Abstract. The aim of the present study was to investigate the phenotype and genotype of plasmid-mediated AmpC (pAmpC) β-lactamase in Klebsiella pneumoniae and its antibiotic resistance. A total of 130 non-repetitive clinical isolates of Klebsiella pneumoniae, obtained from tertiary hospitals, were phenotypically screened for pAmpC β-lactamase production with the cefoxitin disk diffusion test. β-lactamase genes in the screened isolates were detected using multiplex polymerase chain reaction (PCR); carbapenemase genes in pAmpC β-lactamase-producing isolates that were resistant to imipenem were detected using PCR. Out of the 130 isolates of Klebsiella pneumoniae, 62 strains (47.7%) were resistant to cefoxitin, including 14 strains (10.8%) positive for pAmpC β-lactamase (DHA type), among which 12 strains (85.7%) were susceptible to imipenem, and 2 strains, which were carrying Klebsiella pneumoniae carbapenemase (KPC)-2 gene, were resistant to imipenem. The pAmpC β-lactamase-producing Klebsiella pneumoniae isolates from the tertiary hospitals were mainly of DHA-1 genotype, and the majority were susceptible to carbapenems; drug-resistant strains were associated with KPC-2 expression. IntroductionAmpC β-lactamase, which is a type of cephalosporinase (1), is known to be responsible for antimicrobial resistance in gram-negative bacilli (2). The knowledge of potential risk factors for that resistance may help to limit its impact by enabling the implementation of effective control measures and judicious antimicrobial therapy (3). AmpC β-lactamases are either plasmid-or chromosomal-mediated (4). With regard to plasmid-mediated AmpC (pAmpC), no single phenotypic method is satisfactory for its detection; the combined application of phenotypic tests is necessary for the screening and confirmation of the presence of pAmpC-mediated resistance (5). Chromosomal-mediated AmpC hyperproducing Escherichia coli (E. coli) continues to be mostly susceptible to third-generation cephalosporins (6). Polymerase chain reaction (PCR) remains the gold standard for the detection of AmpC β-lactamases (7). As β-lactam antibiotics are widely used, the production of AmpC β-lactamases by bacteria results in considerable antibiotic resistance (8).Resistance to broad-spectrum β-lactams mediated by extended spectrum broad-spectrum β-lactamases (ESBLs) and AmpC β-lactamase enzymes is an increasing problem worldwide (9). In Portugal, 104/124 (83.9%) of Enterobacteriaceae isolates that were resistant to third generation cephalosporins were also resistant to fourth generation cephalosporins (10). In comparison with ESBL-producing gram-negative bacilli, AmpC β-lactamase-producing strains show more extensive antibiotic resistance; 29% of Acinetobacter strains, for example, were found to produce both ESBL and AmpC enzymes (11). To be more specific, the majority of Acinetobacter baumannii isolates were producers of carbapenemase and metallo-β-lactamase (12). In a study of isolates collected between 2002 and 2008, DHA-1 was the most prevalent acquired AmpC (94%)...