# Molecular Evidence of Clonal Salmonella Enteritidis Persistence in Poultry Cold-Chain Environments Under Environmental Stress

## TL;DR

This study shows that Salmonella Enteritidis can persist in poultry cold-chain environments in warm climates, posing a food safety risk.

## Contribution

The study introduces an integrated ISSR/RAPD molecular fingerprinting method for improved strain-level discrimination of Salmonella in cold-chain environments.

## Key findings

- Salmonella Enteritidis was the most prevalent serovar in poultry storage units in Al-Mandaq.
- ISSR/RAPD fingerprinting revealed clonal groupings of S. Enteritidis, indicating cross-contamination and prolonged survival.
- The combined ISSR/RAPD method outperformed single-marker systems for bacterial source differentiation in refrigeration equipment.

## Abstract

Breakdown of cold-chain integrity drives the persistence of foodborne pathogens in poultry supply chains in warm, mountainous climates. This study used Al-Mandaq (Saudi Arabia) as a model to assess genetic diversity and contamination in bacteria from poultry storage units using 16S rRNA sequencing, VITEK 2, selective culturing, and ISSR/RAPD fingerprinting on 150 swabs. The Salmonella enterica complex comprised 15/29 isolates (51.7%), followed by Escherichia spp. 6/29 (20.7%) and Bacillus spp. 3/29 (10.3%). Five Salmonella serovars were identified: Enteritidis (8), Waycross (3), Minnesota (2), Typhimurium (1), and Dublin (1). S. Enteritidis accounted for 8/29 isolates (27.6%) and predominated among Salmonella in supermarket retail samples in Al-Mandaq. Combined ISSR and RAPD cluster analysis revealed highly clonal S. Enteritidis groupings, consistent with cross-contamination and prolonged survival in refrigeration equipment. In resource-limited settings, the combined ISSR and RAPD approach enhanced identification and differentiation of bacterial contamination sources within refrigeration equipment, providing superior strain-level discrimination compared to single-marker systems and improving epidemiological traceability of cross-contamination events. These results highlight the risk of clonal pathogen persistence in poultry cold-chain environments and the value of integrated molecular fingerprinting for surveillance in challenging climates.