I imagine a future in which children grow up with healthy microbial communities. Engineering human microbiomes might actually be achievable in the near future, as we enter an era of hunting for human-adapted bacterial strains and phages. Furthermore, breath metabolites could allow us to track whether a probiotic colonizes persistently or a phage has knocked down a microbe of interest. Recent successes with probiotics, such as bifidobacteria that can break down human milk oligosaccharides, are making a future in which infants are intentionally colonized with health-promoting strains seem less unlikely. Viruses that infect bacteria, bacteriophages, are also important for human health both because of their role in the human microbiome and because of their potential for use in phage therapy. Monitoring the outcome of microbiome-focused interventions with breath volatile sampling is also on the horizon, which could mean real-time tracking of microbial metabolite production. Studies of early life during microbiome assembly, when the potential for effective interventions to reduce disease risk is greatest, are essential.

Cystic fibrosis (CF) is a genetic disease that results in the accumulation of dense, dehydrated mucus in the airways. Unclearable mucus contributes to the establishment of long-term microbial communities in the CF airways. Opportunistic pathogens damage the airways by inducing the host inflammatory response and generating toxic metabolites, contributing to decreased quality of life and lifespan (~40 years) for persons with CF. In order to improve treatment of lung infections, it is necessary to understand the role of microbes and microbial metabolism in triggering CF pulmonary exacerbations (CFPE), periods of worsening lung function. While antibiotics ease CFPE symptoms, infections are almost never fully eradicated. To elucidate antibiotic failure, we must first understand how relevant conditions in the airways, particularly around the time of CFPE, shape the microbiome and its antibiotic sensitivity.

CFPE may be characterized by increases in acidity (1), fermentation metabolites (2), and anaerobic bacteria (3). We characterized CF polymicrobial metabolic interactions between the fermenting microbe, Rothia muciliganosa, and the opportunistic pathogen, Pseudomonas aeruginosa (Chapter 1). Even in nutrient-rich medium designed to mimic sputum content, P. aeruginosa utilized Rothia-derived fermentation products to produce amino acids. As both host inflammation and microbial fermentation often result in drops in pH, we next determined that acidic pH is stressful for another CF opportunistic pathogen, Stenotrophomonas maltophilia, using transcriptomics and metabolomics (Chapter 2). S. maltophilia coped with low pH by expressing stress response genes and catabolizing amino acids to synthesize polyamines. Fermentation and subsequent drops in pH can be the result of hypoxia, which is a predominant condition in CF sputum (4). We used a combination of fluorescence lifetime imaging and spectral microscopy to study bacterial metabolism in oxygen gradients (Chapter 3).

To tackle the question of how antibiotics impact the microbiome, we need objective information about which antibiotics are reaching the infection-site. Given the diverse properties of antibiotics, the type and concentration of each antibiotic should be incorporated. We developed an LC-MS method to detect 18 antibiotics in 171 sputum samples and assessed the specificity of our LC-MS assay relative to subject self-reported usage (Chapter 4).

Volatile organic compounds (VOCs) measured from exhaled breath have great promise for the diagnosis of bacterial infections. However, determining human or microbial origin of VOCs detected in breath remains a great challenge. For example, the microbial fermentation product 2,3-butanedione was recently found in the breath of CF patients; parallel culture-independent metagenomic sequencing of the same samples revealed that Streptococcus and Rothia spp. have the genetic capacity to produce 2,3-butanedione. To investigate whether the genetic capacity found in metagenomes translates to bacterial production of a VOC of interest such as 2,3-butanedione, we fed stable isotopes to three bacterial strains isolated from Cystic Fibrosis (CF) patients: two Gram-positive bacteria, Rothia mucilaginosa and Streptococcus salivarius, and a dominant opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. Culture headspaces were collected and analyzed using a gas chromatographic system to quantify the abundance of VOCs of interest; mass spectroscopy was used to determine whether the stable isotope label had been incorporated. Our results show that R. mucilaginosa and S. salivarius consumed D-Glucose-13C6 to produce labeled 2,3-butanedione. R. mucilaginosa and S. salivarius also produced labeled acetaldehyde and ethanol when grown with 2H2O. Additionally, we find that P. aeruginosa growth and dimethyl sulfide production are increased when exposed to lactic acid in culture. These results highlight the importance VOCs produced by P. aeruginosa, R. mucilaginosa, and S. salivarius as nutrients and signals in microbial communities, and as potential biomarkers in a CF infection.

