The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Catholic University of Louvain

To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates.

Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered at two Belgian hospitals.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing KPC-2 carbapenemase in Belgium.

During a PCR-based surveillance study of β-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumannii isolates were detected at 6 Belgian hospitals. DNA sequencing of the bla(GES) genes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n = 2), Egypt (n = 2), and Palestinian territories (Gaza) (n = 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located bla(GES-11) and bla(GES-12) and a likely chromosomally located novel variant bla(GES-14)) were detected as part of a class 1 integron, also including the aac6'Ib and dfrA7 genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing bla(GES-11) and bla(GES-12). Cloning of the bla(GES) genes in Escherichia coli identified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various bla(GES-like) genes, especially those conferring carbapenem resistance in A. baumannii and its importation in Western Europe from Middle Eastern countries.

GES extended-spectrum β-lactamases in Acinetobacter baumannii isolates in Belgium.

Five multidrug-resistant nonclonally related Enterobacteriaceae isolates were recovered in Belgium in 2010 from three patients who had been hospitalized in Pakistan, Montenegro, and Serbia/Kosovo. New Delhi metallo-β-lactamase (NDM-1) was detected in each of the isolates in addition to several extended-spectrum β-lactamases (CTX-M-15, SHV-12), plasmidic cephalosporinases (CMY-16, CMY-58), rRNA methylases (ArmA, RmtB), and Qnr genes (qnrA6, qnrB1, qnrB2). One patient died from uncontrolled sepsis, while the two others recovered. No secondary cases occurred in any of the hospitals.

Emergence of NDM-1-producing Enterobacteriaceae in Belgium.

A new commercial low-density microarray which identifies common extended-spectrum β-lactamase plasmid-mediated cephalosporinase genes, as well as carbapenemase (bla(KPC) and bla(NDM)) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceae possessing different bla genes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated bla(AmpC), bla(KPC), and bla(NDM) genes using bla gene sequencing as the reference method.

Multicenter evaluation of a new DNA microarray for rapid detection of clinically relevant bla genes from beta-lactam-resistant gram-negative bacteria.

Rapid diagnosis and appropriate empirical antimicrobial therapy before the availability of conventional microbiological results is of pivotal importance for the clinical outcome of ventilator-associated pneumonia (VAP). We evaluated the VAPChip, a novel, closed cartridge molecular tool aiming to identify directly from clinical samples and within a working day the principal bacteria causative of VAP as well as clinically relevant β-lactam resistance genes.

Analytical validation of a novel high multiplexing real-time PCR array for the identification of key pathogens causative of bacterial ventilator-associated pneumonia and their associated resistance genes.

To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum β-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories.

Validation of carbapenemase and extended-spectrum β-lactamase multiplex endpoint PCR assays according to ISO 15189.

A national survey was conducted to determine the prevalence and risk factors of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamases-producing Enterobacteriaceae (ESBLE) and vancomycin-resistant enterococci (VRE) among nursing home residents in Belgium.

Epidemiology of multidrug-resistant microorganisms among nursing home residents in Belgium.

Accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) constitutes a major laboratory diagnostic challenge. We evaluated an electrochemical technique (the BYG Carba test) which allows detection of CPE in less than 35 min. The BYG Carba test was first validated in triplicate against 57 collection isolates with previously characterized β-lactam resistance mechanisms (OXA-48, n = 12; KPC, n = 8; NDM, n = 8; VIM, n = 8; IMP, n = 3; GIM, n = 1; GES-6, n = 1; no carbapenemase, n = 16) and against a panel of 10 isolates obtained from the United Kingdom National External Quality Assessment Service (NEQAS). The test was then evaluated prospectively against 324 isolates referred to the national reference center for suspicion of CPE. The BYG Carba test results were compared with those obtained with the Carba NP test using multiplex PCR sequencing as the gold standard. Of the 57 collection and the 10 NEQAS isolates, all but one GES-6-producing isolate were correctly identified by the Carba BYG test. Among the 324 consecutive Enterobacteriaceae isolates tested prospectively, 146 were confirmed as noncarbapenemase producers by PCR while 178 harbored a carbapenemase gene (OXA-48, n = 117; KPC, n = 25; NDM, n = 23; and VIM, n = 13). Prospectively, in comparison with PCR results, the BYG Carba test displayed 95% sensitivity and 100% specificity versus 89% and 100%, respectively, for the Carba NP test. The BYG Carba test is a novel, rapid, and efficient assay based on an electro-active polymer biosensing technology discriminating between CPE and non-CPE. The precise electrochemical signal (electrochemical impedance variations) allows the establishment of real-time objective measurement and interpretation criteria which should facilitate the accreditation process of this technology.

Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae.

The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array 'Check-MDR CT103 XL' (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs (blaTEM, blaSHV, blaCTX-M, blaBEL, blaPER, blaGES and blaVEB), pAmpCs (blaCMY-2-like, blaDHA, blaFOX, blaACC-1, blaACT/MIR and blaCMY-1-like/MOX) and carbapenemases (blaKPC, blaOXA-48, blaVIM, blaIMP, blaNDM, blaGIM, blaSPM and blaOXA-23, -24 and -58) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for blaIMP. The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents.

Evaluation of a DNA microarray for rapid detection of the most prevalent extended-spectrum β-lactamases, plasmid-mediated cephalosporinases and carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) represents a major challenge for microbiology laboratories. We evaluated the BYG Carba v2.0 using a simplified protocol, which detects CPE in less than 30 minutes. This new procedure reduces the hands-on-time from 5 to one minute and only requires a limited amount of material (one to three colonies) thereby preventing the need for subculturing bacterial isolates to reach a larger amount of pure biomass. This multicentre study involved four European reference laboratories. For the 1181 isolates tested across four centres, BYG Carba v2.0 yielded overall sensitivity and specificity of 96.3% (CI95: 94.5-97.5) and 99.7% (CI95: 98.6-100) respectively. Considering only the 670 consecutive isolates tested prospectively, the BYG Carba v2.0 displayed overall positive and negative predictive values of 99.7% (CI95: 95.4-98.9) and 97.5% (CI95: 94.9-98.8). Regarding time to positivity, 85% of CPE detected were positive within ten minutes. The BYG Carba v2.0 is a new highly simplified, rapid and accurate electrochemical assay discriminating between CPE and non-CPE in less than 30 min. The real-time quantified signal allows objective and traceable interpretation of the results.

Multicentre evaluation of the BYG Carba v2.0 test, a simplified electrochemical assay for the rapid laboratory detection of carbapenemase-producing Enterobacteriaceae.

Pierre Bogaerts

Laboratory of Clinical Microbiology

Catholic University of Louvain

The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Pierre Bogaerts

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Clinical Microbiology

Catholic University of Louvain

The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Laboratory of Clinical Microbiology