Plaque Assay Plaque assays were performed to identify the concentration of phi 6 for filtrate phi 6 bac- teriophages. Next, 10-fold serial dilutions of the phi 6 filtrate were made in 0.02% phos- phate bu¨ered saline (PBS) and Tween (PBST, 100 ml of PBS + 0.02% Tween 20) bu¨er. The remaining filtrate was stored in a refrigerator at 4 °C for later use after wrapping tubes with aluminum foil to protect the phi 6 from light. Next, 1 ml of the diluted phi 6 was mixed with 100 µl of overnight cultures of P. syringae . The mixture was added to a tube containing 3 ml of prewarmed (45–50 °C) TSB soft agar. The soft agar with host and phi 6 was mixed 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, the plaque-forming units were quantified. Persistence Experiment Before the start of the experiment, all items were cut into either square 5 x 5 cm or 10 x 10 cm coupons, depending on the item. Cou- pons were sterilized using either an autoclave for 15 min at 121 °C or by using 70% etha- nol. The inoculum was prepared by adding 5 ml of phi 6 stock to 45 ml of 0.02% PBST bu¨er (10 8 PFU/ml). To inoculate, each cou- pon surface was spot-inoculated with 0.2 ml of inoculum, and L-shaped spreaders were used to distribute the phage on the surfaces evenly. The coupons were air-dried for 1 hr at room temperature (23 ± 2 °C). Alongside the drying process time, TSA soft agar tubes were prepared for overlay by melting prepared TSA soft agar in a 48–50 °C water bath. After drying time, two samples from each surface were taken and placed in a stomacher bag containing a 45 ml or 90 ml of virus buf- fer (0.02% PBST) and homogenized for 2 min. Next, 10-fold dilutions were made and 1 ml from each dilution and 100 μl of the overnight host were added to one melted and tempered soft (3 ml) TSA agar overlay tube and poured onto a TSA plate. Each TSA plate was tilted to ensure that the overlay mix- ture completely coated the plate. The plates were allowed to solidify inside the biosafety cabinet for 30 min before being inverted and placed for 18–24 hr at 22 °C in an incuba- tor. The sterile phage bu¨er (no phi 6) plates were prepared and used as negative control plates to test for potential contamination.
infected traveler from the UK (Leong et al., 2021). In another example, the Diamond Princess cruise ship outbreak resulted in 696 COVID-19 cases in February 2020 (Expert Taskforce for the COVID-19 Cruise Ship Out- break, 2020). Since 2000, there have been sev- eral incidents of acute respiratory illness out- breaks caused by the influenza virus on cruise ships (Brotherton et al., 2003; CDC, 2001; Fernandes et al., 2014). Cleaning and ecient housekeeping have become even more essential for the hotel industry to provide further assurance to guests (Pillai et al., 2021). In response to the pandemic, many major hotel chains imple- mented new safety and cleaning strategies such as increased use of technology (e.g., remote check-in, contactless ordering in res- taurants), noncontact sanitizers, and partner- ships with industry and academic experts (Four Seasons, 2020; Hilton, 2020; Marri- ott International, Inc., 2022). Additionally, Zemke et al. (2015) showed that guests are willing to pay more if the hotel has disinfect- ing programs and shows that the guest rooms receive sucient cleaning. For the purposes of our study, phi 6 bac- teriophages were used as a surrogate for the SARS-CoV-2 virus. Phi 6 is an enveloped virus that poses similar characteristics to respiratory viruses (Aquino de Carvalho et al., 2017; Turgeon et al., 2014) and has been validated as a suitable surrogate for corona- viruses for environmental investigation (Bai- ley et al., 2022; Franke et al., 2021; Serrano- Aroca, 2022). In addition, as a Biosafety Level 3 laboratory is required for use of the SARS- CoV-2 virus (Turgeon et al., 2014), the use of phi 6 allows for replication studies to be conducted without these extra protections. The objectives of our study were to under- stand the extent of respiratory virus survival and persistence on hotel guest surfaces and evaluate the transfer rate between hands and high-touch surfaces to help hotel manage- ment develop better cleaning and disinfecting strategies of contamination hot spots.
room curtains, leather, bathroom faucets, and stainless-steel coupons were purchased from local retail stores. Samples were chosen based on use in previous hotel microbiological stud- ies (Park et al., 2019; Zemke et al., 2015). Bacteriophage and Host Pseudomonas syringae (host) and phi 6 were provided by the Centers for Disease Control and Prevention. The host was cultivated on tryptic soy agar (TSA) and grown in tryptic soy broth (TSB). To prepare the phi 6 stock solutions, propagated phi 6 was suspended in TSB at concentrations of approximately 8 to 10 log PFU/ml. We prepared and stored working stocks of phi 6 at 4 °C, streaked P. syringae on the TSA plate using a plastic inoculation loop from a previously prepared TSA slant, and the plates were incubated for 18 hr at 22 °C . After overnight incubation, a single colony of P. syringae was taken and inoculated in a 250-ml flask containing 50 ml of TSB using a plastic inoculated needle; the flask was then placed in a shaking incubator for 18 hr at 22 °C. After incubation, the density of the culture was determined using a spectrophotometer (Spectronic 20D, Thermo Fisher Scientific). The density of the culture was set to opti- cal density (OD550) wavelength to achieve absorbance between 0.5 and 0.8 on the spec- trophotometer. After preparing a host, 1 ml of room temperature (23 ± 2 °C) TSB was added to the tube containing the lyophilized phi 6 and vortexed for 1 min, followed by the addition of 500 μl of the rehydrated phi 6 to 50 ml of TSB in a 250-ml flask, followed by the addition of 100 μl of overnight growth of P. syringae (host) to the flask containing the virus. The flask containing TSB, the virus, and P. syringae was placed in a shaking incu- bator for 18 hr at 22 °C. Purification of Phi 6 Stock The phi 6 was purified using a 0.22 μm PVDF filter that was attached to a sterile needle-less Millipore SLGV033RS 60 cc syringe. After pulling the plunger out from the syringe, 15 cc of the overnight culture was pipetted into the syringe barrel. The plunger was replaced, the syringe filtered out any bacterial debris, and the phi 6 was dispensed into a sterile polypropylene tube (i.e., centrifuge tube). All procedures were performed inside a biosafety cabinet.
Methods
Reagents and Coupons All media and reagents were purchased from VWR. The bed, carpet, and hotel amenities were provided by a national hotel chain. Light switches, door handles, TV remote controls,
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October 2023 • Journal of Environmental Health
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