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September 22, 2019

Acquisition of resistance to carbapenem and macrolide-mediated quorum sensing inhibition by Pseudomonas aeruginosa via ICE Tn4371 6385

Pseudomonas aeruginosa can cause life-threatening infections in immunocompromised patients. The first-line agents to treat P. aeruginosa infections are carbapenems. However, the emergence of carbapenem-resistant P. aeruginosa strains greatly compromised the effec- tiveness of carbapenem treatment, which makes the surveillance on their spreading and transmission important. Here we characterized the full-length genomes of two carbapenem- resistant P. aeruginosa clinical isolates that are capable of producing New Delhi metallo-ß- lactamase-1 (NDM-1). We show that blaNDM-1 is carried by a novel integrative and conjugative element (ICE) ICETn43716385, which also carries the macrolide resistance gene msr(E) and the florfenicol resistance gene floR. By exogenously expressing msr(E) in P. aeruginosa laboratory strains, we show that Msr(E) can abolish azithromycin-mediated quorum sensing inhibition in vitro and anti-Pseudomonas effect in vivo. We conclude that ICEs are important in transmitting carbapenem resistance, and that anti-virulence treatment of P. aeruginosa infections using sub-inhibitory concentrations of macrolides can be challenged by horizontal gene transfer.

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September 22, 2019

Coexistence of mcr-1, blaKPC-2 and two copies of fosA3 in a clinical Escherichia coli strain isolated from urine.

Here we report the first clinical Escherichia coli isolate co-harboring mcr-1, blaKPC-2 and two copies of fosA3 from China. The five plasmids of the isolate were completely sequenced and analyzed. Gene mcr-1 and blaKPC-2 were located on IncI2 and IncR plasmid, respectively. A variety of other resistance determinants such as fosA3 (two copies), blaCTX-M-123, blaOXA-1 and blaCTX-M-65 were also identified from the rest plasmids. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Long-read sequencing data analysis for yeasts.

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ~41 h to generate a complete and well-annotated genome from ~100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

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September 22, 2019

The presence of colistin resistance gene mcr-1 and -3 in ESBL producing Escherichia coli isolated from food in Ho Chi Minh City, Vietnam.

Colistin is indicated for the treatment of multidrug-resistant gram-negative bacterial infections. However, the spread of colistin-resistant bacteria harbouring an mcr gene has become a serious concern. This study investigated local foods in Vietnam for contamination with colistin-resistant bacteria. A total of 261 extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Escherichia coli isolates from 330 meat and seafood products were analysed for colistin susceptibility and the presence of mcr genes. Approximately, 24% (62/261) of ESBL- or AmpC-producing E. coli isolates showed colistin resistance; 97% (60/62) of colistin-resistant isolates harboured mcr-1, whereas 3% (2/62) harboured mcr-3. As the result of plasmid analysis of two strains, both plasmids harbouring mcr-3 revealed that plasmid replicon type was IncFII. Sequencing analysis indicated that an insertion sequence was present near mcr-3, suggesting that IncFII plasmids harbouring mcr-3 could be transferred to other bacterial species by horizontal transfer of the plasmid or transfer with some insertion sequence. In conclusion, ESBL-producing E. coli and AmpC-producing E. coli have acquired colistin resistance because 24% of such isolates show colistin resistance and 3% of the colistin-resistant strains harbour mcr-3. We reported the present of the mcr-3-carrying ESBL-producing E. coli isolated from pork in Vietnam.

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September 22, 2019

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

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September 22, 2019

Diversity and evolution of the emerging Pandoraviridae family.

With DNA genomes reaching 2.5?Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes.

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September 22, 2019

Unexpected invasion of miniature inverted-repeat transposable elements in viral genomes

Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life.

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September 22, 2019

Parallels between experimental and natural evolution of legume symbionts.

The emergence of symbiotic interactions has been studied using population genomics in nature and experimental evolution in the laboratory, but the parallels between these processes remain unknown. Here we compare the emergence of rhizobia after the horizontal transfer of a symbiotic plasmid in natural populations of Cupriavidus taiwanensis, over 10 MY ago, with the experimental evolution of symbiotic Ralstonia solanacearum for a few hundred generations. In spite of major differences in terms of time span, environment, genetic background, and phenotypic achievement, both processes resulted in rapid genetic diversification dominated by purifying selection. We observe no adaptation in the plasmid carrying the genes responsible for the ecological transition. Instead, adaptation was associated with positive selection in a set of genes that led to the co-option of the same quorum-sensing system in both processes. Our results provide evidence for similarities in experimental and natural evolutionary transitions and highlight the potential of comparisons between both processes to understand symbiogenesis.

