East respiratory syndrome (MERS or MERS- CoV) and severe acute respiratory syndrome (SARS-CoV-1)—can persist on surfaces and potentially cause infection (Aboubakr et al., 2021; Marzoli et al., 2021). MERS and SARS-CoV-1 belong to the same family as SARS-CoV-2 (the virus that caused the recent COVID-19 pandemic) and are characterized as enveloped, single-strand, positive-sense RNA viruses (Yang & Wang, 2020). Thus, the potential survival and transmission of SARS-CoV-2 via fomites at farmers markets are possible. SARS-CoV-2 is difficult to work with, requires careful handling, and necessitates a Biosafety Level 3 laboratory; for these reasons, we used a phi 6 bacteriophage surrogate. Phi 6 regularly is used as a surrogate to study envel- oped viruses in environmental investigations (Aquino de Carvalho et al., 2017; Casanova & Weaver, 2015; Turgeon et al., 2014). There is no literature on the survival and persistence of enveloped viruses on fomites at farmers markets and the risk of viral cross- contamination via fomites. Thus, the objec- tives of our study were to: 1) investigate the survival and persistence of phi 6 on fomites from farmers markets and 2) determine the transfer rate and cross-contamination of phi 6 particles from fomites to produce and hands. Methods Bacteriophage (phi 6) and the host ( Pseudo- monas syringae) were provided by the Centers for Disease Control and Prevention (CDC). We purchased the media and reagents from VWR. Additionally, we purchased four mate- rials (plastic, molded pulp fiber, wicker, and tablecloths) that are widely used at farmers markets from an online retail store. These materials were chosen due to their use in pre- vious studies to assess microbiological persis- tence related to farmers markets (Beiza et al., 2021). We selected bell peppers, cantaloupe, and lettuce as produce samples due to their history of foodborne illness outbreaks and use in microbiological analysis (Centers for Disease Control and Prevention, 2023; Stine et al., 2005). Virus and Host Propagation For safety purposes, we used phi 6 as a sur- rogate for coronavirus (SARS-CoV-2) and P. syringae as a host. The host was cultivated on tryptic soy agar (TSA) and grown in tryptic
soy broth (TSB). The host was prepared by streaking P. syringae on TSA plates from a previously prepared TSA slant using a ster- ile plastic inoculation loop; plates were incu- bated for 18 hr at 22 °C . After incubation, a single colony of P. syringae was activated from overnight growth on TSA and, using a plas- tic inoculated needle, inoculated in a 250-ml flask containing 50 ml of TSB. The flask was then incubated in a shaking incubator for 18 hr at 22 °C. Lastly, the density of the culture was checked using a spectrometer by reading the optical density level at 550 nM to reach an absorbance range of 0.5–0.8 on the spectro- photometer (Spectronic 20D, Thermo Fisher Scientific). Lyophilized Phage Reconstitution After preparing the host, 1 ml of room tem- perature (23 ± 2 °C) TSB was added to the lyophilization tube containing phi 6 and vortexed for 1 min to mix and rehydrate. Next, 500 μl of this rehydrated phi 6 was added to the flask containing 50 ml of TSB, followed by 100 μl of overnight growth of P. syringae (host). The flask containing TSB, phi 6, and P. syringae was placed in a shak- ing incubator for 18 hr at 22 °C. After incu- bation, virus purification was performed using a 0.22-μm PVDF membrane filter attached to a sterile needle-less Millipore SLGV033RS 60-cc syringe. Then the syringe plunger was pulled out from the syringe and 15 cc of the overnight culture was pipetted into the syringe barrel. Lastly, the plunger was replaced, the syringe filtered out bacte- rial debris, and phi 6 bacteriophages were collected in a sterile polypropylene tube (centrifuge tube). All procedures were per- formed inside a biosafety cabinet. Plaque Assay Plaque assays were used to determine the phi 6 concentration for filtrate viruses. A 1-ml aliquot of filtrate phi 6 was used and serially diluted in 0.02% phosphate bu§ered saline (PBS) and Tween (PBST, 100 ml of PBS + 0.02% Tween 20) bu§er. The remaining fil- trate phi 6 was stored in a refrigerator at 4 °C for later use after wrapping tubes with alu- minum foil to protect the phi 6 from light. Next, 1 ml of diluted phi 6 was mixed with 100 μl of the overnight cultures of P. syrin- gae . The mixture was then added to a tube containing 3 ml of prewarmed (45–50 °C)
TSB soft agar. The soft agar with host and phi 6 was quickly poured onto TSA plates and tilted by hand to evenly distribute the soft agar on top. The plates were left to dry for 30 min, inverted, and incubated for 24 hr at 22 °C. After incubation, plaques were enumer- ated by multiplying the number of plaques by dilution factors, and the concentration of phi 6 was determined. Persistence Experiment For our study, we used plastic, cardboard, molded pulp fiber, wicker, and tablecloths that are commonly used at farmers markets. Before the start of the experiment, all mate- rials (except wicker) were manually cut into square 10 cm x 10 cm (100 cm 2 ) coupons. The wicker samples were cut into square 5 cm x 5 cm (25 cm 2 ) coupons. Later, all items were sterilized in an autoclave for 15 min at 121 °C or sterilized using 70% ethanol. Next, 5 ml of the stocked phi 6 was used for inoculation after being diluted in 45 ml of virus bu§er (0.02% PBST). Subsequently, 0.2 ml of the described diluted phi 6 inocu- lum (10 8 PFU/ml) was spotted on the top of each item and spread over the surface using an L-shaped plastic spreader. Afterward, the inoculated items were air-dried for 1 hr at room temperature (23 ± 2 °C) and relative humidity (74 ± 2%). After the drying period, each item underwent microbiological analy- sis to enumerate phi 6 plaques on each plate. Microbiological Analysis Two samples of each surface (fomite) were randomly taken from their respective group and placed into sterile stomacher bags con- taining 45 ml or 90 ml of virus bu§er (0.02% PBST), then homogenized for 2 min. Next, 10-fold-dilutions were made. TSA soft agar tubes were prepared for the overlay process by melting prepared TSA soft agar in a water bath set to 48–50 °C. Then 1 ml from each dilution and 100 μl of the overnight host was added to one melted and tempered soft (3 ml) TSA agar overlay tube and poured onto a TSA plate. The TSA plate was agitated to ensure the overlay mixture completely cov- ered the TSA plates. The plates were then allowed to solidify in a biosafety cabinet for 30 min, inverted, and incubated for 18–24 hr at 22 °C. Negative control plates used sterile phage bu§er (i.e., no virus) to test for poten- tial contamination. After the incubation
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