abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

abstract: The transition to college has been identified as a vulnerable period for weight gain and the onset of obesity. Research has shown that the gut microbiota is different in obese compared to lean individuals, but a period of weight gain has never been studied in free-living individuals. The objective of this longitudinal, observational study was to assess the association between changes in the intestinal microbiota and weight-related outcomes in healthy college students living in on-campus dormitories at Arizona State University (n=39). Anthropometric measures and fecal samples were collected at the beginning and end of the school year, and microbial relative abundance for A. muciniphila, F. prausnitzii, R. gnavus, and L. acidophilus was measured through qPCR analyses. In this population, body mass index (BMI) and waist circumference (WC) increased by 0.97 ± 1.28 kg/m2 and 2.64 ± 4.90 cm, respectively. Wilcoxon-Rank tests revealed that R. gnavus fold change was significantly different between groups of weight loss/maintenance and weight gain ≥ 5% body weight (0.14 [-0.21, 0.64], n=24 vs. -0.14 [-0.92, 0.05], n=15, respectively; p=0.028). Correlation analyses suggested a significant negative association between A. muciniphila fold change and both % WC change and % BMI change (r= -0.66; p<0.01 and r= -0.33; p=0.04, respectively). However, multivariate regression analysis controlling for sex and race/ethnicity showed a significant association between A. muciniphila and % WC change, but not % BMI change (R2= 0.53; p<0.01 and R2= 0.24; p=0.15). F. prausnitzii was not associated with weight-related outcomes in this sample. L. acidophilus was excluded from study analyses after subsequent qPCR trials revealed no amplification in participant samples. Overall, this was the first study to show a relationship between A. muciniphila fold change and weight-related outcomes over a period of weight gain. Specifically, A. muciniphila was strongly negatively associated with WC in this sample. Further research is needed to more accurately describe these associations and potential mechanisms associated with the shift in gut microbiota observed with weight gain. Findings from future research may be used to develop interventions for college students aiming to shift the gut microbiota to prevent weight gain. / Dissertation/Thesis / Masters Thesis Nutrition 2017

The widespread use of antibiotics has led to dramatic increases in the incidence and severity of Clostridium difficile infection (CDI). No group of patients suffers more from CDI than the elderly. Nursing homes (NH) represent the perfect storm of a vulnerable group of frail elders living in confined communities. Nursing home residents suffer from increased morbidity and mortality from CDI and corresponding high rates of C. difficile colonization. Upwards of 40 to 50% of CDI current cases originate from NHs and the prevalence of colonization rates remain high within these facilities, with as many as half of the residents being colonized with C. difficile at any given time. One factor that has become of increasing interest and a target of preventive strategies is the human intestinal microbiome. A healthy, diverse microbiome interacts with the host immune system and contributes to pathogen resistance. In this investigation, we first examine elder specific variables to determine if the associated risks of CDI differ by home living environment (nursing home versus community-dwelling). We then go on explore the relationships of NH environment, frailty, nutritional status, and residents’ age with microbiome composition and potential metabolic function. Finally, we describe the C. difficile colonization patterns among elderly NH residents and the associated risk of colonization based on clinical variables and microbiome determinants. A better understanding of the microbiome’s contribution to C. difficile colonization will provide the basis for informing rational interventions and public health policies to better combat CDI in the nursing home.

