Plasmidome analysis of a hospital effluent biofilm: Status of antibiotic resistance.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

Reconstruction of plasmids by shotgun sequencing from environmental DNA: which bioinformatic workflow?

Plasmids play important roles in microbial evolution and also in the spread of antibiotic resistance. Plasmid sequences are extensively studied from clinical isolates but rarely from the environment with a metagenomic approach focused on the plasmid fraction referred to as the plasmidome. A clear challenge in this context is to define a workflow for discriminating plasmids from chromosomal contaminants existing in the plasmidome. For this purpose, we benchmarked existing tools from assembly to detection of the plasmids by reference-free methods (cBar and PlasFlow) and database-guided approaches. Our simulations took into account short-reads alone or combined with moderate long-reads like those actually generated in environmental genomics experiments. This benchmark allowed us to select the best tools for limiting false-positives associated to plasmid prediction tools and a combination of reference-guided methods based on plasmid and bacterial databases.

Morphology and infraciliature of two new earthworm ciliates, Hoplitophrya polymorphus sp. nov. and Anoplophrya simplex sp. nov. (Ciliophora: Oligohymenophorea: Astomatia).

Morphological and infraciliature studies carried out using pyridinated ammoniacal silver carbonate and the 4',6-diamidino-2-phenylindole (DAPI) staining techniques, led to the identification of two new species of ciliates pertaining to the subclass Astomatia. The first species, Hoplitophrya polymorphus sp. nov., displays two main cellular forms: the elongated form (150-247 µm long and 40-87 µm width) and the stocky form (140-170 µm long and 70-98 µm width). The macronucleus is generally skinny in the elongated forms and ribbon-shaped in the stocky forms. The common feature of the two cellular shapes uniting them in the same species is the identical structure of their skeletal apparatus, a V-shaped element located in an apical depression and bearing skeletal fibres on its ventral face. The second species, Anoplophrya simplex sp. nov., is totally deprived of skeletal apparatus. The cell shape is fusiform and dorsoventrally flattened (105-180 µm long and 65-125 µm width). Six to 12 pulsatile vacuoles form two rows arranged symmetrically on either side of the macronucleus. These two newly identified species highlight the extremely rich diversity of ciliates inhabiting the digestive tract of tropical earthworms.

Ciprofloxacin residue and antibiotic-resistant biofilm bacteria in hospital effluent.

Discharge of antimicrobial residues and resistant bacteria in hospital effluents is supposed to have strong impacts on the spread of antibiotic resistant bacteria in the environment. This study aimed to characterize the effluents of the Gabriel Montpied teaching hospital, Clermont-Ferrand, France, by simultaneously measuring the concentration of ciprofloxacin and of biological indicators resistant to this molecule in biofilms formed in the hospital effluent and by comparing these data to ciprofloxacin consumption and resistant bacterial isolates of the hospital. Determination of the measured environmental concentration of ciprofloxacin by spot sampling and polar organic chemical integrative (POCIS) sampling over 2 weeks, and comparison with predicted environmental concentrations produced a hazard quotient >1, indicating a potential ecotoxicological risk. A negative impact was also observed with whole hospital effluent samples using the Tetrahymena pyriformis biological model. During the same period, biofilms were formed within the hospital effluent, and analysis of ciprofloxacin-resistant isolates indicated that Gamma-Proteobacteria were numerous, predominantly Aeromonadaceae (69.56%) and Enterobacteriaceae (22.61%). Among the 115 isolates collected, plasmid-mediated fluoroquinolone-resistant genes were detected, with mostly aac(6')-lb-cr and qnrS. In addition, 60% of the isolates were resistant to up to six antibiotics, including molecules mostly used in the hospital (aminosides and third-generation cephalosporins). In parallel, 1247 bacteria isolated from hospitalized patients and resistant to at least one of the fluoroquinolones were collected. Only 5 of the 14 species identified in the effluent biofilm were also found in the clinical isolates, but PFGE typing of the Gram-negative isolates found in both compartments showed there was no clonality among the strains. Altogether, these data confirm the role of hospital loads as sources of pollution for wastewater and question the role of environmental biofilms communities as efficient shelters for hospital-released resistance genes.

Mixture of Sodium Hypochlorite and Hydrogen Peroxide on Adhered Aeromonas hydrophila to Solid Substrate in Water: Impact of Concentration and Assessment of the Synergistic Effect.

