京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23332号 / 生博第450号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 永尾 雅哉, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Travail réalisé en cotutelle avec l'université du Littoral côte d'opale, France / Afin de réduire l’usage des fertilisants chimiques et leurs empreintes environnementales, la production de bioinoculants à base de bactéries rhizosphériques solubilisatrices de Phosphore (PSB) est une biotechnologie prometteuse. Lorsque celle-ci est associée à l’apport de phosphate de roche (RP), elle pourrait constituer une alternative écoresponsable d’amélioration de la fertilité des sols rhizosphériques, notamment à travers l’augmentation du phosphore (P) biodisponible. Pour répondre à cet enjeu environnemental majeur, il est indispensable d’étudier la biodiversité microbienne des sols en interaction avec le RP. Ainsi, ce travail de thèse a pour objectif de caractériser, d’un point de vue taxonomique, les communautés bactériennes et fongiques associées aux compartiments racinaires et rhizosphériques de plantes natives d’environnements singuliers, peu explorés, que sont d’anciens sites miniers riches en RP. Le séquençage Illumina MiSeq d’amplicons de gènes ribosomiques des différents groupes microbiens et l’assignation taxonomique des séquences regroupées en ASV, nous ont permis de mettre en évidence des valeurs de richesse et de diversité microbiennes comparables malgré la différence des teneurs en RP et en P des sols étudiés. De même, une grande similarité entre les profils des communautés a été identifiée à haut niveau taxonomique, avec une prévalence marquée des phyla Actinobacteriota et Ascomycota, et de l’ordre Glomerales pour les communautés de champignons mycorhiziens arbusculaires (CMA). À un niveau taxonomique plus bas, un core microbien de 26 ASV mycorhiziennes, persistantes à travers le gradient de RP et de P, a également été mis en évidence. Toutefois, l’analyse statistique des données environnementales nous a permis d’identifier des ASV indicatrices des habitats enrichis versus non enrichis en RP, pour chaque communauté microbienne étudiée. La synthèse de ces résultats originaux montre une influence mineure des teneurs en RP et en P sur le profil des communautés microbiennes des sites étudiés et questionne la signification écologique de la prévalence de certains taxa en lien avec l’histoire minière du site, sa restauration écologique et sa résilience, ainsi que les stratégies de vie et les attributs fonctionnels des taxa dominants. Par ailleurs, une collection de PSB a été réalisée à partir d’isolats racinaires issus des sites miniers étudiés, ou à partir d’isolats issus de spores et d’hyphes mycorhiziens piégés au contact d’apports en RP déposés dans la rhizosphère de plantes d’intérêt agronomique. Ces PSB racinaires ou hyphosphériques, testés par ailleurs pour plusieurs traits additionnels promoteurs de la croissance des plantes, ont été assignés à trois phyla : Proteobacteria, Firmicutes et, dans une moindre mesure, Actinobacteria ; Bacillus et Pseudomonas étaient les genres prédominants. Enfin, à partir de ces isolats, deux consortia bactériens avec différent traits promoteurs de la croissance des plantes (PGP), ont été constitués et testés seuls ou en combinaison avec l’apport de RP et/ou l’ajout d’un inoculum mycorhizien à base de Rhizophagus irregularis, sur la croissance de la tomate, espèce d’intérêt agronomique. Une augmentation du taux de germination, un accroissement de la hauteur des jeunes plantules et des modifications au niveau du système racinaire des plants adultes, en particulier des variations de la densité du tissu racinaire, ont été observés en réponse à l’inoculation simple par les consortia ou à leur interaction double ou tripartite avec le RP et/ou le CMA. Le profil taxonomique du microbiote natif avant et après apport des inoculants et du RP a été caractérisé. En conclusion, l’ensemble de nos travaux s’appuyant sur l’analyse des données environnementales, combinées aux données de laboratoire sur l’inoculation de plantes par des isolats bactériens, notamment d’origine minière, soulignent l’intérêt mais aussi la complexité de l’élaboration d’inoculants microbiens promoteurs de la croissance des plantes, solubilisateurs de P. / In order to reduce the use of chemical fertilizers and their environmental footprint, the production of bioinoculants based on phosphorus solubilizing rhizospheric bacteria (PSB) is a promising biotechnology. When combined with rock phosphate (RP), it could be an eco-responsible alternative to improve rhizospheric soil fertility, especially by increasing bioavailable phosphorus (P). To address this major environmental issue, studying soil microbial biodiversity and its interactions with RP is required. Thus, the objective of this thesis is to characterize, from a taxonomic point of view, the bacterial and fungal communities associated with the root and rhizospheric