Browsing by Subject "Microbiome"

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Advancements In Microfluidics For Biotechnology Applications
(2018-10) Agrawal, Pranav
Microfluidic technology has made a huge impact in the field of biotechnology and life sciences. The advancements can be categorized into three aspects: understanding of physical phenomena at the microscale; development of tools for easy integration of different phenomena; and devising systems for various applications. This thesis highlights the ability of microfluidic technology in manipulating different biological entities by fabricating small feature sizes. In particular, we have focused on the development of new processes for three biotechnology applications – (i) long DNA sample preparation for genomic; (ii) delivery of genetic delivery vehicles for gene and cell therapy; and (iii) an in vitro model to study human gut. Each of these systems is developed in close collaboration with potential users and is aimed towards easy integration with the existing workflow. Long-read genomic applications such as genome mapping in nanochannels require long DNA that is free of small-DNA impurities. Chapter 2 reports a chip-based system based on entropic trapping that can simultaneously concentrate and purify a long DNA sample under the alternating application of an externally applied pressure (for sample injection) and an electric field (for filtration and concentration). In contrast, short DNA tends to pass through the filter owing to its comparatively weak entropic penalty for entering the nanoslit. The single-stage prototype developed here, which operates in a continuous pulsatile manner, achieves selectivity of up to 3.5 for λ-phage DNA (48.5 kilobase pairs) compared to a 2 kilobase pair standard based on experimental data for the fraction filtered using pure samples of each species. The device is fabricated in fused silica using standard clean-room methods, making it compatible for integration with long-read genomics technologies. Non-viral delivery vehicles are becoming a popular choice to deliver genetic materials for various therapeutic purposes, but they need engineering solution to improve and control the delivery process. In Chapter 3, we demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in short transfection time (<1 minute) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. Microfluidic in vitro models are being developed to mimic individual or combination of various human organ functions for systematic studies, and for better predictive models for clinical studies. In Chapter 4, we outline a microfluidic-based culture system to study host-pathogen interaction in the human gut. We demonstrate that the infection of Enterohemorrhagic Escherichia coli (EHEC) in epithelial cells are oxygen dependent and can be used to prolong co-culture of bacterial and epithelial cells. This work presents a large scope to study the factors influencing the infection, especially the commensal microbiome in the human gut. Overall, this thesis shows how the microfluidic system can be useful in solving real-life problems and envision further advancements in the field of biotechnology.
Antimicrobial Efficacy of Passive Ultrasonic and Multisonic Irrigation Techniques on Extracted Teeth with History of Pulp Necrosis
(2022-08) Park, Ki Hong
Introduction: The purpose of this study was to analyze the antimicrobial activity in extracted molars with a history of pulp necrosis after conventional treatment with passive ultrasonic irrigation (PUI) technique or minimal instrumentation with multisonic irrigation. Materials and Methods: Twenty-three extracted mandibular molars were prepared for collection of superficial (crown surface control), pre-treatment and post-treatment samples. The teeth were divided into two groups: Group 1: Conventional treatment with PUI (n=11), Group 2: Minimal instrumentation with multisonic irrigation (n=12). The collected samples were processed with 16S rRNA high-throughput sequencing to measure microbial diversity before and after the antimicrobial treatment. The Chao1 index, and Shannon index evaluated alpha diversity, and the Bray-Curtis dissimilarity test was used to measure beta diversity and was visualized by ordination using principal coordinate analysis (PCoA). Kruskal-Wallis test was performed to evaluate the differences in abundances of genera and abundance of each genera in the samples. Differences in each community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. Results: The total number of reads in biological samples ranged from 2 to 120,298. The numbers of OTUs showed a significant difference from pre-treatment to post-treatment in conventional treatment, whereas no significant difference was observed in minimal instrumentation with multisonic irrigation (p < 0.001). Overall, pre-treatment samples presented greater relative abundance of Lactobacillus, Parvimonas, Prevotella, Lacticaseibacillus in the minimal-instrumentation group than in the conventional group although without statistical significance. After treatment, there was a significantly greater reduction of Eubacterium using conventional treatment with PUI and a significantly greater reduction of Prevotella using minimal-instrumentation with multisonic irrigation (Kruskal-Wallis p=0.007 and p=0.002 respectively). Conclusion: There was no statistically significant difference in pre or post-treatment samples between conventional treatment with PUI, minimal instrumentation with multisonic irrigation, and surface controls. All treatments reduced the level of bacteria on the crown surface and in the root canal system.
