title: 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.
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.
authors.affiliation: Wu Lien-Teh Institute, Harbin Medical University, Harbin, China.|Department of Microbiology, Harbin Medical University, Harbin, China.|Department of Pathology, School of Medicine, Kenyatta University, Nairobi, Kenya.
authors.forename: James O
authors.initials: JO
authors.lastname: Ogutu
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Qingmeng
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Huo
authors.initials: H
authors.lastname: Yan
authors.affiliation: Obstetric Ward, the Fourth Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Lijie
authors.initials: L
authors.lastname: Su
authors.affiliation: Department of Clinical Laboratory, The People's Hospital of He Gang, He Gang, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Gao
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Wenli
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Jizi
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Cai
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian, Liaoning, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Microbiology, Harbin Medical University, Harbin, China.
authors.forename: Wuqi
authors.initials: W
authors.lastname: Song
authors.affiliation: Wu Lien-Teh Institute, Harbin Medical University, Harbin, China.
authors.forename: Xiaobei
authors.initials: X
authors.lastname: Chen
authors.affiliation: Wu Lien-Teh Institute, Harbin Medical University, Harbin, China.|Department of Microbiology, Harbin Medical University, Harbin, China.|Heilongjiang Provincial Key Laboratory of Infection and Immunity, Harbin Medical University, Harbin, China.
authors.forename: Yingmei
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Wu Lien-Teh Institute, Harbin Medical University, Harbin, China.|Department of Microbiology, Harbin Medical University, Harbin, China.|Heilongjiang Provincial Key Laboratory of Infection and Immunity, Harbin Medical University, Harbin, China.
authors.forename: Fengmin
authors.initials: F
authors.lastname: Zhang
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001618:beta-Lactamases