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Compartmentalization of Class 1 Integrons and Incp-1 Plasmids In Compartmentalization of class 1 integrons and IncP-1 plasmids in the Orne river (France), an aquatic ecosystem impacted by urban and industrial anthropogenic pressures Magali de la Cruz Barrón To cite this version: Magali de la Cruz Barrón. Compartmentalization of class 1 integrons and IncP-1 plasmids in the Orne river (France), an aquatic ecosystem impacted by urban and industrial anthropogenic pressures. Ecosystems. Université de Lorraine, 2018. English. NNT : 2018LORR0212. tel-02096872 HAL Id: tel-02096872 https://hal.univ-lorraine.fr/tel-02096872 Submitted on 11 Apr 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. AVERTISSEMENT Ce document est le fruit d'un long travail approuvé par le jury de soutenance et mis à disposition de l'ensemble de la communauté universitaire élargie. Il est soumis à la propriété intellectuelle de l'auteur. Ceci implique une obligation de citation et de référencement lors de l’utilisation de ce document. D'autre part, toute contrefaçon, plagiat, reproduction illicite encourt une poursuite pénale. Contact : [email protected] LIENS Code de la Propriété Intellectuelle. articles L 122. 4 Code de la Propriété Intellectuelle. articles L 335.2- L 335.10 http://www.cfcopies.com/V2/leg/leg_droi.php http://www.culture.gouv.fr/culture/infos-pratiques/droits/protection.htm Ecole Doctorale BioSE (Biologie-Santé-Environnement) Thèse Présentée et soutenue publiquement pour l’obtention du titre de DOCTEUR DE l’UNIVERSITE DE LORRAINE Mention : « Sciences de la Vie et de la Santé » par Magali DE LA CRUZ BARRÓN Compartmentalization of class 1 integrons and IncP-1 plasmids in the Orne river (France), an aquatic ecosystem impacted by urban and industrial anthropogenic pressures 20 décembre, 2018 Membres du jury : Rapporteurs : Mme. Isabelle KEMPF DR1 Anses-DVM, HDR, Laboratoire ANSES, Ploufragan Mme. Sandra DA RE CR1 Inserm, HDR, UMR Inserm 1092 RESINFIT, Limoges Examinateurs : Mme. Sophie PAYOT-LACROIX DR INRA, HDR, Laboratoire DYNAMIC, UMR1128 INRA-UL, Nancy M. Régis GRIMAUD Professeur, HDR, Université de Pau; IPREM UMR5254 - UPPA/CNRS « « M. Christophe MERLIN MCU, HDR, LCPME UMR7564 CNRS-UL, Nancy, directeur de thèse M. Xavier BELLANGER MCU, LCPME UMR7564 CNRS-UL, Nancy, co-directeur de thèse ----------------------------------------------------------------------------------------------------------------------------- ------ LCPME-Laboratoire de Chimie PhysiQue et Microbiologie pour les Matériaux et l'Environnement, UMR 7564 CNRS-Université de Lorraine, 15 avenue du Charmois 54500 Vandoeuvre-les-Nancy, France. ACKNOWLEDGMENTS I would like to start by addressing my sincerely and gratitude for several people who have supported me during my PhD, and all those who contributed to the successful completion of this work. I would like to thank all the jury members for taking the time to be part of the defense and assess of this work. I would like to thank Professor Armand Maul for his kindness in guiding my path in the implementation of statistics, Emmanuelle Montarg̀s-Pelletier for her support, expertise, collaboration and helpful feedback during the course of this project. My sincere gratitude to my supervisors, Christophe Merlin, Xavier Bellanger and Laurence Mathieu for their invaluable advice, guidance and encouragement during these years. I'm very thankful for their support during the hardest moments of my life. My sincere thanks also goes to all members of the LCPME MIC team with special thanks to Hélène, Sylvie, David, Edith, Audrey, Maryse, and to Lorine and Arthur, master students, for their collaboration in this work. I thank to the best officemates, Anaïs, Véronica and Zineb, for their friendship, for all the fun (in and outside the lab), and for their encouragement in the difficult moments. Also, I would like to acknowledge the financial support from the different institutions: the National Council on Science and Technology of Mexico (CONACYT) scholarship (reference 410720), the Agence Nationale de la Recherche ANR (MOBISED project, ANR-14-CE01-0019), the Rhin-Meuse Water Agency (AERM project QUALIORNE), the LTSER-France, and the Lorraine Region through the research network of Zone Atelier Moselle (ZAM). I want to thank ALL my latino-friends who became my family and who made these years away from home, less difficult and very enjoyable, especially to team Mexico represented by Mariana, Carmen. Piña, Marcela, Bibiana, Yara (My favorite mexas in Germany). Team cuba, Claudia, Alejandro, Alexis, Mariana (Brazil) for all the moments we spent