Temporal GWAS identifies a widely distributed putative adhesin contributing to pathogen success in Shigella spp

Bacteria of the genus Shigella are a major contributor to the global diarrhoeal disease burden causing >200,000 deaths per annum globally where S. flexneri is the major pathogenic species. Increasing antimicrobial resistance (AMR) in Shigella and the lack of a licenced vaccine has led WHO to recognise Shigella as a priority organism for the development of new antimicrobials. Understanding what drives the long-term persistence and success of this pathogen is critical for ongoing shigellosis management and is relevant for other enteric bacteria. To identify key genetic drivers of Shigella evolution over the past 100 years, we analysed S. flexneri from the historical Murray collection (n=45, isolated between 1917-1954) alongside a comparatively modern collection (n=262, isolated between 1950-2011) using a novel approach called temporal genome-wide association study (tGWAS). We identified SNPs (n=94), COGs (n=359) and significant kmers within 48 genes significantly positively associated with time. These included T3SS encoding genes, proteins involved in intracellular competition, acquired antimicrobial resistance genes, insertion sequences, and genes of unknown function (28%, 49/172 of those hits investigated). Among the unknown proteins we identified a novel plasmid borne putative adhesin, named Stv. Genomic epidemiological analyses reveal that Stv was associated with clonal expansions of multiple phylogroups of S. flexneri and its acquisition predates multidrug resistance acquisition and the global dissemination of Lineage III S. sonnei . Stv, and close relatives, are widely distributed in other Enterobactericeae and bacteria, indicating that its importance likely extends beyond shigellae. This work highlights the effectiveness of using tGWAS on historical isolate collections for identifying novel contributors to pathogen success over time. This approach is readily translatable to other pathogens and our application in Shigella identified Stv, a putative adhesin and potential drug target that is widely distributed across the AMR priority group Enterobacteriaceae. Author Summary Shigellosis is a leading cause of diarrhoeal disease worldwide and is represented among the multiple Enterobacteriaceae which WHO have declared as priority pathogens for which new antimicrobials are urgently needed. The majority of shigellosis is caused by the species Shigella flexneri and Shigella sonnei . In this study we collated S. flexneri isolates that spanned a 94-year period, encompassing the pre- and post-antibiotic era to implement a novel bioinformatic technique, temporal GWAS (tGWAS), to identify key factors of pathogen success during this time period. Alongside recovering AMR and virulence genes, we also identified a novel, mobilisable putative adhesin, named Stv herein, which appeared to contribute to clonal expansions across multiple Shigella species and is present across the broader Enterobacteriaceae. Our results indicate the potential importance of Stv in controlling Shigella and other infections, and the validity of a tGWAS approach for identifying biological drivers underpinning the evolution and expansion of AMR pathogens over time. ### Competing Interest Statement The authors have declared no competing interest.