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

Infection by extended-spectrum β-lactamase (ESBL)-producingEnterobacteriaceae 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 identifybla SHV ,bla CTX-M-3 -like, andbla CTX-M-14 -like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalentbla SHV genes (bla SHV-1 ,bla SHV-2 ,bla SHV-2a ,bla SHV-5 ,bla SHV-11 , andbla SHV-12 ) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of thebla SHV andbla CTX-M genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40Enterobacter cloacae , 68Escherichia coli , and 91Klebsiella 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 toEnterobacter cloacae , the majority of which produced SHV-type ESBLs,E. coli andK. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of thebla CTX-M-3 -like (CTX-M-15) andbla CTX-M-14 -like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs amongE. coli ,K. pneumoniae , andEnterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.