Estrus synchronization (ES) is a valuable technology that can help beef cow-calf producers to overcome infertility caused by prolonged anestrus. Protocols for ES that use progesterone (P4) supplementation are of particular value to cows with prolonged postpartum anestrus as P4 triggers them to begin cycling and allows them to have fertility similar to that of cycling cows. Supplementation of P4 intravaginally with the use of a controlled internal drug release device (CIDR) improves cycle induction when compared to oral administration of P4. Vaginitis has been reported as a side effect to CIDR use in cattle, which raises concerns about its downstream effects on fertility. More specifically, the effects of CIDR use on the vaginal environment requires exploration, as no studies have explored the changes in vaginal microbiome in response to CIDR based ES protocols. In cattle, the vaginal microbiome has not been widely explored. On the contrary, the human vaginal microbiome is a well-defined environment, rich in bacteria from the genus Lactobacillus, which are responsible for promoting an environment of acidic pH. The dominance of Lactobacillus in the human vagina, however, fluctuates according to steroid hormone concentrations, and disruptions in the vaginal environment will cause depletion of Lactobacillus species, increase in vaginal pH and decreased fertility. Based on this data in humans, our objectives were to describe incidence of vaginitis caused by the CIDR in beef cows, as well as the vaginal microbiome changes in response to CIDR based protocols, and explore their relation to fertility. We found high incidences of vaginitis caused by CIDR use, yet CIDR-induced vaginitis did not negatively affect pregnancy outcomes. However, at CIDR withdrawal, there was decreased bacterial diversity, increased vaginal pH, increased bacterial abundance, and increased vaginal inflammation when compared to what was observed prior to CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed artificial insemination. This important finding suggests that although the vaginal microbiome was disrupted by the use of CIDR, the vaginal microbiome is resilient and capable of restoring its natural conditions without intervention. Finally, cows that ultimately became pregnant were found to have had increased bacterial diversity and decreased vaginal pH prior to protocol initiation, suggesting that the vaginal microbiome may play a role in individual cow fertility. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success. / Doctor of Philosophy / According to the Food and Agriculture Organization, the world population is expected to grow by 51% by the year of 2100. The efficiency of food production must therefore be optimized, given the finite availability of farmable land. In beef production, cow fertility is of great importance, since it will ultimately determine the number of animals available for slaughter. The main reproductive issue that cow-calf producers face is that after calving, cows will undergo a period known as postpartum anestrus, in which cows fail to ovulate. Artificial insemination (AI) can help to optimize beef production efficiency, since it allows for the dispersal of semen from valuable bulls to facilitate the genetic enhancement of herds. The use of estrus synchronization (ES) protocols allows for induction and synchronization of ovulation, which allows AI to be performed at the same time for large groups of cows. Progesterone is often used in ES protocols and is the hormone responsible for inducing cyclicity in postpartum cows. Progesterone can be administered either orally or intravaginally via the use of a controlled internal drug release (CIDR). While the CIDR seems to be more effective at inducing cyclicity of cattle compared to oral progesterone administration, vaginal inflammation as response to the CIDR has been reported in cattle. Little is known about the downstream effects of this inflammation on the normal vaginal microbiota and fertility in cattle. In humans, the vaginal microbiome is predominated by a single genus of bacteria (Lactobacillus), that has an essential role in producing lactic acid, which results in the human vagina being remarkably acidic. In humans, depletion of this bacteria, a condition called bacterial vaginosis (BV), allows for other types of bacteria to grow, which results in an increased vaginal pH and decreased fertility. The bovine vaginal microbiome composition and pH in response to the hormones administered during ES protocols and its relation to fertility have not been widely explored. Our objectives were to document the incidence of vaginitis caused by the CIDR in beef cows and evaluate its effects on the vaginal microbiome changes and fertility. We found high incidences of vaginitis caused by the CIDR, yet no effects of CIDR-induced vaginitis were seen on pregnancy success to the protocol. However, decreased bacterial diversity, followed by increases in vaginal pH, abundance of bacteria and vaginal inflammation are all detected at CIDR withdrawal when compared to before CIDR insertion. Furthermore, abundance of bacteria, vaginal inflammation, and bacterial diversity, but not vaginal pH, were restored to normal values by the day of timed AI, indicating that although the vaginal microbiome was disrupted using CIDR, the vaginal microbiome can restore to natural conditions, and indicate resilience of the vaginal microbiome. Finally, cows that became pregnant to the protocol presented increased bacterial diversity and decreased vaginal pH prior to the protocol. In conclusion, our results indicate that for beef cows a more diverse vaginal microbiome with decreased vaginal pH presents greater resilience of the microbiome towards disruptions caused by an ES protocol, which is translated in greater pregnancy success.

Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

Preterm birth is a leading cause of both maternal and neonatal morbidity and mortality. It occurs in roughly one in every ten pregnancies, and at an even higher rate among Black Americans and residents of underdeveloped nations. Preterm birth can be initiated in response to a maternal or neonatal indication, or can occur spontaneously. Though indications for the former may vary, the most frequent indication for indicated preterm birth is preeclampsia, a disorder of pregnancy marked by high blood pressure and systemic organ damage. While spontaneous preterm birth and preeclampsia account for a substantial fraction of the burden of prematurity, our understanding of the triggers for and pathogenesis of both diseases are lacking. As a result, we are not able to accurately identify women early in pregnancy who are at high risk of having a spontaneous preterm birth or of developing preeclampsia. There is mounting evidence that local and systemic inflammation, infection, and environmental exposures impact the vaginal ecosystem and may be triggers of spontaneous preterm birth and preeclampsia. In this thesis, I explore the role of vaginal microbes, metabolites, and immune factors in spontaneous preterm birth and preeclampsia. After reviewing what is known about the vaginal ecosystem in spontaneous preterm birth and preeclampsia, I present a paired study of the vaginal microbiome and metabolome in a cohort of 232 women, 80 of whom delivered spontaneously preterm, and whose vaginal ecosystems were profiled during the second trimester of pregnancy. In this study I identify several metabolites strongly associated with spontaneous preterm birth, and suggest that many of these may be exogenous in origin. I also use metabolic models to investigate tyramine, a metabolite found to be associated with lower risk of spontaneous preterm birth. Finally, using predictive models I show that vaginal metabolite levels can be used to identify women at risk of spontaneous preterm birth months in advance. I then present a second study of the vaginal microbiome and immune factors in a cohort of 124 women, 62 of whom developed severe preeclampsia, and whose vaginal ecosystems were profiled at the end of the first trimester. In this study, I demonstrate for the first time that the levels of vaginal microbes early in pregnancy as well as genomic variation in the vaginal microbiome are associated with the risk of developing preeclampsia. I also identify that many vaginal immune factors are significantly depleted in the vaginal ecosystem of women who develop severe preeclampsia. I then use predictive models to show that the levels of vaginal microbes are modestly predictive of preeclampsia risk, and that features from the vaginal ecosystem can be used to improve current methods for the identification of women at risk for severe preeclampsia. Finally, I show that the microbiome signature associated with severe preeclampsia replicates in an independent cohort, suggesting that the early pregnancy vaginal microbiome is robustly associated with the diagnosis of preeclampsia months later in pregnancy. Overall, the microbial and molecular signatures that I identify in these studies contribute novel insight to our understanding of the signs and pathogenesis of both spontaneous preterm birth and preeclampsia, and in doing so, suggest novel approaches to intervention and diagnosis.

Immunotherapy has emerged as one of the four “standard” cancer therapies, alongside surgery, chemotherapy, and radiotherapy. Immune checkpoint inhibitor (ICI) therapy is an immunotherapy that blocks inhibitory immune checkpoint interactions, allowing T cells and other immune cells to kill tumor cells. In the tumor microenvironment, there is often overexpression of immune checkpoint proteins, whose binding interaction with cytotoxic T cells and other immune cells results in the dampening of the antitumor response. Programmed cell death protein 1 (PD-1) and T-lymphocyte-associated protein 4 (CTLA-4) are the two most targeted immune checkpoint proteins. Antibodies against PD-1 and CTLA-4, as well as other checkpoint proteins, are approved for clinical use as well as in clinical trials. While ICIs have changed the treatment landscape for many cancers, particularly those with significant immunogenicity, only 20-40% of patients respond to ICI therapy. Many factors are behind the lack of response and resistance, and significant efforts are aimed at improving the response to ICI therapy. One major area is modulating the gut microbiome, as it is well-established that microbial dysbiosis is associated with various human diseases. The concept is that by modulating the microbiome, we might be able to return it to a composition more similar to that seen in healthy individuals or provide microorganisms beneficial to clinical response. In the case of ICI therapy, it is proposed that there is a connection between certain microbial species and the immune system via metabolites and other signaling effects. The microbiome can be manipulated through many methods, including fecal microbiota transplantation (FMT), transferring bacterial isolates or consortia, probiotics, antibiotics, and soluble dietary fiber. For clinical insights, it is important to consider how the pre-treatment microbiome of patients may affect their response to ICI therapy, as well as how their microbiomes can be manipulated to enhance their response. Initial clinical trials have been promising, but this is an emerging field with additional work to be done. Particularly, a better understanding of the microorganisms involved in the response to ICI therapy and the mechanism by which they communicate with the immune system is essential. Future studies will need to be much larger to reduce noise between studies and to allow for emerging computational techniques to be applied.

