Extraintestinal pathogenic Escherichia coli (ExPEC) is a group of Escherichia coli strains that can cause severe infectious diseases outside the gastrointestinal tract, such as urinary tract infections, meningitis, septicemia, etc. We report a case of a calf herd infection by ExPEC with high rates of morbidity and mortality. The research purpose of this study was to thoroughly investigate the characteristics of the ExPEC responsible for the calf herd infection. Specifically, we aimed to understand the mechanisms underlying its multidrug resistance and high pathogenicity. Clinical samples were collected for the isolation and identification of ExPECs, cultured on MacConkey agar, and further tested by PCR for the uidA gene, 16S rRNA gene sequencing, and adhesion patterns on HEp-2 cells. The antimicrobial activity was determined using the disk diffusion method according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The pathogenicity was assessed through the experimental infection of Kunming mice, tracking their survival and weight changes, and performing autopsies for bacterial counts and histopathological analysis. Additionally, whole-genome sequencing (WGS) and a comprehensive analysis were performed, including multilocus sequence typing (MLST), serotyping, drug-resistance gene analysis, virulence factor analysis, metabolic pathway analysis, and enrichment analysis, using various online tools and databases. An ExPEC strain named RZ-13 was responsible for this case and was identified as ST345 and O134: H21. Among the 14 antibiotics tested, 13 showed resistance, indicating that the RZ-13 strain is a multidrug-resistant (MDR) bacterium. The experimental infection of Kunming mice proved the greater pathogenicity of RZ-13 than that of CICC 24186. The comprehensive WGS revealed the presence of 28 antibiotic resistance genes and 86 virulence-related genes in the genome of the strain, corroborating its clinical manifestations of MDR and high pathogenicity. Our study isolated a MDR ExPEC strain, RZ-13, with a strong pathogenicity. This is the first case report of ExPEC leading to severe mortality in calf herds in China, underscoring the need for the rational use of antibiotics to reduce the risk of the generation and transmission of MDR bacteria from food-producing animals to ensure food safety and public health.

Extraintestinal pathogenic Escherichia coli (ExPEC) poses escalating threats to human and veterinary health amid rising antimicrobial resistance. We isolated a highly virulent ExPEC strain RZ-13 (ST345, O134:H21) from diarrheic calves at a large beef cattle farm in Rizhao City, and conducted whole genome sequencing, conjugation experiments, and antimicrobial susceptibility testing to elucidate its genomic architecture and resistance mechanisms. The RZ-13 genome comprises one chromosome and four plasmids. The chromosome harbors virulence factors for adhesion, invasion, biofilm formation, and iron acquisition. Notably, plasmids pRZ13-1 (265,777 bp, IncHI2-IncHI2A) and pRZ13-3 (74,304 bp, IncFII) carry the majority of resistance genes. Plasmid pRZ13-1 carries 25 resistance genes, including bla_CTX-M-55, floR, qnrS1, sul3, and tet(A), as well as a complete tellurite resistance gene cluster, terABCDEFZY1. Its multidrug resistance (MDR) region features an IS26-mediated tandem amplification and an approximately 29 kb inverted structure. Comparative analysis indicated that the MDR region carried by this plasmid is highly prevalent in both animal-derived and human-derived isolates. Plasmid pRZ13-3 harbors an IS91-mediated mobile region that integrates both antimicrobial resistance and stress adaptation genes, which have been repeatedly identified in plasmids from diverse sources, including animals and humans. Conjugation experiments confirmed both pRZ13-1 and pRZ13-3 plasmids are self-transmissible and confer multidrug-resistant phenotypes to recipient strains, with pRZ13-3 exhibiting an exceptionally high transfer frequency of 8.9 × 10⁻², substantially exceeding that of previously reported IncFII plasmids. These findings demonstrate that pRZ13-1 and pRZ13-3 serve as critical vehicles for resistance dissemination through complex mobile genetic element structures and efficient horizontal transfer, highlighting the urgent need for surveillance of livestock-reservoir ExPEC to mitigate public health risks.