Inactivation of MS2 bacteriophage on copper film deployed in high touch areas of a public transport system

Abstract

Although SARS-CoV-2 is primarily an airborne risk, the COVID-19 pandemic also highlighted the need for self-disinfection surfaces that could withstand the demand of high occupant densities characteristic of public transportation systems. The aim of this study was to evaluate the durability and antiviral activity of a copper film deployed for 90 days in two high touch locations within an active metropolitan bus and railcar. The antiviral efficacy of this copper film after being deployed in transit vehicles for 90 days (deployed copper film) was then compared to new (unused) copper film to determine if frequent touches and cleaning protocols could decrease the efficacy of the copper films. Deployed copper film, new copper film, and aluminium foil (positive control) coupons were inoculated with ~1 × 10⁶ MS2 virus particles, allowed a contact time of either 5- or 10-min, and analysed for residual viral infectiousness. On both new and deployed copper films, MS2 was completely inactivated (≥5 log reduction) at both time points. These results suggest that the copper film may provide the durability demanded by high touch public spaces while maintaining the antiviral activity necessary to reduce exposure risk and viral transmission via surfaces in public transportation settings.

Introduction

The early stages of the COVID-19 pandemic raised concerns for millions of commuters utilizing public transportation systems throughout the United States. For example, in New York City it has been estimated that 46–51% of residents use the subway as their primary source of transportation (Sy et al. 2021). Where public transportation is a primary infrastructure, it is characterized by high occupant density in confined spaces, which may increase the risk of disease transmission. Additionally, mass transportation environments contain a significant amount of frequently touched surfaces that may act as fomites for disease transmission by constantly facilitating a flux of microbes among humans and their surroundings (Hsu et al. 2016; Gohli et al. 2019). Although surface contamination is likely a minor contributor to the transmission and exposure risk for SARS-CoV-2 (the virus that causes COVID-19) (Moreno et al. 2021), contaminated surfaces can play a key role in the spread of viral infections (Boone and Gerba 2007). This recognition led to exploratory measures that could reduce viral transmission in mass transportation environments by utilizing self-disinfecting surfaces, such as copper films, placed in high touch areas where routine cleaning may be inconsistent or easily outpaced by the usage demand or frequency of repeated touches.

The antimicrobial properties of copper are well known and have demonstrated efficacy against many bacterial and viral agents (Grass et al. 2011; Poggio et al. 2020). The most widespread public health applications for copper and copper alloys have been to combat the prevalence of hospital-associated infections (HAIs), with copper products often being utilized in water filtration systems or as a self-disinfecting surface (Grass et al. 2011; Schmidt et al. 2016; Vincent et al. 2016). In 2008, the Environmental Protection Agency recognized copper surfaces for their antimicrobial properties (Grass et al. 2011); and as of this writing, copper is the first and only metal to be awarded this status. Approximately 500 copper and copper alloy surfaces have been registered under the U.S. Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) as antimicrobial public health materials that have proven to be effective and pose no risk to public health (Copper Alliance Development Inc 2021). Furthermore, copper and copper alloys have been used to demonstrate antimicrobial effects against five strains of bacteria (Grass et al. 2011; Vincent et al. 2016; Copper Alliance Development Inc 2021).

The current study aimed to investigate the efficacy of a copper film placed in high touch areas in a mass transportation environment for 90 days. MS2, a non-enveloped bacteriophage, was selected for this study because it has been commonly used to assess the efficacy of virucidal decontaminants (Bae and Schwab 2008; D’Souza 2010).

Results and discussion

Copper films placed in two high touch locations (as shown in Fig. 1) were evaluated for residual antiviral activity using MS2 as the test virus. Both post-deployed and new films were attached to aluminium foil and cut into 2- by 4-cm coupons for analysis. Aluminium foil coupons of the same size were used as positive controls. Copper test and aluminium positive control coupons were each inoculated with ~1 × 10⁶ plaque forming unit (PFU) MS2 and initially allowed a 30-min contact time (railcar samples only), but upon observing full MS2 inactivation (data not shown), the copper films were challenged with additional tests using 5- and 10-min contact times. Viricidal efficacy was calculated as the difference between the positive control and test sample Log₁₀ recoveries (De Vries and Hamilton 1999). Virus recoveries from positive control coupons ranged from 6·6 to 6·8 Log₁₀ PFU, while recoveries from both types of copper films were at or below the detection limit (i.e., 1 Log₁₀ PFU), providing an overall log reduction ≥5. This result is significant considering EPA guidelines stating disinfectants with viricidal claims must demonstrate at least a 3-log reduction of the virus on tested surfaces (USEPA 2018) as part of the registration requirements. The analysis method used in this study had a detection limit of 10 PFU per sample, therefore a 1 Log₁₀ recovery (instead of 0 Log₁₀) was reported and utilized in efficacy calculations for samples with no observable plaques to avoid inflating viricidal efficacies.

