Variation in exposure to environmental microbial communities has been implicated in the etiology of allergies, asthma and other chronic and immune disorders. In particular, preliminary research suggests that exposure to a high diversity of microbes during early life, for example through living in highly vegetated environments like farms or forests, may have specific health benefits, including immune system development and stimulation. In the face of rapidly growing cities and potential reductions in urban greenspace, it is vital to clarify our understanding of the relationship between vegetation and microbial communities so that we can better design cities that support human health. To explore whether and how urban airborne bacterial communities vary with the amount and structural diversity of nearby vegetation, I used passive air sampling and culture-independent microbial DNA sequencing combined with more traditional landscape architecture tools, including geographic information systems (GIS) and remote sensing data. The results indicated that locations with little vegetation (i.e., paved parking lots) were marked by significantly different bacterial composition from areas that were heavily vegetated (parks and forests). These differences were largely driven by taxonomic groups and indicator species that were enriched at certain sites. My work also shows that regional agricultural activities during the summer may have a substantial effect on airborne bacterial communities in the Eugene-Springfield metropolitan area (Oregon), specifically through elevated abundance of Sphingomonas faeni, a taxon previously isolated from hay dust. The second part of my work focused on building a conceptual bridge between scientific findings and potential design principles that can be tested in practical application. I performed a narrative review of vegetation-health, vegetation-microbe, and microbe-health relationships, which formed the foundation of a framework to translate scientific findings into design-relevant concepts. Strengthening this linkage between science and design will help ensure that research questions are relevant to design practice and that new scientific knowledge is accessible to designers. This dissertation includes previously published and unpublished co-authored material. / 2020-09-06

The intestinal microbiome (IM) or the community of commensal and pathogenic microbes that inhabit the intestinal tract of fish has long been of interest in aquaculture because of its hypothesized role in nutrient digestion and fish immunity. Artificial improvement to the IM of fish with pre- and probiotics has been shown to have benefits in some studies; however, the mechanisms behind these supplements are poorly understood because of a lack of knowledge on the basic structure of the IM of fish. The research described here aims to characterize the IM of the highly studied and commercially important Icatlurid catfish, channel catfish Ictalurus punctatus and blue catfish I. furcatus. In this study we evaluated differences between the homeostatic IM, across genotype (Objective 1) and developmental ontogeny (Objective 2), using high-throughput sequencing of 16S rRNA gene amplicons. We compared the IM of four selected strains of blue catfish and three selected strains of channel catfish at 193 days post-hatch (DPH) in Objective 1 (n = 40); while the influence of developmental ontogeny was evaluated by observing the IM of a single family of channel catfish at 3, 65, 125, and 193 DPH Objective 2 (n = 20). The bacterial phyla Fusobacteria, Firmicutes, and Proteobacteria were detected in all fish strains and developmental time points; however, at the genus level the abundances of different bacteria varied among experimental units, as well as being rather variable between individuals. At 3 DPH (n = 5) the IM of channel catfish sac-fry larvae showed the most variation between individuals; with bacteria from the genus Bradyrhizobium dominating the IM of two sac-fry (84% and 88%), and Flavobacterium, Lactobacillus, Comamonadaceae dominating the IM of the remaining three sac-fry analyzed (86%, 99%, and 97% respectively). The dominant bacteria in the gut of all other fish analyzed (n = 55), was Cetobacterium somerae (mean ± SD, 41.4% ± 36%), a commensal microbe that is known to produce vitamin B12. Statistical differences in the abundance of three different bacteria within the order Clostridiales (P ≤ 0.039) were detected in Objective 1, with more of these OTUs inhabiting the gut of channel catfish than that of blue catfish. Statistical differences in the abundance of Edwardsiella, a genus of bacteria known to cause disease in catfish, were detected across the two species of Ictalurid catfish in Objective 1 (P = 0.038), and across developmental ontogeny in Objective 2 (P = 0.021); however, these bacteria were detected at low abundance (0.002% - 0.004%). Comparisons of beta diversity showed significant differences between many strains of Ictalurids, with a highly significant difference between channel catfish and blue catfish (P = 0.001); however, few differences were detected when comparing beta diversity across the four time points over the first 193 DPH in Objective 2, with only 3 DPH and 125 DPH showing significant differences (P = 0.022). Overall these data suggest host-genotype, and to a lesser extent developmental ontogeny, influence the structure of the IM of Ictalurid catfish. As the first study to be conducted on the IM of these fish species, these results have performance implications on the culture of these commercially important catfish, while also enhancing our basic knowledge on the dynamics of the fish microbiome.

