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
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Date
2015
Authors
Ogutu, James O.
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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.
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Research paper
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The Journal of Antibiotics (2015) 68, 725–733