Rapid Molecular Pathotyping of Major Salmonella enterica Serotypes Based on Single-nucleotide Polymorphisms (SNPs) in the Adenylate Cyclase (cyaA) Gene

Introduction: Salmonella enterica subsp. enterica serotype Enteriditis (S. Enteriditis) is the leading cause of salmonellosis worldwide, including the USA.  Many S. enterica serotypes known to cause foodborne disease are associated with broiler meat contamination.  While some serotypes are specific to birds (S. enterica serotypes Pullorum, Gallinarum), many represent human zoonotic pathogens (S. enterica serotypes Enteriditis, Heidelberg, Typhimurium).  The survival capabilities of S. enterica serotypes throughout the continuum of poultry production environments (farm to processing facilities) is of vital concern to the poultry industry. Environmental parameters that decrease survival of these Salmonella populations are important to identify.

Purpose: It is essential to rapidly pathotype the various types of Salmonella to determine potential food safety-related issues, especially those that occur within the top 100 serotypes linked to disease and those that have accumulated genetic change that enhances virulence. Serology (O/H antigen testing) and molecular techniques (Premi® Test) have been developed to serotype S. enterica, but they are time consuming and rather expensive.  More rapid molecular-based methods need to be developed to address the food safety concerns of the poultry industry. The adenylate cyclase gene (cyaA) represents an ideal target for these assays due to its necessity in both energy production/metabolism and virulence of S. enterica serotypes. 

Methods: Primers were designed to target a 300-bp region of the cyaA gene, and 5 different SNPs were identified within this region that could distinguish between multiple S. enterica serotypes (3 Enteriditis, Typhi, Typhimurim/Heidelberg, and Kentucky).  Probes were specifically designed to target these SNPs along the amplified cyaA amplicon, and qualitative and quantitative serotyping occurred using a Luminex MagPlex hybridization protocol that we developed and optimized.

Results: The designed hybridization protocol was found to be highly specific to only the target SNP, and thus the target serotype, when testing against an extensive panel of S. enterica serotypes.  Additionally, the protocol effectively analyzed mixed cultures of several combinations of targeted and non-targeted serotypes.  The distribution of these different serotypes, and pathotypes of S. Enteriditis, were found to be differentially affected according to the culture conditions under which the strains were grown, indicating potential biases in these cultural conditions towards certain serotypes/pathotypes.

Significance: These data contribute to a greater understanding of not only the survival of S. enterica serotypes under different cultural conditions, but also the survival dynamics of the separate serotype and virulence types relative to one another.  Expanding this method to include other serotype-specific SNPs within the cyaA gene, or applying it to environmental poultry production samples will allow the poultry industry to not only rapidly determine how much Salmonella is present, but more importantly which serotypes are present, but also which environmental parameters control the survival of the food safety related serotypes.