One of the largest environmental liabilities facing the Canadian and international mining industry includes the effects of acidic drainage to water resources. This thesis sought to determine biogeochemical mechanisms of rare earth element and yttrium (REY) enrichment in mine drainage, linkages between REYs and microbial populations, and whether REYs were present in water or biofilm at mineable quantities or toxic levels. Water and co-occurring biofilm samples were collected from North and South American mining and control sites, and a passive water treatment system in Pennsylvania. REY concentrations within mineralized biofilm were observed to occur at borderline mineable quantities within biofilm in coal mine drainage (1,000 mg/kg dry weight total REYs), where REYs were bound predominately to particulate organic matter, manganese and iron, limiting their bioavailability. Within the passive treatment system, REYs showed the greatest maximum water-biofilm partitioning coefficients after Al and Fe, and a strong inverse relationship with aqueous REY concentration. Photosynthetic eukaryotes were observed to occur within biofilms that contained an abundance of neutrophilic iron oxidizing bacteria.

Candida albicans is the most common fungal pathogen that infects humans. The research described in this thesis focuses on an in-depth characterization of the regulatory pathways controlling white-opaque switching, mating and biofilm formation, and the relationships among these programs in this pathogen. It was demonstrated in 2006 that minority opaque cells of C. albicans release pheromone to signal majority white cells of the opposite mating type to form enhanced biofilms. The white cell biofilms in turn facilitate opaque cell chemotropism, an essential step in mating. The white cell response is a general characteristic of C. albicans, occurring in all tested strains and in all common lab media. By generation of deletion mutants of select genes in the opaque cell mating pathway, it was demonstrated that the pathway regulating the white cell response shares all of the components of the opaque mating pathway, from the pheromone receptor through the MAP kinase cascade with the exception of the downstream transcription factor. In addition, it was demonstrated that a C. albicans-specific region in the first intracellular loop, IC1, of the α-pheromone receptor is required for the white, but not the opaque, pheromone response. Furthermore, the cis-acting element in the promoters of genes induced by pheromone in white cells was identified. The white-specific pheromone response element, WPRE, is important for the regulation of the white pheromone response and induction of white-specific genes by pheromone. Finally, based on a misexpression library screening of transcription factors previously implicated in adhesion, cell wall biogenesis, filamentation or biofilm formation, the transcription factor Tec1 was identified to be the key regulator in the white pheromone response pathway. Tec1 binds to the WPRE in the promoters of genes induced by pheromone in white cells to mediate the white cell response. The white pheromone response pathway appears to be a relatively young pathway that borrowed the upstream components from the opaque mating pathway, the transcription factor from the ancestral filamentation pathway, and the downstream genes from the pathway regulating biofilm formation in a/α cells of C. albicans. Therefore, the configuration of the white response pathway provides a unique glimpse and possibly a paradigm for the evolution of signal transduction pathways in eukaryotes.

Bacterial biofilm infection is a common (~ 2 to 4%) complication for recipients of surgically implanted medical devices. Due to the tremendous increase in antibiotic resistance when these bacteria enter the biofilm phenotype, present treatment requires explantation and replacement of the device, often with multiple surgeries and always with much longer patient recovery time. The specific objective of this study was to quantify the degree of biofilm deactivation from exposure to thermal shock for varying temperature and time durations. While extreme temperature (>150˚C) is routinely used to sterilize (e.g. autoclaves), such temperatures have a severe cost within the body. Despite extensive studies on thermal deactivation of bacteria in the planktonic phenotype over a wide range of temperatures (e.g., pasteurization protocols), surprisingly little is known about the thermal deactivation of biofilms except under extreme conditions. Here, the deactivation of Pseudomonas aeruginosa biofilms is reported. These biofilms were cultured at 37°C for 24 hours in a drip-flow reactor and subjected to heat shocks on the range of 50°C to 80°C for durations of 1 to 30 minutes. Heat shocks were delivered by immersion in thermostatted media for the prescribed time and the resulting concentration of colony forming units (CFU/mL) were quantified using direct enumeration. Up to 6.6 orders of magnitude reduction in CFU concentration was observed, indicating that thermal deactivation is a reasonable approach to biofilm mitigation. Integrating this approach with a magnetic nanoparticle implant coating will result in an innovative treatment for implant infections in situ without explantation or device replacement.

