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1. Use of Ratiometric Probes with a Spectrofluorometer for Bacterial Viability Measurement

Author

Thierry Grard, Denis Watier, Jé r ô me Cleach, Guillaume Duflos, Bruno Le Fur, Thomas Brauge, Philippe Lencel, Université du Littoral Côte d'Opale (ULCO), Plate Forme d'Innovations Nouvelles Vagues, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Pole Metropolitain de la Cote d'Opale council, PFI Nouvelles Vagues, French government, Hauts-de-France region of the CPER 2014-2020 MARCO project, FranceAgrimer, Institut National de la Recherche Agronomique (INRA), Biochimie des Produits Aquatiques (BPA), Université du Littoral Côte d'Opale (ULCO)-Transfrontalière BioEcoAgro - UMR 1158 (BioEcoAgro), Université d'Artois (UA)-Université de Liège-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Université d'Artois (UA)-Université de Liège-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA), and Université catholique de Lille (UCL)-Université catholique de Lille (UCL)

Subjects
0301 basic medicine, Bacterial viability, [SDV]Life Sciences [q-bio], 030106 microbiology, Ionophore, Applied Microbiology and Biotechnology, Rhodamine 123, Fluorescence spectroscopy, Membrane Potentials, 03 medical and health sciences, chemistry.chemical_compound, Escherichia coli, Fluorescence microscope, Fluorescent Dyes, Membrane potential, Microbial Viability, Ionophores, Chemistry, Spectrofluorometer, General Medicine, Fluorescence, Spectrometry, Fluorescence, Biophysics, fluorescence techniques, membrane potential, Cytometry, Biotechnology
Abstract

International audience; Assessment of microorganism viability is useful in many industrial fields. A large number of methods associated with the use of fluorescent probes have been developed, including fluorimetry, fluorescence microscopy, and cytometry. In this study, a microvolume spectrofluorometer was used to measure the membrane potential variations of Escherichia coli. In order to estimate the sensitivity of the device, the membrane potential of E. coli was artificially disrupted using an ionophore agent: carbonyl cyanide 3-chlorophenylhydrazone. The membrane potential was evaluated using two ratiometric methods: a Rhodamine 123/4',6-diamidino-2-phenylindole combination and a JC-10 ratiometric probe. These methods were used to study the impact of freezing on E. coli, and were compared with the conventional enumeration method. The results showed that it was beneficial to use this compact, easy-to-use, and inexpensive spectrofluorometer to assess the viability of bacterial cells via their membrane potential.

Published
2018
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2. Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

Author

Jean-Jacques Joffraud, Souad Christieans, Catherine Denis, Carole Feurer, Xavier Dousset, Marie-France Pilet, Dephine Roux, Hervé Prévost, Stéphanie La Carbona, Aurélie Chaulot-Talmon, Sylvie Lorre, Marina Rivollier, Monique Zagorec, Sabine Leroy, Françoise Leroi, Régine Talon, Mireille Cardinal, Erwann Hamon, Hélène Caekebeke, Marie Hélène Desmonts, Sabrina Macé, Stéphane Chaillou, Marie-Christine Champomier-Vergès, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Plate Forme d'Innovations Nouvelles Vagues, Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Association pour le Développement de l'Industrie de la Viande, ACTALIA [Villers-Bocage], Aérial - Centre de Ressources Technologiques et Institut Technique Agro-Industriel, UMR 1014 SECurité des ALIments et Microbiologie, Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Oniris, PRES Université Nantes Angers Le Mans (UNAM), Institut du Porc (IFIP), Unité de Microbiologie (MIC), Institut National de la Recherche Agronomique (INRA), Biocéane, French Agence Nationale de la Recherche (ANR) within ALIA (Food Research) program ANR-10-ALIA-018-02 VALORIAL AQUIMER, Institut National de la Recherche Agronomique (INRA)-Département Microbiologie et Chaîne Alimentaire (MICA), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-SECurité des ALIments et Microbiologie (SECALIM), Institut du Porc, Microbiologie Environnement Digestif Santé - Clermont Auvergne (MEDIS), Université Clermont Auvergne (UCA)-INRA Clermont-Ferrand-Theix, INRA Clermont-Ferrand-Theix-Université Clermont Auvergne (UCA), Microbiologie Environnement Digestif Santé (MEDIS), and INRA Clermont-Ferrand-Theix-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])

Subjects
Meat, [SDV]Life Sciences [q-bio], Food spoilage, Food Contamination, Microbiology, Polymerase Chain Reaction, Meat spoilage, RNA, Ribosomal, 16S, Food microbiology, Animals, DNA Barcoding, Taxonomic, Food science, Ecology, Evolution, Behavior and Systematics, 2. Zero hunger, biology, Bacteria, Microbiota, food and beverages, 16S ribosomal RNA, biology.organism_classification, Europe, Food waste, Seafood, Food Microbiology, Pyrosequencing, Original Article, Food contaminant
Abstract

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189 +/- 58 operational taxonomic units (OTUs) but dropped to 27 +/- 12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Published
2014
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3. Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

Author

Chaillou S, Chaulot-Talmon A, Caekebeke H, Cardinal M, Christieans S, Denis C, Desmonts MH, Dousset X, Feurer C, Hamon E, Joffraud JJ, La Carbona S, Leroi F, Leroy S, Lorre S, Macé S, Pilet MF, Prévost H, Rivollier M, Roux D, Talon R, Zagorec M, and Champomier-Vergès MC

Subjects
Animals, Bacteria classification, Bacteria genetics, DNA Barcoding, Taxonomic, Europe, Polymerase Chain Reaction, RNA, Ribosomal, 16S genetics, Food Contamination, Food Microbiology, Meat microbiology, Microbiota, Seafood microbiology
Abstract

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Published
2015
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