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Tesis Doctoral 2014 Filogenia Y Evolución De Las Poblaciones Ambientales Y Clínicas De Pseudomonas Stutzeri Y Otras Especies

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Tesis Doctoral 2014 Filogenia Y Evolución De Las Poblaciones Ambientales Y Clínicas De Pseudomonas Stutzeri Y Otras Especies TESIS DOCTORAL 2014 FILOGENIA Y EVOLUCIÓN DE LAS POBLACIONES AMBIENTALES Y CLÍNICAS DE PSEUDOMONAS STUTZERI Y OTRAS ESPECIES RELACIONADAS Claudia A. Scotta Botta TESIS DOCTORAL 2014 Programa de Doctorado de Microbiología Ambiental y Biotecnología FILOGENIA Y EVOLUCIÓN DE LAS POBLACIONES AMBIENTALES Y CLÍNICAS DE PSEUDOMONAS STUTZERI Y OTRAS ESPECIES RELACIONADAS Claudia A. Scotta Botta Director/a: Jorge Lalucat Jo Director/a: Margarita Gomila Ribas Director/a: Antonio Bennasar Figueras Doctor/a por la Universitat de les Illes Balears Index Index ……………………………………………………………………………..... 5 Acknowledgments ………………………………………………………………... 7 Abstract/Resumen/Resum ……………………………………………………….. 9 Introduction ………………………………………………………………………. 15 I.1. The genus Pseudomonas ………………………………………………….. 17 I.2. The species P. stutzeri ………………………………………………......... 23 I.2.1. Definition of the species …………………………………………… 23 I.2.2. Phenotypic properties ………………………………………………. 23 I.2.3. Genomic characterization and phylogeny ………………………….. 24 I.2.4. Polyphasic identification …………………………………………… 25 I.2.5. Natural transformation ……………………………………………... 26 I.2.6. Pathogenicity and antibiotic resistance …………………………….. 26 I.3. Habitats and ecological relevance ………………………………………… 28 I.3.1. Role of mobile genetic elements …………………………………… 28 I.4. Methods for studying Pseudomonas taxonomy …………………………... 29 I.4.1. Biochemical test-based identification ……………………………… 30 I.4.2. Gas Chromatography of Cellular Fatty Acids ................................ 32 I.4.3. Matrix Assisted Laser-Desorption Ionization Time-Of-Flight Mass Spectrometry ...................................................................................... 32 I.4.4. DNA-DNA hybridization and G+C %mol content ......................... 35 I.4.5. Multilocus sequence analysis ....................................................... 35 Objectives …………………………………………………………………………. 37 Chapter 1: Identification and genomovar assignation of clinical strains of Pseudomonas stutzeri .…………………………………………………………….. 41 1.1. Introduction ………………………………………………………………. 43 1.2. Methods …………………………………………………………………... 44 1.3. Results ……………………………………………………………………. 45 1.4. Discussion ………………………………………………………………… 49 1.5. Supplemental material ……………………………………………………. 52 Chapter 2: Whole-cell MALDI-TOF mass spectrometry and multilocus sequence analysis in the discrimination of Pseudomonas stutzeri populations: three novel genomovars ………………………………………………………….. 61 2.1. Introduction ………………………………………………………………. 63 2.2. Methods …………………………………………………………………... 64 2.2.1. Strains studied and growth conditions ……………………………... 64 2.2.2. DNA extraction, PCR and sequencing protocols ………………….. 64 2.2.3. Sequence analysis ………………………………………………….. 66 2.2.4. DNA-DNA Hybridisation …………………………………………. 67 2.2.5. Phenotypic Tests …………………………………………………… 67 2.2.6. WC-MALDI-TOF Mass Spectrometry ……………………………. 67 2.2.7. Nucleotide Sequence Accession Numbers ………………………… 68 2.3. Results ……………………………………………………………………. 68 2.3.1. MLSA ……………………………………………………………… 68 2.3.2. Whole-Cell MALDI-TOF Mass Spectrometry ……………………. 74 2.4. Discussion ………………………………………………………………… 76 2.5. Supplemental material ……………………………………………………. 78 Chapter 3: Concordance between whole-cell matrix-assisted laser- desorption/ ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the genus Pseudomonas ……………………………………………………………………… 99 5 Index 3.1. Introduction ………………………………………………………………. 101 3.2. Methods …………………………………………………………………... 103 3.2.1. Bacterial strains and culture conditions ……………………………. 103 3.2.2. Sequencing conditions and sequence analysis …………………….. 103 3.2.3. WC-MALDI-TOF mass spectrometry …………………………….. 104 3.3. Results ……………………………………………………………………. 106 3.3.1. Multilocus sequence analysis ……………………………………… 106 3.3.2. WC-MALDI-TOF MS analysis ……………………………………. 107 3.3.3. Resolution at the group and subgroup level within the genus Pseudomonas ……………………………………………………………... 111 3.3.4. Resolution at the genus and species levels ………………………… 113 3.4. Discussion ………………………………………………………………… 114 3.5. Supplemental material ……………………………………………………. 116 Chapter 4: Environmental microbiota represents a natural reservoir for dissemination of clinically relevant metallo--lactamases …………………….. 117 4.1. Introduction ………………………………………………………………. 119 4.2. Methods and Results ……………………………………………………… 119 Chapter 5: Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov. … 127 5.1. Introduction ………………………………………………………………. 129 5.2. Methods and Results ……………………………………………………… 130 5.3. Description of Brevundimonas faecalis sp. nov. …………………………. 136 5.4. Supplemental material ……………………………………………………. 