Isolation and Identification of Salmonella spp. from Chicken Processing Facility

A total of 182 samples were collected in triplicate from a chicken processing facility to isolate and identify of Salmonella spp. Every sample was collected from 7 different spots in the chicken skin facility (Yougin, Gyeonggi-do, Korea). Environmental surface, in-line food, and final food sampling were conducted by following the standard sampling guidelines (FDA, 2018). To collect microbial samples from environmental surfaces, such as the cutting board and the conveyor belt, as well those surfaces indirectly contacted, such as the box containing the food piled up in vinyl, the swab test was performed by slowly wiping the identified area (approximately 10 × 10 cm, i.e., 100 cm²) horizontally and vertically with sterilized cotton (Pipette Swab Plus, 3M Korea Ltd., Seoul, Korea). For food sampling, the food products (chicken nugget and chicken karaage [Japanese-style fried chicken nuggets]) from 2 processing lines were transferred by sterile SS tongs to sterile sample bags (Nasco, Fort Atkinson, WI). The swab and food samples were transferred to the laboratory and immediately examined by the Salmonella qualitative test based on the modified method of the Korean Ministry of Food and Drug Safety (MFDS, 2019). For pre-enriching the food samples, 25 g of the sample and 225 mL of 2% buffered peptone water (Oxoid, Thermo Scientific, Lenexa, KS) were placed in a Whirl-Pak filter bag (Nasco) and homogenized for 2 min in stomacher at the maximum speed (Bag Mixer; Interscience, Saint Nom, France). The homogenized food samples and swab samples were then incubated at 37°C for 24 h. Next, 100 μL of each enriched sample was pipetted into 10 mL of Rappaport-Vassiliadis broth (Oxoid, Thermo Scientific, Waltham, MA) and incubated at 42°C for 24 h. Aliquots of the incubated Rappaport-Vassiliadis broth were streaked on xylose−lysine−deoxycholate agar (XLD, Oxoid, Thermo Scientific, Waltham, MA) using a loop, and each plate was incubated at 37°C for 24 h. Presumptive pink colonies with or without black centers on XLD were detected as Salmonella and streaked separately on fresh XLD agar plates for biochemical examination. The isolated strains were cultured in triple sugar iron agar (Difco Laboratories, Sparks, MD) and Christensen urea agar base (Oxoid, Thermo Scientific, Waltham, MA), respectively. The Salmonella-positive bacteria were identified, cultured and then analyzed by PCR to distinguish serotypes and PFGE to identify gene similarity.

The selected Salmonella spp. colony from the agar plate was inoculated into 10 mL of tryptic soy broth (TSB, Difco Laboratories) and incubated at 37°C for 24 h in a shaking incubator (VS-8480, Vision Scientific, Gyeonggi-do, South Korea) maintained at 220 rpm. The cultures in TSB were centrifuged at 11,000 × g, 4°C for 10 min. The pellets were washed and resuspended in Dulbecco's PBS (Oxoid, Basingstoke, UK) solution (pH 7.3 ± 0.2 at 25°C) with 30% glycerol (w/v) to obtain a stock solution that was stored at −80°C until required.