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International audience; It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Despite strong leprosy control measures, including effective treatment, leprosy persists in the Comoros. As of May, 2022, no resistance to anti-leprosy drugs had been reported, but there are no nationally representative data. Post-exposure prophylaxis (PEP) with rifampicin is offered to contacts of patients with leprosy. We aimed to conduct a countrywide drug resistance survey and investigate whether PEP led to the emergence of drug resistance in patients with leprosy.In this observational, deep-sequencing analysis we assessed Mycobacterium leprae genomes from skin biopsies of patients in Anjouan and Mohéli, Comoros, collected as part of the ComLep (NCT03526718) and PEOPLE (NCT03662022) studies. Skin biopsies that had sufficient M leprae DNA (2000 bacilli in 2 μl of DNA extract) were assessed for the presence of seven drug resistance-associated genes (ie, rpoB, ctpC, ctpI, folP1, gyrA, gyrB, and nth) using Deeplex Myc-Lep (targeted next generation deep sequencing), with a limit of detection of 10% for minority M leprae bacterial populations bearing a polymorphism in these genes. All newly registered patients with leprosy for whom written informed consent was obtained were eligible for inclusion in the survey. Patients younger than 2 years or with a single lesion on the face did not have biopsies taken. The primary outcome of our study was the proportion of patients with leprosy (ie, new cases, patients with relapses or reinfections, patients who received single (double) dose rifampicin-PEP, or patients who lived in villages where PEP was distributed) who were infected with M leprae with a drug-resistant mutation for rifampicin, fluoroquinolone, or dapsone in the Comoros.Between July 1, 2017, and Dec 31, 2020, 1199 patients with leprosy were identified on the basis of clinical criteria, of whom 1030 provided a skin biopsy. Of these 1030 patients, 755 (73·3%) tested positive for the M leprae-specific repetitive element-quantitative PCR (qPCR) assay. Of these 755 patients, 260 (34·4%) were eligible to be analysed using Deeplex Myc-Lep. 251 (96·5%) were newly diagnosed with leprosy, whereas nine (3·4%) patients had previously received multidrug therapy. 45 (17·3%) patients resided in villages where PEP had been administered in 2015 or 2019, two (4·4%) of whom received PEP. All seven drug resistance-associated targets were successfully sequenced in 216 samples, 39 samples had incomplete results, and five had no results. No mutations were detected in any of the seven drug resistance-related genes for any patient with successfully sequenced results.This drug resistance survey provides evidence to show that M leprae is fully susceptible to rifampicin, fluoroquinolones, and dapsone in the Comoros. Our results also show, for the first time, the applicability of targeted sequencing directly on skin biopsies from patients with either paucibacillary or multibacillary leprosy. These data suggest that PEP had not selected rifampicin-resistant strains, although further support for this finding should be confirmed with a larger sample size.Effect:Hope, The Mission To End Leprosy, the Fonds Wetenschappelijk Onderzoek, the EU.

Although irrigation water is frequently assessed for the presence of plant pathogens, large spatial and temporal surveys that provide clues on the diversity and circulation of pathogens are missing. We evaluate the diversity of soft rot Pectobacteriaceae (SRP) of the genera Dickeya and Pectobacterium over 2 years in a temperate, mixed-use watershed. The abundance of isolated strains correlates with the agricultural gradient along the watershed with a positive correlation found with temperature, nitrate, and dissolved organic carbon water concentration. We characterized 582 strains by amplification and sequencing of the gapA gene. Multilocus sequence analysis, performed with three housekeeping genes for 99 strains, and core genome analysis of 38 sequenced strains, confirmed for all the strains but one, the taxonomic assignation obtained with the sole gapA sequence. Pectobacterium spp. (549 isolates) were far more abundant than Dickeya spp. (33 isolates). Dickeya spp. were only observed in the lower part of the river when water temperature was >19°C, and we experimentally confirmed a decreased fitness of several Dickeya spp. at 8°C in river water. D. oryzae dominates the Dickeya spp. and P. versatile and P. aquaticum dominate the Pectobacterium spp., but their repartition along the watershed was different, with P. versatile being the only species regularly recovered all along the watershed. Excepting P. versatile, the Dickeya and Pectobacterium spp. responsible for disease outbreak on crops were less abundant or rarely detected. This work sheds light on the various ecological behaviors of different SRP types in stream water and indicates that SRP occupation is geographically structured.

Multiple SARS-CoV-2 variants have successively, or concommitantly spread worldwide since summer 2020. A few co-infections with different variants were reported and genetic recombinations, common among coronaviruses, were reported or suspected based on co-detection of signature mutations of different variants in a given genome. Here we report three infections in southern France with a Delta 21J/AY.4-Omicron 21K/BA.1 “Deltamicron” recombinant. The hybrid genome harbors signature mutations of the two lineages, supported by a mean sequencing depth of 1,163-1,421 reads and mean nucleotide diversity of 0.1-0.6%. It is composed of the near full-length spike gene (from codons 156-179) of an Omicron 21K/BA.1 variant in a Delta 21J/AY.4 lineage backbone. Importantly, we cultured an isolate of this recombinant and sequenced its genome. It was observed by scanning electron microscopy. As it is misidentified with current variant screening qPCR, we designed and implemented for routine diagnosis a specific duplex qPCR. Finally, structural analysis of the recombinant spike suggested its hybrid content could optimize viral binding to the host cell membrane. These findings prompt further studies of the virological, epidemiological, and clinical features of this recombinant.

International audience; We detected SARS-CoV-2 of PANGO lineage R.1 with the spike substitution E484K in three patients. Eleven other sequences in France and 8,831 worldwide were available from GISAID, 92% originating from Japan. The three genome sequences from our institute were phylogenetically closest to another from Guinea-Conakry, where one of the patients had travelled. These viruses did not exhibit any unusual features in cell culture. Spike structural predictions indicated a 1.3-time higher transmissibility index than for the globally spread B.1.1.7 variant but also an affinity loss for gangliosides that might have slowed dissemination. The spread of new SARS-CoV-2 mutants/variants is still not well understood and therefore difficult to predict, and this hinders implementation of effective preventive measures, including adapted vaccines.

International audience; Objectives: There is no specific recommendation about antimicrobial treatment length for documented infections in chemotherapy induced febrile neutropenia (FN). Practices have changed along time in our center regarding length of antibiotic treatment. The aim of this study was to compare long versus short antibiotic course for bloodstream infection (BSI) treatment in acute myeloid leukemia (AML) patients during FN. Methods: This monocentric retrospective comparative study included all consecutive BSI episodes among AML patients with FN for 3 years (2017-2019). Episodes were classified regarding the length of antibiotic treatment, considered as short course if the treatment lasted ≤ 7 days, except for nonfermenting bacteria and Staphylococcus aureus or lugdunensis for which the threshold was ≤ 10 days and ≤ 14 days, respectively. The primary outcome was the number of BSI relapses in both groups within 30 days of antibiotic discontinuation. Results: Among 71 AML patients, 104 BSI episodes were included; 48 (46%) received short course treatment. Only 8 (7.6%) BSI episodes relapsed within 30 days of antibiotic discontinuation, 5 having received short course treatment. No association was found between risk of relapse and short course of antibiotic treatment (p = 0.37). The only risk factor significantly associated with BSI relapse was neutropenia duration (p = 0.005). Conclusion: Antibiotic short course seemed as effective as prolonged treatment for BSI in AML patients during FN, with very few relapses at day 30. These encouraging findings should be confirmed through prospective studies.

