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      Evaluation of MALDI-ToF Mass Spectrometry for Rapid Detection of Cereulide From Bacillus cereus Cultures

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          Abstract

          Bacillus cereus plays an often unrecognized role in food borne diseases. Food poisoning caused by this pathogen is manifested by either diarrhea or emesis. Due to the relatively high prevalence of emetic toxin cereulide associated food poisoning, methods for simple and reliable detection of cereulide producing strains are of utmost importance. Recently, two different studies reported on the application of MALDI-ToF MS for either the differentiation of emetic and non-emetic strains of B. cereus or for direct detection of cereulide from bacterial colony smears. However, for implementation of cereulide detection using MALDI-ToF MS in routine microbiological diagnostics additional investigations on the sensitivity and specificity as well as on the fitting into common workflows for bacterial identification are needed. These aspects prompted us to investigate open issues and to test sample preparation methods, commonly used for microbial identification for their suitability to detect the emetic toxin from bacteria. Based on our experimental findings we propose a workflow that allows identification of B. cereus and sensitive detection of cereulide in parallel, using linear-mode MALDI-ToF MS equipment. The protocol was validated in a blinded study and is based on the well-established ethanol/formic acid extraction method. Cereulide is detected in the ethanol wash solution of samples identified as B. cereus as peaks at m/z 1175 and 1191. Peak position difference of 16 Th (Thomson) indicates detection of the sodium and potassium adducts of cereulide. This sample treatment offers possibilities for further characterization by more sophisticated LC-MS-based methods. In summary, the ease of use and the achieved level of analytical sensitivity as well as the wide-spread availability of MALDI-ToF MS equipment in clinical microbiological laboratories provides a promising tool to improve and to facilitate routine diagnostics of B. cereus associated food intoxications.

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          Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

          Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.
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            Phylogenetic classification and identification of bacteria by mass spectrometry.

            Bacteria are a convenient source of intrinsic marker proteins, which can be detected efficiently by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The patterns of protein masses observed can be used for accurate classification and identification of bacteria. Key to the reliability of the method is a robust and standardized procedure for sample preparations, including bacterial culturing, chemical treatment for bacterial cell wall disruption and for protein extraction, and mass spectrometry analysis. The protocol is an excellent alternative to classical microbiological classification and identification procedures, requiring minimal sample preparation efforts and costs. Without cell culturing, the protocol takes in general <1 h.
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              Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs

              (2016)
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                06 October 2020
                2020
                : 11
                : 511674
                Affiliations
                [1] 1Proteomics and Spectroscopy Unit (ZBS 6), Centre for Biological Threats and Special Pathogens, Robert Koch Institute , Berlin, Germany
                [2] 2Food Chemistry and Molecular Sensory Science, Technical University of Munich , Munich, Germany
                [3] 3Functional Microbiology, Institute of Microbiology, Department of Pathobiology, University of Veterinary Medicine Vienna , Vienna, Austria
                Author notes

                Edited by: Aldo Corsetti, University of Teramo, Italy

                Reviewed by: Sean Doyle, Maynooth University, Ireland; Laura Sanchez, University of Illinois at Chicago, United States

                *Correspondence: Joerg Doellinger, doellingerj@ 123456rki.de

                This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2020.511674
                7709880
                64535334-85e5-4650-a7d4-e1ffd66fda1d
                Copyright © 2020 Doellinger, Schneider, Stark, Ehling-Schulz and Lasch.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 15 November 2019
                : 16 September 2020
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 30, Pages: 12, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                cereulide,b. cereus,maldi-tof,mass spectrometry,toxin detection
                Microbiology & Virology
                cereulide, b. cereus, maldi-tof, mass spectrometry, toxin detection

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