Studying the microbiome in natural populations could improve our understanding of the biological factors that influence microbiome variation. If host genetic variation is important in microbiota assembly, then understanding genetic divergence among natural populations could be informative. Despite advances in sequencing technology, we have not yet taken full advantage of this technology in natural populations. Here we integrate genome-wide population genomic and microbiome analyses in wild threespine stickleback (Gasterosteus aculeatus) fish distributed throughout western Oregon, USA. We found that gut microbiome varied in diversity and composition more among than within wild host populations. Furthermore, this among population variation was better explained by host population genetic divergence than by environment and geography. We also identified a subset of gut microbial taxa that were most strongly sorted both across environments and across genetically divergent populations. We believe this study contributes generalizable methods and findings in host systems. This thesis includes supplemental materials. / 2021-04-30

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Host-microbe interactions at the gastrointestinal interface have emerged as a key component in the governance of human health and disease. Advances in micro-physiological systems are providing researchers with unprecedented access and insights into this complex relationship. These systems combine the benefits of microengineering, microfluidics, and cell culture in a bid to recreate the environmental conditions prevalent in the human gut. Here we present the human-microbial cross talk (HuMiX) platform, one such system that leverages this multidisciplinary approach to provide a representative in vitro model of the human gastrointestinal interface. HuMiX presents a novel and robust means to study the molecular interactions at the host-microbe interface. We summarize our proof-of-concept results obtained using the platform and highlight its potential to greatly enhance our understanding of host-microbe interactions with a potential to greatly impact the pharmaceutical, food, nutrition, and healthcare industries in the future. A number of key questions and challenges facing these technologies are also discussed. (C) 2017 THE AUTHORS. Published by Elsevier LTD on behalf of the Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Plants and animals have evolved unique morpho-physiological adaptions to cope with the harsh and steep environmental gradients that characterise the mangrove ecosystem. However, the capacity of these two main components of the system to thrive, and the extraordinary productivity of mangrove forests in extreme conditions, has been overlooked in terms of the role of the microbiome. By combining approaches that included molecular microbial ecology, biogeochemical analyses, microscopy, raman spectroscopy and microsensor measurements, this thesis aimed to investigate the potential role of bacterial symbiosis in the adaptation of mangrove crabs to their environment and subsequently how these different animals modify their environment. Finally, with a field-based approach monitoring microbial communities, sediment metabolism and plant performance, the thesis aimed to investigate the plant/animal/bacterial dynamics in relation to seasonal environmental changes to contribute to understand the mangrove plant productivity paradox of high productivity under conditions of limited nutrents. Crab species were associated with distinct gill-bacteria communities, that produced carotenoids, according with their level of terrestrial adaptation. These carotenoids may be involved in protecting the gills from oxidative stress during air exposure. The main groups of ecosystem engineering crabs in mangroves had significant but diverse effects on the sediment environment and microbiome predominantly related to their ecology (i.e. filter feeder vs herbivore). Burrows increase aerobic microbial activity in the immediate burrow wall with a cascade effect on sediment microbial communities and nutrient distribution observed consistently across mangroves in different locations and with diverse environmental conditions. Microorganisms play an important role in adapting crabs on their evolutionary path to land and could contribute to the success of their colonization. At high population densities, of more than 50 individuals per square meter in some mangroves, these crabs deeply impact the functioning of the mangrove ecosystem, affecting microbial networks and nutrient recycling in the sediment, which may ameliorate conditions for plant growth. The microbiome is an understudied component of mangroves that lies at the basis of the functioning of these systems, influencing the success of the animal inhabitants (ecosystem engineers) that deeply modify the sediment microbiome, therefore influencing ecosystem functioning and resilience and, potentially, the success of the plants themselves (ecosystem architects).

