Characterization of IS26-bracketed blaCTX-M-65 resistance module on IncI1 and IncX1 plasmids in Escherichia coli ST224 isolated from a chicken in China

Antimicrobial resistance (AMR) poses a significant global health threat, particularly due to increasing bacterial resistance to β-lactam and aminoglycoside antibiotics, primarily mediated by extended-spectrum β-lactamases (ESBLs) and 16S rRNA methylases in Enterobacteriaceae. In this study, a multidrug resistant (MDR) E. coli strain HN257 isolated from chicken belonging to ST224 and serotype O88:H23 was characterized. SNP-based phylogenetic analysis revealed two distinct clades among poultry-associated E. coli ST224 in this study and others from Genbank, with strain HN257 closely related to chicken-derived E. coli YH17148 (serotype O78:H23), from China. The E. coli HN257 harbored four plasmids with 16 resistance determinants. Two bla_CTX-M-65 genes were located on different plasmids with an IS26-bracketed resistance module IS26-traI-fip-∆ISEcp1-bla_CTX-M-65-IS903D-iroN-IS26. The plasmid pHN257–2 belonged to the IncI1 ST71 epidemic lineage and carried bla_CTX-M-65, bla_TEM-1b, rmtB, fosA3, floR, aac(3)-IV and oqxAB, while plasmid pHN257–4 belonged to the non-conjugative IncX1 and carried bla_CTX-M-65 and fosA3. Under experimental conditions, a rmtB-positive conjugative helper IncI1 ST136 plasmid could fuse with the non-conjugative pHN257–4 carrying bla_CTX-M-65, resulting in the formation of a cointegrate pHN257–F mediated by IS26. Importantly, both single and fused plasmids in transconjugants showed minimal impact on bacterial growth. This study highlights the first identification of a non-conjugative IncX1 plasmid carrying bla_CTX-M-65 and fosA3 in MDR E. coli ST224 from poultry, offering critical insights into the presence and transmission dynamics of bla_CTX-M-65.