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<p>The spread of carbapenem-resistant Enterobacteriaceae (CRE) mediated by New Delhi metallo-β-lactamase (NDM) poses a serious challenge to clinicians and has become a major public health concern. NDM has been evolving into variants that possess different hydrolysis activity toward antibiotics, so as to affect treatment strategy. In addition, very few studies on NDM variants have focused on animal-derived bacterial isolates. Our study reports a novel NDM variant, NDM-20, in an isolate of Escherichia coli CCD1 recovered from the food animal swine in China. The isolate that was assigned to ST1114, exhibited high level resistance to all β-lactams tested, including aztreonam and carbapenems. The gene of bla_NDM-20 was located on an IncX3-type plasmid, surrounded by multiple insertion sequences. Sequencing analysis demonstrated that bla_NDM-20 contained three point mutations at positions 262 (G→T), 460 (A→C), and 809 (G→A), compared with bla_NDM-1, and just one point mutation at position 809 (G→A), relative to bla_NDM-5. Functional analysis revealed that the bla_NDM-20 transformant, DH5α+pHSG398/NDM-20, exhibited a higher resistance to ertapenem than that of bla_NDM-1 transformant DH5α+pHSG398/NDM-1. Kinetic parameter analysis showed that NDM-20 had increased enzymatic activity against some penicillins and cephalosporins but decreased carbapenemase activity relative to NDM-5. The identification of NDM-20 further confirms the evolution and prevalence of NDM variants in bacteria of food-animal origin.