Single Nucleotide Polymorphism Typing of Global Salmonella enterica Serovar Typhi Isolates by Use of a Hairpin Primer Real-Time PCR Assay

Salmonella enterica serovar Typhi is highly homogeneous. Single nucleotide polymorphisms (SNPs) have been shown to be valuable markers for molecular typing of S. enterica serovar Typhi. Here, we used a hairpin primer real-time PCR assay for SNP typing of S. enterica serovar Typhi isolates. Forty-two SNPs were selected from a comparison of 19 published S. enterica serovar Typhi genomes and sequences from other studies. The SNPs were used to type 71 global S. enterica serovar Typhi isolates and differentiated these S. enterica serovar Typhi isolates and the 19 genome sequenced strains into 25 SNP profiles. Phylogenetic analysis revealed that these SNP profiles were grouped into six major clusters. These clusters can be identified by using five SNPs, while the full differentiation of the 25 SNP profiles requires a minimum of 24 SNPs. This real-time PCR-based SNP typing method will be useful for global epidemiological analysis.Salmonella enterica serovar Typhi is highly homogeneous (10, 17, 18). The lack of genetic diversity is a major challenge to the development of suitable typing methods to differentiate S. enterica serovar Typhi isolates for both phylogenetic and epidemiological purposes. Single nucleotide polymorphisms (SNPs) are considered the most valuable markers, particularly for studying the evolutionary relationships of isolates of homogeneous pathogenic clones, such as Bacillus anthracis (16), Mycobacterium tuberculosis (4), and Yersinia pestis (1).SNPs have been used as markers for molecular typing of S. enterica serovar Typhi in a large study by Roumagnac et al. (17). A total of 88 SNPs, found from analysis of 200 gene fragments from 105 diverse S. enterica serovar Typhi isolates, could differentiate 481 global S. enterica serovar Typhi isolates into 85 haplotypes (SNP profiles) and five major clusters (17). However, despite the large number of SNPs used, each of the five clusters was supported by only a single SNP and there was little resolution of the relationships of the haplotypes within a cluster. Eighty of the SNPs have also been used to differentiate 140 Indonesian S. enterica serovar Typhi isolates into nine haplotypes (2).We have also shown that genome-wide SNPs are useful for molecular typing and determining the relationships of global S. enterica serovar Typhi isolates (14). Thirty-seven SNPs selected from a comparison of the genomes of S. enterica serovar Typhi strains CT18 (15) and Ty2 (3) were typed using restriction enzyme digestion to differentiate 73 global S. enterica serovar Typhi isolates into 23 SNP profiles and four distinct genetic groups. As the SNPs were selected by comparison of only two S. enterica serovar Typhi genomes, this resulted in a phylogenetic bias which revealed the full path of the last common ancestors connecting strains CT18 and Ty2 but only the node locations for the other SNP profiles (14).Advances in technology, such as high-throughput sequencing, allow SNPs to be discovered to obtain a full resolution of the phylogenetic relationships of isolates. A recent study by Holt et al. (8) utilized 454 and/or Solexa technologies to sequence 19 S. enterica serovar Typhi isolates selected from the five major clusters found by Roumagnac et al. (17). There were more than 1,700 SNPs found, and these gave a fully resolved phylogenetic tree of these isolates. These SNPs are invaluable resources for investigation of the evolutionary history of global S. enterica serovar Typhi isolates. This study aimed to select a better set of SNPs on the basis of the genome tree and the previous SNP studies by Roumagnac et al. (17) to differentiate and establish the phylogenetic relationships of global S. enterica serovar Typhi isolates, using real-time (R-T) PCR assays based on hairpin (HP) primers (6).