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Epidemiological and Molecular Analysis of Virulence and Antibiotic Resistance in Acinetobacter Baumannii

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Epidemiological and Molecular Analysis of Virulence and Antibiotic Resistance in Acinetobacter Baumannii UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE VETERINARIA DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR IV TESIS DOCTORAL Epidemiological and Molecular Analysis of Virulence and Antibiotic Resistance in Acinetobacter baumannii Análisis Epidemiológico y Molecular de la Virulencia y la Antibiorresistencia en Acinetobacter baumannii MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Elias Dahdouh DIRECTORA Mónica Suárez Rodríguez Madrid, 2017 © Elias Dahdouh, 2016 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE VETERINARIA DEPARTAMENTO DE BIOQUIMICA Y BIOLOGIA MOLECULAR IV TESIS DOCTORAL Análisis Epidemiológico y Molecular de la Virulencia y la Antibiorresistencia en Acinetobacter baumannii Epidemiological and Molecular Analysis of Virulence and Antibiotic Resistance in Acinetobacter baumannii MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Elias Dahdouh Directora Mónica Suárez Rodríguez Madrid, 2016 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE VETERINARIA Departamento de Bioquímica y Biología Molecular IV ANALYSIS EPIDEMIOLOGICO Y MOLECULAR DE LA VIRULENCIA Y LA ANTIBIORRESISTENCIA EN Acinetobacter baumannii EPIDEMIOLOGICAL AND MOLECULAR ANALYSIS OF VIRULENCE AND ANTIBIOTIC RESISTANCE IN Acinetobacter baumannii MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Elias Dahdouh Bajo la dirección de la doctora Mónica Suárez Rodríguez Madrid, Diciembre de 2016 First and foremost, I would like to thank God for the continued strength and determination that He has given me. I would also like to thank my father Abdo, my brother Charbel, my fiancée, Marisa, and all my friends for their endless support and for standing by me at all times. Moreover, I would like to thank Dra. Monica Suarez Rodriguez and Dr. Ziad Daoud for giving me the opportunity to complete this doctoral study and for their guidance, encouragement, and friendship. Additionally, I would like to acknowledge the help and support given by Dr. Jesus Mingorance, Dr. Belen Ortaz, Dr. Carmen San Jose, Dr. Bruno Gonzalez Zorn, Dr. Alicia Aranaz, Dña Sonsoles Pacho, Dña. Rosa Gimez-Gil, Dña. Micheline Hajjar, and “los Brunos”. This work would not have been realized if not for the wonderful support given by all these professors, colleagues, family, and friends. Finally, I would like to acknowledge the scientific journals and scientific conferences that have accepted the various parts of this work for publication. En primer lugar, quiero agradecer a Dios por darme la fuerza y determinación que necesitaba para completar este camino. Quiero agradecer también a mi padre, Abdo, mi hermano, Charbel, mi prometida, Marisa, y todos mis amigos por sus apoyos enormes y por estar a mi lado todo el tiempo. Además, quiero agradecer a la Dra. Mónica Suárez Rodríguez y al Dr. Ziad Daoud por darme la oportunidad de hacer un doctorado y por su dirección, animo, y amistad durante este tiempo. A continuación, quiero agradecer al Dr. Jesús Mingorance, a la Dra. Belén Ortgaz, a la Dra. Carmen San José, al Dr. Bruno González Zorn, a