Identification of Host-Specific Bacteroidales 16S rDNA Sequences from Human Sewage and Ruminant Feces

The need to identify the source of fecal contamination of water has led to the development of various fecal source identification methods, a field known as microbial source tracking (MST). One promising method of MST focuses on fecal members of the order Bacteroidales, some of which exhibit a high degree of host-specificity. In order to identify host-specific Bacteroidales genetic markers, a ∼1060 bp section of Bacteroidales 16S rDNA was amplified from human sewage (n = 6), and bovine (n = 6) and ovine fecal (n = 5) samples and used for the generation of three clone libraries. Phylogenetic analysis of sequences from the three clone libraries revealed that the Bacteroidales species found in both human sewage and bovine and ovine feces were a highly diverse group of organisms, many of which were not represented by previously characterised 16S rDNA. Ovine and bovine feces appear to host similar populations of Bacteroidales species and these species were more diverse and less closely related to cultivated species than the Bacteroidales population found in human sewage. Species of Bacteroidales from the ruminant and human feces formed isolated clusters containing putatively host-specific sequences. These sequences were subsequently exploited for the design of host-specific primers which were used in MST studies

Développement d'outils microbiologiques et chimiques permettant d'identifier l'origine des pollutions fécales dans les eaux de baignades

La pollution organique issue des effluents d'élevage et des stations d'épuration urbaines conduit à un problème essentiel de santé publique lié à la contamination des eaux de surface où s'exercent des activités sensibles telles que la baignade. S'il est possible de déterminer les pollutions localisées liées à un dysfonctionnement des systèmes de traitement, il est beaucoup plus difficile d'identifier les pollutions organiques diffuses qui participent pourtant majoritairement à la dégradation de la qualité des eaux de surface. La problématique des pollutions diffuses est d'autant plus importante que la nouvelle réglementation européenne concernant les eaux de baignade (Directive 2006/7/CE) demande de constituer des profils de baignade qui nécessitent une identification et une hiérarchisation des sources de pollutions fécales. Le dénombrement de Escherichia coli et des entérocoques intestinaux stipulé par les textes réglementaires européens, représente actuellement le seul outil analytique permettant la mise en évidence d'une contamination fécale du milieu aquatique, sans toutefois différencier l'origine humaine ou animale de cette contamination. Il est donc nécessaire de développer de nouvelles méthodes de détection de la pollution fécale qui puissent non seulement mettre en évidence une contamination mais aussi en indiquer l'origine. C'est d'ailleurs dans cet objectif que s'est développé depuis quelques années, le concept de "Microbial Source Tracking" ("Traceurs de Sources Microbiennes") qui consiste à identifier à l'aide de marqueurs microbiologiques ou chimiques les sources de pollutions fécales. Dans ce contexte, six laboratoires de recherche se sont associés pour développer des techniques de traçage des contaminations fécales afin de proposer un outil opérationnel utilisable pour différencier les sources de pollution, de leur point d'émission jusqu'au milieu récepteur final que constituent les eaux de surface. Les marqueurs qui ont fait l'objet de cette étude sont des molécules chimiques naturelles (stéroïdes, caféine), des molécules de synthèse retrouvées dans les effluents de stations d'épuration ou des rapports de fluorescence de la matière organique ainsi que des micro-organismes (bactériophages, bactéries). A la suite des développements méthodologiques, plusieurs marqueurs ont été sélectionnés : - bactéries appartenant aux groupes bactériens dominants du tractus intestinal humain (Bifidobacterium adolescentis) et porcin (Lactobacillus amylovorus) ; - Bacteroidales spécifiques des humains, porcins et bovins (HF183, Pig-2-Bac, Rum-2-Bac); - génogroupes humains des bactériophages F ARN spécifiques; - rapports de stéroïdes : coprostanol/(24ethylcoprostanol+coprostanol) (R1) et sitostanol/coprostanol (R2); - caféine, benzophénone et tri(2-chloroethyl)phosphate (TCEP)

Diagnostic moléculaire de l origine des contaminations fécales dans l environnement littoral (développement de marqueurs Bacteroidales spécifiques de l höte)

