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ABSTRACTIntratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land’s cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

Field Portable X-Ray Fluorescence (FPXRF) was investigated as an alternative analysis tool for the detection and quantification of gold in hyperaccumulator plants in the field. First, synthetic gold standards were prepared with materials chosen to best simulate plant material, using microcrystalline cellulose (mcc) or ash by adding known amounts of gold. Powder samples were obtained containing gold in amounts ranging from 10 to 1000 ppm of gold. They were analysed, as well as blank samples of mcc and ash, using a NITON xl3t GOLDD+ FPXRF spectrometer in AllGeo and Soil modes with an analysis time of 60 s. Results were compared to Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) analysis of the synthetic gold standards after digestion in aqua regia. Gold was detected and accurately quantified in synthetic gold standards containing more than 25 ppm of gold in AllGeo mode. At lower concentration, results were much more inaccurate, with no gold being detected at 10 ppm. In Soil mode, similar results to AllGeo mode were obtained with mcc synthetic standards. However, with ash synthetic standard gold quantification was very poor over the whole concentration range and gold was detected in the blank ash sample, which was confirmed as a false positive by the subsequent ICP analysis. Samples of Phyllanthus stipulatus, suspected gold hyperaccumulators, collected on gold-bearing sites in French Guiana were then analysed following the same protocol. The same false positive phenomenon was observed in some of them, with gold being detected by FPXRF in Soil mode but not by ICP analysis. Furthermore, analysis of the FPXRF spectra revealed that none of the characteristic gold emission ray was present. This false positive was attributed to an interference caused by zinc, present in significant amount in the ash of the synthetic standard and the collected plants.

Precision Medicine is being increasingly used in the developed world to improve health care. While several Precision Medicine (PM) initiatives have been launched worldwide, their implementations have proven to be more challenging particularly in low- and middle-income countries. To address this issue, the “Personalized Medicine in North Africa” initiative (PerMediNA) was launched in three North African countries namely Tunisia, Algeria and Morocco. PerMediNA is coordinated by Institut Pasteur de Tunis together with the French Ministry for Europe and Foreign Affairs, with the support of Institut Pasteur in France. The project is carried out along with Institut Pasteur d’Algérie and Institut Pasteur du Maroc in collaboration with national and international leading institutions in the field of PM including Institut Gustave Roussy in Paris. PerMediNA aims to assess the readiness level of PM implementation in North Africa, to strengthen PM infrastructure, to provide workforce training, to generate genomic data on North African populations, to implement cost effective, affordable and sustainable genetic testing for cancer patients and to inform policy makers on how to translate research knowledge into health products and services. Gender equity and involvement of young scientists in this implementation process are other key goals of the PerMediNA project.In this paper, we are describing PerMediNA as the first PM implementation initiative in North Africa. Such initiatives contribute significantly in shortening existing health disparities and inequities between developed and developing countries and accelerate access to innovative treatments for global health.

The stability of beams of laser wakefield accelerated electrons in dielectric capillary tubes is experimentally investigated. These beams are found to be more stable in charge and pointing than the corresponding beams of electrons accelerated in a gas jet. Electron beams with an average charge of 43 pC and a standard deviation of 14% are generated. The fluctuations in charge are partly correlated to fluctuations in laser pulse energy. The pointing scatter of the electron beams is measured to be as low as 0.8 mrad (rms). High laser beam pointing stability improved the stability of the electron beams.

