Entwicklung und Anwendung molekularbiologischer Methoden zur Identifizierung fäkaler Eintragsquellen in Rohwasser (Microbial Source Tracking)

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. ANHANGAlthough 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. ANHAN

Functional Fruits Through Metabolic Engineering

Metabolic engineering is a main focus of many plant biotechnology programs that look for the production of novel plant varieties with improved human health benefits. Among the most interesting goals are those that are focused in the production of functional fruits. A fruit can be considered as functional if it produces additional benefits to human health and well-being, beyond nutrition. Fruits that present higher levels of beneficial compounds such as essential vitamins, antioxidants, and phytochemicals can be considered as functional as those compounds have long-term benefits in reducing the occurrence of certain diseases. Through the expression, silencing, or mutagenesis strategies, many functional fruit crops have been produced during the last 40 years. Novel plants produce higher amount of carotenoids, antocyanins, and folic acid in their fruits, as well as higher color, sweetness, flavor, and aroma. The improvement of postharvest and resistance to biotic and abiotic stress in commercial plants has been also enhanced as it can led to a better fruit production. Taken together, this chapter will present a revision of the main fruits that have been improved by means of metabolic engineering within the framework of functional foods and super foods

Therapeutic drug monitoring of rivastigmine and donepezil under consideration of CYP2D6 genotype-dependent metabolism of donepezil

Background: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil. Objective: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP2D6 genotype or gene dose-dependent metabolism of donepezil. Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose. Results: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (beta=0.465;p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose. Conclusion: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism

Serum Concentrations of Cholinesterase Inhibitors in Patients With Alzheimer's Dementia Are Frequently Below the Recommended Levels

Background: Acetylcholinesterase inhibitors (AChE-I) are recommended for the treatment of cognitive symptoms but also of behavioral and psychological symptoms in dementia. They are widely used not only in Alzheimer's disease, but also in other forms of dementia. Efficacy of treatment might depend on serum concentration of the respective AChE-I. Objective: In patients with mild to moderate Alzheimer's dementia, we measured serum concentrations of hepatically metabolized donepezil and renally excreted rivastigmine and investigated possible modifiers. Additionally, we looked at correlations between serum concentrations and efficacy for both drugs. Methods: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR). Allele specific PCR were performed to determine CYP2D6 genotype and gene dose. Clinical efficacy was assessed by changes of the subtest wordlist delayed recall of the Consortium to Establish a Registry for Alzheimer's Disease-Neuropsychological Assessment Battery (CERAD-NAB). Results: Sixty-seven patients treated with a stable dosage of donepezil 10 mg (n=41) or rivastigmine 9.5 mg (n=26) were included. Mean serum concentration of donepezil and rivastigmine were 41.2 and 6.5 ng/ml, respectively. Serum concentrations were below the recommended range in 73% of the subjects in the donepezil group and in 65% of the participants in the rivastigmine group. When applying a dose-related reference, ranges 63% of patients in the donepezil group and 32% in the rivastigmine group had concentrations below the expected range. Gene dose, sex, and duration of treatment significantly predicted donepezil serum concentration (p=0.046, p=0.001, p=0.030 respectively). Only for rivastigmine did the serum concentration significantly contribute to the regression model predicting changes on the subtest word list delayed recall (β=0.472; p=0.019). Conclusions: Serum concentrations of about two thirds of the patients were below the recommended range. When not looking at absolute values but at the dose-related reference ranges, these numbers improved but still 32%, respectively 63% of patients had low serum concentrations. High serum concentrations of rivastigmine predicted clinical response to cognition. Therapeutic drug monitoring might help to identify the cause of poor clinical response to cognition and behavioral and psychological symptoms in patients with AChE-I treatment

Stakeholders' perspectives on the operationalisation of the ecosystem service concept : Results from 27 case studies

The ecosystem service (ES) concept is becoming mainstream in policy and planning, but operational influence on practice is seldom reported. Here, we report the practitioners' perspectives on the practical implementation of the ES concept in 27 case studies. A standardised anonymous survey (n = 246), was used, focusing on the science-practice interaction process, perceived impact and expected use of the case study assessments. Operationalisation of the concept was shown to achieve a gradual change in practices: 13% of the case studies reported a change in action (e.g. management or policy change), and a further 40% anticipated that a change would result from the work. To a large extent the impact was attributed to a well conducted science-practice interaction process (>70%). The main reported advantages of the concept included: increased concept awareness and communication; enhanced participation and collaboration; production of comprehensive science-based knowledge; and production of spatially referenced knowledge for input to planning (91% indicated they had acquired new knowledge). The limitations were mostly case-specific and centred on methodology, data, and challenges with result implementation. The survey highlighted the crucial role of communication, participation and collaboration across different stakeholders, to implement the ES concept and enhance the democratisation of nature and landscape planning. (C) 2017 Published by Elsevier B.V.Peer reviewe

ARTEFACTS: How do we want to deal with the future of our one and only planet?

The European Commission’s Science and Knowledge Service, the Joint Research Centre (JRC), decided to try working hand-in-hand with leading European science centres and museums. Behind this decision was the idea that the JRC could better support EU Institutions in engaging with the European public. The fact that European Union policies are firmly based on scientific evidence is a strong message which the JRC is uniquely able to illustrate. Such a collaboration would not only provide a platform to explain the benefits of EU policies to our daily lives but also provide an opportunity for European citizens to engage by taking a more active part in the EU policy making process for the future. A PILOT PROGRAMME To test the idea, the JRC launched an experimental programme to work with science museums: a perfect partner for three compelling reasons. Firstly, they attract a large and growing number of visitors. Leading science museums in Europe have typically 500 000 visitors per year. Furthermore, they are based in large European cities and attract local visitors as well as tourists from across Europe and beyond. The second reason for working with museums is that they have mastered the art of how to communicate key elements of sophisticated arguments across to the public and making complex topics of public interest readily accessible. That is a high-value added skill and a crucial part of the valorisation of public-funded research, never to be underestimated. Finally museums are, at present, undergoing something of a renaissance. Museums today are vibrant environments offering new techniques and technologies to both inform and entertain, and attract visitors of all demographics.JRC.H.2-Knowledge Management Methodologies, Communities and Disseminatio

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead