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Erlangung der Würde eines Doktors der Philosophie

vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät

der Universität Basel


Melanie Wicki

aus Basel

Basel, 2011

Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel

edoc.unibas.ch Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 2.5 Schweiz“ lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licences/by-nc-nd/2.5/ch eingesehen werden.

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Marcel Tanner, Prof. Dr. Alfred Metzler, Dr. Richard Felleisen, Dr. Adrian Auckenthaler, Dr. Andreas Baumgartner Basel, den 21.9.2010 Prof. Dr. Martin Spiess Dekan Table of contents 1 Table of contents TABLE OF CONTENTS 1 ACKNOWLEGEMENTS 4 ABBREVIATIONS 6 SUMMARY 7 ZUSAMMENFASSUNG 10







1.5 MICROBIAL SOURCE TRACKING: STATE OF THE SCIENCE 24 Genotypic or phenotypic methods 25 Chemical tests to differentiate sources of contamination 27 Comparison of two or more bacterial population ratios 28 Detection of biological targets indicative for the source 28

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Thanks to the framework of the Swiss Tropical and Public Health Institude (TPH) and the Federal Office of Public Health (FOPH) I had the opportunity to undertake the present thesis.

I would therefore express my gratitude to Prof. Dr. Marcel Tanner (Director) and the executive board of the FOPH who made this collaboration possible. This study was conducted in the laboratories of the Consumer Protection Directorate of the FOPH, and supported by research grant FOPH/07.006623. I would like to thank the FOPH for providing the infrastructure and for financial support of this project. Many people were involved in different ways in this work and I wish to express my sincere gratitude to some of them.

I would like to thank Prof. Dr. Marcel Tanner, faculty representative at Swiss TPH, for support, inspiration and productive discussions during the time of this thesis. I am indebted to my supervisors Dr. Richard Felleisen and Dr. Andreas Baumgartner at FOPH, for continuous supervision and confidence in my work as well as for support and discussions during the time of this thesis. Many thanks are also addressed to Adrian Auckenthaler (Office of Environmental Protection and Energy, Basel-Landschaft) for his interest in the research and productive discussions. I am grateful to Prof. Dr. Alfred Metzler (Institute of Virology, University of Zurich) for his interest in this study and contribution as coreferee.

My gratitude also goes to Fatma Karabulut for the excellent collaboration during field studies and for contribution of valuable data during her MSc thesis. I thank the entire laboratory team at FOPH for the great working atmosphere and for their interest in this thesis. A special thank

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I am very thankful to Maite Muniesa and Anicet R. Blanch (Department of Microbiology, University of Barcelona, Spain) for scientific advice and technical instruction during a stage in Barcelona and for providing reference material.

Moreover, I thank the microbiology team at the Cantonal Laboratory Basel-Landschaft for their support and for their interest in this thesis. I also extend my thanks to Dominik Bänninger for support in data analysis. I thank Dieter Schnell and André Dettwiler, the staff of the waste water treatment plants and the slaughterhouses and all farmers for their cooperation and facilitation of sampling. A special thank goes to Giselle Weiss and Elaine Littler for a critical reading of several parts of the manuscript.

Finally, I am much obliged to Basil, my family and friends for their support, their interest in

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SUMMARY Waterborne disease outbreaks caused by various pathogens have been reported from all over the world. They also play an important role in industrialized countries, despite good sanitation and high standards of hygiene. In Switzerland, waterborne disease outbreaks are uncommon but faecal contamination of springs occurs quite frequently in some areas. As a consequence, microbiological quality of drinking water is not constantly sufficient and there is potential for improvement. Based on the hygiene ordinance, cantonal authorities contribute with control actions to the supply of safe water to consumers. Microbiological quality assessment based on current drinking water guidelines do not discriminate between human and animal sources of contamination. However, methods for discriminating between human and animal faecal contamination are needed to identify sources of contamination and to provide specific scientific data as a basis for risk management measures and for the implementation of remedial interventions.

The main goal of this thesis was to establish approaches for the identification of human and animal faecal contamination in surface and drinking water and to develop concepts for future investigations. A number of methods can be used to help identify sources of faecal contamination in water. The general concept is referred to as microbial source tracking (MST). Different methodologies for MST were previously used mainly for analysis of faecal samples and recreational waters such as streams, lakes and beaches. Prior to this study, MST was not applied in Switzerland.

In the present thesis, source tracking approaches never before used in MST as well as methods previously described in scientific literature were tested for their potential to indicate

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Two bacterial strains (Bacteroides thetaiotaomicron ARABA 84 and B. fragilis ARABA 19) specific for bacteriophages present in human faecal contamination and three strains (B. caccae RBA 63 and RBA 64 as well as B. fragilis KBA 60) specific for bacteriophages indicating animal faecal contamination were isolated from human wastewater and animal faecal specimens. Thereafter they were used to determine the source of surface and spring water faecal contamination.

In addition, the potential of Streptococcus agalactiae was tested as a new human MST indicator. Different methods of detecting these bacteria in domestic and slaughterhouse wastewater were compared. Three DNA extraction methods and five polymerase chain reaction (PCR) assays were tested to identify the most suitable combination. The most sensitive detection method, a LightCycler real-time PCR assay, detected S. agalactiae in human wastewater but not in animal samples and showed that this bacteria is potentially useful as human MST indicator.

Rhodococcus coprophilus was previously used in MST to indicate animal faecal contamination in water. For the detection of this organism, a culture-dependent method and a conventional as well as a TaqMan real-time PCR assay were previously published. The evaluation of these existing approaches, however, did not reveal any satisfactory result. A novel LightCycler real-time PCR assay was therefore designed and validated in the course of the present thesis. Compared with previously used assays, this new molecular approach showed advantages such as improved sensitivity and specificity and was much faster than the

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Sorbitol-fermenting bifidobacteria and phages infecting the Bacteroides host strains GA-17 and GB-124 were previously described to indicate human faecal contamination. Together with the newly developed and validated approaches, these established MST indicators were included in a comparative study and their potential to indicate human or animal faecal contamination in Swiss surface and spring water was tested. Based upon this assessment, sorbitol-fermenting bifidobacteria and phages of the human host strains B. thetaiotaomicron ARABA 84 and B. ovatus GB-124 can be recommended for detecting human faecal contamination in Swiss surface and drinking water.

The above-mentioned approaches give evidence of the faecal input sites of contamination. To localize possible faecal input sites, they can be followed by an additional approach described in this thesis. This approach is based on the screening of multiresistant Escherichia coli and on the characterisation of selected isolates by antibiotic resistance profiles and pulsed field gel electrophoresis in order to identify identical strains. Thereby, a well equipped toolbox could be provided not only to discriminate sources of faecal contamination but also to localize possible faecal input sites.

By offering this toolbox, the present thesis provides the basis to bridge the gap from innovation to application: Recommendations based upon the concepts and protocols for analysis of MST indictors developed in this study will be elaborated and distributed to water

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Pathogene Mikroorganismen, die durch Wasser übertragen werden, führen weltweit häufig zu Krankheitsausbrüchen. Auch in Industrieländern, welche über gute sanitäre Anlagen verfügen und hohe Hygienestandards aufweisen, sind solche Übertragungswege von Bedeutung. In der Schweiz treten durch Trinkwasser verursachte Krankheitsausbrüche selten auf, dennoch sind in einigen Gegenden mikrobiologische Verunreinigungen durch fäkale Indikator Bakterien relativ häufig. Dies hat zur Folge, dass die mikrobiologische Qualität des Trinkwassers nicht immer einwandfrei ist und es besteht Handlungsbedarf, um diesbezüglich Verbesserungen zu erreichen.

Basierend auf den Richtlinien aus der Hygieneverordnung, führen Kantonale Laboratorien Untersuchungen zur Kontrolle des Trinkwassers durch, womit sie einen wichtigen Beitrag für sicheres Trinkwasser leisten. Eine Unterscheidung von menschlichen und tierischen fäkalen Verunreinigungen ist jedoch anhand der gesetzlich vorgeschriebenen mikrobiologischen Kriterien nicht möglich. Methoden, die eine Unterscheidung von menschlichen und tierischen Verunreinigungen erlauben, wären jedoch notwendig, um eine wissenschaftlich fundierte Datengrundlage für ein verbessertes Risiko Management und für die Umsetzung von Sanierungsmassnahmen zu erhalten.

