Rapid detection of blaVIM-1–37 and blaKPC1/2–12 alleles from clinical samples by multiplex PCR-based assays

Abstract VIM and KPC are two major families of carbapenemases involved in nosocomial outbreaks of multidrug-resistant Gram-negative bacilli. To rapidly detect blaVIM - and blaKPC -encoding strains, three multiplex PCR-based methods were designed and validated: (i) a real-time PCR to detect all repor...

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Bibliographic Details
Published in:International journal of antimicrobial agents 2013-07, Vol.42 (1), p.68-71
Main Authors: Frasson, Ilaria, Biasolo, Maria Angela, Bartolini, Andrea, Cavallaro, Antonietta, Richter, Sara N, Palù, Giorgio
Format: Article
Language:English
Subjects:
Anti-Bacterial Agents - pharmacology
Bacterial Proteins - genetics
beta-Lactamases - genetics
Gram-Negative Bacteria - enzymology
Gram-Negative Bacteria - genetics
Gram-Negative Bacterial Infections - microbiology
Humans
Imipenem - pharmacology
Infectious Disease
KPC carbapenemase
Microbial Sensitivity Tests - methods
Molecular Diagnostic Techniques - methods
Molecular methods
Multiplex Polymerase Chain Reaction - methods
Real-time PCR
Sensitivity and Specificity
Thienamycins - pharmacology
VIM-type carbapenemases
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Summary:Abstract VIM and KPC are two major families of carbapenemases involved in nosocomial outbreaks of multidrug-resistant Gram-negative bacilli. To rapidly detect blaVIM - and blaKPC -encoding strains, three multiplex PCR-based methods were designed and validated: (i) a real-time PCR to detect all reported VIM alleles, namely blaVIM-1–19 ,23–37 ; (ii) a real-time PCR to identify blaVIM -type and blaKPC carbapenemases in an ultrarapid single reaction; and (iii) a standard PCR to amplify and sequence all VIM alleles. All three methods detected 33 VIM-positive samples among 107 Gram-negative isolates with imipenem and meropenem minimum inhibitory concentrations ≥1 mg/L. The three methods displayed 100% sensitivity, specificity and concordance. Sequencing of the blaVIM amplicons revealed that 30 samples encoded blaVIM-1 and 3 samples encoded blaVIM-2 . The real-time assay, optimised for the simultaneous detection of blaVIM and blaKPC , identified 3 and 12 isolates positive for both blaVIM / blaKPC and for blaKPC , respectively. The analytical sensitivity of the real-time assays was linear over 6 log dilutions, with a reproducible detection limit of 1 CFU. No cross-reactivity was detected. The developed assays provide powerful tools for rapid identification of VIM and KPC carbapenemase producers, therefore contributing to the prevention and containment of resistance dissemination.
ISSN:0924-8579
1872-7913
DOI:10.1016/j.ijantimicag.2013.03.006