The broad host range IncC plasmid family and the integrative mobilizable Salmonella Genomic Island 1 (SGI1) and its derivatives enable the spread of medically-important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids.IMPORTANCEAntimicrobial resistance has become a major public health issue, particularly with the increase in multidrug resistance (MDR) in both animal and human pathogenic bacteria, and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements such as conjugative plasmids and integrative elements conferring multidrug resistance is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically-important resistance genes (e.g., ESBLs, and carbapenemases). A better knowledge of the complex biology of these broad-host range mobile elements will help to understand the dissemination of antimicrobial resistance genes that occurred across γ-proteobacteria borders.

In bacteria, DNA is not separated in the cell by a membrane, converse to eukaryotes, which have a nucleus. Therefore, DNA must be protected, while remaining accessible to allow for gene expression. The DNA structuring is achieved via proteins called NAPs (standing for "Nucleoid Associated Protein"). Among these NAPs, the Hfq protein is found. Hfq is a pleiotropic factor involved in bacterial physiology. Its best-known function is to regulate RNA-dependent processes. However, its ability to form an amyloid structure allows it to interact with DNA, to form bridges between distant parts of DNA and thus to compact it strongly. If this property was demonstrated in vitro, analyses were lacking in cellulo. This PhD work highlights the direct and indirect effects of Hfq on the compaction of the nucleoid. The complexes or the cells were observed using cryo-(scanning) transmission electron microscopy (cryo-(S)TEM) and cryo-soft X-ray tomography (cryo-SXT) techniques. The latter is a three-dimensional imaging technique that permits to quantify the volume and density of the bacterial nucleoid. This PhD project made possible to characterize the impact of Hfq on the structure of DNA and bacterial morphology, which will make it possible to test in cellulo the effects of new antibiotics, which could be developed in connection with this property in the future., Chez les bactéries, l'ADN n'est pas séparé du reste de la cellule par une membrane, contrairement aux eucaryotes qui possèdent un noyau. Cet ADN a donc besoin d'être à la fois protégé tout en restant accessible pour permettre l'expression génétique. Cette structuration de l'ADN se fait via des protéines appelées NAPs (pour "Nucleoid Associated Protein"). Parmi ces NAPs, on trouve la protéine Hfq. Hfq est un facteur pléiotrope impliqué dans la physiologie bactérienne. Sa fonction la plus connue est de réguler des processus ARNs dépendants. Cependant, sa capacité à former une structure amyloïde lui permet d'interagir avec l'ADN, de former des ponts entre des parties éloignées de l'ADN et ainsi de le compacter fortement. Si cette propriété a pu être démontrée in vitro, les analyses in cellulo manquaient. Ce travail de thèse met en lumière les effets directs et indirects d'Hfq sur la l'état de compaction du nucléoïde. Ainsi les complexes ou les cellules ont pu être observés à l'aide de techniques de cryo-microscopie électronique en transmission (à balayage) (cryo-(S)TEM) et de cryo-tomographie à rayons-X mous (cryo-SXT). Cette dernière, est une technique d'imagerie tri-dimensionnelle qui permet de quantifier le volume et la densité du nucléoïde bactérien. Ce projet de thèse a permis de caractériser l'impact d'Hfq sur la structure de l'ADN et la morphologie bactérienne, ce qui permettra de tester in cellulo l'effet de nouveaux antibiotiques qui pourront être développés en lien avec cette propriété dans le futur.

International audience; The head louse, Pediculus humanus capitis , is a strictly obligate human ectoparasite with a long history of association with humans. Here, 17 ancient head lice nits were recovered from six shrunken human heads (known as tsantsas ) of individuals from the Shuar/Jivaro tribe, a native Amazonian population from Ecuador, South America. Cytochrome b DNA analysis revealed the presence of three known mitochondrial clades. Clade A was the most frequent (52.94%), followed by F (35.29%), and B (11.76%). Eleven haplotypes were found in 17 samples, and nine of the haplotypes were novel, indicating an unusually high genetic diversity. In conclusion, we confirmed the presence of clades A, B and F in South Amerindian population. Moreover, the description of clade F, together with its previous reports in another Amerindian population from French Guiana, strongly support the hypothesis of a native South American origin for this clade, and probably derived from clade B which was carried to America by an ancestral Eurasian Beringian population. Further support to our conclusion and new insights might come from the analysis of a larger collection of modern and ancient native American lice.

Background Cytoadhesion and sequestration of Plasmodium falciparum infected red blood cells (iRBC) in the microvasculature of vital organs are a major cause of malaria pathology. Several studies have provided evidence on the implication of the human host intercellular adhesion molecule-1 (ICAM-1) as a major receptor for iRBCs binding to P. falciparum erythrocyte membrane protein 1 (PfEMP1) in the development of severe and cerebral malaria. The genetic polymorphism K29M in the immunoglobulin-like domain of ICAM-1, known as ICAM-1Kilifi, has been associated with either increased or decreased risk of developing cerebral malaria. Methods To provide more conclusive results, the genetic polymorphism of ICAM-1Kilifi was assessed by PCR and sequencing in blood samples from 215 Beninese children who presented with either mild or severe malaria including cerebral malaria. Results and conclusions The results showed that in this cohort of Beninese children, the ICAM-1kilifi variant is present at the frequencies of 0.27, similar to the frequency observed in other African countries. This ICAM-1kilifi variant was not associated with disease severity in agreement with other findings from the Gambia, Tanzania, Malawi, Gabon, and Thailand, suggesting no evidence of a direct link between this polymorphism and the pathogenesis of severe and cerebral malaria.

International audience

International audience; Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to M. abscessus, than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active Cyclipostins and Cyclophostin analogs (CyC) and provide new insights regarding the CyC lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells; that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus; and that modification of such intracellular localization via disruption of endolysosomal pH strongly affect the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could became efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in presence several other pathogens like P. aeruginosa and S. aureus. Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyC, and the attractiveness of the fluorescent CyC as potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus.

Type I toxin–antitoxin systems (T1TAs) are extremely potent bacterial killing systems difficult to characterize using classical approaches. To assess the killing capability of type I toxins and to identify mutations suppressing the toxin expression or activity, we previously developed the FASTBAC-Seq (Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing) method in Helicobacter pylori. This method combines a life and death selection with deep sequencing. Here, we adapted and improved our method to investigate T1TAs in the model organism Escherichia coli. As a proof of concept, we revisited the regulation of the plasmidic hok/Sok T1TA system. We revealed the death-inducing phenotype of the Hok toxin when it is expressed from the chromosome in the absence of the antitoxin and recovered previously described intragenic toxicity determinants of this system. We identified nucleotides that are essential for the transcription, translation or activity of Hok. We also discovered single-nucleotide substitutions leading to structural changes affecting either the translation or the stability of the hok mRNA. Overall, we provide the community with an easy-to-use approach to widely characterize TA systems from diverse types and bacteria.

International audience; Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps.

Antibiotic resistance is rapidly spreading by horizontal transfer of resistance genes in mobile genetic elements. While plasmids are key drivers of this process, very few integrative phages encode antibiotic resistance genes. Here, we find that phage-plasmids, elements that are both phages and plasmids, often carry antibiotic resistance genes. We found 60 phage-plasmids with 184 antibiotic resistance genes, including broad-spectrum-cephalosporins, carbapenems, aminoglycosides, fluoroquinolones and colistin. These genes are in a few hotspots, seem to have been co-translocated with transposable elements, and are often in class I integrons, which had not been previously found in phages. We tried to induce six phage-plasmids with resistance genes (including four with resistance integrons) and succeeded in five cases. Other phage-plasmids and integrative prophages were co-induced in these experiments. As a proof of principle, we focused on a P1-like element encoding an extended spectrum β-lactamase, blaCTX-M-55. After induction, we confirmed that it’s capable to infect and convert four other E. coli strains. Its re-induction led to further conversion of a sensitive strain, confirming it’s a fully functional phage. This study shows that phage-plasmids carry a large diversity of clinically relevant antibiotic resistance genes that they transfer across bacteria. As plasmids, these elements seem very plastic and capable of acquiring genes from other plasmids. As phages, they may provide novel paths of transfer for resistance genes, because they can infect bacteria distant in time and space from the original host. As a matter of alarm, they may also eventually mediate transfer to other types of phages.ImportanceDissemination of antimicrobial resistances is a major threat to global health. Here, we show that a group of temperate bacterial viruses (=phages), termed phage-plasmids, commonly encode different and multiple types of resistance genes of high clinical importance, often in integrons. This is unexpected since phages typically do not carry resistance genes and, hence, do not confer their hosts with resistance upon infection and genome integration. Our experiments with phage-plasmids isolated from clinical settings confirmed they infect sensitive strains, rendering them antibiotic resistant. The spread of antibiotic resistance genes by phage-plasmids is worrisome because it dispenses cell-to-cell contact, necessary for the canonical plasmid transfer (=conjugation). Furthermore, their integrons are now genetic platforms for the acquisition of novel resistance genes.

Class C β-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs ((64)SXSK, (150)YXN, (315)KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella, Bradyrhizobium, and Parachlamydia) has raised questions about the possible existence of a C2 subclass of β-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC β-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet (150)YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304–306), and in Hafnia alvei and H. paralvei (deletion of residues 289–290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to β-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (ampR, ampD, ampG, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.

Purpose of Review In this review, we report on the state of knowledge about human Q fever in Brazil and on the Guiana Shield, an Amazonian region located in northeastern South America. There is a contrast between French Guiana, where the incidence of this disease is the highest in the world, and other countries where this disease is practically non-existent. Recent Findings Recent findings are essentially in French Guiana where a unique strain MST17 has been identified; it is probably more virulent than those usually found with a particularly marked pulmonary tropism, a mysterious animal reservoir, a geographical distribution that raises questions. Summary Q fever is a bacterial zoonosis due to Coxiella burnetii that has been reported worldwide. On the Guiana Shield, a region mostly covered by Amazonian forest, which encompasses the Venezuelan State of Bolivar, Guyana, Suriname, French Guiana, and the Brazilian State of Amapá, the situation is very heterogeneous. While French Guiana is the region reporting the highest incidence of this disease in the world, with a single infecting clone (MST 117) and a unique epidemiological cycle, it has hardly ever been reported in other countries in the region. This absence of cases raises many questions and is probably due to massive under-diagnosis. Studies should estimate comprehensively the true burden of this disease in the region.

International audience; The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. The effect of gut commensal bacteria on host protein SUMOylation, an essential ubiquitin-like modification involved in various intestinal functions, remains, however, unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in intestinal cells in a pH-dependent manner. We demonstrate that these metabolites inactivate intestinal deSUMOylases and promote the hyperSUMOylation of nuclear matrix-associated proteins. We further show that BCFAs inhibit the NF-κB pathway, decrease pro-inflammatory cytokine expression, and promote intestinal epithelial integrity. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses triggered by the gut microbiota.

International audience; Invasive bacteria colonise their host tissues by establishing niches inside eukaryotic cells, where they grow either in the cytosol or inside a specialised vacuole. These two distinct intracellular lifestyles both present benefits but also impose various constraints on pathogenic microorganisms, in terms of nutrient acquisition, space requirements, exposure to immune responses, and ability to disseminate. Here we review the major characteristics of cytosolic and vacuolar lifestyles and the strategies used by bacteria to overcome challenges specific to each compartment. Recent research providing evidence that these scenarios are not mutually exclusive is presented, with the dual lifestyles of two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, discussed in detail. Finally, we elaborate on the conceptual implications of polyvalence from the perspective of host-pathogen interactions.

International audience

International audience; Ethionamide (ETH) is a second-line antituberculosis drug. ETH resistance (ETH-R) is mainly related to the mutations of the monooxygenase-activating ETH (EthA), the ETH target (InhA), and the inhA promoter. Nonetheless, diagnosing ETH-R is still challenging. We assessed the strategy used for detecting ETH-R at the French National Reference Center for Mycobacteria in 497 MDR-TB isolates received from 2008 to 2016. The genotypic ETH’s resistance detection was performed by sequencing ethA, ethR, the ethA-ethR intergenic region, and the inhA promoter in the 497 multidrug-resistant isolates, whereas the phenotypic ETH susceptibility testing (PST) was performed using the reference proportion method. Mutations were found in up to 76% of the 387 resistant isolates and in up to 28% of the 110 susceptible isolates. Our results do not support the role of ethR mutations in ETH resistance. Altogether, the positive predictive value of our genotypic strategy to diagnose ETH-R was improved when only considering the variants included in the WHO catalogue and in other databases, such as TB-Profiler. Therefore, our work will help to update the list of mutations that could be graded as being associated with resistance to improve ETH-R diagnosis.

The spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide. Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are considered “critical-priority” bacteria by the World Health Organization (WHO) since 2017 taking into account criteria such as patient mortality, global burden disease, and worldwide trend of multi-drug resistance (MDR). Indeed P. aeruginosa can be particularly difficult to eliminate from patients due to its combinatory antibiotic resistance, multifactorial virulence, and ability to over-adapt in a dynamic way. Research is active, but the course to a validated efficacy of a new treatment is still long and uncertain. What is new in the anti–P. aeruginosa clinical development pipeline since the 2017 WHO alert? This review focuses on new solutions for P. aeruginosa infections that are in active clinical development, i.e., currently being tested in humans and may be approved for patients in the coming years. Among 18 drugs of interest in December 2021 anti–P. aeruginosa development pipeline described here, only one new combination of β-lactam/β-lactamase inhibitor is in phase III trial. Derivatives of existing antibiotics considered as “traditional agents” are over-represented. Diverse “non-traditional agents” including bacteriophages, iron mimetic/chelator, and anti-virulence factors are significantly represented but unfortunately still in early clinical stages. Despite decade of efforts, there is no vaccine currently in clinical development to prevent P. aeruginosa infections. Studying pipeline anti–P. aeruginosa since 2017 up to now shows how to provide a new treatment for patients can be a difficult task. Given the process duration, the clinical pipeline remains unsatisfactory leading best case to the approval of new antibacterial drugs that treat CRPA in several years. Beyond investment needed to build a robust pipeline, the Community needs to reinvent medicine with new strategies of development to avoid the disaster. Among “non-traditional agents”, anti-virulence strategy may have the potential through novel and non-killing modes of action to reduce the selective pressure responsible of MDR.