Salmonellosis or “food poisoning” is a foodborne infection brought on by the pathogen Salmonella from the ingestion of the bacterium on contaminated foods such as vegetables. Infection from Salmonella leads to the highest incidence of hospitalizations and deaths each year, compared to any other bacterial foodborne illness. South Florida is the second largest agricultural winter vegetable producer in the United States, and contamination of vegetables is often observed in preharvest practices. A hardy bacterium, Salmonella, has been shown to live up to 6 weeks in soil and water up to 42°C without a host. The Florida Everglades is a tropical wetland that plays a large role in South Florida’s watershed. It can be divided into agricultural, conservation, and urban areas that connect Lake Okeechobee to Florida Bay by canals, swamps, and rivers. Inland canals tightly regulate water levels in South Florida as a means of flood control for residential and agricultural land. With the influences of anthropomorphic run off from agricultural and urban use, we hypothesized that microbial communities would significantly differ between three select sites in western (Collier county) versus three sites in more urban eastern Florida (Broward county): natural standing water, manmade drainage canal in agricultural areas, and manmade drainage canals in urban areas. We also hypothesized that pathogenic like Salmonella would be present in these habitats. Deep sequencing and ecological genetics analyses of the 16s rRNA V4 region yielded a total of 163,320 unique bacterial OTUs from a total of 139 samples collected monthly for one year in 2015 and part of 2016. Salmonella is not considered an abundant taxon within the microbial population. With the knowledge that Salmonella resides within the microbial population isolates were cultured from soil and water samples that were taken monthly from each site using a modified version of the Food and Drug Administration Bacterial Analytical Methods manual (FDA-BAM). The culturing resulted in 234 isolates obtained and 31 different serovars of Salmonella. Culturing showed that Salmonella favored months with high standing water and high-water temperatures that would lead to the ideal environment for survival. The most commonly occurring isolates within the sample set are those associated with agricultural animals. Though Salmonella may be a rare taxon within the microbial population given the correct environmental conditions such as warm temperatures it is possible to observe Salmonella year round within the South Florida environment.

Os microrganismos presentes no intestino de insetos podem desempenhar importantes funções na biologia de seus hospedeiros, como na nutrição, detoxificação de compostos de defesas das plantas, prevenção da infecção por patógenos e produção de semioquímicos importantes nas interações de insetos. O primeiro passo para entender as funções da microbiota do intestino na biologia de insetos consiste na identificação dessas comunidades. Neste trabalho, a diversidade de bactérias do intestino de larvas e adultos da mosca-das-frutas sul-americana, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), foi determinada por métodos baseados ou não no cultivo. O método baseado no cultivo foi utilizado no estudo da diversidade da microbiota de população de laboratório mantida em frutos de mamão, avaliando-se a diversidade presente no intestino de larvas e em duas regiões distintas do intestino de adultos, o papo e o intestino médio+posterior. A análise metagenômica pela avaliação de bibliotecas de 16S rDNA foi aplicada ao estudo da diversidade da microbiota associada a moscas oriundas de diferentes frutos hospedeiros (guabiroba, nêspera, maçã, mamão e pitanga). As análises por cultivo permitiram a identificação de 25 filotipos associados ao intestino de larvas e adultos, sendo a diversidade em larvas bem distinta daquela de adultos. Os Filos Actinobacteria, Bacteroidetes, Firmicutes e Proteobacteria foram representados por 10 famílias, sendo Enterobacteriaceae a mais abundante. Filotipo próximo à enterobacteriacea Cedecea davisae foi o único a ocorrer no intestino de larvas e papo e intestino de adultos. A análise metagenômica dos insetos obtidos em diferentes frutos hospedeiros estendeu a diversidade anteriormente identificada aos Filos Cyanobacteria, Deinococcus, Elusimicrobia, Planctomycetes e Verrucomicrobia. Novamente, Proteobacteria se destacou como o mais diverso. Esse estudo demonstrou que a comunidade microbiana associada ao intestino de A. fraterculus é inteiramente influenciado pelo fruto hospedeiro utilizado, sendo raros os filotipos compartilhados por insetos em diferentes frutos. A composição da microbiota do intestino do adulto é muito menos diversa do que a da larva, independentemente do fruto utilizado. Mas vários filotipos, como aqueles próximos a Acinetobacter bereziniae, Cedecea davisae, Comamonas koreensis, Enterobacter asburiae, Empedobacter brevis e Hydrogenophilus hirschii, além do parasita intracelular Wolbachia pipientis, mantiveram-se associadas ao intestino de larvas e adultos de A. fraterculus, mesmo após a metamorfose. A análise das comunidades bacterianas de A. fraterculus sugere que as variações em suas estruturas estão relacionadas ao substrato alimentar utilizado pelo inseto. / The microorganisms present in the insect gut can play important roles in the biology of their hosts such as nutrition, detoxification of compounds defenses of plants, preventing infection by pathogens and production of important semiochemicals in insect interactions. The first step to understanding the functions of the gut microbiota in insect biology is the identification of these communities. In this work, the diversity of gut bacteria in larvae and adult South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae) , were determined by methods based on the cultivation or not . The method based on the cultivation was used to study the microbial diversity of laboratory population maintained in papaya fruits, evaluating the diversity present in the gut of larvae and in two distinct regions of the intestine of adults, the crop and midgut + hindgut. The analysis of metagenomic libraries for evaluation of 16S rDNA was applied to the study of the diversity of the microbiota associated with fruit flies from different hosts (guabiroba , medlar , apple, papaya and pitanga). The analysis by cultivation allowed the identification of 25 phylotypes associated with the gut of larvae and adults, larvae diversity being quite different from that of adults. The phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were represented by 10 families, Enterobacteriaceae being the most abundant. Filotipo near Enterobacteriacea Cedecea davisae strains was the only one to occur in the gut of larvae and adult chat and intestine. The metagenomic analysis of insects obtained in different fruit hosts extended to the diversity previously identified phyla Cyanobacteria, Deinococcus, Elusimicrobia, Planctomycetes and Verrucomicrobia. Again, Proteobacteria stood out as the most diverse. This study showed that the microbial community associated with the intestine of A. fraterculus is entirely influenced by host fruit used are rare phylotypes shared by insects in different fruits. The composition of the gut microbiota in adults is far less diverse than the larvae, irrespective of the fruit used. But several phylotypes, such as those near Acinetobacter bereziniae, Cedecea davisae strains, Comamonas koreensis, Enterobacter asburiae, Empedobacter brevis and Hydrogenophilus hirschii, besides the intracellular parasite Wolbachia pipientis, remained attached to the gut of larvae and adults of A. fraterculus, even after metamorphosis. The analysis of bacterial communities of A. fraterculus suggests that variations in their structures are related to the food substrate used by the insect.

