et al., 2020; Pressman et al., 2020). These inanimate objects or surfaces, when con- taminated, can spread pathogens and are called fomites (Castaño et al., 2021). Previ- ous studies have investigated the survival of respiratory viruses—such as the Middle East respiratory syndrome (Kampf et al., 2020; van Doremalen et al., 2013) and severe acute respiratory syndrome (Chan et al., 2011; Kampf et al., 2020)—and showed that these viruses can persist on fomites such as metal, glass, or plastic. Their persistence can last from a few hours to a few days depending on the virus, type of surface, and other envi- ronmental factors. Similar studies in hospi- tal settings demonstrated virus survival on fomites and that transmission from these fomites is possible (Kaslo et al., 2021; Otter et al., 2016; Sizun et al., 2000). In general, virus survival rates in the environment depend on many factors, including moisture, relative humidity, tem- perature, and whether a surface is porous or nonporous (Lopez et al., 2013; Tiwari et al., 2006; Whitworth et al., 2020). Studies have shown that SARS-CoV-2 can survive for as long as 3 days on plastic, 2 days on stainless steel, and up to 24 hr on cardboard (Suman et al., 2020). Kampf et al. (2020) conducted a review of persistence of coronaviruses on inanimate surfaces and found evidence that the SARS-CoV virus could survive on inani- mate surfaces such as metal, glass, or plastic for as many as 5 days, 5 days, and 9 days, respectively (Duan et al., 2003; Rabenau et al., 2005). Mouchtouri et al. (2020) reported that SARS-CoV-2 particles were detected on various surfaces, in air sam- ples, and in sewage waste from hospitals and other community settings. One study also showed that under favorable environ- mental conditions, SARS-CoV-2 can persist and stay viable on fomites for up to 21 days (Kaslo et al., 2021). It is essential to understand how long viruses such as SARS-CoV-2 can persist on high-touch surfaces in food service opera- tions and their transmission rates under various conditions, because rates can vary from hours to days (Kampf et al., 2020). In March 2021, the World Health Organization (2021) reported that SARS-CoV-2 was found on frozen and refrigerated food packaging in China. One study reported that SARS-CoV-2 attached on salmon skin could survive and
stay infectious for more than 7 days if stored at 4 °C and 2 days at 25 °C, concluding that SARS-CoV-2 attached to fish and seafood could serve as a source of contamination (Dai et al., 2020). We selected peppers, cantaloupe, and let- tuce samples because all have been associated with foodborne illness outbreaks in the past (CDC, 2023). Moreover, their diverse physi- cal characteristics allow for a comprehensive investigation of contamination persistence and cross-contamination (Stine et al., 2005). These produce previously have been used to study viral surface contamination (Allwood et al., 2004, Cliver et al., 1983; Le Guyader et al., 2004; Stine et al., 2005). The textured surfaces of lettuce (Takeuchi & Frank, 2001) and cantaloupe (Ukuku & Fett, 2002) have been shown to protect bacteria from chemical and physical interventions, while the smooth surfaces of peppers o er a contrast for inves- tigative purposes. These three produce items are regularly eaten raw, bypassing a lethal- ity step that includes cooking above 140 °F (CDC, 2023). The goals of our study were to 1) investi- gate the persistence of phi 6-relevant fomites within food service operations and 2) evalu- ate the cross-contamination and transfer rate from high-touch surfaces to wiping tools, hands, and produce, and from cutting boards to produce.
an inoculation loop from previously prepared TSA slant and incubated for 18 hr at 22 °C . After overnight incubation, a single colony of P. syringae was picked using a sterile loop and inoculated in a 250-ml flask containing 50 ml of TSB. The flask was incubated in a shaking incubator for 18 hr at 22 °C. After incubation, the density of the culture was verified using a spectrophotometer (Spectronic 20D, Thermo Fisher Scientific) at optical density (OD 550 ) and grown until the reading output showed absorbance between 0.5 and 0.8. After preparing the host, 1 ml of room tem- perature TSB was added to the tube contain- ing the lyophilized virus and vortexed for 1 min to mix. Next, 500 μl of the rehydrated virus was added to 50 ml of TSB in a 250- ml flask, followed by adding 100 μl of over- night growth of P. syringae . The flask contain- ing TSB, the virus, and P. syringae was then placed in a shaking incubator and incubated for 18 hr at 22 °C. New Stock Purification After incubation, phi 6 was purified using a 0.22-μm PVDF membrane filter that was attached to a sterile needle-less Millipore SLGV033RS 60-cc syringe. The plunger was pulled out from the syringe and 15 cc of the overnight culture was pipetted into the syringe barrel. After the plunger was reinserted, the syringe filtered out bacterial debris and the virus was dispensed into a sterile polypropylene tube (centrifuge tube). All procedures were performed inside a bio- safety cabinet. Plaque Assay Plaque assays were carried out to identify the concentration of phi 6 for filtrate viruses; 10-fold serial dilutions of the phi 6 filtrate were made in 0.02% of phosphate bu ered saline (PBS) and Tween (PBST, 100 ml PBS + 0.02% Tween 20) bu er. The remaining filtrate was wrapped with aluminum foil and stored in a refrigerator at 4 °C for later use. Next, 1 ml of the diluted phi 6 was mixed with 100 μl of overnight cultures of P. syrin- gae . The mixture was added to a tube con- taining 3 ml of TSB soft agar prewarmed to 45–50 °C. The soft agar with host and phi 6 was mixed quickly in a tube and poured onto TSA plates. The plates were swirled manually to evenly distribute the soft agar. The plates were allowed to dry for 30 min, inverted,
Methods
Reagents and Coupons All media and reagents were purchased from VWR. The sponges, microfiber towels, and cutting boards were purchased from an online retail website. Coupons of lami- nate tabletop, countertop, wooden floor, and stainless steel were purchased from Thermo Fisher Scientific. Bacteriophage and Host Pseudomonas syringae (host) and phi 6 were obtained from the Centers for Disease Con- trol and Prevention. The host was cultivated on tryptic soy agar (TSA) and grown in tryp- tic soy broth (TSB). The virus stock solutions were prepared by suspending propagated phi 6 in TSB at concentrations of 8–10 log plaque forming units (PFU)/ml. Working stocks of phi 6 were prepared and stored at 4 °C. Next, P. syringae were streaked on TSA plates using
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