Thesis advisor: Babak Momeni / Diseases of the upper respiratory tract encompass a plethora of complex multifaceted etiologies ranging from acute viral and bacterial infections to chronic diseases of the lung and nasal cavity. Due to this inherent complexity, typical treatments often fail in the face of recalcitrant infections and/or severe forms of chronic disease, including asthma. Thus, in order to provide improved standard of care, the mechanisms at play in hard-to-treat etiologies must be better understood. More recently, research has demonstrated a significant association between microbiota and many URT diseases. Previous work has also identified species capable of directly inhibiting standard treatments used to control asthma exacerbations. Despite an exhaustive collection of data characterizing microbiota composition in states of both health and disease, our knowledge of what microbiota profiles are observed in what specific disease etiologies is severely lacking. Yet, gaining these insights is crucial for the translation of such data into application. In this thesis I sought to: 1) identify gut microbiota profiles associated with severe and treatment resistant forms of childhood asthma, and 2) formulate a predictive model to facilitate the restructuring of microbiota for desired therapeutic outcomes. To identify gut microbiota and metabolites enriched in severe and treatment resistant childhood asthma, I looked to an ongoing longitudinal human study on vitamin D and childhood asthma. In this study, I find several fecal bacterial taxa and metabolites associated with more severe (i.e., higher wheeze proportion) and treatment resistant asthma in children at age 3 years. Specifically, several Veillonella species were enriched in children with higher wheeze proportion and in children that responded poorly to inhaled corticosteroid treatment (ICS) (i.e., non-responders). Haemophilus parainfluenzae, a species previously identified as enriched in the airway of adults with ICS-resistant asthma, was also uniquely enriched in children considered ICS non-responders in this study. Several metabolic pathways were also distinctly enriched: histidine metabolism was enriched in children with higher wheeze proportion while sphingolipid metabolism was enriched in ICS non-responders. Both metabolic pathways have been previously identified in association with asthma, further corroborating their role in this disease. Yet, this study is the first to identify these taxa and metabolites in children with preexisting and treatment resistant asthma. In the pursuit of improved treatment outcomes for recalcitrant URT diseases, recent efforts have turned towards microbiota-based therapies. While such treatments have proven successful in the treatment of gastrointestinal infections, these methods have not yet been extended to other conditions. Considering this, I ask whether a predictive model describing microbial interactions can facilitate the restructuring of microbiota for desired therapeutic outcomes. For this, I use a community of nasal microbiota to determine when a simply Lotka-Volterra-like (LV) model is a suitable representation for microbial interactions. I then utilize our LV-like model to examine whether environmental fluctuations have a major influence on community assembly and composition. For this, I looked specifically at pH fluctuations. In this study, I found that LV-like models are most suitable for describing community dynamics in complex low nutrient conditions. I also identified simple in vitro experiments that can reliably predict the suitability of a LV-like model for describing outcomes of a two-species community. When our LV-like model was applied to an in silico community of nasal species to determine the impact of environmental fluctuations, I find that nasal communities are generally robust against pH fluctuations and that, in this condition, facilitative interactions are a stabilizing force, and thus, selected for in in silico enrichment experiments. Overall, this thesis further corroborates the association of microbiota with URT diseases and treatment outcomes while also providing unique insight into their association with specific etiologies in childhood asthma. This thesis also provides a framework for developing models able to facilitate the development of future microbiota-based therapies while also determining how, and when, environmental factors impact community assembly and composition. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Sustainable farming is an integrated practice of crop and livestock production system (integrated crop-livestock system; ICLS) that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for better sustainable farming practices. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the ICLS with a broader goal of manipulating these systems to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed the analysis of 16S rRNA-V4 element for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle alternately grazing nontoxic MaxQ and toxic KY-31 tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify microbial species potentially capable of degrading ergot alkaloids for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially at the cellulolytic and saccharolytic guilds. It suppressed the abundances of Fibrobacter, a major ruminal cellulolytic bacterium, as well as those of Pseudobutyrivibrio and Butyrivibrio, and these losses were compensated by increased occurrences of Eubacterium species. Parts of these new communities lingered once developed, and a different guild composition surfaced upon transfer to MaxQ. We also discovered that most of the observations were not evident at the whole microbiome levels but was identified by analyzing the sessile and planktonic fractions separately. Thus, it showcased the need for analyzing sessile and planktonic segments separately while interrogating a heterogenous microbiome. Finally, we identified several potential ergovaline degrading bacteria such as Paraprevotella and Coprococcus. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS) on two commercial dairy farms, to develop strategies to minimize these losses. We first developed a catalog of the archaea and bacteria that were present therein based on the 16S rRNA-V4 amplicons from manure samples collected from several locations and depths of the storages. Then, we inferred the respective metabolic capabilities via PICRUSt2 and literature curation, and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome composition was more complex and exhibited more location-to-location variation in EP compared to CS. Further, the inlet and a location with hard surface crust in EP had unique consortia. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds such as anammox and autotrophic nitrifiers. However, microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction (DNRA) seemed possible. Minor quantity of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. Higher prevalence of nitrate-transforming microbes at the near-surface locations and all depths of the inlet were found as a result of this instance. These findings suggested that ammonia oxidation to nitrate started on the barn floor and as manure is being stored in EP and CS, nitrate was lost to the environment via denitrification. For carbon transformation, hydrogenotrophic Methanocorpusculum species were the primary methane producers, and it exhibited higher abundance in EP. / Doctor of Philosophy / Sustainable farming is an integrated practice of crop and livestock production systems that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another such as the utilization of arable land to grow forages for livestock grazing or the use of manure as organic nitrogen amendments for crops is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for a better sustainable farming practice. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the integrated crop-livestock system with a broader goal of manipulating these to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed bioinformatic analysis of a genetic marker for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle grazing alternately on KY-31 tall fescue, a major grass used in Virginia that carry a toxin-producing fungi, and nontoxic MaxQ tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify potential microbial species capable of degrading the toxin for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially, especially affecting microbes responsible for degrading cellulose and starch. Some of these communities lingered once developed, and a different microbial population surfaced upon transfer to MaxQ. Several potential toxin-degrading bacteria were also identified. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS), to develop strategies to minimize these losses. We first develop a catalog of the archaea and bacteria that were present in the manure samples collected from several locations and depths of the storages based on a genetic marker. Then, we inferred the respective metabolic capabilities and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome exhibited more location-to-location variation in EP compared to CS. Oxygen exposure, continuous addition of fresh manure, and the presence of crust at the storage surface gave rise to these unique populations. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds. However, microbial conversion of nitrate to gaseous N2, NO, and N2O seemed possible. These observations showcased that ammonia is stable during storage. Nitrate, on the other hand, can be converted into volatile nitrogen compounds via various processes. Thus, it is imperative to limit the level of nitrate in manure prior to placement in the storage, which is potentially originating from oxidative processes occurring on the barn floor.

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Surgical preparation reduces commensal bacterial load. Currently, no standardized preoperative ocular preparation method in the dog has been reported. Previous studies use culture-based methods to determine commensal bacterial populations. Recent reports suggest that high-throughput sequencing may be superior to culture techniques to determine bacterial communities in the eye and other tissues. The goal of this study was to describe the conjunctival commensal ocular microbiome and bacterial community using DNA sequencing and aerobic cultures of six normal, healthy dogs and investigate the short and long-term effects of an antiseptic protocol on the ocular microbiome. Samples were obtained prior to, immediately following, 24 hours following, and 4 weeks following ocular preparation. The Mississippi State University microbiology laboratory evaluated aerobic cultures, and the Gastrointestinal Laboratory of Texas A&M University performed DNA sequencing. This is the first study to show short and long-term effects of standard ocular surgical preparation on the ocular surface microbiome.