Aspirin intervention, inflammation and the oral microbiome
(2020-10) Onyeaghala, Guillaume
Specific pro-inflammatory oral taxa have been shown to be increased in the gut microbiome of individuals with colorectal cancer (CRC). Aspirin is associated with decreased risk of colorectal cancer, potentially by modulating the gut and the oral microbiome. However, it remains unclear how pro-inflammatory oral taxa would respond to anti-inflammatory agents such as aspirin. In this dissertation, we aimed to evaluate the effect of aspirin intervention on specific pro-inflammatory oral taxa and inferred functional traits linked to inflammation in a 6-week double-blind placebo-controlled trial.In the first manuscript, we evaluated the effect of aspirin intervention on the relative abundance of pro-inflammatory oral taxa. We found that the change over-time in the relative abundance of 9 out of the 12 pre-specified taxa at the genus level, and 1 out of 2 pre-specified taxa at the family level differed between the aspirin and placebo groups. These preliminary findings suggest that aspirin may change the relative abundance of oral taxa associated with inflammation. In manuscript 2, we evaluated the effect of aspirin intervention on the relative abundance of inferred functional traits linked to the pro-inflammatory bacterial metabolite, lipopolysaccharide (LPS). We did not find an association between aspirin intervention and the change in relative abundance of inferred functional traits for LPS. However, we observed a positive correlation between the relative abundance of pro-inflammatory oral taxa and the relative abundance of inferred functional traits for LPS. These findings are in line with the current literature on bacterial virulence factors. Lastly, we investigated whether inflammation-related oral taxa and inflammation-related gut taxa are correlated and whether oral and gut microbiome communities respond similarly to aspirin. Our results show that aspirin may induce changes in oral and gut alpha diversity in a similar fashion. In addition, our findings of an inverse correlation between SCFA-producing gut taxa and pro-inflammatory oral taxa suggest that studying oral taxa may be important to understanding the link between inflammation and the gut microbiome. Overall, these findings are in line with a growing body of evidence highlighting the role of the oral microbiome in chronic inflammatory disorders of the intestine, including CRC.
Associations Between Nutrition, Gut Microbial Communities, and Health in Nonhuman Primates
(2015-12) Clayton, Jonathan
The primate gastrointestinal (GI) tract is home to trillions of bacteria that play major roles in digestion and metabolism, immune system development, and pathogen resistance, among other important aspects of host health and behavior. In 2009, the Human Microbiome Project was established with the goal of better understanding the role microbial communities play in health and disease. While the research community has made substantial progress in understanding the role microbial communities play in human health and disease, much less attention has been given to host-associated microbiomes in nonhuman primates (NHPs). My research is focused on developing a better understanding of the link between primate microbial communities and the establishment and maintenance of health. I have begun exploring host-associated microbiomes in NHPs, including red-shanked doucs (Pygathrix nemaeus) and mantled howling monkeys (Alouatta palliata), among other species. Some primate species, such as the red-shanked douc, fail to thrive in captivity due to health issues (e.g., gastrointestinal disease). Maintenance of many primate species in captive settings is hindered by critical gaps in our understanding of their natural diet and the enteric microbial adaptations that facilitate the digestive process. By comparing wild and captive animals within the same species, I hope to determine whether shifts in gut microbiota are linked with health in captivity. Microbes can act as indicators for health of the host, thus broad primate microbiome surveys may allow for the development of predictive biomarkers to improve nonhuman primate health and management.
Bacterial communities associated with chronic rhinosinusitis and the impact of mucin degradation on Staphylococcus aureus physiology
(2020-08) Lucas, Sarah
Chronic rhinosinusitis (CRS) is a heterogeneous disease of the paranasal sinuses. Anatomic variation, genetic mutation, viral infection, and allergic response, are all considered to play etiological roles in CRS. Contributors to pathogenesis include immune dysfunction, impaired mucociliary clearance, increased mucus production and stasis, and bacterial virulence mechanisms, however their importance and order of occurrence is not well understood. Microbiological surveys have implicated Staphylococcus aureus as one of the most frequent bacteria in the CRS sinuses. Paradoxically, S. aureus asymptomatically colonizes the nasal passages of ~50% of healthy adults. This work aimed to further describe the diversity of bacterial communities in the paranasal sinuses of patients with CRS using genomic and bioinformatic methods, and reveal new insights into the balance between S. aureus commensalism and pathogenesis. CRS is particularly prevalent in people with the genetic disorder cystic fibrosis (CF). Bacterial infection of the upper airways is thought to be a focus for pulmonary infection, which accounts for the majority of morbidity and mortality in this population. Using 16S rRNA gene sequencing, we described highly similar bacterial communities between sinus mucus and lung sputum in CF patients. CF patients exhibited high interindividual variation, and several instances where a canonical CF pathogen (e.g. S. aureus, Pseudomonas aeruginosa) was seen in the sinus mucus, but not sputum, suggesting this is a location of initial pathogen colonization and adaptation. Further investigation of the bacterial communities in sinus mucus revealed that diversity of the bacterial community was significantly greater in communities with S. aureus as the dominant organism. In contrast, Pseudomonas dominated communities were less diverse, and associated with both increased patient age, and decreased lung function, though not significantly. These results contribute to a growing body of research suggesting a temporal progression of bacterial diversity in the CF airways towards a community characterized by P. aeruginosa dominance. We further hypothesized that the presence of other bacterial taxa could have differential effects on S. aureus physiology in the non-CF CRS. Using 16S rRNA gene sequencing, the bacterial communities were characterized in CRS and non-CRS patient sinus mucus accessed through endoscopic sinus surgery. Compared to the bacterial communities observed in non-CRS mucus, CRS was characterized by an increased proportional abundance of facultative and obligate anaerobic bacterial genera including Streptococcus, Prevotella, and Fusobacterium, and a decrease in Actinobacteria compared. Guided by taxonomy, we computationally predicted the metagenomic content of CRS-associated communities, which suggested an increased functional capacity for the degradation of mucin-glycoproteins - a highly abundant constituent of mucus responsible for its many biological and physical characteristics. Using anaerobic enrichment of CRS sinus mucus bacterial communities on mucins in a minimal medium, we demonstrated a mucin-degradation phenotype, and further described the bacterial communities to include anaerobic genera such as Streptococcus, Prevotella, Veillonella, and Fusobacterium. This work shows that mucins are not efficiently used as a growth substrate by S. aureus, however, mucin degradation and fermentation by CRS-derived bacterial consortia produced metabolites that could augment S. aureus growth. Furthermore, transcriptomic analysis suggests mucin degradation and fermentation products affect S. aureus metabolic state and virulence potential. This work presents high-resolution molecular characterization of the bacterial communities associated with CRS, demonstrates mucin-degradation as a bacterial phenotype carried out by these communities, and suggests a role for mucin-degradation in supporting the growth and virulence of S. aureus.
