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Yolk Peritonitis can lead to a rapid decline in egg production, which seriously affects the health of laying hens and the profitability of chicken farms. Escherichia coli (E. coli) is the most common cause of yolk peritonitis in laying hens. In this study, bacterial samples were collected from the ovaries and fallopian tubes of laying hens with suspected yolk peritonitis from a laying farm in Jiangsu Province, and their pathogenicity and drug resistance were investigated. Initially, morphological and biochemical detection methods were employed to isolate and identify the pathogenic bacteria. The results showed that a total of 16 strains of E. coli were isolated from laying hens with yolk peritonitis. Subsequently, the drug resistance and pathogenicity of a randomly selected E. coli strain were analyzed and predicted by genome sequencing technology, and the drug resistance of E. coli was verified by drug sensitivity test and PCR. Finally, the virulence was verified by infection experiment in mice. The study revealed that the egg-yolk peritonitis in laying hens was caused by E. coli infection, and the genome sequencing analysis revealed that the bacteria had multidrug resistance and high virulence. The drug susceptibility testing indicates that E. coli exhibited resistance to aminoglycosides, β-lactam, macrolides, fluoroquinolones, and sulfonamides. In this study, resistance genes including KdpE, aadA5, APH(3 ")-ID, APH(6)-ID, and TEM-1 were identified, and their expression levels varied across different stages of bacterial growth. The results of virulence analysis indicated a mortality rate of 50% in mice infected with E. coli at a concentration of 2.985 × 107 CFU/mL. E. coli infection resulted in damage to various tissues and organs in mice, with the intestinal tissue structure being the most severely affected. This study provides a reference for the study of drug resistance mechanisms in E. coli and provides valuable insights into the selection of drugs for the treatment of vitelline peritonitis. [ABSTRACT FROM AUTHOR]