This study investigates a Klebsiella pneumoniae isolate that exhibits moderate to high-level resistance to imipenem and meropenem, with MICs of 16 μg/mL for both drugs. The presence of β-lactamase activity was confirmed through inhibition by clavulanic acid. The strain also showed resistance to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing revealed three β-lactamases with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). PCR and DNA sequence analysis confirmed the presence of blaSHV and blaTEM genes. Transformation and conjugation studies demonstrated that the β-lactamase encoded by KPC-1 is located on a 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned into E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. KPC-1 shares 45% amino acid identity with Sme-1, a carbapenem-hydrolyzing β-lactamase from Serratia marcescens. Kinetic studies revealed that KPC-1 has the highest affinity for meropenem and is inhibited by clavulanic acid and tazobactam. The outer membrane proteins of the parent strain were analyzed, showing the absence of detectable levels of OmpK35 and OmpK37, while OmpK36 was present. The study concludes that the carbapenem resistance in K. pneumoniae strain 1534 is primarily due to the production of the novel class A β-lactamase, KPC-1, with potential contributions from altered porin expression.This study investigates a Klebsiella pneumoniae isolate that exhibits moderate to high-level resistance to imipenem and meropenem, with MICs of 16 μg/mL for both drugs. The presence of β-lactamase activity was confirmed through inhibition by clavulanic acid. The strain also showed resistance to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing revealed three β-lactamases with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). PCR and DNA sequence analysis confirmed the presence of blaSHV and blaTEM genes. Transformation and conjugation studies demonstrated that the β-lactamase encoded by KPC-1 is located on a 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned into E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. KPC-1 shares 45% amino acid identity with Sme-1, a carbapenem-hydrolyzing β-lactamase from Serratia marcescens. Kinetic studies revealed that KPC-1 has the highest affinity for meropenem and is inhibited by clavulanic acid and tazobactam. The outer membrane proteins of the parent strain were analyzed, showing the absence of detectable levels of OmpK35 and OmpK37, while OmpK36 was present. The study concludes that the carbapenem resistance in K. pneumoniae strain 1534 is primarily due to the production of the novel class A β-lactamase, KPC-1, with potential contributions from altered porin expression.