abstract: Water quality in surface water is frequently degraded by fecal contamination from human and animal sources, imposing negative implications for recreational water use and public safety. For this reason it is critical to identify the source of fecal contamination in bodies of water in order to take proper corrective actions for controlling fecal pollution. Bacteroides genetic markers have been widely used to differentiate human from other sources of fecal bacteria in water. The results of this study indicate that many assays currently used to detect human-specific Bacteroides produce false positive results in the presence of freshwater fish. To further characterize Bacteroides from fish and human, the fecal samples were cultured, speciated, and identified. As a result, forty six new Bacteroides 16S rRNA gene sequences have been deposited to the NCBI database. These sequences, along with selected animal fecal sample Bacteroides, were aligned against human B. volgatus, B. fragilis, and B. dorei to identify multi-segmented variable regions within the 16S rRNA gene sequence. The collected sequences were truncated and used to construct a cladogram, showing a clear separation between human B. dorei and Bacteroides from other sources. A proposed strategy for source tracking was field tested by collecting water samples from central AZ source water and three different recreational ponds. PCR using HF134 and HF183 primer sets were performed and sequences for positive reactions were then aligned against human Bacteroides to identify the source of contamination. For the samples testing positive using the HF183 primer set (8/13), fecal contamination was determined to be from human sources. To confirm the results, PCR products were sequenced and aligned against the four variable regions and incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped within the human clade. As an outcome of this study, a tool box strategy for Bacteroides source identification relying on PCR amplification, variable region analysis, human-specific Bacteroides PCR assays, and subsequent truncated cladogram grouping analysis has been developed. The proposed strategy offers a new method for microbial source tracking and provides step-wise methodology essential for identifying sources of fecal pollution. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012

Les légumineuses sont capables de développer des organes symbiotiques, les nodules, qui hébergent des bactéries du sol appelées rhizobia. Au sein des nodules les rhizobia intracellulaires se différencient en bactéroïdes capables de réduire l'azote atmosphérique en ammonium au bénéfice de la plante. En contrepartie, la plante alimente la bactérie en sources de carbone. Des études récentes sur le modèle symbiotique Medicago/Sinorhizobium ont montré dans les nodules la forte présence d'une grande diversité de peptides appelés NCR qui sont similaires aux peptides antimicrobiens (AMP) impliqués dans l'immunité innée. Ces NCR sont responsables du maintien de l'homéostasie entre les cellules hôtes et la forte population bactérienne qu'elles contiennent. Bien que certains NCR sont de vrais AMP, capable de tuer des bactéries in vitro, dans les nodules ils induisent plutôt une différenciation terminale caractérisée par une élongation cellulaire, une amplification du génome, une perméabilité membranaire et une perte des capacités de division de la bactérie. Néanmoins le mode d'action des NCR reste à élucider. Au cours de ma thèse j'ai participé à la caractérisation des processus de différenciation dans le modèle Aeschynomene, une légumineuse tropicale, Bradyrhizobium.Dans un premier temps, une nouvelle classe de NCR a été identifiée chez différentes espèces d'Aeschynomene. Ces NCR sont responsables de la différenciation des Bradyrhizobium via un processus similaire à celui décrit chez Medicago. Ces résultats suggèrent une évolution convergente des processus de différenciation chez les Dalbergioïdes (Aeschynomene) et le clade des IRLC (Medicago).Ensuite, pour identifier les fonctions bactériennes requises lors de la différenciation, j'ai criblé 53 mutants Tn5 d'Aeschynomene indica fix- . Huit gènes bactériens dont la mutation inhibe ou affecte le processus de différenciation ont été identifiés. Parmi eux, je me suis focalisé sur la DD-CPase une enzyme de modification du peptidoglycane et sur 2 gènes impliqués dans l'homéostasie du phosphate.La caractérisation du gène DD-CPase1 a permis de démontrer que le remodelage du peptidoglycane est requis pour une différenciation correcte des bactéroïdes chez les plantes hôtes qui produisent des NCR, en général, et chez Aeschynomene en particulier. Ces résultats suggèrent une interaction possible entre DD-CPase1 et des NCR conduisant à l'endoréplication des bactéroïdes.Enfin, j'ai étudié les propriétés physiologiques et symbiotiques des mutants pstC et pstB. Les mutants Tn5 des gènes pstC et pstB de la souche ORS285 de Bradyrhizobium sont sévèrement affectés par la carence en phosphate en culture pure et leurs propriétés symbiotiques (différenciation, réduction de l'azote) sont fortement réduites. Des analyses fonctionnelles plus approfondies de l'opéron Pst devraient permettre