compartments of native plants in singular, little explored environments, located in former mining sites rich in RP. Illumina MiSeq sequencing of ribosomal gene amplicons of the different microbial groups, then taxonomic assignment of the sequences grouped in ASV, allowed us to highlight comparable microbial richness and diversity indexes of soils despite their differences in RP and P contents. As well, a high degree of similarity between the community profiles was identified at a high taxonomic level, with a marked prevalence of the phyla Actinobacteriota and Ascomycota, and of the order Glomerales for the arbuscular mycorrhizal fungi (AMF) community. At a lower taxonomic level, a microbial core of 26 mycorrhizal ASV, persistent across the RP and P gradient, was also documented. However, statistical analysis of the environmental data allowed us to identify indicator ASV for PR-enriched versus non-rich habitats for each microbial community studied. All together, these original results contributed to rule on a minor influence of RP and P levels on the microbial community’s profiles across the studied sites, and raised the questions of the ecological significance of the prevalence of certain taxa regarding the site’s mining history, its ecological restoration, as well as the life strategies and functional attributes of the dominant taxa. In addition, a collection of PSB was made from mine site root isolates, or from isolates associated with spores and mycorrhizal hyphae trapped in contact with PR inputs deposited in the rhizosphere of agronomic plants. These root or hyphospheric PSB, tested elsewhere for several additional plant growth-promoting traits, were assigned to three phyla: Proteobacteria, Firmicutes, and, to a lesser extent, Actinobacteria; Bacillus and Pseudomonas were the predominant genera. Finally, from these isolates, two bacterial consortia with different plant growth promoting (PGP) traits were constituted and tested, alone or in combination with RP inputs and/or a mycorrhizal inoculum based on Rhizophagus irregularis, on the growth of tomato, a species with an agronomic interest. An increase in germination rate or height of young seedlings, and changes in the root system of adult plants, particularly changes in root tissue density, were observed in response to single inoculation by the consortia or to their dual or tripartite interaction with RP and/or AMF. The taxonomic profile of the native microbiota before and after inoculants and RP input was characterized. In conclusion, our work based on the analysis of environmental data, combined with laboratory data from plant inoculation experiments by bacterial isolates, especially of mining origin, underlines the interest but also the complexity of the development of microbial inoculants promoting plant growth, solubilizers of P.

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Objectives: This study aimed to evaluate physiological and microbiome differences in a murine model consuming a Mediterranean (M) v. Western (W) diet. Methods Study design: To investigate the potential of diets in modifying the fecal microbiota, we used 16 ICR mice per diet split evenly between males and females. Mice were acclimated for 5 weeks, consuming regular chow, before switching to M or W diets. Four same sex mice were housed per cage and randomly assigned M or W diets with 2 cages of male and female mice/diet. All animals were weighed weekly, and stool samples were collected, freeze-dried, and ground. An aliquot was analyzed for short-chain fatty acid (SCFA) to determine the fecal fermentation profile (FFP). Diets: All animals were acclimated to their surroundings while consuming the standard mouse chow diet for 5 wks. Experimental diets were from Envigo for 10 wks. M v.. W diets were 24.2v. 17.3% protein, 42.3 v. 48.5% carbohydrate, 13 v. 21.2 % fat, 9 v. 5% fiber, and energy density of 3.6 v. 4.5 kcal/g, and were offered ad libitum. Fecal Fermentation Profile: Fecal samples had SCFA extracted and analyzed using a modified Schwiertz et al. method via gas chromatography. The resulting SCFA profiles were used to determine differences between diets. Statistical Model: General linear models examined main effects of diet, sex, and week while accounting for baseline value and a random effect for cage (SCFA) or mouse (weight). Results: Diet effects for M v. W diets for SCFA were Acetate 50.12 v. 38.45% (p< 0.01), Propionate 14.19 v. 7.98% (p< 0.01), Butyrate 8.37 v. 17.27% (p< 0.01), Caproate 16.19 v. 23.68% (p< 0.01), and Caprillic 0.44 v. 0.68% (p< 0.01). Sex comparisons showed higher percentages of Butyrate (p< 0.05) for males and higher percentages of Isobutyrate (p< 0.01), Isovalerate (p< 0.01), and Propionate (p=0.02) for female mice. On the Mediterranean diet, male mice gained more weight than female mice, 4.96g v. 2.86g compared to baseline (p<0.01). Animals on a Western diet approached significance by gaining more weight (p<0.0871).

Objectives- The purpose of this study was to determine if differences in breast milk (BM) short chain fatty acid (SCFA) and microbiome profiles are correlated to maternal BMI. Our hypothesis is that BM SCFA are a reflection of colonic SCFA distribution and concentration and may reflect microbiome diversity in the maternal gut. Methods- Study design-This was a cohort study in which forty-six Caucasian participants were recruited from BABE Breastfeeding Coalition of Tri-Cities, divided into two groups, one group with normal pre-gravid BMI between 18.5 and 24.9 kg/m2 (n=23) and the other group with overweight or obese pre-gravid BMI greater than 25.0 kg/m2 (n=23). Each participant completed a demographic and health survey and provided 4 ounces of expressed BM. This study was approved by the ETSU IRB (0915.8s-ETSU). 16s rRNA Isolation & Quantification- Microbiome analysis was performed on thirty-four samples (n=13 for overweight/obese, and n=21 for normal weight). Qiagen QIAmp PowerFecal Pro DNA Kit was utilized for isolation of microbiome DNA; Amplicon sequencing of the 16S rRNA region was performed at the University of Tennessee Genomics Core Laboratory utilizing a modified Klindworth et al method. Microbiome Analysis- Operational Taxonomic Unit (OUT) clustering and taxonomic analysis were performed using CLC Genomics Workbench. Alpha diversity indexes were calculated using the Abundance Analysis tool, and the weighted Unifrac metric was used to calculate Beta diversity. Fatty Acid Profile- BM samples were subjected to SCFA extraction and analysis using a modified Schwiertz et al. method. The resulting SCFA profiles were then utilized to determine if there were any significant differences between groups. Results- No significance was observed in BM microbiome between the normal weight and overweight/obese groups for alpha or beta diversity. Significance was detected between the groups for valeric (p=0.02) and isocaproic acids (p=0.05) with the normal weight group higher than the overweight/obese group. No significance was observed for any of the other SCFAs. Conclusions- Although these results are not significant due to low sample size and lack of diversity, they potentially offer insights into the impact of maternal BMI on microbiome and SCFA profiles, which can have implications for infant health and development. Funding Sources- ETSU Small RDC Grant

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Nausea and vomiting are among the most common symptoms of early pregnancy. Its most extreme form Hyperemesis gravidarum often requires hospitalization and has been linked as a risk factor of perinatal depression. The emetic reflex is to a large extent triggered in the intestinal epithelium by the enterochromaffin cells, however the interplay between gut microbiome and pregnancy nausea is yet unclear. The aim of this study is to investigate the variation in gut microbiota diversity on second-trimester pregnant women with different levels of nausea, and to ascertain potential key species involved in that variation. Using shotgun sequencing to capture bacterial diversity from 1078 fecal samples, we found a reduction on species richness on women with strong nausea. There are measurable differences in the gut microbiota community composition based on the strength of nausea although depression seemed to be even more relevant to explain those differences. Our results provide evidence for the association of nausea and perinatal depression, but further studies are needed to elucidate the mechanisms underpinning the gut-brain axis cross-talk role in nausea and perinatal depression. No evidence of variation in species evenness or differential abundance of species were found. Finally, random forests results point at Lactococcus lactis as potentially displaying a key role determining the intensity of the nausea, although better models are needed to infer clear assumptions.

Particle size distribution of diet, feces, and change from diet to feces, as well as apparent digestibility (aDig, %) of selected nutrients, and novel fecal microbiome identification of mature female leopard tortoises (Stigmochelys pardalis, n = 16) fed exclusively one of three, nutritionally complete, pelleted diets were evaluated in a blind, complete randomized design study. Two diets included insoluble fiber (powdered cellulose) consisting of either 2.0 mm or 0.2 mm length. Insoluble fiber provides nutritional and physical benefits to both the animal host and the microorganisms that inhabit the gastrointestinal tract. Insoluble fiber length was used as a means of evaluating a physically effective fiber (peNDF) definition for hindgut-fermenting vertebrates. Numerical trends of each diet particle size distribution indicated a greater amount of particle recovery on the 2.0 mm sieve for the 2.0 mm diet, and a greater particle recovery on the 0.125 mm sieve for the 0.2 mm diet, both as expected based on the added fiber lengths. Fecal particle size distributions were not different between diets, however, distributions of the change in particle size from diet to feces were different between diets. Similar fecal particle size distributions across diets suggests both cellulose lengths are below the peNDF threshold of the leopard tortoise. Apparent digestibility (aDig, %) of dry matter (DM) and organic matter (OM) was not different based on diet, method, or a diet and method interaction; aDig (%) of neutral detergent fiber (aNDF) and sequential acid detergent fiber (sADF) was different based only on diet. These results suggest that while aDig (%) of OM did not change, the source of OM digestibility shifted, as both aNDF and sADF digestibility increased with the cellulose-added diets compared to the control diet. An increase in insoluble fiber digestibility suggests an "effectiveness" of the cellulose lengths. At both bacterial phyla and genera levels, fecal microbiomes were more similar between tortoises fed the cellulose-added diets versus the control diet, suggesting that the hindgut microbial communities adjusted in the hindgut of the tortoises fed the cellulose-added diets by shifting proportions of microbes, based on their role in the hindgut (i.e., cellulose digestion), to accommodate for the addition of cellulose in the two treatment diets. This may explain the similarity among fecal particle size distributions, and suggests that adaptability of the hindgut microbial communities should be considered when defining peNDF for hindgut-fermenting vertebrates.

Background: The human gut microbiota is the complex microbial community that lives in our gut. The gut microbiota has a key role in health and disease and its disruption has been linked to several chronic diseases such as cardiovascular diseases. As antibiotics are well known disruptors of gut microbiota, the aim of this thesis work was to identify associations between previous antibiotic consumption and the abundance of seven gut microbiota species previously linked to subclinical coronary atherosclerosis in the large population-based Swedish CArdioPulmonary bioImage Study (SCAPIS) cohort. Materials and Methods: Faecal samples of 9,794 individuals from the SCAPIS Uppsala and Malmö cohorts were analysed by deep shotgun metagenomics sequencing in a cross-sectional study. Previous antibiotic use was retrieved using the Swedish Drug Prescribed Register and divided into three periods: one year, between one and five years, and between five and nine years before faecal sampling. Associations between antibiotic consumption and the gut microbiota species were evaluated using linear regression adjusted for covariates and corrected for multiple testing. Results: Our results showed that antibiotic consumption was associated with an increased abundance of Ligilactobacillus salivarius, Bifidobacterium dentium, Rothia mucilaginosa, Streptococcus parasanguinis and Streptococcus oralis subsp. oralis. Often these positive associations were present for antibiotic consumed between one and five years before sampling.  The strongest associations were for broad-spectrum antibiotics and lincosamides with L. salivarius, B. dentium, R. mucilaginosa and S. parasanguinis; and for nitrofurantoin with S. oralis subsp. oralis.   Conclusions: This study provides insights on how antibiotic consumption is associated with enrichment and higher abundance of species previously linked with subclinical coronary atherosclerosis in the gut. Hence, this study provides insights on unintended effects of using antibiotics for managing infections, which underscores antibiotic use as not only a concern for development of antibiotic resistance but also for disrupting the gut microbiota, which may contribute to disease development. Knowledge about effect of antibiotics in gut microbiota may help to adequate this therapy according to comorbidities of individual profiles and to design better diagnostic tools for the risk population with the goal of preventing cardiovascular events in the general population.