Introduction: Garlic-derived organosulfur compounds are associated with physiological benefits, including the reduction of cardiovascular disease (CVD) risk, possibly by reducing the risk marker trimethylamine-N-oxide (TMAO). TMAO production in humans is largely influenced by the metabolic activity of the intestinal bacteria on dietary precursors including L-carnitine. Dietary supplementation of bioactive garlic phytochemical allicin has recently been suggested to reduce the formation of TMAO precursor molecule trimethylamine (TMA) from L-carnitine through impact on the intestinal bacteria, thereby limiting the formation of TMAO by the host. Purpose: The objective of this research was to evaluate and compare the efficacy of fresh and aged garlic extracts (rich in alliin and allicin, respectively) in the reduction of circulating TMAO levels produced from L-carnitine metabolism and identify shifts in the abundances of gastrointestinal bacterial genes that may contribute to reduction in circulating TMA levels, which may, in turn, influence the levels of circulating TMAO. Methods: Five-week old female C57BL/6 mice (n = 12) were challenged with L-carnitine to assess the animal's capacity for TMAO production. Animals were gavaged daily with fresh or aged garlic extract dissolved in L-carnitine for 13 days, then challenged with L-carnitine post-treatment to evaluate changes in TMAO production. Whole blood samples were evaluated for TMAO content using UPLC-MS/MS and compared to non-extract consuming control groups. Post-mortem hepatic tissues were collected and analyzed for TMA-oxidizing flavin monooxygenase 3 (Fmo3) gene abundance and protein expression using quantitative real-time PCR (qPCR) and ELISA. Fecal samples collected prior to and following treatment were analyzed using qPCR to quantify shifts in the abundance of L-carnitine metabolizing genes cntAB and grdH. Results: Postprandial and circulating TMAO levels were not significantly affected (p < 0.05) by inclusion of garlic extract in the diet. Dietary intervention with extracts significantly increased L-carnitine-derived proatherogenic CVD risk marker γ-butyrobetaine levels ~28% higher than the increased levels observed in the positive control group supplemented with L-carnitine only. Mice administered garlic extracts had significant increases of, γ-butyrobetaine, relative to negative control mice and mice supplemented with broad-spectrum antibiotics. Mice supplemented fresh garlic extract saw a 25-fold increase in circulating γ-butyrobetaine levels after intervention; mice supplemented aged garlic extract saw a 23-fold increase in circulating γ-butyrobetaine levels after intervention. Furthermore, FMO3 protein expression levels in either extract treatment group were not significantly different (p < 0.05) from controls. Abundances of L-carnitine metabolizing genes in fecal samples of mice fed either garlic extract were not significantly higher than levels observed in positive or negative controls. Interestingly, treatment with broad-spectrum antibiotics significantly increased abundances of L-carnitine metabolizing genes cntAB and grdH when compared with controls. Abundances of hepatic Fmo3 mRNA transcript in mice supplemented garlic extracts were not significantly different from the positive control group when data were normalized to mg of liver used. Mice supplemented aged garlic extracts significantly lowered Fmo3 mRNA transcript levels relative to the negative control. Significance: This research suggests that garlic extract supplementation in conjunction with excess L-carnitine consumption may not be an appropriate dietary intervention strategy to reduce CVD risk. As it stands, garlic extract supplementation may increase CVD risk by promoting the biosynthesis of proatherogenic γ-butyrobetaine. The impact of garlic extract mediated increases in γ-butyrobetaine should be further investigated in tandem with CVD outcomes to confirm the findings presented in this study. / Doctor of Philosophy / Garlic compounds that contain sulfur are associated with many health benefits, including the reduction of heart disease risk, possibly by lowering the amount of risk marker trimethylamine-N-oxide (TMAO) in the body. TMAO is produced when the gut bacteria break down L-carnitine into trimethylamine (TMA), which is then absorbed and converted to TMAO in the liver. Garlic supplementation has recently been suggested to reduce TMAO formation, which may, in turn, reduce heart disease risk. The objective of this research was to evaluate the potential of fresh and aged garlic extracts (which have different sulfur compounds in them) to reduce TMAO levels and identify changes in the gut bacteria that may contribute to this lowering effect. Mice were fed daily with either fresh or aged garlic extract for 13 days, then given L-carnitine to evaluate changes in TMAO levels in the blood. These levels were then compared to mice that did not consume any garlic extract. Liver samples were tested for their ability to turn TMA into TMAO. Fecal samples were tested to determine if there were any changes to gut bacteria caused by the garlic extracts. TMAO levels in the mice were not significantly affected by consuming garlic extracts. Consuming garlic extracts did, however, increase another risk marker of heart disease known as γ-butyrobetaine. Feeding mice garlic extracts did not affect the ability of mice to turn TMA into TMAO, nor did it affect the gut bacteria. This research suggests that garlic extracts may not be an appropriate strategy to reduce heart disease risk. As it stands, garlic extract supplementation may increase heart disease risk by promoting the γ-butyrobetaine formation. The means that garlic extracts increase γ-butyrobetaine levels should be further investigated.

