Whole-Genome Subtyping Reveals Population Structure and Host Adaptation of Salmonella Typhimurium from Wild Birds

Abstract

Within-host evolution of bacterial pathogens can lead to host-associated variants of the same species or serovar. Identification and characterization of closely related variants from diverse host species are crucial to public health and host-pathogen adaptation research. However, the work remained largely underexplored at a strain level until the advent of whole-genome sequencing (WGS). Here, we performed WGS-based subtyping and analyses of Salmonella enterica serovar Typhimurium (n = 787) from different wild birds across 18 countries over a 75-year period. We revealed seven avian host-associated S. Typhimurium variants/lineages. These lineages emerged globally over short timescales and presented genetic features distinct from S. Typhimurium lineages circulating among humans and domestic animals. We further showed that, in terms of virulence, host adaptation of these variants was driven by genome degradation. Our results provide a snapshot of the population structure and genetic diversity of S. Typhimurium within avian hosts. We also demonstrate the value of WGS-based subtyping and analyses in unravelling closely related variants at the strain level.

Keywords: Salmonella enterica serovar Typhimurium; avian hosts; host adaptation; whole-genome sequencing.

FIG 1

Avian isolates of Salmonella Typhimurium used in this study (n = 787). (A) Number of isolates grouped by avian hosts. The “Others” bird type indicates avian hosts without a designated bird type at EnteroBase or any bird types not included in the defined categories. (B) Number of isolates grouped by geographic locations. (C) Number of isolates grouped by collection years. N/A, the collection year is not available. (The figure was created with BioRender.com.)

FIG 2

Population structure of globally sourced Salmonella Typhimurium isolates from avian hosts. (A) NJ tree of the 787 S. Typhimurium isolates from avian hosts (https://enterobase.warwick.ac.uk/ms_tree?tree_id=70709). The NJ tree is constructed based on the Salmonella wgMLST scheme (21,065 loci) at EnteroBase. The scale bar indicates allele differences in 200 wgMLST loci (or genes). Allele differences between isolates are indicated by numbers on the connecting lines. In the legend “Bird type,” the number in brackets indicates the number of isolates from that specific bird type. “Other, not specified” represents avian hosts without a designated bird type at EnteroBase. “Other, specified” represents avian hosts that do not belong to passerine, larid, water bird, duck/goose or pigeon, and the number of isolates from these avian hosts is less than 10. More detailed information on individual bird types and the corresponding isolates can be found in Data Set S1 in the supplemental material. (B) Maximum-likelihood phylogenetic tree of the 207 S. Typhimurium isolates from avian hosts (see Data set collection in the Materials and Methods for the selection criteria for the 207 isolates out of the whole collection of 787 isolates). The tree is built based on 6,310 SNPs in the core genomic regions with reference to S. Typhimurium LT2 and rooted at midpoint. Individual avian host-associated lineages are supported by a bootstrap value of 100%. The color strip “Sequence type” represents the S. Typhimurium multilocus sequence type determined by seven-gene (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) MLST.

FIG 3

Emergence times of avian host-associated Salmonella Typhimurium lineages inferred by Bayesian time-scaled tree. Estimated emergence times of individual lineages are reported as median years with 95% highest posterior probability density (HPD). The red dot at the tree tip represents the reference genome from S. Typhimurium LT2 (collection year: ca. 1948). The posterior probability values of representative divergent events are greater than 95% (not shown in the figure).

FIG 4

Phylogenetic relationship of Salmonella Typhimurium lineages circulating within diverse hosts (n = 290). The legend field at the right of the tree represents the S. Typhimurium lineage (primary host). Broad host range in parentheses indicates that isolates from the corresponding lineage are commonly identified among humans, cattle, pigs, and poultry. The specific host species in parentheses indicates that isolates from the corresponding lineage are primarily from that specific host (herein, the specific host is referred to as primary host). Individual lineages are correlated with their associated primary host species in the tree. Gray shaded host species in the U288 complex lineage and DT204 complex lineage represent minor hosts (herein, if isolates of a specific lineage are occasionally collected from a host species, the host species is then referred to as minor host) other than the primary host.

FIG 5

Genetic diversity of Salmonella Typhimurium lineages from diverse hosts (n = 290). (A) Number of core genes (genes present in ≥99% isolates of the analyzed data set), soft shell genes (genes present in 95 to 99% of isolates in the analyzed data set), shell genes (genes present in 15 to 95% of isolates in the analyzed data set), and cloud genes (genes present in 0 to 15% of isolates in the analyzed data set) per isolate in individual lineages. (B) Number of S. Typhimurium core genes (n = 3,798) and number of core genes that represent a unique core-gene combination in a specific lineage (see colored key). (C) Average number of antimicrobial resistance (AMR) genes per isolate in individual lineages. (D) Average number of plasmid replicons per isolate in individual lineages. The following are the numbers of isolates in each lineage: passerine lineage 1 (n = 59), passerine lineage 2 (n = 26), larid lineage (n = 33), duck/goose lineage (n = 23), pigeon lineage 1 (n = 17), pigeon lineage 2 (n = 21), water bird lineage (n = 28), ST313 lineage (n = 10), DT204 complex lineage (n = 9), U288 complex lineage (n = 20), ST34 lineage (n = 21), DT193 complex lineage (n = 9), and DT104 complex lineage (n = 14). Error bars represent standard error of the average number of a data set.

FIG 6

Virulence gene profiles of Salmonella Typhimurium lineages from diverse hosts (n = 290). (A) Average number of virulence genes per isolate in individual lineages. Error bars represent standard error of the average number of a data set. (B) Number of virulence genes with identical mutations in individual lineages. (C) Heatmap showing the mutation types of virulence genes in individual lineages. The numbers in parentheses indicate the number of isolates from that specific lineage. “Multiple mutations” indicates that several mutations occur in a virulence gene at different positions. The detailed mutation information (mutation type, mutation position, and base pair change) of each virulence gene in individual lineages can be found in Data Set S8 in the supplemental material.