In total, four experiments were conducted to determine the therapeutic and safety effects of the nutraceutical, turmeric and its active ingredient curcumin on canine and equine. Two studies were conducted on client-owned, moderately arthritic canines, studying the therapeutic and safety effect of curcumin’s anti-inflammatory properties. In Exp. 1, two different dosages, 500 mg, SID of 95% curcumin and 250 mg, BID of 95% liposomal-curcumin, were evaluated in ten moderately arthritic dogs over five months. The dogs in the 95% curcumin group had an overall greater significance in pain reduction by Day 60. Exp. 2, was a follow-up experiment to Exp. 1. In Exp. 2, two different dosages, 500 mg, SID or 100 mg, SID of 95% curcumin, were evaluated in ten moderately arthritic dogs over five months. Findings showed that dogs in the 500 mg, SID group had an overall greater significance in pain reduction by Day 60. Experiment 3 and 4 were a two-part project looking at the anti-microbial and anti-inflammatory properties of turmeric, curcumin, and liposomal-curcumin in cecally-cannulated equine. Exp. 3, was a two-part in vitro study, the first part looked at the anti-microbial effects of turmeric, curcumin, and liposomal-curcumin in reducing opportunistic bacteria found in the equine hindgut, including Streptococcus bovis/equinus complex (SBEC) (P = 0.0056), E. coli K-12 (P = 0.5114), Escherichia coli general (P = 0.1083), Clostridium difficile (P < 0.001), and Clostridium perfringens (P = 0.2439). Treatment D, 95% liposomal-curcumin, numerically reduced the concentration of all five opportunistic strains, and was therefore selected for use in the follow-up in vitro experiment. The second in vitro studied the effects of four different dosages, 15 g, 20 g, 25g, and 30 g of 500 mg/g of 95% liposomal-curcumin at reducing the concentration of SBEC (P < 0.0001), E. coli K-12 (P = 0.0124), E.coli general (P = 0.032), C. difficile (P = 0.5608), and C. perfringens (P = 0.4214). In Exp. 4, 500 mg/g of 95% liposomal-curcumin at 15 g, 25 g, and 35 g, were tested in vivo for anti-inflammatory and anti-microbial therapeutic effects. In total, four experiments were conducted to determine the therapeutic and safety effects of the nutraceutical, turmeric, and its active ingredient curcumin on canines and equines. Two studies were conducted on client-owned, moderately arthritic canines, studying the therapeutic and safety effect of curcumin’s anti-inflammatory properties. In Exp. 1, two different dosages, 500 mg, SID of 95% curcumin and 250 mg, BID of 95% liposomal-curcumin, were evaluated in ten moderately arthritic dogs over five months. The dogs in the 95% curcumin group, overall, had a greater reduction in pain by Day 60. Exp. 2, was a follow-up experiment to Exp. 1. In Exp. 2, two different dosages, 500 mg, SID or 100 mg, SID of 95% curcumin, were evaluated in ten moderately arthritic dogs over five months. We observed that dogs in the 500 mg, SID group had an overall greater significance in pain reduction by Day 60. Experiment 3 and 4 were conducted as a two-part project looking at the antimicrobial and anti-inflammatory properties of turmeric, curcumin, and liposomal-curcumin. The purpose of these studies were to investigate both form and dose of turmeric and its active ingredient, curcumin, on reducing opportunistic bacteria found in the equine hindgut. The bacterial strains of interest included Streptococcus bovis/equinus complex (SBEC), Escherichia coli K-12, Escherichia coli general, Clostridium difficile, and Clostridium perfringens. Exp. 3, was a two-part in vitro study; the first part looked at the antimicrobial effects of turmeric, curcumin, and liposomal-curcumin (LIPC) on reducing opportunistic bacteria found in the equine hindgut, including SBEC (P = 0.006), E. coli K-12 (P = 0.50), E. coli general (P = 0.11), C. difficile (P < 0.0001), and C. perfringens (P = 0.24). The follow-up in vitro 24 h batch culture examined four different dosages (15 g, 20 g, 25 g, and 30 g) of 500 mg/g of LIPC, at reducing the concentration of opportunistic bacteria. These results were utilized to determine the dosing rate in vivo. Exp. 3, in vitro, evaluated the efficacy of antimicrobial and anti-inflammatory properties of LIPC dosed at 15, 20, 25, and 35 g. These results were utilized to determine the dosing rate in vivo. Exp. 4, in vivo, evaluated the efficacy of antimicrobial and anti-inflammatory properties of LIPC dosed at 15, 25, and 35 g compared to a control. In vivo, LIPC’s antimicrobial properties, at 15 g, significantly decreased (P = 0.02) SBEC compared to other treatments. In addition, C. perfringens tended (P = 0.12) to decrease as LIPC dose increased. Non-significant results in digestion, blood parameters, and range of motion suggest there were no adverse side effects from oral dosing increasing doses of curcumin. Valerate decreased (P = 0.005) linearly as LIPC dose increased. As LIPC dose increased, butyrate and iso-valerate decreased (P ≤ 0.03) linearly. However, acetate tended (P = 0.10) to increase linearly as the dose of LIPC increased. Treatment did not affect (P ≥ 0.19) any of the other individual VFAs measured, but increasing doses of LIPC tended (P = 0.10) to increase total VFA concentrations. Additionally, LIPC tended (P = 0.11) to increase total VFA concentrations when compared to control. In the future, further work should be conducted examining liposomal-curcumin’s antimicrobial properties in canine and anti-inflammatory properties in equine over a longer period of time