L’impact de l’homme sur l’environnement mondial et sur la diversité et le fonctionnement des écosystèmes terrestres fait l’objet d’une attention croissante. De nombreuses études ont évalué les effets de facteurs du changement global tels sur les processus de cycle de l'azote du sol dans les prairies. Cependant, ces études n'ont pas pris en compte le fait que la modification du régime de précipitations avait également une influence sur le régime de dépôt d'azote. En outre, la réponse du cycle de l'azote dans les sols des prairies à de multiples facteurs du changement global agissant ensemble et parfois en même temps que des perturbations telles que les incendies, doit encore être étudiée. Cela limite fortement notre capacité à comprendre et à prévoir les effets du changement global sur les prairies. Dans ce travail de doctorat, deux expériences ont été menées: (i) une expérience en mésocosme pour évaluer les effets combinés d'une augmentation des dépôts d'azote et de changements dans la quantité et la fréquence des précipitations sur le cycle de l'azote édaphique dans une prairie semi-aride; (ii) une expérience in situ pour évaluer les effets combinés de l'augmentation de la concentration en CO2, du réchauffement, d’une modification des précipitations, du dépôt d'azote et d’un feu sur le cycle de l'azote du sol dans une prairie méditerranéenne. Cela permet d'étudier les effets de la combinaison de plusieurs facteurs de changement global (et d’une perturbation feu) sur l'abondance des communautés microbiennes du cycle de l'azote.Les groupes microbiens étudiés étaient les bactéries et les archées oxydant l'ammoniac (AOB et AOA, respectivement), les réducteurs de nitrite porteuses des gènes nirK ou nirS, et les réducteurs de N2O porteurs des gènes nosZI- et nosZII, plus les bactéries oxydant le nitrite du genre Nitrobacter et Nitrospira pour la prairie méditerranéenne. Les principaux résultats et conclusions sont les suivants: 1) Les réactions des différents groupes de (dé)nitrifiants aux scénarios de changement global différaient fortement quel que soit le type de prairie. Les AOB étaient principalement dépendant de la disponibilité en azote En revanche, dans les deux prairies, les AOA étaient plus sensibles à la dynamique de l'eau du sol que la dynamique de l'azote. L'abondance des Nitrobacter étaient principalement affectée par les facteurs de changement global affectant l'abondance de l'AOB, tandis que l'abondance des Nitrospira était davantage liée aux changements d'abondance des AOA dans la prairie méditerranéenne. 2）Dans la prairie californienne où deux dépôts d'azote élevés avaient lieu chaque année, l'effet de l'azote dominait les effets du changement global. En revanche, dans la prairie chinoise, les dépôts d’azote simulés par des apports chroniques couplés aux événements de précipitation n’augmentaient pas l’abondance des dénitrifiants et ne faisaient que légèrement augmenter les émissions de N2O. 3） Pour les deux prairies, l'interaction entre les facteurs du changement global sur le cycle de l'azote du sol ne pouvait pas être prédite simplement en étudiant les effets d'un ou de deux facteurs. Ces effets interactifs ont pu être expliqués par des effets sur des variables environnementales clés telles que l'humidité du sol, la disponibilité de l'azote minéral, le pH et la croissance des racines. Ces résultats démontrent qu'il est impossible de prédire comment les (dé)nitrifiants et la (dé)nitrification répondent aux scénarios de changement global impliquant de multiples facteurs uniquement à partir de la connaissance d'effets de facteurs étudiés isolément. Cela nécessite donc des études plus approfondies dans le domaine de la biologie des changements globaux. La modélisation et l'évaluation de la généralité de ces effets d'interaction complexes constituent donc une priorité majeure pour les chercheurs qui veulent prédir les réponses du cycle de l'azote dans le sol au changement global et les rétroactions sur le climat. / The impact of global environmental changes on the diversity and functioning of terrestrial ecosystems has received increasing attention. Many studies evaluated the effects of single -and less often multiple- global change factors on soil N cycling processes in grasslands. However, these studies have not recognized that altered precipitation regime also has an influence on wet N deposition regime. Further, the response of grassland soil N cycling to co-occurring multiple global change factors and disturbance like