Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan.
Infection by extended-spectrum beta-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 bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-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 bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-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.
Chia
Ju-Hsin
JH
Department of Clinical Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Chu
Chishih
C
Su
Lin-Hui
LH
Chiu
Cheng-Hsun
CH
Kuo
An-Jing
AJ
Sun
Chien-Feng
CF
Wu
Tsu-Lan
TL
D016972:Enterobacter cloacae / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D004756:Enterobacteriaceae Infections / Q000453:epidemiology / Q000382:microbiology; D004926:Escherichia coli / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007711:Klebsiella pneumoniae / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013624:Taiwan / Q000453:epidemiology; D044366:Transition Temperature; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
C456080:beta-lactamase CTX-M-14; C417271:beta-lactamase CTX-M-3; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases