Genomic diversity of non-typhoidal Salmonella found in patients suffering from gastroenteritis in Norfolk, UK

<i>Salmonella</i> is a significant public health pathogen responsible for a wide spectrum of diseases, ranging from gastroenteritis to invasive non-typhoidal salmonellosis and enteric fever. Although advancements in whole-genome sequencing have improved surveillance and outbreak investigations, traditional single-colony sequencing methods overlook within-host diversity, potentially underestimating the complexity of infections. This study explores the genome-wide diversity of <i>Salmonella</i> strains recovered from stool samples of eight patients, with up to 20 isolates analysed per sample. A total of 156 <i>Salmonella enterica</i> isolates were recovered. All isolates from individual patients displayed consistent serovars and sequence types. Despite the serotype consistency, microevolution was observed in <i>S</i>. Java ST149 and <i>S</i>. Java ST43, with SNP analyses revealing higher diversity (13 and 5 SNP differences, respectively) compared to the clonal populations of other serovars. Phylogenetic analysis of <i>S</i>. Java ST149 isolates from Patient 2 revealed distinct branching, driven by mutations in genes such as <i>secY</i> and <i>cnoX</i>, while <i>S</i>. Java ST43 isolates from Patient 1 displayed multiple clades with notable SNPs affecting transcriptional regulators. Genome structure (GS) analyses using hybrid assemblies identified uniform GS1.0 across all isolates. Antimicrobial resistance (AMR) profiling revealed the presence of multidrug efflux pump genes (<i>mdsA</i> and <i>mdsB</i>) in all isolates. However, <i>S</i>. Typhimurium isolates from Patient 4 exhibited additional AMR genes, including <i>sul2</i>, <i>aph(3'')-Ib</i> and <i>blaTEM-1</i>, associated with an 8.7 kb resistance region. A single isolate from Patient 4 lacked these additional genes due to the deletion of a ~19 kb genomic region, highlighting structural variation as a driver of phenotypic differences. These findings emphasize the genetic diversity of <i>Salmonella</i> within hosts, particularly in serovars such as <i>S.</i> Java, and underscore the limitations of single-colony sequencing in capturing this complexity. The study highlights the utility of hybrid sequencing strategies for comprehensive analysis of genome variation, offering valuable insights into transmission dynamics, AMR and evolutionary processes in <i>Salmonella</i>.