Detection of Klebsiella pneumoniae Carbapenem Resistance Genes by qPCR: Choosing the Right Method for Total DNA Extraction

Abstract

Rapid and accurate detection of Klebsiella pneumoniae carbapenem resistance is important for infection control and targeted antibiotic therapy. PCR-based assay performance heavily depends on the quality and quantity of template DNA. Challenges arise from the necessity to isolate chromosomal and large plasmid-encoded resistance genes simultaneously from a limited number of target cells and to remove PCR inhibitors. qPCRs for the detection of K. pneumoniae strains carrying blaOXA-48, blaNDM-1, blaKPC-2, and blaVIM-1 carbapenemase genes were developed. We compared the performance of template DNA extracted with silica column-based methods, reversed elution systems, and lysis-only methods either from diluted culture fluid or from a synthetic stool matrix which contained PCR inhibitors typically present in stool. The synthetic stool matrix was chosen to mimic K. pneumoniae containing rectal swabs or stool samples in a reproducible manner. For total DNA isolated from culture fluid, resistance gene detection by qPCR was always possible, independent of the extraction method. However, when total DNA was isolated from synthetic stool matrix spiked with K. pneumoniae, most methods were insufficient. The best performance of template DNA was obtained with reversed elution. This highlights the importance of choosing the right DNA extraction method for consistent carbapenem resistance detection by PCR.