Abstract
BackgroundKlebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome. Laboratory identification of KPCs-harboring clinical isolates would be a key to limit the spread of the bacteria. This study would evaluate a rapid low-cost real-time PCR assay to detect KPCs.MethodsReal-time PCR assay based on SYBR GreenIwas designed to amplify a 106bp product of the blaKPC gene from the159 clinical Gram-negative isolates resistant to several classes of -lactam antibiotics through antimicrobial susceptibility testing. We confirmed the results of real-time PCR assay by the conventional PCR-sequencing. At the same time, KPCs of these clinical isolates were detected by the modified Hodge test (MHT). Then we compared the results of real-time PCR assay with those of MHT from the sensitivity and specificity. Moreover, we evaluated the sensitivity of the real-time PCR assay.ResultsThe sensitivity and specificity of the results of the real-time PCR assay compared with those of MHT was 29/29(100%) and 130/130(100%), respectively. The results of the real-time PCR and the MHT were strongly consistent (Exact Sig. (2-tailed) =1. 000; McNemar test). The real-time PCR detection limit was about 0.8cfu using clinical isolates.ConclusionThe real-time PCR assay could rapidly and accurately detect KPCs -harboring strains with high analytical sensitivity and specificity.
Highlights
Klebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome
The specificities of the real-time PCR primers for the detection of blaKPC genes were evaluated by the BLAST search program, available at www.ncbi.nlm.nih.gov
The blaKPC real-time PCR assay was negative with DNA extracted from the following reference bacterial isolates: K.pneumoniae ATCC 13883, extended-spectrumb-lactamase-positive K.pneumoniae ATCC 700603, A. baumanii ATCC 19606, P. aeruginasa ATCC 27853, C. albicans ATCC 90029, E. coli
Summary
Klebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome. Laboratory identification of KPCs-harboring clinical isolates would be a key to limit the spread of the bacteria. This study would evaluate a rapid low-cost real-time PCR assay to detect KPCs. Carbapenems are widely used to treat serious infections caused by multi-resistant Gram-negative bacteria. KPCs are able to hydrolyze the carbapenems, and cause resistance to multiple classes of antibiotics. The failure of automated susceptibility testing systems to detect KPC-mediated carbapenems resistance was previously reported [12,13,14]. In 2009, the Clinical Laboratory Standards Institute (CLSI) guidelines (M100) recommended MHT to detect carbapenemase production. Rapid and sensitive blaKPC assays are critical to control the spread of blaKPC-harboring bacteria in hospitalized patients
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