cleaning and dis-infection, were evaluated for their effectiveness in reducing virus concentration on stainless steel surfaces.

Methods:

Twenty antimicrobial coating products, predominantly composed of organosilane quaternary ammonium compounds, were applied to stainless steel coupons, dried overnight and evaluated for efficacy against Φ6, an enveloped bacteriophage. Additionally, two peel and stick polymer-based films, a copper-based film and three copper alloys were evaluated. Efficacy was determined by comparison of recoveries from uncoated (positive control) and coated (test) surfaces.

Results:

The results indicated that some of the coating products initially demonstrated >3-log reduction of Φ6; no direct correlation of efficacy was observed with an active ingredient or its concentration. The peel and stick films and copper alloys each demonstrated efficacy in initial testing. However, none of the spray-based products retained efficacy after subjecting the coating to abrasion with either a hypochlorite or quaternary ammonium-based solution applied in accordance with EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings. Of the products tested for this durability, only one peel and stick polymeric film retained efficacy; the copper alloys were not tested for their durability in this study.

Conclusions:

These results suggest that while some organosilane quaternary ammonium compound-based products demonstrate antiviral efficacy, more research and development is needed to understand effective formulations with sufficient durability to perform as supplements to routine cleaning and disinfection.

INTRODUCTION

Antimicrobial coatings have long been used to suppress mould and odour-causing bacteria and fungi growth on surfaces (Kozłowski & Walentowska, 2020; Malek & Speier, 1982), and more recently have been of interest for the potential to reduce healthcare-associated infections (Boyce et al., 2014; Ellingson et al., 2020; Murray et al., 2017; Weber & Rutala, 2013). Indeed, surfaces are known to play a significant role in disease transmission (Otter et al., 2013; Weber et al., 2010), however, surface contamination is likely a minor contributor to transmission and exposure risk for the COVID-19 pandemic, owing to the fact that inhalation of aerosolized droplets and particles is the major transmission route (Kampf et al., 2020; Li et al., 2021; Meyerowitz et al., 2021). Nonetheless, as part of a multi-pronged approach to risk reduction, many end-users of antimicrobial products have shown interest in using antimicrobial coatings in areas characterized by large numbers of people and frequent surface touching, such as casinos, hotels and public transport facilities and vehicles. In these areas, it is often impractical to conduct routine cleaning and disinfection on a frequency that out-paces contamination. Utilizing frequent cleaning and dis-infection, followed by application of antimicrobial coating products with residual efficacy, may offer some exposure risk reduction, especially towards reduction of exposure to pathogens with faecal-oral transmission (Boone & Gerba, 2007; Otter et al., 2013). Accordingly, the results of this study have applicability beyond the current COVID-19 pandemic.

There are a number of antimicrobial coating products currently registered with the U.S. EPA as microbiostats, which are within a category of antimicrobial pesticides that suppress the growth of stain- and odour-causing bacteria and fungi. These products do not have EPA-approved public health claims and should not be used for the purposes of controlling or killing disease-causing micro-organisms. Many of these products have an organosilicon active ingredient that was identified in the late 1960s as having potential residual inhibition of the growth of bacteria and fungi when cured on surfaces (Speier & Malek, 1982). The polymerized form of the 3-(trimethoxy)silylpropyl-dimethyloctadecyl ammonium chloride was listed as the active ingredient in an original EPA registration granted to Dow Corning for use on socks (Gettings, 1987). This practice quickly expanded to application on many textiles, using the active ingredient listed above and referred to as Dow Corning 5700 (Speier & Malek, 1982) and later AEM 5700 (Hayes & White, 1984). Products based upon this active ingredient may have broad spectrum claims against many bacteria, yeast, and fungi (Mcgee et al., 1983).

Products registered as microbiostats, in general, have claims that they are durable and can control stain- and odour-causing bacteria and fungi growth on surfaces for several months. However, microbiostats’ registration claims do not apply to public health pathogens, including viruses such as SARS-CoV-2. An EPA-registered disinfectant or sanitizer should be used, in accordance with the product label, for reducing or eliminating public health pathogens on surfaces. During the COVID-19 pandemic, the potential effectiveness of microbiostats as a residual antimicrobial coating with activity against SARS-CoV-2 was raised. In response, a new category of products, designed to provide longer lasting yet supplemental (e.g. to use with routine cleaning and disinfection) antimicrobial activity, was created to provide a registration path for products that could offer sustained surface protection. The testing guidance for registration of this category of product specifies that the product should withstand laboratory abrasions that simulate the abuse the coating would experience when applied in the real world, whether by abrasion through touching or wet abrasion by chemicals such as cleaners or disinfectants (US Environmental Protection Agency, 2021). The guidance also specifies that the testing demonstrate efficacy against the micro-organisms that a company plans to indicate on the label. For example, if claims against SARS-CoV-2 are intended to be made on the product label, then testing should be conducted with SARS-CoV-2 and reviewed by EPA prior to making these claims. This is especially important for products with public health claims.

The current study aimed to investigate numerous categories of antimicrobial coating products: those registered as microbiostats, products registered as dual purpose (both wet disinfectant and microbiostats), and products with potential for registration against pathogens under the new category, as a supplemental antimicrobial surface coating. Importantly, the current study was conducted for research and development purposes only; Φ6, an enveloped bacteriophage, was used for all testing rather than a pathogenic micro-organism. Φ6 is a nonpathogenic virus and is easily manipulated in a biosafety level 1 (BSL-1) laboratory. Efficacy against Φ6 may approximate that of other nonenveloped viruses, however, efficacy should be determined empirically for any other virus of interest. The intended use of the information resulting from this study is to inform future investments and further research into antimicrobial coatings.

MATERIALS AND METHODS

Virus and bacteria

The enveloped bacteriophage Φ6, and its host, Pseudomonas syringae, were obtained from Battelle Memorial Institute, Columbus, OH. Viral stocks were propagated in bacterial host P. syringae, using a modified Tryptic Soy Agar media preparation (tryptone 10 g/L; yeast extract 1 g/L; Dextrose 1 g/L; sodium chloride 8 g/L; magnesium chloride 1 g/L; calcium chloride 0.22 g/L; Agar 14 g/L for plates or 7 g/L for soft agar) and a conventional soft agar overlay method (Kropinski et al., 2009). A P. syringae overnight culture was prepared in 100 ml of modified Tryptic Soy Agar shaking (~260 rpm) at room temperature (20–26°C). Soft agar tubes (14 ml, 352059, Corning) of modified Tryptic Soy Agar media were prepared in batches using an automated media sterilization and dispensing system (Media Clave/Media Jet, Integra), then covered with an autoclavable cap (KIM-KAP^® Caps, 73663–18, DWK Life Sciences) and stored at 4°C until use. On the day of virus propagation, soft agar tubes were autoclaved at 121°C for 15 min to melt the agar, then held at ~48°C until plating. Stock virus aliquots (1 ml, undiluted) were added to a soft agar tube containing ~6 ml of soft agar and 100 μl of a log phase (OD₆₀₀ ~0.9 to 1.5) P. syringae culture. Soft agar was poured onto the surface of a solidified agar plate (made with the modified Tryptic Soy Agar media), then plates were swirled to evenly distribute the soft agar over the solid agar surface. Following overnight incubation at room temperature (20–26°C), sterile cell spreaders were used to gently scrape soft agar overlays from three 100 mm diameter plates into a sterile 50-ml conical tube containing 15 ml of SM buffer (Teknova, S0249). Tubes were vortexed for 1–2 min to break up agar clumps then centrifuged at 7000 × g for 15 min. The super-natant was removed and filtered through a 0.2-micron syringe filter (Corning PES syringe filters, 431229). Aliquots of 1 ml were stored in cryovials (Thermo Fisher Scientific, AY509X33) at −80°C until use.

Antimicrobial coatings and products

Twenty-six commercially available antimicrobial coatings, films or alloy products were evaluated for residual (i.e. at least 1 day after application to surfaces) antiviral activity (Table 1). The majority of the antimicrobial coating products included in this study list the organosilane 3-(trimethoxysilyl)propyl-dimethyloctadecyl ammonium chloride (CAS# 27668-52-6) or 3-(trihydroxysilyl) propyl-dimethyloctadecyl ammonium chloride(CAS # 199111-50-7) as an active ingredient in varying concentrations. These two organosilanes are structurally very similar and when exposed to water, the trimethoxysilyl quaternary ammonium compounds undergo a chemical reaction which leads to the formation of trihydroxysilyl quaternary ammonium compounds (US Environmental Protection Agency, 2007). For this reason, both chemicals are often referred to as a trimethoxysilyl quaternary ammonium compound. For simplicity, these compounds will be singularly referred to as the trimethoxysilyl quaternary ammonium compound in this manuscript. Many antimicrobial coating products contained only the trimethoxysilyl quaternary ammonium compound while others contained a combination of the trimethoxysilyl quaternary ammonium compound as well as additional active ingredients (e.g. nonorganosilane quaternary ammonium compounds). In some cases, products only had nonorganosilane active ingredients listed (e.g. alkyl dimethyl benzyl ammonium chloride). The products shown in Table 1 were selected for this investigation based upon stakeholder interest, market research, and in many cases, products were provided to the EPA by the product vendors through material transfer agreements (US Environmental Protection Agency, 2020).

TABLE 1.

