Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain ofKlebsiella pneumoniae

Abstract

AKlebsiella pneumoniaeisolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence ofbla_SHVandbla_TEMgenes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia colishowed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniaecarbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,bla_KPC-1, was cloned inE. coliand shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescensS6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parentK. pneumoniaestrain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance inK. pneumoniaestrain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.