Pierre Ledormand

Les bactériophages (phages) sont les virus spécifiques des bactéries, naturellement présents partout où il y a présence de leur hôte sur Terre. Ils sont suspectés de jouer un rôle important dans la construction, l’équilibre et les fluctuations des populations microbiennes des différents écosystèmes auxquels ils sont associés. Cependant, les connaissances sur les communautés de phages lytiques (phageomes) ainsi que les mécanismes d’interactions avec les populations microbiennes dans les boissons et aliments fermentés font défaut. Ceci est particulièrement vrai concernant le cidre, une boisson alcoolisée obtenue par fermentation de jus de pomme, importante pour l’économie de la région Normandie. Ce projet de thèse était divisé en trois grands objectifs. Le premier visait à explorer la diversité des phages du cidre, le deuxième à observer le rôle des phages dans les boissons fermentées en se basant sur un modèle de communauté microbienne inspirée du cidre, et le troisième à étudier les interactions phages-hôte à travers la réponse moléculaire d’une souche bactérienne à une infection phagique. Pour répondre au premier objectif, deux cuves de cidre de deux producteurs, un industriel et un artisanal, ont été suivies au cours du premier mois de fermentation. Il s’est avéré que l’occurrence en phages lytiques était très faible, puisqu’aucune méthode testée n’a permis de mettre en évidence le phageome et qu’un seul siphophage lytique (UCMA 21115), ciblant la souche Liquorilactobacillus mali UCMA 16447, a été isolé des échantillons analysés. Il a été montré que les prophages (phages intégrés aux génomes bactériens) semblaient plus abondants, car 1174 prophages putatifs, dont deux complets, ont été identifiés in sillico. Concernant le deuxième objectif, l’exploration du rôle des phages dans un modèle de communauté microbienne inspirée du cidre ayant subi une perturbation, s’est déroulée à 25°C et 15°C. Ce modèle était composé de quatre souches microbiennes (Saccharomyces uvarum UCMA 10446, Oenococcus oeni UCMA 19297, Leuconostoc mesenteroides CIM 3004 et L. mali UCMA 16447) et de deux phages ciblant la souche L. mali UCMA 16447 et la souche Ln. mesenteroides CIM 3004. Les phages avaient un effet dépendant de la température. Ils contribuaient au retour à l’équilibre de la communauté à 25°C, tandis qu’ils amplifiaient le déséquilibre du modèle à 15°C, tant au niveau des dynamiques microbiennes que du métabolisme. Enfin, les interactions phages-hôte ont été étudiées à travers la réponse moléculaire de la souche L. mali UCMA 16447 infectée par son siphophage lytique UCMA 21115 et entourée d’une communauté. L’infection provoquait une importante perturbation cellulaire, et des fonctions comme le métabolisme des glucides ou la motilité étaient fortement impactées. Ces travaux soulignent l’importance d’étudier les phages dans les matrices alimentaires fermentées puisqu’ils peuvent probablement, de manière directe ou indirecte, influencer la qualité du produit fini en modulant les populations microbiennes qui s’y trouvent.

Model microbial communities are often studied to better understand interactions and fluxes during fermentation processes. However, models that take into account the potential impact of bacteriophages (phages), which are recognized as drivers of microbial communities, are scarce, especially in fermented foods. This study aimed at investigating the behavior of a cider model microbial community, which was subjected to disturbance in the presence or absence of phages and at two different temperatures (25 °C and 15 °C). The model microbial community was composed of three lactic acid bacteria (LAB) strains belonging to the species Liquorilactobacillus mali, Leuconostoc mesenteroides and Oenococcus oeni, and of a Saccharomyces uvarum yeast strain. Two phages were selected, targeting L. mali and Ln. mesenteroides strains. In order to follow the behavior of the microbial community model, the phages and microbial strains were enumerated at several time points, and the metabolic signatures (sugar consumption, production of organic acids and volatile organic compounds) of the model microbial community were monitored. At 25 °C, the community with phages (P) was significantly closer to the control condition (C) than to the condition without phages (D). Microbial levels were similar between conditions C and P, which were characterized by high concentrations of compounds such as 2-phenylethanol, ethyl octanoate and isoamyl alcohol, and more globally by a more complex metabolic signature than that of condition D. In condition D, L. mali and Ln. mesenteroides were dominant while S. uvarum and O. oeni were less present, and this condition was characterized by a high concentration of ethyl lactate. At 15 °C, condition P differed from conditions C and D, as Ln. mesenteroides was not detected while the other strains all reached approximately the same levels. The metabolic range of condition P was less important than for conditions C and D. The current study showed that the influence of phages on the model microbial community dynamics and metabolisms after a disturbance phenomenon was temperature-dependent.

Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in fermented foods yet, and even less in fermented beverages. Two approaches were employed to investigate the presence of phages in cider, a fermented beverage made from apple, during a fermentation process of two cider tanks, one from an industrial producer and one from a hand-crafted producer. The phageome (free lytic phages) was explored in cider samples with several methodological developments for total phage DNA extraction, along with single phage isolation. Concentration methods, such as tangential flow filtration, flocculation and classical phage concentration methods, were employed and tested to extract free phage particles from cider. This part of the work revealed a very low occurrence of free lytic phage particles in cider. In parallel, a prophage investigation during the fermentation process was also performed using a metagenomic approach on the total bacterial genomic DNA. Prophages in bacterial metagenomes in the two cider tanks seemed also to occur in low abundance, as a total of 1174 putative prophages were identified in the two tanks overtime, and only two complete prophages were revealed. Prophage occurrence was greater at the industrial producer than at the hand-crafted producer, and different dynamics of prophage trends were also observed during fermentation. This is the first report dealing with the investigation of the phageome and of prophages throughout a fermentation process of a fermented beverage.

Bacteriophages (phages) are considered the most abundant biological entities on Earth. An increasing interest in understanding phage communities, also called viromes or phageomes, has arisen over the past decade especially thanks to the development and the accessibility of Next Generation Sequencing techniques. Despite the increasing amount of available metagenomic data on microbial communities in various habitats, viromes remain poorly described in the scientific literature particularly when it comes to fermented food and beverages such as wine and cider. In this review, a particular attention is paid to the current knowledge on phage communities, with a special focus on fermented food viromes and the methodological tools available to undertake their study. There is a striking lack of available data on the fermented foods and beverages viromes. As far as we know, and although a number of phages have been isolated from wine, no general study has to date been carried out to assess the diversity of viromes in fermented beverages and their possible interactions with microbiota throughout the fermentation process. With the aim of establishing connections between the currently used technologies to carry out the analysis of viromes, possible applications of current knowledge to fermented beverages are examined.