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September 22, 2019

Phylogenomic analysis of Lactobacillus curvatus reveals two lineages distinguished by genes for fermenting plant-derived carbohydrates.

Lactobacillus curvatus is a lactic acid bacterium encountered in many different types of fermented food (meat, seafood, vegetables, and cereals). Although this species plays an important role in the preservation of these foods, few attempts have been made to assess its genomic diversity. This study uses comparative analyses of 13 published genomes (complete or draft) to better understand the evolutionary processes acting on the genome of this species. Phylogenomic analysis, based on a coalescent model of evolution, revealed that the 6,742 sites of single nucleotide polymorphism within the L. curvatus core genome delineate two major groups, with lineage 1 represented by the newly sequenced strain FLEC03, and lineage 2 represented by the type-strain DSM20019. The two lineages could also be distinguished by the content of their accessory genome, which sheds light on a long-term evolutionary process of lineage-dependent genetic acquisition and the possibility of population structure. Interestingly, one clade from lineage 2 shared more accessory genes with strains of lineage 1 than with other strains of lineage 2, indicating recent convergence in carbohydrate catabolism. Both lineages had a wide repertoire of accessory genes involved in the fermentation of plant-derived carbohydrates that are released from polymers of a/ß-glucans, a/ß-fructans, and N-acetylglucosan. Other gene clusters were distributed among strains according to the type of food from which the strains were isolated. These results give new insight into the ecological niches in which L. curvatus may naturally thrive (such as silage or compost heaps) in addition to fermented food.

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September 22, 2019

A short note on dynamic programming in a band.

Third generation sequencing technologies generate long reads that exhibit high error rates, in particular for insertions and deletions which are usually the most difficult errors to cope with. The only exact algorithm capable of aligning sequences with insertions and deletions is a dynamic programming algorithm.In this note, for the sake of efficiency, we consider dynamic programming in a band. We show how to choose the band width in function of the long reads’ error rates, thus obtaining an [Formula: see text] algorithm in space and time. We also propose a procedure to decide whether this algorithm, when applied to semi-global alignments, provides the optimal score.We suggest that dynamic programming in a band is well suited to the problem of aligning long reads between themselves and can be used as a core component of methods for obtaining a consensus sequence from the long reads alone. The function implementing the dynamic programming algorithm in a band is available, as a standalone program, at: https://forgemia.inra.fr/jean-francois.gibrat/BAND_DYN_PROG.git.

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September 22, 2019

A reference genome of the European beech (Fagus sylvatica L.).

The European beech is arguably the most important climax broad-leaved tree species in Central Europe, widely planted for its valuable wood. Here, we report the 542 Mb draft genome sequence of an up to 300-year-old individual (Bhaga) from an undisturbed stand in the Kellerwald-Edersee National Park in central Germany.Using a hybrid assembly approach, Illumina reads with short- and long-insert libraries, coupled with long Pacific Biosciences reads, we obtained an assembled genome size of 542 Mb, in line with flow cytometric genome size estimation. The largest scaffold was of 1.15 Mb, the N50 length was 145 kb, and the L50 count was 983. The assembly contained 0.12% of Ns. A Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis retrieved 94% complete BUSCO genes, well in the range of other high-quality draft genomes of trees. A total of 62,012 protein-coding genes were predicted, assisted by transcriptome sequencing. In addition, we are reporting an efficient method for extracting high-molecular-weight DNA from dormant buds, by which contamination by environmental bacteria and fungi was kept at a minimum.The assembled genome will be a valuable resource and reference for future population genomics studies on the evolution and past climate change adaptation of beech and will be helpful for identifying genes, e.g., involved in drought tolerance, in order to select and breed individuals to adapt forestry to climate change in Europe. A continuously updated genome browser and download page can be accessed from beechgenome.net, which will include future genome versions of the reference individual Bhaga, as new sequencing approaches develop.

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September 22, 2019

Landscape of the genome and host cell response of Mycobacterium shigaense reveals pathogenic features.

A systems approach was used to explore the genome and transcriptome of Mycobacterium shigaense, a new opportunistic pathogen isolated from a patient with a skin infection, and the host response transcriptome was assessed using a macrophage infection model. The M. shigaense genome comprises 5,207,883?bp, with 67.2% G+C content and 5098 predicted coding genes. Evolutionarily, the bacterium belongs to a cluster in the phylogenetic tree along with three target opportunistic pathogenic strains, namely, M. avium, M. triplex and M. simiae. Potential virulence genes are indeed expressed by M. shigaense under culture conditions. Phenotypically, M. shigaense had similar infection and replication capacities in a macrophage model as the opportunistic species compared to M. tuberculosis. M. shigaense activated NF-?B, TNF, cytokines and chemokines in the host innate immune-related signaling pathways and elicited an early response shared with pathogenic bacilli except M. tuberculosis. M. shigaense upregulated specific host response genes such as TLR7, CCL4 and CXCL5. We performed an integrated and comparative analysis of M. shigaense. Multigroup comparison indicated certain differences with typical pathogenic bacilli in terms of gene features and the macrophage response.