une meilleure compréhension du lien entre l'homéostasie du phosphate et l'efficience symbiotique dans l'interaction Aeschynomene-Bradyrhizobium.Mes travaux ont permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant que le modus operandi impliquant des peptides dérivés de l'immunité innée utilisée par certaines légumineuses pour maintenir leur population bactérienne intracellulaire sous contrôle est plus répandue et ancienne qu'on ne le pensait et a été utilisée par l'évolution à plusieurs reprises. De plus différentes cibles bactériennes pouvant participer au processus de différenciation ont également été identifiées. / The legume species are able to form symbiotic organs, the nodules, that house soil bacteria called rhizobia. Within these nodules intracellular rhizobia differentiate into bacteroids, which are able to reduce atmospheric dinitrogen to ammonium for the benefit of the plants. In counterpart, the plants provide carbon sources to the bacteria. Recent studies on symbiotic model Medicago-Sinorhizobium showed that the nodules of M. truncatula produce a massive diversity of peptides called NCRs, which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are responsible in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs, which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroids characterized by cell elongation, genome amplification, membrane permeability and loss of cell division capacity. However, the action mode of NCRs is still an open question. During my PhD thesis I focused on the identification of plant and bacterial functions required for bacteroid differentiation in the Aeschynomene-Bradyrhizobium model.Firstly, a new class of cysteine rich peptides (NCR-like) was identified in tropical aquatic legumes of the Aeschynomene genus, which belong to the Dalbergioid clade. These peptides govern terminal bacteroid differentiation of photosynthetic Bradyrhizobium spp. This mechanism is similar to the one previously described in Medicago suggesting that the endosymbiont differentiation in Dalbergioid and ILRC legumes is convergently evolved.Secondly, in order to identify the bacterial functions involved in bacteroid differentiation, I screened 53 fix- Tn5 mutants of the ORS278 strain on Aeschynomene indica. This screening allowed identify 8 bacterial genes, which inhibit or disorder the bacteroid differentiation. Among these identified genes, I focused on DD-CPase encoding a peptidoglycan-modifying enzyme and two genes pstC and pstB belonging to Pst-system.The characterization of DD-CPase gene demonstrated that the remodeling peptidoglycan enzyme, DD-CPase1, of Bradyrhizobium is required for normal bacteroid differentiation in host legumes that produce NCRs, in general, and in Aeschynomene spp., in particular. This prompts a possibility of direct interaction of DD-CPase1 with NCRs leading to endoreduplication of the bacteroids.Finally, I have investigated the physiological and symbiotic properties of different mutants of pstC and pstB genes. The Tn5 mutants of pstC and pstB genes of Bradyrhizobium sp. strain ORS278 severely affected symbiosis on A. indica and A. evenia. Further functional studies on pst-operon will provide deeper understanding the correlation between phosphate homeostasis and nitrogen fixation efficiency in Aeschynomene-Bradyrhizobium symbiosis.This study broadens our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.

Microbial Source Tracking (MST) is a field of study that attempts to identify the source of fecal contamination in waterways in order to assist with development of remediation strategies. Biologists at Cal Poly Center for Applications in Biotechnology (CAB) are developing a new MST method using microbes from the genus Bacteroides. Bacteroides species are host-specific microorganisms that can theoretically be used to trace back to a single host species. After fecal samples are collected, biologists use Next-Generation Sequencing (NGS) techniques to obtain only the genetic sequences of microorganisms belonging to the phylum Bacteroidetes. Investigators hypothesize that similar sequences belong to the same phlyogenetic group (i.e., the same genus) and can therefore be computationally clustered. Each cluster of related sequences, typically 97% similar, is called an Operational Taxonomic Unit (OTU). Theoretically, an OTU acts as a molecular signature that can be traced back to a specific host genus. This thesis presents LOTUS, the Library of OTUs, a web-based computational tool for the preliminary investigation of the use of the Bacteroides OTU library as an MST method. This work discusses the four contributions of LOTUS: a database design which accurately models OTUs and the underlying relationships necessary for source tracking, a pipeline to create OTUs from raw sequencing reads, a method of assigning taxonomy to OTUs, and a web-based user interface. In preliminary testing for a reference library of twelve samples, LOTUS produced 1,431 OTUs, of which 891 were single-source (OTUs derived from sequences from a single host species). Using these OTUs, LOTUS was able to accurately taxonomically match four of five unknown test samples, showing promise for using OTUs as an MST method.

