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 carbapenemases 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.
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Acknowledgements
The authors would like to thank Daniele Meunier (Public Health England) for providing part of the strain collection that was used in this study and Michael Perry (Public Health Wales) for constructive comments on the manuscript. Results of this study were presented at the 29th ECCMID, Amsterdam, The Netherlands 13-16 April 2019 (Poster n. 1234).
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Mentasti, M., Prime, K., Sands, K. et al. 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. Eur J Clin Microbiol Infect Dis 38, 2029–2036 (2019). https://doi.org/10.1007/s10096-019-03637-5
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DOI: https://doi.org/10.1007/s10096-019-03637-5