UMD Theses and Dissertations
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Item PROBIOTIC, PREBIOTIC, AND SYNBIOTIC APPROACHES IN SUSTAINABLE POULTRY PRODUCTION THROUGH MICROBIOME MODULATION(2023) Tabashsum, Zajeba; Biswas, Debabrata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter is one of the prominent causative agents of acute gastroenteritis in the US and more than 70% of Campylobacter infections, known as Campylobacteriosis, are occurred through raw or undercooked poultry consumption or improper handling of contaminated poultry products. Moreover, reports show that the antibiotic resistance pattern of Campylobacter is persistent and in the absence of sub-therapeutic antibiotic growth promoter, colonization of this bacterial pathogen in poultry gut or on skin and the potential risk of cross-contamination of the finished poultry products are increasing, Therefore, both conventional and organic/pasture poultry farmers are searching for sustainable alternative to synthetic antibiotics which can reduce colonization and cross-contamination of poultry products with poultry-borne bacterial pathogens specifically Campylobacter. On the other hand, due to the consumers’ demand, majority poultry farmers are growing their chicks without sub-therapeutic growth promotor which leads to slow growth and higher mortality rates. Therefore, to make the poultry farming sustainable, farmers need alternative feed or water supplement which can promote poultry health and growth. Probiotics, prebiotics, or a combination of these two referred to as synbiotics, have emerged as a promising natural and alternative approach to sustainable animal farming. Probiotics and their metabolites such as conjugated linoleic acids (CLAs) play a crucial role in improving host health and act as antimicrobials against enteric pathogens. Our lab developed a genetically engineered probiotic, LC+mcra that can convert more CLA by over-expressing the mcra (myosin-cross-reactive-antigen) in Lactobacillus casei (LC). Further, prebiotic-like components such as bioactive phenolic extracts (BPEs) from berry pomace can stimulate the growth of beneficial microbes including LC, competitively inhibit growth of enteric bacterial pathogens, and promote the growth of chickens in a concentration-dependent manner when applied throughout the growth period. In our previous study, we observed that LC+mcra effectively eliminated Campylobacter jejuni (CJ) in co-culture condition as well as the cell free culture supernatants (CFCS) of LC+mcra was effective in growth reduction of CJ. LC+mcra and its CFCSs also reduced the adherence and invasion ability of CJ to both HD-11 and HeLa cells. Physicochemical properties and gene expressions related to CJ virulence were also altered by CFCSs treatments. These findings suggested, LC+mcra can be an alternative in controlling CJ growth along with other beneficial attributes of LC. Then, we aimed to enhance the efficiency of antimicrobial/beneficial activities of LC+mcra by combining BPEs. In mixed culture condition, LC+mcra in the presence of BPE reduced the growth of CJ more efficiently as well as the CFCS of LC+mcra in the presence of BPE. Interaction of CJ with cultured DF-1, HD-11, and HeLa were altered significantly. Further, combined treatments altered the physicochemical properties and expression of multiple virulence genes such as ciaB, cdtB, cadF, flaA, flaB of CJ. This finding indicates that BPE and LC+mcra in combination might be able to prevent colonization of CJ in poultry. So, in our present study at simulated gut conditions, we evaluated combined effect of LC+mcra and BPE in reducing growth of Campylobacter in cecum contents. Cecum contents were collected from chickens pre-inoculated with kanamycin resistant CJ (CJ-Km), incubated over 48h time period, while being supplemented with either BPE, CFCS from LC+mcra, or their combination. It was found that combined treatments were able to reduce both inoculated and naturally colonized Campylobacter more effectively. Microbiome analysis using 16S rRNA sequencing also revealed that combined treatments were capable of altering natural microflora positively within chicken cecum contents. Then, the effect of sustainable probiotics on CJ colonization and gut microbiome composition was evaluated using chicken as a model. A total of 120 chickens were used in duplicate trials to investigate the effectiveness of LC+mcra in decreasing CJ colonization by means of CJ-Km compared to the control group. We observed that LC+mcra could efficiently colonize various parts of the chicken gut and competitively reduce colonization of natural and challenged Campylobacter. Furthermore, 16S rRNA compositional analysis revealed lower abundance of Proteobacteria, higher abundance of Firmicutes, along with enriched bacterial genus diversity in gut of LC+mcra fed chicken. Outcomes of this study reveal high potential of LC+mcra as sustainable approach to decrease colonization of Campylobacter in poultry gut along with other beneficial attributes. So, we further evaluated the combined effect of LC+mcra and a low dose of BPE on Campylobacter colonization in chicken gut using a day-old chick model. Colonization of CJ-Km as well as the natural colonization of Campylobacter was reduced by the combined effect of LC+mcra and BPEs significantly at all time points. In the cecum contents of the LC+mcra and BPEs treatment