Activity of beta-lactamase in Pseudomonas aeruginosa as studied by HPLC

Mutants with increased resistance were selected from a clinical isolate of Pseudomonas aeruginosa using ceftazidime, piperacillin and carbenicillin. The MICs of these antibiotics and of ticarcillin were determined for the parent strain and for the selected mutants. Subsequently, cell-free extracts of the strains were prepared and the rates of hydrolysis of several beta-lactam substrates by the extracts were determined by HPLC procedures. It appeared possible to determine beta-lactamase activities in the crude cell extracts at the low substrate concentrations which may be attainable in the periplasm of Gram-negative bacteria. It is concluded that the increased drug MICs for mutants selected with ceftazidime or piperacillin, but not for those selected with carbenicillin, were caused by increased chromosomal beta-lactamase activity.