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Background: DNA methylation is an epigenetic mechanism involved in the pathogenicity of several major bacterial pathogens. It can decrease the affinity of some transcriptional regulators to their binding site, leading to sub-populations expressing or not various genes, depending on the DNA methylation state. Dam DNA methyltransferase is widespread in Gammaproteobacteria and methylates the adenine of GATC sites. Objectives: The role of Dam was investigated in Photorhabdus luminescens during its symbiosis with a soil nematode and during its pathogenic stage in insects. Methods: SMRT sequencing (PacBio) and Bisulfite-seq were performed to identify the DNA methylation of the whole genome (methylome). In addition, RNAseq and phenotypic analysis were performed in a P. luminescens strain overexpressing Dam. Results: Dam overexpression caused a decrease in motility whereas it increased biofilm formation. While symbiosis ability of the Dam overexpressing strain was not significantly different from that of a control strain, the nemato-bacterial complex displayed an impaired pathogenicity in insect, as also observed after direct insect injection of the bacteria alone. Transcriptomic analysis revealed that the observed phenotypes were related to differences at the transcriptional level. More than 99% of the GATC sites of the genome were found methylated and DNA methylation levels did not change over growth kinetics. However, the Dam-overexpressing strain displayed more methylated GATC sites than the control and most of these sites were located in promoter regions. These sites may be involved in the observed differences in phenotypes and gene expression and provide clues to understand the involvement of Dam DNA methylation in P. luminescens life-cycle.

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex., SCOPUS: ar.j, info:eu-repo/semantics/published

Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila. We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila, we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host., Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium Xenorhabdus nematophila, a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector Steinernema carpocapsae. We found that phenotypic variants cluster in three groups, one of which is composed of lrp defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of X. nematophila with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted.

Background: DNA methylation is an epigenetic mechanism involved in the pathogenicity of severalmajor bacterial pathogens. It can decrease the affinity of some transcriptional regulators to theirbinding site, leading to sub‐populations expressing or not various genes, depending on the DNAmethylation state. Dam DNA methyltransferase is widespread in Gammaproteobacteria andmethylates the adenine of GATC sites.Objectives: The role of Dam was investigated in Photorhabdus luminescens during its symbiosis witha soil nematode and during its pathogenic stage in insects.Methods: SMRT sequencing (PacBio), which allows identification of the DNA methylation of thewhole genome (methylome), RNAseq and phenotypic analysis were performed in a P. luminescensstrain overexpressing Dam.Results: Dam overexpression caused a decrease in motility whereas it increased biofilm formation.While symbiosis ability of the Dam overexpressing strain was not significantly different from that of acontrol strain, the nemato‐bacterial complex displayed an impaired pathogenicity in insect, as alsoobserved after direct insect injection of the bacteria alone. Transcriptomic analysis revealed that theobserved phenotypes were related to differences at the transcriptional level. More than 99% of theGATC sites of the genome were found methylated and DNA methylation levels did not change overgrowth kinetics. However, the Dam‐overexpressing strain displayed more methylated GATC sitesthan the control and most of these sites were located in promoter regions. These sites may beinvolved in the observed differences in phenotypes and gene expression and provide

Dam DNA methylation can modify the affinity of transcriptional regulators for their binding sitesand can therefore lead to the emergence of subpopulations, expressing or not some genes.Because these differential gene regulations can be transmitted to daughter cells, thisphenomenon is considered as an epigenetic mechanism.Xenorhabdus nematophila is an enterobacterium symbiotically associated to a soil nematodeand pathogenic for several insects. Its methylome analysis revealed that >99% of Adenines inGATC motifs of the genome are methylated. One of the rare unmethylated motifs is located in aputative OxyR-binding site in a promoter region. We constructed a Xenorhabdus oxyR mutant, and phenotypic analysis confirmed the role ofOxyR in adaptation to oxidative environments.We are now investigating if some genes are transcriptionally regulated by OxyR in a DNAmethylation-dependent manner.

Dam DNA methylation is an epigenetic mechanism as it can regulate genes expression by reducing the affinity of transcriptional regulators for their binding site (Casadesus and Low, 2006). This DamDNA methylation has been shown to be involved in pathogenicity and phenotypic heterogeneity of several bacteria (Casadesus and Low, 2013).Photorhabdus luminescens is an entomopathogenic bacteria switching from symbiosis, with a nematode, to pathogenicity, in the insect (Boemare et al., 1993). This bacterium displays phenotypic heterogeneity. Because a Dam MTase has been identified in its genome sequence, we investigated its role in the life-cycle of P. luminescens. Methylome analysis revealed that 99% of GATC sites in the genome are methylated in all tested growth conditions. Overexpressing Dam methylates most of the unmethylated sites and causes a decrease in motility and pathogenicity of the bacteria whereas it increases biofilms formation. It does not impair the bacterial ability to perform a mutualistic association with the nematode. Transcriptomic analysis revealed that the observed phenotypes are related to differences at the transcriptional level.Coupling phenotypic, transcriptomic and methylomic analysis provides clues to identify genes transcriptionally regulated by DNA methylation and to understand Dam DNA methylation involvement in P. luminescens life-cycle.

