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International audience; Environmentally induced reductions in fitness components (survival, fecundity) are generally considered as passive, maladaptive responses to stress. However, there is also mounting evidence for active, programmed forms of environmentally induced cell death in unicellular organisms. While conceptual work has questioned how such programmed cell death (PCD) might be maintained by natural selection, few experimental studies have investigated how PCD influences genetic differences in longer-term fitness across environments. Here, we tracked the population dynamics of two closely related strains of the halotolerant microalga Dunaliella salina, following transfers across salinities. We showed that after a salinity rise, only one of these strains displayed a massive population decline (-69% in one hour), largely attenuated by exposure to a PCD inhibitor. However, this decline was followed by a rapid demographic rebound, characterized by faster growth than the non-declining strain, such that sharper decline was correlated with faster subsequent growth across experiments and conditions. Strikingly, the decline was more pronounced in conditions more favourable to growth (more light, more nutrients, less competition), further suggesting that it was not simply passive. We explored several hypotheses that could explain this decline-rebound pattern, which suggests that successive stresses could select for higher environmentally induced death in this system.

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

International audience; Recent studies have highlighted associations between diseases and host microbiota. It is yet extremely challenging-especially under natural conditions-to clarify whether the host microbiota promotes future infections, or whether changes in host microbiota result from infections. Nonetheless, deciphering between these two processes is essential for highlighting the role of microbes in disease progression. We longitudinally surveyed, in the wild, the microbiota of individual fish hosts (Leuciscus burdigalensis) both before and after infection by a crustacean ectoparasite (Tracheliastes polycolpus). We found a striking association between parasite infection and the host microbiota composition restricted to the fins the parasite anchored. We clearly demonstrated that infections by the parasite induced a shift in (and did not result from) the host fin microbiota. Fin microbiota further got similar to that of the adult stage, and the free-living infective stage of the parasite during infection with a predominance of the Burkholderiaceae bacteria family. This suggests that Burkholderiaceae bacteria is involved in a co-infection process and possibly facilitate T. polycolpus infection. We reveal novel mechanistic insights for understanding the role of the microbiota in host-parasite interactions, which has implications for predicting the progression of diseases in natural host populations.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD− recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

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International audience; Hemp field retting is a bioprocess in which the stem cell wall polysaccharide network is degraded by a complex microbial consortium to facilitate further extraction of fibres. This process is carried out by farmers in an empirical way and depends on pedoclimatic conditions,which makes it difficult to control. This work aims to unravel the biodiversity-ecosystem function relationship. Retting is examined by exploring the temporal dynamics of the microbial communities considering 4 ecological indicators (density, diversity, structure, and activity).DNA metabarcoding approach and enzymatic activity are performed. The stem and extracted fiber color, the fiber biochemical composition, and the stem microstructure are evaluated.Results show that the first 2 weeks of retting are characterized by intense microbial colonization and significant biochemical changes in the composition of the fiber cell wall. This study provides the first thorough description of the hemp field retting microbiome.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD.; International audience; Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix−). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix− nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix− nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules. Analyses of Medicago mutants with non-functional nodules highlight the relationship and mechanisms controlling the establishment of the immune and senescence programs during nodule organogenesis.

International audience; The oral microbiota is a highly complex and diversified part of the human microbiome. Being located at the interface between the human body and the exterior environment, this microbiota can deepen our understanding of the environmental impacts on the global status of human health. This research topic has been well addressed in Westernized populations, but these populations only represent a fraction of human diversity. Papua New Guinea hosts very diverse environments and one of the most unique human biological diversities worldwide. In this work we performed the first known characterization of the oral microbiome in 85 Papua New Guinean individuals living in different environments, using a qualitative and quantitative approach. We found a significant geographical structure of the Papua New Guineans oral microbiome, especially in the groups most isolated from urban spaces. In comparison to other global populations, two bacterial genera related to iron absorption were significantly more abundant in Papua New Guineans and Aboriginal Australians, which suggests a shared oral microbiome signature. Further studies will be needed to confirm and explore this possible regional-specific oral microbiome profile.

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International audience; All tested strains of Streptococcus pyogenes (group A streptococcus, GAS) remain susceptible to penicillin. However, GAS strains with amino acid substitutions in penicillin-binding proteins that confer decreased susceptibility to beta-lactam antibiotics have been identified recently. This discovery raises concerns about emergence of beta-lactam antibiotic resistance in GAS. Whole genome sequencing recently identified GAS strains with a chimeric penicillin-binding protein 2X (PBP2X) containing a recombinant segment from Streptococcus dysgalactiae subspecies equisimilis (SDSE). To directly test the hypothesis that the chimeric SDSE-like PBP2X alters beta-lactam susceptibility in vitro and fitness in vivo, an isogenic mutant strain was generated and virulence assessed in a mouse model of necrotizing myositis. Compared with naturally occurring and isogenic strains with a wild-type GAS-like PBP2X, strains with the chimeric SDSE-like PBP2X had reduced susceptibility in vitro to nine beta-lactam antibiotics. In a mouse model of necrotizing myositis, the strains had identical fitness in the absence of benzylpenicillin treatment. However, mice treated intermittently with a subtherapeutic dose of benzylpenicillin had significantly more colony-forming units recovered from limbs infected with strains with the chimeric SDSE-like PBP2X. These results show that mutations such as the PBP2X chimera may result in significantly decreased beta-lactam susceptibility and increased fitness and virulence. Expanded diagnostic laboratory surveillance, genome sequencing, and molecular pathogenesis study of potentially emergent beta-lactam antibiotic resistance among GAS are needed.

International audience; Modern diatom assemblages were investigated in the Ounianga Lakes located in the driest part of the Chadian Sahara. These lakes still exist today due to groundwater seepage. They contain a variety of diatom species, one of which could not be identified. Here we describe a new species of Staurophora, S. ouniangaensis sp. nov. based on detailed light and scanning electron microscopy observations. The new taxon presents all morphological characters attributed to this genus and differs from other Staurophora species mainly by its specific valve outline and the slightly twisted frustules along the transapical axis. We compared this new species with the morphologically resembling Cymbella obtusa which was reported from Chad. The latter taxon has a much coarser stria density than our newly described species. Staurophora ouniangaensis sp. nov. supports that Staurophora species prefer inland saline environments. Indeed, in the Ounianga lakes, the new species was mainly observed in mesosaline to hypersaline waters.

International audience; Hybridization and adaptation to new hosts are important mechanisms of fungal disease emergence. Evaluating the risk of emergence of hybrids with enhanced virulence is then key to develop sustainable crop disease management. We evaluated this risk in Venturia inaequalis, the fungus responsible for the common and serious scab disease on Rosaceae hosts, including apple, pyracantha and loquat. Field isolates from these three hosts and progenies obtained from five crosses between formae speciales isolates collected from pyracantha (f.sp. pyracantha) and apple (f.sp. pomi) were tested for their pathogenicity on the three hosts. We confirmed a strict host specificity between isolates from apple and pyracantha, and showed that most isolates were able to cause disease on loquat. None of the 251 progeny obtained from five crosses between V. inaequalis f.sp. pyracantha and V. inaequalis f. sp. pomi could infect apple. If confirmed on more crosses, the inability of the hybrids to infect apple could lead to a novel biocontrol strategy based on a sexual hijacking of V. inaequalis f.sp. pomi by a massive introduction of V. inaequalis f.sp. pyracantha in apple orchards. This strategy, analogous to the sterile insect approach, could lead to the collapse of the population size of V. inaequalis and dramatically reduce the use of chemicals in orchards.

International audience; Swiss cheeses have been produced for centuries in a traditional way and their typical opening indicates the presence of propionibacteria. These last are indeed present in raw milk, their growth is favored by the specific technology of Swiss-type cheeses, and their high population in the ripened cheese is responsible for its characteristic sensory properties, including the opening and the aroma. In addition to this long history of safe use and consumption, they constitute a source of nutraceuticals. The specific propionic fermentation leads to the production of short chain fatty acids as main final obligatory products. These exert beneficial effects including nourishment of digestive epithelial cells, as well as promotion of differentiation and of apoptotic depletion of colon cancer cells. Propionibacteria also produce vitamins of the B group, which enhances the nutritional value of the food products they ferment. Some selected strains were moreover shown to produce immunomodulins, proteins able to modify the secretion of cytokines in human immune cells. These last were identified as proteinaceous components of propionibacteria surface layer. They also produce extracellular vesicles which modulate in vitro inflammation and the NFκB pathway in human intestinal epithelial cells. Accordingly, such strains modulate the immune response and mitigate inflammation. In mice, they prevent both mucositis and colitis, via such immunomodulins limiting acute inflammatory response and protecting the gut epithelial barrier. In pigs, their consumption also modulates the inflammatory response and the lymphocyte populations. Finally, clinical studies dealing with consumption of propionibacteria whey cultures suggest a beneficial role in the context of IBD. Propionibacteria strains were recently identified in the gut microbiota of healthy newborns, yet not in that of children with necrotizing enterocolitis. Indeed, propionibacteria were detected in human fecal samples, which means that propionibacteria may be traditional cheese starters or next generation probiotics. The growing knowledge of these peculiar actinobacteria, including their great strain-diversity, opens new avenues for the development of functional foods and of food supplements, in a general context of increasing incidence of ailments that include an inflammatory component. Moreover, their ability to ferment both dairy and vegetable matrices is presently investigated and the first promising advances open new perspectives for the development of functional fermented vegetable products.

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International audience; Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

International audience; The predicted increases in drought in many forest ecosystems may alter soil microbial community diversity and activity, which may further depend on tree species richness. Shifts in microbial community composition and activity could engender changes in ecosystem function, notably, in soil greenhouse gas emissions and C storage. Using soils from mono-specific and mixed three-species forest stands from across Europe, we performed a microcosm experiment to test how soil microbial taxonomic and catabolic diversity are affected by repeated drying-rewetting (DRW) cycles and tree species mixing. We used Illumina sequencing and MicroResp™ analyses to explore community-level changes between microbial functional groups. DRW decreased bacterial richness and carbon substrate use diversity and increased fungal Shannon diversity. Additionally, microbial communities exposed to DRW changed their consumption of 11 out of 15 substrates significantly, suggesting microbial functional shifts. The legacy effect of tree species mixing influenced the structure of the microbial communities (i.e. taxonomic differential abundance) although, community weighted mean (CWM) values of absorptive root traits appeared to affect more strongly microbial richness, relative abundance, and Shannon diversity. No significant tree species mixing:DRW interaction was found for most microbial variables, except for the use of certain substrates and potentially differential abundance. Our data from a laboratory experiment with soils from different forest ecosystems underline that drought may cause shifts in microbial taxonomic and catabolic diversity, while tree species influences primarily taxonomic diversity through root traits.

Toxoplasma gondiiis an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate allT. gondiiparasites. We investigated the presence and concentration ofT. gondiiDNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n= 3) or tissue cysts (n = 3) and naturally infected pigs (n= 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA ofT. gondiiwas detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, theT. gondiiburden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive forT. gondiiby MC-qPCR or qPCR, with the meanT. gondiiburden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution ofT. gondiiin the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability ofT. gondiifrom the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources ofT. gondiihuman infections.

Individual RAD sequences files are available in a fq.gz format at the Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra/PRJNA771401). The VCF files Xylosandruscrassiusculus.vcf as well as the popmap used in STACKS' population module and specimens' metadata (e.g. GPS coordinates) are available on Portail Data INRAE (https://doi.org/10.57745/JMT4KD). The Genbank accession numbers for the mitochondrial haplotypes reported in this paper are OK489329 to OK489334.; International audience; Xylosandrus crassiusculus, a fungus-farming wood borer native to Southeastern Asia, is the most rapidly spreading invasive ambrosia species worldwide. Previous studies focusing on its genetic structure suggested the existence of cryptic genetic variation in this species. Yet, these studies used different genetic markers, focused on different geographical areas, and did not include Europe. Our first goal was to determine the worldwide genetic structure of this species based on both mitochondrial and genomic markers. Our second goal was to study X. crassiusculus' invasion history on a global level and identify the origins of the invasion in Europe. We used a COI and RAD sequencing design to characterize 188 and 206 specimens worldwide, building the most comprehensive genetic dataset for any ambrosia beetle to date. The results were largely consistent between markers. Two differentiated genetic clusters were invasive, albeit in different regions of the world. The two markers were inconsistent only for a few specimens found exclusively in Japan. Mainland USA could have acted as a source for further expansion to Canada and Argentina through stepping-stone expansion and bridgehead events. We showed that Europe was only colonized by Cluster 2 through a complex invasion history including several arrivals from multiple origins in the native area, and possibly including bridgehead from the USA. Our results also suggested that Spain .

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

International audience; The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward beta-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux. The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward beta-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections.

The pathogenic bacteriaBacillus cereus,Bacillus anthracisandBacillus thuringiensisform spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in liveB. cereuscells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes duringB. cereussporulation. We used these markers to characterize a subpopulation of engulfment-defectiveB. cereuscells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organismBacillus subtilis, the activity of σKis not strictly linked to the state of forespore development inB. cereus.