together, the shared meals, the dancing nights… I really going to miss you all guys. Last but not least, I would like to express my profound gratitude to my family and to Gonzalo for their love and constant support, for being my strength when I was weak, without them this wouldn’t be possible. I dedicate this thesis to my parents, to my brother César, to my nieces and my nephew, my boyfriend and to my brother Eric, who rests in peace, he is always in my thoughts and in my heart. Gracias familia, los amo con todo mi corazón, ustedes son el viento que impulsa mis alas. ABBREVIATIONS AND ACRONYMS ANOVA Analysis of variance ARG Antibiotic-Resistance Gene ARB Antibiotic Resistant Bacteria CFC Continuous Flow Centrifugation CV Coefficient of Variation DDD Defined Daily Doses DGGE Denaturing Gradient Gel Electrophoresis DNA Deoxyribonucleic acid DREAL Regional Department of the Environment, Planning and Housing DOC Dissolved Organic Carbon EC Electrical Conductivity EEA European Economic Area EDX Energy Dispersive X-ray Spectroscopy EPA Environmental Protection Agency EU European Union FCM Flow Cytometry FSC Forward Scatter Light HGT Horizontal Gene Transfer ICE Integrative Conjugative Element IME Integrative Mobilizable Element intl 1 Class 1 integrons marker LIEC The Interdisciplinary Laboratory for Continental Environments MGE Mobile Genetic Elements MOBISED Modelling the remobilization of sediments and the release of associate contaminants N-PAC Nitrogen Polycyclic Aromatic compounds OD Optical Density O-PAC Oxygenated Polycyclic Aromatic compounds oriT Origin of Transfer PAC Polycyclic Aromatic Compounds PAH Polycyclic aromatic hydrocarbon PCA Principal Component Analysis qPCR Quantitative Polymerase Chain Reaction Rif rifampicin rpm revolution per minute SIERM Syst̀me d’Information sur l’Eau Rhin-Meuse SPM Suspended Particulate Material SSC Side Scatter light Tet Tetracycline trfA IncP-1 plasmid marker UPGMA Unweighted Pair Group Method with Arithmetic Mean WHO World Health Organization WWTP Wastewater Treatment Plant TABLE OF CONTENTS INTRODUCTION ................................................................................................................... 1 1. Antibiotic resistance, more than a health issue ........................................................... 1 2. Mechanisms of antibiotic resistance ............................................................................ 5 3. Dissemination of antibiotic resistance genes between bacteria by horizontal transfer . 8 3.1 Bacterial transformation ....................................................................................... 9 3.2 Transduction ........................................................................................................ 9 3.3 Conjugation ....................................................................................................... 11 4. Mobile genetic elements, agents of DNA mobility ..................................................... 12 4.1 Transposons ...................................................................................................... 12 4.2 Integrons............................................................................................................ 14 4.3 Plasmids ............................................................................................................ 15 4.4 Integrative Conjugative Elements............................................................. 17 5. Anthropic sources of antibiotic resistance in the environment ................................... 18 6. Role of aquatic ecosystems in the spread of ARB and ARGs ................................... 21 7. Co-selection of antibiotic resistance .......................................................................... 24 8. The case of the inherited pollution in the Orne River ................................................. 25 9. Objectives of this work .............................................................................................. 27 MATERIALS AND METHODS ............................................................................................. 29 1. Microbiological assays .............................................................................................. 29 1.1 Bacterial strains and growth conditions .............................................................. 29 1.2 Crystal-violet staining of adhering biomass ........................................................ 31 1.3 Bacterial mating assays ....................................................................................
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