Discriminatory Detection of Inhibitor-Resistant β-Lactamases in Escherichia 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.