Rapid Discriminatory Detection of Genes Coding for SHV β-Lactamases by Ligase Chain Reaction

Abstract

ABSTRACT Ligase chain reaction (LCR) is a recently developed technique that employs a thermostable ligase and allows for the discrimination of DNA sequences differing in only a single base pair. The method has been adapted and applied to differentiation of bla SHV genes. We have developed an LCR typing method to characterize point mutations in genes for SHV-derived extended-spectrum β-lactamases with four different sets of biotinylated LCR primers. To evaluate the applicability of the current technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12. With the LCR typing, seven SHV genes can be distinguished according to their incorporating point mutations. In an attempt to characterize SHV β-lactamases by LCR typing in clinical isolates, 46 strains carrying bla SHV genes (32 Klebsiella pneumoniae , 10 Enterobacter cloacae , and 4 E. coli ) were subjected to antibiotic susceptibility testing, isoelectric focusing, and LCR typing. LCR typing allowed the characterization of β-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of β-lactamase. Therefore, we concluded that LCR typing may permit defining the SHV families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type β-lactamases.