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Background: Among enterobacteria E. coli and Klebsiella spp. are of great concern in health care settings, as these bacteria sometimes may contaminate the milk due to unhygienic practices and poor udder condition which have been associated with various illnesses. Therefore, this study aimed to detect the carbapenem resistant E. coli and Klebsiella spp. of bovine milk origin with regard to the risk of human transfer via the food chain in community. Methods: Total 240 samples were collected from Ayodhya and Sultanpur districts of Eastern Plain Zone of Uttar Pradesh (India). Confirmation of E. coli and Klebsiella spp. isolates was done by using species specific uidA and 16S rRNA gene, respectively. Then, carbapenemase positive E. coli and Klebsiella spp. were confirmend by DDST, MBL E-strip test and PCR analysis by targeting (bla-NDM, bla-OXA-48 and bla-KPC). Antibiogram of all carbapenemase positive isolates was performed against 20 antibiotics of 12 different classes. XResult: In the present study, total 74(30.83%) isolates were identified including 55(22.92%) E. coli and 19(7.92%) Klebsiella spp. by PCR, out of which 12(16.21%) isolates were confirmed as carbapenemase producers comprising 7(12.72%) E. coli and 5(26.31%) Klebsiella spp by DDST and E-strip. All carbapenemase positive E. coli were found 100% sensitive to polymyxin-B and chloramphenicol, while all Klebsiella spp. were 100% sensitive to amikacin and polymyxin-B. Resistance against imipenem, meropenem, cefotaxime, cefpodoxime, ceftazidime, ceftriazone, aztreonam and ampicillin ranged between 80.0%-100%. All carbapenemase positive isolates were found multidrug resistant. Carbapenemase genes bla-NDM and bla-KPC were detected in E. coli while bla-OXA-48 and bla-KPC were detected in Klebsiella spp. [ABSTRACT FROM AUTHOR]