2.2. Sampling and Filtration

The water samples were collected from the 16 flooded and nine unflooded sites within these rural areas of North Carolina on October 7, 2018, after Hurricane Florence (Table S1). Some of the sampling locations were located on the same branch of a river, and some were not connected to any other locations by river flow (Figure S1). In this study, the sampling locations on the same branch were combined into sampling groups, and the other sites were regarded as individual sampling locations. In total, there were five paired sampling groups (flooded and unflooded adjoining locations): U3‐F8‐F7‐F6, U5‐F11, U6‐F12, U7‐F13, and U9‐F16. And, there were 13 individual sampling locations (U1, U2, U4, U8, F1, F2, F3, F5, F9, F10, F14, F15, and F17). All unflooded locations were upstream of the flooded locations within the same group. The unflooded location U4 was at the highest geographical elevation and was not located near swine farms. Therefore, U4 data were set to represent the concentration of Salmonella in a natural water body that had not been disturbed by swine farm discharge or flooding.

At each site, ∼2 L of water was collected using 38 × 19 cm Whirl‐Pak™ Stand‐Up Sample Bag (Nasco). The actual volume of each water sample was measured afterward during the filtration step, which varied from 0.6 to 2.0 L. The volumes of the majority of the water samples were between 1.6 and 2.0 L. At each location, 2–4 samples (labeled as A, B, C, and D) were collected, depending on the accessibility to the sampling sites. The GPS information (Latitude, Longitude) of each sampling location was recorded together with water sampling at each site (Table S1). The water samples were stored in coolers filled with ice packs during transportation. The water samples were stored at 4°C before filtration. Before filtration, the MgCl₂ solution was added to the sample bags to reach a concentration of 25 mmol MgCl₂/L water sample (Ahmed et al., 2020; Uprety et al., 2017). Then the sample bags were shaken for 30 min for flocculation and were then filtered through 1.6 μm glass fiber prefilter (Millipore; Sadik et al., 2017; Uprety et al., 2017). The number of filters used to filter each sample varied depending on the suspended solids load within each sample. Water filters were continuously changed when clogging occurred until the total volume of each sample was filtered. On average, 1.94 filters were used to filter per liter of each water sample. Each filter was then cut into quarters. One quarter was stored at 4°C for Salmonella isolation, one quarter was stored at −80°C for direct DNA extraction, and the remaining two quarters were stored at −80°C for future analysis.