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The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic that emerged in 2019 has been an unprecedented event in international science, as it has been possible to sequence millions of genomes, tracking their evolution very closely. This has enabled various types of secondary analyses of these genomes, including the measurement of their sequence selection pressure. In this work, we have been able to measure the selective pressure of all the described SARS-CoV-2 genes, even analyzed by sequence regions, and we show how this type of analysis allows us to separate the genes between those subject to positive selection (usually those that code for surface proteins or those exposed to the host immune system) and those subject to negative selection because they require greater conservation of their structure and function. We have also seen that when another gene with an overlapping reading frame appears within a gene sequence, the overlapping sequence between the two genes evolves under a stronger purifying selection than the average of the non-overlapping regions of the main gene. We propose this type of analysis as a useful tool for locating and analyzing all the genes of a viral genome when an adequate number of sequences are available.IMPORTANCEWe have analyzed the selection pressure of all severe acute respiratory syndrome coronavirus 2 genes by means of the nonsynonymous (Ka) to synonymous (Ks) substitution rate. We found that protein-coding genes are exposed to strong positive selection, especially in the regions of interaction with other molecules (host receptor and genome of the virus itself). However, overlapping coding regions are more protected and show negative selection. This suggests that this measure could be used to study viral gene function as well as overlapping genes.

The SARS-CoV-2 virus pandemic that emerged in 2019 has been an unprecedented event in international science, as it has been possible to sequence millions of genomes, tracking their evolution very closely. This has enabled various types of secondary analyses of these genomes, including the measurement of their sequence selection pressure. In this work we have been able to measure the selective pressure of all the described SARS-CoV-2 genes, even analyzed by sequence regions, and we show how this type of analysis allows us to separate the genes between those subject to positive selection (usually those that code for surface proteins or those exposed to the host immune system) and those subject to negative selection because they require greater conservation of their structure and function. We have also seen that when another gene with an overlapping reading frame appears within a gene sequence, the overlapping sequence between the two genes evolves under a stronger purifying selection than the average of the non-overlapping regions of the main gene. We propose this type of analysis as a useful tool for locating and analyzing all the genes of a viral genome, when an adequate number of sequences are available.

Pseudomonas aeruginosa ST274 is an international epidemic high-risk clone, mostly associated with hospital settings and appears to colonize cystic fibrosis (CF) patients worldwide. To understand the relevant mechanisms for its success, the biological and genomic characteristics of 11 ST274-P. aeruginosa strains from clinical and non-clinical origins were analyzed. The extensively drug-resistant (XDR/DTR), the non-susceptible to at least one agent (modR), and the lasR-truncated (by ISPsp7) strains showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity and low motility. Furthermore, the XDR/DTR and modR strains presented low pigment production and biofilm formation, which were very high in the lasR-truncated strain. Their whole genome sequences were compared with other 14 ST274-P. aeruginosa genomes available in the NCBI database, and certain associations have been primarily detected: blaOXA-486 and blaPDC-24 genes, serotype O:3, exoS+/exoU− genotype, group V of type IV pili, and pyoverdine locus class II. Other general molecular markers highlight the absence of vqsM and pldA/tleS genes and the presence of the same mutational pattern in genes involving two-component sensor-regulator systems PmrAB and CreBD, exotoxin A, quorum-sensing RhlI, beta-lactamase expression regulator AmpD, PBP1A, or FusA2 elongation factor G. The proportionated ST274-P. aeruginosa results could serve as the basis for more specific studies focused on better antibiotic stewardship and new therapeutic developments.

El incremento de la demanda de alimentos y el cambio climático están obligando a buscar alternativas sostenibles que mejoren la productividad agrícola. Además de los rizobios, existen otras bacterias (Plant Growth Promoting Bacteria; PGPB) menos estudiadas que mejoran el acceso de las plantas a los nutrientes o su tolerancia al estrés incluyendo especies de alto interés comercial como los cereales. Este trabajo se centra en el estudio de la comunidad cultivable y no-cultivable de bacterias endófitas aisladas de diferentes variedades de trigo relacionadas con su domesticación obtenidas en distintas localizaciones geográficas. Nuestros análisis metataxonómicos muestran que la estructura de la comunidad de distintas semillas se encuentra conservada y dominada por un único género bacteriano perteneciente al grupo de las Enterobacterias. Este género forma parte del microbioma core del trigo, ya que es el único presente en todas sus variedades y tejidos, independientemente de su localización geográfica. Además, su abundancia aumenta proporcionalmente de suelo a raíz, tallo y espiga, acumulándose finalmente en las semillas. Este enriquecimiento sugiere un papel en la transferencia vertical de estas bacterias endófitas a través de las semillas. Para comprobar esta hipótesis, flores y semillas de cereales (trigo y Lolium) y Arabidopsis fueron inoculadas con aislados de este género marcados con un plásmido que codifica la enzima ß-glucoronidasa. Semillas de la siguiente progenie fueron germinadas para comprobar, mediante revelado con el sustrato cromogénico X-gluc, la presencia del endófito únicamente en cereales, confirmando su transmisión vertical a través de la línea germinal de forma específica.

