Environmental metabarcoding reveals contrasting microbial communities at two poplar phytomanagement sites

The aim of the present study is to deepen the current understanding of the microbial communities at two poplar phytomanagement sites to reveal the environmental factors that drive the abundance, diversity and composition of microbial communities. A soil analysis revealed that the two soils displayed contrasting physico-chemical characteristics, with significant lower pH and higher Cd, Zn and Mn CaCl2-extractable fractions at Leforest site, compared with Pierrelaye site. The fungal and bacterial community profiles in the poplar roots and soils were assessed through Illumina MiSeq sequencing. Diversity indices and β-diversity measures illustrated that the root microbial communities were well …

X!TandemPipeline: a tool to manage sequence redundancy for protein inference and phosphosite identification

X!TandemPipeline is a software designed to perform protein inference and to manage redundancy in the results of phosphosite identification by database search. It provides the minimal list of proteins or phosphosites that are present in a set of samples using grouping algorithms based on the principle of parsimony. Regarding proteins, a two-level classification is performed, where groups gather proteins sharing at least one peptide and subgroups gather proteins that are not distinguishable according to the identified peptides. Regarding phosphosites, an innovative approach based on the concept of phosphoisland is used to gather overlapping phosphopeptides. The graphical interface of X!Tandem…

High genetic diversity among methicillin-susceptible Staphylococcus pseudintermedius in dogs in Europe.

International audience

Diversity and complexity of microbial communities from a chlor-alkali tailings dump

Abstract Revegetation of the tailings dumps produced by various industrial activities is necessary to prevent dust storms and erosion and represents a great challenge for ecological restoration. Little is known about the microbial colonisation and community structure of revegetated tailings following site exploitation. Here, we report the sequencing of 16S rRNA and internal transcribed spacer (ITS) fungal RNA gene amplicons from chlor-alkali residue and from an adjacent undisturbed soil to define the composition and assembly of the rhizosphere microbial communities. After quality filtering, a total of 72,373 and 89,929 bacterial sequences and 122,618 and 111,209 fungal sequences remained fo…

Combined Bacteriophage and Antibiotic Treatment Prevents Pseudomonas aeruginosa Infection of Wild Type and cftr- Epithelial Cells

International audience; With the increase of infections due to multidrug resistant bacterial pathogens and the shortage of antimicrobial molecules with novel targets, interest in bacteriophages as a therapeutic option has regained much attraction. Before the launch of future clinical trials, in vitro studies are required to better evaluate the efficacies and potential pitfalls of such therapies. Here we studied in an ex vivo human airway epithelial cell line model the efficacy of phage and ciprofloxacin alone and in combination to treat infection by Pseudomonas aeruginosa. The Calu-3 cell line and the isogenic CFTR knock down cell line (cftr-) infected apically with P. aeruginosa strain PAO…

The rise and the fall of a Pseudomonas aeruginosa endemic lineage in a hospital

The biological features that allow a pathogen to survive in the hospital environment are mostly unknown. The extinction of bacterial epidemics in hospitals is mostly attributed to changes in medical practice, including infection control, but the role of bacterial adaptation has never been documented. We analysed a collection of Pseudomonas aeruginosa isolates belonging to the Besançon Epidemic Strain (BES), responsible for a 12year nosocomial outbreak, using a genotype-to-phenotype approach. Bayesian analysis estimated the emergence of the clone in the hospital 5 years before its opening, during the creation of its water distribution network made of copper. BES survived better than the refe…

Deciphering the role of insertion sequences in the evolution of bacterial epidemic pathogens with panISa software

Next-generation sequencing (NGS) is now widely used in microbiology to explore genome evolution and the structure of pathogen outbreaks. Bioinformatics pipelines readily detect single-nucleotide polymorphisms or short indels. However, bacterial genomes also evolve through the action of small transposable elements called insertion sequences (ISs), which are difficult to detect due to their short length and multiple repetitions throughout the genome. We designed panISa software for the ab initio detection of IS insertions in the genomes of prokaryotes. PanISa has been released as open source software (GPL3) available from https://github.com/bvalot/panISa. In this study, we assessed the utilit…

A technical trick for studying proteomics in parallel to transcriptomics in symbiotic root-fungus interactions

We have developed a protocol in which proteins and mRNA can be analyzed from single root samples. This experimental design was validated in arbuscular mycorrhiza by comparing the proteins profiles obtained with those from a classical protein extraction process. It is a step forward to make simultaneous proteome and transcriptiome profiling possible.

Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks

Summary Aim To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. Methods We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. Findings PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates …

Impact of poplar-based phytomanagement on soil properties and microbial communities in a metal-contaminated site

Despite a long history of use in phytomanagement strategies, the impacts of poplar trees on the structure and function of microbial communities that live in the soil remain largely unknown. The current study combined fungal and bacterial community analyses from different management regimes using Illumina-based sequencing with soil analysis. The poplar phytomanagement regimes led to a significant increase in soil fertility and a decreased bioavailability of Zn and Cd, in concert with changes in the microbial communities. The most notable changes in the relative abundance of taxa and operational taxonomic units unsurprisingly indicated that root and soil constitute distinct ecological microbi…

Pyomelanin-producingPseudomonas aeruginosaselected during chronic infections have a large chromosomal deletion which confers resistance to pyocins

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, includin…

The membrane proteome of Medicago truncatula roots displays qualitative and quantitative changes in response to arbuscular mycorrhizal symbiosis

International audience; Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored. In this study, we first examined the qualitative composition of the root membrane proteome of Medicago truncatula after microsome enrichment and subsequent in dep…

Protéome membranaire en réponse à la symbiose mycorhizienne à arbuscules par GeLC-MS/MS

Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?

International audience; Bacterial typing is crucial to tackle the spread of bacterial pathogens but current methods are time-consuming and costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been recently integrated into the microbiology laboratory workflow for a quick and low-cost microbial species identification. Independent research groups have successfully redirected the original function of this technology from their primary purpose to discriminate subgroups within pathogen species. However, identical bacterial subgroups could be identified by unrelated peaks by independent methods, thus limiting their robustness and exportability. We…

Contamination of a hospital plumbing system by persister cells of a copper-tolerant high-risk clone of Pseudomonas aeruginosa

Abstract Background Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that thrives best in the distal elements of plumbing and waste-water systems. Although nosocomial outbreaks of PA have been associated with water sources, the role of the plumbing system of healthcare premises as a reservoir for this pathogen is still unclear. Materials and methods We collected water samples from 12 technical areas, distant from any medical activity, in a teaching hospital in France once a week for 11 weeks. We used a method that resuscitates persister cells because of the nutrient-poor conditions and the presence of inhibitors (e.g. chlorine and copper ions). Briefly, water was sampled i…

Biofilm biodiversity in French and Swiss show caves using the metabarcoding approach: First data.

International audience; In recent decades, show caves have begun to suffer from microorganism proliferation due to artificial lighting installations for touristic activity. In addition to the aesthetic problem, light encourages microorganisms that are responsible for physical and chemical degradation of limestone walls, speleothems and prehistoric paintings of cultural value. Microorganisms have previously been described by microscopy or culture-dependent methods, but data provided by new generation sequencing are rare. The authors identified, for the first time, microorganisms proliferating in one Swiss and in four French show caves using three different primers. The results showed that bo…

Genomic characterization of a local epidemic Pseudomonas aeruginosa reveals specific features of the widespread clone ST395

International audience; Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with several clones being frequently associated with outbreaks in hospital settings. ST395 is among these so-called 'international' clones. We aimed here to define the biological features that could have helped the implantation and spread of the clone ST395 in hospital settings. The complete genome of a multidrug resistant index isolate (DHS01) of a large hospital outbreak was analysed. We identified DHS01-specific genetic elements, among which were identified those shared with a panel of six independent ST395 isolates responsible for outbreaks in other hospitals. DHS01 has the fifth largest chromosome of …

Genome analysis of enterobacteriaceae with non-wild type susceptibility to third-generation cephalosporins recovered from diseased dogs and cats in Europe

Extended-spectrum-β-lactamases (ESBL) and plasmid-mediated cephalosporinases (pAmpC)-producing Enterobacteriaceae isolates are now reported worldwide in humans, animals, and in the environment. We identified the determinants of resistance to β-lactams and associated resistance genes as well as phylogenetic diversity of 53 ESBL- or pAmpC-producing Enterobacteriaceae isolated from dogs and cats in Europe.Of a collection of 842 Enterobacteriaceae isolates that were recovered in 2013 and 2014 from 842 diseased and untreated dogs and cats, for 242 ampicillin or amoxicillin resistant isolates (MIC ≥ 16 mg/L), cefotaxime (CTX) and ceftazidime (CAZ) MICs were determined. Isolates with CTX and/or CA…

Carbapenem-Susceptible OXA-23-Producing Proteus mirabilis in the French Community

International audience; Nineteen Proteus mirabilis isolates producing the carbapenemase OXA-23 were recovered over a 2-year period in 19 French hospitalized patients, of whom 12 had community onset infections. The isolates exhibited a slightly reduced susceptibility to carbapenems. Whole-genome analysis revealed that all 19 isolates formed a cluster compared to 149 other P. mirabilis isolates. Because of its susceptibility to carbapenems, this clone may be misidentified as a penicillinase producer while it constitutes a reservoir of the OXA-23-encoding gene in the community.

