Direct Detection and Genotyping of KPC Carbapenemases from Urine using a new DNA Microarray Test

Abstract

Klebsiella pneumoniae carbapenemases (KPC) are considered a serious threat to antibiotic therapy as they confer resistance to carbapenems, which are used to treat Extended-spectrum beta-lactamase (ESBL) producing bacteria. Here, we describe the development and evaluation of a DNA microarray for detection and genotyping of KPC genes (blaKPC) within 5 hours. To test the whole assay procedure (DNA extraction + DNA microarray assay) directly from clinical specimen, we compared two commercial DNA extraction kits (QIAprep Spin Miniprep Kit (Qiagen), Urine Bacterial DNA Isolation Kit (Norgen)) for the direct DNA extraction from urine samples (dilution series spiked in human urine). A reliable SNP typing from 1×105 CFU/mL urine was demonstrated for Escherichia coli (Qiagen and Norgen) and 80 CFU/mL urine on average for K. pneumoniae (Norgen). The study presents for the first time the combination of a new KPC-microarray with commercial sample preparation for the detection and genotyping of microbial pathogens directly from clinical specimen which paves the way towards tests providing epidemiological and diagnostic data enabling better antimicrobial stewardship.