Nadia Riera
email: nriera@pasteur.edu.uy

Danilo Davyt

Rosario Durán

Gregorio Iraola

Philippe Lemanceau

Natalia Bajsa
email: nbajsa@iibce.edu.uy

Chao Lei

Hao Zhang

An antibiotic produced by Pseudomonas fluorescens CFBP2392 with antifungal activity against Rhizoctonia solani

Keywords: antifungal, Pseudomonas fluorescens, biocontrol, Rhizoctonia solani, specialized metabolites, genome

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Molecular communication among plants and microbes is a key factor establishing both beneficial and pathogenic relationships in the rhizosphere [START_REF] Stringlis | Microbial small molecules-weapons of plant subversion[END_REF]. Bioactive compounds are part of the complex communication that shapes the microbiome of the plant, now known to have a significant role in plant's health [START_REF] Mendes | Deciphering the rhizosphere microbiome for disease-suppressive bacteria[END_REF][START_REF] Turner | The plant microbiome[END_REF][START_REF] Schlaeppi | The plant microbiome at work[END_REF][START_REF] Vandenkoornhuyse | The importance of the microbiome of the plant holobiont[END_REF][START_REF] Zhang | Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome[END_REF].

Fluorescent pseudomonas are ubiquitous in many environments including the rhizosphere. Many strains within this group are plant growth promoting bacteria and biocontrol agents [START_REF] Haas | Biological control of soil-borne pathogens by fluorescent pseudomonads[END_REF][START_REF] Weller | Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years[END_REF][START_REF] Höfte | Fluorescent pseudomonads as biocontrol agents for sustainable agricultural systems[END_REF]. Some of their positive traits include the utilization of plant exudates as a nutrient source, the production of lytic enzymes, their ability to efficiently colonize and multiply in the rhizosphere, activate the systemic immune resistance of the plant and their great genomic potential to produce bioactive small molecules [START_REF] Walsh | Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation[END_REF][START_REF] Raaijmakers | Antibiotic production by bacterial biocontrol agents[END_REF][START_REF] Haas | Biological control of soil-borne pathogens by fluorescent pseudomonads[END_REF][START_REF] Weller | Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years[END_REF][START_REF] Stringlis | Microbial small molecules-weapons of plant subversion[END_REF]. In fact, the production of secondary metabolites from Pseudomonas spp. is closely linked to their ability as biocontrol agents [START_REF] Thomashow | Production of the antibiotic phenazine-1-carboxylic acid by fluorescent Pseudomonas species in the rhizosphere of wheat[END_REF][START_REF] Walsh | Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation[END_REF]. Within the beneficial pseudomonas many are able to produce siderophores, growth promoting substances, volatile organic compounds (VOCs) as well as antibiotics. The latter include: 2,4-DAPG, derivatives of phenazine, pyrrolnitrine, pioluteorin, cyclic lipopeptides (CLP), and cell surface molecules with biosurfactant and antifungal activity [START_REF] Haas | Biological control of soil-borne pathogens by fluorescent pseudomonads[END_REF].

Pseudomonas fluorescens A6, registered in the CIRM-CFBP French Collection for Plant Associated Bacteria 1 as CFBP2392, was originally isolated from the rhizosphere of bean cultivated in a soil showing a low incidence of damping-off [START_REF] Lemanceau | Relations entre les quelques caractéristiques in vitro de 10 Pseudomonas fluorescents et leur effet sur la croissance du haricot (Phaseolus vulgaris)[END_REF] bean rhizosphere. This strain was showed to be a good root colonizer [START_REF] Gamalero | Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy[END_REF], to promote plant growth and root architecture [START_REF] Gamalero | Morphogenetic modifications induced by Pseudomonas fluorescens A6RI and Glomus mosseae BEG12 in the root system of tomato differ according to plant growth conditions[END_REF] and to suppress R. solani can suppress damping-off and root rot [START_REF] Berta | Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis[END_REF]. In the present work, we sought to characterize the antimicrobial spectra of the strain against phytopathogenic fungi and bacteria. We observed a strong antifungal activity against R. solani in dual culture assays. To further understand the mechanism of inhibition, we purified the active compound and performed dual culture assays testing the effect of the pure compound and the strain growing together with R. solani. Using Transmission Electron Microscopy (TEM), a structural effect in R. solani morphology was observed when grown in close proximity of the bacteria and the purified compound.

In recent years, the rapid development of new sequence technologies has revolutionized microbiology. An emerging field from this revolution is the discovery of new drugs using genome mining from the new pool of high-quality genomes available [START_REF] Baltz | Genome mining for drug discovery: progress at the front end[END_REF]. In this work, we used Oxford Nanopore Sequencing, a third-generation sequencing technology based on long reads, to obtain the complete high-quality genome of P. fluorescens CFBP2392. We identified different Biosynthetic Gene Clusters (BGCs) including four non-ribosomal peptide synthases (NRPS) clusters. Furthermore, we used the amino acid sequence of the ABC transporter MacB to predict the chemical nature of the metabolite exported using sequence phylogeny as previously reported [START_REF] Girard | Transporter gene-mediated typing for detection and genome mining of lipopeptideproducing Pseudomonas[END_REF]. In agreement with the BGCs prediction an amphisin-transporter was found in this strain and other phylogenetically similar P. fluorescens. The lokisinlike cluster shares 85% similarity with other lokisins, predicting some structural dissimilarities with other amphisins reported thus far. Our results give relevant structural mechanistic properties of the novel 1 https://cirm-cfbp.fr/ antibiotic compound and provide a genomic background of the putative chemical nature.

Materials and methods

Antifungal activity

Antifungal activity was tested in dual culture petri dishes by inoculating mycelia in the center of the dish and a bacterial strike 4 cm on each side as previously described [START_REF] Geels | Selection of antagonistic fluorescent Pseudomonas spp. and their root colonization and persistence following treatment of seed potatoes[END_REF]. Strain P. fluorescens CFBP2392 was tested against Pythium debaryanum, Fusarium oxysporum, Aspergillus niger, Alternaria sp., and different isolates of Rhizoctonia solani (Supplementary Table 1). All assays were performed in rich (PSFM) and minimal (MMP) media. In each case, a control plate with mycelium alone was incorporated as a control and all plates were incubated at 25°C.

Media

Pseudomonas Agar F Modified is a modified version of Bacto Pseudomonas Agar F (Difco ® ) containing per liter: Tryptone 10 g, Peptone protease 10 g, K 2 HPO 4 1.5 g, MgSO 4 .7H 2 O 1.5 g, glycerol 15 mL, Agar 20 g, and FeCl 3 50 μM. MMP media was done as a modified version of Minimal media for Pseudomonas [START_REF] Loper | Role of fluorescent siderophore production in biological control of Pythium ultimum by a Pseudomonas fluorescens strain[END_REF] containing per liter: K 2 HPO 4 3 g, NaH 2 PO 4 1 g, NH 4 Cl 1 g, MgSO 4 .7H 2 O 1.5 g, FeCl 3 50 μM, and glycerol 15 mL.

Purification of antimicrobial compounds

Strain P. fluorescens CFBP2392 was grown in 50 mL of PSFM medium for 3 days. Four 6-L flasks containing 2 L of PSFM were inoculated with 30 mL of the previous cultures and incubated for 3 days. The 8 L of culture were acidified with TFA to pH 1 and extracted with ethyl acetate. To separate the phases, the emulsion formed was filtered through celite (diatom skeletons). The organic phase was evaporated to dryness and the residue obtained was stored at -20°C.

Sample preparation for microscopy

Samples of young R. solani mycelium were taken from bioassays by cutting pieces of agar from the outer edge of the colony, adjacent to the bacteria or the paper disk. Fixation with 2% glutaraldehyde and 200 mM CaCl 2 in cacodylate buffer was performed overnight at 4°C. After four washes with cacodylate buffer, half of the samples from each treatment were post-fixed with osmium tetroxide in the same buffer for 1 h at 4°C. All the samples were dehydrated using increasing concentrations of ethanol and then the inclusion process was carried out, ensuring that the apices of the hyphae were positioned in the anterior part of the blocks. The samples with double fixation were included in Epon resin and the rest in LR White resin. Semi-thin sections (0.5 μm) were made using a Reicher Ultracut microtome and a glass blade, which were stained with toluidine blue and observed under an optical microscope. Subsequently, ultrathin sections (97 nm) were cut using a 1.2 mm wide diamond blade (Diatome), which were placed on copper or gold grids pre-coated with carbon. All cuts were made with the precaution of sampling the apical zone of the mycelium.

Transmission electron microscopy

Observations were made using a Hitachi 7500 transmission electron microscope at 80 kV, located at INRA (Dijon, France). For each treatment and with each of the revealed ones, two grids (from two different blocks) were analyzed and approximately 15 hyphal sections were examined in each of them.

Measurement of cell wall thickness

Cell wall thickness measurements were made on digital images obtained by DC staining, using the Image-Pro Plus V 4.5.0.19 program (Cellular Biology Laboratory, IIBCE). Approximately 20 measurements per image were carried out and the wall thickness in nanometers was calculated from the average value (correcting for the magnification used in each image). 25-30 cells per treatment, found in two grids from two different blocks, were analyzed. The data were analyzed by ANOVA after a log10 transformation to homogenize the variances. The means were compared with Duncan's test with p < 0.05.

DNA extraction and sequencing

A 2 mL overnight liquid culture from a single colony was used for nucleic acid extraction. DNA was extracted using PureLink ® Genomic DNA Kit following the manual for Gram-negative bacteria cell lysis. Extracted DNA was fragmented to a 10 kb size using Covaris g-TUBE. Final DNA concentration was quantified fluorometrically using the Qubit 3.0 fluorimeter (Thermo Fisher Scientific).

Fragmented DNA was further used to prepare the library following the Oxford Nanopore protocol Ligation sequencing gDNA (SQK-LSK109). Briefly, for DNA repair and end-prep, 500 ng fragmented DNA was treated with NEBNext FFPE DNA Repair (New England Biolabs) followed by Ultra II End Repair (New England Biolabs) in a Bio-Rad T100TM thermal cycler. Oxford Nanopore Adapter Mix (AMX) was ligated to the end-repaired adenylated DNA using Ligation Buffer (Oxford Nanopore Technologies, United Kingdom) and NEBNext Quick T4 DNA ligase at room temperature. In each step, DNA was purified using AMPure XP Reagent beads and quantified with a Qubit 2.0 Fluorometer. The library was loaded in a Flongle flow cell on a MinION Mk1C device and sequenced for 24 h.

Genome assembly and annotation

Sequencing finalized with a total of 779,479 reads that passed quality threshold. High accuracy basecalling and trimming was performed with guppy (V4.0.11). Processed reads were assembled with flye (V2.9) and the final assembly resulted in a genome total length of 6,645,616 bases, in one fragment, and an estimated mean coverage of 111 with a minimum overlap of 5,000. The genome was annotated with prodigal (V2.6.3).

Comparative genome analysis and specialized metabolites prediction

For the comparative analysis, genomes from Pseudomonas fluorescens group (taxon ID = 294) were downloaded from NCBI database with the-assembly-level complete flag. FastANI was used to compare P. fluorescens CFBP2392 with the rest, those genomes with ANI > 97% were selected for further studies (n = 13). Assembled genomes were annotated with prokka (v1.14.6), and pangenome estimation was analyzed with Pewit. 2 Maximum likelihood phylogenetic analysis was performed using Pagoo library in R with a core level of 95%, resulting in 4,560 core genes [START_REF] Ferrés | Protocol for post-processing of bacterial pangenome data using Pagoo pipeline[END_REF]. Prediction of the putative antimicrobial biosynthesis clusters was performed using antiSMASH software (version 7.0; [START_REF] Blin | antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation[END_REF].

Phylogeny of PleB protein

Pseudomonas lipopeptide export B (PleB) protein sequences were downloaded from NCBI database from each representative lipopeptide family factin, bananamide, viscosin, orfamide, poaeamide, amphisin, gacamide, putisolvin, xantholysin, entolysin, tolaasin, and peptin. Accession numbers were as follows AHZ34236.1, SDT21849.1, ESW57247.1, AGL83961.1, AGE25482.1, MBY8934714.1, ABA73958.1, ABW17379.1, AGM14936.1, CAK15812.1, PKA77400.1, and AKF46139.1, respectively. PleB sequence from region 5 of CFBP2392 was included in the analysis. We further compared seven additional PleB proteins from amphisin family from NCBI namely: ABW17379.1, MBY8934714.1, WP_064118557.1, BAF40423.1, QDF82252.1, QNL34620.1, and WP_175554031.1 together with P. fluorescens CFBP2392 PleB MacB_R5_CFBP2392. In all cases, sequences were aligned according to their protein sequence using PROMALS3D with standard parameters (mafft; [START_REF] Pei | PROMALS3D web server for accurate multiple protein sequence and structure alignments[END_REF]. The resulting alignment was used to generate the tree with iqtree (2.1.4-beta).