Determining the Efficacy of a Dairy-Origin Probiotic, Propionibacterium freudenreichii subspecies freudenreichii, against Multidrug-Resistant Salmonella Heidelberg in Turkeys
(2018-02) Valsala Devi Thankappan Nair, Divek
Poultry contributes to more than half of foodborne salmonellosis in the U.S. through contaminated meat, eggs, and other products. Recently, Salmonella Heidelberg (SH) has emerged as a major serovar transmitted to humans via poultry. Cecal colonization of SH and consequent fecal shedding of the pathogen contaminate the environment and carcasses, necessitating interventions to control SH in poultry. Efficacy of a dairy-originated non-host specific probiotic bacterium, Propionibacterium freudenreichii subsp. freudenreichii NRRL 3523 (PF), against a multidrug-resistant (MDR) SH in turkeys has been narrated in this thesis. In vitro experiments revealed that PF reduced the virulence factors of SH associated with its colonization in the host (P<0.05). In addition, PF exhibited anti-SH qualities such as growth reduction, adhesion to intestinal epithelial cells, and survival to low pH and bile salts without possessing pathogenic characters (hemolysis, invasion into the epithelial cells and antibiotic resistance) (P>0.05). Further, the efficacy of PF against SH colonization and its dissemination to internal organs such as liver and spleen in turkeys were validated using in vivo experiments in 2-, 7-, and 12-week-old commercial turkeys. A high PF viability in turkey cecum (3.5-5.0 log10 CFU/ml) was observed in different age group turkeys after supplementing 1010 CFU/ml through drinking water. The PF supplementation resulted in 1.6 to 2.2-, 1.0 to 1.3-, and 1.7- to 2.6- log10 CFU/g reduction of SH in the cecum of turkeys at 2-, 7- and 12-week, respectively (P<0.05), compared to the controls. A reduced dissemination of SH to liver and spleen (P<0.05) also resulted. Populations of PF increased the relative abundance of several production-associated beneficial bacteria, including carbohydrate fermenting and short chain fatty acid producing groups (P<0.05). However, SH inoculation caused an increase in abundance of microflora associated with inflammatory response (P<0.05). Overall the results revealed that PF could be used in turkeys to control MDR SH colonization in the cecum and its invasion of liver and spleen without adversely affecting the cecal microbiome. Whereas SH challenge resulted in an abundance of inflammation associated bacteria, and the condition was effectively alleviated by increased abundance of other beneficial bacteria as a result of PF supplementation.
Development And Application Of Targeted Dna Sequencing Tools To Profile Microbiome-Wide Antimicrobial Resistance And Pathogens Of Public Health Importance
(2024-02) Slizovskiy, Ilya
Antimicrobial resistance (AMR) poses critical health challenges as drivers of frequent and severe global outbreaks. In the U.S. alone, AMR accounts for one infection every eleven seconds, and one death every fifteen minutes. Bacterial antimicrobial resistance genes (ARGs) are the underpinning determinants of AMR, and traditionally prevention and surveillance efforts have focused on cultivating and studying resistant pathogens harboring DNA-encoded ARGs, isolated from human, animal, food, and environmental sources. However, across most compartments of the biosphere, bacteria reside as community members of complex microbial ecosystems with diverse ecological interactions and defined niche profiles. The perspective of the community-level composition and processes that lead to AMR rise and dissemination is rarely accounted for. Though culture-independent methods like PCR have been used for decades, recent advances in the field of metagenomics offers the possibility of directly sequencing the entire genetic milieu across the total microbiota within a sample (i.e. the ‘metagenome’). This technique offers an ecosystem-wide glimpse into bacterial community members, their genes, and their functional potential. However, metagenomic sequencing is rarely adopted in public health surveillance and tracking of pathogens, as well as risk assessment of AMR. The resulting sequencing data offers a low resolution and fragmented view of AMR hazard potential within microbial communities which is not conducive to motivating quality evidence-based decision-making for clinicians, public health practitioners, food producers, and policy makers. This dissertation consists of four integrated studies that attempt to: (1) Formalize the major impediments precluding informative metagenomic sequencing and data analysis in the study of AMR and its hazard potential; (2) Demonstrate an improved metagenomic approach to elucidate epidemiological trends in a major public health context of AMR; (3) Innovate and implement a novel metagenomic sequencing platform and associated bioinformatic tools to address impediments to metagenomic sequencing and enhance risk characterization of AMR; and (4) Extend technical metagenomic innovations for deployment in public health surveillance and monitoring activities. All metagenomic methods involved the use of unique human, animal, environmental, and food safety-related samples, and all studies were conducted using systems in vivo, in vitro, and / or in silico.