To our knowledge, this metagenomics study is the first of its kind to determine how cover crops and tillage management practices affect the soil microbiome in southern Illinois. Seven different cover crops were used over the course of two years from 2014 to 2015, and two different forms of tillage were used: Conventional Tillage (CT) and No-Tillage (NT). Four barcodes were used to generate libraries for the phylogenetic identification of fungi, bacteria, oomycetes, and fusaria: the ITS1, EF1a (Elongation Factor 1-a), and the V4 region of the 16s rRNA subunit. Targeted amplicon sequencing using 250 base pair Paired End (PE) reads yielded 14 x 106 base pair reads in total. Using these amplicons, we successfully unveiled the fungal and bacterial constituents of the studied field plots (database limitations considered) using the QIIME and NCBI Blast protocols. Specifically, this study had three goals 1) to determine if cover crops or tillage had a significant impact on the overall microbial diversity found in bulk soil samples taken from cover crop plots; 2) to determine if the incidence and abundance of individual bacterial or fungal taxa were affected by the cover crop or tillage treatment; 3) perform a bioinformatics methodology comparison for fungal identification using the ITS1 region between Qiime, and MEGAN protocols. Our results indicate many instances of cover crop or tillage interacting with one or more groupings of taxa. Significant whole community differences could be detected to the species (P=0.0335) and family (P=0.0001) taxonomic ranks of fungi using with the three most abundant families based on assigned reads being Mortierellaceae, Trichocomaceae, and Botryosphaeriaceae. Significant whole community interactions between tillage types and year at the level of phylum were observed between bacteria and archaea. Three main phyla constituting bacterial reads were Proteobacteria, Actinobacteria, and Acidobacteria. The primary driver in individual differences in bacterial populations appeared to be the year in which samples were taken either 2014 or 2015 (P=0.0001). This was attributed in part due to drastic fluctuations in weather from November 2014 to November 2015. Whole community differences and shifts could be observed based on cover crop down to the species level using both QIIME and NCBI BLAST protocols. The different dispersions and taxa found between cover crops imply that there is a relationship between certain organisms and the type of plant matter present. Tillage type, year, and cover crop were all found to have some degree of clustering based on reads taken from the four amplicons used. For comparison between NCBI and QIIME methodologies using the ITS1 region, the NCBI BLAST protocol provided the most overlap between taxa at the Order and Class taxonomic rankings. An upwards of 70% complementarity of taxa was found comparing the results after using the NCBI or the QIIME protocols. Whole community analysis using PERMANOVA revealed complementarity shifts based on treatment types when comparing both QIIME and NCBI protocols for taxonomic assignments visualized using PCoA plots. This comparison between the two methods for fungal community analysis using the ITS region, highlights the significant discrepancies as well as the complementarity of the two methodologies when analyzing fungal microbiomes.