Liquid scintillators are a common choice for neutrino physics experiments, but their capabilities to perform background rejection by scintillation pulse shape discrimination is generally limited in large detectors. This paper describes a novel approach for a pulse shape based event classification developed in the context of the Double Chooz reactor antineutrino experiment. Unlike previous implementations, this method uses the Fourier power spectra of the scintillation pulse shapes to obtain event-wise information. A classification variable built from spectral information was able to achieve an unprecedented performance, despite the lack of optimization at the detector design level. Several examples of event classification are provided, ranging from differentiation between the detector volumes and an efficient rejection of instrumental light noise, to some sensitivity to the particle type, such as stopping muons, ortho-positronium formation, alpha particles as well as electrons and positrons. In combination with other techniques the method is expected to allow for a versatile and more efficient background rejection in the future, especially if detector optimization is taken into account at the design level., 27 pages, 14 figures

The aim of this preliminary study was to evaluate the effectiveness of specially trained and supervised paraprofessionals in administering the eye movement desensitization and reprocessing Integrative Group Treatment Protocol (EMDR-IGTP) to reduce work-related posttraumatic stress disorder (PTSD) symptoms. The 2 paraprofessionals in this study were specially selected and trained in the application of the EMDR-IGTP and then provided treatment in an uncontrolled clinical trial to 37 clients from 3 non-governmental organizations in Bolivia. The participants were adult staff members (protective services workers, caregivers, psychologist, lawyers, and social workers) who provided care to children and adolescents with severe interpersonal trauma. Four EMDR-IGTP sessions within a parallel 2-week period were administered for each randomly assigned group. The Posttraumatic Stress Disorder Checklist for DSM-5 (PCL-5) was administered at pretreatment and 30 and 90 days’ posttreatment. A repeated measures analysis of variance (ANOVA) determined that PCL-5 score means differed statistically significantly between time points F(2, 72) = 574.53, p < .001, ηP2 = .94. The study presents preliminary evidence scaling up EMDR therapy in a low- and middle-income country, making it possible to reach larger numbers of people in a shorter time, thereby offering an operational advantage. The study has limitations specially related to the size of the sample, the use of only one measure, and the lack of comparison with a control group or treatment. Further studies are required to present large samples with more measures and comparison of results with another therapy or control group.

Purpose The purpose of this paper is to present a sustainable, community-based action model to improve the care and protection of children between the ages of zero and eight years who have been left behind due to migration. One facet of the program is to train community leaders to teach an experiential program to facilitate and increase the likelihood of the development of life habits in children, caretakers, teachers, mothers, and fathers that will improve the caretaker-infant relationship. Design/methodology/approach Community leaders were evaluated via a pre-training questionnaire and a post-training questionnaire. Findings The evaluation of the training of the community leaders revealed the following statistically significant results: an increase in the level of a number of life habits, which include self-awareness, management of emotions, empathy, assertive communication, decision-making, critical thinking, conflict negotiation and resolution, and teamwork; a reduction in the degree to which psychosocial barriers hinder personal development; an increase in the level of autonomy and self-efficacy; an increase in the level of knowledge that participants have about the topics of the workshops: child development, child-rearing and academic potential, as well as migration; and positive changes in the attitudes that they have regarding certain topics related to child development. Originality/value The program was conducted in Hidalgo, Mexico, a state whose high rates of migration put the children at greater risk for developmental delays if they lack adult support.

Lipid A is a potent endotoxin, and its fatty acids (lauric, myristic, and sometimes palmitic acid) anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of most Gram-negative bacteria. The highly anionic charge of the glucosamine lipid A moiety makes the LPS a powerful attractant for cationic antimicrobial peptides (AMPs). AMPs are major component of innate immunity that kill bacteria by permeabilization of lipid bilayers. Secondary lipid A acylation of Klebsiella pneumoniae, involving the acyltransferase LpxM (formally, msbB or WaaN) that acylates (KDO)2-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis, has been associated with bacterial resistance to AMPs contributing to virulence in animal models. We investigated here the role of the msbB gene of the entomopathogenic bacterium Photorhabdus luminescens in AMP resistance, by functional complementation of the AMP susceptible K. pneumoniae lpxM mutant with the P. luminescens msbB gene. We showed that msbB (lpxM) gene of P. luminescens is able to enhance polymyxin B, colistin and cecropin A resistance of K. pneumoniae lpxM mutant, compared to the non-complemented mutant. However, we could not obtain any msbB mutant of Photorhabdus by performing allelic exchange experiments based on positive selection of sucrose highly resistant mutants. We thus suggest that msbB-mediated Photorhabdus lipid A acylation is essential for outer membrane low-permeability and that modification of lipid A composition, fluidity and osmosis-resistance have an important role in the ability of Photorhabdus to grow in sucrose at high concentrations.

