Since the invention of high-throughput sequencing, the majority of experiments studying bacterial microbiomes have relied on the PCR amplification of all or part of the gene for the 16S rRNA subunit, which serves as a biomarker for identifying and quantifying the various taxa present in a microbiomic sample. Several computational methods exist for analyzing 16S amplicon based metagenomics, but the most commonly used bioinformatics tools are unable to produce quality genus-level or species-level taxonomic calls and may underestimate the degree to which such calls are possible. In this thesis, I have used 16S sequencing data from mock bacterial communities to evaluate the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries used for microbiome analyses, with a focus on measuring the accuracy of species-level taxonomic assignments of 16S amplicon reads. With the efficacy of these tools established, I then applied them in the analysis of data from two studies into human microbiomes. I evaluated the metagenomics analysis tools Qiime 2, Mothur, PathoScope 2, and Kraken 2, in conjunction with reference libraries from GreenGenes, Silva, Kraken, and RefSeq, using publicly available mock community data from several sources, comprising 137 samples with varied species richness and evenness, several different amplified regions within the 16S gene, and both DNA spike-ins and cDNA from collections of plated cells. PathoScope 2 and Kraken 2, both tools designed for whole genome metagenomics, outperformed Qiime 2 and Mothur, which are theoretically specialized in 16S analyses. I used PathoScope 2 to analyze longitudinal 16S data from infants in Zambia, exploring the maturation of nasopharyngeal microbiomes in healthy infants, establishing a range of typical healthy taxonomic profiles, and identifying dysbiotic patterns which are associated with the development of severe lower respiratory tract infections in early childhood. I used Qiime 2 to analyze 16S data from human subjects in a controlled dietary intervention study with a focus on dietary carbohydrate quality. I correlated alterations in the gut microbiome with various cardiometabolic risk factors, and identified increases in some butyrate-producing bacteria in response to complex carbohydrates. I also constructed a metatranscriptomics pipeline to analyze paired rRNA-depleted RNAseq data. My evaluation of 16S methods should improve 16S amplicon analyses by advocating for the modernization of computational tools; my analysis of infant nasopharyngeal microbiomes lays groundwork for future predictive models for childhood disease and longitudinal microbiomic studies; my analysis of gut microbes illuminates the mechanisms through which bacteria can mediate cardiovascular health. Taken together, the research I present here represents a significant contribution to 16S metagenomics and its application to epidemiology, clinical nutritional science.

Objectives: Earlier reports suggested that aggressive periodontitis is common in certain African populations and is associated with the JP2 clone of Aggregatibacter actinomycetemcomitans (Aa). There are few studies that investigated the type of microorganisms that colonize the subgingival sites in young subjects inflicted with a subcategory of aggressive periodontitis that is associated with the Aa-JP2 clone. Hence, the objective of this study was to characterize the subgingival microbiome of JP2 clone-associated aggressive periodontitis. Methods: The study subjects were drawn from a large survey among 14-18 years old schoolchildren in Morocco. The sample included 7 JP2-positive aggressive periodontitis subjects and 14 JP2-negative controls. The controls were selected to be either JP2-positive, JP2-negative (but Aa positive), or Aa-negative. Subgingival samples from these subjects were sequenced for the V1-V3 region (16S rRNA gene) on a Miseq platform. High-quality, non-chimeric merged reads were classified with our previously reported BLASTn-algorithm. Downstream analysis was performed with QIIME and LEfSe. Results: There were no significant differences between the groups in species richness. However, aggressive periodontitis subjects showed significantly lower alpha diversity. The microbiomes of aggressive periodontitis clustered distinctively from the controls. However, there was no significant separation between the subgroups of the control group. Species associated with health included Streptococcus spp., Haemophilus spp., Neisseria spp., Gemella spp., Rothia spp., Fusobacterium nucleatum subsp. polymorphum, Porphyromonas oral taxon 279, Veillonella parvula, Granulicatella adiacens and Lautropia mirabilis. Important periodontal pathogens, including Treponema spp., Fretibacterium spp. P. gingivalis and Tannerella forsythia were significantly enriched in aggressive periodontitis subjects. However, the taxa detected in high abundance and showed strongest association with aggressive periodontitis but not the controls were Pseudomonas oral taxon C61 and Enterobacter cloacae. Conclusions: The results suggest that several periodontal pathogens involved in chronic periodontitis also play a role in aggressive periodontitis. Future studies should investigate the role of Pseudomonas and Enterobacter spp. in the pathogenesis of aggressive periodontitis. / Oral Biology

No description available.