Master of Science / Department of Agronomy / Charles W. Rice / Maize (Zea mays L.) is a major staple crop whose wild ancestor was domesticated about 9,000 years ago (Beadle, 1939). Long-term breeding for more desirable traits ultimately resulted in the maize we see today. This long-term breeding likely impacted the processes within the rhizosphere of maize, however, to what extent is not well understood. This study examined the microbial communities between an inbred maize line (B73), a hybrid of two isogenic lines (B73xMo17), and two genetically modified maize hybrids (DKC63-55RIB and DKC64-69RIB) to determine if the plant’s ability to attract beneficial microbes changed with breeding. The hypothesis was that the isogenic cultivar forms better relationships with bacteria and fungi compared to the newer cultivars, especially in low P soil. It was also expected that the greater the difference between the cultivars the more distinct their soil microbiome. To test these hypotheses, experiments were conducted under greenhouse and field conditions. Analyses consisted of root staining to test symbiotic relationships, phospholipid fatty acid analysis (PLFA) for microbial communities, total plant and root biomass, and nutrient content to understand plant responses. Based on the field results, there was no impact on root and shoot biomass and nutrient content by differences in cultivar. Differences in cultivar did impact arbuscular mycorrhizal fungi (AMF) colonization, which decreased over time and depth for all. Soil AMF also saw a significant effect by cultivar. Other microbial groups were not impacted by cultivar, were greatest in the control, and decreased over time. Greenhouse results showed a cultivar by time interaction for root and shoot biomass. Soil P also impacted shoot biomass, but not root biomass. Shoot nutrient content was greater in high P soil, while roots only saw an impact for root P. No cultivar effect was found for soil microbial groups except for fungi, while all microbial groups were reduced in the control soil. Most soil microbial groups were also impacted by soil P as indicated by reduced concentrations in low P soil. AMF was the only microbial group that was not negatively impacted by limited soil P. In addition, all soil microbial groups increased over time, although fungi saw a decrease at R1. No significances were observed for percent AMF colonization.

Recent studies have uncovered a strong effect of host genetic variation on the composition of host-associated microbiota. Here, we present HOMINID, a computational approach based on Lasso linear regression, that given host genetic variation and microbiome taxonomic composition data, identifies host single nucleotide polymorphisms (SNPs) that are correlated with microbial taxa abundances. Using simulated data, we show that HOMINID has accuracy in identifying associated SNPs and performs better compared with existing methods. We also show that HOMINID can accurately identify the microbial taxa that are correlated with associated SNPs. Lastly, by using HOMINID on real data of human genetic variation and microbiome composition, we identified 13 human SNPs in which genetic variation is correlated with microbiome taxonomic composition across body sites. In conclusion, HOMINID is a powerful method to detect host genetic variants linked to microbiome composition and can facilitate discovery of mechanisms controlling host-microbiome interactions.

archives@tulane.edu / 1 / Caitlin Rose Bumby

Coral reefs are crucial for the ecological sustainability of the oceans, yet, increasing sea surface temperature is threatening these ecosystems globally. Microbial communities associated with corals have become a recent research focus, as the associated microbiome may contribute to coral resilience to environmental stressors, e.g., heat stress. However, research in this area is hampered by the difficulty of working with corals. This study aims to use Aiptasia, a sea anemone, as a tractable laboratory model system to study the role of the coral microbiome. Analyses of the bacterial compositions associated with different Aiptasia strains across two temperatures (25 °C and 32 °C), based on 16S rRNA gene sequencing. This study aims also to identify a “core” microbiome associated with heat stress acclimation, as well as host-specific differences. In general, results showed that bacterial composition associated with Aiptasia strains differs significantly with temperature. Higher bacterial diversity and richness were observed when all Aiptasia strains were placed under heat stress. Moreover, results showed an increase in beta diversity and dispersion of bacterial communities in response to heat stress. These changes in the bacterial composition are in line with the recently described “Anna Karenina principle” for animal microbiomes, which suggests that the microbiomes of unhealthy individuals vary more than healthy and stable individuals. This study further shows that while temperature had the greatest effect on structuring the bacterial compositions, there were some variations better attributed to batch and host effects. This suggests that technical aspects have to be carefully addressed in the framework of microbiome studies. Members of a putative “core” microbiome associated with 32 °C Aiptasia have been identified as indicator species of heat stress (i.e., Francisella sp.,). Previous reports have shown that these indicator taxa are associated with saline environments and can tolerate high temperatures. Putative functional profiles based on taxonomic inference of associated bacterial taxa (i.e., enrichment and depletion of various metabolic processes) were also identified, implying functional differences of the microbiomes associated with Aiptasia strains in response to heat stress. Future studies should more specifically examine how the microbiome influences the animal ability to respond to environmental changes.

