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. Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC). Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189. Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.

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 E scherichia 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.

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.

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.

To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital. OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53. Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%–29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The bla_OXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid. OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period.

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.