The Digestive Tract Microbiome and Cardiometabolic Disease: Exploring Nitric Oxide and Lipopolysaccharide Synthesis as Mechanistic Intermediates
(2023) Bohn, Bruno
Introduction: The role of the digestive tract microbiomes on cardiometabolic health is becoming ever clearer. However, mechanistic remain largely unexplored and methods exploring metabolic outputs are lacking. This dissertation explores a novel approach to quantify the microbiome's lipopolysaccharides (LPS) and nitric oxide (NO) producing potential, two molecules with known health effects. We investigated the association between functional metrics and cardiometabolic health among healthy individuals and heart failure (HF) patients.Methods: Cross-sectional data from adults free of cardiometabolic disease (ORIGINS) and with HF (HFM) were used. Blood pressure was measured and biomarkers or inflammation and/or endotoxemia quantified from blood serum. Oral (saliva) and/or gut microbiomes were characterized with 16S rRNA (HFM/ORIGINS) and/or metagenomic sequencing (ORIGINS). Pathway- and gene-level functional profiles were operationalized as metrics of LPS- and NO-producing potentials. Metrics were compared across sequencing approaches (ORIGINS). For each cohort, multivariable linear regression was used to explore associations between: i) blood pressure and NO-producing potential; ii) inflammation and NO-producing potential; iii) inflammation and LPS-producing potential; and iv) endotoxemia and LPS-producing potential (HFM). Results: In ORIGINS, 253 and 170 participants had 16S and metagenomic data, respectively. A modest positive association was observed between functional metrics, with more features detected through 16S methods. Metagenomic and 16S-derived NO-reducing potential metrics were linked to lower inflammation and blood pressure, respectively. No meaningful associations were observed for LPS-producing potential. In HFM, saliva and gut 16S sequencing data was available for 146 and 128 participants, respectively. No correlations were observed between gut and oral microbiomes. No meaningful associations were observed between NO-producing potential and blood pressure. Gut LPS-producing potential, but not oral, was associated with endotoxemia. Inflammation was not meaningfully linked to either functional feature. Conclusions: A novel approach to utilized microbiome functional profile metrics was explored. Findings were mixed, but highlighted the potential use of these approaches in population-based studied of the human microbiome and cardiometabolic health.
Effect Of Early-Life Management Practices On The Rumen Metagenome Of Beef Cattle
(2023) Diaz Ortiz, Gerardo
Although the rumen microbiome plays a critical role in beef cattle health and productivity, the effect of early-life management practices on the rumen metagenome of beef calves has been scarcely studied. Through two research studies, this thesis investigated the effect of four castration time windows and two weaning strategies on the rumen metagenome of beef calves through a comprehensive metagenomic sequencing approach that included a technical comparison of shotgun metagenomic sequencing and 16S rRNA gene sequencing to study the rumen microbiome. We found that castration timing had limited long-term effects on the rumen microbiome, while weaning strategy showed short-term effects on the microbiome composition and methanogenic gene content of the rumen, but not on the rumen resistome. In addition, our results showed that despite technical discrepancies, 16S rRNA gene sequencing and shotgun metagenomic sequencing methods led to similar ecological inferences about the effect of weaning strategy on the rumen microbiome of beef calves. These studies highlight the importance of considering early-life interventions in beef cattle management and provide insights into the comparability of rumen microbiome sequencing methods. Understanding rumen microbiome dynamics and choosing appropriate sequencing approaches are crucial for advancing rumen microbiome research in beef cattle.
Evaluating the information content of human microbiomes
(2022-03) Hillmann, Benjamin
Microbes vastly outnumber all other organisms on earth and are integral to many aspects of the ecological fitness of the earth’s soils, oceans, animals, and plants. Unfortunately, most of the microbes in these communities cannot be cultured, so to observe these communities’ biological functions, we must study their DNA. After a researcher sequences a microbial community, they utilize informatics methods to correlate the taxonomic and functional profiles to their traits of interest. However, these methods assume that the underlying taxonomic and functional profiling are accurate. If procedures are developed to identify the profiles of a community more accurately, the increased precision will enable higher power testing of hypotheses and detection of these communities’ causal roles. We propose novel, accurate, and data-efficient methods for taxonomic and functional profiles in shotgun metagenomic datasets.
