Non-radioactive PCR–SSCP with a single PCR step for detection of inhibitor resistant β-lactamases in Escherichia coli

Abstract

A method based on PCR–SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) β-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT β-lactamases was evaluated with PCR products digested with RsaI. All the bla TEM genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin–clavulanate (MIC, ≥32 μg/ml), the electrophoretic mobilities of the digested bla TEM PCR products were identical to those of the reference bla TEM-1A (6 strains) and bla TEM-1B (18 strains) genes. The remaining five bla TEM PCR products displayed SSCP profiles different from those of the reference bla TEM genes and their nucleotide sequence identified them as bla TEM-1C in one strain, bla TEM-30/IRT-2 in two strains, bla TEM-37/IRT-8 in one strain, and bla TEM-40/IRT-11 in one isolate. Overexpression of the wild-type bla TEM-1 gene, as detected by high-level resistance to β-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla TEM-1. We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT β-lactamases; identification should be confirmed by sequence data.