No description available.

E. coli and Enterococcus were used to determine the fate and survival of fecal indicator bacteria (FIB) in sand and sediments. The microbial source tracking (MST) methods antibiotic resistance analysis (ARA), Bacteroides human-specific primer test, and fluorometry were compared against the FIBs to determine how reliable each method was in detecting the presence of human fecal contamination. Two phases (Summer 2009 and 2010) were evaluated based on the type of contamination event. A combined sewage overflow (CSO) event was simulated in Phase I, where large amounts of influent were added to sand and bay water columns over 1 to 4 days. In 2010, a low volume sewage leak was simulated in which smaller doses of influent were added to sand and bay water columns over a period of 5 to 15 days. Within each of the phases, both non- and re-circulated columns were also evaluated. Evaluation of FIB survival indicated that Enterococcus was able to stabilize and re-grow in the water and at the sediment/water interface within the Phase I non-circulated columns. E. coli was unable to re-grow and/or stabilize within any environment. Comparisons between the ARA and the FIBs revealed a large majority of isolates identified as coming from either bird or wildlife sources. Human sources were identified but at much lower concentrations than expected. Bacteroides results indicated strong relationships between the increase of FIB concentrations and the presence of the human-specific Bacteroides. Fluorometry results did not indicate any relationship with the FIBs. Unexpectedly, fluorometry readings increased as time progressed indicating that another compound was present that fluoresced at the same wavelength as optical brighteners (OBs). This project was one of the first to study the differences related to two different pollution events (CSO vs. sewage leak) while also evaluating what happens to pollution as it settles into the sediment. It was also unique because it compared bacterial (ARA), molecular (Bacteroides), and chemical (fluorometry) MST methods. / Master of Science

Notre équipe vient de découvrir une méthode originale d’obtention d’extraits de Bacteroides thetaiotaomicron (E) qui préserve sa viabilité. Après culture anaérobie de ce commensal intestinal en milieu gélosé pauvre en facteurs de croissance, puis exposition à l’air, la bactérie semble posséder et générer dans E tout l’équipement de détoxication des espèces réactives de l’oxygène in vitro. Il laisse alors augurer d’un pouvoir thérapeutique à visée anti-inflammatoire.Objectifs et méthodes : Le but est d’abord de caractériser E, aux plans glucidique, lipidique et protéique. Dans ce dernier cas, il s’agit de séparer les protéines produites par la bactérie vivante et contenues dans E par électrophorèse bidimensionnelle et de les identifier par la technique des cartes peptidiques massiques. Les gels (n≥6) sont traités statistiquement (PDQuest®, Bio-Rad). Pour mieux localiser ces protéines dans la bactérie, elles sont comparées avec celles obtenues par destruction de B. thetaiotaomicron et identifiées dans la fraction cellulaire relative à la membrane bactérienne externe. Un travail de microscopie électronique est aussi entrepris pour visualiser les éventuels évènements intervenant pendant l’extraction.Le but est alors de vérifier, in vitro, l’effet antioxydant de l’extrait bactérien standardisé et d’en contrôler l’innocuité en modèles cellulaires utilisant le granulocyte neutrophile. L’effet thérapeutique anti-inflammatoire est ensuite recherché chez l’animal. L’action de E est d’abord évaluée en modèle murin d’inflammation cutanée auriculaire induite par dépôt de chlorure de benzalkonium, sous