fire, and how N cycling response to fire could differ under different global change scenarios, remains unclear. This strongly restricts our ability to understand and predict global change effect on grasslands. In this work, two experiments were conducted: (i) a mesocosm experiment to assess the combined effects of increased N deposition and changes in both the amount and frequency of rainfall on soil N cycling in a semi-arid Monsoon grassland; and (ii) an in situ experiment to assess the combined effects of elevated CO2, warming, increased precipitation, N deposition and fire on soil N cycling in a Mediterranean grassland. This allows studying the -possibly interactive- effects of several global change factors on the abundances of soil N-cycling microbial communities. The microbial groups studied were ammonia oxidizing bacteria and archaea (AOB and AOA, respectively), nirK- and nirS-nitrite reducers, nosZI- and nosZII-N2O reducers, plus Nitrobacter and Nitrospira for the Mediterranean grassland. The main results and conclusions are: 1）The responses of different groups of soil (de)nitrifiers to global change scenarios differed strongly regardless the grassland type. AOB were mostly driven by N. In contrast, AOA were more sensitive to soil water dynamics than N dynamics in both grasslands. Nitrobacter abundance was mostly affected by global change factors through their effects on AOB abundance, whereas Nitrospira abundance was more related to changes of AOA in the Mediterranean grassland. Similarly, nirK- and nirS-harboring nitrite reducers and nosZI-harboring N2O reducers were more sensitive to N deposition than nosZII-harboring N2O reducers, and nirK- and nirS-bacteria positively responded to reduced precipitation. This highlights niche differentiation between them and indicates that the balance between them may be altered in the future; 2）In the Mediterranean grassland, where high N deposition was simulated by two N addition events each year, the N effect dominated global change effects. In contrast, in the Monsson grassland, chronic wet N deposition did not increase denitrifier abundance and only weakly increased soil N2O emissions. This was explained by the efficient capture of added N by the dominant grass species and by the increased plant growth leading to increased transpiration and decreased soil moisture. 3）For both grasslands, the interaction between global change factors on soil N cycling could not be predicted simply by studying the effects of one or two factors. These interactive effects were explained by effects on key environmental variables like soil moisture, mineral N availability, pH and belowground plant growth.These results demonstrates the limitation of predicting how (de)nitrifiers respond to global change scenarios involving multiple factors only from studying single factor effects. Particularly, interactive effects were observed between N deposition, decreased precipitation amount and altered precipitation frequency in the Monsoon grassland; and between fire, N deposition, warming, elevated precipitation and elevated CO2 in the Mediterranean grassland. This calls for more comprehensive studies in the global change biology domain. Modelling and evaluating the generality of these complex interaction effects is thus a high priority for research to predict the responses of soil N cycling processes to global change and feedbacks on climate in the future

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: see item for full text / AFRIKAANSE OPSOMMING: sien item vir volteks

A cultivation-independent approach, sequence analysis of 18S rRNA genes PCR-amplified from environmental DNA, was used to explore the diversity and distribution of eukaryotic microbes inhabiting algal mats in two acidic geothermal streams in Yellowstone National Park. The objectives were to: (1) clarify the identity of mat forming algae in Nymph Creek (2) survey microbial species in the Nymph Creek mat over seasonal intervals along a thermal gradient (3) compare microbial species in the Nymph Creek mat with those in Alluvium Creek mats (4) evaluate microbial species in algal mats formed on different substrates in Alluvium Creek. The results show that a novel red alga dominates high temperature regions (~50ºC) of Nymph Creek and two "Chlorella-like" algae predominate the cooler regions (<38ºC). The predominant algae in Alluvium Creek were distinctly different from those in Nymph Creek. Several stramenophiles and fungi were detected in each algal mat.

La couverture neigeuse arctique peut atteindre jusqu'à un tiers de la surface terrestre. Cet environnement, chimiquement très dynamique, est en interaction avec tous les compartiments environnementaux : l’atmosphère, le sol, les aquifères, et ce influence la biosphère toute entière. Durant les dernières décennies, la neige a été reconnue comme étant un réservoir de microorganismes. Pourtant l’écologie des microbes du manteau neigeux reste mal comprise. L’objectif principal de cette thèse est donc de caractériser le manteau neigeux en tant qu’écosystème fonctionnel, par définition une communauté d’organismes vivants, en conjonction avec la composante non vivante de leur environnement et agissant comme un système. Pour cela, la composition taxonomique et fonctionnelle des communautés microbiennes a été analysée via la technologie de séquençage haut débit pour deux types de modèles de manteau neigeux : une neige saisonnière d’eau douce d’un manteau neigeux terrestre (Ny--‐Alesund, Svalbard) et une couverture neigeuse saline sur la glace de mer (Nuuk, Greenland). Le premier objectif est de caractériser l’hétérogénéité des communautés microbiennes en relation avec les fluctuations conditions environnementales. La composition des communautés microbiennes du manteau neigeux est très variable en fonction de l’avancement dans la saison du printemps vers l’été et en fonction de la profondeur. La corrélation entre les fonctions microbiennes et les conditions environnementales soutient l’hypothèse que les communautés microbiennes interagissent avec les fluctuations des conditions en abiotiques de leur habitat. Le second objectif concerne la spécificité des communautés microbiennes du manteau neigeux ; si le manteau neigeux est un écosystème fonctionnel alors les communautés microbiennes le composant devraient présenter des caractéristiques spécifiques liées à leur adaptation aux conditions de cet habitat, malgré la variabilité. La comparaison de la distribution fonctionnelle entre la neige et des environnements distants (polaires ou non) ainsi que des environnements en interaction proche permet de confirmer une spécificité des communautés microbiennes de la neige. Le troisième objectif se concentre sur la sélection environnementale ; étant donné que l’existence d’une communauté microbienne spécifique implique que des processus de sélection se réalisent au sein du manteau neigeux. La comparaison de la distribution de la structure (quels microorganismes sont présents) et la fonction (que sont-ils capables de faire ?) des communautés microbiennes en fonction de la source des microorganismes au sein d’un manteau neigeux couvrant la glace de mer révèle que la communauté est largement influencée mais diffère de leur source en réponse aux conditions environnementales spécifiques. Les résultats préliminaires des analyses metagénomiques et metatranscriptomiques ont révélé qu’il existe une grande variabilité entre les communautés présentes et potentiellement actives au sein du manteau neigeux. Bien que des limitations conceptuelles et techniques persistent, les méthodes de séquençages haut-débit basées sur les molécules d’ARN sont des outils prometteurs pour décrire les réponses à court terme des communautés microbiennes du manteau neigeux aux variations des conditions environnementales. Finalement, une approche mécanistique préliminaire basée sur la mise en place de microcosmes de neige artificielle et des microorganismes modèles a été développée afin de déterminer les processus de colonisation au sein du manteau neigeux. Alors que de nombreuses questions demeurent concernant l’activité microbienne et les interactions complexes de communautés, les études menées durant cette thèse ont permis de soutenir l’hypothèse que la neige est un écosystème fonctionnel. / The Arctic seasonal snowpack can extend at times over a third of the Earth’s land surface. This chemically dynamic environment interacts with different environmental compartments such as the atmosphere, soil and meltwater, and thus, strongly influences the entire biosphere. During the last decades, snow has been recognized as a microbial reservoir. The ecology of snow microorganisms however remains poorly understood. The main goal of this thesis was to investigate the snow as a functional ecosystem; i.e. a community of living organisms in conjunction with the non--‐living component of their environment and interacting as a system. In order to do so, microbial community taxonomic and functional composition of snow samples from two arctic snowpack models: seasonal snow from terrestrial fresh water snowpack (Ny--‐Alesund, Svalbard) and sea ice snow cover (Nuuk, Greenland) was analyzed using high throughput sequencing technologies. The first objective addressed microbial community heterogeneity in relation with fluctuating environmental conditions. Snow microbial community composition was highly variable during spring season and depth. The relationship between microbial functions and environmental conditions supports the hypothesis that the snow microbial community interacts with the abiotic variability characteristic of their habitat. The second objective addressed snow community specificity; if the snowpack is a functional ecosystem, then the microbial communities inhabiting it should have specific features related to their adaptation to the conditions of this environment, despite variability. The comparison of functional distribution between snow and both remote (polar and non polar) and closely interacting environments provided evidence of snowpack microbial community specificity. The third objective focused on environmental selection, given that the existence of a specific snow microbial community implies that one or more selective processes occur in the snowpack. Comparing the distribution of microbial community structure and function as related to the source of the microorganisms in a sea ice snow cover revealed that snow microbial communities were largely influenced by, yet differed from their seeding sources in response to specific environmental conditions. Mechanistic approaches with model microorganisms in snow microcosms were developed during this thesis and, based on preliminary results, will help to determine colonization processes within snowpack. Finally, preliminary results in the first section of Chapter 4 also showed that a high variability exists between the microorganisms present within the snowpack, and those that are active. Although technical and conceptual issues remain, RNA based high throughput sequencing was evaluated as an encouraging tool to evaluate short--‐term responses of microbial communities to environmental fluctuations. While numerous questions remain about microbial activity and complex community interactions, the results from this thesis support the hypothesis that snow is a functional ecosystem.

