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The study aims at characterizing the circulating clinical isolates of Escherichia coli and Klebsiella pneumoniae in Assam which includes antibiotic susceptibility testing, phenotypic detection of beta-lactamases and the PCR detection of the responsible genes. Further, design a multiplex PCR assay that could detect the common antibiotic-resistant genes in the clinical samples and study the phylogenetic evolution of a few representative isolates through Multi-locus Sequence Typing. This study provides genetic epidemiological data on antibiotic resistant E. coli and K. pneumoniae in the clinical setting of Guwahati, Assam. This allowed for the investigation of antibiotic resistance in the two most commonly circulating bacterial pathogen in this state. This understanding extends the overlying emphasis on the rational use of antibiotics in clinical medicine and innovative means to find newer drug candidates.