Antimicrobial coating product list and active ingredient

Product	EPA registration	Active ingredients	Application	Neutralization methods
A	Microbiostat	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (0.75%)	Used as received; applied by electrostatic sprayer, trigger sprayer, trigger plus wiping postapplication, and submersion	10% Dey-Engley broth in PBS
B	Microbiostat	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (<1%)	Used as received; applied by electrostatic sprayer and trigger sprayer	10% Dey-Engley broth in PBS
C	Microbiostat	3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (<5%); methanol (<1%)	Used as received; applied by electrostatic sprayer and trigger sprayer	10% Dey-Engley broth in PBS
D	Disinfectant (nonresidual), Microbiostat	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (36.60%); n-alkyl (50% C14, 40% C12, 10% C16) dimethyl benzyl ammonium chloride (6.40%); Octyl decyl dimethyl ammonium chloride (4.80%); Didecyl dimethyl ammonium chloride (2.88%); Dioctyl dimethyl ammonium chloride (1.92%)	Diluted 1:1 as requested by manufacturer; applied by airbrush by the manufacturer and electrostatic sprayer	10% Dey-Engley broth in PBS
E	Microbiostat	3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (0.84%)	Used as received; applied by electrostatic sprayer and trigger-sprayer	10% Dey-Engley broth in PBS
F	Microbiostat	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1.3%)	Used as received; applied by electrostatic sprayer and trigger-pull sprayer	10% Dey-Engley broth in PBS
G	Microbiostat	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (1%)	Used as received; applied by trigger-pull sprayer	10% Dey-Engley broth in PBS
H	n/a	3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride (<1%)	Used as received per manufacturer; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
I	Disinfectant (bactericide, fungicide, virucide), 24-h sanitizer (bacteria), Mildewstat	Alkyl* dimethyl benzyl ammonium chloride (*50%C14, 40%C12, 10%C16) (0.200%); Octyl decyl dimethyl ammonium chloride (0.075%); Didecyl dimethyl ammonium chloride (0.150%); Dioctyl dimethyl ammonium chloride (0.075%)	Used as received; applied by trigger-pull sprayer supplied within the product packaging	50% Dey-Engley broth in PBS
J	Microbiostat	3 (Trihydroxysilyl) propyldimethyl octadecyl ammonium chloride (5.0%)	Diluted 1 : 6 as requested by manufacturer; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
K	n/a	Octadecylaminodimethyltrimethoxysilylpropylammonium chloride (<1%); Quaternary ammonium compounds benzyl-C12-C16-Alkyldimethyl, chlorides (<5%); Alkyldimethylbezyl ammonium chloride (<2%); Alkyl (C12-C14) dimethyl(ethylbenzyl) ammonium chloride (<2%)	Used as received per manufacturer; applied by electrostatic sprayer	50% Dey-Engley broth in PBS
L	n/a	3 (Trihydroxysilyl) propyldimethyl octadecyl ammonium chloride (1.0%)	Used as received per manufacturer; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
M	Microbiostat	Octadecylaminodimethyltrihydroxylsilylpropyl ammonium chloride (0.75%) (also named 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride)	Used as received per manufacturer; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
N	Hospital disinfectant, Microbiostat	3-(trimethoxysilylpropyldimethyloctadecyl) ammonium chloride (30%–40%); Alkyl (C12–16) dimethylbenzyl ammonium chloride (4%–8%); Didecyldimethyl ammonium chloride (1%–5%); Decyldimethyloctyl ammonium chloride (1%–5%); Dimethyldioctyl ammonium chloride (1%–5%); methanol (6%–10%); Ethanol (0.5%–2.5%); chloropropyl trimethoxysilane (4%–8%); Diethylene glycol monobutyl ether (20%–30%)	Diluted (0.5 ounce in 1 quart) as per manufacturer’s directions; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
O	Antimicrobial copper alloy	Copper alloy, 99.9%	Coupons cleaned per manufacturer’s instructions prior to use in testing	10 mM EDTA in 10% DE Broth
P	n/a	n-Propanol [n-Propyl alchohol] (35%−45%); Toluene [Methyl-benzene] (35%–45%)	Peel and stick onto stainless steel per manufacturer’s instructions	10% Dey-Engley broth in PBS
Q	n/a	Cylohexane (40%–60%); Naphtha (Petroleum) Hydrotreated light (20%–40%)	Peel and stick onto stainless steel per manufacturer’s instructions	10% Dey-Engley broth in PBS
R	Disinfectant, microbiostat	Octyldecyl dimethyloctyl ammonium chloride (0.0375%); Dioctyl dimethyl ammonium chloride (0.0150%); Didecyl dimethyl ammonium chloride (0.0225%); Alkyl (C14, 50%; C12, 40%; C16, 10%) dimethyl benzyl ammonium chloride (0.0500%); 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (0.3280%)	Used as received; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
S	n/a	Octadecyl dimethyl (3-trihydroxysilyl propyl) ammonium chloride (0.05%); Octyl decyl dimethyl ammonium chloride (0.005%)	Used as received; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
T	Hospital disinfectant, 24-h residual disinfectant (bacteria), Microbiostat	Quaternary ammonium compounds, benzyl-C12–16-alkyldimethyl, chlorides (<1%); Didecyl dimethyl ammonium chloride (<0.5%), Dioctyl dimethyl ammonium chloride (<0.5%), Ethanol (65–80%); polyethylene oxide (<0.1%)	Used as received; applied by trigger-pull sprayer supplied within the product packaging	50% Dey-Engley broth in PBS
U	n/a	Octadecylaminodimethyltrihydroxylsilylpropyl ammonium chloride (~5%) (also named 3-(trihydroxysilyl)propyldimethyloctadecyl ammonium chloride)	Diluted 1:5 as requested by manufacturer; applied by electrostatic sprayer	10% Dey-Engley broth in PBS
V	n/a	3-(Trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (<3%); methanol (<1%); trade secret (<1%); Poly (ethylene oxide) (<0.1%)	Diluted 1:4 as requested by manufacturer; applied by trigger pull sprayer supplied by manufacturer	10% Dey-Engley broth in PBS
W	Antimicrobial copper alloy	Copper alloy, 90%	Coupons cleaned per manufacturer’s instructions prior to use in testing	10 mM EDTA in 10% DE Broth
X	Antimicrobial copper alloy	Copper alloy, 70%	Coupons cleaned per manufacturer’s instructions prior to use in testing	10 mM EDTA in 10% DE Broth
Y	n/a	Alkyl dimethyl benzyl ammonium Chloride (C12–16) (<0.5%)	Used as received; applied by electrostatic sprayer	50% Dey-Engley broth in PBS
Z	Antimicrobial copper alloy	Copper film, 91.3%	Peel and stick onto stainless steel per manufacturer’s instructions	10 mM EDTA in 10% DE Broth

