Clonal and plasmid-mediated dissemination of CTX-M-14–producing Escherichia coli within a single cattle farm in Japan

Ryuichi Nakano^1*Yuki Suzuki¹Akiyo Nakano¹Koichi Yamaguchi¹Saori Horiuchi¹Yasuo Ono²Hisakazu Yano¹

• ¹Nara Medical University, Kashihara, Japan
• ²Teikyo Heisei Daigaku Kenko Medical Gakubu, Toshima, Japan

Introduction: Extended-spectrum β-lactamase (ESBL)–producing Escherichia coli threatens humans and animals. blaCTX-M-14, which is prevalent in Asia, is mainly disseminated via incompatibility group I1 (IncI1) plasmids, as they can efficiently transfer across Enterobacterales. However, direct evidence linking livestock and farmers at the genomic level is limited, and our study addresses this concern. Methods: Fecal samples were collected in 2013 from one parent cattle, three calves, and a farmer on a cattle farm in southern Kyushu, Japan. ESBL-producing E. coli were identified and characterized using hybrid Illumina–Nanopore assemblies, resistance gene profiling, and plasmid replicon typing. Clonal relatedness was assessed using core-genome multilocus sequence typing and core single-nucleotide polymorphism (SNP) analysis. Conjugation assays were used to evaluate plasmid transferability. Results: Seven blaCTX-M-14-positive E. coli isolates representing five sequence types (STs) were recovered. Two ST533 isolates from parent cattle and calf 1 were identical by core-genome ST and differed by only one core SNP, indicating recent clonal transmission. Additionally, ST1148, ST1261, and ST1431 were isolated from other calves, while ST448 isolates from the farmer and calf 3 exhibited a large genetic distance (3,891 core SNPs) and distinct cgSTs. All blaCTX-M-14 genes were located on conserved IncI1 plasmids of approximately 114 kb, showing >99.9% sequence identity, including the IncI1 plasmid. Conjugation frequencies ranged from 10-2 to 10-3. Other resistance genes, including tet(A) and mcr-3.1, were encoded on separate plasmids. Conclusions: These findings underscore the possible role of IncI1 plasmids in bridging resistance gene flow across host boundaries and emphasize the benefit of integrated One Health genomic surveillance to monitor and mitigate antimicrobial resistance transmission.