la Dra. Alicia Aranaz, a Dña. Sonsoles Pacho, a Dña. Rosa Gómez-Gil, a Dña. Micheline Hajjar, y “los Brunos” por sus ayudas y apoyos. Completar este trabajo no habría sido posible sin el apoyo maravilloso de todos estos profesores, colegas, familiares, y amigos. Para finalizar, quiero agradecer a las revistas y congresos científicos que han aceptado partes de este trabajo para sus publicaciones. Da Mónica Suárez Rodríguez, Profesora Titular de Universidad del Departamento de Sanidad Animal de la Facultad de Veterinaria de la Universidad Complutense de Madrid, CERTIFICA: Que la Tesis Doctoral titulada “Análisis Epidemiológico y Molecular de la Virulencia y la Antibiorresistencia en Acinetobacter baumannii”, que presenta el Titulado en Máster en Ciencias Biomédicas, D. Elias Dahdouh, ha sido realizada bajo mi dirección en el Departamento de Sanidad Animal de la Facultad de Veterinaria dentro del programa de tercer ciclo “Bioquímica, Biología Molecular, y Biomedicina”, y estimamos que cumple todos los requisitos necesarios para optar al grado de Doctor por la Universidad Complutense de Madrid. Madrid, Noviembre de 2016 Fdo.: Dra. Mónica Suárez Rodríguez “Ask, and it shall be given you; seek, and ye shall find; knock, and it shall be opened unto you: For every one that asketh receiveth; and he that seeketh findeth; and to him that knocketh it shall be opened.” The Bible - Matthew 7:7-8 To my beloved Missa, amazing father, and wonderful brother. INDEX Ia. RESUMEN 1 Ib. SUMMARY 15 II. LIST OF ABBREVIATIONS 27 III. LIST OF FIGURES 31 IV. LIST OF TABLES 32 V. INTRODUCTION 35 1. CHARACTERISTICS OF Acinetobacter baumannii 37 1.1. History of the Genus Acinetobacter 37 1.2. General Characteristics of Acinetobacter spp. 39 1.3. Identification of Acinetobacter spp. 40 1.4. Natural Habitat of Acinetobacter spp. 44 2. PATHOGENESIS AND VIRULENCE OF Acinetobacter baumannii 44 2.1. Virulence of Acinetobacter baumannii 45 2.1.1. Formation of Biofilms 45 2.1.2. Proteolytic Activity and Siderophore Production 46 2.1.3. Hemolysis and Surface Motility 47 2.1.4. Other Factors Contributing to Virulence in Acinetobacter baumannii 48 2.2. Nosocomial Infections Caused by Acinetobacter baumannii 49 3. TREATMENT OPTIONS FOR Acinetobacter baumannii 51 3.1. Beta-Lactams 52 3.2. Aminoglycosides 54 3.3. Polymyxins 55 3.4. Tetracyclines and Glycylcyclines 56 3.5. Fluoroquinolones 56 3.6. Folic Acid Synthesis Inhibitors 57 3.7. Combination Therapies 58 4. RESISTANCE TO ANTIMICROBIAL AGENTS 59 4.1. Innate Mechanisms of Resistance in Acinetobacter baumannii 59 4.2. Acquired Mechanisms of Resistance in Acinetobacter baumannii 60 4.2.1. Non-Enzymatic Mechanisms of Resistance 60 4.2.1.1. Efflux Pumps 61 4.2.1.2. Down-Regulation of Porins 63 4.2.1.3. Changes in the Target of Antimicrobial Agents 63 4.2.2. Enzymatic Mechanisms of Resistance 65 4.2.2.1. Beta-Lactamases 66 4.2.2.1.1. Ambler Class A Enzymes 67 4.2.2.1.2. Ambler Class B Enzymes 69 4.2.2.1.3. Ambler Class C Enzymes 72 4.2.2.1.4. Ambler Class D Enzymes 73 4.2.2.1.4. In-vitro Detection of Beta-Lactamases 82 4.2.2.2. Aminoglycoside Modifying Enzymes 85 4.3. Resistance to Colistin 86 5. GLOBAL EPIDEMIOLOGY OF RESISTANT Acinetobacter baumannii 89 5.1. Common Molecular Tools Used for Epidemiological Studies 89 5.2. Worldwide Dissemination of Multi-Drug Resistant A. baumannii Clones 92 5.3. Current