Development of Salmonella Heidelberg Mutant Library Using a Transposon Mutagenesis System

Introduction: Salmonella enterica with 1500 serovars has been considered as one of the most prominent foodborne pathogens in poultry industry. In last several years, the incidence of Salmonella Heidelberg was increased significantly in the USA and identified as one of the primary serovars associated with human illness. Although S. Heidelberg exhibits similar antigenic characteristics with S. Typhimurium one of the more common serovars in human diseases, S. Heidelberg has not been investigated nearly to the same extent.

Purpose: The aims of this research were to develop S. Heidelberg mutant library using transposon mutagenesis and identify essential genes for survival and virulence that could be used a potential targets for more effective intervention methods.

Methods: Salmonella Heidelberg ARI-14 isolated from poultry was evaluated for ampicillin and kanamycin susceptibility prior to transposon mutant library construction because the EZ-Tn5 pMOD-6 plasmid DNA possesses both antibiotics genes as selection markers. The EZ-Tn5 pMOD-6 plasmid DNA was transformed to S. Heidelberg strain in advance to improve efficiency through host adaptation followed by development of a transposon mutant library.

Results: Salmonella Heidelberg ARI-14 strain was susceptible ampicillin (100 µg/ml) and kanamycin (60 µg/ml). The efficiency of EZ-Tn5 pMOD-6 plasmid DNA transformation to ARI-14 for host adaptation was 10⁶cells per ml. The excised transposon from host-adapted plasmid DNA was utilized for mutant library.

Significance: The developed S. Heidelberg mutant library can be applied to various growth conditions like low pH and thermal stress to screen essential genes for survival and virulence. Identification of these genes will help to elucidate more effective control measures in food systems.