AIM1. To analyse the clinically important Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and their susceptibility pattern in a tertiary care hospital; 2. To study the epidemiology of the isolates and sensitivity pattern of the Gram negative bacteria in a tertiary care hospital, Madurai, South Tamilnadu. METHODSSamples collected from different wards were subjected for culture, isolation and identification of Gram negative bacterial isolates and antibacterial susceptibility testing by Vitek System and Kirby Bauer method. This study was carried out in Apollo Speciality Hospital, Lab Services, Madurai. RESULTS6900 non-repetitive isolates from 29,275 patients were collected from urine, blood, pus and respiratory samples from January to December 2013-2015. The Gram Positive Cocci and Gram Negative bacterial isolates were 2357 and 4543, respectively. Urine (52%), blood (7%), respiratory samples (21%) and pus (20%) accounted for the samples from which these samples were obtained. Most common isolates in urine were E. coli (69%) and Klebsiella species (16%). Most common isolates in blood were E. coli (38%), Klebsiella species (25%) and Salmonella (13%). Most common isolates in respiratory samples were Klebsiella species (44%), Pseudomonas (28%) and E. coli (16%). Most common isolates in pus samples were E. coli (32%), Klebsiella species (23%) and Pseudomonas (27%). E. coli, Klebsiella species, Pseudomonas from urine, blood, respiratory samples, pus showed high sensitivity to Carbapenems, Piperacillin/Tazobactam, Cefoperazone/Sulbactam and Aminoglycosides. E. coli showed high sensitivity to Nitrofurantoin in urine. E. coli, Klebsiella species, Pseudomonas from urine, blood, respiratory samples and pus showed resistance to 3rd generation Cephalosporins, Fluoroquinolones and Trimeth/Sulfonamides. Overall, ESBL strains increased from 64% to 66.3% in 2014, then decreased to 63.7% in 2015, while Carbapenem resistant strains increased from 2.4% to 3.8%. CONCLUSIONIn this 3 years study we found that there was a progressive increase in antimicrobial resistance in isolates of E. coli, K. pneumoniae and P. aeruginosa, isolated from blood, urine, pus and respiratory culture specimens. ESBL production was seen in the majority of isolates of E. coli and K. pneumoniae. These bacteria showed high sensitivity to Carbapenem, Piperacillin, Tazobactam and Cefoperazone Sulbactam.