anesthésie générale. Des témoins positifs et négatifs de traitement et d’autres ne subissant pas d’irritation sont testés en parallèle. L’épaisseur des oreilles est mesurée toutes les heures pendant 5 h et des coupes histologiques d’oreilles, effectuées au bout de 2 h chez certains animaux. Deux colorations différentes permettent alors d’évaluer la quantité de mastocytes dégranulant localement.L’action de E, administré par voie intra-rectale (IR), est ensuite testée chez des souris subissant les premières phases d’un processus inflammatoire, en modèle de colite aiguë. Celle-ci est induite per os par du dextran sulfate sodium (DSS) ; elle évolue sur 8 jours. Sont considérés en parallèle des témoins positifs et négatifs de traitement et d’autres ne subissant pas de colite. Des scores cliniques et des scores histologiques de sévérité sont établis tous les jours de l’expérience. Des marqueurs de l’inflammation sont suivis dans les tissus murins après autopsie des animaux. [...] / Our team had discovered a new method to obtain extracts of Bacteroides thetaiotaomicron (E) which preserved its viability. This intestinal symbiont was anaerobically grown on an agar medium poorly supplemented in growth factors. After exposure to air, the bacterium seemed to possess and generate in E all the equipment able in vitro to detoxify reactive oxygen species. It let us expect a therapeutic power referred to anti-inflammatory properties.Objectives and methods: The aim was first to characterize E, in terms of carbohydrates, lipids and proteins. To achieve this last-mentioned goal, proteins contained in E coming from living bacteria were separated by two-dimensional electrophoresis and identified by the peptide mass fingerprinting technique. The gels (n ≥ 6) were statistically analyzed (PDQuest®, Bio-Rad). To find the origin of these proteins in bacteria, they were compared with those obtained by destruction of B. thetaiotaomicron (BT) and identified in the cell fraction containing the bacterial outer membrane proteins. Electron microscopy work was also undertaken to visualize any event occurring during extraction.The antioxidative effect of standardized E extracts was checked in vitro. E safety was also controlled in cell models using polymorphonuclear neutrophils. An E anti-inflammatory effect was then searched in animal models. E was first evaluated using a skin irritation mouse model. Inflammation was induced by benzalkonium chloride on ears of anesthetized mice. Positive and negative controls were treated in parallel. The ear thickness was measured every hour for 5 h and histological ear sections were performed after 2h for some animals. Two different staining methods enabled the enumeration of degranulating mast cells in ear sections.The effect of the bacterial extract was next tested locally by intrarectal (IR) instillations in mice undergoing the early stages of inflammation in a dextran sodium sulfate (DSS)-induced colitis. This acute model evolved over 8 days. In parallel, positive and negative animal controls underwent or not the colitis and were treated or not. Clinical and colonic histological severity scores were daily determined. Inflammation markers were measured in mouse colonic tissues after animal autopsy. [...]

