Genetic and biochemical characterization of CAD-1, a chromosomally encoded new class A penicillinase from Carnobacterium divergens.
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 beta-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 beta-lactamases from Bacillus cereus and Staphylococcus aureus, respectively. Alignment of CAD-1 with other class A beta-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A beta-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 microM), clavulanic acid (IC(50) = 4.7 microM), and sulbactam (IC(50) = 43.5 microM). 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.
Meziane-Cherif
Djalal
D
Unité des Agents Antibactériens, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
Decré
Dominique
D
Høiby
E Arne
EA
Courvalin
Patrice
P
Périchon
Bruno
B
D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D016521:Electrophoresis, Gel, Pulsed-Field; D006094:Gram-Positive Bacteria / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010403:Penicillin Resistance; D010405:Penicillinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D047090:beta-Lactams / Q000494:pharmacology
D047090:beta-Lactams; D010405:Penicillinase