|BYERS, SARA - University Of Georgia|
|KRIEBEL, PATRICK - University Of Georgia|
Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/29/2016
Publication Date: 4/1/2016
Citation: Guard, J.Y., Abdo, Z., Byers, S.O., Kriebel, P., Rothrock Jr, M.J. 2016. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA). Foodborne Pathogens and Disease. 13(7)350-362.
Interpretive Summary: Methods that rapidly identify epidemiologically important serovars of Salmonella enterica subspecies I are of vital importance to the poultry industry. To supplement the screening method for serotype known as intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNP) were characterized within the cyaA gene. Adenylate cyclase was chosen as a pivitol housekeeping gene because it is essential for many metabolic functions, attenuates virulence if mutated, and it has only one copy in the genome of Salmonella enterica regardless of serotype. Perusal of the NCBI database found 373 cyaA gene sequences from Salmonella enterica subspecies I that included 42 unique cyaA sequences. Of the 42 sequences, 20 altered amino acid sequence. Five representative serovars, namely Typhimurium, Kentucky, and Enteritidis phagetypes PT4 and PT13a, were differentiated within a microsphere-based fluidics system by using SNPs in cyaA. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serovars yielded a ~89% success rate at identifying the serovar of the isolate. One SNP was shared between two major serogroup B serovars (Typhimurium and Heidelberg) but these two serovars were already distinguishable based on ISR SNPs. Pathotypes and phage lineages of serovar Enteritidis 13a and 4 were successfully identified. Comparative ranking of variance within the cyaA gene to other classes of genes indicated that it was an appropriate target gene for rooting subtype of Salmonella serovars to major epidemiological trends, while other genes such as the diguanylate cyclases may be better for correlating phenotype to genotype after housekeeping functions are established.
Technical Abstract: Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclase (cyaA). The NCBI database had 378 cyaA sequences from Salmonella enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4 and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele specific primer extension (ASPE). Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ~89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases and genes used for multilocus sequence typing (MLST) indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.