Rapid detection of IMP, NDM, VIM, KPC and OXA-48-like carbapenemases from Enterobacteriales and Gram-negative non-fermenter bacteria by real-time PCR and melt-curve analysis

Abstract

Carbapenemase producing microorganisms are increasingly isolated and often associated with treatment failures and outbreaks. The need for reliable and timely detection and/or confirmation of carbapenemase production is paramount, therefore a real-time PCR assay targeting IMP, NDM, VIM, KPC and OXA-48-like carbapenemases was designed and validated. All available allele variants of the above carbepenmases were downloaded from the Beta-Lactamase DataBase (http://bldb.eu/), aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized for the QuantStudio 6-Flex (Applied Biosystems) using the PowerUp SYBR Green Master Mix (Life Technologies) and validated using a panel of 204 characterised strains carrying a wide range of beta-lactamases, sometimes in combination. Melt-curve analysis was used to confirm positive results. The in silico approach allowed primers to be designed in conserved regions of the KPC and NDM alignments, while three primer sets for IMP and two for VIM were necessary to ensure amplification of the different variants. One primer set was designed for OXA-48-like, however it is unlikely to detect all variants. Expected results were obtained for all 204 tested strains, with 100% sensitivity and specificity. Melt-curve analysis showed consistent Tm results for KPC, NDM and OXA-48-like; differences were instead noted for IMP and VIM as likely consequence of higher variability in the PCR target regions. Inhibition was not observed. The assay is rapid, easy to perform and implement. It enables unequivocal detection of IMP, NDM, VIM, KPC and OXA-48-like carbapenemases even when more than one type is present simultaneously.

Evaluation of High-Resolution Melting Curve Analysis (HRM) assay for Detection of Pseudomonas aeruginosa PASGNDM699: A dangerous New Delhi metallo-β-lactamase (NDM) strain

“…This problem gets extremely worse in the MCA that sometimes we lost the long amplicon even at the primer ratio of 1:1. Furthermore, this results agree with Mentasti et al [2]. They proved that the MCA method for the detection of NDM strains has high e ciency and precision, which is best achieved by adjusting the primer dilutions.…”

“…Author details 1 Microbiology department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. 2 Microbiology department, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.…”

“…β-lactam antibiotics make up more than 50% of all commercially prescribed antibiotics for treatment of bacterial infections. The general mechanism of action of β-lactam antibiotics is inhibition of peptidoglycan synthesis which constitutes a major portion of bacterial cell wall synthesis [2]. The attachment of β-lactam antibiotics to penicillin-binding protein leads to the inhibition of transpeptidase that eventually leads to the death of bacteria.…”

“…These strains hydrolyze the β-lactam ring of drug compound, thereby inactivating them. In contrast to serine β-lactamases, MBLs use at least one but more commonly two Zn2+ ions in their active site to catalyze the hydrolysis of β-lactam rings [2,3]. There are various methods for identifying MBL and NDM strains, which fall into two phenotypic and genotypic groups.…”

Treatment Options for <em>K. pneumoniae</em>, <em>P. aeruginosa</em> and <em>A. baumannii</em> Co-resistant to Carbapenems, Aminoglycosides, Colistin and Tigecycline. An Approach Based on the Mechanisms of Resistance to Carbapenems

“…Rapid diagnostic methods, such as nucleic acidbased tests that detect carbapenemase genes, phenotypic assays that detect hydrolysis of carbapenems including MALDI-TOF mass spectrometry, and immunochromatographic assays, allow faster detection of carbapenemase production and many methods can even determine the most prevalent types of carbapenemases (e.g. KPC, VIM, NDM, OXA-48-like) [13,14,[18][19][20][21]. Many of these methods can be implemented directly on spiked blood cultures [21,22], allowing even earlier identification of the mechanisms of resistance and targeted treatment in a timely manner.…”