The dam vaginal microbiota is the first major microbial inoculating community within the neonate. The composition of the dam vaginal microbiota has implications in calf commensal microbiota development. Alterations of the dam microbial community prior to parturition could alter inoculating communities and immune responses in both the dam and calf. Thus, authors aimed to elucidate the microbial community composition of the bovine dam vaginal and calf nasal microbiota post-partum after utilizing betadine lavages (BL). The dam vaginal and calf nasal microbial communities and immune responses were evaluated at 0-, 15-, 30- and 60-day post-partum. Microbiota composition of the dam haircoat, udder, and IgG in the colostrum/calf sera were also evaluated at day 0. Serial BLG prior to parturition did not alter the alpha diversity of the dam-vaginal microbiota but did alter the calf-nasal microbiota at parturition (P = 0.03). Dams receiving BLG prior to calving had increased colostrum IgG concentrations compared to CON dams (P =0.04). These results suggest physiological insults (BLG) prior to parturition led to an increased immune response which altering dam colostrum IgG. Thus, neonatal colostrum consumption could drive immune responses against inoculating bacteria resulting in differing nasal microbial communities between treatment groups. The beta diversity of the calf nasal microbiota was significantly different at day 0 compared to all other timepoints (P = 0.006). The calf nasal beta diversity at day 15 was similar to day 30 (P = 0.38) but significantly different compared to day 60 (P = 0.006). There was no effect of time on altering the alpha (P = 0.60) or beta (P = 0.06) diversity of the dam vaginal microbiota. The calf nasal microbiota was different from the dam vaginal microbiota at all timepoints post-partum, regardless of treatment. At day 15, the alpha and beta diversity of calves was altered compared to day 0, suggestive of a reinoculation timepoint between 0 and 14 days of age. Together, this data contributes to the paucity within beef cattle dam-calf post-partum microbiota literature and provides directionality for future research objectives within this field.

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Mangrove forests are highly productive ecosystems widespread in tropical and subtropical coastlines, with a coverage of 75% of the world’s tropical shorelines. Mangrove plants developed specific physiological and morphological adaptation to thrive in such unique environments. Together with plant adaptations, mangroves develop a tight partnership with microorganisms, mainly bacteria and fungi, that form the so-called mangrove-microbiome. Plant-associated microorganisms are generally recruited by the root system (root tissues and rhizosphere) and the colonization process starts with the release of root-related exudates detected by the surrounding edaphic microorganisms that are attracted in the rhizosphere zone. Then, root surface selects those microorganisms that can enter the tissues as endophytes. The microorganisms recruited belowground can migrate through the plant tissues by using the plant vessels and may colonize the aboveground compartments of the plant. Here, I aimed to evaluate the environmental origin and compartmentalization of the mangrove microbiome. To do this, I sampled bulk sediments, sea water, and mangrove plant compartments (root rhizosphere and endosphere, pneumatophores, shoot, leaves, flowers and propagules) of 20 gray mangrove trees (Avicennia marina L.) across two sites on the Red Sea coast of the King Abdullah University of Science and Technology (KAUST), Saudi Arabia. By high-throughput sequencing of the bacterial 16S rRNA gene, I showed that the bacterial assembly in A. marina plant compartments follows a clear niche partition process in which bacterial communities are actively recruited from the surrounding ecosystem (sediment and sea water) by the root system, and further distributed across the different plant organ and compartments. Moreover, the composition of microbiome detected had many similitudes with others previously described around the world, suggesting that certain bacteria represent a mangrove “core microbiome”. The conservation of microbiome composition, mainly driven by environmental and host selection, that beneficial bacteria provide to the plant and contribute to its growth and fitness by several mechanisms. Thus, the characterization and identification of mangrove microbiome can meliorate our knowledge regarding plant–microbe interactions, as well as put the bases for the development of Nature-based Solution (NBS) to enhance reforestation and rehabilitation of mangrove ecosystems