Discriminatory Detection of Inhibitor-Resistant β-Lactamases inEscherichia coli by Single-Strand Conformation Polymorphism-PCR

ABSTRACT

Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM derivative production, and OXA production, lead to amoxicillin-clavulanic acid resistance in enterobacteria. The ability of the single-strand conformation polymorphism (SSCP)-PCR method to differentiate the genes encoding inhibitor-resistant β-lactamases was evaluated with three bla _TEM primer pairs. Thebla _TEM genes, which were known to be different on the basis of their nucleotide sequences (bla _TEM-1A, bla _TEM-1B,bla _TEM-2, bla _TEM-30,bla _TEM-32, andbla _TEM-35), were identified as different by their electrophoretic mobilities. Thebla _TEM-33, bla _TEM-34,bla _TEM-36, bla _TEM-37,bla _TEM-38, andbla _TEM-39 genes, whose sequence differences have been established by oligotyping, displayed different SSCP profiles for different fragments, suggesting genetic differences in addition to those defined by oligotyping. Confirmed by sequencing, these additional genetic events concerned silent mutations at certain positions and, notably, a G→T transversion at position 1 of the −10 consensus sequence in bla _TEM-34,bla _TEM-36, bla _TEM-37, and bla _TEM-39. Applied to eight clinical isolates of Escherichia coli resistant to amoxicillin-clavulanic acid, the SSCP method detected TEM-1 in three strains and TEM-30, TEM-32, and TEM-35 in three other strains, respectively. A novel TEM derivative (TEM-58) was detected in another strain, and the deduced amino acid sequence showed two substitutions: Arg244Ser, which is known to confer amoxicillin-clavulanic acid resistance in TEM-30, and Val261Ile, which has not been described previously. The eighth strain produced an OXA β-lactamase. Given the discriminatory power and the applicability of SSCP-PCR, this method can be proposed as a means of following the evolution of the frequencies of the different inhibitor-resistant β-lactamases.