The degradation of a mixture of ibuprofen, naproxen, and diclofenac in various effluents by UVC/H2O2 or UVC/S2O82− was studied to assess the impact of the matrix composition and of the oxidant precursor on process efficiency. Experiments were carried out in a 20-L laboratory pilot (a scaled-down version of a full-scale pilot). In effluents collected during dry weather, the rural constructed wetland effluent allowed faster degradation than the urban conventional WWTP effluent, regardless of the nature of the targets or of the oxidant precursor. This was mainly attributed to a three-times higher chemical oxygen demand in the urban effluent, likely to quench the oxidative species. UV fluences to reach 90% degradation of the three compounds were 3,800 and 5,500 mJ cm−2 in the rural effluent, whereas they were 6,600 and 6,100 mJ cm−2 in the urban effluent with H2O2 and S2O82−, respectively. After a rainfall event, the rural effluent composition was not significantly affected compared to that of the urban effluent that underwent the dilution effect. Therefore, the stability of the rural effluent composition allowed comparable degradation efficiency, whereas the dilution effect led to a significant increase in the degradation rate constants in the urban effluent (up to four times higher). HIGHLIGHTS UVC-based process efficiency varies with weather and effluent composition.; Downstream effluent composition seems more consistent in a rural constructed wetland than in an urban WWTP.; UVC-based AOPs are more appropriate to treat effluents with low and consistent organic matter concentration.; allows for faster degradation of NAP and H2O2 for faster degradation of IBU.;

In New Caledonia, a French archipelago situated in the Pacific Ocean and benefiting from a large autonomy, the population, which included natives and non-natives, is diverse depending on how long they have been settled in the territory. Non-natives, who have settled in successive waves since 1855 in the context of colonization and then of the 1960s nickel boom, can be divided between Caldoches, metropolitans and other minorities (especially Asians and Oceanians), despite widespread interbreeding. This diversity is reflected in the territory by a division between inalienable Kanak customary lands and lands transferable to other populations, and by the maintenance of a customary system which was institutionalized in 1999 with the establishment of a customary Senate. The Matignon-Oudinot agreements (1988) thus ratified a division of the archipelago between the Kanaks (Northern Province and the islands) and the Caldoches (Southern Province).

Abstract Background Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem, the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. Results To gain insight into the activity of lysogenic bacteriophages within the context of their host genomes, we performed proximity ligation-based sequencing (Hi-C) in both in vitro and in vivo conditions on the 12 bacterial strains of the OMM12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM12). High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments, and an overall stability over time in the gut of mice. The DNA contacts pointed at 3D signatures of prophages leading to 16 of them being predicted as functional. We also identified circularization signals and observed different 3D patterns between in vitro and in vivo conditions. Concurrent virome analysis showed that 11 of these prophages produced viral particles and that OMM12 mice do not carry other intestinal viruses. Conclusions The precise identification by Hi-C of functional and active prophages within bacterial communities will unlock the study of interactions between bacteriophages and bacteria across conditions (healthy vs disease). Video Abstract

Abstract The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesized that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the C. difficile lipoprotein CD1687 during DCA-induced biofilm formation. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites and some are induced in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and that this localization is required for DCA-induced biofilm formation. Given this localization and the fact that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.

Summary: Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and long non-coding RNAs (lncRNAs), including transcripts coding for antiviral factors, such as interferons (IFNs). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-κB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-κB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

Abstract Since the beginning of the SARS-CoV-2 coronavirus pandemic, genome sequencing is essential to monitor viral mutations over time and by territory. This need for complete genetic information is further reinforced by the rapid spread of variants of concern. In this paper, we assess the ability of the hybridization technique, Capture-Seq, to detect the SARS-CoV-2 genome, either partially or in its integrity on patients samples. We studied 20 patient nasal swab samples broken down into five series of four samples of equivalent viral load from CT25 to CT36+ . For this, we tested 3 multi-virus panel as well as 2 SARS-CoV-2 only panels. The panels were chosen based on their specificity, global or specific, as well as their technological difference in the composition of the probes: ssRNA, ssDNA and dsDNA. The multi-virus panels are able to capture high-abundance targets but fail to capture the lowest-abundance targets, with a high percentage of off-target reads corresponding to the abundance of the host sequences. Both SARS-CoV-2-only panels were very effective, with high percentage of reads corresponding to the target. Overall, capture followed by sequencing is very effective for the study of SARS-CoV-2 in low-abundance patient samples and is suitable for samples with CT values up to 35.

Bismuth vanadate – bismuth molybdate solid-solution was prepared to elaborate ceramics with different amounts of cation vacancies. Dense ceramics with similar microstructures were obtained and the evolution of their melting point, specific heat, thermal diffusivity, and conductivity as a function of the amount of vacancy was evaluated. At room temperature, the thermal conductivity decreases from 1.74 W m−1 K−1 for BiVO4 (x=0) to 1.12 W m−1 K−1 for Bi0.867□0.133Mo0.4V0.6O4 (x=0.4). Moreover, we show that a very small amount of vacancy (1.7%, x=0.05) is enough to provide a large decrease in thermal conductivity by more than 15%, in agreement with a mass fluctuation scattering model. However, the temperature of the melting point also decreases with increasing amount of vacancy. Our results suggest adding only a very small amount of vacancy as the best strategy to obtain superior materials for thermal barriers and thermoelectric devices, with ultra-low thermal conductivity and high-temperature stability.

A dicationic PdII dimer, bis{2-[(diisopropylphosphanyl)methyl]quinoline-8-thiolato}palladium(II) bis(hexafluoridoantimonate) dichloromethane monosolvate, [Pd2(C32H42N2P2S2)](SbF6)2·CH2Cl2, containing a 2-[(diisopropylphosphanyl)methyl]quinoline-8-thiolate pincer ligand, was isolated and its crystal structure determined. The title compound crystallizes in the orthorhombic space group Pbca. A dimeric structure is formed by bridging coordination of the S atoms. The geometry of the butterfly-shaped Pd2S2 core is bent, with a hinge angle of 108.0 (1)° and a short Pd...Pd distance of 2.8425 (7) Å. These values are the lowest measured compared to ten dicationic dimers with a Pd2S2 core featuring sulfur atoms embedded in a chelating ligand. One of the two hexafluoridoantimonate anions is disordered over two sets of positions with site-occupancy factors of 0.711 (5) and 0.289 (5). The crystal structure is stabilized by many C—H...F and C—H...π interactions, forming a supramolecular network.

Oropouche fever is a zoonotic dengue-like syndrome caused by Oropouche virus. In August–September 2020, dengue-like syndrome developed in 41 patients in a remote rainforest village in French Guiana. By PCR or microneutralization, 23 (82.1%) of 28 tested patients were positive for Oropouche virus, documenting its emergence in French Guiana.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses-Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)-with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Abstract Interactions between the prokaryotic microbiome and eukaryotic parasites in the vertebrate gut may affect overall host health and disease. While intertropical areas exhibit a high rate of parasites carriers, such interactions are understudied in these populations. Our objectives were to (1) describe the gut microbiome of individuals living in Madagascar, (2) identify potential associations between bacterial taxa and parasites colonizing the digestive tract and (3) highlight main determinants of the gut microbiota composition in this developing country. Metadata (socioeconomic, diet, clinical) and fecal samples were collected from 219 volunteers from North-West Madagascar (Mahajanga). Fecal microbiome was assessed through 16S rRNA gene sequencing and metabolomics, and related to dietary habits and parasites carriage. We highlight important Malagasy gut microbiome peculiarities. Out of three detected enterotypes, only one is similar to that observed in Westernized countries (Ruminococcus-driven). Functions associated with the two others (Clostridium sensu stricto-driven and Escherichia/Shigella-driven) are mostly directed toward amino acids biosynthesis and degradation, respectively. Diet and protozoan carriage were the main drivers of microbiota composition. High protozoan carriage was associated with higher diversity, richness and microbial functionalities. The gut microbiome of Malagasy strongly differs from that of Westernized countries. Asymptomatic protozoan carriage and dietary habits are the external factors with the deepest impact on gut microbiome. Further studies are needed to understand whether gut microbial richness constitute a predilection niche for protozoans colonization, due to their gazing features, or whether the parasites themselves induce a higher bacterial richness.

