Complete genome sequence of Mycobacterium chelonae type strain CCUG 47445, a rapidly growing species of nontuberculous mycobacteria

Mycobacterium chelonae strains are ubiquitous rapidly growing mycobacteria associated with skin and soft tissue infections, cellulitis, abscesses, osteomyelitis, catheter infections, disseminated diseases, and postsurgical infections after implants with prostheses, transplants, and even hemodialysis procedures. Here, we report the complete genome sequence of M. chelonae type strain CCUG 47445.This work, including the efforts of Antoni Bennasar-Figueras, was funded by Ministerio de Economía y Competitividad (MINECO) (CGL2012-39604).Peer Reviewe

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.publishedVersio

Knockout of Targeted Plasmid-Borne β-Lactamase Genes in an Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile

Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne β-lactamase genes (blaOXA-1, blaTEM-1, and blaCTX-M15) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted β-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the blaCTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of blaOXA-1 and blaTEM-1 affected fewer proteins’ expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss.publishedVersio

Implicaciones ecológicas y clínicas del análisis comparativo de genomas de micobacterias ambientales y patógenas

[spa] El objetivo principal de la presente tesis es realizar un estudio detallado desde el punto de vista genómico de todas aquellas características que pueden beneficiar el carácter patogènico de las micobacterias de crecimiento rápido (MCR), en especial de las especies estrechamente relacionadas Mycobacterium abscessus, Mycobacterium chelonae y Mycobacterium immunogenum. Dichas especies son consideradas como importantes patógenos oportunistas, responsables de infecciones difíciles de tratar por sus características intrínsecas y que complican el estado de salud de los pacientes hospitalizados. Además, con este estudio se pretende compensar el desequilibrio histórico existente en cuanto al estudio del género Mycobacterium, donde los esfuerzos se han centrado fundamentalmente en especies patógenas como Mycobacterium tuberculosis. Incrementar la información disponible de las MCR mencionadas permitirá futuros estudios centrados en la investigación de los elementos de patogenicidad que poseen, así como proponer nuevas dianas para el desarrollo de tratamientos alternativos de las infecciones que provocan. Con el fin de abordar el problema del escaso número de genomas presentes en las bases de datos de las especies mencionadas; se desarrolló un protocolo de extracción de ADN eficaz para micobacterias, consideradas como microorganismos difíciles de lisar. Obtenidas las muestras de ADN, se utilizaron tecnologías de secuenciación de nueva generación para la obtención de grandes cantidades de secuencias (denominadas lecturas) a partir de las cuales se obtuvieron los genomas de las respectivas cepas. Con este fin se utilizaron las herramientas bioinformáticas más óptimas de las disponibles para el ensamblaje, mejora y evaluación de los genomas obtenidos. De esa forma se aseguró la obtención de genomas de alta calidad con las herramientas disponibles en el momento. Los genomas obtenidos, junto con los disponibles en las bases de datos, se utilizaron para el estudio comparativo basado en el cálculo del genoma esencial (que incluye el conjunto de proteínas compartidas por todos los genomas) y pangenoma (conjunto de todos los grupos de proteínas diferentes presentes en un conjunto de genomas). El genoma esencial permitió clarificar las relaciones evolutivas de las diferentes especies consideradas, especialmente en el complejo de subespecies de M. abscessus. El estudio del pangenoma permitió sugerir una alta capacidad adaptativa de las MCR debido a la tendencia abierta detectada en su pangenoma. El mismo resultado se obtuvo en el grupo abscessuschelonae-immunogenum, pero no en la especie M. immunogenum, donde la tendencia cerrada del pangenoma refleja la escasa plasticidad genómica de la especie. Un hito importante conseguido fue la caracterizaron funcional de las proteínas exclusivas de una determinada especie o genoma. En este sentido, se realizó un catálogo de todos aquellos elementos genómicos implicados en la patogenicidad de cada microorganismo. Para ello se utilizaron toda una serie de bases de datos especializadas para la búsqueda de genes de resistencias a antibióticos, factores de virulencia, elementos móviles, proteínas reguladoras y elementos implicados en la percepción del Quórum. De esta forma se determinó un extenso catálogo de elementos genéticos que pueden influir de forma decisiva en la patogenicidad de cada cepa analizada. Finalmente, se caracterizaron los llamados sistemas toxina-antitoxina (STA), cuya funcionalidad puede influir en la patogenicidad de los microorganismos. En este caso no solo se utilizaron toda una serie de bases de datos especializadas y herramientas bioinformáticas para la descripción de estos elementos, sino que también se estableció un protocolo para realizar el ensayo experimental de su funcionalidad, utilizando a Escherichia coli como hospedador y vectores de expresión donde se clonaron los genes de la toxina y la antitoxina por separado. De esta forma se consiguió la descripción completa desde el punto de vista genómico, estructural y funcional de todos los sistemas detectados en las micobacterias objeto de estudio, observando resultados compatibles con un STA en tres ellos.[cat] L’objectiu principal de la present tesis es realizar un estudi detallat desde el punt de vista genòmic de totes aquelles caracteristiques que poden bneficiar el caràcter patogènic de les micobacteris de creixement ràpid (MCR), en especial de les espècies estretament relacionades Mycobacterium abscessus, Mycobacterium chelonae y Mycobacterium immunogenum. Les espècies esmentades es consideren importants patògens oportunistes, responsables d’infeccions difícils de tractar degut a les seves característiques intrínseques i que compliquen l’estat de salut dels pacients hospitalitzats. A més a més, amb aquest estudi es pretén compensar el desequilibri històric existent en quant a l’estudi del gènere Mycobacterium, on l’esforç s’ha centrat fonamentalment en espècies patògenes com Mycobacterium tuberculosis. Incrementar la informació disponible de les MCR esmentades permetrá futurs estudis centrats en la investigació dels elements de patogenicitat que posseeixen, així com proposar noves dianes per al desenvolupament de tractaments alternatius de les infeccions que provoquen. Amb la finalitat d’abordar el problema de l’escàs nombre de genomes presents en els bases de dades de les espècies destacades, es va