Bioinformatics is a unique field in that it incorporates many different disciplines, including biology, computer science, and statistics, to study biological data. There is a vast array of techniques that utilize bioinformatics, including pangenomics, RNASeq, whole genome metagenomics, and 16S sequencing. To study bacterial interactions, we used a model system of species interactions, Myxococcus xanthus. M. xanthus is a soil bacterium that is a known predator of other bacteria. It has one of the largest repertoires of two component systems (TCS) to respond to external stresses. TCS are a pair of proteins, one that senses environmental stress (histidine kinase, HK) and another that usually acts as a transcriptional regulator (response regulators, RR). We studied a class of RRs, NtrC-like, reliant on an alternative sigma factor, sigma54. The oligomerization of NtrC-like RRs is regulated to modulate activation of the protein, which would change the bacterium’s ability to respond to its environment. We studied HsfA, a NtrC-like RR that regulates specialized metabolites. Specialized metabolites are used in bacterial interactions. In predation interactions they are used to kill prey. Our goal was to find genes that might be involved in specialized metabolite production that would aid in predation. We used prediction tools to find putative binding sites of HsfA to find potentially new metabolites. We used two motifs to attempt to predict if the oligomerization of these response regulators is positively or negatively regulated. We found that the presence of a motif in the receiver domain to be associated with negative regulation of oligomerization, but further studies are needed to experimentally confirm this finding. One environment in which bacterial interactions occur is in the gut. The gut microbiome is the consortium of organisms and their genomic content in the gastrointestinal tract. The gut microbiome is sensitive to aspects of a person’s lifestyle, such as diet and medication. Here we studied the effect of two different diets and two drugs on the gut microbiome. Risperidone, an antipsychotic used to treat schizophrenia and bipolar disorder, has been shown to cause obesity and diabetes. We studied the effect of diet and risperidone usage on weight gain and the microbiome using a C57Bl/6J female mouse model. Our results show that diet has a strong impact on the microbial composition of the gut in response to risperidone. As many mental health patients stop and restart their medication, we examined the effect of stopping and restarting risperidone on the microbiome. When risperidone is stopped the microbiome reverts to a state similar to the control group but diverges into a different microbial composition upon restarting treatment. Interestingly, mice did not gain significantly more weight than their control group upon the second risperidone treatment. Further studies are needed to examine the functional changes occurring with the stop and restart of risperidone to determine the mechanism of mice resisting weight gain during the second round of treatment. Captopril is used to treat hypertension, a very common disease in the United States. Here we studied the effect of captopril on weight gain, metabolic phenotypes, and the gut microbiome. Our results showed that captopril caused an increase in resting metabolic rate (RMR) in mice. This occurred through an increase in energy expenditure. This increase in RMR had the effect of captopril-treated mice being resistant to weight gain. Our group has previously shown that the gut microbiome can directly affect RMR. Therefore, we studied the gut microbiome of captopril-treated mice. We observed a shift in their gut microbiome to organisms Akkermansia muciniphila and Lactobacillus, associated with lean body mass. Captopril therefore has the potential to be a better medication to treat patients with both hypertension and obesity. Further studies are needed to determine the effect of captopril on the microbiome in a hypertension mouse model.

Probiotics and lactoferrin are currently being used in neonatal intensive care units in the hopes of reducing rates of sepsis and necrotizing enterocolitis. While studies have shown that these measures can be clinically beneficial to premature babies, little is known about their effect at the level of the intestinal microbiome. We performed a prospective study describing the composition and evolution of the microbiota of premature babies receiving probiotics with and without lactoferrin supplementation. Furthermore, we compared our findings to a piglet model. We found that the addition of lactoferrin did not lead to a distinct microbiome using UniFrac analysis, nor increased diversity or richness as compared to probiotics alone. The relative abundance of probiotic bacterial strains was approximately 3%. At the phylum level Firmicutes and Proteobacteria predominated, while overall Enterobacteriaceae was the most abundant family. We remain the first to describe the microbiome of premature infants receiving lactoferrin.

Chronic inflammation is associated with a dysregulation of the immune response. Inflammation is also associated with decreased reproductive capacity in women, however an exact mechanism has yet to be identified. Physiological states such as obesity and disease states such as endometriosis are both associated with chronic inflammation, an insufficient immune response, and infertility in women; therefore these two conditions serve as excellent models to study the effects of inflammation on reproductive function. Early indications of inflammation may aid in early detection of immune dysregulation associated with different physiological and pathological conditions. One way to measure immune balance between pro- and anti-inflammatory states in the female urogenital tract is by observing changes in the bacterial species that populate the mucosal surface. Commensal bacteria that make up the microbiome play a critical role in the development and maturation of the immune system in humans. Because these microbes and the host’s immune system are constantly influencing each other, several immunological conditions and disease states have been shown to have an altered microbial profile than that of healthy individuals. The goal for this study was to examine how triggers of inflammation alter the peripheral immune response, urogenital microbial communities, and reproductive function. Specifically, our aims were to 1) use an animal model of obesity to determine whether this physiological model of inflammation decreases immune protection of the urogenital microbiome and alters ovarian function; and 2) use endometriosis as a disease model of inflammation to assess whether the presence of endometriotic lesions alters urogenital microbial dynamics, and also whether surgical intervention restores commensal bacterial profiles to that of a non-disease state. The results of this study revealed that the urogenital microbial community dynamics were altered in both our obese and disease models of inflammation compared to their respective controls. In the obesity study, we also found that our obese model had decreased markers of inflammation, which may be due to dietary composition. In the endometriosis study, we observed that patients with disease had a unique urogenital microbiome profile and that surgery had an effect in shifting the urogenital microbial profiles of several patients. Overall, our long-term goal is to determine whether the urogenital microbiome is a good indicator of immune stress and if alternative therapies can alter microbial community dynamics, eliminate immune stress associated with disease. Ultimately we are looking at the microbiome as an indicator of overall immune health and implementing alternative diagnostic and treatment strategies to immune diseases that affect reproductive function in women.

Prior studies have shown that the low-level microbiome in the placenta is most similar to the non-pregnant oral microbiome, suggesting a hematogenous route of bacterial transmission. Based on these studies, we theorized that a disruption of the normal balance of pathogenic and commensal microorganisms in the oral cavity will lead to conditions that favor colonization of the placenta and amniotic cavity, leading to inflammation and preterm birth. We hypothesized that an altered oral microbiome profile will promote preterm birth. Our study aimed to compare metagenomic profiles of saliva and tongue in women delivering preterm to those of women delivering at term. Unstimulated saliva and tongue brushings were collected according to an IRB-approved protocol from patients who delivered preterm, and from age and race-matched patients who delivered at term. Exclusion criteria included obvious risk factors for preterm birth or other major complications (multiple gestation; history of or current cervical cerclage; history of hypertension or diabetes; prior history of preterm birth or preeclampsia; age less than 16 or greater than 45) as well as immunologic problems (HIV, organ transplant, etc). Oral samples were collected within 24-48 hours after delivery. Samples were analyzed using 16S rDNA-based sequencing, where the DNA was extracted and then amplified by PCR using 16S rDNA primers. Data was processed using the UPARSE/SINTAX pipeline, and differentially abundant taxa were determined using the LEfSe method and MaAsLin2. The parent study enrolled 100 patients who delivered preterm and 205 patients who delivered at term. A subset of patients had oral samples collected, and 95 saliva and 70 tongue samples were analyzed using 16S rDNA-based sequencing. Communities from tongue and saliva were significantly different between women who delivered preterm and those who delivered at term, although not between those delivering low birthweight versus normal birthweight infants. When assessing beta diversity using the unweighted unifrac metric, patients who delivered very preterm (VPT, < 32 weeks) had a tongue microbiome that was consistently and statistically different from the tongue microbiome of patients who delivered both term (T, > 37 weeks) and late preterm (LPT, 32-<37). Differences remained significant after controlling for tobacco use. In a three-way comparison of saliva samples between the groups using MaAsLin2, the genus Lachnoanaerobaculum was significantly less abundant in the VPT group. Our studies suggest a tongue and salivary microbiome that differs between women delivering term and late preterm versus very preterm. Future studies are required to prospectively confirm differences and may yield data to design noninvasive tests to predict preterm birth risk. / 2021-06-12T00:00:00Z

<p>The Food Processing and Postharvest Handling Innovation Lab (FPIL) project seeks to reduce food loss and link up consumers with food-to-food fortified instant products that are enriched with micronutrient sources that target vitamin A, zinc, and iron deficiencies. These are mostly maize-based products, but may be combined with other cereals, such as sorghum, and pseudocereals, such as amaranth. The general goal of this thesis study was to facilitate the adoption of extrusion technology to process instant flours, assess the acceptance and willingness to pay (WTP) for these products, and to assess the health impacts of the products on gut health. A low-cost, single-screw extruder was used that was developed at Purdue, and has been placed in different locations in Africa country study sites. The first study aimed to optimize process conditions of a low-cost single-screw extruder, currently done at 35% feed moisture, for African small- to medium-scale entrepreneurs to produce good quality and low-cost pregelatinized instant pearl millet porridge flours and other whole grains by relating feed moisture (27, 29, 31, 33, and 35%) to extrusion energy, drying time and physicochemical properties. We found that we could lower the feed moisture to 27% and still attain good pasting profiles of the porridges, reduce drying time, have better expansion of the extrudates, obtain increased <i>L</i>* color values of the flours, and with a higher extrusion energy but lower drying time. In conclusion, the single screw extruder can be efficiently operated at 27% feed moisture compared to the currently used 35% feed moisture and obtain instant flours with desired quality. It is not known whether higher extrudate energy consumption may be offset by the lower drying time representing lower drying energy. In the second study, we investigated extrusion enhance <i>in vitro</i> fecal fermentation of maize bran, which has been characterized by a poor gut microbiota fermentation property due to its highly crosslinked and densely branched arabinoxylan chemical structure, making it poorly available to the gut microbiota. We hypothesized that this dense cell wall matrix can be opened for better fermentation by applying extrusion. Test conditions of a twin-screw extruder at Purdue were low (200 RPM) and high (400 RPM) shear rates applied to a maize meal and bran mixture (60:40) at different feed moisture conditions (20, 25, 30%). <i>In vitro</i> fermentation of test materials was conducted on stool samples from three donors. Extrusion increased total short chain fatty acids and produced individualized donor effects on the gut microbiota. Some extruder test condition effects were observed on certain bacteria. For example, extrusion at 30% feed moisture and 400 RPM tended to increase genera of <i>Subdoligranulum</i> and <i>Eubacterium hallii</i> and <i>Ruminococcus</i> <i>torques</i> groups in Donor 1 compared to non-extruded bran. There was also a trend of increase in <i>Subdoligranulum</i> and <i>Blautia</i> in extruded compared to non-extruded bran in Donor 2. In Donor 3, <i>Lachnospiraceae NK4A136</i> group was increased at 20 and 25% feed moistures at 200 RPM and 30% feed moisture at 400 RPM compared to non-extruded bran. In the final study, we investigated the acceptance and WTP for instant fortified flours using the Becker-DeGroot-Marschak mechanism when consumers are incrementally given nutrition information and demonstration how to reconstitute instant flours. This study was conducted in Eldoret, Kenya. Participants preferred the fortified thick porridge higher in maize content than fortified thin porridge prepared from the same blend. Contrarily, thin porridge made from fortified flour with higher sorghum content was ranked more highly than for the corresponding thick porridge. Participants were willing to pay more for instant fortified products higher in sorghum when given product name and nutrient composition, even without a practical demonstration of how to reconstitute the flours. For the instant product higher in maize, consumers needed demonstration of how to reconstitute the instant flour for them pay a higher premium. These findings suggest that food-to-food fortified instant porridge flours have the potential to be adopted and can be used as a vehicle to deliver micronutrients to these populations and that extrusion somewhat enhances fermentation of whole grain fibers by the gut microbiome.</p>

The plant nectar microbiome (NMB) primarily composed of fungi and bacteria can qualitatively and quantitively affect floral rewards and ultimately impact plant-pollinator interactions and plant reproductive success. Evidence suggests that changes in microenvironmental conditions across spatial gradients can induce changes in the floral nectar microbiome, leading to microbiome variation within species. Specifically, changes in biotic and abiotic conditions across an elevation gradient (i.e. pollinator community composition, temperature, UV exposure, nutrient availability) have the potential to induce variation NMB composition and abundance. However, this has been little explored. For instance, we could expect that the diversity and abundance of the NMB will decrease with elevation as the growing conditions become less favorable (e.g. lower temperatures). In this study, we evaluate spatial variation in nectar microbiome composition, diversity, and abundance in populations of Rhododendron catawbiense at high (H) and low (L) elevations separated by over 1000ft in Roan Mountain, TN. Nectar samples were collected, plated, quantified and isolated. Fungal ITS sequences were obtained from individual colonies by Sanger sequencing and directly from nectar samples by Nanopore NGS. Preliminary results suggest that elevation can affect fungal abundance and composition in the NMB. We found 10 fungal species inhabiting the nectar of R. catawbiense (H= 4; L= 9). We also observed variation in nectar yeast abundance with more than 30x as many CFUs (colony forming units) (H= 1.89 ±10.64; L=38.43 ±1.39.12) and twice as many RPKMs (reads per kilobase matched) (H= 2.8x107 ± 2.46x107; L=5.5x107 ±3.6x107) on average at the lower elevation. Interestingly, R. catawbiense NMB in the lower elevation had a broader diversity of species (Simpson Diversity Index: H= 0.52; L= 0.95), perhaps as a result of more favorable growing conditions. This trend is largely associated with higher abundance of Metschnikowiaceae yeast OTUs and lower abundance of potentially pathogenic Basidyomycete OTUs. Overall, results indicate that elevation can mediate changes in the composition and abundance of microorganisms in the NMB, which in turn can lead to differences in pollinator community composition and plant reproductive success. These results highlight the need evaluate within-species variation in NMB at large spatial scales and its potential consequences for plant reproductive success, plant-pollinator interactions and plant community dynamics. This is particularly important in the face of human-mediated environmental disturbances that can alter plant-microbe interactions.