Multiplex real-time PCR assay for detection and classification of Klebsiella pneumoniae carbapenemase gene (bla KPC) variants

Abstract

Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla(KPC)) have been reported, with KPC-2 (bla(KPC-2)) and KPC-3 (bla(KPC-3)) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla(KPC) variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla(KPC) variants (bla(KPC-2) to bla(KPC-11)). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla(KPC) variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla(KPC-3) and bla(KPC-2) identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla(KPC) variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.

Fig. 1.

Hypothetical stepwise evolution of bla_KPC variants, based on single nucleotide changes and order of discovery. In this scenario, bla_KPC-2 is the prototype from which all subsequent variants have arisen (44) and gives rise by stepwise nonsynonymous substitution to bla_KPC-3 (46), bla_KPC-5 (44), bla_KPC-6 (33), and bla_KPC-11 (GenBank accession no. HM066995). Similarly, bla_KPC-3 gives rise to bla_KPC-7 (30), bla_KPC-8 (15), and bla_KPC-10 (31), while bla_KPC-4 (27) and bla_KPC-9 (FJ624872) each differ by two nucleotides from bla_KPC-2 and bla_KPC-3, respectively. Alternatively, bla_KPC-10 and bla_KPC-11 may be derived from bla_KPC-5, bla_KPC-4 may be derived from either bla_KPC-5 or bla_KPC-6, and bla_KPC-9 may be derived from bla_KPC-8. Nucleotide sequences for each bla_KPC variant are deposited in GenBank (NCBI) under the following accession numbers: AY034847 (bla_KPC-2), AF395881 (bla_KPC-3), AY700571 (bla_KPC-4), EU400222 (bla_KPC-5), EU555534 (bla_KPC-6), EU729727 (bla_KPC-7), FJ234412 (bla_KPC-8), FJ624872 (bla_KPC-9), GQ140348 (bla_KPC-10), and HM066995 (bla_KPC-11).

Fig. 2.

Real-time PCR amplification results for bla_KPC-2 (A) and bla_KPC-3 (B), obtained after 40 cycles on the Stratagene Mx3005P multiplex quantitative PCR system. The multiplex real-time MB-PCR assay consists of six molecular beacon probes labeled with five different fluorescent dyes, as indicated in the legend.