Abstract:Antibiotic resistance in gram negative bacteria is increasing worldwide in both outpatients as well as hospitalized patients. Most bacterial isolates carry resistance determinants for extended spectrum beta lactamases (ESBL) production on plasmids that can easily spread from organism to organism. This study was conducted to compare the rate of detection of ESBL positive organisms by different methods, to use the clinical and laboratory standard institute (CLSI) detection methods for detecting ESBLs in bacteria other than Enterobacteriaceae and genotypic characterization of these ESBL strains. A total of 100 non repetitive gram negative isolates, which were resistant to ceftriaxone, cefotaxime or ceftazidime were only included in the study. All the 100 screen test positive isolates were tested for ESBL production using the different phenotypic detection methods. Molecular characterization of the strains was done at the Rajiv Gandhi Centre for Biotechnology, Trivandrum. Among total 100 gram negative isolates which were 3 rd generation cephalosporin resistant, 79(79%) were ESBL positive by using CLSI phenotypic confirmatory test. Of this 79%, majority of isolates were detected in blood samples (38%). Of the different methods employed, E-test detected additional 3% and 11% ESBL positive Escherichia coli and Klebsiella pneumoniae respectively. CLSI phenotypic confirmatory test also detected ESBL positive Pseudomonas sp, Acinetobacter sp and Enterobacter cloacae. The genotype characterization of 52 isolates showed 29 with CTX-M and 13 with TEM genes. Thus, the study shows a significant rate of ESBL production in gram negative bacteria emphasizing the need for enhanced infection control and antibiotic stewardship programs to limit the spread of these organisms.