desenvolupar un protocol d’extracció d’ADN eficaç per micobacteris, considerades com a microorganismes difícils de lisar. Un cop obtingudes les mostres d’ADN, s’empraren tecnologies de seqüenciació de nova generació per a l’obtenció de grans quantitats de seqüencies (denominades lectures) a partir de les quals es varen obtindre els genomes de les respectives soques. Amb aquest objectiu es varen utilitzar les eines bioinformàtiques més òptimes de les disponibles per a l’ensamblatje, millora y evaluació dels genomes obtinguts. D’aquesta manera es va asegurar l’obtenció de genomes d’alta qualitat amb les eines disponibles en el moment. Els genomes obtinguts, juntament amb els disponibles en les bases de dades, es varen utilitzar per a l’estudi comparatiu basat en el càlcul del genoma esencial (que inclou el conjunt de proteïnes compartides per tots els genomes) y pangenoma (conjunt de tots els grups de proteïnes diferents presents en un conjunt de genomes). El genoma esencial va permetre aclarir les relacions evolutives de les diferents espècies considerades, especialment en el complexe de subespècies de M. abscessus. L’estudi del pangenoma va permetre sugerir una alta capacitat adaptativa de les MCR degut a la tendència oberta detectada en el pangenoma. El mateix resultat es va obtenir en el grup abscessuschelonae-immunogenum, pero no en la espècie M. immunogenum, en la que la tendència tancada del pangenoma reflecteix la baixa plasticitat genòmica de l’espècie. Un fet important aconseguit va ser la caracterització funcional de les proteïnes exclusives d’una espècie determinada o genoma. En aquest sentit, es va realitzar un catàleg de tots aquells elements genòmics implicats en la patogenicitat de cada microorganisme. Per aconseguir-ho es varen utilitzar tota una sèrie de bases de dades especialitzades per a la recerca de gens de resistència a antibiòtics, factors de virulència, elements mòvils, proteïnes reguladores y elements implicats en la percepció del Quòrum. D’aquesta forma es va determinar un extens catàleg d’elements genètics que poden influir de manera decisiva en la pateogenicitat de cada soca analitzada. Finalment, es caracteritzaren els coneguts com a sistemes toxina-antitoxina (STA), la funcionalitat dels cuals pot influir en la patogenicitat dels microorganismes. En aquest cas no tan sols es varen utilitzar tota una sèrie de bases de dades especialitzades y eines bioinformàtiques per a la descripció d’aquests elements, sinó que també es va establir un protocol per realitzar l’assaig experimental de la seva funcionalitat, emprant a Escherichia coli com hospedador y vectors d’expresió on es varen clonar els gens de la toxina i la antitoxina per separat. D’aquesta manera es va aconseguir la descripció completa des del punt de vista genòmic, estructural i funcional de tots els sistemas detectats en els micobacteris objecte d’estudi, observant resultats compatibles amb un STA en tres dels sistemes detectats.[eng] The main objective of the present thesis is to perform a detailed study from a genomic point of view of all those characteristics that can benefit the pathogenicity of the rapid growing mycobacteria (RGM), especially of the closely related species Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium immunogenum. These species are considered important opportunistic pathogens, responsible of infections that are hard to treat due to their intrinsic characteristics. These infections complicate the health status of the hospitalized patients. In addition, this study pretends to compensate the historical imbalance in the study of the genus Mycobacterium, in which the efforts have been put in the study of pathogenic species as Mycobacterium tuberculosis. Increasing the information available of the RGM mentioned before in the databases will allow future studies to be focused on the research of the pathogenicity elements that they have, as well as propose new targets for the development of alternative treatments of the infections that they provoke. With the objective to increase the actual low number of genomes present in the databases of the species mentioned before, it was developed a DNA extraction protocol effective for mycobacteria, considered as “hard-to-lyse” microorganism. Having obtained the DNA samples, next-generation sequencing technologies were used to obtain massive amounts of sequences (called reads) from which the genomes of the respective strains were obtained. With this purpose, the most suitable and available bioinformatic tools were used for the assembly, improvement and checking of the genomes obtained. Thus, it was ensured the obtention of high-quality genomes with the tools available at that moment. The resulting genomes, along with the genomes available in the databases, were used for a comparative study based on the estimation of the core genome (which includes all the proteins shared by all the genomes) and the pangenome (set of all the groups of different proteins present in all the studied genomes). The core genome allowed to clarify the evolutive relationships among the species considered, especially inside the complex of subspecies of M. abscessus. The determination of the pangenome allowed to suggest a high adaptive capacity of the RGM group due to the open tendency observed in its pangenome. The same result was obtained in the group abscessus-chelonae- immunogenum, but not in the species M. immunogenum, in which the closed tendency reflects the limited genomic plasticity of the species. An important achievement was the characterization of the proteins exclusive of one specific species or genome. In this sense, it was obtained a detailed catalogue of all those genomic elements related with the pathogenicity of each microorganism using specialized databases to find genes related to antibiotic resistances, virulence factors, mobile genetic elements, regulatory proteins and elements related to the Quorum sensing. Consequently, it was determined a large catalogue of genetic elements that can influence decisively in the pathogenicity of each strain. Finally, the elements called toxin-antitoxin systems (TAS) were characterized. The functionality of these elements can be closely related with the pathogenicity of the microorganisms. In this case, in addition to the use of specialized databases and bioinformatic tools for the description of these elements, it was developed a protocol for the experimental assay of their functionality. For this purpose, it was used Escherichia coli as host and expression vectors where the genes of the toxins and antitoxins were cloned separately. With this procedure, the TAS were completely described from the genomic, structural and functional point of view, obtaining results compatible with a TAS in three of them