Antimicrobial susceptibility of nine udder pathogens recovered from bovine clinical mastitis milk in Europe 2015–2016: VetPath results

International audience; VetPath is an ongoing pan-European antimicrobial susceptibility monitoring programme collecting pathogens from diseased cattle, pigs and poultry not recently treated with antibiotics. Non-duplicate isolates (n = 1244) were obtained from cows with acute clinical mastitis in eight countries during 2015-2016 for centrally antimicrobial susceptibility testing according CLSI standards. Among Escherichia coli (n = 225), resistance was high to ampicillin and tetracycline, moderate to kanamycin and low to amoxicillin/clavulanic acid and cefazolin. The MIC50/90 of danofloxacin, enrofloxacin and marbofloxacin were 0.03 and 0.06 μg/mL. For Klebsiella spp. (n = 70), similar resu…

High prevalence and moderate diversity of Pseudomonas aeruginosa in the U-bends of high-risk units in hospital

The presence of P. aeruginosa in water supply is clearly identified as a risk factor for P. aeruginosa infection in critical care units, even if routes of transmission are often unclear and remain a matter of debate. We determined here the frequency of U-bends contaminated with P. aeruginosa in high-risk units and described the population structure of this opportunistic pathogen in a non-outbreak situation. Eighty-seven U-bends from sinks of rooms in five wards were sampled 3 times and P. aeruginosa was detected in 121 of the 261 (46.4%) U-bend samples. We genotyped 123 P. aeruginosa isolates with pulsed-field gel electrophoresis and multilocus sequence typing and found 41 pulsotypes distri…

panISa: ab initio detection of insertion sequences in bacterial genomes from short read sequence data.

Abstract Motivation The advent of next-generation sequencing has boosted the analysis of bacterial genome evolution. Insertion sequence (IS) elements play a key role in prokaryotic genome organization and evolution, but their repetitions in genomes complicate their detection from short-read data. Results PanISa is a software pipeline that identifies IS insertions ab initio in bacterial genomes from short-read data. It is a highly sensitive and precise tool based on the detection of read-mapping patterns at the insertion site. PanISa performs better than existing IS detection systems as it is based on a database-free approach. We applied it to a high-risk clone lineage of the pathogenic spec…

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high p…

The rise and the fall of a Pseudomonas aeruginosa epidemic lineage in a hospital

AbstractThe biological features that allow a pathogen to survive in the hospital environment are mostly unknown. The extinction of bacterial epidemics in hospitals is mostly attributed to changes in medical practice, including infection control, but the role of bacterial adaptation has never been documented. We analyzed a collection of Pseudomonas aeruginosa isolates belonging to the Besançon Epidemic Strain (BES), responsible for a 12-year nosocomial outbreak, using a genotype-to-phenotype approach. Bayesian analysis estimated the emergence of the clone in the hospital five years before its opening, during the creation of its water distribution network made of copper. BES survived better t…

Bacterial diversity associated with poplar trees grown on a Hg-contaminated site: Community characterization and isolation of Hg-resistant plant growth-promoting bacteria

International audience; Industrial waste dumps are rarely colonized by vegetation after they have been abandoned, indicating biological infertility. Revegetation of industrial tailings dumps is thus necessary to prevent wind erosion, metal leaching and has been shown to restore soil functions and ecosystem services. However, little is known about the microbial colonization and community structure of vegetated tailings following the application of restoration technologies. In this study, we investigated the rhizosphere and phyllosphere bacterial communities of a poplar tree plantation within a phytomanagement-based restoration program of a Hg-contaminated site. We used Illumina-based sequenc…

Label-free 1-DE-LC-MS/MS to identify arbuscular mycorrhiza-related membrane proteins

Deep changes in the shape and number of organelles, together with profound modifications in various membrane compartments, are induced within arbuscular mycorrhizal (AM) symbiosis. In this context, to investigate the membrane-associated proteins that are regulated in the model interaction Medicago truncatula – Rhizophagus irregularis, label-free 1DE-LC-MS/MS approach has been employed as alternative to two-dimensional gel electrophoresis. The existence of a correlation between protein abundance and peak areas or number of MS/MS spectra has widened the choice of label-free quantitative proteomics. The results highlighted microsomal protein candidates that could be involved in the symbiotic e…

Genotypic study of Citrobacter koseri , an emergent platelet contaminant since 2012 in France

International audience; Background: Transfusion-transmitted bacterial infection is a rare occurrence but the most feared complication in transfusion practices. Between 2012 and 2017, five cases of platelet concentrates (PCs) contaminated with the bacterial pathogen Citrobacter koseri (PC-Ck) have been reported in France, with two leading to the death of the recipients. We tested the possibilities of the emergence of a PC-specific clone of C. koseri (Ck) and of specific bacterial genes associated with PC contamination.Study design and methods: The phylogenetic network, based on a homemade Ck core genome scheme, inferred from the genomes of 20 worldwide Ck isolates unrelated to PC contaminati…

Production of Norspermidine Contributes to Aminoglycoside Resistance in pmrAB Mutants of Pseudomonas aeruginosa

Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-components systems, that promote addition of 4-amino-4-deoxy-L-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but r…