<p dir="ltr">The vaginal microbiome and female sex hormones are both involved in the development and progression of gynecological pathologies. The individual mechanisms by which the vaginal microbiome leads to disease progression and how female sex hormones are known. However, the mechanisms by which the vaginal microbiome regulates female sex hormones, such as progesterone, are not well understood. This study seeks to understand how the vaginal microbiome regulates progesterone receptor (PGR) expression via secondary analysis of host and vaginal microbiome multi-omics data from the Partners PrEP cohort. This dataset consists of cervicovaginal samples of women enrolled in the Partners PrEP study. Partial Least Squares Regression (PLSR) models were created for each biological data type (microbial composition, metabolomics, metaproteomics) to assess how these factors regulate PGR expression. Significant factors were identified through variable importance of projection (VIP) and correlation analysis. Partial correlation analysis and follow-up PLSR models incorporating clinical and demographic variables were performed to assess the robustness of the vaginal microbiome-PGR associations. The PLSR models indicated lower PGR expression was associated with <i>G. vaginalis,</i> and higher PGR expression was associated with <i>Lactobacillus </i>species. Cytosine, guanine, and tyrosine were among metabolites significantly associated with higher PGR expression and experimentally determined to be produced by <i>Lactobacillus</i> species. Conversely, citrulline and succinate were associated with lower PGR expression and experimentally determined to be produced by <i>G. vaginalis</i>. The models indicated that bacterial metabolic pathways involved in glucose metabolism, such as glucagon signaling and starch and sugar metabolism, may regulate PGR expression. Demographic phenotypes were also considered from the dataset and did not significantly alter the association between the biological explanatory variables and PGR expression. The results indicate that guanine, cytosine, succinate, starch and sucrose metabolism, and glycolysis gluconeogenesis may be regulators of PGR abundance and function. The models suggest vaginal microbiome factors could play a role in gynecological conditions where progesterone signaling is suppressed. Future experimental work is needed to validate the results of these models and support their use as predictive tools to understand the role of the vaginal microbiome.</p>

Over the past century, dust emissions have increased in frequency and intensity due to anthropogenic influences and extended droughts. Dust transports microbes, nutrients, heavy metals and other materials that may then change the biogeochemistry of the receiving environments. The purpose of this study was to find whether unique bacterial communities may provide distinct fingerprints of dust sources in the Western USA. We collaborated with the National Wind Erosion Research Network (NWERN) to identify bacterial core communities (core) of dust from ten NWERN sites, and compared communities to location, soil, and regional characteristics. In order of importance, precipitation levels (F = 43, P = 0.0001, Df = 2, r2 = 0.25), location (F = 16, P = 0.0001, Df = 5, r2 = 0.23), soil texture (F = 14, P = 0.0001, Df = 3, r2 =0.12), seasonality (F = 11, P = 0.0001, Df = 2, r2 = 0.064), and elevation (F = 5.7, P = 0.0002, r2 = 0.033) determined bacterial community composition. Bacterial core communities were defined as taxa present in at least 50% of samples at each site and offered predictable patterns of dust communities in terms of abundant (> 1% relative abundance) and rare (< 1% relative abundance) signatures. We found distinct bacterial core communities that reflected dust source systems, for example, sites contaminated with heavy metals contained Romboutsia, Turicibacter, Clostridium sensu stricto 1, Geodermatophilus, and Microvirga. Sites with association to plants and biocrusts contained Methylobacterium-Methylorubrum, Bradyrhizobium, Paenibacillus thermoaerophilus, Cohnella, and bacterial families Solirubrobacteraceae, Sphingobacteraceae, and Myxococcaceae. The presence of Sphingomonas, Stenotrophomonas, Rhodococcus, and Phenylobacterium were found in hydrocarbon contaminated soils. High stress (UV radiation and desiccation) sites contained Deinococcus, Blastococcus, and Modestobacter. We found that seasonal changes affected microbial community composition in five NWERN sites (CPER, HAFB, Jornada, Red Hills, and Twin Valley) (p < 0.05), while no seasonal effects on bacterial distribution were observed at Moab. Our results identify that the use of core microbiomes may offer a fingerprinting method to identify dust source regions.

