AimTo molecularly characterize clinical isolates of Acinetobacter spp. and Pseudomonas spp. from various clinical samples so as to identify the carbapenemases mechanisms harboured by them. Materials and ResultsA total of 95 carbapenem-resistant, nonduplicate, multi-drug resistant Gram-negative clinical isolates (53 Acinetobacter spp. and 42 Pseudomonas spp.), were collected between July and December 2012. Modified Hodge test (MHT) for the detection of carbapenemases was performed. Inhibitor-based test, EDTA for the detection of metallo--lactamases (MBL) and phenyl boronic acid (PBA) for the detection of Klebsiella pneumoniae carbapenemase (KPC), were performed to distinguish between different classes of -lactamases. Two-tubed multiplex-PCR was performed for genotypic characterization of different classes of carbapenemases ((bla(NDM-1), bla(OXA-48 like), bla(KPC), bla(VIM), bla(IMP)), (bla(OXA-23 like), bla(OXA-24 like), bla(OXA-51 like), bla(OXA-58 like))). Eighty-five per cent (81/95) isolates were carbapenemase producers. Among these, 567% (44) were multiple carbapenemase producers. Furthermore, 4814% (39) were MBLs, 358% (29) were carbapenem hydrolyzing class D -lactamases (CHDLs), 16% (13) had MBLs as well as CHDLs and 147% (14/95) had none of the targeted resistance mechanisms. The overall rate of concordance between phenotypic and genotypic test was 97% and 98% for the detection of carbapenemases and MBL, respectively. ConclusionThis is the first study from Western India which highlights the presence of multiple carbapenemases in nonfermenters Gram-negative bacilli (NFGNB). Co-existence of multiple carbapenemases along with other resistance mechanisms might result in treatment failure. Molecular characterization of the resistance mechanisms of suspected pathogens would help provide appropriate antimicrobial treatment for good clinical outcome. Significance and Impact of the StudyDual-tubed multiplex PCR decreases the time of amplification and thus the turnaround time which is crucial in clinical microbiology; this would be helpful in rapid characterization of CHDLs and MBLs.