Co-evolution of the pangenome, mobile genetic elements, and antimicrobial resistance genes reveals the public health risks of the dominant Salmonella 4,[5],12:i:- ST34 clone.

Salmonella 4,[5],12:i:- (S. 4,[5],12:i:-) has emerged as an important foodborne pathogen, and the continued expansion of its dominant ST34 lineage poses a substantial threat to food safety and public health. However, the dynamic changes in antimicrobial resistance, mobile genetic elements (MGEs), and the accessory genome accompanying ST34 expansion remain incompletely understood. In this study, we integrated 4747 S. 4,[5],12:i:- genomes collected globally and applied population genomic, pangenomic, and Bayesian phylogeographic analyses to characterize ST34 expansion, its associated genomic changes, and their implications for public health. Population analysis revealed a pronounced expansion of ST34, with its effective population size peaking around 2016. This expansion was accompanied by progressive diversification of the resistance repertoire, with 50 antimicrobial resistance genes (ARGs) showing significant temporal increases across eight antimicrobial classes. Notably, an mcr-1.1/blaCTX-M-14-positive ST34 subpopulation emerged during the ST34 population-growth phase. Its most recent common ancestor was traced to approximately 2001 in Guangdong Province, followed by dissemination to neighboring provinces, with the detection rates of both resistance genes peaking around 2017. MGE copy number was significantly positively correlated with ARG burden and accessory-genome size (P < 0.001). ST34 harbored substantially more temporally increasing MGE-associated gene clusters than ST19, among which the highly prevalent and continuously increasing ISEc42 was located within a Gifsy-1-associated prophage region. Pangenome analysis revealed an open pangenome in S. 4,[5],12:i:-, with 3086 gene clusters (20.71%) showing significant temporal associations. A marked expansion of the ST34 pangenome occurred from approximately 2019 to 2024, later than the peak of its population expansion, indicating continued changes in accessory-gene content after the major phase of clonal growth. Among the 1825 temporally increasing gene clusters, prophage-associated genes constituted the largest functional category (19.0%), with additional enrichment of ARGs and genes associated with conjugative transfer, transposition, metal tolerance, and environmental stress responses. Differential genes between ST34 and ST19 were also predominantly associated with prophages, plasmids, ARGs, and conjugative-transfer functions. Host-sharing analysis further showed that swine- and human-derived isolates exhibited the highest similarity in temporally increasing ARGs (Jaccard = 0.65) and the greatest sharing of temporally variable pangenome genes (Jaccard = 0.77). In addition, 2190 pangenome genes were specifically shared between human- and swine-derived isolates, primarily involving prophage-, conjugative-transfer-, and DNA repair-related functions, together with a substantial proportion of genes of unknown function. Overall, the global expansion of ST34 was accompanied by diversification of the resistance repertoire, MGE- and prophage-associated changes, and continued remodeling of the accessory genome. The extensive genetic overlap between swine- and human-derived isolates further highlights the importance of the swine-pork-human interface in ST34 dissemination. These findings provide a genomic basis for the continued surveillance and control of high-risk ST34 populations throughout the food chain.