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Background Emergence of multidrug resistance in Enterobacteriaceae limits the selection of antimicrobials for treatment of infectious diseases. Identification of NDM-1 makes more difficulty in treating multidrug-resistant Enterobacteriaceae infections. Carbapenem-resistant Escherichia coli clinical isolates from a tertiary hospital in Wenzhou, east China, were investigated for NDM-1 production. Results The two tested isolates were negative for modified Hodge test, but positive for a double-disc synergy test used for detecting metallo-β-lactamase production. E. coli WZ33 and WZ51 exhibited discrepant-level resistance to most clinically frequent used antimicrobials, but still susceptible to trimethoprim/sulfamethoxazole, amikacin, fosfomycin, tigecycline and polymyxin B. E. coli WZ33 and WZ51 were positive for blaNDM-1 determined by PCR and DNA sequencing. Other than blaNDM-1, E. coli WZ33 also harbored blaCTX-M-14 and blaCMY-42, while E. coli WZ51 simultaneously harbored blaSHV-12, blaCTX-M-14 and blaCMY-42. Carbapenem resistance for E. coli WZ51 and WZ33 could not be transferred to E. coli recipients through conjugation, but could be transferred to E. coli recipients by chemical transformation. The EcoR1-digested DNA pattern of plasmids from the transformant of E. coli WZ51 was different from that of E. coli WZ51. MLST showed that E. coli WZ33 and WZ51 belonged to an animal-associated clone (ST167). Conclusion The present study is the first report of blaNDM-1 carriage in E. coli ST167 isolates and coexistence of blaNDM-1 and blaCMY-42 in same isolate. Systemic surveillance should focus on the dissemination of blaNDM-1 among Enterobacteriaceae, especially E. coli ST167 clone associated with animal infection. [ABSTRACT FROM AUTHOR]