A history of both clonality and recombination governs the population structure of Alternaria endophyte communities on prairie Dalea
(2021-10) DeMers, Mara
Prairie has become one of the most endangered biomes in North America under changing climates engendered by anthropogenic activity. The patterns of diversity we observe of native prairie species in remnant prairie sites are the outcome of both historical and contemporary processes, and understanding these underlying processes will inform the responses of these populations to future environmental changes. The assembly of fungal endophyte communities within plants depends on the complex interactions of fungal taxa, their host plants, and the abiotic environment. Prairie plant communities provide a unique avenue to explore the interplay of biotic and abiotic factors affecting endophyte communities, since the historical distribution of prairies spans a broad range of temperature and precipitation, while the distances between small fragments of contemporary prairie communities may challenge the dispersal capabilities of these otherwise ubiquitous fungi. We sampled foliar fungal endophytes from two native prairie legumes, purple and white prairie clovers (Dalea purpurea and D. candida), in 17 remnant prairie sites across Minnesota in order to evaluate the relative contributions of abiotic factors, host species, and dispersal limitation to the diversity and structure of these communities. We found that similarity of communities was significantly associated with their location along a temperature and precipitation gradient, and we showed a distance-decay relationship that suggests dispersal limitations only over very large spatial scales. Although the effect of host species was small relative to these other factors, the two Dalea species maintained distinct communities within sites where they co-occur. Our results illustrate the capacity of many of these endophyte taxa to disperse over large distances and across heterogeneous biotic and abiotic environments and suggest that the interplay of biotic and abiotic factors maintains high diversity observed in endophyte communities. Our results showed that community composition of endophytic fungi in Dalea spp. varied along a precipitation and temperature gradient, among hosts, and in apparent response to the abundance of Alternaria spp., but left unexplained patterns of genetic variation among the many isolates assigned to Alternaria alternata. We used genotyping-by-sequencing (GBS) to assess population genetic structure of endophytic Alternaria among sampling sites and between host species, and compare levels of recombination and clonality and assess evidence for sexual or parasexual reproduction in these communities. We found cryptic diversity among sequenced samples, as only a subset aligned well to A. alternata reference genomes. Analysis of sequences closely related to reference A. alternata genomes, treated here as a single species, revealed a high level of clonality, and genotypic diversity shared across populations. However, we also detected evidence of recombination events consistent with sexual or parasexual reproduction, as well as two mating types, MAT1-1 and MAT1-2, which were present in roughly equal frequencies in all but one sampled population. Together, the results suggest recent asexual proliferation after rapid colonization of the sampled sites from an ancestral source population. Recombination likely occurred more frequently in the source population, and is now rare.
Investigating Bi-directional Impacts of the Microbiome and Drinking Water Quality in Drinking Water Distribution System Water Mains and Storage Facilities
(2022-07) Gomez, Christa Kimloi
The microbial communities that live in the biofilms of drinking water distribution system (DWDS) environments can exert significant impacts on drinking water quality before it reaches the consumer. The relatively recent advent and accessibility of powerful culture-independent techniques, such as high-throughput sequencing, have enabled characterization of diverse microbial communities; however, the difficulties of accessing DWDS infrastructure has hindered many efforts to study the health-relevant DWDS microbiome. In this work, high-throughput sequencing and quantitative real-time polymerase chain reaction (PCR) techniques were leveraged to characterize the biofilm communities of simulated and full-scale water mains, as well as the in situ suspended and biofilm communities of elevated water storage towers and underground reservoirs in a chloraminated DWDS. Seasonal variability and drivers of community composition were assessed in the simulated DWDS biofilms and in full-scale drinking water storage facilities. Among other examined drivers of community, the presence and concentration of disinfectant was an important selective pressure that impacted community composition. Communities in the simulated and full-scale DWDS biofilms were generally dominated by bacteria that live preferentially in, and form biofilms, exhibit increased resistance to disinfectant concentrations, or display versatility in substrate-utilization. These included genera that contain opportunistic pathogens, such as Mycobacterium, Pseudomonas, and Stenotrophomonas, genera implicated in microbiologically-caused corrosion of infrastructure (sulfate-reducing Desulfovibrio), - as well as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) that catalyze nitrification processes and can cause reoccurring, problematic decreases in chloramine residual concentrations. Species-level taxonomic resolution of DWDS Mycobacterium, achieved by sequencing a mycobacterial heat shock protein gene, indicated that the bulk of these bacteria were not disease-associated strains. Early stages of community succession occurred rapidly for biofilms on new surfaces that were in proximity to more established biofilms – within a month, the biofilms on new surfaces exhibited similar compositions to neighboring, older biofilms. Apart from early changes in composition indicative of an initialization stage, biofilm communities in water storage facilities were temporally stable, although somewhat spatially heterogeneous. In contrast, suspended communities showed seasonal changes and were heavily influenced by water chemistry. Additionally, suspended communities were spatially homogeneous within a facility, and even at different facilities within the DWDS. In storage facilities that experienced problematic nitrification episodes and decreases in chloramine concentrations, suspended AOB concentrations increased as chloramine concentrations decreased. Notably, decreases in disinfectant were not accompanied by increases in the growth of other bacteria. Rather, as AOB concentrations increased, the total biomass of suspended communities actually decreased. During nitrification events, biofilm and suspended community compositions were most similar, lending further support to the concept that biofilms may act as reservoirs for nuisance and pathogenic bacteria in the DWDS. Abundant taxa were consistent with other studies of DWDSs that maintain chloramine, which provided support for the applicability of these findings to other systems, especially as there are no studies to compare to, to-date, of the microbiome in elevated storage towers.