Dispersion of transferable oxazolidinone resistance genes among enterococci poses a serious problem to human health. Prompt detection of bacteria carrying these genes is crucial to avoid their spread to multidrug-resistant bacteria. The aim of the study was to describe the presence of optrA-positive isolates among enterococci in a Spanish hospital, and to determine their genetic context and location through whole genome sequencing. All enterococci recovered in a Spanish hospital (Hospital El Bierzo; HEB) from February to December 2018 (n = 443), with minimal inhibitory concentrations (MICs) to linezolid (LZD) ≥4 mg/L, were tested by polymerase chain reaction for the presence of cfr, optrA, and poxtA transferable genes. Only four Enterococcus faecalis isolates (0.9%) had LZD MICs ≥4 mg/L and none of them was positive for cfr or poxtA genes. However, the optrA gene was detected in three isolates collected from urine samples of community patients, whose genomes were sequenced and subjected to bioinformatics analysis. These isolates belonged to different clones: ST7, ST480, and ST585. In these three isolates, the optrA gene was located on plasmids, associated with IS1216 in different arrays. In one isolate, the optrA plasmid coexists with a second plasmid, which carried multiple resistance genes for different classes of antibiotics. Detection of optrA-positive E. faecalis isolates in the community is a matter of concern. The spread of these bacteria into hospital settings, particularly in those, such as the HEB, where vancomycin-resistant enterococci are endemic, should be avoided, to preserve the efficacy of the last-resort oxazolidinones.

[Motivation]: Detailed and accurate analysis of mobile genetic elements (MGEs) in bacteria is essential to deal with the current threat of multiresistant microbes. The overwhelming use of draft, contig-based genomes hinder the delineation of the genetic structure of these plastic and variable genomic stretches, as in the case of pipolins, a superfamily of MGEs that spans diverse integrative and plasmidic elements, characterized by the presence of a primer-independent DNA polymerase., [Results]: ExplorePipolin is a Python-based pipeline that screens for the presence of the element and performs its reconstruction and annotation. The pipeline can be used on virtually any genome from diverse organisms and of diverse quality, obtaining the highest-scored possible structure and reconstructed out of different contigs if necessary. Then, predicted pipolin boundaries and pipolin encoded genes are subsequently annotated using a custom database, returning the standard file formats suitable for comparative genomics of this mobile element.

pUO-STmRV1 is an IncC plasmid discovered in the Spanish clone of the emergent monophasic variant of Salmonella enterica serovar Typhimurium, which has probably contributed to its epidemiological success. The sequence of the entire plasmid determined herein revealed a largely degenerated backbone with accessory DNA incorporated at four different locations. The acquired DNA constitutes more than two-thirds of the pUO-STmRV1 genome and originates from plasmids of different incompatibility groups, including IncF (such as R100 and pSLT, the virulence plasmid specific of S. Typhimurium), IncN and IncI, from the integrative element GIsul2, or from yet unknown sources. In addition to pSLT virulence genes, the plasmid carries genes conferring resistance to widely-used antibiotics and heavy metals, together with a wealth of genetic elements involved in DNA mobility. The latter comprise class 1 integrons, transposons, pseudo-transposons, and insertion sequences, strikingly with 14 copies of IS26, which could have played a crucial role in the assembly of the complex plasmid. Typing of pUO-STmRV1 revealed backbone features characteristically associated with type 1 and type 2 IncC plasmids and could therefore be regarded as a hybrid plasmid. However, a rooted phylogenetic tree based on core genes indicates that it rather belongs to an ancient lineage which diverged at an early stage from the branch leading to most extant IncC plasmids detected so far. pUO-STmRV1 may have evolved at a time when uncontrolled use of antibiotics and biocides favored the accumulation of multiple resistance genes within an IncC backbone. The resulting plasmid thus allowed the Spanish clone to withstand a wide variety of adverse conditions, while simultaneously promoting its own propagation through vertical transmission.