Results

Antifungal and antibacterial activity of Pseudomonas fluorescens CFBP2392

We screened the antimicrobial spectrum of P. fluorescens CFBP2392 against different fungal and bacterial isolates. Initially, in vitro antifungal activity was tested against four different fungal species: Alternaria sp., Fusarium oxysporum, Pythium debaryanum, and Rhizoctonia solani AG3. Pseudomonas fluorescens CFBP2392 was able to inhibit fungal growth in vitro in antagonist assay against all species. We further tested the ability to inhibit different strains within the same species and we tested the antifungal activity against three additional R. solani Argentinian isolates: R. solani 118 AG4, R. solani R81 AG4, and R. solani 109. We decided to test two different growth conditions, Pseudomonas minimal media (MMP) and rich media Pseudomonas Agar F Modified (PSFM) to evaluate the antimicrobial activity against these phytopathogens. We observed stronger antifungal activity in vitro against F. oxysporum, Alternaria, and R. solani when microorganisms were grown in rich media (PSFM). Pseudomonas fluorescens CFBP2392 presented inhibitory activity against all tested fungal isolates in vitro (Figure 1; Table 1;Supplementary Table 1).

Fluorescent pseudomonas are a group of ubiquitous well known biocontrol agents. Antibiotic compounds produced by biocontrol strains help to orchestrate microbial communities and structure. In order to explore the antibacterial spectrum of P. fluorescens CFBP2392, we decided to evaluate the effect on Gram positive and Gram negative bacterial phytophatogens. Pseudomonas fluorescens CFBP2392 was able to inhibit Clavibacter michiganensis but no activity was observed against the Gram negative Ralstonia solanacearum Rs8 or Herbaspirillum seropedicae Z69 (Supplementary Table 2; Supplementary Figure 1; Supplementary Methods). In addition, we decided to explore the ability of the strain to produce compounds with antimicrobial activity seen in other Pseudomonas spp. strains. We here show that P. fluorescens CFBP2392 is able to produce hydrogen cyanide (HCN) and exoproteases (Supplementary Figure 2; Supplementary Methods).

Production and purification of antifungal compounds

Cell free supernatant (CFS) from P. fluorescens CFBP2392 grown in PSFM was evaluated for its antifungal activity by incorporating either (1) no CFS (Control), (2) 2.5 mL CFS, and (3) 5 mL CFS in plates where R. solani was later inoculated. After 4 days, the control plate with no added CFS was covered with R. solani mycelia while plates with 2.5 mL were only half covered with mycelia and for plates with 5 mL CFS we observed no fungal growth. We decided to do a first chemical extraction of CFS using ethyl acetate and resuspending the extract in methanol (see methods). We confirmed the organic phase had antifungal activity as compared to control with methanol in plate bioassays (Supplementary Figure 2). For further analysis, we performed the same protocol for chemical extraction from an initial culture of 8 L, and we purified the organic phase with adsorption chromatography. Fractions with activity were evaluated with a bioassay as previously described and further purified with HPLC collecting a single peak (Supplementary Figure 3). To evaluate the impact in R. solani mycelia, the fraction corresponding to a single peak was used in subsequent microscopic analysis.

Pseudomonas fluorescens CFBP2392 induces alterations in Rhizoctonia solani cell wall ultrastructure

Rhizoctonia solani cell wall enlargement due to antifungal exposure was previously reported (Benhamou and Chet, 1993;[START_REF] Bianchi | Ultrastructural studies of the effects of Allium sativum on Phytopathogenic fungi in vitro[END_REF][START_REF] Benhamou | Ultrastructural and cytochemical aspects of the interaction between the mycoparasite Pythium oligandrum and soilborne plant pathogens[END_REF]. We sought to evaluate the modifications in the cell wall thickness in the presence of bacteria or purified compound (PC) or partially purified compound (PPC). The antifungal activity of the purified extract was tested in vitro and samples for microscopy were prepared from these plates (Supplementary Figure 4). A strong enlargement effect was observed when comparing R. solani cell wall thickness in the presence of P. fluorescens CFBP2392 against untreated control, but not in the presence of the purified or partially purified compound (Figure 2). The morphological alterations in cell wall morphology were clear in the presence of P. fluorescens. In some regions, P. fluorescens induced deformations in the fungal cell wall and separation toward the cell interior. These changes were not observed for the purified or partially purified compound. In light of these results, we cannot attribute the changes in cell wall morphology to the purified antibiotic. Further data are needed to explain the cause of P. fluorescens effect on R. solani ultrastructural changes.

Pseudomonas fluorescens CFBP2392 triggers cytoplasmic alterations in

Rhizoctonia solani

Fungal tissue grown in the presence of P. fluorescens CFBP2392 or purified compound (PC) presented strong structural and morphological changes in cytoplasmic organelles as compared to untreated R. solani control (Figure 3A). Heterogeneous regions were abundantly present in the cell, which corresponded with glycogen accumulation (based on silver proteinate-periodic acid and thiocarbohydrazide staining). An increase in the number of vacuoles was observed in the fungal cell tissue when exposed to partially purified compound (PPC) and CP but not in the presence of P. fluorescens CFBP2392 (Figure 3B). In a similar way, PC also produced membranous vesicles with granular content in R. solani. The most significant change induced by PC and PPC in cytoplasm morphology, however, is seen in the mitochondria. The purified fraction of antifungal compound triggered changes in mitochondria morphology resulting in hypertrophy (Figure 3B).

Complete genomic sequencing of Pseudomonas fluorescens CFBP2392

We decided to explore the genomic content of the strain by obtaining the complete genome using Oxford Nanopore Sequencing Technologies. High accuracy base calling and read processing were performed with guppy (4.0.11) and the obtained reads were assembled with flye (V2.9). The resulting sequence mapped to a single circular chromosome with an estimated mean coverage of 111 and a minimum overlap of 5,000 pb (Supplementary Table 3). We speculated that other members of the Pseudomonas fluorescens complex community may have been previously characterized for their ability to produce specialized antibiotics. We collected all complete genomes from Pseudomonas fluorescens from NCBI database and performed a pangenome analysis to study the distribution of core/accessory genes among the group. We obtained 173 genomic sequences from NCBI under the taxon ID 294 and we filtered them by genome quality with CheckM, which resulted in 165 genomes. Due to the high variability among the complex, we further filtered those genomes with high ANI (>85%) with P. fluorescence CFBP2392 and that resulted in a smaller set of 14 genomes. We used the pangenome estimation tool Pewit to visualize the genomic content of this selected group. The core genome of these subsets was composed of 4,560 genes. Principal component analysis of P. fluorescens CFBP2392 accessory genome shows this strain separates from the other in the PC1 and PC2 dimensions (Figure 4).