The SARS-CoV-2 21K/BA.1, 21L/BA.2, and BA.3 Omicron variants have recently emerged worldwide. To date, the 21L/BA.2 Omicron variant has remained very minority globally but became predominant in Denmark instead of the 21K/BA.1 variant. Here we describe the first cases diagnosed with this variant in south-eastern France. We identified thirteen cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travellers returning from Tanzania. Overall, viral genomes displayed a mean (±standard deviation) number of 65.9±2.5 (range, 61-69) nucleotide substitutions and 31.0±8.3 (27-50) nucleotide deletions, resulting in 49.6±2.2 (45-52) amino acid substitutions (including 28 in the spike protein) and 12.4±1.1 (12-15) amino acid deletions. Phylogeny showed the distribution in three different clusters of these genomes, which were most closely related to genomes from England and South Africa, from Singapore and Nepal, or from France and Denmark. Structural predictions pointed out a significant enlargement and flattening of the 21L/BA.2 N-terminal domain surface compared with that of the 21K/BA.2 Omicron variant, which may facilitate initial viral interactions with lipid rafts. Close surveillance is needed at global, country and center scales to monitor the incidence and clinical outcome of the 21L/BA.2 Omicron variant.

Vγ9Vδ2 T cells have been reported to participate to the immune response against infectious diseases such as the Q fever caused by Coxiella burnetii infection. Indeed, the number and proportion of Vγ9Vδ2 T cells are increased during the acute phase of Q fever. Human Vγ9Vδ2 T cell responses are triggered by phosphoantigens (pAgs) produced by pathogens and malignant cells, that are sensed via the membrane receptors butyrophilin-3A1 (BTN3A1) and -2A1 (BTN2A1). Here, by using CRISPR-Cas9 inactivation in THP-1 cells, we show that BTN3A and BTN2A are required to Vγ9Vδ2 T cell response to C. burnetii infection, though not directly involved in the infection process. Furthermore, C. burnetii-infected monocytes display increased BTN3A and BTN2A expression and induce Vγ9Vδ2 T cell activation that can be inhibited by specific antagonist mAb. More importantly, we show that the antimicrobial functions of Vγ9Vδ2 T cells towards C. burnetii are enhanced in the presence of an BTN3A activating antibody. This supports the role of Vγ9Vδ2 T cells in the control of C. burnetii infection and argues in favor of targeting these cells as an alternative treatment strategy for infectious diseases caused by intracellular bacteria.

International audience; Chronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds.

Leptospira interrogans is a bacterial species responsible for leptospirosis, a neglected worldwide zoonosis. Mice and rats are resistant and can become asymptomatic carriers, whereas humans and some other mammals may develop severe forms of leptospirosis. Uncommon among spirochetes, leptospires contain lipopolysaccharide (LPS) in their outer membrane. LPS is highly immunogenic and forms the basis for a large number of serovars. Vaccination with inactivated leptospires elicits a protective immunity, restricted to serovars with related LPS. This protection that lasts in mice, is not long lasting in humans and requires annual boosts. Leptospires are stealth pathogens that evade the complement system and some pattern recognition receptors from the Toll-like (TLR) and Nod-Like families, therefore limiting antibacterial defense. In macrophages, leptospires totally escape recognition by human TLR4, and escape the TRIF arm of the mouse TLR4 pathway. However, very little is known about the recognition and processing of leptospires by dendritic cells (DCs), although they are crucial cells linking innate and adaptive immunity. Here we tested the activation of primary DCs derived from human monocytes (MO-DCs) and mouse bone marrow (BM-DCs) 24h after stimulation with saprophytic or different pathogenic virulent or avirulent L. interrogans. We measured by flow cytometry the expression of DC-SIGN, a lectin involved in T-cell activation, co-stimulation molecules and MHC-II markers, and pro- and anti-inflammatory cytokines by ELISA. We found that exposure to leptospires, live or heat-killed, activated dendritic cells. However, pathogenic L. interrogans, especially from the Icterohaemorraghiae Verdun strain, triggered less marker upregulation and less cytokine production than the saprophytic Leptospira biflexa. In addition, we showed a better activation with avirulent leptospires, when compared to the virulent parental strains in murine BM-DCs. We did not observe this difference in human MO-DCs, suggesting a role for TLR4 in DC stimulation. Accordingly, using BM-DCs from transgenic deficient mice, we showed that virulent Icterohaemorraghiae and Manilae serovars dampened DC activation, at least partly, through the TLR4 and TRIF pathways. This work shows a novel bacterial immune evasion mechanism to limit DC activation and further illustrates the role of the leptospiral LPS as a virulence factor.

International audience

AimTo investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning.MethodsAnimals from a new transgenic rat line expressing green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received at Day-12, near neural diversification, a single oral bolus of mir-320-3p or miR-375-3p, embedded in DiOleyl-Succinyl-Paromomycin (DOSP), and were further early (Day-15) or regularly (Day-30) weaned. Relevant miRNA (miR-320-3p, miR-375-3p, miR-375-5p, miR-16-5p, miR-132-3p, miR-504), polr3d, hspb6, inflammation, enteroendocrine, and circadian clock-related mRNAs, chromatin complexes, and duodenal cell density were assayed at 8h post-inoculation and at Day-45.ResultsThe miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (pConclusionAddressing oral miRNA-320-3p loads to duodenal cell lineage is paving the way for the design of new therapeutics, manipulating long term consequences of early life stress.

Coastal hydrosystems, located at the land-sea interface, are both subject to autochthonous sources of pollution but also form a receptacle for terrigenous inputs from upstream areas. The attractiveness of these environments leads to high population densities, the permanent development of activities and the construction of infrastructures, resulting in the qualitative degradation of coastal aquatic environments. This is particularly true in the case of marinas, which are both places of dense internal activities (yachting, marina, shipyards (careening and maintenance of boats) but also receptacles of waters from the catchment areas loaded with suspended solids and / or contaminants, to which they are connected.The Gulf of Aigues-Mortes (GAM) extends along the western margin of the Rhone delta. The marinas spread over this restricted geographical area present variable management problems depending on their size and location. Siltation or silting phenomena exist and the situation is becoming more and more difficult to manage in order to maintain the depths for most of the harbours. In these marinas that are not dredged regularly, the quantities of potentially contaminated sediments (organic and metallic micro-pollutants) to be extracted represent from a few thousand to several tens of thousands of m3 of sediments on the scale of each port. Four harbours of the GAM have joined together to set up a mutualized dredging project in line with the regional dredging scheme in Occitania, based on the valorization of dredged sediments by distinguishing between ports with sand inputs of wind or current origin and ports confronted with sand and mud inputs, which may be contaminated by the port activity and/or the watershed. The objective of this project is to carry out a diagnosis of the chemical and microbiological contamination of the waters of the 4 ports located in the Gulf of Aigues Mortes. The size of the marinas varies from 130 to 5000 boat places. The volume of dredged sediments is between 5 000 m3 of sand and 30 000 m3 of mud. Regular water samplings were done before, during and after dredging operations. Water column quality/contamination was characterized by major physicochemical water parameters (pH, temperature, dissolved oxygen, salinity, turbidity), inorganic compounds including major and trace metallic elements, organic compounds organotin compounds (including tributyltin). Trace elements were then determined using an ICP-MS, X Series II (Thermo Fisher Scientific), equipped with a collision cell technology (CCT) chamber and the organotin species monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) were determined using a gas chromatograph (Focus GC – Thermo Fisher Scientific) coupled with an inductively coupled plasma mass spectrometer (ICP-MS X Series II-Thermo Fisher Scientific).In addition to chemical parameters, indicators of fecal contamination (BTCF thermotolerant coliforms and intestinal enterococci) were also monitoring at the same frequency. The originality of the results lies in the fact that it will not only make it possible to compare the chemical and microbiological qualities of the waters of the marinas, but also to determine the impact on the quality of the waters of the same type of dredging on the resuspension of contaminants in the water column, thanks to mutualized dredging.Finally, it will make it possible to determine, when contaminant resuspension occurs, the time required for the quality of the water to return to its initial state after dredging operations. The possibility of working on sites with heterogeneous materials and disparate levels of contamination allowed us to take into account the parameters likely to influence the phenomena of sorption/desorption of contaminants (temperature, salinity, pH, porosity, nature and age of the sediments...) and to better understand the impact of the activities of these marinas, including the dredging operations on the whole Gulf of Aigues Mortes.

During cell proliferation, the maintenance of genetic information is essential. In bacteria, replication and segregation are concomitant. Replication starts at the single, bidirectional origin of replication of bacterial chromosomes. Two replication arms are then defined, and replication ends in a region diametrically opposite to the origin, the terminus. As replication progresses, the newly replicated sister chromosomes migrate to opposite cell compartments. However, microscopic observations suggest that there is a delay between replication and segregation, and that this delay varies along the length of chromosomes. The delay between replication and segregation of the sister copies of a genomic position is referred to as sister chromatid cohesion. During my PhD, I used the high-resolution tool that allows for a genome-wide analysis of Sister Chromatid Cohesion (High-SC2) and studied the cohesion profile of the model organism Vibrio cholerae. It has been shown in E. coli that the cohesion responsible for the variation of segregation speed is modulated by Topoisomerase IV, a major decatenating enzyme. One of the identified partners of this decatenase is an SMC complex, MukBEF. Cells carrying a mukB deletion show a production of anucleate cells, and a mispositioned origin of replication. Chromosome segregation is impaired, and therefore sister chromatid cohesion is increased overall. The Topo IV-MukBEF interaction is regulated by MatP, which seems to displace MukBEF from the terminus of replication, facilitating the association of the MukBEF complex with the origin of replication. I therefore decided to investigate the role of MukB, in the formation of the long-range patterns of cohesion in V. cholerae. Using genetic approaches coupled with the High-SC2 assay, I demonstrated that the deletion of mukB leads to an increase in cohesion on Chr1, especially on its left replication arm, far from the origin. These results suggested that MukB does not preferentially act on specific regions and that the differential effect of the mukB deletion on Chr1 and Chr2 is probably linked to differences in their origin of replication and/or partition systems. Previous observations in the lab have in fact shown that a double deletion of MukB and ParAB1 leads to a strong phenotype, thus I investigated its effect on the cohesion profile. My results show an additional increase of cohesion in Chr1 near the ori, suggesting that the partitioning system acts on the decohesion of the ori domain while MukB acts on the chromosomal arms. In addition, it has been shown that MatP kept the sister-copies of the ter domain of Chr1 together until cell division. I used the Hi-SC2 assay to study its role in the increased cohesion of this region. I showed that MatP was responsible for the cohesion of the ter1 domain at cell division not behind the replication fork, unlike MukB. My results have also shown that it is the density of the matS sites located on the ter domain of each chromosome that influence the level of cohesion of these domains., La maintenance de l'information génétique est essentielle pendant la prolifération cellulaire. Chez les bactéries, la réplication et la ségrégation sont concomitantes. La réplication débute à l'origine de réplication bidirectionnelle du chromosome bactérien. Deux bras de réplications sont ensuite définis, et la réplication se termine dans la région diamétralement opposée à l'origine, le terminus. Alors que la réplication progresse, les chromatides sœurs nouvellement répliquées migrent vers des côtés opposés de la cellule. Cependant, des observations par microscopie suggèrent qu'il existe un délai entre la réplication et la ségrégation qui varie le long du chromosome. Ce délai entre la réplication et ségrégation des chromatides sœurs est appelé cohésion des chromatides sœurs. Pendant ma thèse, j'ai utilisé l'outil de haute-résolution qui permet une analyse de la cohésion du génome entier (High-SC2) pour étudier le profil de cohésion de l'organisme modèle Vibrio cholerae. Il a été démontré chez E. coli que la cohésion responsable de la variation de la vitesse de ségrégation est modulée par Topoisomérase IV, une enzyme de décaténation majeure. L'un des partenaires identifiés de cette décaténase est le complexe SMC, MukBEF. Les cellules portant une délétion de mukB montrent une production de cellules anucléées, ainsi qu'une origine de réplication mal positionnée. La ségrégation des chromosomes est affectée, et la cohésion des chromatides sœurs est augmentée. L'interaction Topo IV-MukBEF est régulée par MatP qui chasserait MukBEF du terminus de réplication, facilitant ainsi l'association de MukBEF à l'origine de réplication. J'ai donc décidé d'étudier le rôle de MukB dans la formation des motifs de cohésion chez V. cholerae. Grâce à des approches génétiques couplées à l'outil High-SC2, j'ai pu démontrer que la délétion de mukB mène à une augmentation de la cohésion sur le Chr1, plus précisément sur le bras gauche, assez loin de l'origine. Mes résultats suggèrent que MukB n'agit pas préférentiellement sur des régions spécifiques, mais que ces effets différents sur les deux chromosomes de cet organisme sont dus aux différences dans leurs origines de réplication et/ou leurs systèmes de partition. De précédentes observations dans notre laboratoire ont montré qu'une double délétion de MukB et ParAB1 cause un phénotype sévère, plus important que les délétions individuelles, j'ai donc étudié les conséquences de cette double délétion sur le profil de cohésion. Mes résultats montrent une augmentation additionnelle de la cohésion dans le Chr1 près de l'origine, suggérant ainsi que le système de partition agit sur la décohésion sur le domaine de l'origine pendant que MukB agit sur le reste du chromosome. Il a été également montré que MatP retardait la ségrégation des chromatides soeurs du terminus de réplication du Chr1. J'ai utilisé le même outil qui m'a permis d'étudier le rôle de MatP dans la cohésion de cette région. J'ai pu montrer que MatP était responsable de cette cohésion uniquement au moment de la division cellulaire et non pas pendant la réplication contrairement à MukB. Mes résultats montrent également que la densité des matS présents dans le domaine ter de chaque chromosome qui influent sur la cohésion de ce même domaine.

