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Neonatal calf diarrhoea (NCD) is a major threat to dairy industries. It is a multi-etiology disease with E. coli as the predominant cause. Emergence of antimicrobial resistance is of utmost concern because it has rendered the present day antibiotics less effective. The present study aims for isolation, molecular characterization and antimicrobial resistant profiling of E. coli from neonatal calves. One hundred two fecal samples were collected from neonatal calves for isolation of E. coli. All the isolates showed positive result on biochemical analysis. But only 78.43% (80/102) of them were found positive for uspA (universal stress protein) gene. The nucleotide sequences flanking the gene encoding the uspA and was found to be highly specific for E. coli. The antibiogram of E. coli isolates by Kirby Bauer disc diffusion method revealed highest resistance against kanamycin (97%) and lowest against gentamicin (5.86%). Eighty three percent (85/102) isolates were MDR. 39 (38.24%) isolates were phenotypic ESBL producers (by combination disc diffusion method) and 22 (21.57%) isolates were phenotypic carbapenem resistant (against Ertapenem, Meropenem, Imipenem, Doripenam) Hence, surveillance based study and search for an alternative to antibiotics is needed to address the global issue of the antimicrobial resistance. | Isolation, identification, molecular characterization and antibiogram of E. coli isolates from neonatal calves