Commensal bacteria act as important reservoirs of virulence and resistance genes. However, existing data are generally only focused on the analysis of human or human-related bacterial populations. There is a lack of genomic studies regarding commensal bacteria from hosts less exposed to antibiotics and other selective forces due to human activities, such as wildlife. In the present study, the genomes of thirty-eight E. coli strains from the gut of various wild animals were sequenced. The analysis of their accessory genome yielded a better understanding of the role of the mobilome on inter-bacterial dissemination of mosaic virulence and resistance plasmids. The study of the presence and composition of the CRISPR/Cas systems in E. coli from wild animals showed some viral and plasmid sequences among the spacers, as well as the relationship between CRISPR/Cas and E. coli phylogeny. Further, we constructed a single nucleotide polymorphisms-based core tree with E. coli strains from different sources (humans, livestock, food and extraintestinal environments). Bacteria from humans or highly human-influenced settings exhibit similar genetic patterns in CRISPR-Cas systems, plasmids or virulence/resistance genes-carrying modules. These observations, together with the absence of significant genetic changes in their core genome, suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Nitrogen content of grape musts strongly impacts on fermentation performance and wine metabolite production. As nitrogen is a limiting nutrient in most grape musts, nitrogen supplementation is a common practice that ensures yeast growth during fermentation. However, preferred nitrogen sources -as ammonium- repress the genes related to alternative nitrogen sources consumption, usually involved in aromatic compounds production. Here, we describe the effect of high ammonium doses in Saccharomyces cerevisiae fermentation performance and wine properties, and how it is affected by yeast co-inoculation in mixed (S. cerevisiae + Torulaspora delbrueckii) fermentations. In addition, an RNA-seq analysis allowed us to study the S. cerevisiae transcriptional response to ammonium nutrition and yeast interaction, demonstrating that T. delbrueckii presence affects the global S. cerevisiae transcriptional response, reducing ammonium effects at both phenotypic -fermentation kinetics and metabolite production- and transcriptional levels, under experimental conditions., Ministerio de Economia, Industria y Competitividad. Centro para el Desarrollo Tecnológico Industrial (CDTI), Fundación para el Conocimiento madri+d/Universidad Rey Juan Carlos, Universidad Complutense de Madrid, Hessen State Ministry of Higher Education, Research and the Arts, Federation of European Microbiological Societies(FEMS), Agrovin S.A, Fundação para a Ciência e Tecnologia to Biosystems and Integrative Sciences Institute (BioISI), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Porcine Escherichia coli ST131 isolates are scarcely documented. Here, whole genome sequencing and core genome (CG) and plasmidome analysis of seven isolates collected from diarrheic piglets and four from pork meat were performed. All of the 11 ST131 isolates belonged to serotype O25b:H4 and clade B and showed fimH22 allele or mutational derivatives. The 11 porcine isolates possessed virulence traits that classified the isolates as avian pathogenic, uropathogenic, and extraintestinal pathogenic E. coli–like (APEC-, UPEC-, and ExPEC-like) and constituted virotype D. The CG was performed for all porcine isolates in addition to 73 ST131 reference isolates from different origins. Within clade B, the CG showed nine subclusters, allowing us to describe five new subclades (B6, B6-like, B7, B8, and B9). There was an association between subclade B6, PST43, virotype D2, and food origin, whereas subclade B7 included PST9 isolates with virotype D5 from diarrheic piglets (p = 0.007). The distance between human and porcine isolates from subclades B6 and B7 had an average of 20 and 15 SNP/Mb, respectively. [F2:A-:B1]-IncF, ColE1-like, and IncX plasmids were the most prevalent. Besides, IncF plasmids harbored a ColV region frequent among APEC isolates. Antimicrobial resistance genes conferring resistance to penicillin, tetracycline, quinolones, and colistin were the most common. The mcr-1.1 gene was detected in 5 of 11 porcine isolates, integrated into the chromosome of one isolate and into plasmids in the remainder isolates (two MOBH11/IncHI2-ST4, one MOBP3/IncX4, and one MOBF12/IncF [F2:A-:B1] supposedly cointegrated with an IncHI2). The surrounding environments of the mcr-1 cassette showed variability. However, there were conserved structures within the same plasmid family. In conclusion, CG analysis defined five new subclades. The ST131 porcine isolates belonged to new subclades B6 and B7. Moreover, porcine and clinical human isolates were strongly related. The 11 porcine ST131

The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid spreading across the country. We present the results for 2170 sequences from the first wave of the SARS-Cov-2 epidemic in Spain and representing 12% of diagnosed cases until 14th March. This effort allows us to document at least 500 initial introductions, between early February-March from multiple international sources. Importantly, we document the early raise of two dominant genetic variants in Spain (Spanish Epidemic Clades), named SEC7 and SEC8, likely amplified by superspreading events. In sharp contrast to other non-Asian countries those two variants were closely related to the initial variants of SARS-CoV-2 described in Asia and represented 40% of the genome sequences analyzed. The two dominant SECs were widely spread across the country compared to other genetic variants with SEC8 reaching a 60% prevalence just before the lockdown. Employing Bayesian phylodynamic analysis, we inferred a reduction in the effective reproductive number of these two SECs from around 2.5 to below 0.5 after the implementation of strict public-health interventions in mid March. The effects of lockdown on the genetic variants of the virus are reflected in the general replacement of preexisting SECs by a new variant at the beginning of the summer season. Our results reveal a significant difference in the genetic makeup of the epidemic in Spain and support the effectiveness of lockdown measures in controlling virus spread even for the most successful genetic variants. Finally, earlier control of SEC7 and particularly SEC8 might have reduced the incidence and impact of COVID-19 in our country.

Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations. Here, we analyze genomic composition and pairwise sequence identity for over 10,000 reference plasmids to obtain a global map of the prokaryotic plasmidome. Plasmids in this map organize into discrete clusters, which we call plasmid taxonomic units (PTUs), with high average nucleotide identity between its members. We identify 83 PTUs in the order Enterobacterales, 28 of them corresponding to previously described archetypes. Furthermore, we develop an automated algorithm for PTU identification, and validate its performance using stochastic blockmodeling. The algorithm reveals a total of 276 PTUs in the bacterial domain. Each PTU exhibits a characteristic host distribution, organized into a six-grade scale (I–VI), ranging from plasmids restricted to a single host species (grade I) to plasmids able to colonize species from different phyla (grade VI). More than 60% of the plasmids in the global map are in groups with host ranges beyond the species barrier.

This study was aimed at assessing whether the repeated exposure of 12 strains of Salmonella spp., Escherichia coli, and Listeria monocytogenes to alternative nonthermal decontamination techniques with UV light (UV-C) and nonthermal atmospheric plasma (NTAP) may cause the emergence of variants showing increased resistance to clinically relevant antibiotics (ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin, and colistin). UV-C and NTAP treatments were applied on the surface of inoculated brain heart infusion (BHI) agar plates. Survivors were recovered and after 24 h of growth in BHI broth were again subjected to the decontamination treatment; this was repeated for 10 consecutive cycles. A total of 174 strain/decontamination technique/antibiotic combinations were tested, and 12 variant strains with increased resistance to one of the antibiotics studied were identified, with the increases in the MICs in Mueller-Hinton broth ranging from 2- to 256-fold. The variant strains of Salmonella spp. isolated were further characterized through phenotypic screenings and whole-genome sequencing (WGS) analyses. Most changes in susceptibility were observed for antibiotics that act at the level of protein synthesis (aminoglycosides, tetracyclines, and glycylcyclines) or DNA replication (fluoroquinolones), as well as for polymyxins. No changes in resistance to -lactams were detected. WGS analyses showed the occurrence of sequence alterations in some antibiotic cellular targets (e.g., gyrA for ciprofloxacin-resistant variants, rpsL for a streptomycin-resistant variant), accompanied by variations in stress response regulators and membrane transporters likely involved in the nonselective efflux of antibiotics, which altogether resulted in a low- to mediumlevel increase in microbial resistance to several antibiotics.Importance. The emergence and spread of antibiotic resistance along the food chain can be influenced by the different ant

Self-synthesizing transposons are integrative mobile genetic elements (MGEs) that encode their own B-family DNA polymerase (PolB). Discovered a few years ago, they are proposed as key players in the evolution of several groups of DNA viruses and virus–host interaction machinery. Pipolins are the most recent addition to the group, are integrated in the genomes of bacteria from diverse phyla and also present as circular plasmids in mitochondria. Remarkably, pipolins-encoded PolBs are proficient DNA polymerases endowed with DNA priming capacity, hence the name, primer-independent PolB (piPolB). We have now surveyed the presence of pipolins in a collection of 2,238 human and animal pathogenic Escherichia coli strains and found that, although detected in only 25 positive isolates (1.1%), they are present in E. coli strains from a wide variety of pathotypes, serotypes, phylogenetic groups and sequence types. Overall, the pangenome of strains carrying pipolins is highly diverse, despite the fact that a considerable number of strains belong to only three clonal complexes (CC10, CC23 and CC32). Comparative analysis with a set of 67 additional pipolin-harboring genomes from GenBank database spanning strains from diverse origin, further confirmed these results. The genetic structure of pipolins shows great flexibility and variability, with the piPolB gene and the attachment sites being the only common features. Most pipolins contain one or more recombinases that would be involved in excision/integration of the element in the same conserved tRNA gene. This mobilization mechanism might explain the apparent incompatibility of pipolins with other integrative MGEs such as integrons. In addition, analysis of cophylogeny between pipolins and pipolin-harboring strains showed a lack of congruence between several pipolins and their host strains, in agreement with horizontal transfer between hosts. Overall, these results indicate that pipolins can serve as a vehicle for genetic transfer

Microorganisms strongly influence and are required to generate the selective substrate that provides nutrients and support for fungal growth, and ultimately to induce mushroom fructification under controlled environmental conditions. In this work, the fungal and bacterial microbiota living in the different substrates employed in a commercial crop (compost phase I, II and III, flush 1 and 2, and casing material on day 1, 6 and 8 after compost casing and during flush 1 and 2) have been characterized along the different stages of cultivation by metataxonomic analysis (16S rRNA and ITS2), analysis of phospholipid fatty acid content (PLFAs) and RT-qPCR. Additionally, laccase activity and the content of lignin and complex carbohydrates in compost and casing have been quantified. The bacterial diversity in compost and casing increased throughout the crop cycle boosted by the connection of both substrates. As reflected by the PLFAs, the total living bacterial biomass appears to be negatively correlated with the mycelium of the crop. Agaricus bisporus was the dominant fungal species in colonized substrates, displacing the pre-eminent Ascomycota, accompanied by a sustained increase in laccase activity, which is considered to be a major product of protein synthesis during the mycelial growth of champignon. From phase II onwards, the metabolic machinery of the fungal crop degrades lignin and carbohydrates in compost, while these components are hardly degraded in casing, which reflects the minor role of the casing for nourishing the crop. The techniques employed in this study provide a holistic and detailed characterization of the changing microbial composition in commercial champignon substrates. The knowledge generated will contribute to improve compost formulations (selection of base materials) and accelerate compost production, for instance, through biotechnological interventions in the form of tailored biostimulants and to design environmentally sustainable bio-based casing

Twitter is one of the most popular social media networks that, in recent years, has been increasingly used by researchers as a platform to share science and discuss ongoing work. Despite its popularity, Twitter is not commonly used as a medium to teach science. Here, we summarize the results of #EUROmicroMOOC: the first worldwide Microbiology Massive Open Online Course taught in English using Twitter. Content analytics indicated that more than 3 million users saw posts with the hashtag #EUROmicroMOOC, which resulted in over 42 million Twitter impressions worldwide. These analyses demonstrate that free Microbiology MOOCs shared on Twitter are valuable educational tools that reach broad audiences throughout the world. We also describe our experience teaching an entire Microbiology course using Twitter and provide recommendations when using social media to communicate science to a broad audience.

Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation.

[Abstract] Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes., [Author summary] The Central Dogma of Biology schematically highlights the transmission of genetic information stored in DNA, through RNA, to the formation of proteins. This general flow implicates RNA export from the nucleus to the cytoplasm and proper protein localization within the eukaryotic cell. Ultimately, proteins are the cell’s structural and catalytic functional units. As a result, cells differentiate into one cell type or another (such as epithelial, muscle, neuron…) and exhibit specific shape and functionality. Here we describe, in a C. elegans model, how mutations in genes involved in a general and ubiquitous mechanism, such as mRNA export, may result in tissue-specific developmental phenotypes that show up in processes that are highly demanding of cytoplasmic transcripts like epithelialization and gamete formation. A deep understanding of the mechanisms underlying the "connectors" shown in the Central Dogma of Biology is key both to unravel the general genetic control of an organism’s development and, at the same time, contribute to a better understanding of tissue-specific diseases.

Plasmids are devices for gene propagation and, thus, fundamental agents of horizontal gene transfer (HGT) in bacteria. The myriad plasmids carry many differences in genetic structure, which are probably adaptive. Their genetic structure, together with environmental cues and barriers to plasmid dissemination, also limit the hosts they can infect. Determining the boundaries of plasmid host range is essential to understand the role of HGT in bacterial evolution and, more specifically, the dissemination of antibiotic resistance. The task is complicated by the difficulty in organizing plasmids into discrete, functionally equivalent categories. We analyzed the prokariotic plasmidome to determine whether plasmid genomes cluster together in genomically defined groups. Pairwise comparisons revealed that plasmids form discrete, coherent recombination groups, functionally equivalent to ¿molecular species¿. By analyzing the distribution of plasmid groups, we unveil a vast roadmap for HGT in enterobacteria. We also start to understand the functional roles of key plasmid genetic adaptations.

Carbapenem-resistant Enterobacteriaceae, including the increasingly reported OXA-48 Escherichia coli producers, are an emerging public health threat worldwide. Due to their alarming detection in our healthcare setting and their possible presence in the community, seven OXA-48-producing, extraintestinal pathogenic E. coli were analysed by whole genome sequencing as well as conventional tools, and tested for in vivo virulence. As a result, fve E. coli OXA-48-producing subclones were detected (O25:H4-ST131/PST43-fmH30-virotype E; O25:H4-ST131/PST9-fmH22-virotype D5, O16:H5-ST131/ PST506-fmH41; O25:H5-ST83/PST207 and O9:H25-ST58/PST24). Four ST131 and one ST83 isolates satisfed the ExPEC status, and all except the O16:H5 ST131 isolate were UPEC. All isolates exhibited local infammatory response with extensive subcutaneous necrosis but low lethality when tested in a mouse sepsis model. The blaOXA-48 gene was located in MOBP131/IncL plasmids (four isolates) or within the chromosome (three ST131 H30-Rx isolates), carried by Tn1999-like elements. All, except the ST83 isolate, were multidrug-resistant, with additional plasmids acting as vehicles for the spread of various resistance genes. This is the frst study to analyse the whole genome sequences of blaOXA-48-positive ST131, ST58 and ST83 E. coli isolates in conjunction with experimental data, and to evaluate the in vivo virulence of blaOXA-48 isolates, which pose an important challenge to patient management

