Rapid screening of epidemiologically important Salmonella enterica subsp. enterica serovars by Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) mass spectrometry

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Moury, Frédérique | Gassilloud, Benoît | Py, Jean-Sébastien | Amandine, Wilhelm | Ribeyre, Antonin | Piquet, Christine | Morel, Viviane | Lailler, Renaud | Six, Sabrina Cadel

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International audience. IntroductionIn the last decade the use of MALDI-TOF mass spectrometry for the identification of bacteria has been successfully integrated in laboratories of clinical microbiology. This revolutionary technique allows making the diagnosis of pathogens more easily and quickly compared to conventional methods. The aim of this study was to evaluate the potential of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for identification of epidemiologically important Salmonella enterica subsp. enterica serovars. Indeed conventional serotyping, which is considered the gold standard for classification of Salmonella, is delicate procedure that is laborious, time-consuming and expensive. Material and methodsTwenty strains of Salmonella enterica subsp. enterica corresponding to 20 different serovars, were analyzed in 3 separate series after grow on 3 different culture medium at 36°C during 24 hours. The mass spectrometry MALDITOF identification was realized with a Microflex LT® (Bruker Daltonics) after proteomic extraction with ethanol, formic acid and acetonitrile. To explore the impact of the extraction proceed on the spectra generated, either isolated colonies or aggregate colonies were used separately for the proteomic extractions. The volumes of the chemical products used for the extractions were adapted within the range required by the provider and 8 sample deposits for each strain were carried out in 96 microwells plate. The HCCA matrix (Acide α-cyano- 4-Hydroxycinnamique), was used for all the analyses and especially prepared during each series of analyses to avoid an aging effect. The 8 deposits were run 4 independent times to acquire 32 spectra for each strain in analytical reproducibility requirement. An operator effect was included in the analytical sequence to include a possible bias related to the preparation. References spectra for each serovars and medium tested were generated using Flex analysis® software and store in a specific data base (DB). The spectra analysis and their comparison were operated using Maldi BioyperTM, and Clinprotool® software. To test the robustness of the database built and the reproducibility of the protocol developed blind tests were realized.ResultsANSES Laboratory of Nancy (France) developed a data base of spectra of 20 strains of Salmonella enterica subsp. enterica corresponding to 20 different serovars, the most frequently isolated in France. The results obtained show that it is possible to fast identify the serovars which presented characteristic spectra as S. Senftenberg, S. Agona,S. Tenessee or S Napoli. For others serovars the discrimination is more complex and needs mathematical algorithms for the discovery of specific biomarkers. Repeatability and reproducibility assays are necessary to avoid misinterpretation.ConclusionFirst results on the 20 serovars analyzed show that MALDI-TOF mass spectrometry could be a rapid method for prescreening S. enterica subsp. enterica to identify epidemiologically important serovars. However discrimination between serovars requires exploring the presence or absence of specific biomarker-peaks and should integrate analysis of reproducibility to avoid false interpretation. Impact of the media used for the culture of the bacteria will be also included in the study in the aim to enhance reliability of the serovars discrimination scheme.

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