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Journal of Clinical Microbiology, September 2005, p. 4486-4491, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4486-4491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Multiplex PCR and SHV Melting-Curve Mutation Detection System for Detection of Some SHV and CTX-M ß-Lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan

Ju-Hsin Chia,1 Chishih Chu,3 Lin-Hui Su,1 Cheng-Hsun Chiu,2 An-Jing Kuo,1 Chien-Feng Sun,1 and Tsu-Lan Wu1*

Department of Clinical Pathology, Chang Gung Memorial Hospital,1 Department of Pediatrics, Chang Gung Children's Hospital, Taoyuan, Taiwan,2 Department of Applied Microbiology, National Chiayi University, Chiayi, Taiwan3

Received 14 October 2004/ Returned for modification 9 December 2004/ Accepted 13 June 2005

Infection by extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify blaSHV, blaCTX-M-3-like, and blaCTX-M-14-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent blaSHV genes (blaSHV-1, blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-11, and blaSHV-12) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the blaSHV and blaCTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the blaCTX-M-3-like (CTX-M-15) and blaCTX-M-14-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

* Corresponding author. Mailing address: Department of Clinical Pathology, Chang Gung Memorial Hospital, 5 Fu-Hsin St., Kweishan 333, Taoyuan, Taiwan. Phone: 886 3 3281200, ext. 2736. Fax: 886 3 3971827. E-mail: wutsulan{at}adm.cgmh.org.tw.

Journal of Clinical Microbiology, September 2005, p. 4486-4491, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4486-4491.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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