Genome mining predicts four putative biosynthesis gene clusters of NRPSs and primary metabolites with potential antibiotic potential

In recent years, genome mining and next generation sequencing proved to be useful tools to predict putative specialized metabolites. Using antiSMASH (v7.0.0), we mined for putative biosynthetic gene clusters within the genome, as a result, 10 putative regions were predicted (Figure 5). Two of these regions were predicted to encode ribosomally synthesized and post-tranlationally modified peptides (RIPP)-like antibiotics, three NRPS and one NRPS-like, one betalactone, one siderophore, one RRE-containing, and one arylpolyene (Supplementary Table 4).

One region shows a high percentage of identity with lokisin antibiotic (85%), the rest presented low identity to known metabolites or no predicted metabolites at all (Supplementary Figure 5). The predicted BGCs in the genome are in accordance with the other 13 genomes of P. fluorescens with high sequence identity to P. fluorescens CFBP2392 (Supplementary Figure 6). We decided to compare the amino acid sequence of P. fluorescens CFBP2392 MacB with other genomically related genomes together with representative sequences from genetically or chemically known MacB families. We used the MacB sequence of the other 13 strains selected for the pangenome analysis together with representative members of different lipopeptide families. Therefore, we obtained sequences from the amphisin, bananamide, entolysin, factin, gacamide, orfamide, peptin, poaeamide, putisolvin, tolaasin, viscosin, and xantholysin families and we compared them using the structure-based algorithm PROMALS3D (PROfile Multiple Alignment with predicted Local Structures and 3D constraints; Figure 6). As expected, based on MacB phylogeny, P. fluorescens CFBP2392 as well as the other 13 MacB sequences, clustered with amphisin family of lipopeptides with a sequence similar to Pseudomonas fluorescens MBY8934714.1, P. fluorescens WP_064118557.1, and Peudomonas sp. QNL34620.1 (Figure 6; Supplementary Table 5).

AntiSMASH also predicted several saccharides and fatty acids in P. fluorescens primary metabolites (Supplementary Table 4). Of note, the algorithm predicted a fatty acid biosynthesis region with 100% identity to koreenceine (region 4), a polyketide produced by Pseudomonas koreencis. Recently, this chemical analog of plants tetrahydropyridine alkaloids, was identified as responsible for the inhibition of the Gram negative bacteria Flavobacterium johnsoniae [START_REF] Lozano | Bacterial analogs of plant tetrahydropyridine alkaloids mediate microbial interactions in a rhizosphere model system[END_REF].

Discussion

Antimicrobial activity

The antifungal activity of P. fluorescens CFBP2392 was tested against R. solani, F. oxysporum, P. debaryanum, and Alternaria sp. evaluating in each case the growth of the mycelium vs. a control without bacterial inoculum. We found inhibition of the fungal strains, consistent with previous literature of P. fluorescens CFBP2392 against pathogenic fungi [START_REF] Bajsa | New antifungal compounds isolated from biocontrol Pseudomonas fluorescens and their effects on Rhizoctonia solani at cytological level[END_REF][START_REF] Berta | Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis[END_REF]. In this work, we observed greater inhibition of R. solani growth as compared to the other species of phytopathogenic fungi. For this reason, a second antagonism assay was carried out, testing the ability to inhibitory of P. fluorescens CFBP2392 against different isolates of R. solani. We selected four different isolates, belonging to two anastomosis groups (AG3 and AG4). It was observed that the greatest inhibition occurred with the AG3 LEM strain. Based on these results, R. solani was used to evaluate the morphological effect using TEM and to corroborate the production of the metabolite and its presence or absence during the purification process.

The antimicrobial activity of P. fluorescens CFBP2392 was observed against the Gram positive actinomycete C. michiganensis but not against the plant growth promoting H. seropericae. In a previous work from our lab, activity of the P. fluorescens UP61 strain was reported in vitro against bacteria [START_REF] Fuente | Pseudomonas Fluorescens UP61 isolated from birds foot trefoil rhizosphere produces multiple antibiotics and exerts a broad Spectrum of biocontrol activity[END_REF]. Pseudomonas fluorescens UP61 activity was screened against 12 phytopathogenic bacteria and detected against six of them: Agrobacterium tumefaciens, C. michiganensis subspecies michiganensis, Corynebacterium indiosum, Pseudomonas syringae pv. atrofaciens, Pseudomonas syringae pv. syringae, and Xanthomonas campestris pv. mavacearum [START_REF] Fuente | Pseudomonas Fluorescens UP61 isolated from birds foot trefoil rhizosphere produces multiple antibiotics and exerts a broad Spectrum of biocontrol activity[END_REF].

For the antifungal and antibacterial activity presented, the inhibitory capacity of the strain was tested, not the pure antibiotic. This implies that the observed inhibition may also be due to other active compounds produced by the strain. Most of the bacteria within the Pseudomonas genus have more than one inhibition mechanism [START_REF] Haas | Biological control of soil-borne pathogens by fluorescent pseudomonads[END_REF][START_REF] Arora | Diverse mechanisms adopted by fluorescent Pseudomonas PGC2 during the inhibition of Rhizoctonia solani and Phytophthora capsici[END_REF]. We show that this particular strain is capable of producing pyoverdine, hydrocyanic acid (HCN), and proteases. It was taken into account that pyoverdine synthesis is inhibited in iron-rich culture media and that hydrocyanic acid is too volatile to be responsible for inhibition. For all cases in this work, culture media with added iron were used.