Background It is debated whether a low total diversity of the gut microbiota in early childhood is more important than an altered prevalence of particular bacterial species for the increasing incidence of allergic disease. The advent of powerful, cultivation-free molecular methods makes it possible to characterize the total microbiome down to the genus level in large cohorts. Objective We sought to assess microbial diversity and characterize the dominant bacteria in stool during the first year of life in relation to atopic eczema development. Methods Microbial diversity and composition were analyzed with barcoded 16S rDNA 454-pyrosequencing in stool samples at 1 week, 1 month, and 12 months of age in 20 infants with IgE-associated eczema and 20 infants without any allergic manifestation until 2 years of age (ClinicalTrials.gov ID NCT01285830). Results Infants with IgE-associated eczema had a lower diversity of the total microbiota at 1 month (P = .004) and a lower diversity of the bacterial phylum Bacteroidetes and the genus Bacteroides at 1 month (P = .02 and P = .01) and the phylum Proteobacteria at 12 months of age (P = .02). The microbiota was less uniform at 1 month than at 12 months of age, with a high interindividual variability. At 12 months, when the microbiota had stabilized, Proteobacteria, comprising gram-negative organisms, were more abundant in infants without allergic manifestation (Empirical Analysis of Digital Gene Expression in R [edgeR] test: P = .008, q = 0.02). Conclusion Low intestinal microbial diversity during the first month of life was associated with subsequent atopic eczema. / <p>Funding Agencies|BioGaia AB, Stockholm, Sweden||Ekhaga Foundation, the Heart and Lung foundation||Research Council for the South-East Sweden|F2000-106|Olle Engqvist Foundation||Swedish Asthma and Allergy Association||Swedish Research Council||University Hospital of Linkoping||Soderberg Foundation||Vardal Foundation for Health Care Science and Allergy Research, Sweden||BioGaia AB||</p>

Der Nachweis von Fäkalindikatorbakterien (wie z. B. E. coli oder intestinale Enterokokken) gibt zwar einen Hinweis auf eine fäkale Belastung im Gewässer, erlaubt jedoch keinen Rückschluss auf den Ursprung. Im Rahmen dieser Dissertation wurden verschiedene Methoden zur Identifizierung von fäkalen Eintragsquellen (Microbial Soruce Tracking, MST) geprüft. Hierzu zählen Kultur-basierte Verfahren, wie der Nachweis von Toxingenen in E. coli-Isolaten, und Kultur-unabhängige Methoden wie die Denaturierende-Gradienten-Gelelektrophorese oder der Nachweis von DNA-Abschnitten, die spezifisch für menschlichen oder tierischen Kot sind. Die Untersuchung von E. coli-Isolaten aus der aquatischen Umwelt auf Toxingene erwies sich in Hinblick auf die Identifizierung von fäkalen Eintragsquellen als nicht zielführend. Problematisch war neben dem hohen Zeitaufwand vor allem das seltene Vorkommen dieser Virulenzfaktoren im untersuchten Gewässer. Enteropathogene E. coli-Bakterien werden nur von infizierten Wirten abgegeben, daher ist der Nachweis der Virulenzgene in Gewässern stark eingeschränkt. Im nächsten Schritt wurden Datenbank-unabhängige Verfahren für die Untersuchungen im Einzugsgebietsmaßstab etabliert. Methoden zur spezifischen Detektion von fäkalen Einträgen durch Menschen, Rinder (Wiederkäuer), Schweine, Hunde, Schafe, Hühner und Pferde standen anschließend für die Identifizierung von fäkalen Einträgen in verschiedenen Einzugsgebieten zur Verfügung. Die Ergebnisse der Untersuchungen belegen, dass mit den etablierten MST-Verfahren sehr empfindliche und spezifische Werkzeuge für die Identifizierung von fäkalen Einträgen im Einzugsgebietsmaßstab zur Verfügung stehen. Durch die Untersuchung von Wasserproben auf das Vorkommen von MST-Markern konnten Aussagen über Auftreten und Stärke fäkaler Belastungen in städtisch und ländlich geprägten Einzugsgebieten gemacht werden. Kontaminationsquellen wurden identifiziert und Vorschläge für gezielte Management¬maßnahmen im Einzugsgebiet abgeleitet. Des Weiteren wurde die MST-Verfahren erfolgreich dazu genutzt Erkenntnisse über den Ursprung von Antibiotikaresistenzgenen im Tai-See (China) zu