Molecular typing and evolutionary relationships of Salmonella enterica serovar Typhi

Abstract

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.