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Estudio Molecular De Poblaciones De Pseudomonas Ambientales

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Estudio Molecular De Poblaciones De Pseudomonas Ambientales Universitat de les Illes Balears ESTUDIO MOLECULAR DE POBLACIONES DE PSEUDOMONAS AMBIENTALES T E S I S D O C T O R A L DAVID SÁNCHEZ BERMÚDEZ DIRECTORA: ELENA GARCÍA-VALDÉS PUKKITS Departamento de Biología Universitat de les Illes Balears Palma de Mallorca, Septiembre 2013 Universitat de les Illes Balears ESTUDIO MOLECULAR DE POBLACIONES DE PSEUDOMONAS AMBIENTALES Tesis Doctoral presentada por David Sánchez Bermúdez para optar al título de Doctor en el programa Microbiología Ambiental y Biotecnología, de la Universitat de les Illes Balears, bajo la dirección de la Dra. Elena García-Valdés Pukkits. Vo Bo Director de la Tesis El doctorando DRA. ELENA GARCÍA-VALDÉS PUKKITS DAVID SÁNCHEZ BERMÚDEZ Catedrática de Universidad Universitat de les Illes Balears PALMA DE MALLORCA, SEPTIEMBRE 2013 III IV Index Agradecimientos .................................................................................................... IX Resumen ................................................................................................................ 1 Abstract ................................................................................................................... 3 Introduction ............................................................................................................ 5 I.1. The genus Pseudomonas ............................................................................................ 7 I.1.1. Definition ................................................................................................................ 7 I.1.2. Pseudomonas genus history ................................................................................... 7 I.1.3. Phenotypic characteristics of Pseudomonas .......................................................... 9 I.1.4. Genotypic characteristics of Pseudomonas ........................................................... 9 I.1.5. Ecology .................................................................................................................. 11 I.2. Pseudomonas aeruginosa ......................................................................................... 13 I.3. Taxonomy of the genus Pseudomonas ..................................................................... 16 I.4 The rpoD gene ............................................................................................................ 21 I.4.1. Design and specifity test of the primer set PsEG30F/PsEG790R .......................... 21 I.4.2. Detection of Pseudomonas in environmental DNA .............................................. 23 I.4.3. Analysis, comparison and selection of genes for phylogenic studies of the genus Pseudomonas .............................................................................................. 24 I.5. Next-Generation Sequencing Technology (NGST) .................................................... 26 I.5.1. Illumina HiSeq 2000 technology ........................................................................... 26 I.5.2. Roche 454/GS FLX Sequencing technology .......................................................... 26 Objectives ............................................................................................................. 30 Chapter 1: Population structure of environmental and clinical Pseudomonas aeruginosa isolates from the same geographical area ............................................ 34 Abstract ........................................................................................................................... 36 1.1. Introduction ............................................................................................................. 38 1.2. Material and Methods ............................................................................................. 39 1.2.1. P. aeruginosa isolates .......................................................................................... 39 1.2.2. Antibiotic resistance ............................................................................................ 39 1.2.3. DNA extraction, PCR amplification and DNA sequencing conditions .................. 40 1.2.4. MultiLocus Sequence Typing ............................................................................... 40 1.2.5. Phylogenetic reconstruction ................................................................................ 40 1.2.6. Population structure ............................................................................................ 41 1.2.7. Genetic Diversity index ........................................................................................ 41 1.2.8. Clonality and recombination tests ....................................................................... 41 1.3. Results ...................................................................................................................... 42 1.3.1. P. aeruginosa isolates .......................................................................................... 42 1.3.2. Antibiotic resistance pattern ............................................................................... 42 1.3.3. MultiLocus Sequence typing ................................................................................ 42 1.3.4. MLST genes phylogeny ........................................................................................ 47 1.3.5. Population structure ............................................................................................ 47 V 1.3.6. Genetic diversity index ........................................................................................ 51 1.3.7. Clonality and recombination tests ....................................................................... 51 1.4. Discussion ................................................................................................................ 51 1.5. Supplementary data ................................................................................................ 54 Chapter 2: Genome analysis of environmental and clinical P. aeruginosa isolates from Sequence Type ST-1146 ................................................................................. 58 Abstract ........................................................................................................................... 60 2.1. Introduction ............................................................................................................. 62 2.2. Materials and Methods ........................................................................................... 64 2.2.1. P. aeruginosa isolates .......................................................................................... 64 2.2.2. De novo assembled genome analysis .................................................................. 64 2.2.3. Average Nucleotide Index (ANIb) and conserved DNA ........................................ 64 2.2.4. Core and Pan-genome analysis ............................................................................ 64 2.2.5. Gene prediction and clustering analysis .............................................................. 65 2.2.6. Genome mutational profiles ................................................................................ 65 2.2.7. Polymorphic sites and nucleotide substitutions .................................................. 65 2.2.8. Phylogenetic reconstructions .............................................................................. 65 2.3. Results ...................................................................................................................... 66 2.3.1. Whole genome characteristics of isolates in ST-1146 ......................................... 66 2.3.2. Average Nucleotide Index (ANIb) and conserved DNA ........................................ 66 2.3.3. Core and Pan-genome analysis ............................................................................ 66 2.3.4. Gene comparisons with CD-HIT ........................................................................... 70 2.3.5. Genome mutational profiles and allele comparisons .......................................... 72 2.3.6. Nucleotide polymorphisms ................................................................................. 76 2.3.7. Pathogenic factors ............................................................................................... 77 2.3.8. Phage related genes ............................................................................................ 81 2.4. Discussion ................................................................................................................ 83 2.5. Supplementary data ................................................................................................ 89 Chapter 3: Assessment of the species diversity of Pseudomonas by culture- dependent and -independent methods in a water sample from the river Woluwe ............................................................................................................... 101 Abstract ......................................................................................................................... 103 3.1. Introduction ........................................................................................................... 105 3.2. Material and methods ........................................................................................... 106 3.2.1. Water Sampling ................................................................................................. 106 3.2.2. Total cell count by DAPI ....................................................................................
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