Carbapenem-resistant Enterobacteriaceae, including the increasingly reported OXA-48 Escherichia coli producers, are an emerging public health threat worldwide. Due to their alarming detection in our healthcare setting and their possible presence in the community, seven OXA-48-producing, extraintestinal pathogenic E. coli were analysed by whole genome sequencing as well as conventional tools, and tested for in vivo virulence. As a result, five E. coli OXA-48-producing subclones were detected (O25:H4-ST131/PST43-fimH30-virotype E; O25:H4-ST131/PST9-fimH22-virotype D5, O16:H5-ST131/PST506-fimH41; O25:H5-ST83/PST207 and O9:H25-ST58/PST24). Four ST131 and one ST83 isolates satisfied the ExPEC status, and all except the O16:H5 ST131 isolate were UPEC. All isolates exhibited local inflammatory response with extensive subcutaneous necrosis but low lethality when tested in a mouse sepsis model. The bla OXA-48 gene was located in MOBP131/IncL plasmids (four isolates) or within the chromosome (three ST131 H30-Rx isolates), carried by Tn1999-like elements. All, except the ST83 isolate, were multidrug-resistant, with additional plasmids acting as vehicles for the spread of various resistance genes. This is the first study to analyse the whole genome sequences of bla OXA-48-positive ST131, ST58 and ST83 E. coli isolates in conjunction with experimental data, and to evaluate the in vivo virulence of bla OXA-48 isolates, which pose an important challenge to patient management

PLACNET is a graph-based tool for reconstruction of plasmids from next generation sequence pair-end datasets. PLACNET graphs contain two types of nodes (assembled contigs and reference genomes) and two types of edges (scaffold links and homology to references). Manual pruning of the graphs is a necessary requirement in PLACNET, but this is difficult for users without solid bioinformatic background. PLACNETw, a webtool based on PLACNET, provides an interactive graphic interface, automates BLAST searches, and extracts the relevant information for decision making. It allows a user with domain expertise to visualize the scaffold graphs and related information of contigs as well as reference sequences, so that the pruning operations can be done interactively from a personal computer without the need for additional tools. After successful pruning, each plasmid becomes a separate connected component subgraph. The resulting data are automatically downloaded by the user.

Carbapenem-resistant Enterobacteriaceae, including the increasingly reported OXA-48 Escherichia coli producers, are an emerging public health threat worldwide. Due to their alarming detection in our healthcare setting and their possible presence in the community, seven OXA-48-producing, extraintestinal pathogenic E. coli were analysed by whole genome sequencing as well as conventional tools, and tested for in vivo virulence. As a result, five E. coli OXA-48-producing subclones were detected (O25:H4-ST131/PST43-fimH30-virotype E; O25:H4-ST131/PST9-fimH22-virotype D5, O16:H5-ST131/PST506-fimH41; O25:H5-ST83/PST207 and O9:H25-ST58/PST24). Four ST131 and one ST83 isolates satisfied the ExPEC status, and all except the O16:H5 ST131 isolate were UPEC. All isolates exhibited local inflammatory response with extensive subcutaneous necrosis but low lethality when tested in a mouse sepsis model. The bla gene was located in MOB/IncL plasmids (four isolates) or within the chromosome (three ST131 H30-Rx isolates), carried by Tn1999-like elements. All, except the ST83 isolate, were multidrug-resistant, with additional plasmids acting as vehicles for the spread of various resistance genes. This is the first study to analyse the whole genome sequences of bla -positive ST131, ST58 and ST83 E. coli isolates in conjunction with experimental data, and to evaluate the in vivo virulence of bla isolates, which pose an important challenge to patient management.

Plasmid conjugation is the preferred route of dissemination of important adaptive traits in bacteria, such as antibiotic resistance genes. If conjugation could be tamed and controlled at will, we will have a strong hand to control antibiotic resistance acquisition by human pathogens. To achieve this objective, we determined the dynamics of plasmid conjugation using various plasmid prototypes. We could distinguish between density-dependent and frequency-dependent conjugation dynamics. Broad-host range plasmids, which show inherently high conjugation rates (R0 >1), are able to invade recipient populations. This is in contrast to repressed plasmids, such as members of the MOBF12 / IncF complex, which show R0 <1. Comparative genomics analysis shows that the transfer systems of MOBF12 / IncF are adapted to their host bacteria and show interesting variations in their regulatory systems. Besides, and using plasmid R388 as a model, we determined how interactions with other plasmids and host chromosomes greatly affect the plasmid rate of transfer. Additionally, we screened a large series of compounds as conjugation inhibitors and investigated the mechanisms by which they affect conjugation. These inhibitors are able to eliminate plasmid invasion of recipient populations. Finally, we developed a genome analysis tool, called PLACNET, which helps in reconstructing plasmids from whole genome sequences. PLACNET has now been updated as a web tool that can be run from a PC. Using PLACNET, we were able to show that E. coli ST131, sometimes considered as an E. coli clone, contains nearly as much plasmid diversity as the whole enterobacteria. All these results taken together suggest new approaches to control plasmid conjugation and, thus, the dissemination of antibiotic resistance to human pathogens.