The human oral microbiome is the total amount of microbial biodiversity present in the oral cavity and, given its relevance to human health and disease, has recently become a foci for study. By analyzing dental calculus, and sequencing the bacterial DNA, it is possible to reconstruct and examine the oral microbiomes of past individuals. In this study, dental calculus was sampled from (N=4) skeletons recovered from the cemetery of the mid 19th- 20th, century Mississippi State Asylum in Jackson, MS. Bacterial DNA isolation and shotgun sequencing were successful, with 16S analyses yielding an average of 96 identified species. All samples were significantly different from each other at all taxonomic levels (p <0.0001). Targeted examinations for opportunistically pathogenic oral bacteria were performed, but no detectable bacterial DNA was found in the samples. This study is the first to reconstruct the oral microbiomes of a subsample of an historic institutionalized population.

No description available.

The Appalachian Highlands Region is a biodiverse temperate forest that scales through a multitude of states along the eastern United States. This region contains microbial-rich soil with more than 10,000 different species of bacteria in one gram of soil and is still almost entirely undiscovered with very little known about the microbes that call this region home. The goals of this study are to 1) characterize the microbiome of this unique region and 2) potentially find uncharacterized microbes that are capable of producing their own natural antibiotics. Our research team utilized Buffalo Mountain as a training ground to collect soil samples at differing locations such as creek banks, bases of trees, overlooks, forested areas, bald ridges, and many more. With these samples, the team was able to grow and isolate various microorganisms that were characterized to have specific roles in this ecosystem. These included those microbes that have roles in degradation of pollutants, aid in the health of the plant species native to the region, belong to the biome of regional insects, and still others that were able to produce antimicrobial compounds. Through the process of isolation, purification, and 16s rRNA sequencing of the microorganism’s DNA, our team was able to identify many of the regional microbes and begin to characterize them, as well as, testing them against known pathogens such as Staphylococcus, Bacillus, and Escherichia. Two of the isolated microorganisms, PAP6, a previously unidentified and uncultured bacterium, and BK1, identified as Pseudomonas vancouverensis, exhibited zones-of-inhibition towards Gram positive bacteria indicating potentially uncharacterized antimicrobials. Taken together, these results prove that there are numerous culturable microorganisms with numerous unique abilities and qualities in the Southern Appalachian Mountains. Future work from this project will continue to sample the various mountain ranges in the regions, identify and characterize these microbes, investigate potential antimicrobial producers, and map the unique microbiome of our region.