The synergistic effects of the combined treatments of NaOCl and H2O2 on the elimination of A. hydrophila adhered to polythene under static and dynamic conditions were evaluated. The concentrations 0.1, 0.2, and 0.3‰ NaOCl and 0.5, 1, and 1.5‰ H2O2 were used. The contact periods were 180, 360, 540, and 720 minutes. The abundance of cells adhered reached 2.47 and 2.27 units (log (CFU/cm²)), respectively, under static and dynamic conditions after action of the mixture of disinfectants, whereas it reached 2.41 and 3.39 units (log (CFU/cm²)) after action of NaOCl and H2O2 alone, respectively. Increase in the incubation period resulted in a significant decrease in the abundance of cells adhered when the mixture of 0.3‰ NaOCl and 1.5‰ H2O2 was used (P < 0.01). For each cell growth phase, there was a significant difference amongst the mean densities of cells adhered after action of the mixture of disinfectants (P < 0.05). Although the Freundlich isotherm parameters relatively varied from one experimental condition to another, the K f value registered in the exponential growth phase was relatively higher in static state than in dynamic regime; cells adhered under dynamic condition seem more sensitive to the synergistic action than those adhered under static condition.

Microcalorimetry: a powerful and original tool for tracking the toxicity of a xenobiotic on Tetrahymena pyriformis.

Fighting against water pollution requires the ability to evaluate the toxicity of pollutants such as herbicides. Tetrahymena pyriformis are ubiquitous ciliated protozoans commonly used in ecotoxicological research. Microcalorimetry can be used in many biological investigations as a universal, non-destructive and highly sensitive tool that provides a continuous real-time monitoring of the metabolic activity. This technique based on the thermal power output was applied to evaluate the influence of herbicide diuron on cultures of T. pyriformis. The heat flux produced upon addition of 0, 3.5, 7.0, 14.0, 28.0, and 56.0 µg mL⁻¹ of diuron was monitored. The biomass change during the growth was also determined by flow cytometry. The results confirmed that the growth of T. pyriformis is progressively inhibited as the concentration of diuron increases and revealed that the state of the living system is severely altered at a concentration of 56.0 µg mL⁻¹. The IC50 was estimated at 13.8 µg mL⁻¹ by microcalorimetry and at 18.6 µg mL⁻¹ by flow cytometry. It was shown that microcalorimetry is not only a very effective tool for the determination of the growth rate constant but that it is also a valuable probe for a rapid detection of the metabolic perturbations and, in ultimate cases, of the critical alterations of the living system under the action of a toxic agent.

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.

Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river.

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems.

Characterization and evolution of natural aquatic biofilm communities exposed in vitro to herbicides.

River biofilms are assemblies of autotrophic and heterotrophic microorganisms that can be affected by pollutants such as those found in watersheds and wastewater treatment plants. In the laboratory, experimental biofilms were formed from river water, and their overall composition was investigated. Denaturing gradient gel electrophoresis (DGGE) and cytometry were used to assess the richness and diversity of these communities. The software Cytostack (available on request) was developed to treat and analyze the cytometric data. Measurements of chlorophyll-a and carotenoids were used to assess the global composition of the photoautotrophic community, whereas proteins, polysaccharides (PS) content, and esterase activities were used to assess overall changes in the mixed communities. We evaluated the effects that 3 weeks of treatment with the herbicides diuron and glyphosate (10 µg L(-1)) had on these biofilms. Exposed to diuron, bacterial communities adapted, changing their composition. Glyphosate inhibited growth of one autotrophic community but caused no chlorophyll deficit. As a whole, the biofilm acted as a micro-ecosystem, able to regulate and maintain a constant level of photosynthetic pigment through the structural adaptation of the autotrophic community. These results are one more proof that microbial diversity of aquatic biofilms is influenced by chemical stresses, potentially leading to disturbances within the ecosystems.

Comparative effects of the herbicides chlortoluron and mesotrione on freshwater microalgae.

