Validated small Col-like accessory elements in a near-clonal same-host stool–blood Salmonella pair

BACKGROUND: Nontyphoidal Salmonella can cause bloodstream infection in vulnerable hosts, yet the bacterial differences that remain when paired stool and blood isolates are already near-clonal are not well defined. Core-genome SNP analysis is useful for assessing relatedness, but may overlook small circular accessory elements that are difficult to resolve using standard assembly workflows. This study investigated whether a clinically linked same-host stool-blood Salmonella pair contained validated small Col-like accessory elements not apparent from core SNP analysis alone. METHODS: We analyzed a temporally adjacent same-host stool-blood Salmonella index pair, FB1-XY1, together with two short-interval stool-stool comparator pairs. The analysis integrated strict reads-backed core SNP filtering, accessory-element analysis, Illumina and ONT read-level support, PCR/Sanger/qPCR validation and public-genome background screening. RESULTS: Strict reads-backed filtering retained only four core SNPs in the FB1-XY1 pair, whereas both stool-stool comparator pairs had no retained strict core SNPs. Despite this minimal core-genome divergence, the blood isolate XY1 carried two validated small Col-like accessory elements. The first was an assembly-resolved 13.8-kb ColRNAI-like plasmid containing ColRNAI-like replicon hits and duplicated cka/colicin K-like annotations, with a Proteinase K-sensitive inhibitory phenotype. The second was a 5,241-bp Col156-like circular element, contig_40, which was not represented as a near-full-length replicon in the final XY1 closed-assembly output but was supported by full-length XY1 Illumina and ONT read coverage, 116 ONT junction-spanning reads, PCR, Sanger sequencing, multi-colony screening and qPCR. FB1 Illumina reads showed no corresponding support for contig_40. In a screened public Salmonella background of 16,456 genomes, strict full-length hits were rare for both elements: 2 hits for the 13.8-kb plasmid and 15 hits for contig_40. Neither element had a strict full-length hit among 402 screened Muenster genomes. CONCLUSIONS: These findings identify small Col-like circular accessory elements as a validated, assembly-sensitive layer of genomic variation in a near-clonal same-host stool-blood Salmonella pair. By showing that clinically linked stool-blood isolates can differ at the level of small circular accessory elements despite minimal core-genome divergence, this study extends paired-isolate interpretation beyond core SNP relatedness alone. Integrating read-level evidence, molecular validation and public-genome context may improve the interpretation of small circular elements in clinical microbial genomics.