Evolution of Salmonella enterica serotype Typhimurium driven by anthropogenic selection and niche adaptation

Abstract

Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteraemia worldwide, and a model organism for the study of host-pathogen interactions. Two S. Typhimurium strains (SL1344 and ATCC14028) are widely used to study host-pathogen interactions, yet genotypic variation results in strains with diverse host range, pathogenicity and risk to food safety. The population structure of diverse strains of S. Typhimurium revealed a major phylogroup of predominantly sequence type 19 (ST19) and a minor phylogroup of ST36. The major phylogroup had a population structure with two high order clades (α and β) and multiple subclades on extended internal branches, that exhibited distinct signatures of host adaptation and anthropogenic selection. Clade α contained a number of subclades composed of strains from well characterized epidemics in domesticated animals, while clade β contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and β was reflected by the distinct distribution of antimicrobial resistance (AMR) genes, accumulation of hypothetically disrupted coding sequences (HDCS), and signatures of functional diversification. These observations were consistent with elevated anthropogenic selection of clade α lineages from adaptation to circulation in populations of domesticated livestock, and the predisposition of clade β lineages to undergo adaptation to an invasive lifestyle by a process of convergent evolution with of host adapted Salmonella serotypes. Gene flux was predominantly driven by acquisition and recombination of prophage and associated cargo genes, with only occasional loss of these elements. The acquisition of large chromosomally-encoded genetic islands was limited, but notably, a feature of two recent pandemic clones (DT104 and monophasic S. Typhimurium ST34) of clade α (SGI-1 and SGI-4).

Fig 1. Phylogenetic relationship of the ST19 Salmonella Typhimurium phylogroup.

(A) Maximum likelihood phylogenetic tree and based on sequence variation (SNPs) in the core genome with reference to S. Typhimurium strain SL1344. The root was identified using S. Heidelberg (accession number NC_011083.1) as the outgroup. 1^st (α and β) and 3^rd (α11–19 and β1–7) are indicated (vertical bars). Phage type complexes associated with the third-level clusters are indicated (bold type) colour coded with the lineages and representative strains from third level clusters (italicized type). The source of each isolate in the tree is indicated by filled boxes colour coded as indicated in the inset key (arrow). The presence of replicon sequence (grey box), antimicrobial resistance genes (blue box) and hypothetically disrupted coding sequence (HDCS) of virulence related genes (red box) in short read sequence data are indicated. HDCS in nfsA and nfsB resulting in resistance to nitrofuran antibiotics and ns SNPs resulting in substitutions in the QRDR of GyrA are indicated (light blue boxes). (B) Bars indicate the number of ancestral (black), phage or insertion sequence elements (grey), chromosomal gene (colour coded with lineages in Fig 1A) HDCS in the genome of representative strains from each third level clade. (C) Box plots indicate the mean Δbitscore (DBS: bitscore SL1344 –test strain bitscore) of proteomes in third level clades. (D) Box plot indicates the percentage of the proteome of the proteome of isolates from each third level clade with a non-zero bitscore (bitscore SL1344 –test strain bitscore >0 or <0) as an estimate of function divergence. (E) Box plots indicate the mean invasiveness index per genome, the fraction of random forest decision trees voting for an invasiveness phenotype based on training on the DBS of a subset of the proteome of ten gastrointestinal and extraintestinal pathovar serotypes.

Fig 2. The pan genome of 131 S. Typhimurium isolates.

Gene families were identified based on sequence alignment with a cut off of 90% sequence identity and assigned to non-prophage chromosomal (red), prophage (green), plasmid (blue), or undefined (grey), based on their genome context in eleven annotated reference genomes from each third level clade. (A) Number of genome families in the core, softcore, shell and cloud components of the pangenome. (B) Number of genome families of each pan genome component in isolates from each S. Typhimurium third level clade. (C) Accessory genome (shell and cloud) in each isolate. Gene families present in more than 130 or less than 5 strains were excluded. Maximum Likelihood tree based on variation (SNPs) in the core genome with reference to S. Typhimurium SL1344. Third-level clades are indicated in colour coded in common with the phylogeny vertical bars.

Fig 3. Clustering of prophage genes based on sequence identity indicates related families and potential recombination.

Genes from all prophage identified in complete and closed whole genome sequence of eleven reference strains of S. Typhimurium were assigned to families based on sequence identity (>90% identity). Prophage genes (columns) were clustered to identify related prophage. The presence of a gene is indicated with a box predicted function based on in silico annotation are colour coded based on annotation, terminase (black), capsid (green), recombinase/integrase (purple), tail fibre (blue), other phage associated (red), and hypothetical protein (grey). A cladogram showing the relationship of prophage is based on the pattern of gene presence or absence is indicated (top).

Fig 4. Genome alignment and phylogenetic relationship of complete and closed reference strains of S. Typhimurium or used in this study.

Sequence with >90% nucleotide sequence identity are indicated where this is direct alignment (green) or reverse and complement (red). The location of prophage sequence (red bars) or integrative elements (blue bars) are indicated. A maximum likelihood tree based on sequence variation (SNPs) in the core genome with reference to S. Typhimurium strain SL1344 (left) is annotated with the most likely order of acquisition (black arrow) or loss (red arrow) of prophage and integrative elements, based on the principle of parsimony.

Fig 5. Recombination inferred by high SNP density in 131 S. Typhimurium strains.

Regions of high SNP density (red) are indicated for each of the 131 isolates in the S. Typhimurium ST19 cluster with reference to the S. Typhimurium strain SL1344 genome. Recombination is shown with reference to the population structure and phylogeny of Typhimurium shown in Fig 1. The position of predicted prophage (blue) in the S. Typhimurium strain SL1344 genome are indicated (top).