Antibiotics have been one of the greatest scientific discoveries for humanity. Their great success has been hindered by the increasing of antibiotic resistance events. Conventional wisdom considers antibiotic resistance as a strictly clinical issue. In reality, more and more studies have proven that non-clinical environments, especially aquatic environments, are critical factors for the spread of antibiotic resistance. Antibiotic resistance genes (ARGs) are usually located on mobile genetic elements, which can disseminate among taxonomically unconnected species. Therefore, environmental bacteria can serve as hotspots for the development and dissemination of antibiotic resistance. The aim of this thesis is to determine how human originated pollution affects the antibiotic resistance abundance in the surrounding water environments. The case studies were three example environments, wastewater treatment plants (WWTPs), receiving river bodies and combined sewer overflows (CSOs). Molecular microbiology methods have been applied to analyse the resistance levels of bacterial communities from WWTP, CSOs and wastewater affected environments at different European geographical locations. Additionally, a novel approach consisting in an amplicon sequencing is described, in order to be able to investigate and asses the composition of a significant amount of integron gene cassettes. Findings consistently indicate that effluents originated from WWTPs and CSOs have a significant impact on the levels of ARGs of the receiving water bodies. Moreover, this thesis suggests that gene blaOXA-58, could be utilized as a proxy to investigate the spread of ARGs. Its occurrence has been reported to show, consistently throughout the chapters, lower concentrations upstream, but at higher concentrations in the WWTP effluent, CSOs and downstream of the effluent. In conclusion, although antibiotic resistance genes and integrons are part of the environmental resistome, water environments that are affected by anthropogenic wastewater display high levels of the above-mentioned genetic elements. These findings clearly suggest the need to limit pollution levels, as well as the need to establish a more responsible policy in antibiotic prescriptions. This must take place in order to be able to perform efficient risk assessments and to establish acceptable levels of antibiotic and ARGs pollution.:List of Figures VII List of Tables IX List of Abbreviations X 1. Introduction 11 1.1 Antibiotic resistance: an increasing problem 11 1.2 Intrinsic antibiotic resistance and horizontal gene transfer 13 1.2.1 Transformation 13 1.2.2 Transduction 13 1.2.3 Conjugation 14 1.3 Gene transfer elements 14 1.4 Antibiotic resistance and anthropogenic impact on surrounding environments 16 1.5 Study goals 17 2. Genetic variations of the resistome of wastewater treatment plants driven by the seasonality of antibiotic prescription 18 2.1 Introduction 18 2.2 Materials and methods 19 2.3 Results and discussion 20 3. Assessment of inter-laboratory variations of quantitative analyses of antibiotic resistance genes 27 3.1 Introduction 27 3.2 Materials and methods 27 3.3 Results and discussion 30 3.4 Conclusions 36 4. Antibiotic resistance genes in treated wastewater and in the receiving water bodies: A pan-European survey of urban settings 37 4.1 Introduction 37 4.2 Materials and methods 38 4.3 Results 44 4.4 Discussion 49 4.5 Conclusions 53 5. Analysis of integron gene cassettes composition in treated wastewater 55 5.1 Introduction 55 5.2 Materials and methods 55 VI 5.3 Results 56 5.4 Discussion 57 6. Role of combined sewer overflows in the dissemination of antibiotic resistance in surrounding environments 59 6.1 Introduction 59 6.2 Materials and methods 60 6.3 Results 61 6.4 Discussion 66 7. Closing Conclusions 68 References 70 List of publications 88 Note on the commencement of the doctoral procedure 89 Acknowledgments 90

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L’optimisation du système d’assainissement et la réduction des rejets d’eaux résiduaires urbaines non traitées est devenue un enjeu majeur pour de nombreuses collectivités dans le but d’atteindre les objectifs de qualité des milieux aquatiques fixés par la Directive Cadre européenne sur l’Eau (DCE 2000/60/CE). Pour cela, une parfaite connaissance du système d’assainissement est nécessaire. L’objectif de cette thèse, financée par la CDAPP et l’Agence de l’Eau Adour Garonne, était l’étude de la dynamique du système d’assainissement de la CDAPP et de sa contribution sur les flux de polluants dans le milieu récepteur (le Gave de Pau). La première étape du travail a été consacrée à la caractérisation hydraulique et physicochimique du système d’assainissement par temps sec et par temps de pluie. Une étude hydraulique a été tout d’abord réalisée et a permis de mieux appréhender la dynamique des déversements via les déversoirs d’orage (DO) en fonction de la nature des évènements pluvieux. D’autre part, la caractérisation physico chimique des eaux usées (matières en suspension ou MES, demande chimique en oxygène ou DCO, métaux, hydrocarbures aromatiques polycycliques) a mis en évidence que pour l’ensemble de ces paramètres, une nette augmentation des flux par temps de pluie était observée en particulier en début d’événement (augmentation d’un facteur de 2 à 10). Ce phénomène s’explique par l’apport de polluant par les eaux de ruissellement et l’érosion des dépôts accumulés par temps sec dans les réseaux. Seul l’azote total se comporte différemment puisqu’il est majoritairement présent sous forme dissoute. Afin de mieux appréhender la dynamique des paramètres polluants réglementaires (MES, DCO et azote), un suivi haute fréquence (au pas de temps de cinq minutes) a été mis en place durant un an à l’aide de sondes de turbidité et de conductivité. Ce suivi en continu constitue la troisième partie de ce travail. Des corrélations (r² ≈ 0,9) ont été établies entre d’une part, les paramètres polluants DCO et MES, et la turbidité et d’autre part, entre la conductivité et l’azote total. Ces enregistrements ont permis une meilleure compréhension du fonctionnement du système d’assainissement : mise en évidence du phénomène de first flush, estimation des flux polluants déversés via les DO, étude des phénomènes de stockage dans les réseaux. La dernière partie de cette thèse vise à l’étude de la contribution des rejets d’assainissement dans le milieu récepteur. Elle a permis de démontrer la contribution modérée du rejet de sortie de STEP (entre 1 et 15 %) par temps sec. Par temps de pluie, la contribution du système d’assainissement via les DO est extrêmement variable suivant les conditions hydro-climatiques (de < 1 % à plus de 50 %). Ce travail a permis d’une part d’apporter des résultats utilisables par le gestionnaire de l’assainissement pour optimiser la gestion des eaux usées de la CDAPP. D’autre part, ce travail apporte des résultats plus fondamentaux relatifs à une meilleure connaissance de la dynamique hydrologique et physicochimique des eaux résiduaires urbaines et des polluants associés tels que la mise en évidence, à l’aide d’outils statistiques, des paramètres influençant les déversements et les concentrations en polluants par temps de pluie. / Optimization of sewer system and reduction of untreated wastewater discharges has become a key issue for many communities in order to achieve the good quality of aquatic environments set by the European Framework Directive (WFD 2000/60/EC). For this, a perfect knowledge of sanitation is required. The objective of this thesis, funded by CDAPP and Adour Garonne Water Agency, was the study of the dynamics of the CDAPP sanitation and its contribution to the pollutants fluxes in the receiving environment (the river Gave de Pau). The first step of the work was devoted to the hydraulic and physicochemical wastewater characterization during dry and wet weather. A hydraulic study was first carried and helped to better understand the dynamics of discharges through the combined sewer overflow (CSO) according to the rainfall events characteristics. On the other hand, the physico-chemical characterization of wastewater (suspended solids or TSS, chemical oxygen demand or COD, metals, polycyclic aromatic hydrocarbons) showed that for all these parameters, a clear increase of pollutant fluxes was observed at the beginning of the rainfall event (increase by a factor of 2 to 10). This phenomenon is explained by the runoff contribution and erosion of sediments accumulated in the networks during dry weather periods. Only total nitrogen behaves differently because it’s mostly dissolved. To better understand the dynamics of pollutants parameters (TSS, COD and nitrogen), high frequency monitoring (every five minutes) has been established for one year with turbidity and conductivity sensors. This continuous monitoring is the third part of this work. Correlation functions (r² ≈ 0.9) were found between, the pollutant parameters COD and TSS, and turbidity, and secondly, between conductivity and total nitrogen. These records allowed a better understanding of sanitation system: highlighting the first flush phenomenon, estimation of pollutant loads discharged by CSO, study of storage networks phenomenon. The last part of this thesis aims to study the contribution of wastewater discharges to the receiving environment. It demonstrated the moderate contribution of rejection output STEP (between 1% and 15%) in dry weather. In rainy weather, the contribution of sanitation through CSO is extremely variable depending on the hydro-climatic conditions (<1% to over than 50%). This work has led to provide usable results for the sanitation manager to optimize CDAPP wastewater treatment. Moreover, this work provides most fundamental results for a better understanding of the hydrological and physicochemical dynamics of urban wastewater and associated pollutants such as highlighting, using statistical tools, the parameters influencing pollutant concentrations during rainfall events.

Les bassins périurbains, constitués de zones urbaines, agricoles et naturelles, sont des bassinsversants complexes à étudier. L’augmentation des surfaces imperméables et les modifications deschemins d’écoulement par les réseaux d’assainissement influencent leur hydrologie. Ces modificationssont notamment liées aux choix de modes de gestion des eaux pluviales : réseaux unitaires,réseaux séparatifs, infiltration à la parcelle, etc. La modélisation hydrologique spatialisée, quirend compte de l’hétérogénéité des bassins versants, est un outil permettant d’évaluer les différentsenjeux en termes d’occupation du sol et de gestion des eaux pluviales. Cependant, peu demodèles ont été construits pour être appliqués aux bassins périurbains, à l’échelle des gestionnaires(˜ 100 km2) et pour des simulations sur de longues périodes (> 10 ans). La modélisationhydrologique doit donc être adaptée afin de mieux capter les spécificités des milieux périurbainstelles que l’hétérogénéité de l’occupation du sol et la connexion de certaines zones urbaines à unréseau d’assainissement.Ce travail de thèse a consisté à développer un nouvel outil de modélisation adapté à ces problématiques: le modèle distribué horaire J2000P. Ce modèle simule les processus hydrologiquesen milieux ruraux et urbains et prend en compte les réseaux d’assainissement, les connexionsà ces réseaux et les déversements des déversoirs d’orage (DO). Le modèle a été mis en oeuvresur le bassin périurbain de l’Yzeron (˜ 130 km2), situé à l’ouest de Lyon. L’évaluation, effectuéeà l’exutoire de différents sous-bassins de tailles et d’occupations du sol différentes, montre desrésultats très encourageants. Le modèle a tendance à sous-estimer le débit mais la dynamiquedes pics est bien représentée tout comme le déversement des DO. Suite aux résultats de l’évaluation,une analyse de sensibilité « pas à pas » du modèle a été réalisée et différentes hypothèsesde fonctionnement du bassin ont été formulées pour améliorer la compréhension du modèle etdes processus représentés. Le modèle a ensuite été utilisé pour tester l’impact de modificationsde l’occupation des sols et/ou de la gestion des eaux pluviales sur la réponse hydrologique. Lemodèle montre que la gestion de l’occupation du sol a moins d’influence sur l’hydrologie dubassin que la gestion du réseau d’assainissement. / Growing urbanization and related anthropogenic processes have a high potential to influencehydrological process dynamics. Typical consequences are an increase of surface imperviousnessand modifications of water flow paths due to artificial channels and barriers (combined and separatedsystem, sewer overflow device, roads, ditches, etc.). Periurban catchments, at the edgeof large cities, are especially affected by fast anthropogenic modifications. They usually consistof a combination of natural areas, rural areas with dispersed settlements and urban areas mostlycovered by built zones and spots of natural surfaces. Spatialized hydrological modeling tools, simulatingthe entire hydrological cycle and able to take into account the important heterogeneityof periurban watersheds can be used to assess the impact of stormwater management practiceson their hydrology.We propose a new modeling tool for these issues : the hourly distributed J2000P model.This model simulates the hydrological processes in rural and urban areas and takes into accountthe sewerage networks, connections to these networks and overflows from sewer overflow devices(SOD). The application site is the Yzeron catchment (˜ 130 km2), located in the West of Lyon.The evaluation, conducted at the outlet of different sub-basins with different sizes and landuse, shows very encouraging results. The model tends to underestimate the discharge but thedynamics of the peaks and the SOD overflows are well simulated. The model is also used to testthe impact of changes in land use and/or stormwater management on the hydrological response.The results show that land use management has less impact on the hydrology of the catchmentthan stormwater management.

Urban waste water collecting systems are designed to convey domestic, industrial and storm water. When sizing sewer network, heavy rainfall must be considered to provide the needed hydraulic capacity for collection. Maintenance is also required in order to avoid anomalies such as inflow, infiltration and unusual polluted discharges from Combined Sewer Overflows (CSOs). Inflow and infiltration decrease the treatment yield at the Waste water Treatment Plant (WWTP) and participate in hydraulic overloads and overflows. CSOs have a direct impact on the pollution of water bodies and must be strictly sized and monitored. Detecting sizing and maintenance anomalies is crucial to ensure public health and a good status of our natural environment. Today, numerical hydraulic models support consulting engineers in assessing overflows then in choosing the best technical and cost-effective scenario. The objectives of this paper are to review the dysfunctions of collecting system and to understand how a numerical hydraulic model is constructed, calibrated and then used to establish a Corrective Action Plan (CAP). My master thesis is based on a working project achieved at SAFEGE (Group SUEZ Environnement) in the Urban Hydraulics Department in Lyon (Rhône-Alpes, France) between September 2013 and February 2014. The paper will present some outcomes obtained from an Urban Hydraulic Project at Chazelles-sur-Lyon (Rhône-Alpes, France) in the South West of Lyon. Mike Urban is the software - developed by the company DHI Water- used for the numerical hydraulic modeling.

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京都大学 / 新制・課程博士 / 博士(工学) / 甲第23179号 / 工博第4823号 / 新制||工||1754(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 清水 芳久, 講師 中田 典秀 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Les rejets urbains par temps de pluie dégradent l’état écologique des écosystèmes aquatiques et peuvent induire une exposition des populations humaines aux contaminants chimiques et microbiens (bactéries, virus, parasites). L’objectif de ce travail de thèse était d’évaluer les effectifs et de prédire le devenir de bactéries pathogènes introduites dans les milieux aquatiques par une source majeure comme les eaux usées rejetées par des dispositifs tels que les déversoirs d’orage (DO) et les lagunes d’épuration (WWTL). La répartition d’un agent pathogène fortement liée aux milieux hydriques, Pseudomonas aeruginosa, a été comparée avec celles observées pour des indicateurs de contaminations fécales (E. coli et les entérocoques intestinaux), mais également avec celle de l’espèce pathogène Aeromonas caviae. La dangerosité des formes retrouvées dans ces milieux a été évaluée par approches moléculaires (PFGE et MLST). Les résultats obtenus montrent un fort apport en P. aeruginosa via les eaux usées, avec un effet significatif sur les effectifs observés en fonction de l’intensité des pluies et des périodes de temps sec, et les fluctuations du régime hydrologique et des paramètres physico-chimiques. Une grande diversité infra-spécifique des P. aeruginosa, et la capacité de certains génotypes à s’installer durablement dans ces milieux (macrophytes et périphyton) ont été observées. Certaines souches ont par ailleurs montré une parenté avec des lignées d’infections communautaires, ou encore des clones épidémiques majeurs (PA14 et C). Des études en microcosme ont été effectuées pour valider les interactions observées avec certains macrophytes, et identifier des propriétés d’adhérence bactérienne (dont les lectines) impliquées dans ces interactions. Ces travaux ont impliqué une analyse de la distribution des gènes lecA et lecB, codant des lectines chez P. aeruginosa, et une étude de leurs ligands. Le gène lecA a été localisé dans une zone de forte plasticité génomique. Ces travaux ont permis la description d’une nouvelle structure de l’adhésine LecB / Urban wet-weather discharges degrade the ecological status of aquatic ecosystems and may expose human populations to chemical and microbial contaminants (bacteria, viruses, parasites). The aim of this thesis was to evaluate the numbers and predict the fate of pathogenic bacteria introduced into aquatic ecosystems by a major source like wastewater from devices such as combined sewer overflows (CSO) and wastewater treatment lagoons (WWTL). The distribution of a human pathogen closely linked to hydric environments, Pseudomonas aeruginosa, was compared with those observed for fecal indicators (E. coli and intestinal enterococci), but also with that of Aeromonas caviae pathogenic species. Dangerousness of strains found in these environments was evaluated by molecular approaches (PFGE and MLST). Obtained results showed a high contribution of wastewater in P. aeruginosa release, with a significant effect of rainfall intensity and preceding dry periods, in addition to changes in hydrological regime and physico-chemical parameters on recorded data. A large infra-specific diversity was observed within P. aeruginosa and the ability of some genotypes to colonize permanently aquatic surfaces (macrophytes and periphyton) were observed. Some strains showed a kinship with lineages of community infections or major epidemic clones (PA14 and C). Microcosm studies were performed to validate observed interactions with macrophytes, and to identify bacterialadhesion properties (including lectins) involved in these interactions. These investigations involved analysis of the distribution of lectin encoding loci lecA and lecB within P. aeruginosa, and a study of their ligands. lecA was located in a highly unstable genomic region. This work allowed the description of a new structure of the adhesin LecB

Made available in DSpace on 2016-06-02T20:00:16Z (GMT). No. of bitstreams: 1 DissAVF.pdf: 3142723 bytes, checksum: d32fec625d6a407ea812f6bfdadc0c46 (MD5) Previous issue date: 2006-02-28 / The main purpose of this work was to identify and to estimate the rainfall derived infiltration and inflow (IADP) into the sewer in a small city and to do an analysis of their influence on this collection system. This work was composed by field measurements on the sewage flowrate output and volume arriving to the Sewage Pumping Plant Station, simultaneously to the collection and measurement of rainfall on the corresponding same urban watershed area of the Borborema, in São Paulo State. The existing methodologies were divided in the follow steps: choosing and characterization of the working field; data collection of the sewage volume and flowrate; collection of the rainfall data; choosing and characterization of the data collected; employment of existing models and methodologies useful to the present available data, in order to derive the underground water infiltration rate and the overflow occurring in the sewage colletion network. The volumetric and percentile IADP were quantified. The constant unitary rate (TUC) was also estimated, by several ways. The results allowed obtaining mathematical formulations to derive the IADP and TUC of the Borborema s sewage network. Such mathematical formulations are suitable to be applied in urban areas of cities of a similar to that of Borborema. It was estimated that the IADP can generate flowrates 2,5 times greater than the sewage flows in dry weather and infiltration rates in the sewage network similar to those adopted for system design. The work allowed to estimate how complex are the infiltration and inflow of stormwater in the sewer network. The methodologies applied were quite simple and may be easily applied in other cities. / O objetivo precípuo desta pesquisa foi identificar e estimar a infiltração e o afluxo devidos da precipitação (IADP) na rede de esgoto sanitário em município de pequeno porte e efetuar uma análise da sua influência no sistema de esgotamento sanitário. Este trabalho foi realizado através da medição em campo de vazão e volume de esgoto sanitário a montante da EEE Estação Elevatória de Esgoto, simultaneamente com a coleta e medição da chuva precipitada na correspondente área urbanizada da bacia hidrográfica da cidade de Borborema/SP. As metodologias empregadas foram divididas nas seguintes fases: escolha e caracterização do local da pesquisa; coleta de dados de vazão e volume dos efluentes de esgotos sanitários e coleta de dados de precipitação de chuva no local do estudo; seleção e caracterização dos dados coletados; aplicação das metodologias e modelagens existentes possíveis de aplicar em função dos dados disponíveis, para a obtenção da taxa de infiltração das águas subterrâneas e da sobrecarga na rede de esgoto sanitário. Quantificou-se o IADP volumétrica e percentualmente. Calculou-se também a taxa unitária constante (TUC), sob diferentes formatos. Os resultados desta pesquisa permitiram a obtenção de equações matemáticas para avaliação da IADP e da TUC do sistema de esgoto da cidade de Borborema que possivelmente poderão ser aplicadas em áreas urbanizadas de outras cidades do mesmo porte. Estimou-se que a IADP pode gerar volumes 2,5 vezes maiores que o volume de esgotos em tempo seco, bem como taxas de infiltração na rede coletora próximas àquelas adotadas para cálculo da rede. O trabalho possibilitou avaliar quão complexos são a infiltração e o afluxo de águas de chuva na rede de esgoto sanitário. Os métodos empregados, tanto de coleta quanto de modelagem, foram muito simples e podem ser aplicados em outras cidades com certa facilidade.