Since similar findings were observed with both strains in the in vitro studies, S. Heidelberg 1904 was used for further investigations. To avoid potential competition from the skin and meat microflora, the strain was induced for resistance to nalidixic acid at 50 µl/mL nalidixic acid sodium salt (NA; CAS No. 3374-05-8, Alfa Aesar, Haverhill, MA) for selective enumeration as previously described (Nair and Kollanoor Johny, 2017b). After the growth of culture to 9 log10 CFU/mL was confirmed by plating, an overnight culture was pelleted by centrifugation (3,600 x g for 15 min at 4°C) and suspended in sterile PBS to obtain a final concentration of 4 log10 CFU/50 µl.

Samples were prepared from 100% all-natural (non-enhanced) chicken drumsticks and breast fillets purchased from a retail store. The skin samples from drumsticks were separated from the underlying muscle and cut into 1 square inch pieces using a sterile scalpel (Nair and Kollanoor Johny, 2017b). Breast fillets were also aseptically cut into 2 g pieces using a sterile scalpel (Wagle et al., 2017). Both skin and meat samples were first sterilized by UV light for 5 min to eliminate background microorganisms before inoculation. Each skin or meat sample was spot inoculated with 50 µl of the inoculum containing 4 log10 CFU S. Heidelberg 1904. The samples were subsequently kept under the biosafety cabinet at room temperature for 30 min to facilitate Salmonella attachment.

The dip treatments were prepared by adding LGEO to sterile deionized (DI) water at concentrations of 0, 0.5, 1, or 2% (v/v). The temperature of treatment solutions was maintained at 4°C using a refrigerator and 54°C using a water bath to simulate the chilling and scalding stages of poultry processing, respectively. The treatment water was vortexed for 30 s before dipping the samples. Each skin or meat sample was immersed in separate 20 mL of the dip solution containing one of the 4 concentrations of LGEO for either 2, 3, or 5 min before microbiological analysis.

Preparation of dip treatments and temperature of treatment solutions was similar to that of the individual scalding and chilling experiments. For this experiment, each skin or meat sample was first dipped in a separate 20 mL dip solution containing one of the 4 concentrations of LGEO for 2 min at the scalding temperature. The samples were then aseptically moved to a dip treatment containing their corresponding LGEO concentrations at chilling temperature for 2 min before microbiological analysis. Additionally, dip treatments at both temperatures were also analyzed for surviving S. Heidelberg.

Each skin and meat sample was transferred to a sterile Whirl-Pak bag containing 10 mL PBS to enumerate surviving S. Heidelberg attached to skin or meat after LGEO dip treatments. The samples were subsequently homogenized for 2 min at 200 rpm using a stomacher (100/125V, 50/60Hz; Neutec Group Inc., Farmingdale, NY) followed by 10-fold serial dilution with PBS before plating 100 µl from appropriate dilutions on XLD+NA plates. Additionally, 100 µl from dipping solutions used in the sequential scalding and chilling treatments were also plated on XLD+NA to enumerate surviving pathogens in the treatment water. All XLD+NA plates were incubated at 37°C for 24 h before bacterial enumeration. Dipping solutions and sample homogenates were enriched with 10 mL of Selenite Cysteine Broth (SCB; Hardy Diagnostics, Santa Maria, CA, United States) and streaked on XLD+NA plates after 8 h of incubation to detect any surviving Salmonella that was not observed with initial plating.

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