DISCRIMINATORY DETECTION OF INHIBITOR-RESISTANT BETA-LACTAMASES IN ESCHERICHIA-COLI BY SINGLE-STRAND CONFORMATION POLYMORPHISM-PCR

Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM deriv ative production, and OXA production, lead to amoxicillin-clavulanic a cid resistance in enterobacteria. The ability of the single-strand con formation polymorphism (SSCP)-PCR method to differentiate the genes en coding inhibitor-resistant beta-lactamases was evaluated with three bl a(TEM) primer pairs. The bla(TEM) genes, which were known to be differ ent on the basis of their nucleotide sequences (bla(TEM-1A), bla(TEM-1 B), bla(TEM-2), bla(TEM-30), bla(TEM-32), and bla(TEM-35)), were ident ified as different by their electrophoretic mobilities. The bla(TEM-33 ), bla(TEM-34), bla(TEM-36), bla(TEM-37), bla(TEM-38), and bla(TEM-39) genes, whose sequence differences have been established by oligotypin g, displayed different SSCP profiles for different fragments, suggesti ng genetic differences in addition to those defined by oligotyping. Co nfirmed by sequencing, these additional genetic events concerned silen t 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 E scherichia coli resistant to amoxicillin-clavulanic acid, the SSCP met hod 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 showe d two substitutions: Arg244Ser, which is known to confer amoxicillin-c lavulanic acid resistance in TEM-30, and Val261Ile, which has not been described previously. The eighth strain produced an OXA beta-lactamas e. Given the discriminatory power and the applicability of SSCP-PCR, t his method can be proposed as a means of following the evolution of th e frequencies of the different inhibitor-resistant beta-lactamases.