The lifespan of people living in westernized countries is increasing, suggesting we are getting healthier; however, we are now struggling with a new health problem: the increasing incidence of chronic disease. We have only recently begun to appreciate the association of microbial colonization with health, and how changes in our lifestyles (i.e. diet, urbanization, sanitation) are affecting this association. Changes in microbial community composition have been linked with numerous acute and chronic disease states. Despite the microbiome’s strong correlation with health, there is still much work to be done in understanding the ideal structure and function of these human associated microbial communities.Humans first experience with microorganisms occurs during birth, when we are exposed to the maternal microbiome. Cervicovaginal microbes and their metabolic by-products critically influence the structuring and functioning of the early infant microbiome and immune system; however, global assessments of maternal microbes and metabolites are lacking. In Chapter 1, my co-authors and I address this knowledge gap by sequencing cervicovaginal fluid from 18 healthy mothers longitudinally throughout their pregnancy. Parallel to microbiome sequencing, we examined the vaginal metabolomes using GF-TOF and LC-MS/MS to broadly capture metabolites within this system. Additionally, we analyzed the metabolomes of offspring through their first year of life. We report strong metabolic biomarkers of microbiome composition within the cervicovaginal tract, which may ultimately be useful in future diagnostic and therapeutic endeavors for vaginal diseases like bacterial vaginosis (BV). We show a strong correlation between mannitol and the presence of Lactobacillus crispatus and speculate as to why a homofermentative vaginal lactobacillus may be generating mannitol via fructose reduction for redox purposes. In chapter 1, I focus on describing the early microbes and metabolites infants are exposed to during pregnancy and birth. Throughout our lives, our microbiomes are under constant selection by intrinsic (e.g., phage predation) and extrinsic (e.g., antibiotic use) factors. One strong architect of gut microbial composition is diet. Dietary fiber is a favored substrate of microbes deep within the colon, which is able to resist digestion by endogenous enzymes in order to feed the dense bacterial communities throughout the gut. In chapter 2, we conducted a two-week diet intervention study, asking whether an increase in dietary fiber intake through whole foods was capable of changing the gut microbial communities and associated metabolome. We show on a short time scale, increasing dietary fiber in the gut alters the microbial composition significantly, but does not change the abundance of metabolic by-products of fiber fermentation (short-chain fatty acids). While more work needs to be done to disentangle the relationship between how diet influences bacterial metabolites, our work illustrates how malleable gut microbial communities are to a change in diet. Finally, in chapter 3, I contributed context to a growing body of microbiome research that is woefully lacking microbiome characterizations from non-industrialized populations. We teamed up with anthropologists to study the microbiome of a remote community of people deep within the Ecuadorian Amazon. Our findings show that the microbiomes from these Conambo people are distinct from the microbiomes of industrialized individuals, containing a high abundance of VANISH taxa (volatile and/or associated negatively with industrialized societies of humans). Notably, nearly 40% of the average fecal metagenome consisted of reads mapping to Prevotellaceae. The Conambo people have unique diets, with a significant portion of their caloric intake coming from a cultural variant of cassava beer (“chicha”). Chicha is made by mastication and re-mastication of yuca root by women within each household. We show that while the initial inoculums of chicha are household specific, over the course of fermentation the communities converge on a more similar microbiome irrespective of household origin. These data contribute vitally towards the open question: how has industrialization shaped the microbiome?

Enterococcus can be both a friend and a foe in the human microbiome. As an opportunistic pathogen, Enterococcus is normally benign. However, Enterococcus is responsible for many hospital-acquired infections and has shown rising rates of vancomycin resistance. Bacteriophages (phages) could be an alternative to antibiotics to target these antibiotic resistant bacteria, but the evolutionary and phenotypic outcomes of phage-bacteria interactions need to be investigated more thoroughly.

We begin by investigating the gut microbiome of preterm infants that had been exposed to antibiotics to learn about the scenarios in which Enterococcus blooms occur in the gut (Chapter 1). We show that antibiotic exposed gut microbiomes are dominated by facultative anaerobes such as Enterococcus and Enterobacteriaceae. Further, we show that the bacterial composition is correlated to the overall metabolite profile. Metabolomics is a powerful tool for investigating microbial metabolism, and we contribute to the effort of developing standardized practices for metabolomics in the human microbiome by showing that freezing microbial samples required for long term storage (Chapter 2).