Modifications in the cell wall of Rhizoctonia solani

In the observed hyphae cuts, it was verified that the portions of sampled mycelium contained active cells, corresponding to the growth zone of the fungus. The presence of the bacteria or the antifungal compound did not significantly change the cellular state of R. solani, which was estimated by evaluating the presence of symptoms of mycelial aging (less electron dense cytosol, greater presence of vacuoles, and less abundance of organelles such as mitochondria and ribosomes). Alterations in the cell wall of the fungus produced by P. fluorescens CFBP2392 were also observed during the interaction of these two microorganisms on tomato roots [START_REF] Lherminier | Reduction of the chemical inputs in a vegetable crop by the use of beneficial rhizospheric microorganisms[END_REF]. A thickening of the wall of R. solani that doubled the width with respect to the control was reported when the fungus was treated with a garlic plant extract [START_REF] Bianchi | Ultrastructural studies of the effects of Allium sativum on Phytopathogenic fungi in vitro[END_REF] and after exposure to plant chitinases [START_REF] Benhamou | Ultrastructural and cytochemical aspects of the interaction between the mycoparasite Pythium oligandrum and soilborne plant pathogens[END_REF]. In contrast with our observations, the antibiotic phenazine 1 carboxamide was reported to produce thinning and deformations in R. solani cell wall [START_REF] Xiang | Effects and inhibition mechanism of phenazine-1-carboxamide on the mycelial morphology and ultrastructure of Rhizoctonia solani[END_REF]. Deformations similar to those produced by P. fluorescens CFBP2392 were observed during the interaction of R. solani with the mycoparasite Pythium oligandrum [START_REF] Benhamou | Ultrastructural and cytochemical aspects of the interaction between the mycoparasite Pythium oligandrum and soilborne plant pathogens[END_REF].

In Mucor mucedo (Zygomycete), a thickening of the cell wall was also observed induced by the fungicides pentachloronitrobenzene (PCNB) and terrazole, although in the latter case it appears to be a non-specific effect reflecting damage to mitochondria [START_REF] Casperson | Effect of terrazol on the ultrastructure of Mucor mucedo[END_REF][START_REF] Lyr | Mode of action of terrazoleon Mucor mucedo[END_REF]. However, in studies carried out with Phytophthora cactorum, PCNB did not cause any deformation of the wall [START_REF] Casperson | Effect of terrazol on the ultrastructure of Mucor mucedo[END_REF]. Cell wall distortions could not be attributed to the action of the compound purified from P. fluorescens CFBP2392 and thus could be possibly related to the production of other antifungal factors by the bacteria such as hydrolytic exoenzymes. In this work, we report that the strain is able to produce exoproteases and hydrocyanic acid, and it had previously been reported that produces lipases and chitinases [START_REF] Andreucci | Reduction of the chemical inputs in a vegetable crop by the use of beneficial rhizospheric microorganisms[END_REF]. Similarly, Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacea CACua-24 strains were reported to degrade R. solani cell wall as observed by Scanning Electron Microscopy (SEM; [START_REF] Chávez-Ramírez | Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: a proposal for biocontrol of phytopathogenic fungi[END_REF]. In other Pseudomonas spp. strains, the importance of hydrolytic exoenzymes in their antagonistic activities was et al., 1994). The production of chitinases and β-1,3 glucanases to digest fungal cell wall had been also reported [START_REF] Gooday | The ecology of chitin degradation[END_REF][START_REF] Leah | Biochemical and molecular characterization of three barley seed proteins with antifungal properties[END_REF][START_REF] Fridlender | Global regulation of expression of antifungal factors by a Pseudomonas fluorescens biological control strain[END_REF].

Cytoplasmic alterations in Rhizoctonia solani

After treatment with P. fluorescens CFBP2392, and to a lesser degree with partially purified compound, an increase in glycogen accumulation was observed in the cytosol of R. solani. In a study of the effect of pentachloronitrobenzene (PCNB) on the ultrastructure of Mucor mucedo and Phytophthora cactorum, an increase in vacuole formation was also observed [START_REF] Casperson | Effect of terrazol on the ultrastructure of Mucor mucedo[END_REF]. Another effect observed in this work was the appearance of membranous vesicles with unknown granular content. The most noticeable action produced by the purified antibiotic was on the appearance of the mitochondria of R. solani. These were observed larger than in the control and with their disorganized membranes.

Similar to the effect caused by the purified metabolite from P. fluorescens CFBP2392 on mitochondria is the one caused by that produced by cereulide, a cyclic dodecadepsipeptide produced by Bacillus cereus strains. This toxin causes loss of motility in pig spermatozoa and an increase in the size of their mitochondria, but without depleting cellular ATP (allows phosphorylation at the substrate level) or damaging the plasma membrane. These symptoms are similar to those produced by the ionophores valinomycin and gramicidin, indicating that cereulide acts as a channel-forming ionophore, damaging mitochondria, and blocking oxidative phosphorylation in these organelles, necessary for sperm motility [START_REF] Andersson | A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores[END_REF]. TEM observations of the purified antibiotic phenazine 1 carboxamide in R. solani cells show twisted septum, organelles difficult to discern, and mitochondria disappear [START_REF] Xiang | Effects and inhibition mechanism of phenazine-1-carboxamide on the mycelial morphology and ultrastructure of Rhizoctonia solani[END_REF]. In the study of Xiang and coworkers, gene expression of complex I of the mitochondria electron transporter chain and chitin synthase was decreased in the presence of phenazine 1 carboxamide, consistent with the observed effect on both the mitochondria and the cell wall.

Two fungal strains, Alternaria longipes and Epicoccum nigrum, had also been reported to inhibit R. solani and evoke an effect in the cell wall [START_REF] Lahlali | Screening, identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants[END_REF]. In addition, there are two fungicides PCNB and terrazole whose main effect is on mitochondria, as observed by TEM. PCNB causes diffuse lysis of the internal structure of these organelles in Mucor mucedo and Phytophthora cactorum, and also an increase in the perinuclear space [START_REF] Casperson | Effect of terrazol on the ultrastructure of Mucor mucedo[END_REF]. In contrast, terrazole attacks the inner membrane of Mucor mucedo mitochondria leading to complete lysis of said organelles. Other affected membranes are the plasma membrane, which elongates and invaginates from the cell wall but remains intact, and the nuclear envelope, which shows vesicles between the membranes [START_REF] Casperson | Effect of terrazol on the ultrastructure of Mucor mucedo[END_REF]. The observed effects are induced by a release of phospholipases within mitochondria, and perhaps other membranes. The decrease in phosphorylation activity in mitochondria had as a side effect a pathological thickening of the cell wall [START_REF] Lyr | Mode of action of terrazoleon Mucor mucedo[END_REF].

Genome mining and specialized metabolite prediction

In the recent years, the development of new sequencing technologies together with the emergence of new and improved genome mining techniques has revolutionized the search of new natural compounds [START_REF] Ziemert | The evolution of genome mining in microbes-a review[END_REF][START_REF] Albarano | Genome mining as new challenge in natural products discovery[END_REF][START_REF] Baltz | Genome mining for drug discovery: progress at the front end[END_REF][START_REF] Blin | antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation[END_REF]. The high quality finished-genomes obtained with long-read sequencing third generation techniques such as Oxford Nanopore highly improves the prediction of biosynthetic gene cluster (BGCs). We found that P. fluorescens CFBP2392 harbors different BGCs clusters across its genome. In particular, we identified a lokisinlike NRPS and a koreenceine-like polyketide cluster that were previously reported to have antimicrobial activity [START_REF] Lozano | Bacterial analogs of plant tetrahydropyridine alkaloids mediate microbial interactions in a rhizosphere model system[END_REF][START_REF] Omoboye | Pseudomonas cyclic Lipopeptides suppress the Rice blast fungus Magnaporthe oryzae by induced resistance and direct antagonism[END_REF].

Lokisin is a well described lipopeptide with plant growth promoting activities. Lokisin can induce systemic resistance against rice blast disease [START_REF] Omoboye | Pseudomonas cyclic Lipopeptides suppress the Rice blast fungus Magnaporthe oryzae by induced resistance and direct antagonism[END_REF], reduce P. ultimun infection in tomato [START_REF] Hultberg | Suppression of disease in tomato infected by Pythium ultimum with a biosurfactant produced by Pseudomonas koreensis[END_REF], and has reported antifungal activity [START_REF] Gu | Genomic insights into a plant growth-promoting Pseudomonas koreensis strain with cyclic lipopeptide-mediated antifungal activity[END_REF]. This lipopeptide is part of the amphisin family as classified according to the total sequence length of the peptide and the size of the macrocycle [START_REF] Girard | Transporter gene-mediated typing for detection and genome mining of lipopeptideproducing Pseudomonas[END_REF]. Lipopeptides are transported from the periplasm to the outer cell compartment by a tripartite efflux pump formed with a MacB ABC transporter, a MacA adaptor protein in the periplasm, and a TolC outer membrane protein that exports the molecule outside the cell in Gram negative bacteria [START_REF] Greene | Antibiotic resistance mediated by the MacB ABC transporter family: a structural and functional perspective[END_REF]. MacB, recently proposed to be renamed PleB [START_REF] Girard | Transporter gene-mediated typing for detection and genome mining of lipopeptideproducing Pseudomonas[END_REF], is conserved in the ATP-binding domain and the integral membrane helical domain but varies significantly in the domain binding the lipopeptide. Recently, due to the high variability in the substrate binding site, PleB phylogeny has been proposed as a good indicator of the chemical nature of lipopeptides exported out of the cell [START_REF] Girard | Transporter gene-mediated typing for detection and genome mining of lipopeptideproducing Pseudomonas[END_REF]. In accordance with the genetic similarity found in the lokisin-cluster by antiSMASH, we found that PleB indeed clustered with the amphisin family suggesting a similar binding substrate as those previously reported.

Conclusion

The strain Pseudomonas fluorescens CFBP2392 was previously proposed as a promising biocontrol strain. Here we report the antifungal activity of the strain against F. oxysporum, P. debaryanum, Alternaria sp., and four R. solani, isolates. In addition, we observed antimicrobial activity against the Gram positive phyhtopathogen C. michiganensis in vitro. We purified an active compound from P. fluorescens CFBP2392 and evaluated its effect by TEM against R. solani. We observed that the strain and the purified chemical compound could produce morphological changes in R. solani morphology. Dual culture assay with the strain produced a thickening of the fungal cell wall. The purified compound produced alterations in the organelles including hypertrophy in mitochondria, membranous vesicles and a larger number of vacuoles per cell. The complete genome sequence of P. fluorescens CFBP2392 was sequenced using long-read technology Oxford Nanopore sequencing. We explore genomes 10.3389/fmicb.2023.1286926 Frontiers in Microbiology 10 frontiersin.org from other members of the Pseudomonas fluorescens complex in comparison with that of P. fluorescens CFBP2392. The profile of BGCs of P. fluoresces CFBP2392 was similar to that of other members of the complex with high ANI, all of them showing a lokisin-like cluster. The lokisin-like cluster from P. fluorescens CFBP2392 predicts a cyclic decapeptide NRPS with a MacAB transport system. Based on antiSMASH prediction as well as MacB phylogeny the predicted metabolite seems to follow in the amphisin family of cyclopeptides with previously reported plant growth-promoting properties. We further identified a koreenceine-like cluster that could potentially explain some of the antimicrobial activity. Our results give new insights in P. fluorescens CFBP2392 antimicrobial activity and provide a genomic background to explore its metabolic potential. Further experiments are needed to elucidate the chemical structure of the antifungal compound.

FIGURE 1

 1 FIGURE 1Antifungal activity of Pseudomonas fluorescens CFBP2392 against fungal phytopathogens. Antagonist assays against (A) Fusarium oxysporum, (B) Alternaria alternata, (C) Pythium debaryanum, and (D) Rhizoctonia solani, reveal growth defects when grown in proximity to P. fluorescens CFBP2392. For each case, left shows control fungal growth, right shows antagonist assay with P. fluorescens CFBP2392.

FIGURE 2

 2 FIGURE 2Effect of Pseudomonas fluorescens CFBP2392 in Rhizoctonia solani cell wall thickness. (A) Pseudomonas fluorecsens CFBP2392 induces deformations (d) and separation (s) of R. solani cell wall (cw). Glycogen accumulation is also observed (g). Images were obtained with TEM using periodic acid-silver thiocarbohydrazide-proteinate (PATAg) staining. (B) Cell wall thickness for P. fluorescens, purified compound (PC), partially purified compound (PPC) 1/2 dilution, and PPC 1/10 dilution measured in 20 points. Values represent the average thickness of 25-30 sections from two different grids. ANOVA was calculated (n = 135) after a log10 transformation to homogenize standard deviation.

FIGURE 3

 3 FIGURE 3Morphological changes in Rhizoctonia solani observed in the presence of Pseudomonas fluorescens CFBP2392 or its purified compound (PC). (A) Images were obtained with TEM using double contrast staining. m, mitochondria; cw, cell wall; hm, hypertrophied mitochondria; mv, membranous vesicles; hz, heterogeneus zones; and v, vacuoles. (B) Summary of ultrastructural changes with high (red), medium (pink), or no alterations (blank) observed in R. solani. All changes expressed as compared to untreated R. solani growth.

FIGURE 4

 4 FIGURE 4 Pangenome analysis with 14 selected strains of Pseudomonas fluorescens. (A) Heatmap represents presence and absence of genes within each genome clustered by phylogeny based on predicted core genes (n = 4,560). (B) Biplot visualization of the two main components of the PCA.

FIGURE 5

 5 FIGURE 5 Pseudomonas fluorescens CFBP2392 genome analysis predicts 10 putative BGCs. (A) Four NRPS regions are predicted within P. fluorescens CFBP2392 genome. (B) Chemical structure of lokisin as reported in MIBiG.

FIGURE 6

 6 FIGURE 6Pseudomonas fluorescens CFBP2392 and other genomically related P. fluorescens MacB amino acid sequence presents high homology with the amphisin family of Pseudomonas export lipopeptides proteins. (A) MacB sequences from the selected genomically related P. fluorescens (gray) cluster with the family amphisin of lipopeptides (red). (B) Amino acid sequence from six genetically or chemically characterized ABC transporters from the amphisin family (red) with nine sequences from selected P. fluorescens (gray).

TABLE 1

 1 Antifungal activity of Pseudomonas fluorescens CFBP2392 against Alternaria alternata, Fusarium oxysporum, Pythium debaryanum, and four strains of Rhizoctonia solani.

	Isolate	Inhibition PMSF Inhibition MMP
	Alternaria sp.	++	+
	Fusarium oxysporum	+	+
	Pythium debaryanum	++	+
	Rhizoctonia solani AG3	++
	Rhizoctonia solani 118 AG4	+	+
	Rhizoctonia solani R81 AG4	++	+
	Rhizoctonia solani 109	+	+

Inhibition observed in dual culture plate assay. (++) High inhibition, (+) Inhibition.

Frontiers in Microbiology

Frontiers in Microbiology 03 frontiersin.org

Frontiers in Microbiology 07 frontiersin.org

10.3389/fmicb.2023.1286926 Frontiers in Microbiology 08 frontiersin.org

Acknowledgments

The authors are grateful to the C. Arnould and J. Lherminier for their major contribution to the TEM observations; these observations were performed at Plateforme DimaCell, INRAE Dijon (France).

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was partly supported by the PEDECIBA and INCO-DC Program ERBIC18CT970180.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1286926/ full#supplementary-material


Pierre-Alexandre Lacotte

Sandrine Denis-Quanquin

Isabelle Martin-Verstraete

Thomas Candela

INTRODUCTION

C. difficile is an anaerobic, motile and spore-forming bacterium, responsible for 15 to 25% of post-antibiotic diarrhea and 95% of pseudomembranous colitis. While its toxins are described to be the major virulence factors in C. difficile infections, there is an increasing interest in the role of non-toxin factors in pathogenesis and virulence. In many other pathogens, cell wall glycopolymers influence the virulence. In C. difficile, three major carbohydrates are described : the polysaccharide I (PSI), the polysaccharide II (PSII) and the lipoteichoic acid (LTA). Dalanylation of surface polysaccharides reduces the affinity and efficacy of cationic antimicrobial compounds (CAMPs) on the bacterial surface. In C. difficile, the localization of D-alanylation is unknown and its implication in antibiotic resistance is not elucidated.

AIMS OF THE STUDY

The aim of our study was to determine the site of D-alanylation in C. difficile and investigate its role in antibiotic susceptibility.

RESULTS

CONCLUSION -PERSPECTIVES



  D-alanylation in C. difficile is involved in sensitivity to antibiotics and CAMPs  1 H NMR analysis of LTA and PSII from the 630 and the 630 ΔdltDABC mutant strains  D-alanylation was found in the LTA of the 630, but not in the 630 ΔdltDABC mutant strain  D-alanylation is exclusively found in the LTA  No modifications in susceptibility to β-lactams and cephalosporines  Modification in susceptibility of antibiotics having a deep interaction or complete binding to the bacterial membrane Location of the D-alanylation at the C. difficile surface The 630 ΔdltDABC mutant strain is more susceptible to bacitracin, DLT-1 inhibitor was then tested  Survival to bacitracin of the 630 and 630 ΔdltDABC mutant strains  Test of DLT-1 inhibitor  Survival of the 630 strain to bacitracin in the presence of DLT-1 From Heloïse Coullon  D-alanylation is induced by the presence of lysozyme MIC (µg.mL -Significant decrease in survival to bacitracin of the ΔdltDABC mutant in comparison with the 630 strain  DLT-1 decreases the D-alanylation rate  DLT-1 did not change the survival of the 630 stain against bacitracinIn this study, we report the specific D-alanylation of C. difficile LTA. It is to note that the level of D-alanylation of both the UK1 and the 078 strains is reduced approximately 2-fold compared to that of the 630 strain, hinting at either a lowered expression of the dltDABC operon or lower quantities of LTA. We also confirmed the role of D-alanylation in the sensitivity to CAMPs and antibiotics such as vancomycin and bacitracin. All these antibiotics have a deep interaction or complete binding to the bacterial membrane, but these compounds differ from CAMPs in their structure and mechanisms of action. In addition, we did not observe modifications of susceptibility to the β-lactams families. The D-alanylation of LTA could create a steric hindrance at the surface reducing the sensitivity for antibiotics targeting or directly binding membrane. Our data suggest that the design of specific inhibitors for C. difficile represents an opportunity to impact C. difficile way of life and an additional tool for managing CDI.


Christine Lors

Jean-François Ponge

Denis Damidot

Environmental hazard assessment of coal tar polluted soils through a multidisciplinary approach combining chemistry, ecotoxicology and microscopy

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Institutions

. LORS Christine (1) PONGE J.F. (2) DAMIDOT Denis (1) (1) IMT Lille Douai (Douai, France)

(2) Muséum National d'histoire Naturelle (Paris, France)

Industrial activities have led to the discharge of a wide range of hazardous chemicals in soils, such as coal tars which are always composed of hundreds of organic compounds (PAHs, BTEX,…), but also of some inorganic compounds. Environmental hazard assessment of soils contaminated with coal tar are generally investigated by a multidisciplinary approach coupled chemical and ecotoxicological analysis. However, a direct correlation between chemical analysis and results of ecotoxicological bioassays is generally not observed. The aim of this work is to deepen the relationships between PAHs concentration and ecoscores caculated from a battery of specifically selected bioassays including solid and liquid phase bioassays (Lors et al., 2010 & 2011). First, PAHs distribution is measured on several fraction of soils, and, second, parameters governing the release of PAHs to soil water are investigated by microscopic techniques.

