A Polymerase Chain Reaction (PCR) Method to Detect Emerging Multidrug-Resistant Salmonella Infantis Harboring the pESI Plasmid in Seafood

Abstract

Background Salmonella Infantis is an emerging multidrug-resistant pathogen worldwide due to the acquisition of a megaplasmid pESI (Plasmid of Emerging Salmonella Infantis). Reported initially from poultry, the distribution of pESI-harbouring S. Infantis in other food types, including seafood, is unknown. Objective This study aimed to develop and optimize a PCR assay for detecting pESI plasmid in Salmonella and non-Salmonella Enterobacterales. Methods A duplex PCR targeting the hilA gene and a pESI-associated gene of S. Infantis was designed, and the PCR conditions were optimized. The specificity and sensitivity of the assay were established using 119 Salmonella serovars and 51 non-Salmonella bacterial strains. Results All Salmonella isolates yielded hilA PCR product, while only pESI S. Infantis was positive for both hilA and pESI genes. No amplification product was obtained with the DNA of 51 non-Salmonella bacterial strains. The detection limit of the duplex PCR was 104 CFU/ml of pure culture of pESI S. Infantis. The sensitivity of detection in artificially spiked shrimp meat was 1 CFU/g after 6 h of enrichment in lactose broth, followed by 12 h of selective enrichment in the Rappaport-Vassiliadis medium. Conclusions The duplex assay will help screen seafood for Salmonella in general and pESI S. Infantis in particular. Given its high sensitivity, the PCR will be a valuable tool for seafood quality assurance. This approach decreases the typical 3-6 days’ identification time of Salmonella to less than 24 hours. Highlights S. Infantis carrying the highly transmissible megaplasmid (pESI) is a significant food safety concern. Given its rapid geographical spread and high antimicrobial resistant traits, it is necessary to have a molecular tool that detects pESI-harbouring Salmonella. This study successfully developed a duplex PCR assay that simultaneously detects Salmonella enterica and pESI S. Infantis. This molecular tool will help understand the distribution, sources, and spread of MDR plasmid in the food environment.