The genus Staphylococcus contains at least 47 species and 23 subspecies. Bacteria in this genus are ubiquitous; many are commensals on human and animal skin and can be opportunistic pathogens. In dairy cattle, staphylococci are the leading cause of intramammary infections (IMI) and mastitis. Mastitis is the inflammation of the mammary gland, and is one of the leading infectious diseases causing production losses in the dairy industry. Based on the ability to clot blood plasma in vitro, members of the genus can be divided into two groups: coagulase positive staphylococci (CPS) and coagulase negative staphylococci (CNS). In the dairy industry, Staphylococcus aureus is the most common CPS causing mastitis and is considered a major mastitis pathogen compared to the CNS, which as a group have been described as minor mastitis pathogens. The CNS species are increasingly recognized as an important cause of bovine mastitis, although the relative role of some species is still uncertain. Our understanding of the local and global epidemiology of CNS mastitis is improving with application of more accurate DNA sequence-based species identification methods and techniques to discriminate between strains within species. These factors have led to a shift in perspective, with the CNS being recognized as a heterogeneous group where some species are more important than others in bovine mastitis. The major goals of this thesis were to describe Staphylococcus mastitis epidemiology, and to identify phenotypes that may contribute to persistence in various niches on selected dairy farms in Vermont. We conducted 2 field studies on 2 groups of farms in Vermont. In the first study, we collected S. aureus isolates from bulk tank milk of 44 certified organic dairy farms. In the second field study, we completed quarter milk, cow skin, and environmental sampling of 5 herds that make farmstead cheeses. In both studies, we used non-selective and selective agar medium to isolate staphylococci from the farm sources. From these studies, we collected 1,853 Staphylococcus spp. isolates. We used PCR-amplicon sequence-based species identification to describe Staphylococcus species diversity on these selected Vermont dairy farms. S. aureus isolates were strain-typed using an established Multilocus Sequence Typing (MLST) scheme. A novel MLST scheme was developed to investigate the molecular epidemiology of S. chromogenes, one of the leading CNS species causing bovine mastitis in this and other studies. We also evaluated antibiotic resistance and biofilm formation phenotypes and genotypes of staphylococci to test the hypothesis that these phenotypes may be associated with strain types. In the study of organic dairy farms, 20 S. aureus strain types (STs) were identified, including ten novel STs. The majority of STs belonged to lineages or clonal complexes (CCs) previously identified as cattle adapted (e.g. CC97 and CC151). Associations between ST and carriage of beta-lactam resistance and biofilm forming capacity were identified among the S. aureus isolates from these farms. In the 5-herd study, a total of 27 different staphylococci species were identified from various niches including humans, but only five species; S. chromogenes, S. aureus, S. haemolyticus, S. simulans, and S. xylosus were commonly identified to cause IMI. S. aureus and S. chromogenes strain types were niche specific.