Figure 1.

Photographs of representative copper film deployment locations on hand railing in a bus (left) and a railcar (right).

Recoveries (expressed as Log₁₀ virus PFU recovered) and antiviral efficacy data (expressed as Log₁₀ Reduction) for MS2 for both the new and post-deployed copper films are shown in Fig. 2. Railcar and bus deployed copper films were tested on separate days, and recovery results indicate that the post-deployed copper film maintained antiviral activity similar to new, unused copper film.

Figure 2.

Efficacy of new and post-deployed copper film against MS2. Recovery (bars) and efficacy (symbols) of new and post-deployed (deployed) copper film, from the railcar location and bus location are reported as mean Log₁₀ PFU (±standard deviation) for recoveries (left y-axis), and mean Log₁₀ reduction (±pooled standard error) for efficacy (right y-axis). Black bars represent recovery following a 5-min contact time, gray bars represent recovery following a 10-min contact time, black squares represent efficacy following a 5-min contact time, and gray circles represent efficacy following a 10-min contact time. The horizontal line at 1·0 indicates the analytical limit of detection for the assay (i.e., 1 Log₁₀ PFU recovered).

Mass transit systems are critical to everyday life for millions of people, and the potential role in the transmission of infectious disease, as well as bioterrorism risk, is significant (Zhang et al. 2016; Gohli et al. 2019). As a result, there is a widespread interest in tools for disease and bioterrorism surveillance in major transportation systems. A recent metagenomic analysis of surfaces was used to characterize the microbial diversity and live bacterial communities existing across a city-wide mass transit environment (Afshinnekoo et al. 2015). Results showed that most of the recovered DNA sequences matched organisms from taxa commonly associated with human skin, but nearly half (48%) of the DNA detected did not match any known organism. Further, swab samples were used to recover viable organisms which could be cultured on standard media, and of those recoveries, ~28% were resistant to common antibiotics (kanamycin, chloramphenicol, and ampicillin). Such baseline data demonstrate the important role of surfaces in capturing, harbouring, and potentially spreading microorganisms, including pathogens or select biological agents. Therefore, consideration of pre-emptive mitigation measures such as placement of self-sanitizing surfaces in public spaces has received a renewed interest. For example, a 2012 ten-week study measured bacterial levels on brass (copper alloy), glass, and wood surfaces (e.g. railings and door pulls) located in Grand Central Terminal (New York City, NY) (Michels and Michels 2016). Significant reductions (84–97%) in bacterial loads were reported for the copper alloy surfaces when compared to the non-copper materials. In this case, long-standing terminal infrastructure containing copper alloys appeared to maintain antimicrobial characteristics even after decades of deployment in high touch areas. Similarly, the data reported here illustrate that the antiviral activity of copper films used in high touch surface areas can be sustained over relatively short periods of time (up to 90 days) despite the potentially heavy microbial burden and frequent surface cleaning.

Other environments that may benefit from surfaces equipped with copper films include hospitals and health care facilities. In 2002, hospitalized patients in the United States were estimated to have a 4·5% risk of developing a hospital acquired infection (HAI), resulting in nearly 99 000 estimated deaths (Klevens 2007) and adding an estimated $28–45 billion (2007 dollars) to annual health-care costs (Scott 2009; Stone 2009). Although the idea that environmental contaminants are responsible for HAIs is debated, the possibility that touching of contaminated surfaces may be a source of transfer to patients must be considered (Casey et al. 2010). As a result, the CDC has guidelines and recommendations for surface cleaning and disinfection in hospitals and other health care settings (Rutala and Weber 2008), and surfaces that incorporate copper or copper alloys may provide added antimicrobial benefit between routine cleanings. Some studies have shown that the use of copper surfaces in health care settings may significantly reduce the microbial burden over sustained periods (Casey et al. 2010; Schmidt et al. 2012; Michels and Michels 2016). And at least one study demonstrated that placing copper alloy surfaces onto six high touch objects in ICU rooms reduced the risk of HAI by more than 50% for all study sites (Salgado et al. 2013). These studies and the data presented herein suggest that copper film could be an effective option for controlling or reducing microbial transmission from surfaces in areas that experience high occupant density and/or frequent touches. Additional studies should be performed to evaluate the antiviral efficacy of these surface coatings over a longer deployment period, with shorter contact times that may represent intervening time between separate passenger touches, under varied environmental conditions, against wet- and dry-deposited contaminants, and in public locations which may be subjected to more frequent surface cleanings that would further explore the promising findings of this initial investigation.

Materials and methods

Bacteriophage propagation

The non-enveloped bacteriophage MS2 (ATCC 15597-B1) and its host, Escherichia coli (ATCC-15597), were obtained from the American Type Culture Collection. Viral stocks were propagated in the log phase bacterial host (OD₆₀₀ = 0·6–0·9), Escherichia coli using Luria-Bertani Broth (Difco, 244610, Sparks, MD) and a conventional soft agar overlay method (Kropinski et al. 2009). Following overnight incubation at 35°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, Hollister, CA). Tubes were vortexed for 1–2 min to break up agar clumps then centrifuged at 7000 g for 15 min. Supernatant was removed and filtered through a 0·2-micron syringe filter (Corning PES syringe filters, 431229, Corning, NY). Aliquots of 1 ml were stored in cryovials (Thermo Fisher Scientific, AY509X33, Waltham, MA) at −80°C until use.

Field test conditions

Commercially available, self-adhesive copper films (CuVerro^® V, 91·3% Copper, EPA registration #853535, East Alton, IL) were placed on the hand railing and stanchions of both buses and railcars operating within the Los Angeles Metropolitan Transportation Authority system, as shown in Fig. 1. The films were left in place for 90 days (February through April 2021) and were subject to cleaning with a direct spray and wipe using either Turbo Kill and/or Monofoil M when the vehicles were out of service, usually at night. While ridership during the copper film deployment period was down an average of approximately 55% across the overall system, the estimated weekday ridership still averaged over 12 500 000 per month (Los Angeles Metro 2021). After 90 days, a subset of the deployed copper film was removed and shipped to the EPA RTP Microbiology Laboratory in Durham, NC for analysis. Copper films were adhered to aluminium foil for transport, then cut into individual coupons (2- by 4-cm) prior to inoculation and analysis.

Sample inoculation and analysis

New and unused CuVerro^® V copper film (stored at room temperature in the laboratory) was adhered to aluminium foil, then cut into 2- by 4-cm coupons to remain consistent with the handling of the deployed copper film. Copper film test coupons and positive control aluminium foil coupons were inoculated in triplicate with 10 μl of an MS2 virus preparation (concentration 1 × 10⁸ MS2 virus particles per ml) stabilized in 5% fetal bovine serum (FBS) (10082139, Gibco, certified heat inactivated). The 10 μl inoculum was delivered by pipetting the inoculum in a line lengthwise along the centre of the coupon, then using the pipette tip to spread the inoculum over the surface of the coupon. The inoculated coupons were stored in covered petri-dishes at ambient conditions within a Type 2 Biological Safety Cabinet (~23°C, 54–57% RH) for the entirety of the contact time. After the prescribed contact time (5- or 10-min), virus particles were recovered from both test and control coupons by continuously vortexing each coupon for 2 min in a 50-ml conical tube containing 10 ml of extraction buffer composed of 10 mmol l⁻¹ ethylenediaminetetraacetic acid (EDTA) in 10% Dey-Engley (DE) Broth (BD 281910, Difco, Fisher Scientific, Waltham, MA) prepared in 1X phosphate buffered saline (PBS) (P0196, 10X solution, Teknova, Hollister, CA). Inoculation controls were prepared by directly inoculating 10 ml of extraction buffer. Negative controls were prepared by placing a non-inoculated 2- by 4-cm coupon for each material type (new copper, deployed copper, and aluminium foil) in extraction buffer and processing them in same manner as the test coupons.

Sample plating and analysis

Optical density measurements of the E. coli cultures were taken before plating as indicators of bacterial host log phase growth (OD₆₀₀ = 0·6–0·9). A tenfold dilution series was prepared as appropriate in 1X PBS for each sample. Undiluted and diluted aliquots of either 100 μl or 1 ml were plated with 100 μl of log phase E. coli in triplicate using a conventional soft agar overlay method (Kropinski et al. 2009). Plates were incubated overnight at 35°C and manually enumerated the following day. The inoculum titer was determined each test day by performing a tenfold dilution series directly from the inoculum, plating 100 μl aliquots of each dilution in triplicate, and enumerating plaques the following day.

Post-test neutralization assays

Post-test neutralization assays were performed to verify that the extraction buffer was effectively chelating the copper and quenching residual antimicrobial activity. These assays were performed by removing 5 ml aliquots from non-inoculated samples (i.e. negative controls) and directly inoculating the aliquots with 10 μl of the virus preparation (concentration 1 × 10⁸ MS2 virus particles per ml). Inoculation controls were prepared by directly inoculating extraction buffer. Following inoculation, samples were vortexed to mix, incubated at room temperature for ≥10-min, then plated. For effective neutralization, test sample recoveries should approximate control recoveries. Recoveries from these assays demonstrated effective neutralization and were as follows: Log₁₀ 6·3, 6·4, 6·4 and 6·3 for the inoculum titer, inoculation controls, deployed copper film, and new copper film, respectively. These data verify the effectiveness of the neutralizer.

Significance and Impact of the Study:

The use of commercially available copper films placed in high touch areas could be an effective supplemental measure for reducing the exposure risk and transmission of infectious diseases in mass transportation environments.

Data Availability Statement

All data supporting this study and manuscript are available on data.gov.