Development and evaluation of a real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase genes - PubMed (original) (raw)

Development and evaluation of a real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase genes

Justin M Cole et al. J Clin Microbiol. 2009 Feb.

Abstract

We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of bla(KPC) genes. The TaqMan real-time PCR assay amplified a 399-bp product from the bla(KPC) gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum beta-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the bla(KPC) PCR assay was used to confirm the presence of bla(KPC) genes in any meropenem-resistant Klebsiella spp. The PCR assay detected bla(KPC) in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of bla(KPC) in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two bla(KPC) gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with bla(KPC-2) and bla(KPC-3), respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Real-time PCR assay for _bla_KPC. Real-time PCR using a TaqMan probe generates a 399-bp amplicon for all _bla_KPC genes. Amplicons from positive samples can be digested with BstNI and RsaI to detect the nucleotide polymorphisms in the PCR amplicon reported for _bla_KPC-1, _bla_KPC-2, and _bla_KPC-3 at positions 650 and 944 corresponding to GenBank accession no. AF297554. Based on the above sequence polymorphisms, restriction endonuclease digestion of the PCR amplicons should yield fragments of 359 bp and 40 bp for KPC-2 and 295 bp, 64 bp, and 40 bp for KPC-3. The KPC-1 amplicon is not cleaved by BstNI and RsaI.

References

    1. Alba, J., Y. Ishii, K. Thomson, E. S. Moland, and K. Yamaguchi. 2005. Kinetics study of KPC-3, a plasmid-encoded class A carbapenem-hydrolyzing beta-lactamase. Antimicrob. Agents Chemother. 494760-4762. - PMC - PubMed
    1. Anderson, K. F., D. R. Lonsway, J. K. Rasheed, J. Biddle, B. Jensen, L. K. McDougal, R. B. Carey, A. Thompson, S. Stocker, B. Limbago, and J. B. Patel. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 452723-2725. - PMC - PubMed
    1. Bradford, P. A., C. Urban, N. Mariano, S. J. Projan, J. J. Rahal, and K. Bush. 1997. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41563-569. - PMC - PubMed
    1. Bratu, S., S. Brooks, S. Burney, S. Kochar, J. Gupta, D. Landman, and J. Quale. 2007. Detection and spread of Escherichia coli possessing the plasmid-borne carbapenemase KPC-2 in Brooklyn, New York. Clin. Infect. Dis. 44972-975. - PubMed
    1. Bratu, S., D. Landman, R. Haag, R. Recco, A. Eramo, M. Alam, and J. Quale. 2005. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch. Intern. Med. 1651430-1435. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources