Less than 10% of the world’s domestic wastewater is disinfected prior to discharge into surface waters; therefore, human exposure to diverse wastewater-related pathogens results in millions of cases of illness each year. Among the enteric pathogens, viruses represent an important group of emerging pathogens and are frequently the cause of food- and water-borne outbreaks of illness. Although the World Health Organization and many government agencies mandate the use of bacterial indicators to identify poor microbial water quality, it is well known that these indicators poorly correlate with fecal pollution contamination events and risk of disease. The field of public health-related environmental microbiology has made significant advances over the last twenty years; however, there is still a need for improved methods to identify poor microbial water quality and manage health risks associated with water-related activities (e.g., recreation, shellfish harvesting, irrigation). Furthermore, it is imperative to effectively detect fecal pollution in the environment as well as determine the extent of pathogen removal during (waste)water treatment to meet the Sustainable Development Goals associated with water and food security as well as the water reuse recommendations by the U.S. National Research Council. This dissertation directly addressed the need to identify an improved viral indicator by exploring the application of pepper mild mottle virus (PMMoV), a virus of dietary origin that is extremely abundant in human feces, as a surrogate for enteric viruses in diverse settings and contexts. Using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method, PMMoV was compared with other indicators, index surrogates, and reference pathogens for assessing surface water quality in a developed country (Appendix A) and developing country context (Appendix B and C). In addition, the applicability of PMMoV as a (waste)water treatment process indicator was demonstrated in natural treatment systems in developing countries (Appendix C and D) and artificial treatment systems (Appendix E). In all settings, PMMoV always co-occurred with at least one reference pathogen, index organism, and/or indicator; additionally, PMMoV was detected more frequently and in higher concentrations than other reference pathogens and indicators. Three investigations also associated PMMoV detection with predicted human health risks greater than the health benchmark for recreation and water reuse activities (Appendix A, C, and D). Additionally, PMMoV facilitated an improved understanding of virus-particle interactions in wastewater treatment pond systems (Appendix D) and allowed for an improved understanding of virus removal with respect to riverbank filtration systems and wastewater reuse in agriculture (Appendix C). PMMoV was established as a valuable component of the microbial source tracking toolbox in Costa Rica (Appendix B) and appeared to be useful in Bolivia (Appendix C). Finally, a paradigm shift in (waste)water management is occurring, in which routine monitoring is being replaced by a more holistic approach that includes sanitary surveys, targeted water quality monitoring, and exploratory quantitative microbial risk assessment. To support and complement this paradigm shift, field-based, laboratory-free methods are needed to identify and/or infer the presence of enteric viruses (Appendix F). Collectively, all the investigations presented here confirm the use of PMMoV as a surrogate for enteric viruses; however, its utility depends on the context and research question.

In Virginia, over one million households rely on private water supplies (e.g. well, spring, cistern). Previous literature acknowledges bacterial contamination in private water supplies as a significant public health concern in the United States. The present study tested private wells and springs in 20 Virginia counties for total coliforms (TC) and E. coli (EC) along with a suite of chemical contaminants. Sample collection was organized by the Virginia Household Water Quality Program (VAHWQP), a Virginia Cooperative Extension effort managed by faculty in the Biological Systems Engineering Department. Microbial and chemical source tracking were used to identify possible sources of contamination. A logistic regression was employed to investigate potential correlations between TC contamination and chemical parameters (e.g. NO3-, turbidity) as well as homeowner provided survey data describing system characteristics and perceived water quality. TC and EC contamination were quantified via the Colilert (www.idexx.com) defined substrate method for most probable number (MPN) of EC and TC per 100 mL of water. Of the 538 samples collected, 41% (n=221) were positive for TC and 10% (n=53) for EC. Chemical parameters were not statistically predictive of microbial contamination. Well depth, water treatment, and farm location proximate to the water supply were factors in a regression model that predicted presence/absence of TC with 74% accuracy. Microbial and chemical source tracking techniques (Polymerase Chain Reaction (PCR) and fluorometry, respectively) identified 4 of 26 samples as likely contaminated with human wastewater. Application of these source-tracking analyses on a larger scale will prove useful in defining remediation strategies. / Master of Science

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

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Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-05-11T17:59:22Z No. of bitstreams: 1 Dissertação_Kayo_Bianco.pdf: 5086210 bytes, checksum: 14083186869c48adebe043f3e9e02a6c (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-05-11T17:59:44Z (GMT) No. of bitstreams: 1 Dissertação_Kayo_Bianco.pdf: 5086210 bytes, checksum: 14083186869c48adebe043f3e9e02a6c (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-05-11T17:59:54Z (GMT) No. of bitstreams: 1 Dissertação_Kayo_Bianco.pdf: 5086210 bytes, checksum: 14083186869c48adebe043f3e9e02a6c (MD5) / Made available in DSpace on 2015-05-11T17:59:54Z (GMT). No. of bitstreams: 1 Dissertação_Kayo_Bianco.pdf: 5086210 bytes, checksum: 14083186869c48adebe043f3e9e02a6c (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A contaminação fecal de corpos hídricos é uma das principais causas de doenças entéricas veiculadas pela água no mundo, responsáveis pela morte de mais de dois milhões de crianças por ano. As águas das bacias do rio São Joao e do rio Guandu vêm sendo destinadas ao abastecimento público da Região dos Lagos e da região metropolitana do estado do Rio de Janeiro. Os indicadores de contaminação fecal usualmente empregados na avaliação da qualidade microbiológica da água quanto a presença de patógenos entéricos são os coliformes termotolerantes, representados pela Escherichia coli, os quais estão associados com matéria fecal humana e de outros animais de sangue quente. Entretanto, para um gerenciamento mais efetivo deve ser identificada a fonte de contaminação fecal. Microrganismos anaeróbios, como a ordem Bacteroidalese as archaeas metanogênicas, vêm sendo apontados como bioindicadores alternativos de contaminação fecal hospedeiro-especifico. O presente estudo tem como objetivo avaliar a qualidade das águas das bacias do rio São João e do rio Guandu por meio da aplicação de biomarcadores moleculares capazes de revelar contaminações fecais suína, humana, equina e de ruminantes. Esta abordagem representa uma importante ferramenta na detecção de contaminação hospedeiro-específica de ambientes aquáticos, o que poderá contribuir para as ações preventivas de vigilância ambiental em saúde. / Fecal contamination of water bodies is a major cause of waterborne enteric diseases in the world, responsible for the deaths of more than two million children per year. The waters from São João river basin and Guandu river basin have been intended for public supply of the Lakes Region and the metropolitan region of the State of Rio de Janeiro. Indicators of fecal contamination usually employed to evaluate the microbiological quality of water for the presence of enteric pathogens are coliforms, represented by Escherichia coli, which are associated with human faecal matter and other warm-blooded animals. However,for a more effective management the source of fecal contamination should be identified. Anaerobic microorganisms, such as the order Bacteroidales and methanogenic archaea, have been identified asalternative biomarkers for host-specific fecal contamination. The present study aims to evaluate the water quality of São João river basinand Guandu riverbasinthrough the application of molecular biomarkers able to reveal swine, human, equine and ruminants fecal contamination. This approach represents an important tool in the detection of host-specific contamination of aquatic environments, which may contribute to prevention efforts for environmental health surveillance.

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Les virus entériques sont à l’origine de pathologies liées au péril fécal et dans l’état actuel des connaissances, la recherche des indicateurs de pollution fécale conventionnels (i.e. Escherichia coli, entérocoques) peut s’avérer inefficace pour évaluer le danger viral. La définition d’autres indicateurs pour gérer le danger lié à la présence des virus entériques dans les matrices hydriques et alimentaires est aujourd’hui nécessaire. Parmi eux, les bactériophages ARN F-spécifiques (FRNAPH) présentent plusieurs intérêts. Ces virus d’origine entérique sont présents en quantité importante dans les eaux usées. Très proches des virus entériques en termes de structure, ces microorganismes présentent l’avantage d’être facilement cultivables. Ils sont enfin souvent étudiés pour déterminer l’origine d’une pollution fécale (i.e. humaine ou animale). Certaines limites leur sont cependant fréquemment associées, que ce soit en termes de corrélation avec les pathogènes entériques ou concernant leur potentiel pour discriminer l’origine d’une pollution. Dans ce contexte, l’objectif du travail présenté ici était de préciser l’intérêt des FRNAPH en tant qu’indicateurs de pollution fécale mais aussi en tant qu’indicateurs de pollution virale dans l’environnement et les coquillages. Ces travaux ont permis dans un premier temps d’améliorer la capacité des FRNAPH à identifier les contaminations d’origine humaine. Nos résultats soulignent par ailleurs la plus-value apportée par la recherche des FRNAPH en cas de pollution fécale massive, en particulier si on s’intéresse à la contamination des coquillages. En effet, contrairement aux indicateurs bactériens, l’accumulation des FRNAPH ainsi que leur persistance dans ces aliments est très comparable à celles des virus entériques (i.e. norovirus). Enfin, en utilisant des méthodes de détection comparables, une forte corrélation entre la présence des FRNAPH d’origine humaine et celle des norovirus a été observée dans les coquillages. Compte tenu de ces résultats, une méthode de détection assurant la détection sensible des FRNAPH infectieux d’origine humaine dans différents types de matrices hydriques ou alimentaires (e.g. eaux de surface, fruits de mer, fruits rouges, salades) est proposée pour améliorer la gestion du danger viral / Enteric viruses are a leading cause of fecal-oral route transmitted diseases and currently, conventional fecal indicator bacteria (i.e. Escherichia coli, enterococcus) fail to assess this kind of hazard. In this context, the use of more efficient indicators to assess the hazard linked to viruses in water or foodstuff is required. F-specific RNA bacteriophages (FRNAPH) present numerous benefits for this purpose. Of enteric origin, these viruses are found in high concentrations in wastewater. Sharing many structural similarities with pathogenic enteric viruses, FRNAPH are easily cultivable and their potential to track the origin of the pollution is also often investigated. However, some limits are still associated with these indicators, regarding to their ability to track the origin of the pollution or concerning the lack of correlation with pathogens. In this context, the aim of this work was to make clear the potential of FRNAPH as fecal and as viral indicators in environmental waters and shellfish. As a first step, their ability to track human pollution was optimized. In addition, our results underlined the gains bringing by FRNAPH detection, especially when focusing on shellfish microbiological quality management. Indeed, unlike fecal indicator bacteria, the accumulation of FRNAPH and their persistence in shellfish have been found to be close to that of enteric viruses (i.e. norovirus). Furthermore, when using comparable methods for their detection, high correlation was observed between human FRNAPH and norovirus in shellfish. Taking into account these observations, a sensitive method allowing the detection of infectious FRNAPH of human origin was developed to improve viral hazard management in water and food commodities (e.g. environmental waters, shellfish, soft fruits, leaf)

La microbiologia de l’aigua és un camp científic que ha ajudat enormement a millorar la seguretat sanitària de les aigües destinades als usos humans. De fet, augmentar el coneixement sobre les poblacions microbianes presents a l’aigua tant pel que fa a les poblacions autòctones com a les al•lòctones, pot ser de gran ajuda de cara a garantir els estàndards de qualitat, que cada vegada són més exigents. Tanmateix, la gran heterogeneïtat dels sistemes aquàtics i de les problemàtiques lligades als seus usos, fan necessari un estudi individualitzat de les seves poblacions microbianes. Així doncs, el propòsit d’aquesta tesi doctoral va ser estudiar diferents poblacions microbianes associades a diferents sistemes aquàtics: aigües minerals naturals, aigües recreatives i aigües regenerades. Pel que fa a les aigües minerals naturals, es van estudiar les poblacions bacterianes aeròbies heterotròfiques autòctones de tres fonts a 22ºC i 37ºC durant l’hivern i l’estiu. Totes les soques aïllades es van fenotipar mitjançant les microplaques PhP-48 del Phene-Plate System®. Es van poder aïllar un elevat nombre de gèneres poc descrits en aquests ambients. Això afegit als canvis de composició observats entre estacions, confirma que es tracta d’ambients complexos, i que encara hi ha un gran desconeixement sobre la seva composició i dinàmica. D’altra banda, es va confirmar que cada aigua mineral natural conté una microbiota autòctona pròpia, l’estructura i composició de la qual és prou específica per poder diferenciar-la amb mètodes de caracterització fenotípica de la d’altres aigües minerals naturals. D’altra banda es va avaluar la capacitat de la ISO 16266:2006 per identificar Pseudomonas aeruginosa en aigües minerals naturals. En general, el procediment de la ISO 16266:2006 identifica adequadament la major part dels aïllats de P. aeruginosa, llevat dels casos de soques no pigmentades. Els resultats recomanen la incorporació dels assaigs de creixement en agar King A i a 4°C i 42°C en les anàlisis rutinàries d’aigües envasades, i s’aconsella l’ús de les galeries API®20NE en la confirmació d’aquells aïllats que generin dubtes en base als criteris de la ISO 16266:2006. En l’àmbit de les aigües recreatives, es van caracteritzar les poblacions microbianes associades a quatre piscines naturalitzades d’ús privat. Tres d’elles presentaven a l’estiu concentracions d’E. coli o enterococs superiors als límits establerts a les recomanacions. La contaminació fecal relacionada amb la fauna salvatge s’apunta com un contribuïdor significatiu en les piscines naturalitzades privades. A més, el sistema de depuració natural de les piscines naturalitzades sembla ser força limitat alhora de disminuir la contaminació fecal a les piscines, cosa que posa en dubte la seva aplicabilitat en piscines de major volum i obertes al públic. Pel que fa a les aigües regenerades, es van avaluar la capacitat del fenotipatge bioquímic de coliforms fecals i enterococs per determinar l’origen de la contaminació fecal en dues basses d’aigua regenerada utilitzades en la irrigació d’un camp de golf. La caracterització bioquímica i l’estudi d’inactivació van proporcionar evidències que la contaminació fecal detectada a les basses a l’estiu no tenia relació amb les poblacions que entraven a través de l’aigua regenerada, sinó que semblava estar relacionat amb una aportació de material fecal animal, probablement ocells, generat al propi sistema. El fenotipatge bioquímic d’enterococs i coliforms fecals és una eina útil per a l’estudi de l’origen de la contaminació fecal en aquells casos on la càrrega fecal és baixa, fet que és rellevant, tenint en compte que molts altres mètodes presenten problemes en ser aplicats per sota d’aquestes concentracions. En general, els estudis realitzats donen peu a continuar aprofundint en l’estudi de la diversitat microbiana en diferents ambients aquàtics, tant pel que fa a les poblacions autòctones com a les al•lòctones. / "Characterization and diversity of microbial populations associated to with natural mineral waters, recreational waters and reclaimed waters" SUMMARY: Microbial populations from natural mineral waters, recreational waters and reclaimed waters were characterized to increase the knowledge about their indigenous and introduced microorganisms. Regarding natural mineral waters, we characterized the aerobic heterotrophic bacteria from three sources at 22ºC and 37ºC during summer and winter. The study of the similarities showed that growing temperatures and seasons caused significant differences in structures and composition. In addition, several bacterial species were isolated and identified, some of them rarely isolated in natural mineral waters, revealing the complexity and lack of knowledge of these ecosystems. Furthermore, we evaluated the procedure included in ISO 16266:2006 to identify presumptive P. aeruginosa isolates in natural mineral waters. The results showed that ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained. Additional test assayed such as King A medium, growth tests at 4 °C and 42 °C and API® 20NE correctly discriminated all the studied strains. As far as recreational waters are concerned, we analyzed the microbial populations in four private natural swimming pools. Three of them exceeded the E. coli or enterococci limits stated in the recommendations for natural swimming pools. The concentrations of P. aeruginosa and aerobic heterotrophic bacteria were acceptable. The results suggest that wildlife was an important source of fecal pollution in the pools. Since there is a lack of regulations on these systems, and the health risks are higher than in conventional swimming pools, further research is needed to establish the parameters for ensuring safe bathing in private and public natural swimming pools. Finally, we aimed to identify the origin of the fecal contamination in two reclaimed-water open-air ponds used to irrigate a golf course. Although the concentration of fecal bacterial indicators was low, the biochemical fingerprinting used provided evidence that the origin of the fecal contamination in the ponds was not related to the reclaimed water. Furthermore, a mesocosm assays indicated that none of the microbial fecal indicators was able to regrow in the ponds. Finally, the study highlighted the fact that reclaimed water may be recontaminated in open-air reservoirs.