No description available.

No description available.

Soil microorganisms have a variety of beneficial and deleterious effects on plants, impacting such processes as plant growth, soil nutrient cycling, crop yield, disease resistance and tolerance to an array of biotic and abiotic stressors. The disruption of soil microbial community structures, particularly when beneficial soil biota are altered, has been shown to reduce crop yield and leave plants susceptible to disease. Long-term disruption of microbial communities may occur with repeated fumigation, being the application of gaseous pesticides, in agricultural soils. For this reason, we characterized bacterial, fungal, oomycete and nematode populations in paired fumigated and nonfumigated potato fields located in Idaho, Oregon, Washington and Minnesota. Samples were taken at three distinct timepoints: one before a fall fumigation event and two others at important stages in potato production, row closure and vine death. Soil biota populations were assessed by targeting the 16S, 18S and ITS1 gene regions. FunGuild, a database capable of guild and trophic assignment of fungal lineages, was used to sort fungal OTUs in different trophic modes. Fungal analyses indicated an increase in relative abundances of saprotrophic fungal populations and a decrease in pathotrophic fungal populations, both during row closure. Principally, the fungal genera of Humicola and Mortierella were responsible for the increase of saprotrophs while Alternaria decreased the most for pathotrophs. Other fungi occupying multiple trophic modes, such as Fusarium, also decreased during row closure. We found that fumigation treatments, in combination with various pesticide and fertilizer applications, alter both alpha- and beta- bacterial soil diversity although certain treatments, i.e. chloropicrin, may alter bacterial populations more than other treatment types such as metam-sodium. Nematode populations were likewise distinct at each location with soils from Boardman, OR, Minidoka, ID and Pine Point, MN with these having higher levels of nematodes associated with better soil health, i.e. Dorylaimidae. Conversely, nematodes associated with plant pathogenesis were found in higher relative abundances at Minidoka, ID and Quincy, WA. In this study, we characterize the populations of bacteria, fungi, oomycetes and nematodes with an emphasis on fungal taxa. We found that relative abundances of fungal trophic modes vary temporally. Additionally, we catalogue several other high abundance taxa with seasonal differential abundances whose functional capacity in potatoes remain uncharacterized.

The human gut microbiota contributes to host metabolic processes. Diverse microbial metabolic enzymes can affect therapeutic agents, resulting in chemical modifications that alter drug efficacy and toxicology. These interactions may result in ineffective treatments and dose-limiting side effects, as shown by bacterial modifications of the cardiac drug digoxin and chemotherapy drug irinotecan, respectively. Yet, few drug-microbiome interactions have been characterized. Here, a platform is developed to screen for drug-microbiome interactions, validated by the isolation of a gut bacterium capable of inactivating the antineoplastic drug doxorubicin. Two hundred gut strains isolated from a healthy patient fecal sample were cultured in the presence of antibiotic and antineoplastic drugs to enrich for resistance and possible inactivation. Raoultella planticola was identified for its ability to inactivate doxorubicin anaerobically through whole cell and crude lysate assays. This activity was also observed in other Enterobacteriaceae and resulted in doxorubicin inactivation by the removal of its daunosamine sugar, likely mediated by a molybdopterin-dependent enzyme. Other potential drug-microbiome interactions were identified in this screen and can be analyzed further. This platform enables the identification of drug-microbiome interactions that can be used to study drug pharmacology, improve the efficacy of therapeutic treatments, and advance personalized medicine. / Thesis / Bachelor of Science (BSc) / The collection of microbes in the human intestinal tract, referred to as the gut microbiome, can modify therapeutic agents and change the efficacy of drug treatments. Identifying these interactions between drugs and the microbiome will help the study of drug metabolism, provide explanations for treatment failure, and enable more personalized health care. For this project, a platform was developed to isolate gut bacteria from human fecal samples and characterize bacteria that are capable of inactivating various antibiotics and anticancer drugs. Through this platform, the gut bacterium Raoultella planticola was found to inactivate doxorubicin, a commonly used anticancer drug. These results suggest that doxorubicin may be inactivated in the gut and demonstrates how this platform can be used to identify drug-microbiome interactions.