137 Conclusions ……………………………………………………………………….. 149 References ………………………………………………………………………… 153 6 Acknowledgments Agradecimientos Quisiera agradecer en general a todas las personas que han estado presentes durante en esta etapa de mi vida. Quiero agradecer por orden histórico al Dr. Rafael Bosch por sus valiosas clases que fueron mi primer “enganche” a la Microbiología, el me transmitió su pasión por allá en el 2004 cuando cursaba Métodos y Técnicas de Microbiología y me dio la oportunidad de colaborar en algunos proyectos con Celia. A Jorge Lalucat por ser mi director de tesis y encontrar un momento para mí incluso siendo decano de la Universidad. Gracias por aportar siempre una solución a cualquier problema que se me presentaba, por respetar mis decisiones y mi manera de trabajar y sobre todo por enseñarme a ser autónoma. Siento una profunda admiración por ti porque logras guiarnos a todos y sacar lo mejor de cada uno para alcanzar un proyecto común. Gracias también por tu aliento y paciencia en estos últimos años alejada del laboratorio. A Elena por las charlas y discusiones que empezaban con genomovares y terminaban en recetas de empanadas e historias de hijos y madres. Tengo el recuerdo de tu despacho lleno de papeles entre los cuales siempre encontrabas “esa” lista de gliceroles o “ese” artículo que estaba necesitando… Gracias. Gracias a Toni Bennasar por enseñarme el lado más moderno de la microbiología, buscando nuevos experimentos y técnicas por aplicar, por insistirme en leer mucho y tenerme paciencias en mis comienzos. Gracias a los demás profesores del laboratorio por las enseñanzas no solo teóricas sino del trabajo diario en el laboratorio. Gracias Balbi por obligarme a usar la bata e inculcarme buenos hábitos de trabajo y por organizar tan enriquecedores seminarios. Y bueno, les llega el turno a los compañeros del laboratorio. Qué les puedo decir que no sepan ya. Qué los adoro! Qué buen equipo humano tenemos en el labo! Siempre nos ayudamos y apoyamos entre todos. Gracias por ser no solo compañeros de laboratorio sino amigos. Fue una suerte poder trabajar con todos ustedes. Con Cris y más tarde con Claudia P. siempre liadas con los muestreos… colapsando la cabina; con Magda y David peleándonos por los turnos de PCR, que risas siempre en el labo cuando hacíamos PCRs con Mariana y cantábamos las cantidades de primers como si fuera el sorteo de navidad… Tengo infinitos recuerdos y experiencias inolvidables con todos (no me olvido de Joseph, Mariuchi, Joaneta, Sebas, Lady, Arantxa, Toni B., Zoyla, Celia, Laura, Mariette, Pau, Farith, Mohamed, etc). Gracias a todos. Muchas gracias a Sebastián Albertí, Antonio Oliver, Carlos Juan y Antonio Ramirez por su colaboración en el estudio de aguas fecales de Son Llàtzer. 7 Acknowledgments Muchas gracias Francisca Barceló y Jesús Prades por abrirme las puertas de la investigación en UIB y enseñarme lo que es la tenacidad y constancia. Gracias a Aina y Andreu en la recepción del edificio aportando una sonrisa cada mañana al llegar, a las chicas de limpieza y los mozos que nos ayudaron siempre a que todo funcione bien en el laboratorio. Muchas gracias a toda mi familia y amigos por su apoyo, por entender mis tantas ausencias. Dejo para lo último y no por eso menos importante a mi querida amiga y directora Margarit. No me alcanzan las palabras para agradecerte todo los que me has dado en estos años a nivel profesional y personal. Esta tesis no hubiera sido posible sin tu ayuda, has estado en cada detalle, con la ayuda precisa y más. El tiempo que me has dedicado no tiene precio…has sido mi cable a tierra estos últimos años de maternidad y esta tesis te la dedico a ti. Gracias por aportarme tantas cosas buenas y sobre todo por aguantarme! Quiero agradecer a la Conselleria d’Economia, Hisenda i Innovacio del Govern de les Illes Balears por brindarme la oportunidad de realizar esta Tesis con una beca predoctoral para la formación de personal investigador (FPI2007) y a la Dirección general de Investigación por el proyecto CGL2006-09719/BOS en el cual se ha enmarcado esta tesis. 8 Abstract Abstract: “Phylogeny and Evolution of Environmental and Clinical Populations of Pseudomonas stutzeri and related species”. Pseudomonas stutzeri is a widely distributed species with very high genetic diversity and metabolic capacities, occupying many diverse ecological niches. In the first chapter, the identification of Pseudomonas stutzeri clinical isolates through conventional phenotypic methods was compared with identification through partial rpoD gene sequencing. We observed that commercial phenotypic systems easily confuse P. stutzeri with other Pseudomonas species. We also demonstrated that most of the clinical strains of P. stutzeri herein studied (79%) belonged to genomovar 1 of the species. We propose the use of partial rpoD gene sequence
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