Phenotypic study of resistance of beta-lactamase-inhibitor-resistant TEM enzymes which differ by naturally occurring variations and by site-directed substitution at Asp276

Abstract

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 beta-lactamase-inhibitor-resistant (IRT) beta-lactamases. The effect of the Asn276-->Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to beta-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 Asn276-->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 beta-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp276 can form two salt bonds with Arg244 close to the penicillin-binding cavity. The addition of the Asp276 mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in beta-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM beta-lactamases by virtue of the use of beta-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.

FIG. 1

MICs for E. coli producing TEM-1 wild-type enzymes, TEM-1 variant enzymes obtained by site-directed mutagenesis, and natural IRT enzymes. The effects of clavulanate and tazobactam combined with amoxicillin were determined. The following enzymes were used: wild-type TEM-1 enzymes R111 (this study), pBSKS (this study), and pBR322 (33); TEM-1 variants pMBS276D with Asn276Asp (this study) and pBR322 with Trp165Arg (33); natural IRT enzymes (this study) P30 producing IRT-5 (Met69Leu), P9 producing IRT-6 (Met69Val), P11 producing IRT-7 (Met69Val plus Asn276Asp), and P12 producing IRT-8 (Met69Ile plus Asn276Asp); and Pey producing IRT-4 (Met69Leu plus Asn276Asp) (7) and P37 producing IRT-14 (Met69Leu plus Arg275Gln) (10). , amoxicillin; , amoxicillin plus clavulanate; □, amoxicillin plus tazobactam.

FIG. 2

Molecular modeling of the Asn276Asp mutant of TEM-1: stereo view of the environment of Asp²⁷⁶ and interactions with Arg²⁴⁴. Hydrogen bonds are indicated by dashed lines. The backbones of α-helices h1, h2, and h10 are shown (at the bottom) as thin lines.