Investigating the Effects of Antidepressants on Intestinal Bacteria
(2024-04-16) Lebakken, Sophia; Basting, Christopher M; Bailey, Melisa; Schroeder, Ty; Broedlow, Courtney A; Guerrero, Candace; Hemmila, Charlotte; Klatt, Nichole R
Introduction: The gut-brain axis (GBA) involves bidirectional communication between the gastrointestinal tract and brain, which contains many species of bacteria that play an important role in this communication. Major depressive disorder is often treated with antidepressant medications (ADMs) that pass through the gastrointestinal tract; however, the possible adverse effects of ADMs on the gut microbiome are not well characterized. Methods: This project investigates the impact of three selective serotonin reuptake inhibitors, sertraline, fluoxetine, citalopram; one norepinephrine and dopamine reuptake inhibitor, bupropion; and one tetracyclic antidepressant, mirtazapine, on the growth of eight species of gut bacteria, Bacteroides fragilis, Bifidobacterium longum, Bacteroides uniformis, Collinsella aerofaciens, Prevotella copri, Escherichia coli, Akkermansia muciniphila, and Lactobacillus plantarum. Bacteria were treated with various concentrations of each ADM to determine potential impact on growth. We calculated the concentration of drug needed to inhibit growth by 50% (IC50) using spectrophotometry. Results Several ADMs inhibited gut bacterial growth. At 50% bacterial growth inhibition, the most prominent was sertraline (28.742 μM), followed by bupropion (43.976 μM), then fluoxetine (76.449 μM). Citalopram (244.738 μM) and mirtazapine (294.316 μM) exhibited far less inhibition. Discussion These findings suggest ADMs have antibiotic effects that disturb the microbiome resulting in potential consequences for microbiota-GBA interactions. Building on these results, future experimentation will measure uptake and metabolism of ADMs by exposing bacteria to each drug longitudinally. Metabolites will be characterized using liquid chromatography-mass spectrometry. Conclusion Given the profound impact of the gut microbiome on the gut-brain axis, these data provide novel insights into potential mechanisms by which ADMs could have unintended consequences on the gut that may perpetuate, instead of treat, mood disorders thus the microbiome should be further investigated in relation to ADMs.
INVESTIGATION OF THE REMOVAL OF CONTAMINANTS OF EMERGING CONCERN AND THE MICROBIOME IN BIOFILTERS
(2019-04) Ma, Ben
The removal and fate of contaminants of emerging concern (CECs) in water treatment systems is of interest given the widespread occurrence of CECs in water supplies. Biofiltration, which granular media filters are inhabited by viable bacteria, has the potential of providing long-term, sustainable CECs removal in drinking water treatment. Bacterial communities in biofilters can be beneficial through biodegradation of contaminants but also pose potential risks by harboring and releasing detrimental microbes into water distribution systems. In this work, the removal of eight CECs, including atenolol, atrazine, carbamazepine, fluoxetine, gemfibrozil, metolachlor, sulfamethoxazole and tris(2-chloroethyl) phosphate, was investigated in pilot-scale granular activated carbon (GAC)-sand and anthracite-sand biofilters. The effects of water quality and engineering decisions on the biofilter microbiome and the effect of biofilters on the microbiome in filter effluent were evaluated in the aforementioned pilot-scale biofilters. In addition, the geographic patterns of the biofilter microbiome were investigated by sampling filter media from full-scale biofilters at fourteen treatment plants throughout North America. The CECs concentrations in the filter influent and effluent were determined using liquid chromatograph tandem mass spectrometry, and the bacterial abundance and community composition in the biofilters were determined using real-time quantitative polymerase chain reaction (qPCR) and Illumina HiSeq high-throughput sequencing of PCR amplicons. GAC-sand biofilters provided superior CECs removal for all compounds (mean removal efficiencies: 49.1-94.4%) compared to anthracite-sand biofilters (mean removal efficiencies: 0-66.1%) due to a combination of adsorption and biodegradation. Adsorption was determined to be the dominant removal mechanism for most selected CECs in GAC-sand biofilters. A multiple linear regression based empirical relationship considering water quality, engineering decisions, and CECs chemical properties was developed to predict CECs removal in the GAC-sand biofilters. The microbiome in the pilot-scale and full-scale biofilters contained genera that are commonly found in the freshwater environments and water distribution systems, such as Limnohabitans, Flavobacterium, Nitrospira, and, Hydrogenophaga. The microbiome in the pilot-scale biofilters exhibited temporal variations, and varied with media type (GAC vs. anthracite), backwash strategy (chloraminated vs. non-chloraminated), and bed depth. The pilot-scale biofilters effectively removed biomass (~70%) from the water, but only marginally impacted the microbiome in the filter effluent. Significant inter-filter variations were observed in the full-scale biofilter investigation that followed a weak but highly significant distance-decay relationship. The water quality characteristics exhibited a stronger influence on the microbiomes in the full-scale biofilters than the geographic distance according to a multiple regression on matrix analysis. Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) were generally more abundant than ammonia oxidizing archaea in the full-scale and pilot-scale biofilters. The ratios of nitrite oxidizing bacteria to AOB exceeded the theoretical ratio for conventional two step nitrification in most full-scale biofilters (12 of 14 biofilters) and in the pilot-scale biofilters for most of the operation. This work should be beneficial to drinking water treatment facilities in improving CECs removal performance using biofilters, as well as to environmental engineers and scientists in understanding the microbiome in biofilters.
