No isolate, no problem: Using a novel insertion sequence PCR to link rats to human shigellosis cases in an underserved urban community

Introduction: During an investigation into a cluster of Shigella flexneri serotype 2a cases in an underserved community, we assessed the relatedness of human and rat S. flexneri isolates utilizing a novel PCR targeting insertion sites (IS-PCR) of mobile elements in the Shigella genome characteristic of the cluster strain. Methods: Whole genome sequences of S. flexneri (n=50) associated with the cluster were analyzed. de novo genome assemblies were analyzed by a Geneious V10.2.6 motif search, and 2 unique IS were identified in all human Shigella sequences of the local cluster. Hydrolysis probe PCR assays were designed to detect these sequences consisting of forward and reverse primers to amplify across each insertion site, and a hydrolysis probe spanning the insertion site. IS-PCR was performed for three Shigella PCR-positive culture-negative rat intestine specimens from this community. Results: Both insertion sites were detected in the de novo genome assemblies of all clinical S. flexneri isolates (n=50). Two of the three PCR-positive culture-negative rat samples were positive for both unique IS identified in the human S. flexneri isolates, suggesting that the rat Shigella spp. strains were closely related to the human strains in the cluster. The cycle threshold (Ct) values were >35, indicating that the bacterial load was very low in the rat samples. Conclusions: Two unique IS were identified in clinical isolates from a community S. flexneri cluster. Both IS targets were identified in PCR-positive (Shigella spp.), culture-negative rat tissue and clinical isolates from humans, indicating relatedness. ### Competing Interest Statement SDC is a shareholder in BugSeq Bioinformatics Inc. The other authors report no relevant conflicts of interest.