The human gut microbiome exists as a community of microorganisms in symbiosis with its host. Prebiotics are functional compounds that modulate this microbial community, promoting the growth and activity of bacteria that are beneficial to human health. Resistant starches (RS), a subclass of prebiotics, are compounds linked to a number of host-beneficial effects when included in human diets. Understanding how RS shapes gut flora composition and function is crucial to understanding these effects; however, these effects are clouded by the complexity of the microbiome’s interactions. Comprehensively characterizing microbiome shifts as the result of prebiotics is an intriguing bioanalytical problem. In the thesis project, I hypothesize that: RS changes microbiome biochemical pathway expression community-wide and at different taxonomic levels; that RS forms will affect microbiome bacterial taxonomic distribution; and that a linear programming optimization approach can parsimoniously distribute ambiguous peptide abundances amongst their constituent species, leading to different interpretations of functional and structural characteristics in microbiome metaproteomics data. To address these hypotheses, the thesis project utilizes a combined metaproteomics and bioinformatics approach. The Figeys lab-developed RapidAIM bioanalytical assay is deployed to generate label-free mass spectrometry metaproteomics data, testing for these effects experimentally. Further, Cerberus, a bioinformatics platform for microbiome metaproteomics analyses, was developed to integrate workflows from different software sources into a unified pipeline. Cerberus also implements a novel linear optimization approach addressing the shared-peptide problem. Through experimental data analyses using Cerberus, microbiomes encountering RS showed concerted taxonomic shifts, general and specific functional modulations linked to these taxonomic changes, and a significantly variable pathway expression profile for host-beneficial microbiome processes. The peptide-species linear optimization procedure demonstrates how naïve approaches to the shared-peptide problem greatly skew downstream taxonomic and functional analyses in metaproteomics experiments, marking an important consideration for microbiome studies seeking to resolve taxon-specific alterations.

Progestins are synthetic progestogens that prevent pregnancy by thickening the mucous of the cervix to prevent sperm entry and by disrupting implantation via alteration of the timing of endometrial changes occurring during a normal menstrual cycle. Various hormonal birth control methods utilize progestins, with some of the most effective types of birth control methods being long-acting reversible contraceptives. These include hormonal injections such as depot medroxyprogesterone acetate (DMPA), hormonal implants such as Nexplanon, and hormone-releasing intrauterine devices (IUDs) such as Mirena. Although there have been many studies on the safety and effectiveness of these methods, fewer studies have examined how these hormonal methods may impact the bacterial environment of the vagina, better known as the vaginal microbiome. The health of the vagina relies heavily on the bacteria composing the microbiome. Changes in species composition correlate with higher risk of sexually transmitted infections (STIs) and adverse pregnancy outcomes. When women select their preferred hormonal contraceptive method, they should know if it will impact their vaginal microbiome and increase susceptibility to disease. Twenty-one patients enrolled in this study, with one patient initiating DMPA, 14 initiating levonorgestrel (LNG) IUD, and 6 initiating the etonogestrel subdermal implant (ESI). At initiation, 3 months post initiation, and 6 months post initiation, no differences were seen in the vaginal microbiomes of each of the women enrolled in the study. Some differences in the vaginal microbiota of postpartum women and those who were not postpartum were seen. More specifically, enrichment of three families, Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae, was seen in women who were more than 12 weeks postpartum, but the effects of those differences remain unclear. Although our sample size was small, the lack of changes in the vaginal microbiome in women initiating long-acting progestin contraception is reassuring; further study in this area is needed.