Phenotypic Study of Resistance of β-Lactamase-Inhibitor-Resistant TEM Enzymes Which Differ by Naturally Occurring Variations and by Site-Directed Substitution at Asp(276)

At this time an amino acid substitution at position 276 in the TEM-1 enzyme is associated with an additional substitution at position 69 in natural β-lactamase-inhibitor-resistant (IRT) β-lactamases. The effect of the Asn(276)→Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to β-lactamase inhibitors, but the MICs of amoxicillin combined with clavulanic acid or tazobactam were strikingly different for E. coli strains producing the Asn276Asp variant and those producing naturally occurring IRTs with single or double substitutions. The inhibitory effects of clavulanic acid and tazobactam were the same in IRTs with substitutions at position 69 (IRT-5 and IRT-6). The effect of clavulanic acid on the MICs of amoxicillin for the Asn276Asp variant was greater than that of tazobactam. In IRTs with double substitutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 plus 275 (IRT-14), tazobactam was a more potent inhibitor than clavulanic acid. The effect of the Asn(276)→Asp substitution on the values of the kinetic constants and the concentration required to inhibit by 50% the hydrolysis of benzylpenicillin confirms that this single mutation is responsible for resistance to β-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp(276) can form two salt bonds with Arg(244) close to the penicillin-binding cavity. The addition of the Asp(276) mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in β-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM β-lactamases by virtue of the use of β-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.