Genomic diversity and metabolic potential of marine Pseudomonadaceae

Recent changes in the taxonomy of the Pseudomonadaceae family have led to the delineation of three new genera (Atopomonas, Halopseudomonas and Stutzerimonas). However, the genus Pseudomonas remains the most densely populated and displays a broad genetic diversity. Pseudomonas are able to produce a wide variety of secondary metabolites which drives important ecological functions and have a great impact in sustaining their lifestyles. While soilborne Pseudomonas are constantly examined, we currently lack studies aiming to explore the genetic diversity and metabolic potential of marine Pseudomonas spp. In this study, 23 Pseudomonas strains were co-isolated with Vibrio strains from three marine microalgal cultures and rpoD-based phylogeny allowed their assignment to the Pseudomonas oleovorans group (Pseudomonas chengduensis, Pseudomonas toyotomiensis and one new species). We combined whole genome sequencing on three selected strains with an inventory of marine Pseudomonas genomes to assess their phylogenetic assignations and explore their metabolic potential. Our results revealed that most strains are incorrectly assigned at the species level and half of them do not belong to the genus Pseudomonas but instead to the genera Halopseudomonas or Stutzerimonas. We highlight the presence of 26 new species (Halopseudomonas (n = 5), Stutzerimonas (n = 7) and Pseudomonas (n = 14)) and describe one new species, Pseudomonas chaetocerotis sp. nov. (type strain 536T = LMG 31766T = DSM 111343T). We used genome mining to identify numerous BGCs coding for the production of diverse known metabolites (i.e., osmoprotectants, photoprotectants, quorum sensing molecules, siderophores, cyclic lipopeptides) but also unknown metabolites (e.g., ARE, hybrid ARE-DAR, siderophores, orphan NRPS gene clusters) awaiting chemical characterization. Finally, this study underlines that marine environments host a huge diversity of Pseudomonadaceae that can drive the discovery of new secondary metabolites

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization, direct viable counting and solid phase cytometry

Aims: We developed an improved Fluorescent In Situ Hybridization FISHbased method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. Methods and Results: The method includes a direct viable count (DVC) assay, multi-probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC-FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. Conclusion: The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. Significance and Impact of the Study: The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments

Dynamique des légionelles dans un fleuve côtier méditerranéen, le Tech.

Plus d un quart de siècle après leur découverte, les légionelles constituent toujours un problème de santé publique et suscitent un grand intérêt au sein de la communauté scientifique. Le comportement de cette bactérie hydrotellurique dans son environnement naturel est aujourd hui méconnu. L objectif de ce travail a été d apporter les premiers éléments pour mieux comprendre l écologie complexe des légionelles notamment en déterminant les facteurs responsables de leur occurrence et de leur persistance en milieu fluviatile. Les difficultés inhérentes à la détection des légionelles par les techniques classiques de mise en culture ont nécessité le développement de méthodes alternatives adaptées au dénombrement des Legionella pneumophila dans les eaux de surface et à l analyse de la diversité génétique des Legionella spp.. Les travaux réalisés ont permis le développement d une méthode rapide et directe d immunodétection des L. pneumophila combinée à la cytométrie en phase solide et couplée au marquage de l activité estérase des cellules, afin de quantifier les cellules viables potentiellement pathogènes dans les eaux et les sédiments fluviatiles. La méthode d empreinte moléculaire, de CE-SSCP1, développée dans ce travail, a permis d étudier l évolution au cours d un cycle annuel de la structure génétique des populations de légionelles présentes en rivière. Les résultats de cette étude réalisée sur un fleuve côtier méditerranéen, le Tech, ont permis de confirmer le caractère ubiquitaire des légionelles, présentes en quantité importante et récurrente de l amont à l aval du fleuve. L étude de la dynamique des L. pneumophila a mis en évidence (i) des variations spatiales des concentrations en L. pneumophila dans les eaux fluviales, (ii) l existence d un gradient de concentration de l amont vers l aval, et (iii) des variations saisonnières, avec des maxima en été et des minima en hiver. L étude de la diversité génétique des légionelles a montré des évolutions différentes et notamment une composition génétique très diversifiée des populations légionelles dès l amont du fleuve et une absence de gradient amont/aval des espèces présentes. Par ailleurs, l impact des rejets anthropiques sur l activité physiologique des L. pneumophila et sur la structure génétique des populations de légionelles a été démontré, et principalement ceux des rejets des établissements thermaux utilisant des eaux chaudes. La température a été estimée comme étant un des facteurs abiotiques clés dans la prolifération et la survie des légionelles en rivière. 1 CE-SSCP : Capillary Electrophoresis Single Strand Conformation PolymorphismPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family Enterobacteriaceae in Freshwater and Drinking Water

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10(8) nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed

Rapid quantification of viable Legionella in nuclear cooling tower waters using filter cultivation, fluorescent in situ hybridization, and solid phase cytometry

International audienceTo develop a rapid and sensitive method to quantify viable Legionella spp. in cooling tower water samples. A rapid, culture-based method capable of quantifying as few as 600 Legionella microcolonies per litre within 2 days in industrial waters was developed. The method combines a short cultivation step of microcolonies on GVPC agar plate, specific detection of Legionella cells by a fluorescent in situ hybridization (FISH) approach, and a sensitive enumeration using a solid-phase cytometer. Following optimization of the cultivation conditions, the qualitative and quantitative performance of the method was assessed and the method was applied to 262 nuclear power plant cooling water samples. The performance of this method was in accordance with the culture method (NF-T 90-431) for Legionella enumeration. The rapid detection of viable Legionella in water is a major concern to the effective monitoring of this pathogenic bacterium in the main water sources involved in the transmission of legionellosis infection (Legionnaires' disease). The new method proposed here appears to be a robust, efficient and innovative means for rapidly quantifying cultivable Legionella in cooling tower water samples within 48 h

Genetic diversity and phenotypic plasticity of AHL-mediated Quorum sensing in environmental strains of Vibrio mediterranei

International audienceN-Acyl homoserine lactone (AHL)-mediated Quorum sensing (QS) is one of the most studied social behavior among Proteobacteria. However, despite the current knowledge on QS-associated phenotypes such as bioluminescence, biofilm formation, or pathogenesis, the characterization of environmental factors driving QS in realistic ecological settings remains scarce. We investigated the dynamics of AHL and AHL-producing Vibrio among 840 isolates collected fortnightly from the Salses-Leucate Mediterranean lagoon in spring and summer 2015 and 2016. Vibrio isolates were characterized by gyrB gene sequencing, Enterobacterial repetitive intergenic consensus polymerase chain reaction, and genome sequencing, and AHL production was investigated by a biosensors-based UHPLC–HRMS/MS approach. Our results revealed, for the first time, a succession of V. mediterranei isolates with different AHL production phenotypes over time and this dynamics was observed in a single genotype (average genomic nucleotide identity >99.9). A multivariate DistLM analysis revealed that 83.4% of the temporal variation of V. mediterranei QS phenotypes was explained by environmental variables. Overall, our results suggest that isolates of a single genotype are able to change their QS phenotypes in response to environmental conditions, highlighting the phenotypic plasticity of bacterial communication in the environment

Evidence of a Large Diversity of N-acyl-Homoserine Lactones in Symbiotic Vibrio fischeri Strains Associated with the Squid Euprymna scolopes

Vibrio fischeri possesses a complex AHL-mediated Quorum-sensing (QS) system including two pathways, LuxI/R (3-oxo-C6-HSL and C6-HSL) and AinS/R (C8-HSL), which are important for the regulation of physiological traits. Diverse QS-dependent functional phenotypes have been described in V. fischeri; however, AHL diversity is still underestimated. In the present study, we investigated AHL diversity in five symbiotic V. fischeri strains with distinct phenotypic properties using UHPLC-HRMS/MS. The results obtained (1) revealed an unexpectedly high diversity of signaling molecules, (2) emphasized the complexity of QS in V. fischeri, and (3) highlight the importance of understanding the specificity of AHL-mediated QS.status: publishe

Evidence of a Large Diversity of <i>N</i>-acyl-Homoserine Lactones in Symbiotic <i>Vibrio fischeri</i> Strains Associated with the Squid <i>Euprymna scolopes</i>

International audienc

Spatiotemporal dynamics of total viable Vibrio spp. in a NW Mediterranean Coastal Area

A cellular approach combining Direct Viable Counting and Fluorescent In Situ Hybridization using a one-step multiple-probe technique and Solid Phase Cytometry (DVC-FISH-SPC) was developed to monitor total viable vibrios and cover the detection of a large diversity of vibrios. FISH combined three probes in the same assay and targeted sequences located at different positions on the 16S rRNA of Vibrio and Aliivibrio members. We performed a 10-month in situ study to investigate the weekly dynamics of viable vibrios relative to culturable counts at two northwestern Mediterranean coastal sites, and identified the key physicochemical factors for their occurrence in water using a multivariate analysis. Total viable and culturable cell counts showed the same temporal pattern during the warmer season, whereas the ratios between both methods were inverted during the colder seasons (<15°C), indicating that some of the vibrio community had entered into a viable but non-culturable (VBNC) state. We confirmed that Seawater Surface Temperature explained 51-62% of the total variance in culturable counts, and also showed that the occurrence of viable vibrios is controlled by two variables, pheopigment (15%) and phosphate (12%) concentrations, suggesting that other unidentified factors play a role in maintaining viability.SCOPUS: ar.jinfo:eu-repo/semantics/publishe