Rapid and sensitive detection of bla KPC gene in clinical isolates of Klebsiella pneumoniae by a molecular real-time assay

Adriana Mosca,Laura Pagani,Giuseppe Miragliotta,Raffaele Del Prete,Gerasimos Tzakis,Francesco Bruno,Aurora Piazza,Lidia Dalfino,Luisa Miragliotta,Roberta Migliavacca

doi:10.1186/2193-1801-2-31

Abstract

BackgroundThe aim of this study was the rapid identification of blaKPC gene in 38 Klebsiella pneumoniae clinical isolates with reduced susceptibility to carbapenems. The modified Hodge Test (MHT) was carried out to phenotypically determine whether resistance to carbapenems was mediated by a carbapenemase. The detection of the blaKPC gene was performed by real-time acid nucleic sequence-based amplification (NASBA™™), specifically designed for the detection of KPC RNA target.ResultsThirty-two/38 isolates evaluated by MHT showed the production of carbapenemases, while all the strains exhibited the production of KPC by inhibition test with phenylboronic acid (the combined disk test with IPM/IPM plus phenylboronic acid). The detection of blaKPC gene by Nuclisens EasyQ KPC yielded positive results in 38/38 (100%) strains. The presence of blaKPC gene was confirmed in all K. pneumoniae isolates when tested by the gold standard PCR assay.ConclusionsIn consideration of the serious challenge represented by infections due to K. pneumoniae it appears necessary the rapid identification of carbapenemases in clinical settings as it is made possible by the use of NASBA™ assay.

Highlights

Over the last decade carbapenemase-producing Enterobacteriaceae have emerged and these multidrug-resistant pathogens became a problem in the clinical care of patients
We evaluated the performance of a new molecular assay (NASBATM, NucliSens EasyQKPC, bioMérieux, France), for the rapid detection of blaKPC gene in isolates of K. pneumoniae from patients hospitalized in ICU, as well as in Medical and Surgical wards of the teaching hospital Policlinico of Bari, Bari, Italy
The isolates were further investigated by combined disk test with IPM and IPM plus phenylboronic acid (PBA) or ethylenediaminetetraacetic acid (EDTA) as inhibitors of Klebsiella pneumoniae carbapenemase (KPC) or metallo beta-lactamases (MBLs), respectively (Tsakris et al 2009)

Summary

Introduction

Over the last decade carbapenemase-producing Enterobacteriaceae have emerged and these multidrug-resistant pathogens became a problem in the clinical care of patients. Among Enterobacteriaceae, Klebsiella pneumoniae carbapenemase (KPC)-producing strains of K. pneumoniae broadly disseminated worldwide (Nordmann et al 2009). Misidentification of KPC-producing bacteria is common with standard susceptibility testing (Nordmann et al 2009), whereas the presence of a KPC may cause MIC elevations that remain within the susceptible or patients hospitalized in ICU, as well as in Medical and Surgical wards of the teaching hospital Policlinico of Bari, Bari, Italy. The aim of this study was the rapid identification of blaKPC gene in 38 Klebsiella pneumoniae clinical isolates with reduced susceptibility to carbapenems. The detection of the blaKPC gene was performed by real-time acid nucleic sequence-based amplification (NASBATM), designed for the detection of KPC RNA target

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