Global Rates of Resistant Acinetobacter baumannii 100 6. RELATIONSHIP BETWEEN VIRULENCE AND RESISTANCE 103 6.1. Effects of Resistance on Bacterial Fitness and Virulence 104 6.2. Biological Cost of Resistance in Acinetobacter baumannii 107 VI. OBJECTIVES AND JUSTIFICATION 111 VII. RESULTS 115 1. Phenotypic and Genotypic Characterization of Acinetobacter baumannii Strains 117 Isolated from a Spanish Hospital 1.1. Abstract 117 1.2. Introduction 119 1.3. Materials and Methods 120 1.3.1. Bacterial Strains 120 1.3.2. Antimicrobial Susceptibility Testing 121 1.3.3. Polymerase Chain Reactions 121 1.3.4. Pulsed Field Gel Electrophoresis 122 1.3.5. Surface Motility 122 1.3.6. Biofilm Formation 122 1.3.7. Hemolytic Activity 122 1.3.8. Proteolytic Activity 123 1.3.9. Siderophore Production 123 1.3.10. Growth Curves 123 1.3.11. Statistical Analysis 124 1.4. Results 124 1.4.1. Distribution of Bacterial Isolates 124 1.4.2. Antibiotic Susceptibility Profiles 124 1.4.3. Detection of Carbapenemases and Virulence Genes 125 1.4.4. Clusters, Clones and Carbapenemases 125 1.4.5. Determination of the Virulence Factors and Association with Clonality 128 and Carbapenemases 1.5. Discussion 131 1.6. Conclusions 133 2. Phenotypic and Genotypic Characterization of Acinetobacter baumannii Strains 137 Isolated from a Lebanese Hospital 2.1. Abstract 137 2.2. Introduction 139 2.3. Materials and Methods 140 2.3.1. Bacterial Strains 140 2.3.2. Antimicrobial Susceptibility Testing 140 2.3.3. Polymerase Chain Reactions 141 2.3.4. Growth Curves 141 2.3.5. Biofilm Formation 141 2.3.6. Hemolytic Activity 141 2.3.7. Siderophore Production 142 2.3.8. Surface Motility 142 2.3.9. Proteolytic Activity 142 2.3.10. Statistical Analysis 142 2.4. Results 143 2.4.1. Bacterial Isolates 143 2.4.2. Antibiotic Susceptibility 143 2.4.3. Dissemination of Carbapenemases and International Clones 144 2.4.4. Virulence Determinants in Relation with Clonality and Carbapenem 144 Susceptibility 2.4.5. Associations between Virulence and Resistance 147 2.5. Discussion 147 2.6. Conclusions 149 3. Genomic and Phenotypic Characterization of two Colistin Resistant Acinetobacter baumannii Clinical Isolates in Comparison to their Sensitive 153 Counterparts 3.1. Abstract 153 3.2. Introduction 155 3.3. Materials and Methods 156 3.3.1. Bacterial Strains 156 3.3.2. Determination of Clonality 156 3.3.3. Sequencing of the pmrCAB Operon 156 3.3.4. Polymerase Chain Reactions 157 3.3.5. Full Genome Sequencing 157 3.3.6. Growth Curves 158 3.3.7. Hemolysis 158 3.3.8. Biofilm Formation 158 3.3.9. Motility 158 3.3.10. Proteolytic Activity 159 3.3.11. Siderophore Production 159 3.4. Results 159 3.4.1. Patient History 159 3.4.2. Sequencing of the pmrCAB Operon 161 3.4.3. Detection of Carbapenemases by PCR 161 3.4.4. Clonality Analysis 161 3.4.5. Whole-Genome Sequencing 162 3.4.6. Generation times and in-vitro Virulence 163 3.5. Discussion 164 3.6. Conclusions 166 4. Different Patterns and Kinetics of Biofilms Produced by Acinetobacter 169 baumannii clinical isolates with Different Antibiotic Susceptibility Profiles 4.1. Abstract 169 4.2. Introduction 171 4.3. Materials and Methods 172 4.3.1. Bacterial Strains 172 4.3.2. Biofilm Experimental System 173 4.3.3. Cell Recovery and Counting 173 4.3.4. Siderophore Determination in CAS Solution 174 4.3.5.
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