The availability of high-throughput sequencing technologies has enabled metagenomicinvestigations into complex bacterial communities with unprecedented resolution andthroughput. The production of dedicated data sets for metagenomic analyses is, however, acostly process and, frequently, the first research questions focus on the study species itself. Ifthe source material is represented by fecal samples, target capture of host-specific sequencesis applied to enrich the complex DNA mixtures contained within a typical fecal DNA extract.Yet, even after this enrichment, the samples still contain a large amount of environmentalDNA that is usually left unanalysed. In my study I investigate the possibility of using shotgunsequencing data that has been subjected to target enrichment for mtDNA from the hostspecies, Grauer’s gorilla (Gorilla beringei graueri), for further analysis of the microbialcommunity present in these samples. The purpose of these analyses is to study the differencesin the bacterial communities present within a high-altitude Grauer’s gorilla, low-altitudeGrauer’s gorilla, and a sympatric chimpanzee population. Additionally, I explore the adaptivepotential of the gut microbiota within these great ape populations.I evaluated the impact that the enrichment process had on the microbial community by usingpre- and post-capture museum preserved samples. In addition to this, I also analysed the effectof two different extraction methods on the bacterial communities.My results show that the relative abundances of the bacterial taxa remain relatively unaffectedby the enrichment process and the extraction methods. The overall number of taxa is,however, reduced by each additional capture round and is not consistent between theextraction methods. This means that both the enrichment and extraction processes introducebiases that require the usage of abundance-based distance measures for biological inferences.Additionally, even if the data cannot be used to study the bacterial communities in anunbiased manner, it provides useful comparative insights for samples that were treated in thesame fashion.With this background, I used museum and fecal samples to perform cluster analysis to explorethe relationships between the gut microbiota of the three great ape populations. I found thatpopulations cluster by species first, and only then group according to habitat. I further foundthat a bacterial taxon that degrades plant matter is enriched in the gut microbiota of all threegreat ape species, where it could help with the digestion of vegetative foods. Another bacterialtaxon that consumes glucose is enriched in the gut microbiota of the low-altitude gorilla andchimpanzee populations, where it could help with the modulation of the host’s mucosalimmune system, and could point to the availability of fruit in the animals diet. In addition, Ifound a bacterial taxon that is linked with diarrhea in humans to be part of the gut microbiotaof the habituated high-altitude gorilla population, which could indicate that this pathogen hasbeen transmitted to the gorillas from their interaction with humans, or it could be indicative ofthe presence of a contaminated water source.

Our gut microbiota is involved in human development, nutrition, and the pathogenesis of gut disorders, but has more recently been implicated as a possible mechanism in the pathophysiology of several brain disorders, including disorders of mood and affect, such as depression. Researchers have referred to this dynamic, bidirectional signaling pathway between the gut and the brain as the “gut-brain axis.” However, most research on this axis has been limited to rodent studies, and there has been little insight into the mechanism behind it. I propose that the kynurenine pathway, where tryptophan is converted to kynurenine, is a compelling mechanism mediating the gut microbiota’s influence on depression. Kynurenine is a metabolite associated with depression, and this pathway has been shown to be manipulated through probiotic (Lactobacillus reuteri) consumption. I propose to study a probiotic intervention in humans, which would assess tryptophan metabolism along the kynurenine pathway by measuring metabolites downstream of this pathway. Urine, feces and blood samples would be collected from two groups, control and probiotic treatment, on day zero and day thirty. Colonic biopsies would be obtained on day thirty, and various analyses would be run to measure metabolite concentrations from the collected samples. The results from this study will help clarify a mechanistic connection between gut microbes and depression via the kynurenine pathway. Additionally, findings could indicate that a probiotic intervention has the ability to influence depressive behavior via a two-pronged approach originating from the kynurenine pathway.

Les algues non-indigènes ont des impacts sur les communautés naturelles à une échelle mondiale: elles affectent la biodiversité et le fonctionnement des écosystèmes ainsi que les services écosystémiques, avec des conséquences économiques et sociales. Parmi les conséquences de ces introductions sont le déplacement d'espèces indigènes, des compétitions avec des espèces en danger et des effets sur les trajectoires éco-évolutives des espèces des communautés envahies. Bien que crucial pour anticiper le devenir des espèces non-indigènes, comprendre les mécanismes d'acclimatation et d'adaptation agissant post-introduction reste un challenge chez les algues. Au cours de cette thèse, nous avons étudié certains processus d'acclimatation, en particulier le rôle des bactéries associées, et leurs effets, en utilisant comme modèle d'étude Sargassum muticum. Cette algue brune originaire d'Asie, est une espèce emblématique parmi les algues introduites avec une présence du Mexique à l'Alaska en Amérique et du Maroc à la Norvège en Europe. Nous avons effectué une analyse bibliographique des traits et caractéristiques écologiques qui pourraient expliquer son succès. Nous avons ensuite montré que l'acidification n'a pas d'effets significatifs sur les bacteries associées, bien que des changements saisonniers du microbiome aient été observés. Enfin, nous avons utilisé un modèle de niche écologique, intégrant la phénologie, pour prédire la distribution de S. muticum sous deux scénarios de changement climatique. Selon nos résultats, d'ici 2100, la distribution de S. muticum devrait se déplacer vers le nord dans l'hémisphère nord avec des régressions dans certaines zones actuellement occupées. / Non-indigenous seaweeds impact natural communities worldwide, affecting biodiversity, ecosystem functioning, and ecosystem services, resulting in significant economic and social consequences. Among major impacts are the displacement of native species, a threat to endangered species, and effects on ecological and evolutionary processes within the invaded communities. While critical to predict the fate of introduced species, understanding the mechanisms of acclimation and adaptation following introduction represents a great challenge in seaweeds. In this thesis, we investigated some acclimation processes, with an emphasis on the role of associated microbiota, and examined their effects with Sargassum muticum as a model species. This brown seaweed native to Asia, is an emblematic invader among seaweeds, with a distribution now ranging from Mexico to Alaska in America and from Morocco to Norway in Europe. We first reviewed the competitive advantages and traits that may contribute to its invasiveness. We then showed that acidification has no significant effects on associated bacteria, although seasonal changes in the microbiome have been observed. Finally, we applied ecological niche modelling, but innovatively accounting for phenology, to project the distribution of S. muticum under two future climate change scenarios. According to our projections, by 2100 the distribution of S. muticum is expected to shift northwards along its European, North American and Asian distributions with partial retreat from the currently occupied areas.

