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We determined the prevalence and molecular characteristics of blaCTX-M-55-positive Escherichia coli (E. coli) isolated from duck–fish polyculture farms in Guangzhou, China. A total of 914 E. coli strains were isolated from 2008 duck and environmental samples (water, soil and plants) collected from four duck fish polyculture farms between 2017 and 2019. Among them, 196 strains were CTX-M-1G-positive strains by PCR, and 177 (90%) blaCTX-M-1G-producing strains were blaCTX-M-55-positive. MIC results showed that the 177 blaCTX-M-55-positive strains were highly resistant to ciprofloxacin, ceftiofur and florfenicol, with antibiotic resistance rates above 95%. Among the 177 strains, 37 strains carrying the F18:A-:B1 plasmid and 10 strains carrying the F33:A-:B- plasmid were selected for further study. Pulse field gel electrophoresis (PFGE) combined with S1-PFGE, Southern hybridization and whole-genome sequencing (WGS) analysis showed that both horizontal transfer and clonal spread contributed to dissemination of the blaCTX-M-55 gene among the E. coli. blaCTX-M-55 was located on different F18:A-:B1 plasmids with sizes between ~76 and ~173 kb. In addition, the presence of blaCTX-M-55 with other resistance genes (e.g., tetA, floR, fosA3, blaTEM, aadA5 CmlA and InuF) on the same F18:A-:B1 plasmid may result in co-selection of resistance determinants and accelerate the dissemination of blaCTX-M-55 in E. coli. In summary, the F18:A-:B1 plasmid may play an important role in the transmission of blaCTX-M-55 in E. coli, and the continuous monitoring of the prevalence and transmission mechanism of blaCTX-M-55 in duck–fish polyculture farms remains important. [ABSTRACT FROM AUTHOR]