NEHA June 2023 Journal of Environmental Health

ADVANCEMENT OF THE SCIENCE

TABLE 1

Persistence of Phi 6 on Restaurant Surfaces Over 30 Days

Day

Mean Log PFU/cm 2 and Standard Deviation on the Surface of Each Fomite a

Sponge

Microfiber Towel 5.2 ± 0.3 3.2 ± 0.5 2.4 ± 0.1 2.1 ± 0.3 1.6 ± 0.1 1.2 ± 0.1 0.8 ± 0.5 0.5 ± 0.2 0.4 ± 0.2

Stainless Steel

Floor

Tabletop

Countertop Cutting Board Light Switch

1 2 3 4 7

5.7 ± 0.2 4.0 ± 0.7 2.7 ± 0.1 2.3 ± 0.2 1.9 ± 0.2 1.5 ± 0.2 1.3 ± 0.2 0.7 ± 0.6 0.5 ± 0.2 0.4 ± 0.2

5.4 ± 0.1 3.1 ± 0.8 2.6 ± 0.2 2.3 ± 0.3 1.7 ± 0.3 1.2 ± 0.2 1.0 ± 0.2 0.5 ± 0.2 ND ± 0.3

5.6 ± 0.4 3.5 ± 0.5 2.7 ± 0.1 2.2 ± 0.1 1.6 ± 0.3 1.3 ± 0.1 0.8 ± 0.5 0.4 ± 0.2

5.5 ± 0.3 3.4 ± 0.4 2.8 ± 0.1 2.4 ± 0.1 1.9 ± 0.2 1.5 ± 0.2 1.1 ± 0.3 0.8 ± 0.3 0.5 ± 0.2

5.6 ± 0.4 3.8 ± 0.2 2.6 ± 0.1 2.2 ± 0.1 1.7 ± 0.3 1.3 ± 0.2 0.9 ± 0.4 0.7 ± 0.4

5.5 ± 0.5 3.5 ± 0.5 2.6 ± 0.1 2.1 ± 0.2 1.7 ± 0.2 1.3 ± 0.2 1.1 ± 0.2 0.6 ± 0.3 0.4 ± 0.2

5.3 ± 0.3 3.3 ± 0.4 2.5 ± 0.1 2.2 ± 0.4 1.7 ± 0.4 1.4 ± 0.1 0.9 ± 0.5 0.5 ± 0.2

10 13 16 19 22 25 28 30

ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0

ND ± 0 ND ± 0 ND ± 0 ND ± 0

ND ± 0 ND ± 0 ND ± 0

a Mean and standard deviation of survival of phi 6 on each surface of each item over 30 days (N = 6). The greatest reduction of phi 6 occurred within the first 3 days postinoculation. Note. ND = none detected.

and incubated for 24 hr at 22 °C. PFUs were quantified after incubation.

were made and 1 ml from each dilution and 100 μl of the overnight host were added to one melted and tempered TSA soft agar (3 ml) overlay tube and poured onto TSA plates, which were tilted to ensure that the soft agar mixture completely coated the TSA plates. The plates were allowed to solidify in a biosafety cabinet for 30 min before they were inverted and incubated for 18–24 hr at 22 °C in the incubator. Negative control plates were prepared using sterile phage buer (no phi 6) to test for potential contamination. After the incubation period, plaques on each plate were quantified and recorded as PFUs. For each of three biological replicates under similar experimental conditions, the above sample plating procedures were carried out on days 1, 2, 3, 4, 7, 10, 13, 16, 19, 22, 25, 28, and 30. After 30 days of sampling, no phi 6 was detected; therefore, 30 days was chosen as the sampling plan for our study. Simulation Experiment We conducted a simulation experiment to understand the potential for phi 6 cross-con- tamination in a food service operation setting and quantify the rate of cross-contamination

from surfaces to wiping tools, and from hands or cutting boards to produce. The experi- ments were performed using high and low (10 7 and 10 3 PFU/cm 2 , respectively) phi 6 concentrations to simulate dierent contami- nation levels. In total, three biological repli- cates were conducted. Contamination of Surfaces With a High or Low Level of Phi 6 For the first scenario, 0.2 ml of phi 6 suspen- sion (10 7 and 10 3 PFU/ml, respectively) was inoculated onto tabletop, countertop, and stainless steel (5 cm x 5 cm) coupons and held at room temperature (23 ± 2 °C) for 1 hr to facilitate attachment. Next, a sponge or microfiber towel was used to swab each sur- face. Then, each sponge or microfiber towel was placed into a stomacher bag and mixed using a stomacher lab blender for 2 min. Each item was then subjected to microbiological analysis as described in the previous section.

Persistence Experiment

Sample Preparation and Inoculation of Fomites Before the start of the experiment, all coupons were cut into either 5 x 5 cm or 10 x 10 cm squares, depending on the item. Coupons were sterilized using an autoclave for 15 min at 121 °C or by using 70% ethanol. The inoc- ulum was prepared by adding 5 ml of phi 6 stock to 45 ml of 0.02% PBST buer (10 8 PFU/ ml). Each coupon surface was spot-inoculated with the inoculum and an L-shaped spreader was used to evenly distribute the phage. The coupons were air-dried for 1 hr at room tem- perature (23 ± 2 °C) in a biosafety cabinet. During the drying time, TSA soft agar tubes were prepared for overlay by melting prepared TSA soft agar in a 48–50 °C water bath. After the coupons were air-dried, two inoc- ulated coupon samples for each surface were taken and placed in a stomacher bag contain- ing either 90 ml or 45 ml of buer (0.02% PBST) and homogenized using a stomacher lab blender for 2 min. Next, 10-fold dilutions

Cross-Contamination From Surfaces to Hands

Hands were washed for 30 s using soap and warm water (40 °C), dried using paper tow-

Volume 85 • Number 10