Prolonged culture and long lasting infections select for poorly transmitted bacterial variants. MicrobiOccitanie 2019- Rencontre des Microbiologistes Région Occitanie

Whole Genome DNA Methylation (Methylome) Analysis and Role of Dam DNA Methyltransferase in the Entomopathogenic Bacterium Photorhabdus luminescens. MicrobiOccitanie 2019- Rencontre des Microbiologistes Région Occitanie

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (P-mdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The P-mdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the P-mdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the P-mdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

Photorhabdus luminescensis an entomopathogenic bacterium found in symbiosis with the nematodeHeterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, includingP. luminescens,whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation inP. luminescenssymbiosis withH. bacteriophora. We constructed a strain overexpressingdamby inserting an additional copy of thedamgene under the control of a constitutive promoter in the chromosome ofP. luminescensand then achieved association between this recombinant strain and nematodes. Thedamoverexpressing strain was able to feed the nematodein vitroandin vivosimilarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50was observed during insect infestation with the nematode associated with thedamoverexpressing strain. These results suggest that theP. luminescensDam plays a role in the pathogenicity of the nemato-bacterial complex.

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that the P. luminescens Dam plays a role in the pathogenicity of the nemato-bacterial complex.

International audience; Entomopathogenic nematodes (EPNs) form specific mutualistic associations with bioluminescent enterobacteria. In Heterorhabditidis indica, Ochrobactrum spp. was identified beside the symbiont Photorhabdus luminescens but its involvement in the symbiotic association in the EPNs remains unclear. This study describe the population structure and the diversity in Ochrobactrum natural populations isolated from EPNs in the Caribbean basin in order to question the existence of EPN-specialized clones and to gain a better insight into Ochrobactrum-EPNs relationships. EPN-associated Ochrobactrum and Photorhabdus strains were characterized by multi-locus sequence typing, Pulsed-Field Gel Electrophoresis fingerprinting and phenotypic traits. Population study showed the absence of EPN-specialized clones in O. intermedium and O. anthropi but suggested the success of some particular lineages. A low level of genetic and genomic diversification of Ochrobactrum isolated from the natural population of Caribbean nematodes was observed comparatively to the diversity of human-associated Ochrobactrum strains. Correspondences between Ochrobactrum and P. luminescens PFGE clusters have been observed, particularly in the case of nematodes from Dominican Republic and Puerto Rico. O. intermedium and O. anthropi associated to EPNs formed less biofilm than human-associated strains. These results evoke interactions between Ochrobactrum and the EPN symbiotic system rather than transient contamination. The main hypothesis to investigate is a toxic/antitoxic relationship because of the ability of Ochrobactrum to resist to antimicrobial and toxic compounds produced by Photorhabdus.

Antibiose et antibiorésistance: tuer et ne pas être tué

Evaluation des nématodes entomopathogènes et leurs bactéries symbiotiques pour lutter contre les taupins. Natural Products and Biocontrol et Journée Elicitra

La collection de Nématodes Entomopathogènes : Ressources biologiques utilisables comme agents de Bio-contrôle. 3. International Congress Natural Products and Biocontrol

The yields and production rates of the radioisotopes [superscript 9]Li and [superscript 8]He created by cosmic muon spallation on [superscript 12]C, have been measured by the two detectors of the Double Chooz experiment. The identical detectors are located at separate sites and depths, which means that they are subject to different muon spectra. The near (far) detector has an overburden of ∼120 m.w.e. (∼300 m.w.e.) corresponding to a mean muon energy of 32.1 ± 2.0 GeV (63.7 ± 5.5 GeV). Comparing the data to a detailed simulation of the [superscript 9]Li and [superscript 8]He decays, the contribution of the [superscript 8]He radioisotope at both detectors is found to be compatible with zero. The observed [superscript 9]Li yields in the near and far detectors are 5.51 ± 0.51 and 7.90 ± 0.51, respectively, in units of 10[superscript −8]μ[superscript −1]g[superscript −1]cm[superscript 2]. The shallow overburdens of the near and far detectors give a unique insight when combined with measurements by KamLAND and Borexino to give the first multi-experiment, data driven relationship between the 9Li yield and the mean muon energy according to the power law Y=Y[subscript 0](⟨E[subscript μ]⟩/1 GeV)[superscript ¯ over α], giving [¯over α]=0.72±0.06 and Y0 = (0.43 ± 0.11) × 10[superscript −8]μ[superscript −1]g[superscript −1]cm[superscript 2]. This relationship gives future liquid scintillator based experiments the ability to predict their cosmogenic [superscript 9]Li background rates. Open image in new window Keywords Neutrino Detectors and Telescopes (experiments), National Science Foundation (U.S.), United States. Department of Energy

International audience; Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.