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

Vietnam Journal of Science and Technology - Tập 62 Số 3 - Trang 521-529 - 2024
Le Thi Thuy1, Do Quang Minh1, Le Thi Thu Hang1, Dong Van Quyen2, Anne-Laure Bañuls3, Nguyen Quang Huy1
1LMI 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
2Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Street, Cau Giay District, Ha Noi, Viet Nam
3UMR MIVEGEC (University of Montpellier- IRD-CNRS), 911 Av Agropolis, 34394 Montpellier, France

Tóm tắt

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.

Từ khóa

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

Tài liệu tham khảo

Ventola C. L., - The antibiotic resistance crisis: part 1: causes and threats, P T. 40 (4) (2015) 277-83. PMCID: PMC4378521

Codjoe F. S., Donkor E. S. - Carbapenem Resistance: A Review, Med Sci (Basel). 6 (1) (2017). DOI: 10.3390/medsci6010001

Kumar H., Chen B-H., Kuca K., Nepovimova E., Kaushal A., Nagraik R., Bhatia S. K., Dhanjal D. S., Kumar V., Kumar A., Upadhyay N. K., Verma R., Kumar D., - Understanding of Colistin Usage in Food Animals and Available Detection Techniques: A Review, Anima (Basel). 10 (10) 1892 (2020). https://doi.org/10.3390/ani10101892

Liu Y.Y., Wang Y., Walsh T. R., Yi L-X., Zhang R., Spencer J., Doi Y., Tian G., Dong B., Huang X., Yu L-F., Gu D., Ren H., Chen X., Luchao L., He D., Zhou H., Liang Z., Liu J-H., Shen J., - Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study, Lancet Infect Dis. 16 (2) 161-82016 (2016). DOI: 10.1016/S1473-3099(15)00424-7

Gao R., Hu Y., Li Z., Sun J., Wang Q., Lin J., Ye H., Liu F., Srinivas S., Li D., Zhu B., Liu Y-H., Tian G-B., Feng Y., - Dissemination and Mechanism for the MCR-1 Colistin Resistance, PLoS Pathog. 12 (11) e1005957 (2016). DOI: 10.1371/journal.ppat.1005957

Ewa K., Tomasz D., Grażyna M., - Rapid detection of NDM, VIM, KPC and IMP carbapenemases by real-time PCR, J Bacteriol Parasitol. 7 (6) (2016). DOI: 10.4172/2155-9597.1000299

Liu J., Zhang Z., Feng Y., Hu H., Yu Y., Qiu L., Liu H., Guo Z., Huang J., Du C., Qiu J., - Molecular Detection of the mcr Genes by Multiplex PCR, Infect Drug Resist. 13 3463-3468 (2020). doi: 10.2147/IDR.S256320

Monteiro J., Widen R. H., Pignatari A. C., Kubasek C., Silbert S., - Rapid detection of carbapenemase genes by multiplex real-time PCR, J Antimicrob Chemother. 67 (4) 906-9 (2012). DOI: 10.1093/jac/dkr563

Jaggi N., Chatterjee N., Singh V., Giri S. K., Dwivedi P., Panwar R., Sharma A. P., - Carbapenem resistance in Escherichia coli and Klebsiella pneumoniae among Indian and international patients in North India, Acta Microbiol Immunol Hung. 66 (3) 367-376 (2019). DOI: 10.1556/030.66.2019.020

Park B.Y., Mourad D., Hong J. S., Yoon E. J., Kim D., Lee H., Jeong S. H., - Performance Evaluation of the Newly Developed BD Phoenix NMIC-500 Panel Using Clinical Isolates of Gram-Negative Bacilli, Ann Lab Med. 39 (5) 470-477 (2019). DOI: 10.3343/alm.2019.39.5.470

Mustapha A., Singh P., - Multiplex assay for detection of bacterial species in biological samples, (2015). Patent US10190177B2

Subirats J., Royo E., Balcázar J. L., Borrego C. M., - Real-time PCR assays for the detection and quantification of carbapenemase genes (bla (KPC), bla (NDM), and bla (OXA-48)) in environmental samples, Environ Sci Pollut Res Int. 24 (7) 6710-6714 (2017). DOI: 10.1007/s11356-017-8426-6

Zaman T. U., Aldrees M., Johani S. M. A., Alrodayyan M., Aldughashem F. A., Balkhy H. H., Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia, Int J Infect Dis. 28 186-92 (2014).

Mustapha A., Singh P., Multiplex assay for detection of bacterial species in biological samples, (2019). Patents US10190177B2

Tenover F. C., Kalsi R. K., Williams P. P., Carey R. B., Stocker S., Lonsway D., Rasheed J. D., Biddle J. W., McGowan J. E., Hanna B., - Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing, Emerg Infect Dis. 12 (8) 1209-13 (2006). doi: 10.3201/eid1208.060291

Elnifro E. M., Ashshi A. M., Cooper R. J., Klapper P. E., - Multiplex PCR: optimization and application in diagnostic virology, Clin Microbiol Rev. 13 (4) 559-70 (2000). doi: 10.1128/cmr.13.4.559-570.2000

Lund M., Petersen M. B., Jørgensen A. L., Paulmann D., Wang M., - Rapid real-time PCR for the detection of IMP, NDM, VIM, KPC and OXA-48 carbapenemase genes in isolates and spiked stool samples, Diagn Microbiol Infect Dis. 92 (1) 8-12 (2018). DOI: 10.1016/j.diagmicrobio.2018.04.002

Tolosi R., Apostolakos I., Laconi A., Carraro L., Grilli G., Cagnardi P., Piccirillo A., - Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples, J Appl Microbiol. 129 (6) 1523-1529 (2020). DOI: 10.1111/jam.14738

Thông tin
Thông tin xuất bản

Vietnam Journal of Science and Technology

Tập 62 Số 3

521-529

Thông tin tác giả