Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Identification of Mycobacteria in Routine Clinical Practice

Background: Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed. Methodology: We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10 5 colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25M. avium and 12 non-tuberculosis clinical isolates with identification scores $2 within 2.5 hours. Conclusions: Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heatinactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories i

Solubilisation and binding characteristics of a recombinant beta(2)-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals

The number of substances with beta-agonistic activity, illegally introduced in meat production or in sports doping as anabolic or beta-blocking agents is increasing. Analytical methods suited for their multianalyte detection are thus necessary. In this perspective, receptor assays were developed. The research activities undertaken in this study describe the solubilisation of a recombinant human beta(2)-adrenergic receptor produced in the inner membrane of genetically modified Escherichia coli, using the detergent n-dodecyl-beta-D-maltoside. Its potential to detect the presence of beta-agonists or beta-blockers in biological samples was evaluated. The solubilised beta(2)-adrenergic receptor retained its binding affinity in a radio-receptor assay based on the competition for the binding to receptors between a ligand (beta-agonist or antagonist) and the radioligand [I-125]iodocyanopindolol. The IC50 values ranged from 5 +/- x 10(-8) M (clenbuterol) to 8 +/- 2 x 10(-6) M (isoxsuprine) for the beta-agonists tested and from 1.5 +/- 0.2 x 10(-10) M (carazolol) to 1.2 +/- 0.2 x 10(-5) M (metoprolol) for the beta-blockers tested. It was shown to have a lower limit of detection than a radio-receptor assay using the solubilised beta(2)-adrenoceptor expressed in a mammalian cell line. The solubilised recombinant human beta(2)-adrenoreceptor expressed in E. coli would be a useful tool to develop non radioactive multianalyte screening methods. (c) 2007 Elsevier B.V. All rights reserved

Adaptation of a microbiological method to detect antimicrobial residues in shrimp tissue from Vietnam

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