Genomic Characterization of the RyC collection: 50 Multidrug Resistant Clinical Isolates of Escherichia coli and Klebsiella spp.

Antimicrobial resistance (AMR) is a major global public health threat, and Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) represent some of the most common and concerning pathogens in clinical settings. Importantly, the dissemination of these resistance mechanisms is largely driven by mobile genetic elements (MGEs), particularly plasmids. Advancing our understanding of AMR evolution through experimentation requires moving beyond domesticated laboratory strains and towards clinically relevant isolates. However, despite the abundance of genomic data in public repositories, there is a lack of well-characterised clinical collections available for experimental work. Here, we characterise the RyC collection, which includes 50 multidrug-resistant, ESBL-producing Escherichia coli and Klebsiella spp. strains isolated from the gut microbiota of hospitalised patients at Hospital Universitario Ramon y Cajal (Madrid, Spain). We generated high-quality genome assemblies for all strains using a combination of short- and long-read sequencing technologies. From these data, we performed a comprehensive characterisation of the pangenome, mobilome, resistome and defensome of the collection. We present the RyC collection as a robust and experimentally tractable resource to study AMR evolution and MGEs dynamics in clinically relevant bacterial backgrounds. Impact statementAntimicrobial resistance (AMR) is a growing global health threat driven by the rapid dissemination of resistance genes among clinically relevant bacteria. A major challenge in studying AMR evolution is the reliance on domesticated laboratory strains, which poorly represent the complexity of pathogens circulating in hospitals. Here, we introduce the RyC collection, a set of well-characterised, multidrug-resistant Enterobacterales isolates obtained from hospitalised patients. By combining high-quality genome sequencing with detailed analyses of their gene content and mobile genetic elements (MGEs), this collection provides a realistic and experimentally tractable system to study how resistance evolves and spreads. The RyC collection will facilitate research on AMR dynamics, plasmid biology and host-MGEs interactions, ultimately contributing to the development of more effective strategies to combat antibiotic-resistant infections.