Escherichia coli is a commensal microorganism of the gastrointestinal tract of animals and humans and it is an excellent model organism for the study of antibiotic resistance mechanisms. The resistance transmission and other characteristics of bacteria are based on different types of gene transfer occurring throughout the bacterial evolution. One of which is horizontal gene transfer that allows us to understand the ability of bacteria to acquire new genes. One dimensional and two dimensional electrophoresis (2-DE) techniques were performed in order to identify and characterize the proteome of two E. coli strains: Electromax DH10B, a transformation-ready strain; and TF-Se20, the Electromax DH10B that contains the aac(6′)-Ib-cr4-harboring pMdT1 plasmid. After 2-DE and subsequent analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), it was possible to identify 76 distinct proteins on the TF-Se20 strain, whereas 71 had a known function. From Electromax DH10B strain, 72 different proteins were identified of which 71 were associated with a biological process. The protein of interest, aminoglycoside N-(6′)-acetyltransferase type 1, was identified by MALDI-TOF MS. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was performed to determine its sequence. Seventy six percent of the acetylase sequence was reconstructed only in the TF-Se20 strain, representing the single protein associated to antibiotic resistance. MALDI-TOF MS and LC-MS/MS approaches allowed us to determine the total proteome of both strains, as well as the acetylase sequence. Both of them enhance the ability to obtain more accurate information about the mechanisms of antimicrobial resistance. The pMdT1 plasmid brings a new perspective in understanding the metabolic processes that lead to antibiotic resistance. Biological significance This study highlights the importance of proteomics and bioinformatics in understanding mechanisms of gene

The F plasmid is the foremost representative of a large group of conjugative plasmids, prevalent in Escherichia coli, and widely distributed among the Enterobacteriaceae. These plasmids are of clinical relevance, given their frequent association with virulence determinants, colicins, and antibiotic resistance genes. Originally defined by their sensitivity to certain male-specific phages, IncF plasmids share a conserved conjugative system and regulatory circuits. In order to determine whether the genetic architecture and regulation circuits are preserved among these plasmids, we analyzed the natural diversity of F-like plasmids. Using the relaxase as a phylogenetic marker, we identified 256 plasmids belonging to the IncF/ MOBF12group, present as complete DNA sequences in the NCBI database. By comparative genomics, we identified five major groups of F-like plasmids. Each shows a particular operon structure and alternate regulatory systems. Results show that the IncF/MOBF12 conjugation gene cluster conforms a diverse and ancient group, which evolved alternative regulatory schemes in its adaptation to different environments and bacterial hosts.

[Background]: Metagenomics has recently been used to analyze the ensemble of antimicrobial resistance genes in a particular environment (resistome). All metagenomic open and closed formats exhibit low sensitivity and lack of specificity to uncover the size and diversity of minor fractions of total metagenomes. To achieve better sensitivity and specificity in resistome mining, we developed a targeted sequence capture panel (ResCap v.01) based on SeqCapEZ technology (NimblegeneRoche), designed to enrich sequences from genes encoding resistance to antibiotics, biocides and heavy metals. [Material & Methods]: ResCap is composed by a panel of approximately 79,000 non-redundant genes (80 Mb) including 7,963 well-known antibiotic resistance genes, 30,740 biocide & heavy metal resistance genes, and 2,517 relaxases, signature proteins of plasmid conjugation. Design of the probes was based on a non-redundant database (DB), constructed by mixing curated DBs for antibiotic resistance (CARD, ARG-ANNOT and RED-DB). To improve the detection of “novel” AbR genes, a Hidden Markov Model (HMM) from each AbR family was constructed for detection of homologous sequences by using hammer3 software against Uniref100 DB. Sequences coding for resistance to heavy metals and antiseptics were recruited from BacMet DB, which includes both experimentally confirmed and predicted antibacterial biocide- and metal-resistance genes. Relaxases were retrieved from the ConjDB repository. ResCap workflow consisted on i) whole-metagenome shotgun library construction, ii) hybridization, and iii) capture. All steps were performed according Nimblegene standard protocols for Illumina platforms. To evaluate ResCap efficiency, samples were sequenced before and after capture. ResCap was validated by analysing fecal samples from 9 humans and 8 swine. Robustness of the platform was tested by comparative analysis of two technical replicates of two samples. Bioinformatic analysis was performed by mapping sequenced rea

