Biochemical Characterization of a Novel Extended-Spectrumβ-Lactamase fromPseudomonas aeruginosa802

Abstract

Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam. It produced a pI 7.6 extended-spectrum β-lactamase (ESBL). The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column. The LBT 802 enzyme had a molecular mass of 30 kDa. It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam. The highest hydrolytic efficiency (V_max/K_m) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin. Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome. LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam. It showed its lowest K_i values for clavulanic acid, imipenem and sulbactam.