International audience; In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the first food consumed on long-term microbiota trajectory and host response are still largely unknown. This study aimed to investigate the influences of (i) the timing of first solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the first exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable fibers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion influenced to a greater extent the development of bacterial communities of the appendix vermiformis, whereas life course polysaccharides ingestion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccharides ingestion as two different tools to modulate microbiota implantation and functionality. IMPORTANCE: Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe–host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals.

Salmonella entericaserovars are invasive gram-negative bacteria, causing a wide range of diseases from gastroenteritis to typhoid fever, representing a public health threat around the world.Salmonellagains access to the intestinal lumen after oral ingestion of contaminated food or water. The crucial initial step to establish infection is the interaction with the intestinal epithelium. Human-adapted serovars such asS.Typhi orS.Paratyphi disseminate to systemic organs and induce life-threatening disease known as typhoid fever, whereas broad-host serovars such asS.Typhimurium usually are limited to the intestine and responsible for gastroenteritis in humans. To overcome intestinal epithelial barrier,Salmonelladeveloped mechanisms to induce cellular invasion, intracellular replication and to face host defence mechanisms. Depending on the serovar and the respective host organism, disease symptoms differ and are linked to the ability of the bacteria to manipulate the epithelial barrier for its own profit and cross the intestinal epithelium.This review will focus onS.Typhimurium (STm). To better understand STm pathogenesis, it is crucial to characterize the crosstalk between STm and the intestinal epithelium and decipher the mechanisms and epithelial cell types involved. Thus, the purpose of this review is to summarize our current knowledge on the molecular dialogue between STm and the various cell types constituting the intestinal epithelium with a focus on the mechanisms developed by STm to cross the intestinal epithelium and access to subepithelial or systemic sites and survive host defense mechanisms.

International audience; The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its mechanism by mutational studies. Our data demonstrate that Tba ADH547 can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 o C) and exhibit similar thermostablilty for oxidization and reduction reactions. However, Tba ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn 2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe 2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H234 to Ala result in the significant activity loss of Tba ADH547, suggesting that residues D195, H199, H262 and H234 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.

Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) have been classified in the group of resistant bacteria of highest priority. We determined the prevalence of ESBL-E collected in feces from household and shelter pets in Guadeloupe (French West Indies). A single rectal swab was taken from 125 dogs and 60 cats between June and September 2019. The prevalence of fecal carriage of ESBL-E was 7.6% (14/185, 95% CI: 4.2-12.4), within the range observed worldwide. The only risk factor associated with a higher prevalence of ESBL-E rectal carriage was a stay in a shelter, suggesting that refuges could be hotspots for their acquisition. All but one (Klebsiella pneumoniae from a cat) were Escherichia coli. We noted the presence of a blaCTX–M–1/IncI1-Iγ/sequence type (ST3) plasmid in 11 ESBL-producing E. coli isolates belonging to ST328 (n = 6), ST155 (n = 4) and ST953 (n = 1). A blaCTX–M–15 gene was identified in the three remaining ESBL-E isolates. The blaCTX–M–1 and most of the antimicrobial resistance genes were present in a well-conserved large conjugative IncI1-Iγ/ST3 plasmid characterized by two accessory regions containing antibiotic resistance genes. The plasmid has been detected worldwide in E. coli isolates from humans and several animal species, such as food-producing animals, wild birds and pets, and from the environment. This study shows the potential role of pets as a reservoir of antimicrobial-resistant bacteria or genes for humans and underlines the importance of basic hygiene measures by owners of companion animals.

International audience; Advances in nanotechnology have made it possible in recent years to develop strategies for rapid and sensitive detection of pathogenic bacteria using new nanomaterials and the development of electronic nano-sensors. The detection of bacteria still faces problems such as long analysis time and complexity of the process. An alternative method is the use of biosensors, which combines a biological recognition mechanism with a physical transduction technique. Thus, a number of methods and manufacturing technologies have been developed in order to achieve performance in sensitivity, detection limit, label-free detection or real-time analysis. This review aims to focus on the state of the art of biosensors for the recognition elements of Escherichia coli in label-free biosensors with a particular focus on the beneficial use of nanomaterials and nano-objects for detection. Among the recent related biosensors based on nano-objects for E. coli detection, the technologies and measurement techniques are detailed by comparing their performances detection in terms of concentration range and detection limit. Detection becomes more sensitive and more flexible using nanoparticles as markers, and real-time electrical detection methods are dominant in comparison with optical ones. The lowest detection limit can be achieved for sensors based on metal (gold or silver nanoparticles) with optical detection techniques in contrast with electrical detection methods using measurement conductance.

International audience; The high sensitivity of the automated tests used for Toxoplasma gondii serology can yield false-positive IgM results due to aspecific reactions. On the other hand, specific therapy can delay IgG production and, therefore, the diagnosis of seroconversion. There is a need for confirmation tests to early detect seroconversions during pregnancy. We conducted a multicentre study to evaluate the diagnostic accuracy of the Toxo II IgG and a new, not yet commercialised Toxo II IgM western blot (WB) (LDBio diagnostics Lyon France) on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity was 97.8% for IgM WB and 98.9% for IgG WB. Specificity was 89.7% and 100%, respectively. The concordance between IgM and IgG Toxo WB with the final diagnosis was very good, K = 0.89 and K = 0.99, respectively. In 5 cases (5.4%), the appearance of IgM, and in 55 cases (59.1%), the appearance of IgG was recorded by WB earlier than by traditional tests. In 10 cases (10.8%), IgM was detected after the traditional tests and in 2 cases (2.2%) for IgG. The association of IgG and IgM WB on the same sample not only detected all seroconversions but also correctly identified most of the false-positive results.

International audience; Most children are less severely affected by coronavirus-induced disease 2019 (COVID-19) than adults, and thus more difficult to study progressively. Here, we provide a neonatal nonhuman primate (NHP) deep analysis of early immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in blood and mucosal tissues. In addition, we provide a comparison with SARS-CoV-2-infected adult NHP. Infection of the neonate resulted in a mild disease compared with adult NHPs that develop, in most cases, moderate lung lesions. In concomitance with the viral RNA load increase, we observed the development of an early innate response in the blood, as demonstrated by RNA sequencing, flow cytometry, and cytokine longitudinal data analyses. This response included the presence of an antiviral type-I IFN gene signature, a persistent and lasting NKT cell population, a balanced peripheral and mucosal IFN-γ/IL-10 cytokine response, and an increase in B cells that was accompanied with anti-SARS-CoV-2 antibody response. Viral kinetics and immune responses coincided with changes in the microbiota profile composition in the pharyngeal and rectal mucosae. In the mother, viral RNA loads were close to the quantification limit, despite the very close contact with SARS-CoV-2-exposed neonate. This pilot study demonstrates that neonatal NHPs are a relevant model for pediatric SARS-CoV-2 infection, permitting insights into the early steps of anti-SARS-CoV-2 immune responses in infants.

