Screening of all clinical isolates was done according to CLSI method.16 click here The detection of carbapenemase production was performed

by phenotypic test using imipenem-EDTA disc method as described earlier.17 The test organism was inoculated onto Mueller–Hinton agar (MHA, Himedia, Mumbai, India) and an increase of 7 mm or more in zone diameter in the presence of EDTA compared to imipenem tested alone was considered to be a positive test for the presence of a carbapenemase. All of the isolates phenotypically positive for carbapenemase were checked for carbapenemase genotypically by PCR. PCR analysis for metallo β-lactamase genes was carried out using the previously reported methods.18 and 19 The sequence of oligonucleotide primers has been shown in Table 1. All of the primers were procured from Sigma Aldrich Chemicals Private Limited, Bangalore, India. For PCR amplifications, about 200 pg of DNA was added to 20 μl mixture containing 0.5 mM of dNTPs, 1.25 μM of each primer and 3.0 U of Taq polymerase (Bangalore Genei) in 1X

PCR buffer. Amplification was performed in an Eppendorf thermal cycler (Germany). The amplified products were separated in 1.5% agarose gel containing 4 μl of 10 mg/ml of ethidium bromide. The gel was run at 70 V for 1 h. The gel images were taken under ultraviolet light using gel documentation system (Bio-Rad, USA). A 100 bp 17-AAG ladder molecular weight marker (Bangalore Genie) was used to measure the molecular weights of amplified products. DNA isolation from the clinical isolates was conducted using the alkaline lysis method.20 The antimicrobial susceptibility testing of the drugs were determined by the disc diffusion method according to the Clinical Laboratory Standards all Institute method (CLSI).16 Quality controls (QC) were performed on each day of testing using Pseudomonas aeruginosa ATCC 27853, Stenotrophomonas maltophilia ATCC 13636 as the reference strain throughout study. All of the clinical isolates obtained from various clinical specimens

were identified as A. baumannii based on their morphological and biochemical characterization. Out of the 454 clinical isolates of A. baumannii, 371 (81.71%) were found to be carbapenemase producing. The maximum carbapenemase producers were found in urine specimen 87.27% (144/165) followed by blood 84.55% (115/136), respiratory secretion 80% (12/15), pus 73.40% (69/94), and fluid 70.45% (31/44). Genotypic screening of carbapenemase producing isolates revealed that 86.5% (321/371) isolates were carbapenemase positive via PCR method (Table 2 and Table 3). Table 4 shows the prevalence of carbapenemase in different clinical specimens of A. baumannii isolates. The highest percentage of carbapenemase producers were confirmed genotypically in isolates obtained from urine 95.1% (137/144) followed by respiratory secretion 91.6% (11/12), blood 82.6% (95/115), pus 79.