ARS | Publication request: Mutational Mapping and Location of Single Nucleotide Polymorphisms in Salmonella Enteritidis Isolates that Vary in Virulence

Submitted to: United States Animal Health Association Proceedings
Publication Type: Abstract
Publication Acceptance Date: August 8, 2006
Publication Date: October 15, 2006
Citation: Bouldin, J.G. 2006. Mutational Mapping and Location of Single Nucleotide Polymorphisms in Salmonella Enteritidis Isolates that Vary in Virulence. United States Animal Health Association Proceedings. p.586-587.

Technical Abstract: Salmonella enterica subspecies I enterica serotype Enteritidis (S. Enteritidis) is currently the leading cause of salmonellosis worldwide and the second leading cause in the United States. The Centers for Disease Control (CDC) and the USDA Food Safety Inspection Service (FSIS) have recently described epidemiological trends that suggest that this pathogen could be increasing in incidence in people and in broiler chickens. Research is needed to identify small scale genetic change that correlates with the ability of S. Enteritidis to cause food borne outbreaks because methods such as DNA-DNA hybridization microarrays and pulsed field gel electrophoresis (PFGE) have failed to differentiate between strains that vary in virulence phenotype. The objectives of this project are to identify single nucleotide polymorphisms (SNPs) that differentiate the genomes of two isolates that were obtained from a single parent strain but that nonetheless had different pathological outcomes in laying hens. To locate SNPs, mutational mapping was performed by comparative genome sequencing (CGS), which is a commercially available service (Nimblegen, Inc). CGS requires that a genomic database be available to generate overlapping primers that resolve sequence to a single base pair. Phage type (PT) 4 S. Enteritidis genome sequence is available from the Pathogen Sequencing Group at the Sanger Institute (http://www.sanger.ac.uk/Projects/Salmonella/). DNA was extracted from three isolates of S. Enteritidis, one of which was a PT4 isolate used as a template to generate the overlapping primers. The other two isolates submitted for CGS were PT13a isolates that varied in their ability to contaminate eggs. One of these isolates could contaminate eggs, grow to high cell density, and produce a capsular LPS molecule at 25°C and it was designated wt S. Enteritidis. The other PT13a isolate was orally invasive, produced biofilm but could not contaminate eggs or grow to high cell density or produce much capsular LPS at 25°C. It was designated bf S. Enteritidis. Both strains were descended from a single parent strain. Results (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=search&term=) were that 409 SNPs out of the 4.686 million base pairs in the genome, or less than 0.01% of the genome, differentiated the two PT13a isolates that varied in virulence potential. There was an average of 8.5 SNPs per 100,000 bp. Areas of the genome that had lysogenic phage could not be compared in this assay, because primers made to the PT13a specific bacteriophage Fels-2 were absent for lack of template in the PT4 genome and primers made to the PT4 specific phage, ST64b, were lacking target DNA in the PT13a strains. Thus, SNPs that occur within phage genes that differ between PT4 and PT13a strains are not included in the total and will require manual sequencing. The virulence plasmid from the two PT13a strains differed by 5 SNPs. All classes of genes had SNPs, although genes involved in metabolism were most heavily represented and included more than 30% of the genes identified. Curvilinear analysis of SNPs with identity to PT4 in every 100kb revealed that the PT4 genome under investigation had SNPs occurring between genes rrfC and yhjO (about 2/5ths of the genome) that were preponderantly similar to bf PT13a S. Enteritidis; however, the rest of the genome was more similar to the wt PT13a isolate. As compared to the two PT13a strains, the PT4 genomic database was genetically a dimorphic hybrid of the wt and bf PT13a isolates, which agreed with previous results obtained by pan-genomic phenotype microarray (Biolog, Inc.). Thus, the PT4 genome sequenced by the Sanger Institute exhibits a mixture of phenotypes from a single genome in response to environmental signals. We conclude that very little genetic change is required for Salmonella Enteritidis to alter its virulence phenotype and that the ability of bacteria to mutate rapidly obscures identification of those SNPs that are most closely linked to outbreaks of salmonellosis. Furthermore, epidemiological investigations that are based on fingerprinting methodology are inadequate for detecting evolutionary trends due to SNPs that impact the virulence potential of the Salmonellae. The current problem of food borne illness associated with S. Enteritidis may have originated when a single bacterial cell was co-infected by incompatible lysogenic bacteriophage. This single cell may have rapidly split into two phage lines that nonetheless had only slightly different pathogenic potential to begin with and that overtime evolved adaptations to selection pressures present in different regions and niches within the on-farm environment. 1U. S. Department of Agriculture, Agricultural Research Service, Athens, GA, USA