Rapid Detection of blaKPC Carbapenemase Genes by Real-Time PCR

Carbapenem resistance among Enterobacteriaceae, in particular among Klebsiella pneumoniae and Escherichia coli, is an emerging problem worldwide (14, 16, 20, 22, 25, 26). Several resistance mechanisms have been reported to circumvent the efficacy of carbapenems, and carbapenemases (carbapenem-hydrolyzing β-lactamases) are the most prominent enzymes that neutralize carbapenems (17, 18). Class A carbapenemases, which include blaKPC, NMC, SME-1 to -3, IMI-1, and GES, have been characterized in several genera of the family Enterobacteriaceae (17). Other carbapenem resistance mechanisms, including porin changes and changes in penicillin-binding proteins, have also been implicated in carbapenem resistance (23, 27, 28). blaKPC enzymes, so called because they have been identified mainly in K. pneumoniae, have been reported in Klebsiella oxytoca, Serratia spp., Enterobacter spp., Salmonella spp., Citrobacter freundii, and Pseudomonas aeruginosa (3, 8, 10, 11, 15, 24, 28). The genes encoding the blaKPC enzymes are usually flanked by transposon-related sequences that have been identified on transferable plasmids, thus giving them the potential to disseminate rapidly. Three different blaKPC genes have been reported to date: blaKPC-2, blaKPC-3, and blaKPC-4; blaKPC-1 and blaKPC-2 are identical (27). blaKPC-producing bacteria are usually resistant to virtually all classes of antibiotics—β-lactam agents, including penicillins, cephalosporins, monobactams, and carbapenems (1, 27, 28)—leaving physicians with limited antibiotic choices for treating infected patients. In order to control the spread of blaKPC-containing bacteria in hospitalized patients, effective infection control measures and controlled antibiotic usage must be complemented by the utilization of rapid and sensitive blaKPC diagnostic assays (13). The utilization of such diagnostic tools will help in rapidly isolating colonized or infected patients and assigning them to cohorts. In this report, we describe the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes. The assay was validated by comparing q-PCR with regular bacterial culturing on MacConkey agar-plus-carbapenem disks of routine surveillance perianal/rectal swabs obtained from the Sheba Medical Center intensive care units during an outbreak of one strain of K. pneumoniae harboring blaKPC-3. Moreover, two automated bacterial DNA extraction methodologies were compared: the bioMerieux NucliSENS easyMAG system and Roche MagNA Pure LC DNA isolation kit III (bacteria, fungi).