Shaqiu Zhang ^1,2,3 Yanxi Shu ¹ Zhechen Yang ¹ Zhijun Zhong ³ Mingshu Wang ^1,2,3 Renyong Jia ^1,2,3 Shun Chen ^1,2,3 Mafeng Liu ^1,2,3 Dekang Zhu ^1,2,3 Xinxin Zhao ^1,2,3 ying wu ^1,2,3 Qiao Yang ^1,2,3 Juan Huang ^1,2,3 Xumin Ou ^1,2,3 di sun ^1,2,3 Bin Tian ^1,2,3 zhen wu ^1,2,3 yu he ^1,2,3 Anchun Cheng ^1,2,3*

• ¹ Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan Province, China
• ² Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University,, Chengdu, Sichuan Province, China
• ³ Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan Province, China

Escherichia coli (E. coli) serves as a critical indicator microorganism for assessing the prevalence and dissemination of antibiotic resistance, notably harboring various antibiotic resistant genes (ARGs). Among these, the emergence of the blaNDM gene represents a significant threat to public health, especially since carbapenem antibiotics are vital for treating severe infections caused by Gram-negative bacteria. This study aimed to characterize the antibiotic resistance features of blaNDM-5-positive E. coli strains isolated from waterfowl in several regions of China and to elucidate the dissemination patterns of the blaNDM-5 gene. We successfully isolated 103 blaNDM-5-positive E. coli strains from 431 intestinal fecal samples obtained from waterfowl across five provincial-level units in China, with all strains exhibiting multidrug resistance (MDR). Notably, the blaNDM-5 gene was identified on plasmids, which facilitate efficient and stable horizontal gene transfer (HGT). Our adaptability assays indicated that while the blaNDM-5-positive plasmid imposed a fitness cost on the host bacteria, the NDM-5 protein was successfully induced and purified, exhibiting significant enzymatic activity. One strain, designated DY51, exhibited a minimum inhibitory concentration (MIC) for imipenem of 4 mg/L, which escalated to 512 mg/L following exposure to increasing imipenem doses. This altered strain demonstrated stable resistance to imipenem alongside improved adaptability, correlating with elevated relative expression levels of the blaNDM-5 and overexpression of efflux pumps. Collectively, this study highlights the horizontal dissemination of the blaNDM-5 plasmid among E. coli strains, confirms the associated fitness costs, and provides insights into the mechanisms underlying the stable increase in antibiotic resistance to imipenem. These findings offer a theoretical framework for understanding the dissemination dynamics of blaNDM-5 in E. coli, which is essential for developing effective strategies to combat carbapenem antibiotic resistance.