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September 22, 2019

Characteristics of carbapenem-resistant Enterobacteriaceae in ready-to-eat vegetables in China.

Vegetables harboring bacteria resistant to antibiotics are a growing food safety issue. However, data concerning carbapenem-resistant Enterobacteriaceae (CRE) in ready-to-eat fresh vegetables is still rare. In this study, 411 vegetable samples from 36 supermarkets or farmer’s markets in 18 cities in China, were analyzed for CRE. Carbapenemase-encoding genes and other resistance genes were analyzed among the CRE isolates. Plasmids carrying carbapenemase genes were studied by conjugation, replicon typing, S1-PFGE southern blot, restriction fragment length polymorphism (RFLP), and sequencing. CRE isolates were also analyzed by pulsed-field gel electrophoresis (PFGE). Ten vegetable samples yielded one or more CRE isolates. The highest detection rate of CRE (14.3%, 4/28) was found in curly endive. Twelve CRE isolates were obtained and all showed multidrug resistance: Escherichia coli, 5; Citrobacter freundii, 5; and Klebsiella pneumoniae, 2. All E. coli and C. freundii carried blaNDM, while K. pneumoniae harbored blaKPC-2. Notably, E. coli with blaNDM and ST23 hypervirulent Klebsiella pneumoniae (hvKP) carrying blaKPC-2 were found in the same cucumber sample and clonal spread of E. coli, C. freundii, and K. pneumoniae isolates were all observed between vegetable types and/or cities. IncX3 plasmids carrying blaNDM from E. coli and C. freundii showed identical or highly similar RFLP patterns, and the sequenced IncX3 plasmid from cucumber was also identical or highly similar (99%) to the IncX3 plasmids from clinical patients reported in other countries, while blaKPC-2 in K. pneumoniae was mediated by similar F35:A-:B1 plasmids. Our results suggest that both clonal expansion and horizontal transmission of IncX3- or F35:A-:B1-type plasmids may mediate the spread of CRE in ready-to-eat vegetables in China. The presence of CRE in ready-to-eat vegetables is alarming and constitutes a food safety issue. To our knowledge, this is the first report of either the C. freundii carrying blaNDM, or K. pneumoniae harboring blaKPC-2 in vegetables. This is also the first report of ST23 carbapenem-resistant hvKP strain in vegetables.

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September 22, 2019

Whole genome sequencing of Klebsiella pneumoniae strain unravels a new model for the development of extensive drug resistance in Enterobacteriaceae.

Increased incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide. The WHO warns about the imminent risk to global health if the spread of resistant bacteria is not contained.Here, single molecule real time sequencing was used to analyse the whole genome and resistome of SKGH01, a strain of Klebsiella pneumoniae .The data showed that SKGH01 was resistant to all commercially available antibiotics. A complete account of extensively drug-resistant (XDR) CRE at a genomic level and the entire location map of all antibiotic resistance components are here presented. Additionally, this work proposes a model of XDR acquisition in Enterobacteriaceae.

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September 22, 2019

Mosaic structure as the main feature of Mycobacterium bovis BCG genomes

Background: The genome stability of attenuated live BCG vaccine preventing the acute forms of childhood tuberculosis is an important aspect of vaccine production. The pur- pose of our study was a whole genome comparative analysis of BCG sub-strains and identification of potential triggers of sub-strains’ transition. Results: Genomes of three BCG Russia seed lots (1963, 1982, 2006 years) have been sequenced, and the stability of vaccine sub-strain genomes has been confirmed. A com- parative genome analysis of nine Mycobacterium bovis BCG and three M. bovis strains revealed their specific genome features associated with prophage profiles. A number of prophage-coded homologs to Caudovirales ORFs were common to all BCG genomes. Prophage profiles of BCG Tice and BCG Montreal genomes were unique and coded homologs to herpes viruses ORFs. The data of phylogenetic analysis of BCG sub-strain groups based on whole genome sequences and genome restriction maps were in con- gruence with prophage profiles. The only fragmentary similarity of specific prophage sequences of BCG Tice, BCG Montreal, and BCG Russia 368 in pair-wise alignments was observed, suggesting the impact of prophages on mosaic structure of genomes. Conclusions: The whole genome sequencing approach is essential for genomes with mosaic structure, harboring numerous prophage sequences. Tools for prophage search are effective instruments in this analysis.

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