More than 12 years have passed since the seminal observation that meningococcus, a pathogen causing epidemic meningitis in humans, occasionally associated with infectious vasculitis and septic shock, can promote the translocation of β-arrestins to the cell surface beneath bacterial colonies. The cellular receptor used by the pathogen to induce signalling in host cells and allowing it to open endothelial cell junctions and reach meninges was unknown. The involvement of β-arrestins, which are scaffolding proteins regulating G protein coupled receptor signalling and function, incited us to specifically investigate this class of receptors. In this perspective article we will summarize the events leading to the discovery that the β2-adrenergic receptor is the receptor that initiates the signalling cascades induced by meningococcus in host cells. This receptor, however, cannot mediate cell infection on its own. It needs to be pre-associated with an “early” adhesion receptor, CD147, within a hetero-oligomeric complex, stabilized by the cytoskeletal protein α-actinin 4. It then required several years to understand how the pathogen actually activates the signalling receptor. Once bound to the N-terminal glycans of the β2-adrenergic receptor, meningococcus provides a mechanical stimulation that induces the biased activation of β-arrestin-mediated signalling pathways. This activating mechanical stimulus can be reproduced in the absence of any pathogen by applying equivalent forces on receptor glycans. Mechanical activation of the β2-adrenergic receptor might have a physiological role in signalling events promoted in the context of cell-to-cell interaction.

International audience

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

International audience; Abstract The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein’s in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

In nature, bacteria form biofilms in very diverse environments, involving a range of specific properties and exhibiting competitive advantages for surface colonization. However, the underlying mechanisms are difficult to decipher. In particular, the contribution of cell flagellar motility to biofilm formation remains unclear. Here, we examined the ability of motile and nonmotile E. coli cells to form a biofilm in a well-controlled geometry, both in a simple situation involving a single-species biofilm and in the presence of co-colonizers. Using a millifluidic channel, we determined that motile cells have a clear disadvantage in forming a biofilm, exhibiting a long delay as compared to nonmotile cells. By monitoring biofilm development in real time, we observed that the decisive impact of flagellar motility on biofilm formation consists in the alteration of surface access time. Implementing a simple mathematical model to calculate surface access velocity for both motile (diffusion) and nonmotile (settling) cells, we discovered that the competitive advantage strongly depends on the geometry of the environment to be colonized. Interestingly, this advantage reverts for the smallest geometries, with a crossover at a height of 100 μm. We also report that the difference between and nonmotile cells in the ability to form a biofilm diminishes in the presence of cocolonizers, which could be due to motility inhibition through the consumption of key resources by the co-colonizers. We conclude that the impact of flagellar motility on surface colonization closely depends on the environment geometry and the population features, suggesting a unifying vision of the role of cell motility in surface colonization and biofilm formation.

Introduction: Like other countries, France has invested in a national medical genomics program. Among the four pilot research studies, the DEFIDIAG project focuses on the use of whole genome sequencing (WGS) for patients with intellectual disability (ID), a neurodevelopmental condition affecting 1–3% of the general population but due to a plethora of genes. However, the access to genomic analyses has many potential individual and societal issues in addition to the technical challenges. In order to help decision-makers optimally introduce genomic testing in France, there is a need to identify the socio-economic obstacles and leverages associated with the implementation of WGS.Methods and Analysis: This humanities and social sciences analysis is part of the DEFIDIAG study. The main goal of DEFIDIAG is to compare the percentage of causal genetic diagnoses obtained by trio WGS (including the patient and both parents) (WGST) to the percentage obtained using the minimal reference strategy currently used in France (Fragile-X testing, chromosomal microarray analysis, and gene panel strategy including 44 ID genes) for patients with ID having their first clinical genetics consultation. Additionally, four complementary studies will be conducted. First, a cost-effectiveness analysis will be undertaken in a subsample of 196 patients consulting for the first time for a genetic evaluation; in a blinded fashion, WGST and solo (index case, only) genomic analysis (WGSS) will be compared to the reference strategy. In addition, quantitative studies will be conducted: the first will estimate the cost of the diagnostic odyssey that could potentially be avoidable with first-line WGST in all patients previously investigated in the DEFIDIAG study; the second will estimate changes in follow-up of the patients in the year after the return of the WGST analysis compared to the period before inclusion. Finally, through semi-directive interviews, we will explore the expectations of 60 parents regarding genomic analyses.Discussion: Humanities and social sciences studies can be used to demonstrate the efficiency of WGS and assess the value that families associate with sequencing. These studies are thus expected to clarify trade-offs and to help optimize the implementation of genomic sequencing in France.Ethics Statement: The protocol was approved by the Ethics Committee Sud Méditerranée I (June 2019)—identification number: 2018-A00680-55 and the French data privacy commission (CNIL, authorization 919361).Clinical Trial Registration: (ClinicalTrials.gov), identifier (NCT04154891).

Cytological analysis of body fluids (BF) provides important information for diagnosis in various medical conditions. We evaluated the analytical performance of the UF-4000 BF mode for ascitic, cerebrospinal, pleural, synovial and continuous ambulatory peritoneal dialysis fluids compared to light microscopy counting (LM).223 consecutive BF were analyzed by UF-4000 and results were compared using Pearson's correlation, Bland-Altman analysis, and contingence tests at relevant cut-off values. This study also included the evaluation of precision, linearity, and carryover.For white and red blood cells (WBC, RBC) counts in all BF, correlation was excellent with Pearson's coefficients RUF-4000 BF mode is a good alternative to manual LM for BF cell counting. This automated method gives rapid and accurate results which is important for therapeutic decisions.

International audience; Background: High rates of COVID-19 vaccination uptake are required to attain community immunity. This study aims to identify factors associated with COVID-19 vaccine uncertainty and refusal among young adults, an underexplored population with regards to vaccine intention generally, in two high-income settings: Canada and France.Methods: A cross-sectional online survey was conducted from October to December 2020 among young adults ages 18-29 years (n = 6663) living in Canada (51.9%) and France (48.1%). Multinomial logistic regression analyses were performed to identify the sociodemographic and COVID-19-related measures (e.g., prevention behavior and perspectives, health-related concerns) associated with vaccine uncertainty and refusal. We conducted weighted analyses by age, gender and province/region of residence.Results: Intention to accept vaccination was reported by 84.3% and 59.7% of the sample in Canada and France, respectively. Higher levels of vaccine uncertainty and refusal were observed in France compared to Canada (30.1% versus 11%, 10.2% versus 4.7%). In both countries, we found higher levels of vaccine acceptance among young adults who reported COVID-19 prevention actions. Vaccine uncertainty and refusal were associated with living in a rural area, having lower levels of educational attainment, not looking for information about COVID-19, not wearing a face mask, and reporting a lower level of concern for COVID-19's impact on family. Participants who had been tested for COVID-19 were less likely to intend to refuse a vaccine.Conclusions: COVID-19 vaccine acceptance was high among young adults in Canada and France during a time in which vaccines were approved for use. Targeted interventions to build confidence in demographic groups with greater hesitance (e.g., rural and with less personal experience with COVID-19) may further boost acceptance and improve equity as vaccine efforts continue to unfold.