group, there was notable change at phylum level microbiome compared to the control group. At genus level colonization of Lactobacillus was significantly higher (1.7 folds), Campylobacter colonization was reduced significantly (6.3 folds), and other microflora remained balanced due to the combined treatment of LC+mcra and BPEs. Therefore, LC+mcra with BPEs could be an alternative to improve the safety of poultry products and reduce campylobacteriosis in humans sustainably. The application period of this synbiotic compositions could be extended to improve the poultry growth rate as an additional benefit of the LC+mcra and BPEs.Item Predicting Cancer Prognosis and Drug Response from the Tumor Microbiome(2021) Hermida, Leandro Cruz; Ruppin, Eytan; Patro, Robert; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tumor gene expression is predictive of patient prognosis in some cancers. However, RNA-seq and whole genome sequencing data contain not only reads from host tumor and normal tissue, but also reads from the tumor microbiome, which can be used to infer the microbial abundances in each tumor. Here, we show that tumor microbial abundances, alone or in combination with tumor gene expression data, can predict cancer prognosis and drug response to some extent – microbial abundances are significantly less predictive of prognosis than gene expression, although remarkably, similarly as predictive of drug response, but in mostly different cancer-drug combinations. Thus, it appears possible to leverage existing sequencing technology, or develop new protocols, to obtain more non-redundant information about prognosis and drug response from RNA-seq and whole genome sequencing experiments than could be obtained from gene expression or mutation data alone.Item Viromics and biogeography of estuarine virioplankton(2021) Sun, Mengqi; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Viruses are the most abundant biological entity in the ocean, and they can influence microbial mortality, evolution and biogeochemical cycles in marine ecosystems. Virioplankton communities in oceans have been studied extensively using viral metagenomics (viromics), but the estuarine viromes remain relatively unexplored. Estuaries are a complex and dynamic ecosystem. My dissertation is dedicated to understanding the composition and distribution of the virioplankton community in the Delaware Bay and Chesapeake Bay by investigating 16 viromes collected from these two bays. A total of 26,487 viral populations (contigs > 5kb) were identified in the two bays, establishing a high quality viromic dataset. The vast majority of the dominant viral populations are unclassified viruses. Viral sequences obtained from marine single cell genomes or long read single molecule sequencing comprised 13 of the top 20 most abundant viral populations, suggesting that we are still far from understanding the diversity of viruses in estuaries. Abundant viral populations (top 5,000) are significantly different between the Delaware Bay and Chesapeake Bay, indicating a strong niche adaptation of the viral community to each estuary. Surprisingly, no clear spatiotemporal patterns were observed for the viral community based on water temperature and salinity. The composition of known viruses (i.e. phages infecting Acinetobacter, Puniceispirillum, Pelagibacter, Synechococcus, Prochlorococcus, etc.) appeared to be relatively consistent across a wide range of salinity gradients and different seasons. Overall, the estuarine viral community is distinct from that in the ocean according to the composition of known viruses. N4-like viruses belong to a newly established viral family and have been isolated from diverse bacterial groups. Marine N4-like viruses were first found in the Chesapeake Bay, but little is known about their biogeographic pattern in the estuarine environment. N4-like viruses were confirmed to be rare in the estuary, and relatively more abundant in the samples from lower water temperature. Viruses which infect SAR11 bacteria (pelagiphage) are one of most abundant viral groups in the open ocean. We found that the abundance and community profile of pelagiphage in the estuaries is similar to that in the open ocean, and has no correlation with environmental factors.Item Reference-guided assembly of metagenomes(2020) Cepeda, Victoria P; Pop, Mihai; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microorganisms play an important role in all of the Earth's ecosystems, and are critical for the health of humans [1], plants, and animals. Most microbes are not easily cultured [2]; yet, Metagenomics, the analysis of organismal DNA sequences obtained directly from an environmental sample, enables the study of these microorganisms. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. The two main paradigms for this method are de novo assembly (i.e., reconstructing genomes directly from the read data), and reference-guided assembly (i.e., reconstructing genomes using closely related organisms). Because the latter paradigm has a high computational cost—due to the mapping of tens of millions of reads to thousands of full genome sequences—Metagenomic studies have primarily relied on the former paradigm. However, the increased availability of high-throughput sequencing technologies has generated thousands of bacterial genomes, making reference-guided assembly a valuable resource regardless of its computational cost. Thus, this study describes a novel metagenome assembly approach, called MetaCompass, that combines reference-guided assembly and de novo assembly, and it is organized in the following stages: (i) selecting reference genomes from a database using a metagenomic taxonomy classification software that combines gene and genome comparison methods, achieving species and strain level resolution; (ii) performing reference-guided assembly in a new manner, which uses the minimum set cover principle to remove redundancy in a metagenome read mapping while performing consensus calling; and (iii) performing de novo assembly using the reads that have not been mapped to any reference genomes. We show that MetaCompass improves the most common metrics used to evaluate assembly quality—contiguity, consistency, and reference-bases metrics—for both synthetic and real datasets such as the ones gathered in the Human Microbiome Project (HMP) [3], and it also facilitates the assembly of low abundance microorganisms retrieved with the reference-guided approach. Lastly, we used our HMP assembly results to characterize the relative advantages and limitations of de novo and reference-guided assembly approaches, thereby providing guidance on analytical strategies for characterizing the human-associated microbiota.Item SOFTWARE INFRASTRUCTURE FOR VISUAL AND INTEGRATIVE ANALYSIS OF MICROBIOME DATA(2018) Wagner, Justin; Corrada Bravo, Hector; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microbiome sequencing allows researchers to reconstruct bacterial community census profiles at resolutions greater than previous methodologies. As a result, increasingly large numbers of these taxonomic community profiles are now generated, analyzed, and published by researchers in the field. In this work, I present new methods and software infrastructure for visualization and sharing of microbiome data. The overall goal is to enable a researcher to complete cycles of exploratory and confirmatory analysis over metagenomic data. I describe Metaviz, an interactive statistical and visual analysis tool specifically designed for effective taxonomic hierarchy navigation and data analysis feature selection. I next detail the incorporation of Metaviz into the Human Microbiome Project Data Portal. I then show a novel method to visualize longitudinal data across multiple features built as an extension over Metaviz. Finally, previous work has shown that specific subjects in an experimental cohort can be identified using their microbiome data. I developed software using a secure multi-party computation library to complete comparative analyses of metagenomic data across cohorts without directly revealing feature count values for individuals.Item BIOLOGICAL EFFICACY, MECHANISMS OF ACTIONS OF SOY-DERIVED PHYTOALEXIN GLYCEOLLINS IN PREVENTION OF CHRONIC DISEASES(2014) Huang, Haiqiu; Yu, Liangli; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cardiovascular disease (CVD) is the leading cause of deaths worldwide. Prostate cancer is the most prevalent cancer in U.S. male population. Diet-induced hypercholesterolemia and chronic inflammation promote the development of both CVD and prostate cancer. Glyceollins are a group of soy phytoalexins possessing a variety of biological activities. This research project focused on characterizing glyceollins' bioactivities in alleviating cholesterol dysregulation, prevention of prostate cancer, and regulating gut microbiome. The first part of the project aimed to evaluate glyceollins' cholesterollowering effect in-vivo. Male golden Syrian hamsters were fed high-fat diet with or without glyceollins supplementation for 28 days. Glyceollins supplementation led to a significant reduction of plasma VLDL, hepatic cholesterol esters and total lipid content. Consistent with changes in circulating cholesterol, glyceollins supplementation also altered expression of the genes related to cholesterol metabolism in the liver. The second part of the study aimed to evaluate glyceollins' effect in reducing prostate cancer tumor growth in a xenograft model. An initial delayed appearance of tumor was observed in a PC-3 xenograft model. However, no difference in tumor sizes was observed in a LNCaP xenograft model. Extrapolation analysis of tumor measurements indicated that no difference in sizes was expected for both PC-3 and LNCaP tumors. Glyceollins had no effect on the androgen responsive pathway, its proliferation, cell cycle, or on angiogenesis genes in tumor and xenobiotic metabolism, cholesterol transport, and inflammatory cytokine genes in liver. Glyceollins' low bioavailability might have led to the ineffectiveness in reducing tumor growth in-vivo. The microbiome has emerged as an important and integral part of the human physiology with a significant role in human health and disease. The third part of the study aimed to evaluate the effect of glyceollins on the gut microbiome in mice. Fecal and cecal samples collected from mouse feeding studies were analyzed for microbial population and composition. Glyceollins supplementation did not alter gut bacteria groups in cecal sample examined in this study. Glyceollins significantly affected total Enterobacteriaceae and Ruminococcus population in fecal samples collected at 24 h, indicating the impact and importance of time of collection in interpreting gut microbiome data in fecal analysis.