Ecosystems are highly variable in space and time. Understanding how spatial and temporal scales influence the patterns and processes occurring across watersheds presents a fundamental challenge to aquatic ecologists. The goal of this research was to elucidate the importance of spatial scale on stream structure and function within the Oksrukuyik Creek, an Arctic watershed located on the North Slope of Alaska (68°36’N, 149°12’W). The studies that comprise this dissertation address issues of scale that affect our ability to assess ecosystem function, such as: methodologies used to scale ecosystem measurements, multiple interacting scales, translation between scales, and scale-dependencies. The first methodological study examined approaches used to evaluate chlorophyll a in ethanol extracts of aquatic biofilms. Quantification of chlorophyll a is essential to the study of aquatic ecosystems, yet differences in methodology may introduce significant errors to its determination that can lead to issues of comparability between studies. A refined analytical procedure for the determination of chlorophyll a was developed under common acidification concentrations at multiple common reaction times. The refined procedure was used to develop a series of predictive equations that could be used to correct and normalize previously evaluated chlorophyll a data. The predictive equations were validated using benthic periphyton samples from northern Alaska and northwestern Vermont, U.S.A. The second study examined interaction and translation between scales by examining how normalization approaches affect measurements of metabolism and nutrient uptake in stream sediment biofilms. The effect of particle size and heterogeneity on rates of biofilm metabolism and nutrient uptake was evaluated in colonized and native sediments normalized using two different scaling approaches. Functional rates were normalized by projected surface area and sediment surface area scaling approaches, which account for the surface area in plan view (looking top-down) and the total surface area of all sediment particles, respectively. Findings from this study indicated that rates of biogeochemical function in heterogeneous habitats were directly related to the total sediment surface area available for biofilm colonization. The significant interactions between sediment surface area and rates of respiration and nutrient uptake suggest that information about the size and distribution of sediment particles could substantially improve our ability to predict and scale measurements of important biogeochemical functions in streams. The final study examined how stream nutrient dynamics are influenced by the presence or absence of lakes across a variety of discharge conditions and how catchment characteristics can be used to predict stream nutrients. Concentrations of dissolved organic carbon (DOC) and other inorganic nutrients were significantly greater in streams without lakes than in streams in with lakes and DOC, total dissolved nitrogen (TDN), and soluble reactive phosphorus concentrations increased as a function of discharge. Catchment characteristic models explained between 20% and 76% of the variance of the nutrients measured. Organic nutrient models were driven by antecedent precipitation and watershed vegetation cover type while inorganic nutrients were driven by antecedent precipitation, landscape characteristics and reach vegetation cover types. The developed models contribute to existing and future understanding of the changing Arctic and lend new confidence to the prediction of nutrient dynamics in streams where lakes are present.

Pseudomonas aeruginosa bacterial biofilms are the leading cause of mortality among cystic fibrosis (CF) patients. Biofilms contain bacteria attached to a surface and encased in a protective matrix. Since bacteria within a biofilm are less susceptible to antibiotics, a new approach is to use dispersion compounds that cause the biofilms to release free-swimming bacteria. Our approach has focused on combining nutrient dispersion compounds with antibiotics to increase eradication of bacteria within biofilms. This approach takes advantage of the enhanced susceptibility of free-swimming bacteria to antibiotics, compared to bacteria within biofilms. Ultimately, this research will guide the development of an aerosol therapy containing both antibiotic and dispersion compounds to treat bacterial biofilm infections. To study the effect of antibiotic and dispersion compound treatments on biofilm eradication, a high-throughput screening assay was used to assess the effect on young Pseudomonas aeruginosa biofilms. In addition, a Lab-Tek chambered coverglass system imaged via confocal microscopy was used to assess the effect on mature Pseudomonas aeruginosa biofilms. Seven antibiotics (amikacin disulfate, tobramycin sulfate, colistin sulfate, colistin methanesulfonate (CMS), polymyxinB sulfate, erythromycin, and ciprofloxacin hydrochloride) were tested alone or in combination with four nutrient dispersion compounds (sodium citrate, succinic acid, xylitol, and glutamic acid) to assess the level of eradication of bacteria within biofilms. For young biofilms, 15 of 24 combinations significantly eliminated more live bacteria within the biofilms (measured in colony forming units per milliliter) compared to antibiotics alone. In the more mature biofilm system, only 3 out of 26 combinations resulted in a higher percentage of live biofilm bacteria being eliminated compared to antibiotics alone, showing the importance of biofilm age in the effectiveness of these potential combination therapies. To aid in confocal microscopic analysis of biofilms, an automated quantification program called STAINIFICATION was developed. This new program can be used to simultaneously investigate connected-biofilm bacteria, unconnected bacteria (dispersed bacteria), the biofilm protective matrix, and a growth surface upon which bacteria are grown in confocal images. The program contains novel algorithms for the assessment of bacterial viability and for the quantification of bacteria grown on uneven surfaces, such as tissue. The utility of the viability assessments were demonstrated with confocal images of Pseudomonas aeruginosa biofilms. The utility of the uneven surface algorithms were demonstrated with confocal images of Staphylococcus aureus biofilms grown on cultured human airway epithelial cells and Neisseria gonorrhoeae biofilms grown on transformed cervical epithelial cells. Finally, a proof-of-concept study demonstrated that dry powder aerosols containing both antibiotic and nutrient dispersion compounds could be developed with properties optimized for efficient deposition in the lungs. A design of experiments study showed that solution concentration was the most significant parameter affecting aerosol yield, particle size, and in vitro deposition profiles. Collectively this work demonstrated that bacterial dispersion from biofilms can enhance antibiotic susceptibility and can be better quantified using the new STAINIFICATION software. Formulation of dispersion compounds and antibiotics into a dry powder aerosol could enable more effective treatment of biofilm infections in the lungs.

Microbial biofilms are significant in a variety of settings including the human microbiome, infectious disease, industrial processes, and environmental remediation. Due to the ubiquitous nature of biofilms, there is a great interest in understanding cellular activities within the biofilm matrix. Biofilm cells are able to better withstand environmental stress, experience increased horizontal gene transfer, and live longer. The purpose of this research is to grow Pseudomonas sp. strain ADP as a biofilm and examine the chemical and physical characteristics the microbe undertakes in a sticky extracellular matrix. ADP is the organism of choice because of its ability to metabolize atrazine. Cells are grown in a drip biofilm reactor and flow cells under varying time lapse to gain insight to biofilm formation. Some cells are grown with atrazine as the sole nitrogen source, while others are grown in a nutrient-rich medium to compare cells response under nutrient-limited conditions with atrazine particles in the matrix. As a positive control, Escherichia coli are grown in a similar manner. Raman spectroscopy was the main analytical technique used to evaluate the chemical and molecular characteristics of this system. Scanning electron microscopy is used to examine cellular distribution, and several assays are performed for molecular composition analysis. Raman analysis in the fingerprint region revealed distinct differences between free cells and cells in biofilm. Soluble extracellular polymeric substances (EPS) were found to be more prevalent than tightly bound EPS and lightly bound EPS in the biofilm matrix. Comparison of relative peak intensity ratios suggests that it is possible to track atrazine degradation by means of intermediates using Raman spectroscopy. SEM micrographs revealed EPS role as an immobilizing agent when in contact with compounds, such as atrazine. Further research is needed to determine if atrazine can bind to EPS fractions outside the presence of cells and whether its affinity to EPS is mostly attributed to physical conditions, due to the architecture of biofilm, or chemical, based on functional groups presents. The results obtained from this research will contribute to the development of a less invasive microscale approach to address the acquisition and induction of biotransformation activity occurring in xenobiotic degrading systems. The extracellular interactions observed can be used to further characterize biofilm-mediated bioremediation. Results have contributed to the Raman spectra library for microorganisms and organic compounds. .

Alkaline conditions induced by algae growth in wastewater stabilization ponds create deprotonated ammonium ions that result in ammonia gas (NH3) volatilization. If algae are utilized to remediate wastewater through uptake of phosphorus, the resulting nitrogen loss will hinder this process because algae generally require a stoichiometric molar ratio of N16P1. Lower ratios of N/P due to loss of ammonia gas will limit the growth and yield of algae, and therefore will reduce phosphorus removal from the water phase into the algae phase. In order to reduce nitrogen loss through volatilization, an ammonium selective zeolite, clinoptilolite, can be used to sequester nitrogen from the water phase as ammonium ion and in a form that is bioavailable for uptake and growth of algae. A novel algae biofilm rotating photo bioreactor (RPB) with clinoptilolite integrated to the outermost surface as the substratum for algae biofilm attachment and growth has been designed, constructed, and tested for ammonium capture and algae biomass production, with simultaneous removal of the algal nutrient phosphorus from water. The clinoptilolite‐based RPB (cRPB) provides algal biomass that can serve as feedstock for biofuel production through uptake of zeolite‐based nitrogen and water phase phosphorus.

Orientador: Marlise Inêz Klein / Resumo: A cárie dentária é um problema de saúde pública mundial causada por uma interação entre dieta rica em açúcar e microrganismos em biofilmes dentais. Abordagens para controlar esses microrganismos em biofilmes são necessárias. Portanto, o objetivo foi avaliar potencial antimicrobiano (cultura planctônica) e antibiofilme de doze extratos de Casearia Sylvestris (0,50 mg/mL) de diferentes biomas brasileiros (Mata Atlântica, Cerrado, Caatinga, Pampa e Pantanal) e variedades (sylvestris, lingua e intermediária) contra duas espécies encontradas em biofilmes cariogênicos (Streptococcus mutans e Candida albicans). A caracterização química dos extratos brutos foi feita via cromatografia. A atividade antimicrobiana foi determinada pela população microbiana (UFC/mL) enquanto a atividade antibiofilme pela população microbiana (UFC/mL) e biomassa dos biofilmes tratados. Para os extratos ativos para S. mutans, foi avaliado o efeito na formação inicial da matriz de glucanos (atividade de GtfB), o desprendimento após a adesão desta bactéria à película salivar e à matriz inicial de glucanos e citotoxicicidade via ensaio MTT. Também foi avaliado o potencial antimicrobiano contra S. mutans de três frações (metanol, acetato de etila e hexano; 0,25 mg/mL) oriundas dos doze extratos. Três extratos do bioma Mata Atlântica e variedade sylvestris reduziram em >50% (ou >3 logs) as contagens de células viáveis de S. mutans (vs. veículo; p <0,0001), enquanto dois extratos, pertencentes ao mesmo bioma e va... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dental caries is a worldwide public health problem caused by an interaction between a diet rich in sugar and microorganisms in dental biofilms. Approaches to control these microorganisms in biofilms are necessary. Therefore, the objective of this study was to evaluate the antimicrobial (planktonic) and antibiofilm potential of twelve Casearia sylvestris extracts (0.50 mg/mL) from different Brazilian biomes (Atlantic Forest, Cerrado, Caatinga, Pampa and Pantanal) and variety (sylvestris, lingua and intermediate) against two species found in cariogenic biofilms (Streptococcus mutans and Candida albicans). The chemical characterization of crude extracts was done via chromatography. The antimicrobial activity was determined by the viable microbial population (CFU/mL) and the antibiofilm activity by the viable microbial