Development and Evaluation of Multiplex PCR for the Detection of Carbapenemase-Producing Enterobacteriaceae

시현 김†1   일권 배†2   나영 김34   새암 손3   선주 김5   조셉 정6   정환 신*34   

1 Department of Clinical Laboratory Science, Semyung University, Jecheon,
2 Department of Dental Hygiene,Silla University
3 Department of Laboratory Medicine, Inje University College of Medicine,
4 Paik Institute for Clinical Research, Inje University College of Medicine, Busan
5 Department of Laboratory Medicine, Gyeongsang National University College of Medicine, Jinju,
6 Department of Laboratory Medicine, Ulsan University Hospital,University of Ulsan College of Medicine, Ulsan,

Abstract

Background: The isolation of carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly common. Continuous surveillance for these organisms is essential because their infections are closely related to outbreaks of illness and are associated with high mortality rates. The aim of this study was to develop and evaluate multiplex PCR as a means of detecting several important CPE genes simultaneously.

Methods: We aimed to develop a multiplex PCR that could detect seven CPE genes simultaneously. The multiplex PCR was composed of seven primer sets for the detection of KPC, IMP, VIM, NDM-1, GES, OXA-23, and OXA-48. We designed different PCR product sizes of at least 100 bp. We evaluated the performance of this new test using 69 CPE-positive clinical isolates. Also, we confirmed the specificity to rule out false-positive reactions by using 71 carbapenem- susceptible clinical strains.

Results: A total of 69 CPE clinical isolates showed positive results and were correctly identified as KPC (N=14), IMP (N=13), OXA-23 (N=12), OXA-48 (N=11), VIM (N=9), GES (N=5), and NDM (N=5) by the multiplex PCR. All 71 carbapenem-susceptible clinical isolates, including Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, showed negative results.

Conclusion: This multiplex PCR can detect seven CPE genes at a time and will be useful in clinical laboratories. (Ann Clin Microbiol 2019;22:9-13)

Keywords

Carbapenem   Carbapenem-resistant Enterobacteriaceae   Carbapenemase-producing Enterobacteriaceae   Enterobacteriaceae   Multiplex PCR   


Acknowledgements

This research was supported by a fund (2017-ER5402-01) by Research of Korea Centers for Disease Control and Prevension.

Figures & Tables

Fig. 1. Multiplex PCR for the detection of seven carbapenemaseproducing Enterobacteriaceae genes. Abbreviations: M, molecular weight marker; N, negative control. Lane: IMP (180 bp), VIM (306 bp), NDM (410 bp), OXA-23 (505 bp), OXA-48 (602 bp), KPC (747 bp), and GES (855 bp).


Figures & Tables

Table 1. Clinical strains of carbapenemase-producing Enterobacteriaceae used in this study

Carbapenemase type and speciesNo
GES5
Klebsiella pneumoniae4
Serratia marcescens1
IMP13
Enterobacter spp.1
Enterobacter cloacae2
Pseudomonas aeruginosa10
KPC14
Enterobacter aerogenes1
Escherichia coli2
Klebsiella pneumoniae11
NDM5
Escherichia coli2
Klebsiella pneumoniae3
OXA-2312
Acinetobacter baumannii11
Acinetobacter nosocomialis1
OXA-4811
Escherichia coli3
Klebsiella pneumoniae8
VIM9
Enterobacter cloacae1
Pseudomonas aeruginosa4
Pseudomonas fulva1
Pseudomonas monteilii1
Pseudomonas putida2
Total69