Genetic and Biochemical Characterization of CAD-1, a Chromosomally Encoded New Class A Penicillinase from Carnobacterium divergens

Abstract

ABSTRACT Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A β-lactamase named CAD-1. The bla CAD-1 gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with β-lactamases from Bacillus cereus and Staphylococcus aureus , respectively. Alignment of CAD-1 with other class A β-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A β-lactamases. Escherichia coli harboring bla CAD-1 exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM −1 s −1 , respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC 50 ] = 0.27 μM), clavulanic acid (IC 50 = 4.7 μM), and sulbactam (IC 50 = 43.5 μM). The bla CAD-1 gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.