Bacterial antimicrobial resistance (AMR) is one of the major global health challenges. The 2024 WHO Bacterial Priority Pathogens List highlights Enterobacterales as top-priority pathogens in which horizontal transfer of AMR genes via conjugative plasmids drives resistance spread. While plasmid research is increasingly shifting from well-characterized model plasmids toward plasmids isolated from clinical settings, access to clinically relevant plasmids remains limited. Moreover, studying plasmids in their native context is challenging: the presence of multiple plasmids within one host and heterogeneous genetic backgrounds complicates the attribution of phenotypes to individual plasmids and their comparison across bacterial hosts.
To address these limitations, we established the Enterobacteriaceae Clinical Plasmid Reference Collection (ECPRef), a publicly available set of 60 conjugative AMR plasmids isolated from hospital patients. All plasmids are maintained in an isogenic host (E. coli MG1655), enabling direct comparison of plasmid-associated traits across the collection, and the inclusion of well-characterized reference plasmids further enables comparison with plasmids beyond ECPRef. Bioinformatic analyses show that ECPRef plasmids are diverse: they range from 38-240 kb in size, span 16 Inc groups and many more subtypes, and form mating pairs of type I, F, or T. Some plasmids encode exclusively beta-lactamases or carbapenemases, while others are multidrug-resistance plasmids additionally conferring pan-aminoglycoside or fluoroquinolone resistance. While only eight plasmids carry virulence factors, most encode one or more bacterial immunity and anti-immunity systems. Phenotypic characterization of the collection is ongoing. Preliminary results demonstrate striking variation spanning of several orders of magnitude in conjugation rates, as well as differences in fitness costs and in segregation loss rates. This work represents the initial release of ECPRef, a standar