International audience; Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a nonreplicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.

The increase in antibiotic-resistant avian-pathogenic Escherichia coli (APEC), the causative agent of colibacillosis in poultry, warrants urgent research and the development of alternative therapies. This study describes the isolation and characterization of 19 genetically diverse, lytic coliphages, 8 of which were tested in combination for their efficacy in controlling in ovo APEC infections. Genome homology analysis revealed that the phages belong to nine different genera, one of them being a novel genus (Nouzillyvirus). One phage, REC, was derived from a recombination event between two Phapecoctavirus phages (ESCO5 and ESCO37) isolated in this study. Twenty-six of the 30 APEC strains tested were lysed by at least one phage. Phages exhibited varying infectious capacities, with narrow to broad host ranges. The broad host range of some phages could be partially explained by the presence of receptor-binding protein carrying a polysaccharidase domain. To demonstrate their therapeutic potential, a phage cocktail consisting of eight phages belonging to eight different genera was tested against BEN4358, an APEC O2 strain. In vitro, this phage cocktail fully inhibited the growth of BEN4358. In a chicken lethality embryo assay, the phage cocktail enabled 90% of phage-treated embryos to survive infection with BEN4358, compared with 0% of nontreated embryos, indicating that these novel phages are good candidates to successfully treat colibacillosis in poultry. IMPORTANCE Colibacillosis, the most common bacterial disease affecting poultry, is mainly treated by antibiotics. Due to the increased prevalence of multidrug-resistant avian-pathogenic Escherichia coli, there is an urgent need to assess the efficacy of alternatives to antibiotherapy, such as phage therapy. Here, we have isolated and characterized 19 coliphages that belong to nine phage genera. We showed that a combination of 8 of these phages was efficacious in vitro to control the growth of a clinical isolate of E. coli. Used in ovo, this phage combination allowed embryos to survive APEC infection. Thus, this phage combination represents a promising treatment for avian colibacillosis. ispartof: MICROBIOLOGY SPECTRUM vol:11 issue:3 ispartof: location:United States status: Published online

International audience; Introduction - International guidelines recommend high doses of β-lactams for most cases of infective endocarditis (IE). Therapeutic drug monitoring (TDM) is increasingly used to adjust β-lactam dose based on plasma concentrations, although there are no comparative studies to support this practice. The benefit of amoxicillin TDM during IE was evaluated. Methods - An observational, retrospective, cohort study of adults treated with high-dose amoxicillin for enterococcal or streptococcal IE was conducted in two referral centers. Patients with, or without TDM were compared. The primary outcome was mean daily amoxicillin dose. Results - A total of 206 cases of streptococcal (n=140, 68%) or enterococcal (n=66, 32%) IE were included. IE occurred on prosthetic valves in 77 (37%) cases, and on intracardiac devices in 28 (14%) cases. Aortic valve was involved in 136 (66%) cases. There were 154 men (75%), mean age was 70 ± 14 years, valve surgery was performed in 81/206 (39%) patients, and in-hospital mortality was 8% (17/206). All patients in the TDM group and most patients in the group without TDM received amoxicillin as continuous infusion. Amoxicillin TDM was performed for 114 patients (55.3%), with a mean of 4.7 ± 2.3 measures per patient, a mean plasma steady-state concentration of 41.2 ± 19 mg/L, most (82/114, 72%) being within the therapeutic target (20-80 mg/L). Mean amoxicillin dose was lower in patients with TDM (10.0 ± 3.3 g/day) than those without TDM (11.3 ± 2.0 g/day) (P=0.003). Conclusion - Amoxicillin TDM was associated with a reduction in daily doses, with no impact on adverse events and prognosis. Individualized treatment of IE through TDM may contribute to decreased use of antibiotics.

Plasmodium falciparum, the most virulent agent of human malaria, spread from Africa to all continents following the out-of-Africa human migrations. During the transatlantic slave trade between the 16thand 19thcenturies, it was introduced twice independently to the Americas where it adapted to new environmental conditions (new human populations and mosquito species). Here, we analyzed the genome-wide polymorphisms of 2,635 isolates across the currentP. falciparumdistribution range in Africa, Asia, Oceania, and the Americas to investigate its genetic structure, invasion history, and selective pressures associated with its adaptation to the American environment. We confirmed that American populations originated from Africa with at least two independent introductions that led to two genetically distinct clusters, one in the North (Haiti and Columbia) and one in the South (French Guiana and Brazil), and the admixed Peruvian group. Genome scans revealed recent and more ancient signals of positive selection in the American populations. Particularly, we detected positive selection signals in genes involved in interactions with host (human and mosquito) cells and in genes involved in resistance to malaria drugs in both clusters. We found that some genes were under selection in both clusters. Analyses suggested that for five genes, adaptive introgression between clusters or selection on standing variation was at the origin of this repeated evolution. This study provides new genetic evidence onP. falciparumcolonization history and on its local adaptation in the Americas.

Background As the epidemiology of encephalitis varies from one country to another, international travel may be an important clue for the diagnostic workout of this puzzling disease. Methods We performed an ancillary study using the ENCEIF prospective cohort conducted in 62 clinical sites in France from 2016 to 2019. All cases of encephalitis in adults that fulfilled a case definition derived from the International Encephalitis Consortium were included. Travellers were defined as patients who spent at least one night in a foreign country within the last six months. Results Of the 494 encephalitis patients enrolled, 69 (14%) were travellers. As compared to non-travellers, they were younger (median age, 48 years [interquartile range, 36–69] vs 66 [49–76], P Conclusion The two primary causes of encephalitis in travellers are arboviruses and HSV. Empirical treatment of encephalitis in travellers must include acyclovir. Pre-travel advice and vaccination may decrease the risk of encephalitis in travellers.

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International audience; Plague is an often fulminant and fatal disease caused by the bacterial pathogen Yersinia pestis. Three major historical plague pandemics have heavily influenced human societies, and recent evidence demonstrates that plague has also influenced human biology. Plague is still endemic in several continents and remains a potential public health risk. In this article, we examine the current situation of plague around the world and the different players that participate in plague ecosystems. We discuss in particular the recent research that allows us to understand plague in pre-historical times, as well as how past pandemics have modulated the evolution of human immune genes. We also highlight future challenges that await us in the study of this deadly but fascinating disease.

