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.
"The plate was incubated overnight at 37°C. Zone of inhibition of the Meropenem 10µg tablet was compared to the zone of inhibition of each Meropenem + inhibitor tablets (Van der Bij et al., 2011). Antibiotic tablets/cartridges were purchased from Roche Diagnostics, U.S. "
[Show abstract][Hide abstract] ABSTRACT: An aquo-alcoholic (1:1) extract of Camellia sinensis (PTRC-31911-A), containing 17.95±0.02 μg of quercetin / mg of dried extract exhibited [Flavonoid/ Polyphenol: F/P (Quercetin %) ~ 0.26 (1.68%)], taken as standardized indicator. The bioactivity fingerprint profile of PTRC-31911-A includes IC50 ratio [DPPH/NOS] = 0.092 as functional standardized value having IC50 (DPPH Scavenging) = 30.332 ± 0.5 μg/ml and IC50 (Nitric Oxide Scavenging) = 326.723 ± 0.5 μg/ml respectively. The reducing ability and anti-lipid peroxidation equivalent (Extract: Standard) of PTRC-31911-A with respect to standard was estimated to be 10.09 (Ascorbic acid) and 1.96 (Quercetin) respectively. The results also showed that the Minimum Inhibitory Concentration (MIC) of PTRC-31911-A extract against New Delhi Metallo-beta-lactamase-1 (NDM-1) Escherichia coli was 500μg/ml. While the respective zones of inhibition against the food borne pathogens i.e., Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahemolyticus, Salmonella typhimurium, E. coli O157:H7 and Shigella sonnei were in the effective range of 12-23 mm. Efficacy in terms of MIC against selected food borne pathogens was found to be in the range of 100 – 350 μg/ml. This broad spectrum antimicrobial activity of PTRC-31911-A could most possibly be attributed to its high total contents of phenolic compounds, flavonoids such as quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) as identified in this study. Further pre-clinical efficacy studies in animal system studies are warranted.
International Journal of Biological & Pharmaceutical Research 01/2015; 6(8):606-616.
[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.
[Show abstract][Hide abstract] ABSTRACT: Clin Microbiol Infect 2012; 18: E369–E372
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.
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