The laboratory diagnosis of Pseudomonas aeruginosa that produce metallo-β-lactamases in a Dutch tertiary care centre.
ABSTRACT The laboratory detection of Pseudomonas aeruginosa that produce metallo-β-lactamases (MBLs) is not well defined in regions with a low prevalence of these enzymes. We report a study that developed ethylenediaminetetraacetic acid (EDTA) disk screen tests using doripenem, imipenem and meropenem and investigated the prevalence of these enzymes among clinical isolates of imipenem-resistant P. aeruginosa in Rotterdam during 2008-2009.
Using strains with well-characterized β-lactamases and the Clinical and Laboratory Standards Institute (CLSI) disk methodology similar to extended-spectrum β-lactamase (ESBL) detection, inhibition zone diameters were determined in tests with doripenem, imipenem, and meropenem, alone and in combination with 370 μg of EDTA. These tests were compared with the MBL E-test. A positive test was a ≥5 mm increase in zone diameter in the presence of EDTA.
The imipenem EDTA disk screen test showed a sensitivity of 100% and a specificity of 90% in 96 recent clinical isolates. Imipenem in combination with doripenem performed better than imipenem alone, meropenem, and the MBL E-test (sensitivity of 100%; specificity of 95%). The majority of clinical isolates were isolated from patient respiratory specimens. Of the 96 imipenem-resistant P. aeruginosa isolated, 35 (36%) were positive for bla(VIM) genes.
The EDTA imipenem/doripenem disk test showed accurate and reproducible results with excellent sensitivity and specificity. It is simple to perform and interpret and can be easily introduced into the workflow of a clinical laboratory to screen for MBLs in imipenem-resistant P. aeruginosa. Due to its high specificity the test is also suitable for regions with a low prevalence of these enzymes.
SourceAvailable from: Lieuwe Roorda[Show abstract] [Hide abstract]
ABSTRACT: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.BMC Infectious Diseases 01/2014; 14(1):27. DOI:10.1186/1471-2334-14-27 · 2.56 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Recently, the first outbreak of clonally related VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in a Dutch tertiary-care centre was described. Subsequently, a nationwide surveillance study was performed in 2010-2011, which identified the presence of VIM-2 MBL-producing P. aeruginosa in 11 different hospitals. Genotyping by multiple-locus variable-number tandem-repeat analysis (MLVA) showed that the majority of the 82 MBL-producing isolates found belonged to a single MLVA type (n = 70, 85%), identified as ST111 by multilocus sequence typing (MLST). As MBL-producing isolates cause serious infections that are difficult to treat, the presence of clonally related isolates in various hospitals throughout the Netherlands is of nationwide concern.Clinical Microbiology and Infection 06/2012; 18(9):E369-72. DOI:10.1111/j.1469-0691.2012.03969.x · 5.20 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa is a growing issue across the globe. Fast and reliable diagnostic tools are needed for appropriate implementation of infection control measures. In this study we evaluated the performance of three commercial combined disk tests, two EDTA based in-house combined disk tests and the Carba NP test in comparison to molecular detection of MBL genes on 133 meropenem non-susceptible non-duplicate P. aeruginosa clinical isolates. The meropenem/DPA based commercial KPC + MBL-confirm ID kit (Rosco Diagnostica, Denmark) and the MASTDISCS™ ID carbapenemase (Enterobacteriaceae) detection disc set (MAST Diagnostics, UK) showed sensitivities of 31.1 % and 28.8 % and specificities of 69.3 % and 79.6 %, respectively. The total MBL confirm kit (Rosco Diagnostica, Denmark) contains imipenem/DPA and imipenem/EDTA combination disks. Evaluation of the single disk combinations revealed 84.4 % sensitivity and 81.8 % specificity for the imipenem/DPA assay and 86.7 % sensitivity and 51.1 % specificity for the imipenem/EDTA test. Applying both tests simultaneously resulted in a slightly higher sensitivity of 88.9 % but a lower specificity of 48.9 % when compared to the single tests alone. The Carba NP test showed 93.3 % sensitivity and 96.6 % specificity. All phenotypic combined disk tests lacked either sensitivity or specificity for the detection of MBL in P. aeruginosa. The Carba NP test showed excellent test properties, but suffers from drawbacks in handling and high costs. The optimal diagnostic approach needs to be chosen depending on the epidemiological situation, laboratory resources and availability of molecular confirmation tests.European Journal of Clinical Microbiology 01/2014; DOI:10.1007/s10096-014-2059-1 · 2.54 Impact Factor