title: Dual-tubed multiplex-PCR for molecular characterization of carbapenemases isolated among Acinetobacter spp. and Pseudomonas spp.
abstract: OBJECTIVE
  To molecularly characterize clinical isolates of Acinetobacter spp. and Pseudomonas spp. from various clinical samples so as to identify the carbapenemases mechanisms harboured by them.
  
  
  RESULTS
  A 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-β-lactamases (MBL) and phenyl boronic acid (PBA) for the detection of Klebsiella pneumoniae carbapenemase (KPC), were performed to distinguish between different classes of β-lactamases. Two-tubed multiplex-PCR was performed for genotypic characterization of different classes of carbapenemases ((blaNDM-1 , blaOXA-48 like , blaKPC , blaVIM , blaIMP ), (blaOXA-23 like , blaOXA-24 like , blaOXA-51 like , blaOXA-58 like )). Eighty-five per cent (81/95) isolates were carbapenemase producers. Among these, 56.7% (44) were multiple carbapenemase producers. Furthermore, 48.14% (39) were MBLs, 35.8% (29) were carbapenem hydrolyzing class D β-lactamases (CHDLs), 16% (13) had MBLs as well as CHDLs and 14.7% (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.
  
  
  CONCLUSIONS
  This 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.
  
  
  CONCLUSIONS
  Dual-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.
authors: Department of Microbiology, P. D. Hinduja National Hospital and Medical Research Centre, Mahim, Mumbai, India.
authors: M
authors: M
authors: Kazi
authors: C
authors: C
authors: Nikam
authors: A
authors: A
authors: Shetty
authors: C
authors: C
authors: Rodrigues
mesh_terms: D000150:Acinetobacter / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000151:Acinetobacter Infections / Q000382:microbiology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology; D006801:Humans; D007194:India; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D011552:Pseudomonas Infections / Q000382:microbiology; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D001618:beta-Lactamases; C063912:carbapenemase
keywords: New-Delhi Metallo-B-lactamases; carbapenem hydrolyzing class D β-lactamases; metallo-β-lactamases; multiple carbapenemases; multiplex-PCR; non fermenters Gram-negative bacilli