PHENOTYPIC STUDY OF RESISTANCE OF BETA-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 e nzyme is associated with an additional substitution at position 69 in natural beta-lactamase-inhibitor-resistant (IRT) beta-lactamases. The effect of the Asn(276)-->Asp substitution On resistance was assessed w ith the Asn276Asp variant, generated by site-directed mutagenesis, The mutant was resistant to beta-lactamase inhibitors, but the MICs of am oxicillin combined with clavulanic acid or tazobactam were strikingly different for E, coli strains producing the Asn276Asp variant and thos e producing naturally occurring IRTs with single or double substitutio ns. 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,vith double subs titutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 pl us 275 (IRT-14), tazobactam was a more potent inhibitor than clavulani c 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 mutatio n is responsible for resistance to beta-lactamase inhibitors. Molecula r modeling of the Asn276Asp mutant shows that Asp(276) can form two sa lt bonds with Arg(244) close to the penicillin-binding cavity, The add ition of the Asp(276) mutation to that preexisting at position 69 conf ers a higher selective advantage to bacteria, as shown by the reductio n in p-lactamase inhibitor efficiencies of the double variants. Theref ore, the emergence of multiple mutations in TEM beta-lactamases by vir tue of the use of beta-lactamase inhibitors increases selection pressu re resulting in the convergent evolution of resistant strains.