Plasmid bioinformatics and two applications in biotechnology

Los plásmidos, como Elementos Genéticos Móviles, son una de las fuerzas clave que moldean la evolución de las bacterias a corto plazo. Esta capacidad, que es la que está detrás de la explosión de resistencias a antimicrobianos de las últimas décadas, también puede ser coaptada para proporcionar características deseables a bacterias de interés biotecnológico. Este trabajo describe una serie de herramientas bioinformáticas para el estudio de plásmidos, entre las que destaca un novedoso método de clasificación basado en el concepto de PTU (Unidad Taxonómica Plasmídica) equivalente al de especie bacteriana. El uso de estas herramientas se ejemplifica con la descripción de dos plasmidomas de interés agrotecnológico: el plasmidoma de Xylella fastidiosa, por la reciente alarma sanitaria de este fitopatógeno en la zona mediterranea, y el plasmidoma de la familia Erwiniaceae, a la que pertenece el género Pantoea y portado habitualmente por las semillas del trigo. Estudios recientes relacionan capacidades bioestimulantes con los plásmidos de Pantoea, abriendo su uso para aplicaciones agrotecnológicas.Plasmids, as Mobile Genetic Elements, are one of the key forces shaping the short-term evolution of bacteria. This ability, which is behind the explosion of antimicrobial resistance in recent decades, can also be coapted to provide desirable characteristics to bacteria of biotechnological interest. This work describes a series of bioinformatics tools for the study of plasmids, among which stands out a novel classification method based on the concept of PTU (Plasmid Taxonomic Unit) equivalent to bacterial species. The use of these tools is exemplified by the description of two plasmidomes of agrotechnological interest: the Xylella fastidiosa plasmidome, due to the recent health alarm caused by this phytopathogen in the Mediterranean area, and the plasmidome of the Erwiniaceae family, to which the genus Pantoea belongs and usually carried by wheat seeds. Recent reports have associated biostimulant capacities with Pantoea plasmids, opening its use in agrotechnological applications.Esta tesis ha sido financiada a través del Programa Estatal de Promoción del Talento y su Empleabilidad, en el marco del Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016 – Ayudas para la formación de doctores en empresas “Doctorados Industriales” – en la convocatoria de 2017, con fondos procedentes del Ministerio de Economía, Industria y Competitividad (DI-17-09164). Para la realización de este trabajo, SANTIAGO REDONDO SALVO también ha recibido la ayuda del Programa de Doctorados Industriales de la Universidad de Cantabria, en la convocatoria 2018, con fondos aportados por el Banco Santander, la Dirección General de Innovación, Desarrollo Tecnológico y Emprendimiento Industrial de la Consejería de Innovación, Industria, Turismo y Comercio del Gobierno de Cantabria y fondos propios de la Universidad de Cantabria (UC)

Aplicación a un receptor GPS de correcciones diferenciales recibidas mediante redes móviles (DGPS-IP)

El principal objetivo del presente proyecto ha consistido en el desarrollo de una aplicación que permite el estudio pormenorizado de los sistemas DGPS sobre IP. La aplicación proporciona los medios para conectarse a diversos receptores, recibiendo datos de estos equipos, habilitando su configuración y proporcionándole las correcciones diferenciales que habilitan el funcionamiento de los receptores diferenciales. En concreto este proyecto se ha centrado en los modelos de receptores Placer y Lassen de la casa Trimble, implementándose el protocolo TSIP para realizar las labores de configuración y toma de datos. El envío de las correcciones diferenciales al receptor se realizó mediante el protocolo RTCM, obtenendose dichas correcciones de servidores conectados por TCP/IP mediante la implementación de la parte cliente del protocolo NTRIP. La aplicación desarrollada proporciona un marco en el que desarrollar pruebas con diversos receptores GPS, permitiendo un funcionamiento interactivo con el que realizar una toma de contacto con los diversos escenarios o un uso automatizado que permite la toma de datos en escenarios más complejos. Estas características hacen de ella una competente herramienta auxiliar que se puede utilizar como entrenador de prácticas. Para la comprobación del funcionamiento de la aplicación se desarrollaron una serie de pruebas con las que caracterizar el comportamiento de los sistemas DGPS cuando reciben las correcciones mediante redes de datos inalámbricas. Dichas pruebas han consistido en la caracterización del error cuando se utilizan correcciones diferenciales correspondientes a estaciones situadas a distintas distancias, cuanticando la mejora de prestaciones respecto a la utilización del GPS sin correcciones, y el estudio del comportamiento de dichos sistemas frente a distintos retardos en la llegada de las correcciones