Posté le 17 septembre 2021.; International audience; ObjectivesA nationwide cross-sectional epidemiological survey was conducted to capture the true extent of coronavirus disease 2019 (COVID-19) exposure in Senegal.MethodsMulti-stage random cluster sampling of households was performed between October and November 2020, at the end of the first wave of COVID-19 transmission. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies were screened using three distinct ELISA assays. Adjusted prevalence rates for the survey design were calculated for each test separately, and thereafter combined. Crude and adjusted prevalence rates based on test performance were estimated to assess the seroprevalence. As some samples were collected in high malaria endemic areas, the relationship between SARS-CoV-2 seroreactivity and antimalarial humoral immunity was also investigated.ResultsOf the 1463 participants included in this study, 58.8% were female and 41.2% were male; their mean age was 29.2 years (range 0.20–84.8.0 years). The national seroprevalence was estimated at 28.4% (95% confidence interval 26.1–30.8%). There was substantial regional variability. All age groups were impacted, and the prevalence of SARS-CoV-2 was comparable in the symptomatic and asymptomatic groups. An estimated 4 744 392 (95% confidence interval 4 360 164–5 145 327) were potentially infected with SARS-CoV-2 in Senegal, while 16 089 COVID-19 RT-PCR laboratory-confirmed cases were reported by the national surveillance. No correlation was found between SARS-CoV-2 and Plasmodium seroreactivity.ConclusionsThese results provide a better estimate of SARS-CoV-2 dissemination in the Senegalese population. Preventive and control measures need to be reinforced in the country and especially in the south border regions.

International audience; Clostridioides difficile is an opportunistic pathogen that causes by a high rate of recurrent infections. Persistence in the gastrointestinal tract is thought to be mediated by sporulation and/or biofilm formation. There is an increase interest in C. difficile biofilm formation and recent findings have provided a framework to model surface-attached biofilm formation. For in vitro biofilm formation, C. difficile must undergo a metabolic reprogramming as it enters stationary phase. This helps maintain long-term viability and increases responsiveness to signals leading to biofilm formation. Metabolic reprogramming and biofilm formation requires several regulatory factors and these overlap with the sporulation cascade. Despite recent advances, further research is needed to answer outstanding questions in the field.

High throughput sequencing in microbiological diagnosis The applications of next-generation sequencing (NGS) in health have recently become more widespread, at first in research studies and then in clinical microbiology. The use of NGS has mainly been developed in bacteriology and mycology, especially based on phylogenetic markers (hypervariable regions of rDNA 16S, 18S, or 28S) which allow by metataxonomy, to describe the physiopathological variations of microbiota. By non-targeted metagenomics approaches, the comparison of whole genome NGS data to international databases allows the detection of genomic antimicrobial resistance which is often correlated with the resistance phenotype. Similarly, different strategies to study virulence factors have been developed to compare the genomes of clinical isolates to reference strains and thus to analyze genomic islands. NGS has also shown its efficiency in determining an inter-individual transmission cluster, to identify the source patient or to improve collective management, to search for the etiological agent of an infection. Even though structures such as the Cofrac have taken NGS into account, the lack of standardization limits the routine development of these approaches, due to the need for bioinformatics., Les applications du séquençage haut débit (NGS) en santé se sont récemment démocratisées, d’abord en recherche puis en microbiologie clinique. L’utilisation du NGS a surtout des applications en bactériologie et mycologie, principalement basées sur les marqueurs phylogénétiques documentés (régions hypervariables du 16S, 18S, ou 28S) permettant, par métataxonomie, de décrire les variations physiopathologiques des microbiotes. En métagénomique non ciblée, la comparaison des données de séquençage de génomes complets à des banques de données internationales permet de détecter une antibiorésistance génomique, souvent corrélée au phénotype de résistance. De même, différentes stratégies d’étude des facteurs de virulence ont été développées pour comparer les génomes des souches isolées en clinique à des souches de référence et ainsi analyser les îlots génomiques. Le NGS a aussi montré son efficacité à déterminer une chaîne de transmission interindividuelle, afin de remonter au patient source ou d’améliorer la prise en charge collective. L'approche métagénomique a permis de rechercher l'agent étiologique d'une infection. Malgré la prise en compte par des structures comme le Comité français d'accréditation, l’absence de standardisation limite le développement en routine de ces approches, en particulier du fait des besoins en bioinformatique.

Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and in various biological processes.Escherichia coliand many α, β, and γ proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) is mainly used under aerobiosis, while (demethyl)menaquinones ((D)MK) are mostly used under anaerobiosis. Yet, we recently established the existence of an anaerobic O2- independent UQ biosynthesis pathway controlled byubiT, ubiU,andubiVgenes. Here, we characterize the regulation ofubiTUVgenes inE. coli.We show that the three genes are transcribed as two divergent operons that are both under the control of the O2sensing Fnr transcriptional regulator. Phenotypic analyses using amenAmutant devoid of (D)MK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and for uracil biosynthesis under anaerobiosis, while it contributes, though modestly, to bacterial multiplication in the mouse gut. Moreover, we showed by genetic study and18O2labelling that UbiUV contribute to hydroxylation of ubiquinone precursors through a unique O2- independent process. Last, we report a crucial role ofubiTin allowingE. colito shift efficiently from anaerobic to aerobic conditions. Overall, this study uncovers a new facet of the strategy used byE. colito adjust its metabolism upon changing O2levels and respiratory conditions. This work links respiratory mechanisms to phenotypic adaptation, a major driver in the capacity ofE. colito multiply in gut microbiota, and of facultative anaerobic pathogens to multiply in their host.ABSTRACT IMPORTANCEEnterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated to various inflammatory bowel diseases. Our study focuses on biosynthesis of ubiquinone (UQ), a key player in respiratory chains, under anaerobiosis. The importance of this study stems from the fact that UQ usage was for long considered to be restricted to aerobic conditions. Here we investigated the molecular mechanism allowing UQ synthesis in the absence of O2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, i.e. enzymes able to insert a O atom in the absence of O2. We also found that anaerobically synthesized UQ can be used for respiration on nitrate and synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens (Salmonella, Shigella, Vibrio) and will help in unravelling microbiota dynamics.

The Paramyxoviridae family is a large family of non-segmented negative RNA viruses, some of which are pathogenic to humans or domestic animals. Their small genome comprises six transcription units, including the one coding for the phosphoprotein, which is polycistronic and allows the production of both the phosphoprotein, an essential component for the functioning of the RNA synthesis machinery, and one or more accessory proteins, notably involved in the escape from the immune system. In particular, the genome of most members of the Orthoparamyxovirinae subfamily contains a superimposed gene in the sequence of the gene coding for the phosphoprotein but in a different reading frame. Cells infected with these viruses thus express an accessory protein C by a mechanism of alternative translation initiation. This protein is multi-functional and is involved in regulating viral RNA synthesis, antagonising the innate immune response and budding new viral particles. For some viruses, this protein is a virulence factor. However, we lack information on the structure and mechanisms of action of protein C.The aim of my work was to characterise the structure of the C protein of two paramyxoviruses, alone or in complex with cellular partners, in order to better understand how this protein can act in infected cells.Bioinformatics studies confirmed the conservation of a common architecture to all Orthoparamyxovirinae C proteins and the existence of a disordered N-terminal region and a helix-structured domain in the C-terminal part of the protein. We expressed and purified the C protein of Tupaia paramyxovirus (TPMV) as well as a C-terminal fragment and determined the crystallographic structure of the latter. Despite low sequence similarity, the three-dimensional structure indicates a folding similar to that of the Sendai virus C-terminal domain. Using small-angle X-ray scattering data, we were able to establish an overall model of the entire protein. In collaboration, we have shown that the TPMV C protein inhibits the RNA synthesis machinery of Nipah virus. In particular, it has been shown in the case of measles virus that protein C inhibits RNA synthesis through an interaction with the phosphoprotein. With this in mind, we undertook the structural characterisation of the TPMV phosphoprotein and investigated whether it interacts with the protein C.We used different strategies to express and purify the Nipah virus C protein, alone or in complex with cellular partners. Using a high-throughput screening method, we defined several soluble constructs and used one of these proteins to generate llama nanobodies to use as crystallisation chaperones. In collaboration, we have shown by yeast two-hybrid and TAP-TAG MS methods that NiV C protein binds to the VPS37B (Vacuolar Protein Sorting 37 Homolog B) protein of the ESCRT-I cellular subcomplex of the ESCRT (Endosomal Sorting Complexes Required for Transport) machinery. We co-expressed the protein C with the ESCRT-I subcomplex and showed that the protein C associates with these proteins.In conclusion, my work demonstrates that the C proteins of the Orthoparamyxovirinae subfamily share a common structure and can cross-act with species belonging to different genera suggesting that they have a common origin.; La famille des Paramyxoviridae est une grande famille de virus à ARN négatif non-segmenté dont certains sont pathogènes pour l’homme ou les animaux domestiques. Leur génome de taille réduite comprend six unités de transcription, dont celle codant pour la phosphoprotéine P qui est polycistronique et permet la production, d’une part, de la phosphoprotéine, un composant essentiel pour le fonctionnement de la machine de synthèse d’ARN, et d’autre part, d’une ou de plusieurs protéines accessoires, notamment impliquées dans l’échappement au système immunitaire. En particulier, le génome de la plupart des membres de la sous-famille des Orthoparamyxovirinae contient un gène surimprimé dans la séquence du gène codant pour la phosphoprotéine mais dans un cadre de lecture différent. Les cellules infectées par ces virus expriment ainsi une protéine accessoire C par un mécanisme d’initiation alternative de la traduction. Cette protéine est multi-fonctionnelle, elle est impliquée dans la régulation de la synthèse d’ARN viral, dans l’antagonisme de la réponse immunitaire innée et dans le bourgeonnement de nouvelles particules virales. Pour certains virus, cette protéine est un facteur de virulence. Mais, nous manquons d’information sur la structure et les mécanismes d’action de la protéine C.Mon travail avait pour objectif de caractériser la structure de la protéine C de deux paramyxovirus, seule ou en complexe avec des partenaires cellulaires, afin de mieux comprendre comment cette protéine peut agir dans les cellules infectée.Des études bio-informatiques ont confirmé la conservation d’une architecture commune à l’ensemble des protéines C des Orthoparamyxovirinae et l’existence d’une région N-terminale désordonnée et d’un domaine structuré en hélice- dans la partie C-terminale de la protéine. Nous avons exprimé et purifié la protéine C du paramyxovirus de la toupaye (TPMV) ainsi qu’un fragment C-terminal et nous avons déterminé la structure cristallographique de ce dernier. En dépit d’une faible similarité de séquence, la structure tridimensionnelle indique un repliement similaire à celui du domaine C-terminal du virus Sendai. A partir de données de diffusion de rayons X aux petits angles, nous avons pu établir un modèle d’ensemble de la protéine entière. En collaboration, nous avons montré que la protéine C du TPMV inhibe la machinerie de synthèse d’ARN du virus Nipah. Il a notamment été démontré dans le cas du virus de la rougeole que la protéine C inhibe la synthèse d’ARN par une interaction avec la phosphoprotéine. Dans cette optique, nous avons entrepris la caractérisation structurale de la phosphoprotéine du TPMV et nous avons cherché à déterminer si elle interagit avec la protéine C.Nous avons utilisé différentes stratégies pour exprimer et purifier la protéine C du virus Nipah, seule ou en complexe avec des partenaires cellulaires. Par une méthode de criblage à haut débit, nous avons défini plusieurs constructions solubles et nous avons utilisé une de ces protéines pour générer des nanobodies de lama qui seront utilisés comme chaperons de cristallisation. En collaboration, nous avons montré par les méthodes de double hybride en levure et de TAP-TAG MS que la protéine C du NiV se lie à la protéine VPS37B (Vacuolar Protein Sorting 37 Homolog B) du sous-complexe cellulaire ESCRT-I de la machinerie ESCRT (Endosomal Sorting Complexes Required for Transport I). Nous avons co-exprimé la protéine C avec le sous-complexe ESCRT-I et montré que la protéine C s’associe à ces protéines.En conclusion, mes travaux démontrent que les protéines C des Orthoparamyxovirinae partagent une structure commune et peuvent agir de manière croisée entre espèces appartenant à des genres différents suggérant qu’elles ont une origine commune.

Aneuploidy is a frequent occurrence in fungal species where it can alter gene expression and promote adaptation to a variety of environmental cues. Multiple forms of aneuploidy have been observed in the opportunistic fungal pathogen Candida albicans, which is a common component of the human gut mycobiome but can escape this niche and cause life-threatening systemic disease. Using a barcode sequencing (Bar-seq) approach, we evaluated a set of diploid C. albicans strains and found that a strain carrying a third copy of chromosome (Chr) 7 was associated with increased fitness during both gastrointestinal (GI) colonization and systemic infection. Our analysis revealed that the presence of a Chr 7 trisomy resulted in decreased filamentation, both in vitro and during GI colonization, relative to isogenic euploid controls. A target gene approach demonstrated that NRG1 , encoding a negative regulator of filamentation located on Chr 7, contributes to increased fitness of the aneuploid strain due to inhibition of filamentation in a gene dosage–dependent fashion. Together, these experiments establish how aneuploidy enables the reversible adaptation of C. albicans to its host via gene dosage–dependent regulation of morphology.

We report a case of severe tick-borne encephalitis in a pregnant woman, leading to a prolonged stay in the intensive care unit. She showed minor clinical improvement >6 months after her presumed infection. The patient was not vaccinated, although an effective vaccine is available and not contraindicated during pregnancy. Keywords: France; Germany; TBEV; intensive care; long-term sequelae; meningitis/encephalitis; pregnant woman; tick-borne encephalitis; tick-borne encephalitis virus; ticks; viruses; zoonoses.

International audience; Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

International audience

Vibrio cholerae is a gram-negative bacillus. It is the pathogenic agent of cholera and responsible for more than 100,000 deaths per year, which motivated its study. The first observations of V. cholerae showed that it had a curved rod shape with a flagellum at one end. It was later showed that the positioning of the flagellum at one end was related to the polarization of the intracellular organisation of the vibrio cell. Studies of factors involved in this polarization suggest that the intracellular organisation of newly formed bacteria is derived from their mother. The objective of my thesis was to understand if and how the intracellular organization of the vibrio cell can be established de novo in V. cholerae. To do this, I first had to find situations in which cellular organisation was lost. Two reversible non-proliferative states are described in V. cholerae: the first is the stationary phase in which the bacterium enters when it lacks nutrients. The second non-proliferative state occurs when the bacterium is subjected to certain stresses such as treatment with antibiotics or exposure to cold temperatures. This second state is associated with a loss of the rod shape and the cell wall: the bacteria adopt a spherical shape. We speak of spheroplasts. I established the cellular organisation of these two states via massive sequencing and fluorescence microscopy techniques, and studied the replicative profile of the cells, the precise localization of factors involved in polarity, chromosome segregation and cell division. The cells in stationary phase were found to retain the organisation of growing cells, despite the absence of division. On the contrary, I observed a notable modification of the cellular organisation of the spheroplasts. These cells show an increased number of genome copies, a disassembly of the division machinery and an inactivity of some polarity and segregation actors. I took advantage of the discovery that the presence of L-arabinose (L-ara) in the culture medium induces the transformation of V. cholerae cells into spheroplasts. An advantage of this method is that cells readily resume proliferation when L-ara is removed, allowing us to study the de novo recovery of cellular organisation by video microscopy. I observed that this recovery invariably follows different steps in a chronological manner. My observations allowed me to describe the dynamics of organisational factors at different stages of the process. Thus, I observed that replication resumes from the first steps, accompanied by a strong activity of proteins involved in the synthesis of the bacterial cell wall. Then the cell begins a morphological transition, the sphere lengthens and forms branches in which the genomes are present. During these stages, the activity of the polarity protein HubP, responsible for the correct localization of the chromosomes, can be noted. Finally, these branches undergo division events allowing the return of the initial rod shape of the bacterium. Once the chronology of the actors was established, we tried to determine if genetic modifications could alter this process. No mutations for various known non-essential candidates involved in maintaining cell organisation, such as the HubP protein, prevented successful recovery. A more systematic strategy based on comparing the contents of a mutant library before and after return also failed to reveal any new candidates. Finally, essential candidates such as the division protein FtsZ or actors involved in wall synthesis (PBPs, MreB) could be tested using specific inhibitors or via heat-sensitive alleles. Only the alteration of the proper functioning and positioning of the wall synthesis machinery is able to prevent the return to a proliferative form, showing the crucial importance of this structure for the de novo re-establishment of the rod shape and the intracellular organization of V. cholerae.; Vibrio cholerae est un bacille à Gram négatif. C'est l'agent pathogène du choléra. Il est responsable de plus de 100 000 morts par an, ce qui a motivé son étude. Les premières observations de V. cholerae ont montré qu'il avait la forme de bâtonnet incurvée avec un flagelle à une extrémité. Il a par la suite montré que le positionnement du flagelle à une seule extrémité était lié à la polarisation de l'organisation cellulaire du vibrion. Les études de facteurs impliqués dans cette polarisation suggèrent que l'organisation cellulaire des bactéries nouvellement formées découle de celle de leur « mère ». L'objectif de ma thèse était de comprendre si et comment l'organisation cellulaire du vibrion peut être est établie de novo chez V. cholerae. Pour cela, il me fallait dans un premier temps trouver des situations dans lesquelles l'organisation cellulaire était perdue. Deux états non prolifératifs réversibles sont décrits chez V. cholerae : le premier est la phase stationnaire dans laquelle la bactérie entre lorsqu'elle manque de nutriments. Le second état non prolifératif intervient lorsque la bactérie est soumise à certains stress comme le traitement aux antibiotiques ou l'exposition à des températures froides. Ce second état est associé à une perte de la forme en bâtonnet et de la paroi cellulaire : les bactéries adoptent une forme sphérique. On parle de sphéroplastes. J'ai établi l'organisation cellulaire de ces deux états via des techniques de séquençage massif et de microscopie à fluorescence et j'ai étudié le profil réplicatif des cellules, la localisation précise de facteurs impliqués dans la polarité, la ségrégation des chromosomes ainsi que la division cellulaire. J'ai observé une modification notable de l'organisation cellulaire des sphéroplastes. Ces cellules présentent un plus grand nombre de copie du génome, un désassemblage de la machinerie de division ainsi qu'une inactivité de certains acteurs de la polarité et de la ségrégation.J'ai profité pour mes études de la découverte que la présence du L-arabinose (L-ara) dans le milieu de culture induit la transformation des cellules de V. cholerae en sphéroplastes. Un avantage de cette méthode est que les cellules repartent facilement en prolifération quand le L-ara est enlevé, ce qui nous a permis d'étudier le rétablissement de novo de l'organisation cellulaire par vidéo microscopie. J'ai observé que ce rétablissement suit invariablement différentes étapes de manière chronologique. Mes observations m'ont permis de décrire la dynamique de facteurs organisationnels aux différentes étapes du processus. Ainsi, j'ai observé que la réplication reprend dès les premières étapes, accompagnée par une forte activité des protéines impliquées dans la synthèse de la paroi bactérienne. Puis la cellule commence une transition morphologique, la sphère s'allonge et forme des branches dans lesquelles les génomes sont présents. On peut noter durant ces étapes l'activité de la protéine de polarité HubP, responsable de la bonne localisation des chromosomes. Enfin ces branches subissent des évènements de division permettant le retour de la forme en bâtonnet initiale de la bactérie. Une fois la chronologie des acteurs établie, une stratégie systématique basée sur la comparaison du contenu d'une banque de mutant avant et après retour n'a pas permis de mettre en évidence des candidats essentiels au retour. Finalement, les candidats essentiels comme la protéine de division FtsZ ou encore des acteurs impliqués dans la synthèse de la paroi (PBPs, MreB) ont pu être testés en utilisant des inhibiteurs spécifiques ou via des allèles thermosensibles. Seule l'altération du bon fonctionnement et positionnement des machineries de synthèse de la paroi est en mesure d'empêcher le retour à une forme proliférative, montrant l'importance capitale de cette structure pour le rétablissement de novo de la forme en bâtonnet et de l'organisation cellulaire de V. cholerae.

The chloroplast signal recognition particle (CpSRP) receptor (CpFTSY) is a component of the CpSRP pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in the co-translational incorporation of chloroplast-encoded subunits of photosynthetic complexes into the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. The lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of photosystem II (PSII), reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanisms may partially compensate for the effect of a disrupted CpSRP pathway.

International audience; Tuberculosis infection (TBI) is defined as a state of infection in which individuals host live Mycobacterium tuberculosis with or without clinical signs of active TB. It is now understood as a dynamic process covering a spectrum of responses to infection resulting from the interaction between the TB bacilli and the host immune system. The global burden of TBI is about one-quarter of the world's population, representing a reservoir of approximately 2 billion people. On average, 5-10% of people who are infected will develop TB disease over the course of their lives, but this risk is enhanced in a series of conditions, such as co-infection with HIV. The End-TB strategy promotes the programmatic management of TBI as a crucial endeavor to achieving global targets to end the TB epidemic. The current development of new diagnostic tests capable of discriminating between simple TBI and active TB, combined with novel short-course preventive treatments, will help achieve this goal. In this paper, we present the current situation and recent developments of management of TBI and the operational challenges.

L’INRAE d’Aurillac mène des recherches sur la microbiologie et la technologie des fromages du Massif Central avec pour objectifs la compréhension des processus de fabrication et d’affinage des fromages et la recherche de solutions microbiennes et technologiques pour l’amélioration de leurs qualités. Leurs recherches ont ainsi contribué au maintien des prairies naturelles du Massif Central, au développement de l’activité économique, la création d’emplois et la reconnaissance internationale des fromages au lait cru. Cette étude a été conduite selon la méthode ASIRPA ex-post.; The INRAE of Aurillac carries out research on the microbiology and technology of cheeses from the Massif Central with the aim of understanding the manufacturing and ripening processes of cheeses and the search for microbial and technological solutions to improve their qualities. Their research has thus contributed to the maintenance of the natural meadows of the Massif Central, the development of economic activity, the creation of jobs and the international recognition of raw milk cheeses. Cette étude a été conduite selon la méthode ASIRPA ex-post.

International audience; The FilmArray Blood Culture Identification 2 panel (BCID2; bioMérieux) is a fully automated PCR-based assay for identifying bacteria, fungi, and bacterial resistance markers in positive blood cultures (BC) in about 1 h. In this multicenter study, we evaluated the performance of the BCID2 panel for pathogen detection in positive BC. Conventional culture and BCID2 were performed in parallel at four tertiary-care hospitals. We included 152 positive BC-130 monomicrobial and 22 polymicrobial cultures-in this analysis. The BCID2 assay correctly identified 90% (88/98) of Gram-negative and 89% (70/79) of Gram-positive bacteria. Five bacterial isolates targeted by the BCID2 panel and recovered from five positive BC, including three polymicrobial cultures, were missed by the BCID2 assay. Fifteen isolates were off-panel organisms, accounting for 8% (15/182) of the isolates obtained from BC. The mean positive percent agreement between the BCID2 assay and standard culture was 97% (95% confidence interval, 95 to 99%), with agreement ranging from 67% for Candida albicans to 100% for 17 targets included in the BCID2 panel. BCID2 also identified the blaCTX-M gene in seven BC, including one for which no extended-spectrum β-lactamase (ESBL)-producing isolate was obtained in culture. However, it failed to detect ESBL-encoding genes in three BC. Two of the 18 mecA/C genes detected by the BCID2 were not confirmed. No carbapenemase, mecA/C, or MREJ targets were detected. The median turnaround time was significantly shorter for BCID2 than for culture. The BCID2 panel may facilitate faster pathogen identification in bloodstream infections. IMPORTANCE Rapid molecular diagnosis combining the identification of pathogens and the detection of antibiotic resistance genes from positive blood cultures (BC) can improve the outcome for patients with bloodstream infections. The FilmArray BCID2 panel, an updated version of the original BCID, can detect 11 Gram-positive bacteria, 15 Gram-negative bacteria, 7 fungal pathogens, and 10 antimicrobial resistance genes directly from a positive BC. Here, we evaluated the real-life microbiological performance of the BCID2 assay in comparison to the results of standard methods used in routine practice at four tertiary care hospitals.

International audience; The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by highperformance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

International audience; Although a strong link between viruses and prokaryotes is commonly known to exist in aquatic systems, few studies have investigated their relationship in spring waters. In the French Massif Central, certain springs are known to exhibit varying levels of naturally occurring radioactivity. Therefore, the aim of this study was to examine the standing stock of viruses together with prokaryotes, and determine the potential environmental factors influencing them in springs characterized by contrasted radioactivity gradient. Among the investigated 15 spring habitats, flow cytometry analyses indicated that both viral (VA) and prokaryotic abundances (PA) varied by an order of magnitude accompanied by virus-to-prokaryote ratio ranging between 8 and 144.2. Significant differences in VA was evident among springs where low abundances (mean ± SD: 21.3 ± 7.0 x 107 VLP l-1) corresponded to high (mean ± SD: 1911 ± 814.0 nGy.h-1) gamma (γ) dose radiation (222Rn) and vice versa. Adverse effect of water radioactivity (222Rn) on VA could perhaps corroborate our hypothesis of drastic effect of ionizing radiations on viruses, which might explain for lack of significant relationship between PA and VA. In such a scenario of prevailing low VA, the adopted phage life strategies could define their existence in these specialized ecosystems.

