Whole Genome Sequence Analysis of Salmonella Typhi Isolated in Thailand before and after the Introduction of a National Immunization Program

Abstract

Vaccines against Salmonella Typhi, the causative agent of typhoid fever, are commonly used by travellers, however, there are few examples of national immunization programs in endemic areas. There is therefore a paucity of data on the impact of typhoid immunization programs on localised populations of S. Typhi. Here we have used whole genome sequencing (WGS) to characterise 44 historical bacterial isolates collected before and after a national typhoid immunization program that was implemented in Thailand in 1977 in response to a large outbreak; the program was highly effective in reducing typhoid case numbers. Thai isolates were highly diverse, including 10 distinct phylogenetic lineages or genotypes. Novel prophage and plasmids were also detected, including examples that were previously only reported in Shigella sonnei and Escherichia coli. The majority of S. Typhi genotypes observed prior to the immunization program were not observed following it. Post-vaccine era isolates were more closely related to S. Typhi isolated from neighbouring countries than to earlier Thai isolates, providing no evidence for the local persistence of endemic S. Typhi following the national immunization program. Rather, later cases of typhoid appeared to be caused by the occasional importation of common genotypes from neighbouring Vietnam, Laos, and Cambodia. These data show the value of WGS in understanding the impacts of vaccination on pathogen populations and provide support for the proposal that large-scale typhoid immunization programs in endemic areas could result in lasting local disease elimination, although larger prospective studies are needed to test this directly.

Fig 1. Genomic analysis of Thai S. Typhi.

(A) Maximum likelihood phylogenetic tree (outgroup rooted). Strains are labelled with their three digit name code, year of isolation (pink shading indicates post-vaccine isolates); source location (shaded by city, as indicated in panel B); and plasmid content (any antibiotic resistance genes are indicated in italics). Branch lengths are indicative of the number of SNPs. (B) Locations from which S. Typhi were isolated in Thailand. (C) Total number of positive blood cultures of S. Typhi (black) and Paratyphi A (grey) between 1970 and 1985; immunization period is indicated in pink; reproduced using data from reference (14).

Fig 2. Zoomed in phylogenies showing relationships of Thai S. Typhi to global isolates.

Maximum likelihood trees including S. Typhi isolates from the Thai and global collections are shown, for each genotype that was observed amongst the Thai isolates. (A) Genotype 2.0.0 tree. (B) Genotype 2.1.7 tree. (C) Genotype 2.2.0 tree. (D) Genotype 2.3.4. tree (E) Genotype 2.4.0. tree (F) Genotype 3.0.0 tree (G) Genotype 3.1.2. tree (H) Genotype 3.2.1 tree. (I) Genotype 3.4.0 tree. (J) Genotype 4.1.0 tree. Colored branches and nodes indicate country of origin, according to the inset legend. Year of isolation is shown to the right; pink and red, Thai isolates obtained before and after the introduction of the immunization program; grey and black, non-Thai isolates obtained before and after the introduction of the immunization program. Thai isolates are also labelled to indicate their city of origin: L, Loi; B, Bangkok; S, Srakaew; N, Nonthaburi. SNP distances between isolates as well as AMR plasmids are labelled, with any resistance genes indicated in italics. Branch lengths are indicative of the number of SNPs.

Fig 3. SNP distances for Thai and global collection isolates.

SNP distance between post-vaccine Thai isolates and their closest pre-vaccine Thai and post-vaccine global collection relatives, colored points indicate country of origin.

Fig 4. Blast comparison of novel plasmid pTy004_01 with pHCM2 (AL513383).

Shaded regions indicate areas of sequence homology, intensity of shading indicates relative nucleotide similarity. Arrows represent protein coding genes, direction indicates coding strand.

Fig 5. Blast comparison of novel phages observed in Thai S. Typhi isolates to nearest known phage sequences.

(A) Novel phage STYP1 compared to Shigella sonnei phage fiAA91-ss (NC_022750). (B) Novel phage STYP2 compared to Shigella flexneri phage SfIV (NC_022749). Shaded regions indicate areas of sequence homology, intensity of shading indicates relative nucleotide similarity. Arrows represent protein coding genes (direction indicates coding strand), colored by encoded protein functions: red, DNA packaging module; orange, virion morphogenesis module; yellow, cargo genes; blue, DNA replication and lysogenic cycle maintenance; green, lysis module.