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Promotionsarbeit 2004 Untersuchungen zur Einsatzmöglichkeit der Dreikantmuschel Dreissena polymorpha als biologischer Filter und Wasserhygienemonitor Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät für Mathematik und Naturwissenschaften Technische Universität Dresden von Dipl.-Biol. Ute Schröter-Bobsin geboren am 13.02.1971 in Dresden Gutachter: 1. Prof. Dr. rer. nat. Isolde Röske, TU Dresden 2. Prof. Dr. med. Enno Jacobs, TU Dresden 3. PD Dr. rer. nat. Juan López-Pila, UBA Berlin Eingereicht am: 28.01.2005 Tag der Verteidigung: 25.04.2005 INHALTSVERZEICHNIS Inhaltsverzeichnis I ABKÜRZUNGSVERZEICHNIS IV VERZEICHNUNG DER TABELLEN VI VERZEICHNIS DER ABBILDUNGEN X 1 EINLEITUNG UND ZIELSETZUNG 1 1.1 Wasser als Ressource, Abwasserreinigung, Wasseraufbereitung 1 1.2 Dreissena polymorpha (Pallas 1771) 3 1.3 Konventionelle, bakterielle Hygieneindikatoren und Legionellen 6 1.4 Bakteriophagen 11 1.5 Enterale Viren 16 1.5.1 Enterovirus (Picornaviridae) 17 1.5.2 Adenovirus (Adenoviridae) 17 1.6 Cryptosporidien und Giardien (Protozoa) 18 1.7 Zielstellung der Arbeit 20 2 MATERIAL UND METHODEN 22 2.1 Untersuchungsgewässer, Probenentnahme und -transport 22 2.2 Präparation der Dreikantmuscheln 23 2.3 Übersicht der mikrobiologischen Untersuchungen 24 2.4 Methodenentwicklungen und kulturelle Nachweise 25 2.4.1 Übersicht der verwendeten Organismen 25 2.4.2 Übersicht der mikrobiologischen Kulturverfahren 26 2.4.3 Elution der Mikroorganismen aus dem Muschelfleisch 28 2.4.3.1 Auswahl des geeigneten Elutionspuffers 28 2.4.3.2 Einfluss der Adsorptionszeit und der Zentrifugation auf die Elution der am Muschelfleisch adsorbierten Mikroorganismen 29 2.4.3.3 Wiederfindung der Bakterien und Phagen aus dem Muschelfleisch (Elutionseffizienz) 29 2.4.4 Hygienemonitoring (in situ) der Muscheln und Wasserproben 30 2.4.5 Akkumulationsverhalten und Eliminationsleistung von Dreissena polymorpha gegenüber Mikroorganismen im Batchversuch (in vitro) 31 2.4.6 Untersuchungen zur Sedimentbelastung unter dem Muschelbett (in situ) 33 I INHALTSVERZEICHNIS 2.5 Molekularbiologische Analysen- und Nachweismethoden 35 2.5.1 Nachweis der Nukleinsäuren 35 2.5.1.1 DNA Extraktion 35 2.5.1.2 DNA Amplifikation mittels Polymerasekettenreaktion (PCR) 36 2.5.1.3 Reverse - Transkriptase - PCR (RT - PCR) 37 2.5.1.4 Aufreinigung der PCR – Produkte 38 2.5.1.5 Gelelektrophorese 39 2.5.1.6 DNA – Sequenzierung 39 2.5.2 Nukleinsäureanalytik 40 2.5.2.1 16S rDNA Genbanken von Muscheln und Wasserproben 40 2.5.2.2 Nachweis von Legionellen in Muscheleluat- und Wasserproben 42 2.5.2.3 Nachweis Stx-Gen tragender Bakteriophagen in Muscheleluaten und in Wasserproben 43 2.5.2.4 Nachweis enteraler Viren in Muscheleluat- und Wasserproben 45 2.5.2.5 Nachweis von Cryptosporidien und Giardien in Muscheleluaten und Wasserproben 47 3 ERGEBNISSE UND DISKUSSION 51 3.1 Kulturelle Analytik 51 3.1.1 Entwicklung eines effizienten Elutionsverfahrens für die mit dem Muschelgewebe assoziierten Mikroorganismen 51 3.1.1.1 Auswahl des geeigneten Elutionspuffers 51 3.1.1.2 Einfluss der Zentrifugalkraft und der Adsorptionszeit auf die Elution der Mikroorganismen vom Muschelfleisch 52 3.1.1.3 Wiederfindung