In southern France, cases of community-acquired meningitis syndrome (CAM) are typically clustered as outbreaks with determinants which remain unknown. This 61-month retrospective investigation in Nîmes and Marseille university hospital laboratories, yielded 2,209/20,779 (10.63%) documented CAM cases caused by 62 different micro-organisms, represented by seasonal viral etiologies (78.8%), including Enterovirus, Herpes Simplex Virus (HSV), and Varicella-Zoster Virus (VZV; 1,620/2,209 = 73.4%). Multi correspondence analysis revealed an association of infection with age and sex, with the risk of infection being relatively higher in young men, as confirmed by Fisher’s exact test (p −3). Bacterial meningitis accounted for 20% of cases, mostly caused by Streptococcus pneumoniae (27.4% of cases), Neisseria meningitidis (12.5%), and Haemophilus influenzae (9.5%) with bacteria/virus coinfection (0.9%), and only six cases of documented fungal meningitis. In total, 62.6% of cases, of which 88.7% were undocumented, arose from 10 outbreaks. 33.2% of undocumented cases were aged >60 years compared to 19.2% of documented cases (p p

International audience; Forests are ecosystems critical to understanding the global carbon budget, due to their carbon sequestration potential in both aboveground and belowground compartments, especially in species-rich forests. Soil carbon sequestration is strongly linked to soil microbial communities, and this link is mediated by the tree community, likely due to modifications of microenvironmental conditions (i.e., biotic conditions, soil properties, and microclimate). We studied soil carbon concentration and the soil microbial biomass of 180 local neighborhoods along a gradient of tree species richness ranging from 1 to 16 tree species per plot in a Chinese subtropical forest experiment (BEF-China). Tree productivity and different tree functional traits were measured at the neighborhood level. We tested the effects of tree productivity, functional trait identity, and dissimilarity on soil carbon concentrations, and their mediation by the soil microbial biomass and microenvironmental conditions. Our analyses showed a strong positive correlation between soil microbial biomass and soil carbon concentrations. In addition, soil carbon concentration increased with tree productivity and tree root diameter, while it decreased with litterfall C:N content. Moreover, tree productivity and tree functional traits (e.g., fungal root association and litterfall C:N ratio) modulated microenvironmental conditions with substantial consequences for soil microbial biomass. We also showed that soil history and topography should be considered in future experiments and tree plantations, as soil carbon concentrations were higher at sites where historical (i.e., at the beginning of the experiment) carbon concentrations were high, themselves being strongly affected by the topography. Altogether, these results implied that the quantification of the different soil carbon pools is critical for understanding microbial community–soil carbon stock relationships and their dependence on tree diversity and microenvironmental conditions.

Group A Streptococcus (GAS) M and M-like proteins are essential virulence factors and represent the primary epidemiological marker of this pathogen. Protein sequences encoding 1054 M, Mrp and Enn proteins, from 1668 GAS genomes, were analysed by SplitsTree4, partitioning around medoids and co-occurrence. The splits network and groups-based analysis of all M and M-like proteins revealed four large protein groupings, with multiple evolutionary histories as represented by multiple edges for most splits, leading to ‘M-family-groups’ (FG) of protein sequences: FG I, Mrp; FG II, M protein and Protein H; FG III, Enn; and FG IV, M protein. M and Enn proteins formed two groups with nine sub-groups and Mrp proteins formed four groups with ten sub-groups. Discrete co-occurrence of M and M-like proteins were identified suggesting that while dynamic, evolution may be constrained by a combination of functional and virulence attributes. At a granular level, four distinct family-groups of M, Enn and Mrp proteins are observable, with Mrp representing the most genetically distinct of the family-group of proteins. While M and Enn protein families generally group into three distinct family-groups, horizontal and vertical gene flow between distinct GAS strains is ongoing.

Atmospheric nitrogen (N) deposition is a worldwide environmental problem and threatens biodiversity and ecosystem functioning. Understanding the responses of community dynamics and assembly processes of abundant and rare soil bacterial taxa to anthropogenic N enrichment is vital for the management of N-polluted forest soils.Soil microbial responses to anthropogenic nitrogen (N) enrichment at the overall community level has been extensively studied. However, the responses of community dynamics and assembly processes of the abundant versus rare bacterial taxa to N enrichment have rarely been assessed. Here, we present a study in which the effects of short- (2 years) and long-term (13 years) N additions to two nearby tropical forest sites on abundant and rare soil bacterial community composition and assembly were documented. The N addition, particularly in the long-term experiment, significantly decreased the bacterial alpha-diversity and shifted the community composition toward copiotrophic and N-sensitive species. The alpha-diversity and community composition of the rare taxa were more affected, and they were more closely clustered phylogenetically under N addition compared to the abundant taxa, suggesting the community assembly of the rare taxa was more governed by deterministic processes (e.g., environmental filtering). In contrast, the abundant taxa exhibited higher community abundance, broader environmental thresholds, and stronger phylogenetic signals under environmental changes than the rare taxa. Overall, these findings illustrate that the abundant and rare bacterial taxa respond distinctly to N addition in tropical forests, with higher sensitivity of the rare taxa, but potentially broader environmental acclimation of the abundant taxa.IMPORTANCE Atmospheric nitrogen (N) deposition is a worldwide environmental problem and threatens biodiversity and ecosystem functioning. Understanding the responses of community dynamics and assembly processes of abundant and rare soil bacterial taxa to anthropogenic N enrichment is vital for the management of N-polluted forest soils. Our sequence-based data revealed distinct responses in bacterial diversity, community composition, environmental acclimation, and assembly processes between abundant and rare taxa under N-addition soils in tropical forests. These findings provide new insight into the formation and maintenance of bacterial diversity and offer a way to better predict bacterial responses to the ongoing atmospheric N deposition in tropical forests.

International audience; Evolutionarily early-branching xanthomonads, also referred to as clade-1 xanthomonads, include major plant pathogens, most of which colonize monocotyledonous plants. Seven species have been validly described, among them the two sugarcane pathogens Xanthomonas albilineans and Xanthomonas sacchari, and Xanthomonas translucens, which infects small-grain cereals, diverse grasses, but also asparagus and pistachio trees. Single-gene sequencing and genomic approaches indicated that this clade likely contains more, yet undescribed species. In this study, we sequenced representative strains of three novel species using long-read sequencing technology. Xanthomonas campestris pv. phormiicola strain CFBP 8444 causes bacterial streak on New Zealand flax, another monocotyledonous plant. Xanthomonas sp. strain CFBP 8443 has been isolated from common bean and Xanthomonas sp. strain CFBP 8445 originated from banana. Complete assemblies of the chromosomes confirmed their unique phylogenetic position within clade 1 of Xanthomonas. Genome mining revealed novel genetic features, hitherto undescribed in other members of the Xanthomonas genus. In strain CFBP 8444, we identified genes related to the synthesis of coronatine-like compounds, a phytotoxin produced by several pseudomonads, which raises interesting questions about the evolution and pathogenicity of this pathogen. In addition, strain CFBP 8444 was found to contain a second, atypical flagellar gene cluster in addition to the canonical flagellar gene cluster. Overall, this research represents an important step toward better understanding the evolutionary history and biology of early-branching xanthomonads.