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

Abstract

Infection by extended-spectrum ␤-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 toEnterobacter 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.

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

“…DNA template was prepared according to a previously reported method 22 with a slight modification. A single colony of each strain was subcultured in 2 ml of Luria-Bertani broth (Oxoid, Hampshire, UK) for 16 h at 37°C with shaking at 200 r.p.m.…”

Association of antibiotic resistance with SHV-12 extended-spectrum β-lactamase in Enterobacter cloacae

“…SHV-type extended-spectrum β-lactamases (ESBLs), encoded by the bla SHV gene confer significant resistance to β-lactam antibiotics (3). SHV-type ESBLs have >140 variants, and the SHV-12-type ESBL is one of the most prevalent types in Asia (4,5).…”

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

“…For this reason, several HRM assays were developed to detect mutations or alleles responsible for this phenotype. Chia et al 49 developed a multiplex PCR to identify bla SHV , bla CTX-M-3 -like and bla CTX-M-14 -like genes and also modified a previously described SHV melting-curve mutation detection (MCMD) method 50 to rapidly differentiate the 6 bla SHV genes prevalent in Taiwan. Specific detection of bla VIM and bla IMP metallo-β-lactamase genes in a single real-time PCR was tested by Bisilkis et al 51 .…”