Extensive use of herbicides in agriculture is accompanied by the risk of environmental contamination of aquatic ecosystems. The present study shows the effects of the herbicides chlortoluron and mesotrione on three microalgae species: two chlorophyceae (Pediastrum tetras, Ankistrodesmus fusiformis) and one diatom (Amphora coffeaeformis). The authors calculated the IC50 for one chlorophyceae and the diatom. The order of toxicity (median inhibitory concentration [IC50]) for mesotrione was A. coffeaeformis (13.1 mg/L) > A. fusiformis (56.1 mg/L) and A. fusiformis (0.05 mg/L) > A. coffeaeformis (0.08 mg/L) for chlortoluron. The impact of herbicides applied at 0.2 mg/L was then examined in Erlenmeyer flasks by monitoring for growth, pigment content, and metabolic activity. Algal responses varied widely according to species and herbicide. For example, chlortoluron showed a significant inhibitory effect on the growth of A. coffeaeformis, whereas mesotrione induced an increase in cellular density in A. fusiformis. Other cellular parameters, such as pigment content in P. tetras, were stimulated by both herbicides. The results obtained confirmed that microalgae cultures are clearly affected by acute and chronic exposition to herbicides. Further monitoring should be carried out in the field to assess the impact of sublethal levels of toxicity and the growth-enhancing effects of mesotrione and chlortoluron on natural algae communities.

Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia.

BACKGROUND: The sub-membranous skeleton of the ciliate Paramecium, the epiplasm, is composed of hundreds of epiplasmic scales centered on basal bodies, and presents a complex set of proteins, epiplasmins, which belong to a multigenic family. The repeated duplications observed in the P. tetraurelia genome present an interesting model of the organization and evolution of a multigenic family within a single cell. RESULTS: To study this multigenic family, we used phylogenetic, structural, and analytical transcriptional approaches. The phylogenetic method defines 5 groups of epiplasmins in the multigenic family. A refined analysis by Hydrophobic Cluster Analysis (HCA) identifies structural characteristics of 51 epiplasmins, defining five separate groups, and three classes. Depending on the sequential arrangement of their structural domains, the epiplasmins are defined as symmetric, asymmetric or atypical. The EST data aid in this classification, in the identification of putative regulating sequences such as TATA or CAAT boxes. When specific RNAi experiments were conducted using sequences from either symmetric or asymmetric classes, phenotypes were drastic. Local effects show either disrupted or ill-shaped epiplasmic scales. In either case, this results in aborted cell division. Using structural features, we show that 4 epiplasmins are also present in another ciliate, Tetrahymena thermophila. Their affiliation with the distinctive structural groups of Paramecium epiplasmins demonstrates an interspecific multigenic family. CONCLUSION: The epiplasmin multigenic family illustrates the history of genomic duplication in Paramecium. This study provides a framework which can guide functional analysis of epiplasmins, the major components of the membrane skeleton in ciliates. We show that this set of proteins handles an important developmental information in Paramecium since maintenance of epiplasm organization is crucial for cell morphogenesis.

Identification of a new protein in the centrosome-like "atractophore" of Trichomonas vaginalis.

The human parasite Trichomonas vaginalis has specific structural bodies, atractophores, associated at one end to the kinetosomes and at the other to the spindle during division. A monoclonal antibody specific for a component of this structure was obtained. It recognizes a protein with a predicted molecular mass of 477 kDa. Sequence analysis of this protein shows that P477 belongs to the family of large coiled-coil proteins, sharing a highly versatile protein folding motif adaptable to many biological functions. P477-might act as an anchor to localize cellular activities and components to the golgi centrosomal region. It may represent a new class of structural proteins, since similar proteins were found in many protozoans.

Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina) form a new family of flagellates (Collodictyonidae) with tubular mitochondrial cristae that is phylogenetically distant from other flagellate groups.

Comparative electron microscopic studies of Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina) showed that they share a distinctive flagellar transitional zone and a very similar flagellar apparatus. In both species, the basic couple of basal bodies and flagella #1 and #2 are connected to the dorsal and ventral roots, respectively. Collodictyon triciliatum has two additional basal bodies and flagella, #3 and #4, situated on each side of the basic couple, each of which also bears a dorsal root. The horseshoe-shaped arrangement of dictyosomes, mitochondria with tubular cristae and the deep ventral groove are very similar to those of Diphylleia rotans. These two genera have very specific features and are placed in a new family, Collodictyonidae, distinct from other eukaryotic groups. Electron microscopic observation of mitotic telophase in Diphylleia rotans revealed two chromosomal masses, surrounded by the nuclear envelope, within the dividing parental nucleus, as in the telophase stage of the heliozoan Actinophrys and the helioflagellate Dimorpha. Spindle microtubules arise from several MTOCs outside the nucleus, and several microtubules penetrate within the dividing nucleus, via pores at the poles. This semi-open type of orthomitosis is reminiscent of that of actinophryids. The SSU rDNA sequence of Diphylleia rotans was compared with that of all the eukaryotic groups that have a slow-evolving rDNA. Diphylleia did not strongly assemble with any group and emerged in a very poorly resolved part of the eukaryotic phylogenetic tree.