Antimicrobial Resistance Determinants in Genomes and Plasmids from Acinetobacter baumannii Clinical Isolates

Acinetobacter baumannii is a Gram-negative coccoid rod species, clinically relevant as a human pathogen, included in the ESKAPE group. Carbapenem-resistant A. baumannii (CRAB) areconsidered by the Worl Health Organization (WHO) as a critical priority pathogen for the research and development of new antibiotics. Some of the most relevant features of this pathogen are its intrinsic multidrug resistance and its ability to acquire rapid and effective new resistant determinants against last-resort clinical antibiotics, mostly from other ESKAPE species. The presence of plasmids and mobile genetic elements in their genomes contributes to the acquisition of new antimicrobial resistance determinants. However, although A. baumannii has arisen as an important human pathogen, information about these elements is still not well understood. Current genomic analysis availability has increased our ability to understand the microevolution of bacterial pathogens, including point mutations, genetic dissemination, genomic stability, and pan- and core-genome compositions. In this work, we deeply studied the genomes of four clinical strains from our hospital, and the reference strain ATCC®19606TM, which have shown a remarkable ability to survive and maintain their effective capacity when subjected to long-term stress conditions. With that, our aim was presenting a detailed analysis of their genomes, including antibiotic resistance determinants and plasmid composition.This research was funded by ‘Plan Nacional de I+D+i and Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias PI16/01103 to J.R.-V.), Subdirección General de Redes y Centros de Investigación Cooperativa, Spanish Ministry of Economy and Competitiveness, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015) and (REIPI RD16/0016) co-financed by the European Development Regional Fund “A way to achieve Europe” ERDF and Ministerio de Ciencia e Innovación, Acciones de dinamización «Redes de Investigación» RED2018-102469-T

Conjugation inhibitors effectively prevent plasmid transmission in natural environments

Plasmid conjugation is a major route for the spread of antibiotic resistance genes. Inhibiting conjugation has been proposed as a feasible strategy to stop or delay the propagation of antibiotic resistance genes. Several compounds have been shown to be conjugation inhibitors in vitro, specifically targeting the plasmid horizontal transfer machinery. However, the in vivo efficiency and the applicability of these compounds to clinical and environmental settings remained untested. Here we show that the synthetic fatty acid 2-hexadecynoic acid (2-HDA), when used as a fish food supplement, lowers the conjugation frequency of model plasmids up to 10-fold in controlled water microcosms. When added to the food for mice, 2-HDA diminished the conjugation efficiency 50-fold in controlled plasmid transfer assays carried out in the mouse gut. These results demonstrate the in vivo efficiency of conjugation inhibitors, paving the way for their potential application in clinical and environmental settings. IMPORTANCE The spread of antibiotic resistance is considered one of the major threats for global health in the immediate future. A key reason for the speed at which antibiotic resistance spread is the ability of bacteria to share genes with each other. Antibiotic resistance genes harbored in plasmids can be easily transferred to commensal and pathogenic bacteria through a process known as bacterial conjugation. Blocking conjugation is thus a potentially useful strategy to curtail the propagation of antibiotic resistance. Conjugation inhibitors (COINS) are a series of compounds that block conjugation in vitro. Here we show that COINS efficiently block plasmid transmission in two controlled natural environments, water microcosms and the mouse gut. These observations indicate that COIN therapy can be used to prevent the spread of antibiotic resistance.Acknowledgments: The work performed by the de la Cruz research group was supported by the European Union Seventh Framework Program (FP7-HEALTH-2011-single-stage) “Evolution and Transfer of Antibiotic Resistance” (EvoTAR), grant agreement number 282004. The work performed by C.P.-G. and M.G. was supported by Ph.D. fellowships funded by the University of Cantabria. The work performed by the B.G.-Z. laboratory was supported by The EFFORT project (www.effort-against-amr.eu) FP7-KBBE-2013-7, grant agreement 613754

Whole-genome sequence of Acinetobacter pittii HUMV-6483 isolated from human urine

Acinetobacter pittii strain HUMV-6483 was obtained from urine from an adult patient. We report here its complete genome assembly using PacBio singlemolecule real-time sequencing, which resulted in a chromosome with 4.07 Mb and a circular contig of 112 kb. About 3,953 protein-coding genes are predicted from this assembly

COPLA, a taxonomic classifier of plasmids

Background: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. Results: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. Conclusions: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.This work was supported by the Spanish Ministry of Science and Innovation [PID2020-117923GB-I00 to FdlC]; the Spanish Ministry of Economy, Industry and Competitiveness [DI-17-09164 to SR-S]; and USA Centers for Disease Control and Prevention [200-2019-06679 to FdlC]. The funders had no role in the design of the study, nor in the collection, analysis, and interpretation of data, nor in writing the manuscript included in this submission

Diseño de modelos basados en aprendizaje para la estimación de pares articulares para la rehabilitación motora mediante exoesqueletos

El presente Trabajo Fin de Máster se enmarca en el proyecto HYPER-Consolider, en el que el Grupo de Robótica de la Universidad de Zaragoza pretende desarrollar exoesqueletos robóticos que ayuden a la rehabilitación funcional de pacientes con trastornos motores. En este sentido, uno de los elementos necesarios para su consecución es la estimación del par de fuerzas producido durante el movimiento de las articulaciones a partir de las señales de electromiografía superficial (sEMG) generadas por la activación de los músculos. La razón de ello es que, una vez conseguido predecir el movimiento voluntario que pretende realizar el paciente, se posibilita el que pueda ser ajustado por el exoesqueleto de forma que contribuya más eficientemente a su rehabilitación. En este trabajo se han desarrollado modelos de caja negra basados en redes neuronales y otras técnicas de aprendizaje automático, diseñando su estructura y definiendo sus variables de entrada, de forma que se permita comparar el rendimiento de las distintas opciones con el obtenido anteriormente a partir de un modelo paramétrico basado en las técnicas de Hill. Como conclusión final se propondrá el modelo que mejor se ajuste a las necesidades del proyecto, valorándose sus posibilidades de generalización e indicándose sus limitaciones

Plasmid classifications

Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs

Biofilm formation in <em>Hafnia alvei</em> HUMV-5920, a human isolate

Hafnia alvei is a Gram-negative, rodshaped, facultative anaerobic bacterium of the family Enterobacteriaceae that has been isolated from various mammals, fish, insects and birds. In humans, case reports of Hafnia-associated enteric infections have been chiefly reported in Spain. Although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens little is known about the factors that contribute to its pathogenesis or virulence factors and regulatory circuits that may enhance the establishment and survival of H. alvei in the environment. The goal of the present study was to analyze the capacity of a H. alvei clinical isolate (strain HUMV-5920) to form biofilms. Biofilm formation by this strain increases during growth at 28 °C compared to 37 °C. Investigation of multicellular behavior by confocal microscopy, crystal violet and calcofluor staining in this strain showed biofilm formation associated with the production of cellulose. Importantly, several genes related to cellulose production including bcsABZC and yhjQ are present in the H. alvei HUMV-5920 chromosome. The ability of H. alvei to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment or food processing environments, increasing the probability of causing infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes