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J Antibiot (Tokyo). 2015 Dec;68(12):725-33. doi: 10.1038/ja.2015.68. Epub 2015 Jun 24.

Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains.

Author information

1
Wu Lien-Teh Institute, Harbin Medical University, Harbin, China.
2
Department of Microbiology, Harbin Medical University, Harbin, China.
3
Department of Pathology, School of Medicine, Kenyatta University, Nairobi, Kenya.
4
Obstetric Ward, the Fourth Affiliated Hospital, Harbin Medical University, Harbin, China.
5
Department of Clinical Laboratory, The People's Hospital of He Gang, He Gang, China.
6
Department of Biotechnology, Dalian Medical University, Dalian, Liaoning, China.
7
Heilongjiang Provincial Key Laboratory of Infection and Immunity, Harbin Medical University, Harbin, China.

Abstract

Resistance to β-lactam antibiotics through β-lactamase production by Enterobacteriaceae continues to burden the health-care sector worldwide. Traditional methods for detection of β-lactamases are time-consuming and labor-intensive and newer methods with varying capabilities continue to be developed. The objective of this study was to develop a multiplex PCR (M-PCR) system for the detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes and to apply it in clinical Klebsiella pneumoniae and Escherichia coli strains. To do this, we used group-specific PCR primers in singleplex reactions followed by optimization into multiplex reactions. Specificity and sensitivity of the M-PCR were then evaluated using 58 reference strains before its application to detect bla group genes in 203 clinical Enterobacteriaceae strains. PCR amplicons were sequenced to determine the β-lactamase subtypes. The M-PCR system exhibited 100% specificity and sensitivity. In all, 83.7% of K. pneumoniae and 89.8% of E. coli clinical strains harbored bla group genes with 46.9%, 40.1%, 15.0%, 21.1% and 6.1% of K. pneumoniae having blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively, whereas 12.2%, 77.6%, 22.4%, 36.7% and 8.2% of E. coli had blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes, respectively. BlaSHV-1, blaSHV-11, blaSHV-27, blaSHV-33, blaSHV-144, blaTEM-1, blaTEM-135, blaOXA-1, blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, blaCTX-M-65 and blaCTX-M-104 were detected. In conclusion, the M-PCR system was efficient and versatile with an advantage of simultaneously detecting all the targeted bla group genes. Hence, it is a potential candidate for developing M-PCR kits for the screening of these genes for clinical or epidemiological purposes.

PMID:
26104141
DOI:
10.1038/ja.2015.68
[Indexed for MEDLINE]

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