Abstract Viruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficult. The proper identification of termini is crucial, as it helps in characterizing the packaging mechanism of bacteriophages and provides information on various aspects of phage biology. Here, we introduce PhageTermVirome (PTV) as a tool for the easy and rapid high-throughput determination of phage termini and packaging mechanisms using modern large-scale metagenomics datasets. We successfully tested the PTV algorithm on a mock virome dataset and then used it on two real virome datasets to achieve the rapid identification of more than 100 phage termini and packaging mechanisms, with just a few hours of computing time. Because PTV allows the identification of free fully formed viral particles (by recognition of termini present only in encapsidated DNA), it can also complement other virus identification softwares to predict the true viral origin of contigs in viral metagenomics datasets. PTV is a novel and unique tool for high-throughput characterization of phage genomes, including phage termini identification and characterization of genome packaging mechanisms. This software should help researchers better visualize, map and study the virosphere. PTV is freely available for downloading and installation at https://gitlab.pasteur.fr/vlegrand/ptv .

Prematurity is a risk factor for dysbiosis of the gut microbiota due to particular birth conditions and frequent prolonged hospitalization of neonates. Although gut microbiota colonization after birth and its establishment during the hospitalization period have been studied in preterm infants, data on gut microbiota following discharge, particularly during early childhood, are scarce. The present study investigated the relationship between gut microbiota at 1 month after birth (hospitalization period) and 3.5 years of age in 159 preterm children belonging to the French EPIFLORE prospective observational cohort study. Analysis using bacterial 16S rRNA gene sequencing showed that the gut microbiota of preterm neonates at 1 month was highly variable and characterized by six distinct enterotypes. In contrast, the gut microbiota of the same children at 3.5 years of age showed less variability, with only two discrete enterotypes. An absence of association between enterotypes at 1 month and 3.5 years of age was observed. While the alpha diversity of gut microbiota significantly increased between 1 month and 3.5 years of age, for both alpha and beta diversities, there was no correlation between the 1-month and 3.5-years time points. Comparison at 3.5 years between children born either preterm (n = 159) or full-term (n = 200) showed no differences in terms of enterotypes, but preterm children harbored a lower Shannon diversity index and a different overall composition of microbiota than full-term children. This study suggests that the characteristics of the early gut microbiota of preterm children are not predictive of the microbial community composition at 3.5 years of age. However, the impact of gestational age is still noticeable on the gut microbiota up to 3.5 years of age.

Ionization-induced electron injection was investigated experimentally by focusing a driving laser pulse with a maximum normalized potential of 1.2 at different positions along the plasma density profile inside a gas cell, filled with a gas mixture composed of 99%H2+1%N2. Changing the laser focus position relative to the gas cell entrance controls the accelerated electron bunch properties, such as the spectrum width, maximum energy, and accelerated charge. Simulations performed using the 3D particle-in-cell code WARP with a realistic density profile give results that are in good agreement with the experimental ones. The interest of this regime for optimizing the bunch charge in a selected energy window is discussed.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σF, during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σF, σE, σG, σK) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.

Since the 1990s, the Christmas markets have become unavoidable festive events during the season of Advent in Alsace, and have staged “the magic of Christmas” to develop. Thanks to the transformation of a medieval German cultural tradition into event tourism, the region is now attracting a growing number of visitors during a slack period of the year. This form of event tourism uses specific temporalities and scales in the “ordinary” territories of the city centers, and gives international visibility to Alsace as a travel destination. But, more and more difficulties are raised by the saturation of space, increasing commodification and territorial conflicts.

In the Ile-de-France area, suburban forests still exist, but their size, uses and exploitations are different compared to other French forests. These suburban forests can in many respects be described as margins. First, they constitute “natural” spaces in an urban area. Moreover, they have a social, ecological and recreational role. The activities they are the site of are only marginal in the French productive system: forestry, hunting, mushroom picking. However the level of marginality increases from Paris to the periphery and from forest edges to forest interior.

Abstract The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5′cos (Lambda), 3′cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).

Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are over-expressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1. dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1. Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread.

ABSTRACT Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291.