der Bakterien und Phagen aus dem Muschelfleisch 54 3.1.2 Hygienemonitoring (in situ) mit der Dreikantmuschel und dem Umgebungswasser 56 3.1.2.1 Hygienemonitoring in der Elbe 56 3.1.2.2 Hygienemonitoring in der KA Reichenau Teich 1 63 3.1.2.3 Hygienemonitoring in der KA Reichenau Teich 2 67 3.1.2.4 Korrelationen zwischen dem Nachweis mikrobieller Indikatoren im Gewebeeluat von Dreissena polymorpha und im Umgebungswasser 69 3.1.3 Eliminations- und Akkumulationsverhalten der Muscheln im Batchversuch (in vitro) 70 3.1.3.1 Entwicklung der Intestinalen Enterokken mit und ohne Filtrationstätigkeit der Muscheln 71 II INHALTSVERZEICHNIS 3.1.3.2 Entwicklung der PhiX 174 Coliphagen mit und ohne Filtrationstätigkeit der Muscheln 73 3.1.4 Untersuchungen zur Sedimentbelastung unter dem Muschelfilter (in situ) 76 3.2 Molekularbiologische Analytik 78 3.2.1 16S rDNA Klonbank vom Verdauungstrakt der Muscheln und dem korrespondierenden Wasser 78 3.2.2 Nachweis von Legionellen in Muscheln und Wasser 81 3.2.2.1 PCR - Nachweis der Legionellen in Muscheleluaten der Monitoringproben 81 3.2.2.2 Vergleichsstudie zum Nachweis der Legionellen (PCR) in Muscheleluaten und in konzentrierten Wasserproben 83 3.2.2.3 16S rDNA Klonbank für den Genus Legionellacea von Muscheleluat- und Wasserkonzentratproben 85 3.2.3 Nachweis Stx - Gen tragender Bakteriophagen in Muscheln und Wasser 87 3.2.4 Nachweis von enteralen Viren (Adenoviridae, Picornaviridae) in Muscheln und Wasser 91 3.2.4.1 Die Gattung Mastadenovirus 91 3.2.4.2 Die Gattung Enterovirus 92 3.2.4.3 Vergleich des Bakteriophagenmonitoring mit dem Vorkommen enteraler Viren 95 3.2.5 Nachweis von Giardien und Cryptosporidien in der Muschel und im Wasser 97 3.2.5.1 Monitoring in der Elbe 98 3.2.5.2 Monitoring im Schönungsteich 1 der KA Reichenau 99 3.2.5.3 Monitoring im Schönungsteich 2 der KA Reichenau 100 3.2.5.4 Monitoring im Waldbad Langebrück 101 3.2.5.5 Vergleich des Parasitennachweises mit bakteriellen und virologischen Indikatoren in Muscheln und Wasser 103 4 ZUSAMMENFASSUNG UND AUSBLICK 105 5 LITERATURVERZEICHNIS 108 TABELLENANHANG XIII PUBLIKATIONEN DANKSAGUNG III ABKÜRZUNGSVERZEICHNIS Abkürzungsverzeichnis AdV Adenovirus AFDW Aschefreie Trockenmasse (ashfree dry weight) ATCC American Type Culture Collection AZV Abwasserzweckverband BGM Buffalo Green Monkey bp Basenpaar CPE Cytopathischer Effekt DEV Deutsche Einheitsverfahren DGL Deutsche Gesellschaft für Limnologie DNA Desoxyribonukleinsäure dNTP Desoxynucleosidtriphosphat DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen EDTA Ethylendiamintetraacetat EWG Einwohnergleichwert EV Enterovirus FL-Zellinie humane Amnionzellinie nach Fogh /Lund HAV Hepatitis A Virus HPLC High Performance Liquid Chromatography IKSE Internationale Kommission zum Schutz der Elbe ISO International Organization for Standardization KA Kläranlage kb Kilobasen(paare) KBE Kolonie bildende Einheiten MPN Most probable number MW Mittelwert n.b. nicht bewertbar NWG Nachweisgrenze O. D. Optische Dichte (photometrisch) PBS Phosphat – gepufferte Kochsalzlösung PEG Polyethylenglykol PFU Plaque forming units IV ABKÜRZUNGSVERZEICHNIS PP Polypropylen Rei T 1 (Kläranlage) Reichenau Zufluss Schönungsteich 1 Rei T 2 (Kläranlage) Reichenau Abfluss Schönungsteich 2 RKI Robert Koch Institut RNA Ribonukleinsäure Rt Raumtemperatur SBK Säurebindungskapazität Stabw Standardabweichung TS Trockensubstanz ü. N. über Nacht UpM Umdrehungen pro Minute # kein Messwert V TABELLENVERZEICHNIS Tabellen Textteil Tabelle 1: Mikroorganismen und Krankheitssymptome sogenannter "neuer Erreger" wasserbürtiger Krankheitsfälle (nach KÖSTER ET AL., 2002) 7 Tabelle 2: Übersicht zu Konzentrationen von Bakteriophagen in verschiedenen Wasserressourcen 15 Tabelle 3: Verwendetete Referenzstämme in den Bakterienuntersuchungen 25 Tabelle 4: Verwendete Referenzstämme in den Bakteriophagenuntersuchungen 26 Tabelle 5: Verwendete Referenzstämme in den Parasitenuntersuchungen 26 Tabelle 6: Kulturelle Nachweismethoden 27 Tabelle 7: DNA - Extraktionsmethoden 35 Tabelle 8: Erfassung der durschnittlichen Anreicherungsfaktoren in Dreissena polymorpha im Vergleich zum Wasser der Elbe 63 Tabelle 9: Erfassung der durchschnittlichen Anreicherungsfaktoren in Dreissena polymorpha im Vergleich zum Wasser der KA Rei T 1 66 Tabelle 10: Erfassung der durchschnittlichen Anreicherungsfaktoren in Dreissena polymorpha im Vergleich zum Wasser der KA Rei T2 68 Tabelle 11: Akkumulationsfaktoren für Enterokokken in Dreissena polymorpha unter verschiedenen Temperaturbedingungen 73 Tabelle 12: Akkumulationsfaktoren für somatische Coliphagen in Dreissena polymorpha unter verschiedenen Temperaturbedingungen 75 Tabelle 13: PCR - Nachweis von Legionellen im Muscheleluat und Umgebungswasser 83 Tabelle 14: Screening Stx- Gen tragender Coliphagen 89 Tabelle 15: Anteil der Adenoviren in den Muschelproben 91 Tabelle 16: Anteil der Enteroviren in Muscheln und Wasser 93 Tabelle 17: PCR Nachweis von Giardia sp. und Cryptosporidium sp. aus Muschel- und Wasserproben der Elbe (REICHARDT, 2003, modifiziert) 98 VI TABELLENVERZEICHNIS Tabelle 18: PCR Nachweis von Giardia sp. und Cryptosporidium sp. aus Muschel- und Wasserproben des Schönungsteiches 1 der KA Reichenau (REICHARDT, 2003, modifiziert) 99 Tabelle 19: PCR Nachweis von Giardia sp. und Cryptosporidium sp. aus Muschel- und Wasserproben des Schönungsteiches 2 der KA Reichenau (REICHARDT, 2003, modifiziert) 100 Tabelle 20: PCR Nachweis von Giardia sp. und Cryptosporidium sp. aus Muschel- und Wasserproben des Waldbades Langebrück (REICHARDT, 2003, modifiziert) 101 Tabelle 21: Erfassung des Parasitenanteils im Vergleich zu mikrobiellen Indikatoren aus Dreissena polymorpha und ihrem Umgebungswasser (REICHARDT, 2003, modifiziert) 103 Tabellen Anhang Tabelle A 1: Kurzcharakteristik der Untersuchungsgewässer XIII Tabelle A 2: Übersicht der verwendeten Primer XIV Tabelle A 3: Übersicht der verwendeten PCR - Protokolle XVII Tabelle A 4: Übersicht RT-PCR Protokoll XIX Tabelle A 5: Vergleich der Elutionsmittel bezüglich ihrer Kapazität zur Elution der Indikatororganismen (n = 3) XX TabelleA 6: Einfluss der Zentrifugalkraft auf denNachweis von Mikroorganismen aus dem Muschelfleischeluat XXI Tabelle A 7: Einfluss der Adsorptionszeit auf die Elution von Mikroorgansimen vom Muschelfleisch XXII Tabelle A 8: Erfassung der prozentualen Wiederfindungsrate für Bakterien und Phagen
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