Researchers identified two small, circular Col-like accessory elements in a blood isolate of Salmonella that were absent from a paired stool isolate from the same patient, despite the two bacteria being nearly identical at the core genome level. Published in BMC Microbiology, the study demonstrates that clinically linked stool and blood isolates can differ in their accessory genome content even when core-genome single nucleotide polymorphism (SNP) analysis suggests they are near-clonal. The findings highlight a specific layer of genomic variation that standard assembly workflows often miss, requiring integrated read-level and molecular validation to detect.
The investigation focused on a temporally adjacent same-host pair, designated FB1 and XY1, recovered from a pediatric patient with acute lymphoblastic leukemia. FB1 was isolated from stool on November 14, 2025, while XY1 was isolated from blood on November 15, 2025. Both isolates belonged to Salmonella serovar Muenster. To provide context for within-host stability, the researchers also analyzed two short-interval stool-stool comparator pairs: FB301 and FB3, recovered five days apart and belonging to serovar Infantis, and FB4 and FB401, recovered seven days apart and belonging to serovar Agona. These comparator pairs helped establish a baseline for expected genetic variation over short time intervals in the same host.
Strict reads-backed filtering of core-genome SNPs revealed minimal divergence between the stool and blood isolates. Only four core SNPs were retained in the FB1-XY1 pair after filtering out variants in repetitive regions, low-depth sites, and other unreliable locations. In contrast, both stool-stool comparator pairs had zero retained strict core SNPs. The four retained SNPs in the index pair included one synonymous variant, one intergenic variant, and two missense variants in the putA and sgrR genes. This low level of core-genome divergence confirms that the isolates are near-clonal, raising the question of what other bacterial features might distinguish the invasive blood isolate from the intestinal stool isolate.
Accessory-element analysis identified a 13,795-bp ColRNAI-like plasmid in the blood isolate XY1 that was not detected in the stool isolate FB1. This plasmid was resolved in the XY1 closed assembly and contained ColRNAI_1 replicon hits and duplicated cka/colicin K-like annotations. No acquired antimicrobial-resistance genes were detected on this plasmid. In a spot-on-lawn assay, the induced supernatant of XY1 showed an inhibition halo against an E. coli indicator strain, an effect that was abolished by Proteinase K treatment, suggesting a proteinaceous inhibitory activity. The FB1 isolate and control samples showed no such inhibition. This functional phenotype provides biological context for the presence of the Col-like plasmid in the blood isolate.
The second element, designated contig_40, was a 5,241-bp Col156-like circular element also found only in XY1. Unlike the 13.8-kb plasmid, contig_40 was not recovered as a near-full-length replicon in the final XY1 closed assembly output, highlighting a limitation of standard assembly workflows for small circular elements. However, full-length coverage was supported by XY1 Illumina reads with a mean depth of 4,682.87x and Oxford Nanopore (ONT) reads with a mean depth of 123.96x. Mapping of FB1 Illumina reads showed no support for contig_40, confirming its absence in the stool isolate. The element contained a Col156_1 replicon hit and five predicted hypothetical open reading frames, with no detected antimicrobial-resistance cargo.
To validate the presence and circular nature of contig_40, the researchers performed multiple molecular assays. PCR targeting an internal region and the predicted circular junction of contig_40 produced expected amplicons in XY1 but not in FB1. Sanger sequencing of these PCR products confirmed their identity, with the junction product crossing the contig boundary, supporting circular structure. Copy-number qPCR estimated approximately 6.2 copies per chromosome equivalent in XY1, while FB1 showed only late, non-reproducible amplification. Multi-colony screening further confirmed that independently picked XY1 colonies carried the element, whereas FB1 colonies did not. These results provide robust evidence that contig_40 is a genuine, sample-specific accessory element rather than a sequencing or assembly artifact.
Public-genome screening placed these XY1-associated elements in a broader genomic context. Across a background of 16,456 Salmonella genomes, strict full-length hits were rare. The 13.8-kb ColRNAI-like plasmid had only two strict full-length hits, both belonging to serovar Derby. The contig_40 Col156-like element had 15 strict full-length hits distributed across several serovars, including Typhimurium and Javiana. Notably, neither element had a strict full-length hit among 402 screened Salmonella Muenster genomes, indicating that these specific Col-like elements are not common background features in this serovar. An expanded targeted raw-read sensitivity analysis of 106 public records found no additional assembly-negative, raw-read-positive candidates for contig_40, reinforcing the rarity of these elements in the public database.
The study underscores the importance of integrating read-level evidence and molecular validation into clinical microbial genomics. Core-genome SNP analysis alone may overlook small circular accessory elements that are difficult to resolve using standard assembly workflows. The inhibitory activity of the 13.8-kb plasmid provides a potential mechanism for bacterial competition, but its role in invasion is not yet established.
Limitations of the study include its reliance on a single index pair, which limits the generalizability of the findings to other clinical scenarios. The function of contig_40 remains unknown, and the qPCR copy-number estimates are chromosome-normalized values rather than exact single-cell counts. DNA extraction and library preparation methods may under-represent short extrachromosomal elements, meaning that absence from an assembly does not definitively prove absence from the genome without read-level confirmation. The public-genome frequencies reported are assembly-detected frequencies and should not be interpreted as true prevalence estimates in the global Salmonella population.
These findings extend the interpretation of paired-isolate comparisons beyond core SNP relatedness, identifying small Col-like circular accessory elements as a validated, assembly-sensitive layer of genomic variation. By demonstrating that clinically linked isolates can differ in their accessory genome despite minimal core-genome divergence, the study highlights the need for comprehensive genomic approaches that account for small circular elements. Integrating read-level evidence, molecular validation, and public-genome context may improve the interpretation of small circular elements in clinical microbial genomics.
Subject of Research: Validated small Col-like accessory elements in a near-clonal same-host stool–blood Salmonella pair
Article Title: Validated small Col-like accessory elements in a near-clonal same-host stool–blood Salmonella pair
Article References: Tong, K., Zhou, J., Wang, J., Huang, Z., Ye, M., & Chen, H. (2026). Validated small Col-like accessory elements in a near-clonal same-host stool–blood Salmonella pair. BMC Microbiology, 26(1), Article 807. https://doi.org/10.1186/s12866-026-05599-z
Image Credits: AI Generated
DOI: 10.1186/s12866-026-05599-z
Keywords: Validated, small, Col-like, accessory, elements, near-clonal, same-host, stool, blood, Salmonella, pair, scientific research
News Source: Drew Townsend. (November 10, 2026). Small Col-like Elements Distinguish Near-Clonal Salmonella Isolates. Scienmag.
