Abstract：Yak pathogenic Escherichia coli (YPEC) is the primary causative agent of diarrhea, enterotoxemia, and mastitis in yaks (Bos grunniens) on the Qinghai-Tibet Plateau. To identify the pathogens causing diarrhea in yaks at a farm in Qinghai Province, and to analyze the phylogenetic grouping, antimicrobial resistance status and virulence characteristics of YPEC. Fecal samples from diarrheic yak calves were collected under aseptic conditions in this study. Pathogens were identified through isolation and culture, morphological observation, biochemical identification, and PCR detection of the E. coli specific phoA gene and 16S rRNA. Subsequently, phylogenetic tree construction, evolutionary grouping, K-B method antimicrobial susceptibility testing, PCR identification of resistance and virulence genes, and mouse (Mus musculus) pathogenicity tests were employed to analyze their biological characteristics. The results showed that 4 E. coli strains were isolated, identified, and named Qinghai-1 (QH-1), Qinghai-2 (QH-2), Qinghai-3 (QH-3) and Qinghai-4 (QH-4). Phylogenetic tree results showed QH-1, QH-2, QH-3, and QH-4 clustered into a small group, suggesting they likely evolved from a common ancestor with close phylogenetic relationships. Among them, QH-2 and QH-4 belonged to the same branch and were most closely related. Phylogenetic grouping results indicated that QH-1 and QH-4 belonged to group D, while QH-2 and QH-3 were classified in group B2. 4 E. coli strains exhibited multidrug resistance (resistance to ≥11 drugs), with 100% resistance to sulfonamides and glycopeptides. Among the drug resistance genes, 4 E. coli strains showed detection of resistance genes in 7 major classes of antimicrobial agents, excluding amides. Notably, the positive rates of the resistance genes blaCIT, blaTEM, and VanA were 100%. Among the virulence genes, 4 E. coli strains carried 7 virulence genes including F17, csgA and irp2, whereas 11 virulence genes such as K88, ST1, and LT1 were all negative. The challenge test results showed that mice began to exhibit clinical symptoms 12 h post infection, and all animals in the experimental group died within 96 h, indicating that the 4 E. coli strains were all YPEC. Histopathological examination revealed varying degrees of pathological changes in the spleen and lung tissues of mice. Taken together, this study confirmed that the pathogen responsible for diarrhea in yaks at a yak farm in Qinghai Province was YPEC. The 4 isolated YPEC strains carried multiple virulence and antimicrobial resistance genes, exhibited multidrug resistant phenotypes and pathogenicity, and phylogenetic homology analysis suggested a potential transmission risk of YPEC within yak farms in Qinghai Province. This study provides a scientific basis for the precise use and effective control of YPEC antibiotics in pastoral areas of the Tibetan Plateau.
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