The Microbiome and Fermentation: From Human Evolution to Human Transformation
(2023-05) Guse, Kylene
There has been a recent surge in both the production and consumption of fermented foods in Western culture. Particularly, lacto-fermented vegetables (LFVs) such as sauerkraut, kimchi and other fermented vegetables have become popular due to their proposed health benefits, including potential probiotic effects in the gut microbiome. However, there is limited data on the mechanisms by which LFV exerts health benefits. This thesis documents how LFV can impact human health by analyzing microbiomes from a systems perspective; from microbes in the soil, to fermented vegetables, to the human gut and even human culture.
Microbiome and Immune Response to Salmonella enterica serovar Typhimurium and Lawsonia intracellularis Infection in Swine
(2018-05) Leite, Fernando Lopes Leivas
Salmonella enterica is a leading cause of foodborne illness world-wide. In the US alone Salmonella is responsible for over 1 million cases of disease a year in humans and causes an estimated loss of more than 3.5 billion dollars annually. Pork is frequently associated with food borne illnesses caused by S. enterica in humans, many of which are attributed to Salmonella enterica serovar Typhimurium. Efforts to reduce the incidence of salmonellosis due to meat consumption have mainly remained ineffective. This study extends previous findings that pigs are more susceptible to colonization by Salmonella enterica when co-infected with the pathogen Lawsonia intracellularis. We determined the composition of the porcine gut microbiome in response to co-infection to determine how potential disturbances caused by L. intracellularis could favor S. Typhimurium. This analysis revealed that L. intracellularis led to a decreased abundance of Clostridium species and Clostridium butyricum in addition to other changes that may favor S. Typhimurium. We also investigated if vaccination against L. intracellularis could have an effect on the shedding S. Typhimurium and found that vaccination significantly reduced S. Typhimurium shedding in animals co-infected with L. intracellularis. To better understand the host response to L. intracellularis, we performed transcriptome analysis of infected mucosal tissue and found that infection induced a signature of genes associated with inflammation and proliferation in the gut. We then tested zinc supplementation, which is known to impact immune function, and found that zinc amino acid supplementation led to a significant reduction of lesions caused by L. intracellularis. Finally, we investigated whether co-infection of enterocytes in vitro caused increases in certain inflammatory cytokines. We found that L. intracellularis up regulated expression of IL-8 and TNFα, two pro-inflammatory cytokines crucial to the pathogenesis of S. Typhimurium infection. This research suggests that increased inflammation mediated by L. intracellularis along with changes in microbiome composition are likely responsible for enhancement of S. Typhimurium infection in swine. We have also identified that L. intracellularis vaccination and zinc amino acid complex supplementation are two promising alternatives to the use of antimicrobials in swine.
Prebiotic Dietary Fibers Must Achieve a Threshold of Beneficial Gut Bacteria in Order to Prevent Adiposity and Fatty Liver in Rats Fed High Fat Diets.
(2020-08) Abernathy, Breann
Introduction Prebiotic dietary fibers are dietary fibers that are highly fermented in the large intestine, produce beneficial changes in the gut microbiome, and impart a health benefit to the host. Using reactive extrusion, we have synthesized a novel dietary fiber that is an oligosaccharide of polymerized lactose, which we term polylactose. Here we report on two studies feeding polylactose to rats to determine its prebiotic potential. Methods In Exp. 1, the polylactose preparation contained 51% dietary fiber, 20% free lactose, 5% glucose, and 24% other materials. Rats were fed high fat diets containing 9% total dietary fiber, including cellulose (C, 9%), polylactose (PL, 6%), polydextrose (PD, 6%), and fructooligosaccharide (FOS, 6%). In Exp. 2, the polylactose preparation contained 75% dietary fiber, 9% lactose, 3% glucose, and 13% other materials. Rats were again fed high fat diets containing 9% total dietary fiber, including C (9%), polylactose (6% or 3%), PD (6%), and galactooligosaccharides (GOS, 6%). In both experiments, rats were fed for 10 weeks, then ceca (empty), cecal contents, livers, and epididymal fat pads were collected. Results In both experiments, final body weight and daily energy intake did not differ among the groups. In Exp. 1, feeding PL greatly increased cecum weight (an indicator of fermentation), cecal Bifidobacterium and Lactobacillus species abundance, increased cecal acetate and propionate, and reduced liver lipids and fat pad weight, compared to the HFC group. PD and FOS increased probiotic species and short chain fatty acids slightly (compared to HFC), but not to the same extent as PL, and neither PD or FOS reduced fatty liver or adiposity. In Exp. 2, 6% PL increased cecum weight relative to 3% PL, PD and GOS, all of which were greater than HFC. The cecal microbiome was similar among PL (both 3 and 6%), PD, and GOS, all of which differed from HFC. Liver lipids, fat pad weight, and body composition did not differ among any of the groups in Exp. 2. Conclusions The prebiotic activity of polylactose differed depending on the preparation, for unknown reasons. However, our results suggest there is a threshold of probiotic bacteria abundance that must be attained before beneficial effects are imparted on the host by prebiotics.