International audience; Infections of the central nervous system are among the most serious infections1,2, but the mechanisms by which pathogens access the brain remain poorly understood. The model microorganism Listeria monocytogenes (Lm) is a major foodborne pathogen that causes neurolisteriosis, one of the deadliest infections of the central nervous system3,4. Although immunosuppression is a well-established host risk factor for neurolisteriosis3,5, little is known about the bacterial factors that underlie the neuroinvasion of Lm. Here we develop a clinically relevant experimental model of neurolisteriosis, using hypervirulent neuroinvasive strains6 inoculated in a humanized mouse model of infection7, and we show that the bacterial surface protein InlB protects infected monocytes from Fas-mediated cell death by CD8+ T cells in a manner that depends on c-Met, PI3 kinase and FLIP. This blockade of specific anti-Lm cellular immune killing lengthens the lifespan of infected monocytes, and thereby favours the transfer of Lm from infected monocytes to the brain. The intracellular niche that is created by InlB-mediated cell-autonomous immune resistance also promotes Lm faecal shedding, which accounts for the selection of InlB as a core virulence gene of Lm. We have uncovered a specific mechanism by which a bacterial pathogen confers an increased lifespan to the cells it infects by rendering them resistant to cell-mediated immunity. This promotes the persistence of Lm within the host, its dissemination to the central nervous system and its transmission.

Mycobacteria possess type VII secretion systems (T7SS), also known as ESX. Recently, our laboratory has demonstrated that the ESX-4 system of M. abscessus is an essential actor in the intracellular survival of M. abscessus Furthermore, mutation of the gene coding for a structural protein of ESX-4 leads to a decrease in the secretion of several proteins, two belonging to the esx-4 locus, EsxU and EsxT. For this project, we studied the role of EsxU and EsxT proteins in M. abscessus. We first demonstrated the localization of these proteins in M. abscessus, EsxU and EsxT are associated with the bacterial membrane and are also secreted. Furthermore, EsxU and EsxT form a complex characteristic of proteins secreted by mycobacterial T7SS. EsxU and EsxT proteins are able to interact with artificial membranes to form pore-like structures in vitro. In an intramacrophage context, esxU and esxT absence does not influence the intracellular behaviour of the ΔesxUT mutated strain, but prevents the disruption of the phagosomal membrane, keeping M. abscessus ΔesxUT in an unacidified phagosome. Finally, we identify a hypervirulent phenotype associated with ΔesxUT deletion characterized by increased animal mortality and bacterial growth in mice and zebrafish. All our results suggest that the absence of both substrates improves the survival and persistence of M. abscessus in animals. We also wanted to study the immunogenic potential of these proteins. The T cell response to synthetic peptides of the EsxU and EsxT proteins did not identify epitopes that induce a T response. This suggests that EsxU and T are not immunogenic after subcutaneous inoculation of M. abscessus in the mouse model suggesting that EsxU and EsxT are not good markers of infection. The secretome and a bioinformatics analysis based on the search for secretion patterns identified other potential substrates of SST7 in M. abscessus. Two proteins with a proline-glutamate (PE) an d proline-glutamate-glutamate (PPE) motif have peptides that induce a Th1-type immune response. However, further experiments are needed to determine whether these proteins represent good diagnostic targets in patients and whether they are able to discriminate M. abscessus infections from infections caused by other mycobacteria., Les mycobactéries possèdent des systèmes de sécrétion de type VII (SST7) aussi nommé ESX. Récemment, notre laboratoire a démontré que le système ESX-4 de M. abscessus est un acteur essentiel à sa survie intracellulaire. De plus, la mutation du gène codant pour une protéine structurale de l’ESX-4 entraîne une diminution de la sécrétion de plusieurs protéines dont deux appartenant au locus esx-4, EsxU et EsxT. Pour ce projet, nous avons étudié le rôle des protéines EsxU et EsxT chez M. abscessus. Nous avons d'abord démontré la localisation de ces protéines chez M. abscessus. EsxU et EsxT sont associées avec la membrane bactérienne et secrétées dans le milieu extérieur. De plus, elles forment un complexe caractéristique des protéines sécrétées par les SST7 des mycobactéries et sont capables in vitro d’interagir avec des membranes artificielles pour former des structures semblables à des pores. Dans les macrophages, leur absence n'influence pas le comportement intracellulaire de la souche mutée ΔesxUT, mais empêche la rupture de la membrane phagosomale, maintenant M. abscessus ΔesxUT dans un phagosome non acidifié. Enfin, nous identifions un phénotype hypervirulent associé à ΔesxUT, caractérisé par une augmentation de la mortalité et de la croissance bactérienne chez la souris et le poisson zèbre. L’ensemble de nos résultats suggèrent que l'absence des deux substrats améliore la survie et la persistance de M. abscessus dans ces modèles animaux. Nous avons aussi voulu étudier le potentiel immunogène de ces protéines. La réponse lymphocytaire T, vis-à-vis de peptides synthétiques des protéines EsxU et EsxT n’a pas permis d’identifier d’épitopes induisant une réponse T. Ceci suggère qu’EsxU et T ne sont pas immunogènes après inoculation de M. abscessus par voie sous-cutanée dans le modèle souris ce qui laisse penser qu’EsxU et EsxT ne représentent pas de bons marqueurs de l’infection. Le sécrétome ainsi qu’une analyse bio-inform atique basée sur la recherche de motifs de sécrétion ont permis d’identifier d’autres substrats potentiels de SST7 chez M. abscessus. Deux protéines avec un motif proline-glutamate (PE) et proline-glutamate-glutamate (PPE) ont des peptides qui induisent une réponse immunitaire Th1. Néanmoins des expériences supplémentaires sont nécessaires pour déterminer si ces protéines représentent de bonnes cibles de diagnostic chez les patients et si elles sont capables de discriminer les infections à M. abscessus des infections causées par d’autres mycobactéries.

Cattle are carriers, without clinical manifestations, of enterohemorrhagic Escherichia coli (EHEC) O157:H7 responsible for life-threatening infections in humans. A better identification of factors playing a role in maintaining persistence of such strains in cattle is required to develop more effective control measures. Hence, we conducted a study to identify farms with a persistent circulation of EHEC O157:H7. The EHEC O157:H7 herd status of 13 farms, which had previously provided bovine EHEC O157:H7 carriers at slaughter was investigated. Two farms were still housing positive young bulls, and this was true over a 1-year period. Only one fecal sample could be considered from a supershedder, and 60% of the carriers shed concentrations below 10 MPN/g. Moreover, EHEC O157:H7 represented minor subpopulations of E. coli. PFGE analysis of the EHEC O157:H7 strains showed that persistent circulation was due either to the persistence of a few predominant strains or to the repeated exposure of cattle to various strains. Finally, we compared fecal microbial communities of shedders (S) (n = 24) and non-shedders (NS) (n = 28), including 43 young bulls and nine cows, from one farm. Regarding alpha diversity, no significant difference between S vs. NS young bulls (n = 43) was observed. At the genus level, we identified 10 amplicon sequence variant (ASV) indicators of the S or NS groups. The bacterial indicators of S belonged to the family XIII UCG-001, Slackia, and Campylobacter genera, and Ruminococcaceae NK4A21A, Lachnospiraceae-UGC-010, and Lachnospiraceae-GCA-900066575 groups. The NS group indicator ASVs were affiliated to Pirellulaceae-1088-a5 gut group, Anaerovibrio, Victivallis, and Sellimonas genera. In conclusion, the characteristics enhancing the persistence of some predominant strains observed here should be explored further, and studies focused on mechanisms of competition among E. coli strains are also needed.