population (CFU/mL) and biomass of the biofilms treated. For the extracts active for S. mutans, the effect on the initial formation of the glucan matrix (GtfB activity), the release after adhesion of this bacterium to the salivary film and the initial matrix of glucans and cytotoxicity via the MTT assay was evaluated. In addition, the antimicrobial potential against S. mutans of three fractions (methanol, ethyl acetate and hexane, 0.25 mg/mL) from the twelve extracts was also evaluated. Three extracts of the Atlantic Forest biome and sylvestris variety reduced the counts of viable S. mutans cells (vs. vehicle, p <0.0001) by >50% (or >3 logs), while two extracts bel... (Complete abstract click electronic access below) / Mestre

Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs. / Meeresschwamm-assoziierte Actinomyceten stellen ein Reservoir für verschiedene natürliche Produkte mit neuartigen biologischen Aktivitäten dar. Ihr antibiotisches Potenzial gegenüber einer Reihe von Gram-negativen und -positiven Bakterien ist bereits intensiv erforscht worden. Wenig ist allerdings über ihre antiinfektive und antivirulente Wirksamkeit gegenüber menschlichen Pathogenen bekannt. Ziel dieser Doktorarbeit war es, die antiinfektiven Fähigkeiten (anti-Shiga-Toxin und anti-Biofilm) der aus Schwämmen isolierten Actinobakterien zu untersuchen. Hierfür wurden bioaktive Metabolite der Actinobakterien identifiziert und isoliert und abschließend wurde ihr Wirkmechanismus mit Hilfe einer Transkriptomanalyse charakterisiert. Diese Arbeit ist in drei Studien gegliedert, welche alle zum Ziel hatten die antiinfektive Wirksamkeit von aus Actinomyceten gewonnenen Extrakten und Komponente(n), welche möglicherweise als zukünftige Therapeutika dienen könnten, zu untersuchen. Die erste Studie befasst sich mit den anti-Shiga-Toxin Effekten der Meeresschwamm- assoziierten Actinomyceten. Durchfallinfektionen stellen in vielen Entwicklungsländern aber auch in Industrieländern eine große Gefahr dar. Durchfallerkrankungen die durch enterohämorrhagische Escherichia coli (EHEC) hervorgerufen werden, können sich zu lebensbedrohlichen Komplikationen wie Gastroenteritis oder dem hämolytisch urenischen Syndrom (HUS) weiterentwickeln. Das von den EHEC Stämmen produzierte Shiga-Toxin (Stx) stellt hierbei den Haupt Virulenz Faktor dar, welcher die eukaryotische Proteinsynthese menschlicher Zellen negativ beeinflusst, was wiederum den Zelltod durch Apoptose zur Folge hat. Die Behandlung der EHEC-Patienten mit Antibiotika wird nicht empfohlen, da dies zu einem Anstieg von freigesetztem Stx der zersetzen Bakterien führen könnte, wodurch das Risiko für die Entwicklung des HUS ansteigt. Aus diesem Grund werden effektive Medikamente dringen benötigt, welche die Stx Produktion blockieren ohne das Wachstum der Bakterien zu beeinflussen. In dieser Studie wurden 194 Extrakte und einige isolierte Komponenten von aus Schwämmen gewonnenen Actinomyceten auf ihren negativen Einfluss auf die Stx Produktion des EHEC Stammes EDL933 mit der Hilfe des Ridascreen® Verotoxin ELISA Kits untersucht. Es konnte gezeigt werden, dass die Zugabe der Extrakte keinen signifikanten Einfluss auf die Stx Produktion hatte. Strepthonium A auf der anderen Seite, welches aus Streptomyces sp. SBT345 isoliert wurde (vom mediterranen Schwamm Agelas oroides) konnte die Stx Produktion von EDL933 bei einer Konzentration von 80 µM reduzieren ohne das Wachstum des EHEC Stammes zu beeinflussen. Die Struktur von Strepthonium A wurde mittels spektroskopischer Analyse (1D- und 2D-NMR), sowie mittels ESI-HRMS und ESI-HRMS2 Experimenten entschlüsselt. Basierend auf diesen Ergebnissen könnte Strepthonium A eine mögliche Alternative oder Zusatz