ADVANCEMENT OF THE SCIENCE
period, plates were enumerated and plaques were recorded as PFU/cm. The procedures for counting the phi 6 were carried out on days 1, 2, 3, 4, 7, 10, 13, 16, 19, 22, 25, 28, and 30 days, at which time no phi 6 was detected. The same procedures were repeated for each of three biological replicates under similar experimental conditions. Simulation Experiment A simulation experiment was conducted to determine the potential of phi 6 contami- nation and cross-contamination at farmers markets and the transfer rate from the fomi- tes to hands and produce (i.e., bell pepper, cantaloupe, and lettuce). The simulation was performed using two dierent levels of phi 6 concentrations (high level = approximately 10 7 log PFU/ml; low level = approximately 10 3 log PFU/ml) in two separate experiments with three biological replicates and duplicate samples each. Cross-Contamination From Inoculated Surfaces With High or Low Level Phi 6 to Produce A 0.2 ml of phi 6 suspension (10 7 log PFU/ ml or 10 3 log PFU/ml) was inoculated onto the surfaces of each fomite: plastic, molded pulp fiber, and wicker. Each fomite was held at room temperature (23 ± 2 °C) for 1 hr to facilitate attachment. Next, the produce items were placed on top of each inoculated surface after a portion of the produce was marked using a red marker. This marked area was left in touch with the fomite for 1 hr. The marked portion of each produce item was swabbed individually using an alginate cotton swab and placed into a tube contain- ing 5 ml of virus buer. A 1-ml aliquot from each collected sample and 100 µl of the over- night host were added to a tube containing 3 ml of soft TSA. The contents were mixed by hand and quickly poured onto TSA plates. The plates were allowed to solidify before being inverted and incubated for 24 hr at 22 °C. Following incubation, the plaques were counted and recorded as PFU/cm 2 . Cross-Contamination From Inoculated Surfaces With a High or Low Level Phi 6 to Hands To start, proper handwashing was performed for 20 s with soap and warm water (40 °C), and hands were dried with paper towels. Hands were then sprayed with 70% ethanol and allowed to air-dry. Next, one hand touched
TABLE 1
Survival and Persistence of Phi 6 on the Surface of Farmers Market Fomites
Day
Mean Log PFU/cm 2 and Standard Deviation on Surface of Each Fomite a Plastic Cardboard Molded Pulp Fiber Wicker Tablecloth
1 2 3 4 7
5.8 ± 0.2 3.7 ± 0.2 2.5 ± 0.3 2.3 ± 0.2 1.8 ± 0.5 1.4 ± 0.3 1.1 ± 0.4 0.7 ± 0.2 0.5 ± 0.4
5.1 ± 0.5 2.7 ± 0.1 2.4 ± 0.3 1.8 ± 0.4 0.8 ± 0.6 0.5 ± 0.4 0.5 ± 0.3
5.5 ± 0.5 3.1 ± 0.2 2.4 ± 0.1 2.0 ± 0.3 0.7 ± 0.2 0.4 ± 0.3 0.7 ± 0.1 0.4 ± 0.2
5.7 ± 0.2 3.7 ± 0.1 2.6 ± 0.2 2.2 ± 0.2 1.8 ± 0.2 1.5 ± 0.1 1.3 ± 0.1 1.2 ± 0.2 0.8 ± 0.3 0.7 ± 0.1 0.5 ± 0.2 0.4 ± 0.2
4.9 ± 0.1 1.7 ± 0.2 0.8 ± 0.2 0.5 ± 0.3
ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0 ND ± 0
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 ND ± 0 ND ± 0 ND ± 0
ND ± 0
a Means and standard deviation of survival of phi 6 on each farmers market fomite over 30 days ( N = 6). Note. ND = none detected.
the inoculated surfaces one at a time with the index finger (primary transfer) for 20 s. Lastly, samples from the hand were collected using a glove-juice method (Larson et al., 1980; Sirsat et al., 2013) with some modifications. In detail, the index finger from each hand touched the inoculated items for 20 s, and then the subject wore a sterile surgical glove containing 1 ml of sterile 0.02% PBST virus buer in the index finger section of the glove. Next, the hand with the glove on was vortexed for 60 s. The sample was then transferred from the glove index finger region to a sterile 10-ml conical tube using a sterile pipette. Finally, 1 ml from each collected sample and 100 µl of the overnight host were added to a tube con- taining 3 ml of soft TSA, shaken by hand, and quickly poured onto TSA plates. The plates were allowed to solidify before being inverted and incubated for 24 hr at 22 °C. Following incubation, the plaques were counted and recorded as PFU/cm 2 . Data Analyses The enumerated plaques were converted into log10, and the survival curve was created
using Microsoft Excel. Next, the persistence results were recorded for each fomite as PFU/ cm 2 . We calculated the transfer rates using the formula below:
Percent transfer rates = (log PFU/cm 2 of phi 6 on exposed subject [hand or produce] / log PFU/cm 2 of phi 6 on the original fomite) × 100
The transfer rates were calculated and the results were compared to determine the potential cross-contamination from farmers market fomites to produce or hands.
Results and Discussion
Persistence of Phi 6 on the Surface of Farmers Market Fomites Table 1 shows the recovery of phi 6 on the surfaces of the plastic, cardboard, molded pulp fiber, wicker, and tablecloth coupons. The rapid reduction of phi 6 was observed on days 1 and 2, where a >2 log PFU/cm 2 reduc- tion was recorded on all surfaces. The results observed in this experiment were similar to those of a pervious study where persistence of MS2 bacteriophage (a surrogate for norovi-
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Volume 86 • Number 1
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