Port Everglades Inlet is one of the busiest ports in the country and is a point source of pollution to surrounding beaches and offshore corals from heavy boat traffic and urban runoff. Understanding fluctuations of bacterioplankton communities in major port inlets is important due to their impacts on surrounding marine environments. To understand annual microbial fluctuations, the 16s rRNA V4 hypervariable region was sequenced using Illumina high-throughput DNA sequencing technology. Surface samples were taken weekly for one year to generate baseline fluctuations in the microbial community. Total reads of 1.4 million were generated with a final count of 16,384 Operational Taxonomic Units. The dominant phyla were Proteobacteria, Cyanobacteria, Bacteroidetes, and Actinobacteria. Pathogenic genera were detected at low abundances during peak shipping and tourist months (November –April). Results indicate significant differences in alpha diversity when comparing microbial communities in August with other times. This was likely caused by low community richness and abundance, and below-average August rainfall levels. Differences in beta diversity were significant when comparing monthly and seasonal changes. Rainfall, temperature, and nutrient trends may have affected microbial composition, specifically during the dry season that was warmer and wetter than historical averages for 2013-2014. Increased nitrogen and phosphorous concentrations were observed in the dry season months of October, December, and January potentially creating optimal bacterial growth conditions. These results can be compared with historical and future data regarding inlet microbial communities to determine underlying baselines of bacterioplankton communities and monitor the health of marine and recreational environments they impact. This study represents the first to characterize at this scale and use Illumina MiSeq technology to analyze water samples from Port Everglades.

The last half-century has seen a dramatic and alarming rise in the incidence of autoimmune disease in industrialized nations too rapid to be accounted for by genetics alone. Among those, Inflammatory Bowel Disease (IBD) has risen from a western disease affecting industrialized populations to an emerging global threat affecting diverse populations around the world. IBD is a complex disease that combines genetic susceptibility and environmental exposure, but one aspect appears to be clear: the involvement of the gut microbiome. Current thought holds that IBD is an autoimmune attack on commensal microbiota, causing extensive collateral damage to the host intestinal tissues in the process. However, it has remained unclear in the field whether the changes observed in the IBD microbiome are causative in nature or whether the microbiome is responding to already-underway inflammatory processes within the host. This dissertation investigates one host factor in particular with regard to the microbiome and the development of inflammation: sodium-hydrogen exchange at the brush border, mediated by sodium hydrogen exchanger 3 (NHE3). NHE3 is inhibited during active IBD, but its loss in knockout animals is also enough to promote spontaneous colitis in a microbiome-dependent fashion. This dissertation investigates the specific contribution of the microbiome in NHE3 knockout animals to determine whether loss of NHE3 may be mediating the onset of colitis through pro-inflammatory changes in the microbiome. Our results suggest that the microbiome fostered in an NHE3-deficient environment may accelerate the onset and severity of experimental colitis, though likely in concert with additional host factors.