Ziel der vorliegenden Arbeit war, Verfahren zu etablieren, die eine Unterscheidung von menschlichen und tierischen fäkalen Kontaminationen in Oberflächengewässern und Trinkwasser ermöglichen. Zudem sollten Konzepte für zukünftige Anwendungen der Methoden entwickelt werden. Das Konzept des „Microbial Source Tracking“ (MST) umfasst verschieden Ansätze, zur Identifizierung des Ursprungs fäkaler Kontamination. Verschiedene

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fäkaler Proben oder zur Untersuchung von Wasserproben aus Badegewässern wie Flüsse, Strände oder Seen verwendet wurden. Im Rahmen der vorliegenden Arbeit wurde das Konzept des MST zum ersten Mal in der Schweiz verwendet. Dabei wurden neue, selbst entwickelte Ansätze sowie bereits früher beschriebene MST Methoden im Hinblick auf ihr Potential für die Detektion von menschlichen oder tierischen Kontaminationen in der Schweiz getestet.

Zwei Bakterienstämme (Bacteroides thetaiotaomicron ARABA 84 und B. fragilis ARABA 19), die spezifisch Bakteriophagen menschlichen Ursprungs detektieren, und drei Stämme (B. caccae RBA 63 und RBA 64 sowie B. fragilis KBA 60), die spezifisch Phagen von tierischer Herkunft erkennen, wurden aus menschlichem Abwasser und tierischen Fäzes isoliert und im Folgenden verwendet, um die Herkunft fäkaler Verunreinigungen in einem Oberflächengewässern und Quellwasser zu bestimmen.

Zudem wurde das Potential von Streptococcus agalactiae als neuer, menschlicher MST Indikator untersucht, indem verschiedene Methoden zum Nachweis dieser Bakterien in menschlichem und tierischem Abwasser getestet wurden. Drei Extraktionsmethoden und fünf PCR-Assays wurden verglichen, um die bestmögliche Kombination zu finden. Mit der sensitivsten Methode, einer LightCycler real-time PCR, konnte S. agalactiae in menschlichem Abwasser, aber nicht in tierischen Proben nachgewiesen und so die Eignung dieser Bakterien als menschlicher MST Indikator gezeigt werden.

Rhodococcus coprophilus wurde bereits früher als tierischer MST Indikator verwendet. Zur Detektion dieser Bakterien wurde in früheren Publikationen ein kultureller Nachweis, eine

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Ansätze ergab jedoch keine zufriedenstellenden Resultate. Deshalb wurde im Rahmen dieser Dissertation eine neue LightCycler real-time PCR entwickelt und validiert, welche im Vergleich zu früher beschriebenen Methoden sensitiver und spezifischer und zudem viel schneller ist als der kulturelle Nachweis.

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B. thetaiotaomicron GA-17 und B. ovatus GB-124 infizieren, wurden als menschliche MST Indikatoren beschrieben. Zusammen mit den neu entwickelten und validierten Ansätzen wurden diese MST Indikatoren in einer Vergleichsstudie untersucht und ihr Potential zur spezifischen Identifizierung von menschlichen oder tierischen fäkalen Verunreinigungen in Schweizer Oberflächengewässern und Quellwasser getestet. Auf Grund der im Rahmen der Arbeit durchgeführten umfassenden Untersuchungen können Sorbitol fermentierende Bifidobakterien, die Wirtsstämme B. thetaiotaomicron ARABA 84 und B. ovatus GB-124 für einen Nachweis von menschlichen Kontaminationen im Schweizer Oberflächengewässern und Quellwasser empfohlen werden.

Die oben erwähnten MST Indikatoren geben Hinweise auf den Ursprung fäkaler Kontaminationen. Um den Ort des Eintrags zu lokalisieren, kann in der Folge ein weiterer Ansatz, der in dieser Arbeit beschrieben wird, verwendet werden. Dieser Ansatz basiert auf

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Charakterisierung durch Antibiogramme und Pulsfeldgelelektrophorese um genetisch identische Stämme zu finden.

In der vorliegenden Arbeit wurde ein Methodenset ausgearbeitet, mit dessen Hilfe einerseits

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