Single nucleotide polymorphism (SNP)-based differentiation of Shigella isolates by pyrosequencing

► We applied single nucleotide polymorphism (SNP) discovery and analysis for distinguishing Shigella serogroups. ► Twenty four informative SNPs were located in nine genes by in silico analysis of eight Shigella whole genome sequences. ► SNPs were verified by pyrosequencing. ► The combination of the 24 SNPs resulted in the identification of 26 SNP genotypes among the four Shigella serogroups. ► Phylogenetic tree based on data from the 24 SNPs constructed identified six lineages among the 26 SNP profiles. Analysis of single nucleotide polymorphisms (SNPs) is an important genetic tool that provides molecular markers for rapid differentiation of closely related strains. We have applied SNP discovery and analysis for distinguishing each of the four Shigella serogroups (Boydii, Dysenteriae, Flexneri, and Sonnei) and for discriminating individual strains within the same serogroup by using 24 SNPs selected from nine genes. Five SNPs were identified from sequence analysis of two housekeeping genes (gapA and thrB) used previously in our lab to differentiate Shigella isolates into distinct lineages. The remaining 19 SNPs were identified by in silico analyses of eight Shigella genomes and are within the genes lpxC, sanA, yaaH, ybaP, ygaZ, yhbO, and ynhA. A total of 118 Shigella strains comprising 20 Boydii, 29 Dysenteriae, 42 Flexneri, and 27 Sonnei isolates were analyzed using the SNP typing scheme reported here. The combination of the 24 SNPs resulted in the identification of 26 SNP genotypes among the four Shigella serogroups and also provided some discriminatory resolution among individual strains within the same serogroup. The SNPs presented here should prove useful in identifying Shigella using PCR amplification and rapid sequence typing strategies.