Les effluents d origine urbaine ou agricole sont les principales sources decontamination microbiologique participant à la dégradation des eaux et des coquillages. Les indicateurs classiques de contamination fécale, Escherichia coli et les entérocoques, permettent de mettre en évidence une contamination mais pas d en identifier l origine. L objectif principal de ce travail de thèse était de développer et de valider une approche basée sur la PCR quantitative en temps réel, pour permettre l identification de l origine des contaminations dans les eaux et les coquillages à partir de la cible Bacteroidales. L analyse phylogénétique des séquences de gène codant les ARNr 16S des Bacteroidales a permis de développer quatre marqueurs, pour identifier les contaminations fécales humaines, porcines et ruminants. L étude de la persistance des marqueurs porcs, en microcosmes d eau de rivière, a montré que sous les conditions les plus défavorables, saturation en oxygène dissous et température de 20C, les marqueurs persistaient au moins 16 jours. Ces résultats suggèrent que ces marqueurs peuvent persister assez longtemps dan les eaux pour étre identifiés: Cette hypothèse a été confirmée par leur quantification dans des eaux provenant du bassin versant de l estuaire de Daoulas et de l estuaire de l Elorn (Finistère, Bretagne). Les marqueurs Bacteroidales spécifiques de l hôte ont également été recherchés dans les coquillages naturellement contaminés où ils ont pu étre quantifiés. Cette étude, confirme l intérêt d utiliser des marqueurs développés à partir de la cible Bacteroidales, pour identifier l origine des contaminations fécales au niveau des zones de baignade et conchylicoles.Human and animal faecal pollution affects environmental water in inland and coastal areas, with negative implications for recreational uses, public safety and shellfish sanitary status. The faecal microbiological indicators used in these regulations, Escherichia coli and enteroccoci, cannot distinguish between human and animal faecal contamination. The main objective of this study was to develop and to validate a molecular approach based on quantitative real-time PCR with the Bacteroidales target, in order to identify the origin of faecal contamination in water and shellfish. Phylogenetic analysis of the partial Bacteroidales 16S rRNA gene sequences allowed the development of 4 host-specific Bacteroidales markers to identify human, porcine and ruminant faecal contaminations. The study of the pig markers persistence in river mater microcosms showed that under unfavourable conditions, aerobic and temperature of 20 C, marker persisted for at least 16 days. These results suggested that Bacteroidales markers could still ha identified after extend period of time in river waters. This hypothesis was confirmed with their detection and quantification in river waters fro the Daoulas catchment estuary and the Elorn estuary (Finistère, Brittany). Host-specific Bacteroidales markers were also quantify in naturally contaminated oysters. Thus, this study confirais the interest of the host-specific Bacteroidales markers developed from the Bacteroidales target to identify faecal contaminations in bathing waters and shellfish harvesting areas.BREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Ecology of the forest microbiome: Highlights of temperate and boreal ecosystems

Due to land use history, most of the current temperate and boreal forests are developed on nutrient-poor and rocky soils, keeping fertile soils for agriculture. Consequently, the conditions occurring in forest ecosystems strongly differ from those of other terrestrial environments, giving importance to the access of nutritive elements and their recycling for the long-lasting development of forest ecosystems. In this review, we present an overview of the recent findings on the relationships between bacterial and fungal communities and their tree hosts at both the taxonomic and functional levels. We highlighted the common and different deterministic drivers of these microbial communities, focusing on the tree species effect, the different interfaces existing between the trees and their environment, the impact of tree byproducts (decaying wood and litter), the impact of soil and seasonal changes, and lastly, the consequences of forestry practices. Depicting both taxonomic and functional diversity based on cultivation dependent and -independent analyses, we highlight the distribution patterns and the functional traits characterizing bacterial and fungal communities. We also discuss the importance of bridging environmental microbiology to genomics and how to integrate the interactions between microorganisms for a better understanding of tree growth and health

Acidisoma silvae sp. nov. and Acidisoma cellulosilytica sp. nov., Two Acidophilic Bacteria Isolated from Decaying Wood, Hydrolyzing Cellulose and Producing Poly-3-hydroxybutyrate