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function. To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH < 1) and hot (temperature > 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the > 100,000 mass spectra collected during an MS experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth.

The ecological niche of the vagina contains a large number of different microbes that are constantly interacting with each other and the host. Culture methods have not been sufficient in order to resolve the complexity of the normal vaginal flora. Further, the methods for delineating normal flora from not normal flora are not easily handled and are traditionally not based on culture but on microscopy of elements of the vaginal fluid. In the work presented in this thesis, an international collaboration was established that pin-pointed some of the difficulties in classifying vaginal floras, including staining, sampling, and discordance when lactobacilli are few in number, and that emphasized the importance of the size of the vision field in microscopes. As lactobacilli are prominent members of the normal vaginal flora they need to be carefully classified if further work towards more robust scoring tools is to be achieved. Phenotypic methods have not been able to separate the closely related Lactobacillus species of the vagina. Progress in molecular biology has provided possibilities to characterize these lactobacilli, which are mainly from the Lactobacillus acidophilus group. In this work a large number of strains collected by true random sampling were subjected to RAPD-PCR, TTGE and multiplex PCR for species identification. The major species found were L. crispatus, L. gasseri and L. jensenii and the recently described L. iners. The presence of L. iners has not been detected in previous studies due to its special nutrient requirements. Development of pyrosequencing technology also made it possible to match signatures of the two variable regions V1 and V3 of the 16S rRNA gene of the vaginal lactobacilli and identify them to the species level in a high throughput manner. The study confirmed that the dominating flora in women with normal vaginal flora comprises the four species mentioned previously. Repetitive sampling during IVF-treatment with highly varying oestrogen levels demonstrates changes that possibly occur during changes in the natural life cycle. Furthermore, L. iners was found to be the first species to be established after spontaneously resolved or treated Bacterial Vaginosis. These findings can be of help in developing new strategies for regaining and retaining the normal vaginal flora.

Gastrointestinal (GI) health is an important contributor to one’s overall well-being. In the past decade, the focus of this aspect of health has been on the organisms that inhabit the intestines: gut microbes. A concept central to understanding bacterial behavior for health or disease promotion is biological film (biofilm) formation. The predominant form of growth for bacteria is biofilm formation, an as yet poorly described phenomenon for gut microbes. In order to better understand bacterial behavior in response to nutrients that pass through the GI system, a high throughput system to assess biofilm formation was developed. Gastrointestinal-associated microbes, Escherichia coli and Enterococcus faecalis, were assayed for biofilm formation in 96-well plates after 24 hours of incubation. Nutrients, inter-and intrakingdom signaling molecules such as monosaccharides, calcium, insulin, endocannabinoids, and AI-1, AI-2 like signaling compounds, respectively, were added to cultures in order to test their effects on biofilm formation. Biofilm was quantified spectrophotometrically by the measurement of optical density of each well measured at 580nm following crystal violet staining of adherent biofilm. Values were expressed as means ± standard error of the mean (SEM); differences between means were assessed using t-testing and ANOVA using GraphPad Prism, with mean differences considered significant at P < 0.05. Results showed that biofilm formation by E. coli was enhanced by glucose, galactose, lactose, AI-1 like signaling compound and insulin at 50 and 100µU/ml, while AI-2 like compound and calcium inhibited biofilm formation. Biofilm formation by E. faecalis was also enhanced by AI-1 like compound and insulin at 50µU/ml in RPMI medium and inhibited in cultures grown in BHI medium or with added calcium. We conclude that gastrointestinal-associated microbes are influenced by nutrients as well as various factors, including the culture medium, signaling compounds, as well as host-signaling compounds such as insulin and calcium. This study provides a platform required for future studies involving nutrient effect on biofilm formation.