Mutant TEM beta-lactamase producing resistance to ceftazidime, ampicillins, and beta-lactamase inhibitors

A derivative of the TEM-1 P-lactamase producing clinically significant levels of resistance to ceftazidime and beta-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection. This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to beta-lactamase inhibitors. Although the Glu104Lys; substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime. Kinetic studies of the purified beta-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (k(cat)/K-m) of ceftazidime. The increases in the K-i values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibillity of beta-lactam resistance with these inhibitors.