Precision Medicine Approaches to Immunosuppression Using Pharmacogenomics and the Microbiome
(2022-08) Saqr, Abdelrahman
In this thesis, I provide two examples of precision medicine applications to currently unsolved drug related problems. In chapter 1, the influence of the microbiome on the enterohepatic recirculation of mycophenolate mofetil (MMF) in hematopoietic stem cell transplant recipients (HCT) is described. There is substantial unexplained interindividual variability in MMF pharmacokinetics. This work illustrates that variability in the gut microbiome composition is associated with enterohepatic recirculation of the mycophenolic acid (MPA), the active metabolite of MMF, and consequently differences in drug exposure. In chapter 2, the influence of CYP3A4 and CYP3A5 genotypes on the magnitude of the drug-drug interaction between tacrolimus and steroids in kidney transplant recipients is described. This drug-drug interaction, while well-known, is unpredictable. Some individuals have an induction of tacrolimus clearance in the presence of steroids and others have little to no changes in clearance. This analysis shows that individuals who carry a loss of function allele such as CYP3A5*3 have a minor and clinically insignificant drug-drug interaction whereas individuals who express CYP3A5 and carry at least one CYP3A5*1 allele have a significant tacrolimus-steroid interaction which results in higher tacrolimus dose requirements during steroid use.
The relationship of gut microbiota in standard and overweight children, before and after probiotic administration
(2019) Linhardt, Carter A; Clayton, Jonathan B; Hoops, Suzie; Amin-Nordin, Syafinaz; Knights, Dan
The usage of probiotic foods and supplements has been widely considered part of a healthy diet by supplementing the gut microbiome with beneficial bacteria. Although the usage of probiotics is a common dietary accessory, there is limited reproducible evidence showing bacterial colonization, thus limiting long term effectiveness. We administered Yakult, a commercial probiotic composed of Lactobacillus paracasei strain Shirota, to overweight and standard weight school children in Malaysia. Using a crossover intervention study design, two groups of school children were administered the probiotic supplement or continued their typical diet in sequential 5-week intervention periods, separated by a 5-week washout period. Fecal samples were collected every five weeks over the course of the 15-week study period. The gut microbiome of each subject was analyzed using 16S rRNA gene sequencing. We observed significant differences in Lachnospiraceae, Coproccus, Roseburia, Pyramidobacter, and Bacteroides ovatus between weight classes. However, differences in overall microbiome diversity between weight classes were not found to be significant. Subjects clustered according to their relative abundance of well-known genera Bacteroides and Prevotella, regardless of age, gender, or weight class. Overall, individual-to-individual variation overshadowed trends in gut microbiome composition associated with probiotic administration.
The Social Microbiome. An Exploration of Bacteria in Vertical and Horizontal Systems in Humans and Baboons
(2024-08) Allert, Mattea
The collection of bacteria, fungi and viruses that comprise the microbiome can have large effects on the host over the course of its lifetime. Changes in microbiome composition are associated with various human diseases, including diabetes, colorectal cancer, and inflammatory bowel disease. Many factors can influence the composition of the gut microbiome, including diet, host genetics, host social interactions, and even the maternal microbiome. Here, I focus on two factors that have been shown to influence microbiome composition: maternal transmission via breast milk, and horizontal transmission via host social interactions. Although the maternal microbial contribution to the infant’s initial gut microbiome assembly has been studied extensively, with emphasis on the role of the mother’s oral, intestinal and vaginal microbiomes, the human breast milk microbiome remains largely understudied. To investigate the maternal milk microbiome taxonomic composition and functional potential in relation to the infant gut microbiome, we collected metagenomic shotgun samples from breast milk and infant stools postpartum, as part of the Mother and Infants LinKed for health (MILK) cohort. A total of 507 samples were collected from 195 healthy and gestational diabetes mother-infant pairs. Mothers with gestational diabetes and their infants did not significantly differ in terms of species composition and functional potential from healthy mother-infant pairs. Overall, the infant gut microbiome was dominated by the presence of Bifidobacteria (mostly B. longum, B. breve and B. bifidum), a pattern which is also reflected in the paired maternal milk samples. We identified both commensal and pathogenic bacterial strains that are shared between the maternal milk and the infant’s gut. Finally, we wanted to better understand the underlying dynamics of social transmission of gut microbiota. Since long-term microbiome transmission studies are impractical in humans, we turned to a longitudinal study on wild populations of baboons from Amboseli National Park, Kenya. We analyzed metagenomic shotgun sequencing samples from two baboon social groups, Mica and Viola. Preliminary results indicate that there are differences in the microbial species composition between the two social groups. With these two projects, we are able to start to disentangle the complex relationship of social connections and environmental influences on gut microbiota.

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