Initial establishment of a multiplex real-time PCR assay for simultaneous detection of common colistin  and carbapenemase genes

Le Thi Thuy; Do Quang Minh; Le Thi Thu Hang; Dong Van Quyen; Anne-Laure Bañuls; Nguyen Quang Huy

doi:10.15625/2525-2518/19130

Author affiliations

Authors

Le Thi Thuy LMI DRISA, University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam https://orcid.org/0009-0002-6130-8247
Do Quang Minh LMI DRISA, University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam https://orcid.org/0009-0006-9313-0438
Le Thi Thu Hang LMI DRISA, University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam
Dong Van Quyen Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam
Anne-Laure Bañuls UMR MIVEGEC (University of Montpellier- IRD-CNRS), 911 Av Agropolis, 34394 Montpellier, France https://orcid.org/0000-0002-2106-8667
Nguyen Quang Huy LMI DRISA, University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam https://orcid.org/0000-0002-3452-1811

DOI:

https://doi.org/10.15625/2525-2518/19130

Keywords:

colistin resistance, multidrug resistance, carbapenems resistance, blaOXA-48, blaKPC, blaNDM, blaIMP-1, mcr-1, multiplex Real-time PCR

Abstract

Carbapenem and colistin are often used as last-resort treatment for Gram-negative multi-drug resistant (MDR) bacteria. Nevertheless, co-resistance of these drugs is threatening the global healthcare system. Rapid and accurate detection of carbapenem and colistin resistant bacteria is critical for adequate antibiotic therapy and infection control, particularly in the context of an outbreak. The presence of blaNDM, blaKPC, blaIMP-1 and blaOXA-48 is responsible for greater than 95% phenotypic resistance in carbapenem-resistant Enterobacteriaceae, while mcr-1 is the most prevalent and well disseminated of all mcr genes in colistin-resistant strains. In this study, we aim to develop a multiplex real time-PCR assay for simultaneous detection of the five genes blaNDM, blaKPC, blaIMP-1, blaOXA-48 and mcr-1. The melting curve-based multiplex real time PCR assay was established with the dissociation temperature range extended from 76°C to 87°C. The whole process is completed within one hour and half, allowing rapid screening of the five genes in cultured bacteria samples with a limit of detection of 10 CFU/ml. The proposed multiplex real-time PCR assay is a robust, reliable and rapid method for the detection of bacterial strains carrying blaOXA-48, blaIMP, blaNDM, blaKPC and mcr-1 gene individually or in cocktail of genes. This assay will be a valuable tool for surveillance and monitoring of MDR bacteria additionally resistant to either carbapenem or colistin or both drugs.

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