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Journal of Clinical Microbiology, February 2009, p. 322-326, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01550-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes{triangledown}

Justin M. Cole,{dagger} Audrey N. Schuetz,{ddagger} Charles E. Hill, and Frederick S. Nolte*

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

Received 11 August 2008/ Returned for modification 8 October 2008/ Accepted 19 November 2008

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 blaKPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the blaKPC 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 β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the blaKPC PCR assay was used to confirm the presence of blaKPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected blaKPC 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 blaKPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two blaKPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with blaKPC-2 and blaKPC-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.

* Corresponding author. Present address: Medical University of South Carolina, Pathology and Laboratory Medicine, 165 Ashley Ave., Suite 309, P.O. Box 250908, Charleston, SC 29425. Phone: (843) 792-5020. Fax: (843) 792-7060. E-mail: nolte{at}musc.edu

{triangledown} Published ahead of print on 26 November 2008.

{dagger} Present address: University of Washington, Department of Laboratory Medicine, Seattle, WA 98109.

{ddagger} Present address: The New York-Presbyterian Hospital-Weill Cornell Medical College, New York, NY 10065.

Journal of Clinical Microbiology, February 2009, p. 322-326, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01550-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Toye, B., Krajden, S., Fuksa, M., Low, D. E., Pillai, D. R. (2009). Carbapenem resistance in Canada. CMAJ 180: 1225-1226 [Full Text]  


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