Liquid-based products were applied in accordance with the directions for use on the product labels and/or instructions from the product vendors, using either an electrostatic sprayer (Protexus, PX200ES, 60-micron nozzle), common trigger-pull hand-held sprayer (McMaster-Carr, 4398T1), submersion, or a spray-then-wipe application. For product applications (except submersion), coupons were arranged horizontally in an array on a sterile 36 × 36 cm stainless steel plate, with each coupon elevated by a common hex nut.

Prior to product applications, sterile stainless-steel coupons (2 × 4 cm) were cleaned using a lab detergent (Liquinox, 1201–1, Alconox, Inc.) then rinsed with deionized water and allowed to dry at ambient laboratory conditions (20–26°C, 15%–55% RH). Coupons were sterilized by autoclaving at 121 °C for 60 min.

For electrostatic sprayer applications, coupons were sprayed for 10 s from a 0.9–1.2-m distance with the electrostatic sprayer pointed towards the array of coupons at a ~0° to 30°angle and then allowed to dry overnight at ambient laboratory conditions, uncovered and inside a laboratory fume hood.

For trigger-pull sprayer applications, coupons were fully wetted by spraying from a ~30 cm distance and allowed to dry overnight at ambient laboratory conditions, uncovered and inside a laboratory fume hood. For submersion application, coupons were fully submerged in a liquid product for 3 min, then removed. Excess liquid was drained from the coupons and coated coupons were dried overnight under ambient laboratory conditions, uncovered and inside a laboratory fume hood. The spray-then-wipe application was conducted by spray-applying product to the coupons according to the trigger-pull method described above. After 3 min, the coupon surface was wiped with a laboratory towelette (Kimberly Clark, 34155).

Metal alloy material products were cut into 2.5 × 2.5-cm pieces (coupons). Prior to testing, coupons were cleaned and de-greased by submerging them for at least 30 s in a 3.5% NaOH solution heated to 71°C, rinsing them with DI water, dipping them in a 5% H₂SO₄ solution for at least 5 s, then rinsing them with DI water. Antimicrobial film type products were cut into either 2.5 × 2.5-cm or 2 × 4-cm pieces (coupons) and adhered to the same size stainless steel coupons prior to testing.

Efficacy

Antiviral efficacy of products was assessed using Φ6 as the test virus. A 10-μl droplet of inoculum, containing ~10⁶ viral particles, was pipetted on each coated (test) or noncoated (control) coupon. The inoculum was spread using a gel loading pipette tip to distribute the virus over the coupon surface (Figure 1c) and initiate the contact time. Coupons remained in covered petri-dishes at ambient laboratory conditions within a Type 2 Biological Safety Cabinet for the entirety of the contact time. Initial efficacy screening tests utilized a 2-h contact time. Age and humidity tests utilized both 0.5-and 2-h contact times, and durability assessment tests utilized a 2-h contact time. Immediately following the prescribed contact time, coupons were placed into 50-ml conical tubes containing 10 ml of neutralization and recovery buffer, then vortexed for 2 min at maximum setting to remove the virus particles from the coupon surface. The effectiveness of the neutralization buffer was evaluated prior to efficacy tests, and specific buffers for each antimicrobial product are provided in Table 1. Infectious virus particles were enumerated using a conventional soft agar overlay method (Kropinski et al., 2009). Tubes of soft modified Tryptic Soy Agar and a P. syringae culture were prepared as previously described (see the Virus and bacteria section). On the day of testing, soft agar tubes were autoclaved at 121°C for 15 min to melt the agar, then held at ~48°C until plating. Tenfold dilution series were prepared in 1X phosphate buffered saline (P0196, 10X PBS solution, Teknova) for each test sample; both serial diluted (100 μl) and undiluted (1 ml and 100 μl) aliquots were then used in plating. Test sample aliquots were added to a soft agar tube containing ~6 ml of soft agar and 100 μl of a log phase (OD₆₀₀ ~0.9 to 1.5) P. syringae culture. Soft agar was poured onto the surface of a solidified agar plate (made with the modified Tryptic Soy Agar media), then plates were swirled to evenly distribute the soft agar over the solid agar surface. Plates were incubated overnight at ambient temperature (20–26°C) and manually enumerated the following day.

FIGURE 1.

Photos of representative stainless steel coupons used for surface-based efficacy testing. (a) coupon array prior to application of antimicrobial coatings, (b) representative 2 cm × 4 cm stainless steel coupon 1 day following application of Product B, (c) control 2 cm × 4 cm stainless steel coupon after addition and spreading of 10 μl inoculum, (d) array of 1 in ×1 in brushed stainless steel coupons following antimicrobial coating application by electrostatic sprayer, (e) 1 in ×1 in brushed stainless steel coupon following application of antimicrobial coating Product H, (f) 1 in ×1 in brushed stainless steel coupon with antimicrobial coating Product H following wet abrasion with 2000 ppm sodium hypochlorite

Effects of coating age and humidity

Antimicrobial coatings were applied to stainless steel coupons (2 × 4 cm), and stored for either 3 or 12 weeks at one of three environmental conditions; ambient laboratory temperature and humidity (20–26°C, 15%–65% RH), ambient temperature and low humidity (20–26°C, ≤15% RH), or ambient temperature and high humidity (20–26°C, ~99% RH) to evaluate the effects of age (time since coating was applied) and environmental conditions during ageing on antimicrobial coating efficacy. The ambient condition coupons were stored in covered petri dishes on a laboratory benchtop. Low and high humidity condition coupons were stored in uncovered petri-dishes enclosed within a plastic bin lined either with desiccant (low humidity condition) or ~1-inch of DI water (high humidity condition) and sealed. HOBO loggers (Onset, UX100–003) were placed with each set of coated coupons and set to record the temperature and % RH every 2-h. Following the 3- or 12-week storage period, the antiviral efficacy of the aged coatings and noncoated control coupons was assessed according to the methods described above.

Coating durability assessments

A subset of antimicrobial products was assessed for resistance to abrasion, in accordance with the EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings (US Environmental Protection Agency, 2021). Briefly, antimicrobial coatings were applied to 2.5 × 2.5-cm brushed stainless steel coupons and allowed to dry overnight before being subjected to an abrasion treatment: either A, C or D. Treatment B was not evaluated. For Treatments A and C, the sponge side of a 3 M Scotch Brite Nonscratch Scrub sponge (C05068, St. Paul) which had been sterilized by autoclaving at 121°C for 20 min, was saturated with 20 ml of the test chemical (either 2000 ± 100 ppm sodium hypochlorite for Treatment A, or the use-dilution of a quaternary ammonium compound disinfectant, Virex II 256 for Treatment C). The saturated sponge was then used to abrade the coated (test) or noncoated (control) coupons, using a Gardco (D10V) wearability tester (Gardco Inc.), through 10 cycles of 8 passes (80 total passes). Each cycle was calibrated to perform 8 passes within 16–20 s, with a 30-min wait time observed between each cycle. Following the 10 cycles, coupons were rinsed three times for 3–5 s with filter-sterilized diH₂O, then allowed to dry overnight in uncovered petri-dishes lined with filter paper (Whatman No. 2), prior to assessing efficacy. For Treatment D, the sponge side of a dry 3 M Scotch Brite Non-Scratch Scrub Sponge was used to abrade coupons through 10 cycles of 16 passes (160 total passes). Each of these cycles was calibrated to perform 16 passes within 32–40 s, with a 30-min wait time observed between each cycle. For Treatments A and C, a 454 g weight was affixed to the top of the sponge boat (holder), and the sponge was replaced with a new sponge after five cycles. The 454 g weight was not used for Treatment D. Following abrasion, efficacy was assessed for abraded, coated coupons (n = 3), abraded, noncoated control coupons (n = 3) and nonabraded, coated coupons (n = 3). Efficacy was determined by comparison of recoveries (log₁₀ PFU) of abraded, coated coupons and abraded, noncoated coupons to that of noncoated, nonabraded coupons (n = 3). Efficacy is reported in terms of log₁₀ reduction (i.e. log reduction).

RESULTS

Twenty-six antimicrobial products, including spray-applied coatings, peel and stick films, and metal alloy materials, were screened for antiviral activity using Φ6 as the test virus and a 2-h contact time for virus-product contact. Virus recovery from positive control (noncoated) coupons following the 2-h period ranged from 3.7 to 6.5 log₁₀ PFU. Efficacy for the recently applied coatings (coatings applied within 3 days) ranged from 0 to 4.4 log reduction (Figure 2). The upper limit of log reduction values was dependent upon, and controlled by, the positive control recovery. Owing to the 1 log₁₀ PFU detection limit of the virus recovery assay, when positive control recoveries were 4.0 log₁₀ PFU, efficacy was recorded as a 3.0 log reduction when no virus was recovered from coated coupons. When the recovery from positive controls was <4.0 log₁₀ PFU, a 3-log reduction could not be achieved. This is important to note since the EPA Interim Guidance specifies a 3-log reduction within a 2-h contact time as a performance metric for registration under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (US Environmental Protection Agency, 2021).

FIGURE 2.

Screening of antimicrobial coating product (A to Z) efficacy against Φ6. Coatings were typically 1-day (≤3 days) old when evaluated for efficacy against Φ6, applied to stainless steel in a manner consistent with the product’s label, and efficacy was assessed after a 2-h contact time

Several products (e.g. A, B, D) were screened for efficacy multiple times to understand the variability between tests and across application methods. Overall, there was little variability (i.e. ≤1 log reduction) in efficacy across application methods (e.g. trigger-pull sprayer versus electrostatic sprayer application), between repeated tests on different test days, and between replicate test coupons.

Five antimicrobial products (D, E, F, H and V) were selected, based upon initial screening results and stakeholder interest, and evaluated for the longevity of their antiviral activity against Φ6. Coated and noncoated control coupons were stored for 3 or 12 weeks at one of three different environmental conditions, prior to subjecting them to the efficacy evaluation. Contact times of 0.5- and 2.0-h were administered for these efficacy tests. Efficacy ranged from 1.2 to >4.9 log reduction when a 0.5-h contact time was used and ranged from 1.4 to >4.5 log reduction for a 2.0-h contact time (Figure 3). In only one instance (Product H, ambient conditions, 0.5-h contact time) was viable virus recovered from coated coupons following 3 weeks of storage. For 12-week-old coatings, several products demonstrated reduced efficacy. This reduction was observed in all three environmental conditions. Product F was the only product that resulted in no viable Φ6 recovery from coated coupons for all treatments and contact times. Low positive control recoveries (3.7 log₁₀ PFU) resulted in low efficacy for the 2.0-h contact time, 3-week-old coatings, low humidity condition. Nonetheless, all products achieved full inactivation for that test, resulting in an efficacy of >2.7 log reduction for all products. Similarly, for the 3-week, humid condition, low positive control recoveries (3.9 log₁₀ PFU) resulted in efficacies of >2.9 log reduction, even though no virus was recovered from any sample following a 2-h contact time.

FIGURE 3.

Evaluation of antimicrobial efficacy against Φ6 following storage for 3 or 12 weeks at low humidity (<15% RH), ambient humidity (15%–65% RH), or high humidity (~99% RH) conditions. Efficacy values preceded by a greater than symbol indicate no viable virus was recovered from test samples following the contact time (i.e. full kill)

A subset of antimicrobial coating products was assessed for durability following abrasion, consistent with the methods described above and in the EPA Interim Guidance (US Environmental Protection Agency, 2021). One-day-old coatings on brushed stainless steel coupons were assessed for efficacy after being subjected to abrasion cycles with a dry sponge, or sponges wetted with either 2000 ppm sodium hypochlorite or the use-dilution of a quaternary ammonium compound EPA-registered disinfectant (Virex II 256). Dry abrasion did not reduce product efficacy as compared to nonabraded, coated coupons (Figure 4, Panel a). Product H and Product Q achieved 4 and 4.7 log reductions following dry abrasion respectively (Figure 4, Panel a). However, efficacy was reduced to near zero for all products other than Product Q following wet abrasion treatments with either 2000-ppm hypochlorite or quaternary ammonium compound disinfectant (Figure 4, Panels b and c). Products P and Q were the only film-type coatings included in the durability testing; all other products were spray-applied.

FIGURE 4.

Antimicrobial coating efficacy against Φ6 following abrasions. Efficacy of antimicrobial coatings against Φ6 for (a) dry abrasion with 3 M sponge (b) 2000 ppm sodium hypochlorite abrasion, and (c) quaternary ammonia disinfectant (Virex II 256) abrasion. Black bars represent efficacy of noncoated, abraded controls samples, gray bars represent efficacy of coated, nonabraded control samples, and white bars represent efficacy of coated, abraded test samples. All tests utilized 1-day-old coatings, on brushed stainless steel coupons, and utilized a 2-h contact time

DISCUSSION

The U.S. Environmental Protection Agency holds the unique responsibility for regulating pesticides within the United States, under FIFRA. Antimicrobial products such as disinfectants and sanitizers are required to meet specified performance metrics and undergo a safety assessment prior to registration and subsequent sale and use. In 2020, to fill a perceived critical need during the COVID-19 pandemic, the U.S. EPA developed a new category of antimicrobial products called Antimicrobial Surface Coatings. Antimicrobial surface coatings are considered a “supplemental” product; they are intended for use within a normal cleaning and disinfection regimen, not as stand-alone products. Accordingly, products registered in this category must not only demonstrate efficacy against the micro-organism(s) targeted in the registration, but they must do so after being subjected to abrasion to show their durability. Abraded coatings must demonstrate a 3-log reduction within a 2-h contact time for registration.

In contrast to the wet testing procedures for liquid sanitizers and disinfectants, antimicrobial coatings are evaluated as a polymerized film. Here, the antimicrobial coating is applied to the surface and allowed to dry, then the microbial challenge is added onto the coated surface and allowed up to a 2-h contact time. For laboratory efficacy testing to predict the real-world performance of these products, it is important that the coating be applied in a manner that is realistic of its intended use. In the current study, an electrostatic sprayer was used to apply products to stainless steel surfaces (coupons), from a consistent distance and using a spray duration that could be feasibly implemented in a real-world setting. Further work is needed to understand how under-and over-application of the product may impact its performance.

The purpose of the current study was not to generate data suitable for product registration, but rather to understand the antimicrobial efficacy potential of surface coatings using Φ6 as a model virus to predict efficacy against other enveloped viruses. Products that achieved a 3-log reduction, or greater, on nonabraded, newly applied coatings were considered for further evaluation of efficacy following ageing or abrasion.

Numerous products included in this study demonstrated efficacy against Φ6 following a 2-h contact time, when the coatings were new and nonabraded. Of the 26 products included in this study, twelve have the trimethoxysilyl quaternary ammonium compound listed as the only active ingredient at use concentrations ranging from 0.75% to 5%. The efficacy results for these products (A, B, C, E, F, G, H, J, L, M, U and V) ranged from highly ineffective (less than 0.5 log reduction) to effective (>3 log reduction) against Φ6. There was no correlation between antiviral efficacy and percentage of this active ingredient. However, the hydrophobicity, and potentially other characteristics of trimethoxysilyl quaternary ammonium compound polymers, can vary by altering initial hydrolysis or polymerization conditions (El Ola et al., 2004). Another potential reason for the differences observed in antimicrobial performance of these similar products is that each product likely has a unique mix of inert ingredients that may impact product performance, yet are not required to be listed on the product label.

Several of the products (D, I, N, R and T) included in this study are registered as dual purpose products (i.e. disinfectant and residual microbiostat) and contain additional or different quaternary ammonium compounds; these products, in general all exhibited >3 log reduction against Φ6. While quaternary ammonium compounds are known to inactivate viruses and bacteria through membrane disruption (Gerba, 2015), these activities are typically described for the liquid form of products rather than surface films. One notable exception was Product N, which contains both the trimethoxysilyl quaternary ammonium compound and additional quaternary ammonium compounds yet achieved low efficacy. Products K, S, V and Y are all quaternary ammonium compound-based formulations that are not currently registered, have different active ingredients, and all exhibited greater than a 3-log reduction.

In addition to the above antimicrobial coatings, copper alloy coupons, a copper film and a polymeric film were also included in this study. The copper alloys (O, W and X), copper film (Product Z) and polymeric peel and stick films (Products P and Q) all resulted in >3 log reduction of Φ6. Indeed, the antimicrobial properties of copper have been well-studied and known for thousands of years (Grass et al., 2011). Copper alloys have been used to reduce hospital-associated infections, in water filtration systems, or as a self-disinfecting surfaces (Grass et al., 2011; Schmidt et al., 2012; Vincent et al., 2016).

Five antimicrobial coatings products (D, E, F, H and V) that were effective (>3-log reduction of Φ6) in the initial evaluation (tested within a day after coating cured on the surface) were tested for longevity. These products were representative of the types of antimicrobial coatings used in this study (microbiostats and dual-purpose products) and had different active ingredients. After 3 weeks at each relative humidity condition (ambient, dry and humid), all products remained effective against Φ6 within a 2-h contact time. After 12 weeks, Product H was observed to have reduced efficacy (less than 3-log reduction) after storage at ambient relative humidity conditions. One possibility was that the minor daily cycles of increasing and decreasing relative humidity impacted the effectiveness of this coating.

None of the spray-applied antimicrobial coating products tested were effective against Φ6 after exposure to the wet abrasion cycles (Treatment A or Treatment C) in accordance to EPA Interim Guidance (US Environmental Protection Agency, 2021). Treatments A and C appeared to remove the coating from the treated surfaces. Of the two antimicrobial film products evaluated against the wet abrasion protocol (Products P and Q), Product Q demonstrated a higher resistance to abrasion. Product Q retained its effectiveness (>3-log reduction against Φ6) after Treatment A and Treatment C.

This study demonstrated the potential antiviral efficacy of many antimicrobial coating products, but the data generated herein (with Φ6) would not be sufficient for product registration as a supplemental antimicrobial coating under FIFRA against a pathogenic micro-organism such as SARS-CoV-2. According to EPA Interim Guidance (US Environmental Protection Agency, 2021), the product must achieve a 3-log reduction when tested with the micro-organism targeted in the registration, after the coating has undergone laboratory simulated abrasions by a dry sponge (dry abrasion, Treatment D), a 2000-ppm hypochlorite-soaked sponge (wet abrasion, Treatment A), a hydrogen peroxide/peroxyacetic acid-based disinfectant-soaked sponge (wet abrasion, Treatment B), and a quaternary ammonium-containing disinfectant-soaked sponge (wet abrasion, Treatment C), all as separate treatments on replicated coated and noncoated coupons. Only the antimicrobial film (Product Q) demonstrated durability in accordance with Treatment A and Treatment C. It should also be noted that Treatment B was not included in the current study. In addition, the copper alloys and copper film were not subjected to the wet or dry abrasion testing.

Low recoveries (i.e. <4 log₁₀ PFU) from positive control coupons were observed during several of the temperature and humidity tests (dry and humid test condition, 2-h contact time only). These instances are apparent where full kill (i.e. >) was achieved, yet less than a 3-log reduction dynamic range was available. These low recoveries were attributed to either a change in buffer or FBS lot, a change in ambient laboratory humidity during facility maintenance, or a change in coupon washing and rinsing procedures. After investigation into the issue, and the use of longer, more thorough rinses during the coupon washing procedures, the low recoveries were resolved. Nonetheless, these results underscore the importance of adequate positive control recoveries, as low recoveries negatively impact the assay’s dynamic range. In addition, demonstration of neutralizer effectiveness for quenching postcontact time antiviral activity and eliminating cytotoxic effects on the host cells are also critical for assay validity. In the current study, PBS amended with 10% or 50% Dey-Engley broth was found effective for all tested products, with the exception of the copper-based products. For complete neutralization of copper alloys’ antimicrobial activity in sample extracts, PBS with 10% Dey-Engley broth and 10 mM EDTA was used. Results demonstrated that neutralized samples from antimicrobial coating extracts, spiked with the virus after neutralization and held at ambient conditions for 10 min, showed equivalent recoveries to control samples without extracts from antimicrobial coated coupons. Similarly, calculations of efficacy (log reductions) should utilize the method detection limit (1.0 log₁₀ PFU in the current study), rather than zero when no virus is recovered in test samples following treatment. For example, if control samples demonstrated a 5-log recovery and test samples were nondetect, then our study would calculate efficacy as 5-log minus 1-log (detection limit), for a 4-log reduction. Utilizing zero as the experimental recovery value for nondetects would improperly inflate the efficacy value.

Overall, the results of this study demonstrate that many antimicrobial coating products have antiviral activity, as demonstrated in the initial screening tests against Φ6, an enveloped bacteriophage. These results should be considered an indication of potential activity against similar viruses and should not be used to justify or support off-label use of the products tested. Most products within the subset of those products tested for longevity of activity, maintained antiviral efficacy against Φ6 after 3 and 12 weeks. However, only one product maintained high antiviral efficacy (i.e. >3 log reduction) following either of the abrasion treatments that include a wetted sponge. These results suggest that product efficacy in the real world may be quickly diminished following routine cleaning, disinfection with liquid chemicals, or potentially by simply wetting the treated surfaces. More research is needed to compare real-world abrasions scenarios to laboratory abrasions meant to simulate potential worst-case scenarios.

DATA AVAILABILITY STATEMENT

All data reported in this manuscript are available at data.gov.