in der Behandlung einer EHEC Infektion darstellen. In der zweiten Studie wurde der Einfluss der Meeresschwamm-assoziierten Actinomyceten auf die Biofilmbildung von Staphylokokken bewertet. Medizinische Produkte wie Kontakt Linsen, metallische Implantate, Katheter, Herzschrittmacher, usw. stellen optimale ökologische Nischen für die Ausbildung von bakteriellen Biofilmen dar, wodurch Infektionen im Menschen hervorgerufen werden können. Bakterien in einem Biofilm sind deutlich toleranter gegenüber der Immunantwort ihres Wirtes sowie gegenüber konventionellen Antibiotika und sind daher schwer zu bekämpfen. In dieser Studie wurde das anti-Biofilm Potential eines organischen Extrakts der flüssigen Fermentation von Streptomyces sp. SBT343 (vom mediterranen Schwamm Petrosia ficiformis) ermittelt. In vitro Ergebnisse zeigten, dass das organische Extrakt anti-Biofilm (gegenüber Staphylococci) Fähigkeiten besitzt und nicht toxisch für Maus Makrophagen (J774.1), Fibroblasten (NIH/3T3) und humane korneale Epithelzellen ist. Zudem konnte gezeigt werden, dass das SBT343 Extrakt die Ausbildung eines Biofilms von Staphylokokken auf den Oberflächen von Polystyrol, Glass und Kontaktlinsen unterbinden konnte ohne das bakterielle Wachstum zu beeinflussen. Die hochauflösende Fouriertransformation-Massenspektrometrie (HR-MS) Analyse konnte die Komplexität sowie die chemische Vielfalt an Komponenten im Extrakt aufzeigen. Eine vorläufige, physio-chemische Charakterisierung deutet darauf hin, dass die aktive Komponente im Extrakt hitzestabil und nicht proteinartiger Natur ist. Abschließend konnte durch Fraktionierungsexperimente gezeigt werden, dass die biologische Aktivität auf synergistischen Effekten mehrerer Komponenten im Extrakt beruht. In einer dritten Studie wurden 50 organische Extrakte, welche aus fester und flüssiger Fermentierung von 25 verschiedenen aus Meeresschwämmen isolierten Actinomyceten gewonnen wurden, auf anti-Biofilm-Aktivität untersucht. Hierbei wurde die anti-Biofilm Aktivität des organischen Extrakts der Festkultur von Streptomyces sp. SBT348 (vom mediterranen Schwamm Petrosia ficiformis) identifiziert. Eine Bioassay gestützte Fraktionierung führte zu der Identifikation der aktiven Fraktion Fr 7 im SBT348 Extrakt. Durch weitere Aufreinigung des Extrakts mit einer semipräparativen HPLC, konnten die bioaktiven Sub-Fraktionen SKC1, SKC2, SKC3, SKC4 und SKC5 isoliert werden. Die Sub- Fraktion SKC3 hatte den stärksten anti-Biofilm Effekt und bestand aus einer reinen Verbindung mit BIC90 und MIC Werten von 3,95 µg/ml und 31,25 µg/ml gegen S. epidermidis RP62A. SKC3 zeigte weder erkennbare Toxizität gegenüber Zelllinien in vitro noch gegenüber den Larven der großen Wachsmotte Galleria melonella in vivo. SKC3 war Hitze- und Enzym-resistent, was auf eine nicht proteinartige Natur hindeutet. Eine HR-MS Analyse ergab, dass die Masse von SKC3 1258,3 Da beträgt. Die Strukturanalyse von SKC3 durch 1D und 2D-NMR ist zurzeit in Bearbeitung. Um weiteres Verständnis über den anti-Biofilm Wirkmechanismus von SKC3 auf S. epidermidis RP62A zu erlangen, wurde eine RNA Sequenzierungsanalyse durchgeführt. Die Transkriptomanalyse zeigte, dass SKC3 von RP62A nach einer 20-minütigen Inkubationszeit erkannt wird und dass SKC3 den zentralen Metabolismus des Staphylokokken Stammes nach 3 h negativ beeinflusst. Zusammengenommen deuten die Ergebnisse darauf hin, dass SKC3 als Leitstruktur für die Entwicklung neuer anti- Staphylokokken Medikamente dienen könnte. Zusammenfassend heben die Ergebnisse dieser Arbeit die antiinfektiven Eigenschaften der Meeresschwamm-assoziierte Actinomyceten hervor und bieten eine Möglichkeit für die Nutzung dieser in Wirkstoffentwicklungsprogrammen.