Rapid Detection of NDM, VIM, KPC and IMP Carbapenemases by Real-Time PCR

Abstract

Carbapenems are the most potent beta-lactams, characterized by broad spectrum of activity against Gramnegative and Gram-positive bacteria. Unfortunately, the dynamic dispersal of carbapenem resistance among nonfermentative bacteria and Enterobacteriaceae is an over-increasing problem and might lead to dangerous limitation of treatment options. Among three different mechanisms of resistance the enzyme production is of special importance. In this case, only one small gene is enough to express carbapenem resistance. The carbapenemase genes are often a part of integrons, which carry diverse arrays of resistance gene cassettes and just one transfer event is enough to disseminate multidrug resistance. Moreover, carbapenemase genes are often located within Mobile Genetic Elements. For these reasons carbapenemases are the most epidemiologically importance. Early detection and identification of carbapenemase producers among clinical isolates can avoid nosocomial infections. We have developed a multiplex Real-Time PCR assay based on the TaqMan technology for rapid detection and identifications of the most common carbapenemases in Europe NDM, VIM, KPC and IMP. There were tested 31 isolates Enterobacteriaceae (n=15) and non-fermentative Gram-negative bacillary (n=16), which acquire NDM, VIM, KPC, IMP, GIM or OXAs carbapenemases. The whole elaborated experiment, including DNA isolation and PCR cycling, lasts up to 2 h.