[Background]: Lateral gene transfer plays an essential role in the bacterial evolution The genomes of most opportunistic pathogens differ in their accessory genome which include determinants associated with virulence, colonization or antibiotic resistance, among other adaptive traits. It exists a wide range of bioinformatics tools for typing, core genome analysis or phylogenetics but only a few tools for analysing the accessory genomes. We present AccNET (Accesory Constelation Network)), a new tool, non-computational expensive, based on bipartite networks that is able to analyse simultaneously tens of genomes.[Material & Methods]: AccNET uses the kClust software to customly detect highly-related homologous genes, by modifying the identity threshold. The core genome, defined as the subset of proteins present in all genomes, is removed from the analysis. Then, AccNET builds a bipartite network of the accessory genome with two kind of nodes: genomes and proteins. The edges that link the nodes represent the presence of a protein in a given genome. The edge-weight is determined as a phylogenetic distance between the proteins included in the cluster. To calculate such distance, we use a pipeline: First, we align all the proteins belonged to the cluster using MUSCLE. Then, we optimize the multiple alignment using Trimal and finally we calculate the distance matrix using ProtDist, (PHYLIP package). For each protein the phylogenetic distance for edge-weight is determined by the mean of the distance with all of the remaining proteins. Final output network is compatible with most of the network viewer software such as Cytoscape or Gephi. We used ForceAtlas2 layout from Gephi to arrange networks. To compare the structure of the accessory distribution with the phylogenetic relationships, we build the core-genome phylogenetic tree using the previous defined core-genome. First, we aligned each one of the core genes using MUSCLE. Then, we concatenated all the multiple alignments an

Pathogenic ESBL-producing E. coli lineages occur frequently worldwide, not only in a human health context but in animals and the environment, also in settings with low antimicrobial pressures. This study investigated the fitness costs of ESBL-plasmids and their influence on chromosomally encoded features associated with virulence, such as those involved in the planktonic and sessile behaviors of ST131 and ST648 E. coli. ESBL-plasmid-carrying wild-type E. coli strains, their corresponding ESBL-plasmid-“cured” variants (PCV), and complementary ESBL-carrying transformants were comparatively analyzed using growth curves, Omnilog® phenotype microarray (PM) assays, macrocolony and biofilm formation, swimming motility, and RNA sequence analysis. Growth curves and PM results pointed toward similar growth and metabolic behaviors among the strains. Phenotypic differences in some strains were detected, including enhanced curli fimbriae and/or cellulose production as well as a reduced swimming capacity of some ESBL-carrying strains, as compared to their respective PCVs. RNA sequencing mostly confirmed the phenotypic results, suggesting that the chromosomally encoded csgD pathway is a key factor involved. These results contradict the hypothesis that ESBL-plasmid-carriage leads to a fitness loss in ESBL-carrying strains. Instead, the results indicate an influence of some ESBL-plasmids on chromosomally encoded features associated with virulence in some E. coli strains. In conclusion, apart from antibiotic resistance selective advantages, ESBL-plasmid-carriage may also lead to enhanced virulence or adaption to specific habitats in some strains of pandemic ESBL-producing E. coli lineages.

There are two types of evolutionary events that impinge on bacterial genomes: microevolution (mutations and homologous recombination), which produces point mutations, and macroevolution, which results in the acquisition, loss, or rearrangement of large DNA segments. Macroevolutionary events ultimately arise from the import of adaptive traits by means of mobile genetic elements (MGEs). MGEs are basically plasmids, phages, and their integrated counterparts (integrative and conjugative elements [ICEs] and prophages). Genomic islands are fossil remnants of MGEs, since they generally lost mobility by rearrangement after the initial insertion event. Nevertheless, they can sometimes still be mobilized by the recombinogenic activity of IncF plasmids, among other possibilities. In summary, the dynamics of bacterial genomes can be interpreted as resulting from the microevolution of a core genome and the macroevolution of the accessory genome, i.e., the horizontal gene transfer events that give rise to the concept of a pangenome.

Salmonellosis is one of the most common and widely distributed foodborne diseases. The emergence of Salmonella strains that are resistant to a variety of antimicrobials is a serious global public health concern. Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) is one of these emerging epidemic multidrug resistant strains. Here we collate information from the diverse and comprehensive range of experiments on Salmonella proteomes that have been published. We then present a new study of the proteome of the quinolone-resistant Se20 strain (phage type DT104B), recovered after ciprofloxacin treatment and compared it to the proteome of reference strain SL1344. A total of 186 and 219 protein spots were recovered from Se20 and SL1344 protein extracts, respectively, after two-dimensional gel electrophoresis. The signatures of 94% of the protein spots were successfully identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Three antimicrobial resistance related proteins, whose genes were previously detected by polymerase chain reaction (PCR), were identified in the clinical strain. The presence of these proteins, dihydropteroate synthase type-2 (sul2 gene), aminoglycoside resistance protein A (strA gene) and aminoglycoside 6'-N-acetyltransferase type Ib-cr4 (aac(6')-Ib-cr4 gene), was confirmed in the DT104B clinical strain. The aac(6')-Ib-cr4 gene is responsible for plasmid-mediated aminoglycoside and quinolone resistance. This is a preliminary analysis of the proteome of these two S. Typhimurium strains and further work is being developed to better understand how antimicrobial resistance is developing in this pathogen.

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<. 0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24. h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.

The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated viru­ lence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than oth

The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated viru­ lence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than oth