Rapid discriminatory detection of genes coding for SHV beta-lactamases by ligase chain reaction.
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 beta-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 beta-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 beta-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of beta-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 beta-lactamases.
Kim
J
J
Department of Microbiology, College of Medicine, Dankook University, Cheonan, Korea. minkim@anseo.dankook.ac
Lee
H J
HJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D011088:DNA Ligases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D011088:DNA Ligases