Ecotoxicological analyses

Chemical analyses Microscopical analyses

Impregnation

Characterization of soils

Biotreatment decreased PAHs concentration resulting in decreased ecotoxicity.

Soil 2 was much less ecotoxic than soil 3 despite amount of PAHs (3.7 g.kg -1 ).

Soil 3 and Soil 3T:

before and after biotreatment

SOILS Soil 2: after biotreatment

In all the soils, the fraction 200-2000 µm (coarser particles) contains the majority of TOC and PAHs.

2-4/5-6 ratio is expected to decrease with the advancement of degradation.

Ecoscores do not correlate with [16PAHs], but increase almost linearly with [16 PAHs] / [TOC].

This ratio estimates [16 PAHs] of coal tar particles.

Bioavailability of PAHs is linked to their release from coal tar particles to soil pore water, depending to diffusion process. The pore network evolves during biodegradation process: an increase of the amount of interconnected porosity is observed due to the microbial consumption of organic compounds in coal tar particles (Soil 3T compared to Soil 3).

Microscopic observations

Soil 2 Soil 3 Soil 3T

Diffusion of PAHs from coal tar particles to soil water depends on the gradient of PAHs content between coal tar particles and soil water and on the surface of coal tar particles in contact with soil water.

Conclusions

The environmental hazard of soils is depending on the chemical properties of organic particles, but also the amount of open porosity of the particles.

  .lors@imt-lille-douai.fr -IMT Lille Douai, CERI Materials & Process, Douai (France)

  of the soil fraction with epoxy resin in circular molds Polishing the surface of samples with several diamond-covered disks Observations under a scanning electron microscope Separation of soil into 6 fractions: < 2, 2-20, 20-50, 50-200, > 2000 µm T.O.C. (TOCmeter) 16 (US-EPA) PAHs (ASE + HPLC-UV detector) Ecoscores calculated from the results of bioassays selected by Lors et al. (2011): Microtox, springtail avoidance, algal growth, lettuce germination, springtail reproduction, micronucleus test

  coal tar particles present a network of connected internal pores open to the surface, but in variable amounts depending on the origin of the coal tars.

Microscopy can be a complementary method to chemical and ecotoxicological analysis to assess the best strategy of remediation.


à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

  Réduire l'appréhension de la douleur liée au soin en pédiatrie : Etude préliminaire avec des logiciels de réalité virtuelle et mixte Stéphane Lille, Laurine Cléret, Thomas Colette, Isaias LopezArtiz, Pauline Petitjean, Prescillia Jany, Victoria Haas, Alexandre Usclade, Emmanuelle Rochette, Etienne Merlin, et al. To cite this version: Stéphane Lille, Laurine Cléret, Thomas Colette, Isaias LopezArtiz, Pauline Petitjean, et al.. Réduire l'appréhension de la douleur liée au soin en pédiatrie : Etude préliminaire avec des logiciels de réalité virtuelle et mixte. 19ème Congrès National de la SFETD, Nov 2019, Strasbourg (67), France. hal-P-094 Réduire l'appréhension de la douleur liée au soin en pédiatrie : Etude préliminaire avec des logiciels de réalité virtuelle et mixte Financement : PEPS CNRS INSIS « Sciences de l'Ingénierie pour la Santé pour accompagner des projets translationnels », 2017, projet AVACM L'Oculus Rift TM est un casque de réalité virtuelle. Il permet d'être en immersion totale dans un jeu. Deux manettes, les Oculus Touch, détectées par des capteurs, sont disponibles afin que l'enfant puisse interagir dans le jeu. Il peut ainsi effectuer un jeu de puzzle ou de tir lors du soin; son attention est alors détourné de l'acte infirmier. cube et du jeu simultanément. La distraction par le jeu pour la réduction de la douleur est alors amoindri. L'utilisation de la RV semble plus pertinente dans la population pédiatrique. En effet, son utilisation est faisable et appréciée par les patients. Cet axe de recherche sera donc favorisé. Le casque Microsoft HoloLens TM est un casque de réalité mixte proposé en France depuis fin 2016. Des informations 3D (hologrammes) sont projetées sur la visière semi-transparente du casque, tout en permettant de voir l'environnement réel. L'enfant porte un bracelet détecté par le casque ; un cube 3D opaque est alors visualisé par l'enfant autour de la zone de soin. Un petit jeu de puzzle est proposé à proximité du cube. une pose de cathéters, une certaine appréhension de la douleur est perçue chez les enfants. De plus en plus, des technologies telles que la réalité virtuelle (RV) ou la réalité mixte (RM) se développent permettant ainsi d'améliorer la prise en charge des patients. que tu aimerais avoir à nouveau le casque pour jouer la prochaine fois que tu te feras soigner à l'hôpital ? Aurais-tu préféré jouer avec une tablette ou un ordinateur ? Avais-tu déjà utilisé un casque de RM ou RV ? Les inconvénients du casque : * casque trop lourd (20%) * casque qui ne tenait pas en place (10%) * douleur aux yeux (10%) 22% 78% Changement de comportement de l'enfant par rapport au standard ? Lors de l'acte infirmier, un casque de RV ou RM a été utilisé chez des enfants âgés de 7 à 14 ans (n = 10, âge moyen = 10 ± 2 ans). L'enfant pouvait choisir entre un jeu de puzzle et un jeu de tir sur des robots ou des cibles, ces jeux ayant la particularité d'être adaptés au temps nécessaire à l'acte de soin. Ces applications ont été développées par des étudiants en informatiques. Hololens® Ecran Caméra RÉSULTATS EN RV : Le ressenti des enfants CONCLUSION ET PERSPECTIVES Stéphane LILLE 1 , Laurine CLERET 2 , Thomas COLETTE 2 , Isaias LOPEZ ARTIZ 2 , Pauline PETITJEAN 1 , Prescillia JANY 1 , Victoria HAAS 1 , Alexandra USCLADE 3 , Emmanuelle ROCHETTE 3 , Etienne MERLIN 3 , Emilie GADEA 1 , Christophe LOHOU 2 CH Emile Roux (Le Puy-en-Velay) 1 , IUT: département Informatique -Informatique Graphique (Université Clermont Auvergne, Le Puy-en-Velay) 2 , CHU de Clermont-Ferrand (Clermont-Ferrand) 3