Insect pests are the most widespread disturbance in Canadian forests, but resulting impacts of forest defoliation on stream ecosystem functions are poorly understood. This study investigated the effects of a spruce budworm outbreak on water quality and the structure and function of microbial communities in streams of 12 catchments across a gradient of cumulative defoliation severity in the Gaspésie Peninsula, Québec, Canada. Bi-weekly stream habitat sampling was conducted spring to fall 2019-2021, with stream flow rates measured and water samples collected and analyzed for water chemistry parameters, nutrients, and dissolved organic matter (DOM) structure and quality. Algal communities were assessed at the same time by measuring in-situ biomass. Bacteria and fungi communities on leaf packs were assessed by incubating six leaf packs for five weeks (mid-August- late September) in one stream reach per watershed. Microbial community composition of leaf packs was determined using metabarcoding of 16S and ITS rRNA genes, and functions were examined using extracellular enzyme assays, leaf litter decomposition rates, and taxonomic functional assignments. This study determined that cumulative defoliation increased stream temperatures, flow rates, and SUVA (DOM aromaticity), but not nutrients. It increased algal biomass and altered microbial community composition, with a stronger influence on bacteria than fungi. The observed increases in SUVA and algal biomass corresponded with changes to bacteria carbon cycling functions, which indicated that microbes were preferentially selecting carbohydrates produced by algae rather than the aromatic compounds from increased terrestrial inputs. There were no changes to other bacteria or fungi functions and no changes to taxonomic or functional diversity. Overall, results indicate that forest pest outbreaks alter carbon inputs to streams and the structure and function of stream microbial communities associated with carbon cycling. / Thesis / Master of Science (MSc) / Terrestrial and aquatic landscapes are tightly linked, and forest disturbances can influence stream ecosystems. Insect pests defoliate millions of hectares of forests each year, but the resulting impacts on stream ecosystems are poorly understood. This study investigated the effects of a spruce budworm outbreak on water quality and microbial communities in streams in Gaspésie, QC, Canada. Microbial communities are critical to the functioning of stream ecosystems as they convert energy (e.g., carbon) into useable forms for other organisms. Results indicate that defoliation altered stream flow rates, temperatures, and carbon composition, as well as the microbial communities involved in carbon cycling processes. Carbon is essential to aquatic food webs and this improved understanding of how carbon flow is altered by a widespread forest disturbance can inform pest management decisions for spruce budworm outbreaks.

Analyses of human oral microbiomes reveal substantial amounts of information about health, diet, and diseases of individuals and their communities within the archaeological record. In order to examine bacterial genomes from the past, specific archaeological samples that contain remnants of the microbial communities in question must be utilized. Recent developments in high-throughput, next-generation DNA sequencing have enabled the characterization of entire oral microbiomes and genomes from the remains of the bacteria trapped in calcified dental plaque. This project analyzed samples of ancient human dental calculus from North and Central America, which were examined for the changes within the oral microbiome in relation to the adoption of agriculture. Additionally, the conditional presence of pathogens associated with an increasingly agricultural and carbohydrate-rich diet was examined. The overarching goal was to examine and determine the level of microbial shifts within the past oral microbiomes of North and Central America, and by virtue of using the associated archaeological reports and analyses, place the data into the proper context. Three distinct sets of dental calculus were used for this Dissertation; The first is from Indigenous samples (N=56), spanning from the Archaic to the Mississippian, recovered from excavations in the Guntersville Basin in Northern Alabama. The second set is from a Late-Terminal Classic Maya city center and satellite village in the Upper Belize Valley (N=11). The final sample set comes from an archaeologically recovered early 20th century Cemetery near Jackson Mississippi (N=12). After individual analyses, they are all examined along a temporal axis to examine the effect of agriculture on the human oral microbiome. The findings from this study have shown that oral microbiomes of the Americas were affected by the introduction of agriculture, but remained biologically diverse. Because various subsistence strategies can shape and affect the oral microbiome, the composition is seen to change over time. Our understanding of the evolution of oral microbiomes throughout human history is more complex than previously thought; there is no global trend for the oral microbiome, but is highly location dependent.

Manure nitrogen in dairy cattle represents a substantial economic and environmental loss to the industry. Current strategies used to improve N efficiency (Neff) have shown limited progress and thus, warrant more effective approaches. Considering that the gastrointestinal bacterial community has been associated with various phenotypes of economic importance, the objective of this project was to evaluate the associations between the rumen and fecal bacteriomes and Neff phenotypes in dairy cows. Results showed similarities in the overall bacterial community composition and structure of cows differing in Neff. However, the relative abundance of specific bacterial ASV differed between low and high Neff cows. Furthermore, bacterial ASV strongly correlated with Neff could be involved in processes such as nutrient supply, performance, and feed efficiency. These suggest that the gastrointestinal bacterial community is a factor influencing Neff in dairy cows and specific bacterial members can potentially serve as markers of Neff phenotypes.

No description available.