Comparative Genomics of Clinical Isolates of the Emerging Tick-Borne Pathogen Neoehrlichia mikurensis

Tick-borne ‘Neoehrlichia (N.) mikurensis’ is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding ‘N. mikurensis’ genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three ‘N. mikurensis’ strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10× Chromium technology, and sequencing by Illumina Hiseq-4500. ‘N. mikurensis’ was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that ‘N. mikurensis’ was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced ‘N. mikurensis’ strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium

Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds

Stutzerimonas balearica (Pseudomonas balearica) has been found principally in oil-polluted environments. The capability of S. balearica to thrive from the degradation of pollutant compounds makes it a species of interest for potential bioremediation applications. However, little has been reported about the diversity of S. balearica. In this study, genome sequences of S. balearica strains from different origins were analyzed, revealing that it is a diverse species with an open pan-genome that will continue revealing new genes and functionalities as the genomes of more strains are sequenced. The nucleotide signatures and intra- and inter-species variation of the 16S rRNA genes of S. balearica were reevaluated. A strategy of screening 16S rRNA gene sequences in public databases enabled the detection of 158 additional strains, of which only 23% were described as S. balearica. The species was detected from a wide range of environments, although mostly from aquatic and polluted environments, predominantly related to petroleum oil. Genomic and phenotypic analyses confirmed that S. balearica possesses varied inherent capabilities for aromatic compounds degradation. This study increases the knowledge of the biology and diversity of S. balearica and will serve as a basis for future work with the species

Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants

The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C.michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)—that grouped Chilean strains in three distinct clusters—and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified

Genomic and Proteomic Characterization of the Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain

Escherichia coli strain CCUG 78773 is a virulent extended-spectrum β-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718–161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent β-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two β-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment

Beware of False 'Type Strain' Genome Sequences

[eng] With this letter, we warn users of bacterial DNA sequence data about recent cases misusing the term "type strain" in bacterial genome sequence reports and highlight the importance that the term is used in the correct context

Staphylococcus borealis sp. nov., isolated from human skin and blood

When analysing a large cohort of Staphylococcus haemolyticus, using whole-genome sequencing, five human isolates (four from the skin and one from a blood culture) with aberrant phenotypic and genotypic traits were identified. They were phenotypically similar with yellow colonies, nearly identical 16S rRNA gene sequences and initially speciated as S. haemolyticus based on 16S rRNA gene sequence and MALDI-TOF MS. However, compared to S. haemolyticus, these five strains demonstrate: (i) considerable phylogenetic distance with an average nucleotide identity Staphylococcus borealis sp. nov. is proposed. The novel species belong to the genus Staphylococcus and is coagulase- and oxidase-negative and catalase-positive. The type strain, 51-48T, is deposited in the Culture Collection University of Gothenburg (CCUG 73747T) and in the Spanish Type Culture Collection (CECT 30011T)