gewinnen. Die Ergebnisse dieser Arbeit zeigen, dass die MST-Methoden basierend auf wirtsspezifischen Markern für den Einsatz in der Praxis geeignet sind. Zusammen mit der Betrachtung der örtlichen Gegebenheiten ist MST ein hilfreiches und kostengünstiges Werkzeug bei der Ursachen-forschung und der Ermittlung der Herkunft fäkaler Belastungen in der aquatischen Umwelt. Damit trägt es wesentlich zu einer zielorientierten Bewirtschaftung des Gewässers bei.:ABKÜRZUNGSVERZEICHNIS ABBILDUNGSVERZEICHNIS TABELLENVERZEICHNIS FORMELVERZEICHNIS 1. HINTERGRUND 1.1 INDIKATORORGANISMEN 1.2 SOURCE TRACKING-METHODEN 1.2.1 Datenbank-abhängige Methoden 1.2.2 Datenbank-unabhängige Methoden 1.2.3 Source Tracking mit chemischen Substanzen 1.3 ANTIBIOTIKARESISTENZEN IN DER UMWELT 2. ZIELSETZUNG DER ARBEIT 3. MATERIAL UND METHODEN 3.1 MONITORING MIKROBIOLOGISCHER PARAMETER – KULTURVERFAHREN 3.1.1 Coliforme Bakterien und Escherichia coli 3.1.2 Enterokokken 3.1.3 Clostridium perfringens-Sporen 3.2 MOLEKULARBIOLOGISCHE ANALYTIK 3.2.1 PCR-Untersuchung von E. coli-Isolaten auf Virulenzgene 3.2.2 Molekularbiologische Untersuchung von Wasserproben 3.3 UNTERSUCHUNGEN ZUR ERFASSUNG DER CHARAKTERISTIKA DER KULTUR-UNABHÄNGIGEN MICROBIAL SOURCE TRACKING-VERFAHREN 3.3.1 Nachweisempfindlichkeit 3.3.2 Spezifität und Sensitivität 3.3.3 Charakterisierung möglicher Eintragsquellen 3.3.4 Untersuchungen zur Stabilität von Microbial Source Tracking-Markern 3.4 STATISTISCHE AUSWERTUNG 4. BESCHREIBUNG DER UNTERSUCHUNGSGEBIETE 4.1 RHEIN 4.2 GALLUSQUELLE 4.3 EINZUGSGEBIETE DER BERLINER WASSERBETRIEBE 4.3.1 Wasserwerk Tiefwerder 4.3.2 Wasserwerk Kaulsdorf 4.4 EINZUGSGEBIETE DER WSW ENERGIE & WASSER AG 4.4.1 Talsperre Herbringhausen 4.4.2 Talsperre Kerspe 4.5 TAI-SEE 4.6 VERGLEICH DER UNTERSUCHUNGSGEBIETE 61 5. ERGEBNISSE UND DISKUSSION 63 5.1 NACHWEIS VON VIRULENZGENEN ALS MICROBIAL SOURCE TRACKING-WERKZEUG 5.2 DATENBANK-UNABHÄNGIGE UND KULTUR-UNABHÄNGIGE MICROBIAL SOURCE TRACKING-VERFAHREN 5.2.1 Etablierung Datenbank-unabhängiger und Kultur-unabhängiger Microbial Source Tracking-Verfahren 5.2.2 Nachweisempfindlichkeit 5.2.3 Spezifität und Sensitivität der Marker 5.2.4 Charakterisierung möglicher Eintragsquellen 5.2.5 Stabilität von Microbial Source Tracking-Markern 5.3 UNTERSUCHUNG IM EINZUGSGEBIETSMAßSTAB 5.3.1 Gallusquelle 5.3.2 Wasserwerke Tiefwerder und Kaulsdorf 5.3.3 Talsperren Herbringhausen und Kerspe 5.4 EINSATZ VON MICROBIAL SOURCE TRACKING-METHODEN ZUR IDENTIFIZIERUNG DER HERKUNFT VON ANTIBIOTIKARESISTENZEN 6. ZUSAMMENFASSUNG UND AUSBLICK 7. EIGENE VERÖFFENTLICHUNGEN 8. FINANZIELLE FÖRDERUNG 9. LITERATURVERZEICHNIS 10. ANHANG / Although evidence of fecal indicator bacteria (such as E. coli or intestinal enterococci) indicates fecal contamination in the aquatic environment, it does not allow any conclusion regarding the origin of the pollution. With the use of so-called microbial source tracking methods it is possible to trace the origin of such contaminations. In this dissertation, various microbial source tracking methods were tested. These include culture-based methods such as the detection of toxin genes in E. coli isolates, and culture-independent methods such as denaturing gradient gel electrophoresis or the detection of defined DNA sections specific for human or animal feces using quantitative real-time PCR. Analysis of E. coli isolates from the aquatic environment on toxin genes proved to be ineffective in identifying fecal sources of input. In addition to the high effort of time, the problem was the rare occurrence of these virulence factors in the examined water. Enteropathogenic E. coli bacteria carry virulence genes which are only released from infected hosts. Therefore, the detection of virulence genes in weakly to moderately contaminated waters is severely limited. In the next step, culture-independent PCR procedures were established. Afterwards, methods for the specific detection of fecal entries by humans, cattle (ruminants), pigs, dogs, sheep, chickens and horses were available for the identification of fecal entries in various catchment areas. The results of these investigations show that established MST methods provide very sensitive and specific tools for the identification of fecal entries at the catchment scale. Investigation using culture-independent methods provided information on the origin and severity of fecal pollutions in urban and rural catchment areas. Origins of contaminations were identified and concrete recommendations for management action plans in the catchment area were derived. Overall, the results show the potential and the suitability for practical application of molecular biological microbial source tracking methods. Furthermore, the MST methods have been successfully used to gain knowledge about the origin of antibiotic resistance genes in Tai Lake (China). The results of this thesis show that MST methods based on host-specific markers are suitable for practical use. Together with the consideration of local conditions, MST is a helpful and cost-effective tool for causal research and the determination of the origin of fecal contamination in the aquatic environment. Thus it contributes significantly to a goal-oriented management of the water body.:ABKÜRZUNGSVERZEICHNIS ABBILDUNGSVERZEICHNIS TABELLENVERZEICHNIS FORMELVERZEICHNIS 1. HINTERGRUND 1.1 INDIKATORORGANISMEN 1.2 SOURCE TRACKING-METHODEN 1.2.1 Datenbank-abhängige Methoden 1.2.2 Datenbank-unabhängige Methoden 1.2.3 Source Tracking mit chemischen Substanzen 1.3 ANTIBIOTIKARESISTENZEN IN DER UMWELT 2. ZIELSETZUNG DER ARBEIT 3. MATERIAL UND METHODEN 3.1 MONITORING MIKROBIOLOGISCHER PARAMETER – KULTURVERFAHREN 3.1.1 Coliforme Bakterien und Escherichia coli 3.1.2 Enterokokken 3.1.3 Clostridium perfringens-Sporen 3.2 MOLEKULARBIOLOGISCHE ANALYTIK 3.2.1 PCR-Untersuchung von E. coli-Isolaten auf Virulenzgene 3.2.2 Molekularbiologische Untersuchung von Wasserproben 3.3 UNTERSUCHUNGEN ZUR ERFASSUNG DER CHARAKTERISTIKA DER KULTUR-UNABHÄNGIGEN MICROBIAL SOURCE TRACKING-VERFAHREN 3.3.1 Nachweisempfindlichkeit 3.3.2 Spezifität und Sensitivität 3.3.3 Charakterisierung möglicher Eintragsquellen 3.3.4 Untersuchungen zur Stabilität von Microbial Source Tracking-Markern 3.4 STATISTISCHE AUSWERTUNG 4. BESCHREIBUNG DER UNTERSUCHUNGSGEBIETE 4.1 RHEIN 4.2 GALLUSQUELLE 4.3 EINZUGSGEBIETE DER BERLINER WASSERBETRIEBE 4.3.1 Wasserwerk Tiefwerder 4.3.2 Wasserwerk Kaulsdorf 4.4 EINZUGSGEBIETE DER WSW ENERGIE & WASSER AG 4.4.1 Talsperre Herbringhausen 4.4.2 Talsperre Kerspe 4.5 TAI-SEE 4.6 VERGLEICH DER UNTERSUCHUNGSGEBIETE 61 5. ERGEBNISSE UND DISKUSSION 63 5.1 NACHWEIS VON VIRULENZGENEN ALS MICROBIAL SOURCE TRACKING-WERKZEUG 5.2 DATENBANK-UNABHÄNGIGE UND KULTUR-UNABHÄNGIGE MICROBIAL SOURCE TRACKING-VERFAHREN 5.2.1 Etablierung Datenbank-unabhängiger und Kultur-unabhängiger Microbial Source Tracking-Verfahren 5.2.2 Nachweisempfindlichkeit 5.2.3 Spezifität und Sensitivität der Marker 5.2.4 Charakterisierung möglicher Eintragsquellen 5.2.5 Stabilität von Microbial Source Tracking-Markern 5.3 UNTERSUCHUNG IM EINZUGSGEBIETSMAßSTAB 5.3.1 Gallusquelle 5.3.2 Wasserwerke Tiefwerder und Kaulsdorf 5.3.3 Talsperren Herbringhausen und Kerspe 5.4 EINSATZ VON MICROBIAL SOURCE TRACKING-METHODEN ZUR IDENTIFIZIERUNG DER HERKUNFT VON ANTIBIOTIKARESISTENZEN 6. ZUSAMMENFASSUNG UND AUSBLICK 7. EIGENE VERÖFFENTLICHUNGEN 8. FINANZIELLE FÖRDERUNG 9. LITERATURVERZEICHNIS 10. ANHANG

No description available.