Clonal Lineage, Not Functional Linkage, Shapes Antimicrobial Resistance Gene, Plasmid Replicon, and Virulence Gene Co-Occurrences in Clinical Cronobacter sakazakii from the United States

Cronobacter sakazakii is a rare, life-threatening neonatal pathogen. In this study, we profiled antimicrobial resistance genes (ARGs), plasmid replicons, virulence genes, sequence types (STs), and core-genome phylogeny across all publicly available clinical C. sakazakii genomes from the United States (n = 116). All isolates harbored exclusively chromosomally encoded blaCSA variants. IncFIB and rep cluster 574 co-occurred in 93.1% of isolates as a conserved backbone among 17 replicon types. Among 49 virulence genes, 13 formed a universal core (flagellar motility and type VI secretion); accessory genes such as the yersiniabactin cluster were sporadic. ARG, plasmid replicon, and virulence gene contents were strongly structured by ST, dominated by ST4, ST1, ST8, and ST13. Stratified testing shows most pairwise associations were confounded by clonal lineage, except the IncFIB-rep cluster 574 co-carriage and an IncFII-hcp/tssD association, significant after ST stratification but not multiple testing correction. Core-genome phylogenetics identified 16 clusters, from a large ST4-dominated group to nine singletons, corroborated by ST monophyly; a genomically identical ST1 pair, nine years apart, indicated long-term clonal persistence. Ancestral state reconstruction distinguished stable single acquisitions from features with repeated turnover. Core-genome and gene-content phylogenies demonstrated substantial discordance despite significant overall correlation. These findings provide a genomic framework for clinical C. sakazakii population structure in the United States.