International audienceTwo novel strains, HW T2.11 T and HW T5.17 T , were isolated from decaying wood (forest of Champenoux, France). Study of the 16S rRNA sequence similarity indicated that the novel strains belong to the genus Acidisoma. The sequence similarity of the 16S rRNA gene of HW T2.11 T with the corresponding sequences of A. tundrae and A. sibiricum was 97.30% and 97.25%, while for HW T5.17 T it was 96.85% and 97.14%, respectively. The DNA G+C contents of the strains were 62.32-62.50%. Cells were Gram-negative coccobacilli that had intracellular storage granules (poly-3-hydroxybutyrate (P3HB)) that confer resistance to environmental stress conditions. They were mesophilic and acidophilic organisms growing at 8-25 °C, at a pH of 2.0-6.5, and were capable of using a wide range of organic compounds and complex biopolymers such as starch, fucoidan, laminarin, pectin and cellulose, the latter two being involved in wood composition. The major cellular fatty acid was cyclo C19:0ω8c and the major quinone was Q-10. Overall, genome relatedness indices between genomes of strains HW T2.11 T and HW T5.17 T (Orthologous Average Nucleotide Identity (OrthoANI) value = 83.73% and digital DNA-DNA hybridization score = 27.5%) confirmed that they belonged to two different species. Genetic predictions indicate that the cyclopropane fatty acid (CFA) pathway is present, conferring acid-resistance properties to the cells. The two novel strains might possess a class IV polyhydroxyalcanoate (PHA) synthase operon involved in the P3HB production pathway. Overall, the polyphasic taxonomic analysis shows that these two novel strains are adapted to harsh environments such as decaying wood where the organic matter is difficult to access, and can contribute to the degradation of dead wood. These strains represent novel species of the genus Acidisoma, for which the names Acidisoma silvae sp. nov. and Acidisoma cellulosilytica sp. nov. are proposed. The type strains of Acidisoma silvae and Acidisoma cellulosilytica are, respectively, HW T2.11 T (DSM 111006 T ; UBOCC-M-3364 T) and HW T5.17 T (DSM 111007 T ; UBOCC-M-3365 T)

Scleromatobacter humisilvae gen. nov., sp. nov., a novel bacterium isolated from oak forest soil

A novel bacterial strain, designated BS-T2-15T, isolated from forest soil in close proximity to decaying oak wood, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences as well as phylogenomic analyses based on coding sequences of 340 concatenated core proteins indicated that strain BS-T2-15T forms a distinct and robust lineage in the Rubrivivax–Roseateles –Leptothrix–Azohydromonas –Aquincola–Ideonella branch of the order Burkholderiales . The amino acid identity and the percentage of conserved proteins between the genome of strain BS-T2-15T and genomes of closely related type strains ranged from 64.27 to 66.57% and from 40.89 to 49.27 %, respectively, providing genomic evidence that strain BS-T2-15T represents a new genus. Its cells are Gram-stain-negative, aerobic, motile by a polar flagellum, rod-shaped and form incrusted white to ivory colonies. Optimal growth is observed at 20–22 °C, pH 6 and 0% NaCl. The predominant fatty acids of strain BS-T2-15T are C16 : 1 ω7c, C16 : 0 and C14 : 0 2-OH. Its polar lipid profile consists of a mixture of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol and its main respiratory quinone is ubiquinone 8. The estimated size of its genome is 6.28 Mb with a DNA G+C content of 69.56 mol%. Therefore, on the basis of phenotypic and genotypic properties, the new strain BS-T2-15T represents a novel genus and species for which the name Scleromatobacter humisilvae gen. nov., sp. nov., is proposed. The type strain is BS-T2-15T (DSM 113115T=UBOCC-M-3373T)

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2017.02.013The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensisK.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifyingtheir organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the actionmechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability,membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10 days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3 days incubation in control supernatant,while itwas prevented for up to 10 days by the active supernatant. Interestingly, between 66 and 99% of yeast cellswere under a viable but non-cultivable (VNC) state despite an absence ofmembrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products