Next, we investigated how phages could be used as therapeutics for treating Enterococcus blooms and infections (Chapters 3, 4, and 5). When Enterococcus is grown with its phages in vitro, it evolves resistance to phage infection by mutating exopolysaccharide synthesis genes. These mutations alter the exopolysaccharides on the surface of the bacterial cell to prevent binding of phage. Further, this mechanism of resistance appears to be a general mechanism leading to resistance against a diverse array of phages. This work demonstrates that experimental evolution is a powerful tool for characterizing interactions between bacteria and phages.

Phage therapy is often administered as a cocktail of multiple phages, but there are no rules or best practices described for combining phages to be most effective. We show that in vitro, Enterococcus phage cocktails are more effective at preventing the growth of phage-resistant mutants, but the composition of the cocktail is important. Genetically diverse phage cocktails performed better than cocktails of related phages. My work demonstrates some of the outcomes of phage-Enterococcus interactions and will move us closer to applying phage therapy to treat Enterococcus infections.

As the world continues to industrialize, there has been a decreased incidence of infectious diseases and an increased incidence in non-communicable chronic diseases, such as obesity and cancer. One potential causal factor is the human microbiome, which refers to the collection of commensal bacteria, fungi, viruses, archaea, and other microorganisms that inhabit our bodies. The human microbiome has a collective genome which outnumbers human genes by 150-to-1, and it produces an expansive repertoire of metabolites which affect health. As such, the gut microbiome has major implications in digestion, educating the host immune system, preventing the colonization of pathogenic bacteria by occupying intestinal niches, and more.

Several components of the industrialized lifestyle have been known to alter the gut microbiome, leading to negative health consequences. The result is a microbiome that promotes inflammation and is associated with non-communicable chronic diseases. As these diseases become more prevalent in industrialized countries, there is an urgency to understand the role of the microbiota in human health. To investigate this issue, my research seeks to answer the following three questions: (1) What is the role of the industrialized gut microbiome in the development of colorectal cancer? (2) How does diet affect the gut microbiome and inflammation in people with myeloproliferative neoplasms? (3) How is dietary fiber utilized by the gut microbiota of US and Moroccan individuals in vitro?

To answer the first question, samples were collected at the UCI Medical Center from 140 subjects during and after colonoscopy to characterize the microbiome associated with colorectal polyps. I used a combination of amplicon and shotgun metagenomic sequencing approaches to understand the effect of sampling method on microbial composition, and to describe the microbiome associated with healthy tissue and two types of colorectal polyps. I discovered that sampling method significantly explained 10-15% of the variation observed in microbiome composition. Additionally, using samples obtained from the colon mucosa, I was able to find associations with microbiome composition and colorectal polyps derived from the serrated pathway of colorectal carcinogenesis, such as a depletion in the lignan-degrading microbe, Eggerthella lenta. Lastly, I was able to use the microbiome to inform machine learning classifiers to accurately distinguish between healthy and polyp-bearing samples (Area under curve = 0.87-0.99).

To address the second question, we collaborated with Dr. Angela G. Fleischman, who conducted a dietary intervention in subjects afflicted with myeloproliferative neoplasms, a class of blood cancer. In this 15-week clinical trial, 28 individuals were assigned to receive dietary counseling following either a Mediterranean style eating pattern or one following U.S. guidelines. Blood and fecal samples were collected to examine inflammation and the gut microbiome, respectively. Using shotgun metagenomic sequencing, I discovered no significant alterations in gut microbiome diversity and composition due to a Mediterranean diet. I did find a significant association between the gut microbiome and myeloproliferative neoplasm subtype, explaining approximately 6% of the variance in microbiome composition. Lastly, I found several significant correlations between microbial species, function, and cytokine concentrations.

My first two chapters suggested a link between the microbiome and dietary fiber, thus, in my final chapter I explored the effect of dietary fiber on microbial growth in vitro. In these experiments, I took the feces of 15 healthy individuals from the US and 15 Moroccans and cultured them anaerobically for 24 hours in the absence and presence of inulin, pectin, or psyllium husk. I found significant cohort effects on the microbiome, with US samples becoming dominated with Bifidobacterium and Moroccan samples becoming dominated with Clostridia. Furthermore, I demonstrate that pectin and psyllium husk perform differential enrichment of microbes and their associated carbohydrate-active enzymes.

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