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Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay, pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after re-annotation of this dataset and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are co-expressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role.ImportanceFilamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed towards the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

The alternative sigma factor B (σ(B)) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σ(B) loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σ(B) or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σ(B)). However, σ(B) becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σ(B) loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σ(B) loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σ(B) in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

International audience; In summer 2019, widespread occurrence of crown gall disease caused byAgrobacterium spp. was observed on commercially grown ornamental plants in south-ern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resemblingthe members of Xanthomonas were also associated with crown gall tissues on weep-ing fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present studywas to characterize the crown gall-associated Xanthomonas strains using plant inocula-tion assays, molecular-phylogenetic analyses, and comparative genomics approaches.Pathogenicity tests showed that the Xanthomonas strains did not induce diseasesymptoms on their host of isolation. However, the strains induced hypersensitive reac-tion on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration.Multilocus sequence analysis suggested that the strains belong to clade IA ofXanthomonas, phylogenetically close to Xanthomonas translucens, X. theicola, and X.hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization valuesbetween the whole-genome sequences of the strains isolated in this study and refer-ence Xanthomonas strains are far below the accepted thresholds for the definition ofprokaryotic species, signifying that these strains could be defined as two new specieswithin clade IA of Xanthomonas. Comparative genomics showed that the strains iso-lated from crown gall tissues are genetically distinct from X. translucens, as almost allthe type III secretion system genes and type III effectors are lacking in the formergroup. The data obtained in this study provide novel insight into the breadth ofgenetic diversity of crown gall-associated bacteria and pave the way for research ongall-associated Xanthomonas-plant interactions.

International audience; Bacteria have to process several levels of gene regulation and coordination of interconnected regulatory networks to ensure the most adequate cellular response to specific growth conditions. Especially, expression of complex and costly fitness and pathogenicity-associated traits is coordinated and tightly regulated at multiple levels. We studied the interconnected regulation of the expression of the colibactin and yersiniabactin polyketide biosynthesis machineries, which are encoded by two pathogenicity islands found in many phylogroup B2 Escherichia coli isolates. Comparative phenotypic and genotypic analyses identified the BarA-UvrY two-component system as an important regulatory element involved in colibactin and yersiniabactin expression. The carbon storage regulator (Csr) system controls the expression of a wide range of central metabolic and virulence-associated traits. The availability of CsrA, the key translational regulator of the Csr system, depends on BarA-UvrY activity. We employed reporter gene fusions to demonstrate UvrY- and CsrA-dependent expression of the colibactin and yersiniabactin determinants and confirmed a direct interaction of CsrA with the 5' untranslated leader transcripts of representative genes of the colibactin and yersiniabactin operons by RNA electrophoretic mobility shift assays. This posttranscriptional regulation adds an additional level of complexity to control mechanisms of polyketide expression, which is also orchestrated at the level of ferric uptake regulator (Fur)-dependent regulation of transcription and phosphopantetheinyl transferase-dependent activation of polyketide biosynthesis. Our results emphasize the interconnection of iron- and primary metabolism-responsive regulation of colibactin and yersiniabactin expression by the fine-tuned action of different regulatory mechanisms in response to variable environmental signals as a prerequisite for bacterial adaptability, fitness, and pathogenicity in different habitats.

Four b acteriophage i nsensitive m utants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following a challenge with the Brussowvirus SW13. The BIMs displayed an altered, sedimentation phenotype. Whole genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family-2-glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated r hamnose- g lucose p olymer (Rgp) biosynthesis locus (designated here as the rgp gene cluster). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis p oly s accharide p ellicle (PSP) initiator, WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescent labelling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide which is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. Importance: Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements which specify the surface receptor(s) is apparent. Our research identifies a single gene which is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C(6) and C(8) alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to “plant cell wall degradation,” despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.

International audience; The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all threeproteases—PrtS, HtrA, and SepM—are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wildtype strain.IMPORTANCE Streptococcus thermophilus is a lactic acid bacterium used widely as a starter in the dairy industry. Due to its “generally recognized as safe” status and its weak cell surface proteolytic activity, it is also considered a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications

The objective of the present study was to determine genomic characteristics of expanded-spectrum cephalosporin (ESC)-resistant Escherichia coli spreading in healthy broilers in Lebanon in 2018. Rectal swabs (n = 280) from 56 farms were screened for the presence of ESC-resistant E. coli isolates. Antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL)/AmpC production were determined by the disk diffusion method. Whole-genome sequencing (WGS) of 102 representative isolates of E. coli was performed to determine their phylogenetic diversity, serotypes, sequence types (ST), acquired resistance genes, and virulence-associated genes. Fifty-two out of 56 farms housed broilers carrying ESC-resistant E. coli isolates. These farms had large and recurrent antimicrobial practices, using, for some of them, critically important antibiotics for prophylactic and therapeutic purposes. Among the 102 sequenced multidrug-resistant (MDR) E. coli isolates, the proportion of ESBL, plasmid-mediated AmpC β-lactamase (pAmpC) producers, and ESBL/pAmpC coproducers was 60%, 27.6%, and 12.4%, respectively. The most prevalent ESBL/pAmpC genes were blaCMY-2, blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-14b (n = 42, n = 31, n =15, n = 9, and n = 7, respectively). These ESBL/pAmpC producers were distributed in different STs, most being well-known avian-associated and sometimes pathogenic STs (ST-10, ST-48, ST-93, ST-115, ST-117, and ST-457). Phylogenetic single nucleotide polymorphism (SNP) analysis confirmed their genetic diversity and wide dispersion across the Lebanese territory. Most isolates were also resistant to ciprofloxacin (101/102 with 3 QRDR mutations), and 19/102 isolates from 11 unrelated STs also carried the mobile resistance gene mcr-1. This survey illustrates the alarming prevalence of MDR E. coli resistant to medically important antibiotics in broilers in Lebanon. This advocates the need for surveillance programs of antimicrobial resistance in Lebanon and the reduction of excessive use of antibiotics to limit the spread of MDR E. coli in food-producing animals. IMPORTANCE Poultry production is a main contributor of the global trend of antimicrobial resistance arising from food-producing animals worldwide. In Lebanon, inappropriate use of antibiotics is frequent in chickens for prophylactic reasons and to improve productivity, resulting in an alarming prevalence of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli, also resistant to other medically important antibiotics (i.e., colistin and ciprofloxacin). Their complex genomic epidemiology highlighted by an important genetic diversity suggests that these resistance determinants are largely spreading in enteric bacteria in Lebanese poultry. Further molecular surveillance is needed to understand the country-specific epidemiology of ESBL/AmpC and mcr-1 genes in Lebanese poultry production. In addition, decisive interventions are urgently needed in order to ban the use of critically important antibiotics for human medicine in food-producing animals and limit the spread of antibiotic resistance in Lebanon.

MSystems 6(3), e01300-20 (2021). doi:10.1128/mSystems.01300-20, Published by American Society for Microbiology, Washington, DC

International audience; Glycosomes are peroxisome-related organelles of trypanosomatid para-sites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nu-cleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophos-phorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and pro-cyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycoso-mal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxy-kinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glyco-somes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are func-tional, since the lethality of the knockdown UGP mutant cell line ((RNAi)UGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle ((RNAi)UGP/(EXP)rUGP-GPDH, where GPDH is glycerol-3-phosphate dehy-drogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the (RNAi)UGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes ((RNAi)UGP/(EXP)rUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones.IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukar-yotes are the only organisms that sequestered glycolysis inside peroxisomes (glyco-somes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

The complete genome sequence of a type III strain of Mycobacterium avium subsp. paratuberculosis was determined. The genome size for this pathogen of sheep is 4,895,755 bp with no plasmid DNA. The chromosome contains 19 copies of the hallmark IS900 element, which is routinely used to identify this subspecies.

International audience; The extensive use of antibiotics in hospitals and in the animal breeding industry has promoted antibiotic resistance in bacteria, which resulted in the emergence of a large number of antibiotic resistance genes in the intestinal tract of human and farmed animals. Genetic exchange of resistance genes between the two ecosystems is now well documented for pathogenic bacteria, but the repertoire of shared resistance genes in the commensal bacterial community and by which genetic modules they are disseminated are still unclear. By analyzing metagenomics data of human and pig intestinal samples both collected in Shenzhen, China, a set of 27 highly prevalent antibiotic resistance genes was found to be shared between human and pig intestinal microbiota. The mobile genetic context for 11 of these core antibiotic resistance genes could be identified by mining their carrying scaffolds constructed from the two datasets, leading to the detection of seven integrative and conjugative/mobilizable elements and two IS-related transposons. The comparison of the relative abundances between these detected mobile genetic elements and their associated antibiotic resistance genes revealed that for many genes, the estimated contribution of the mobile elements to the gene abundance differs strikingly depending on the host. These findings indicate that although some antibiotic resistance genes are ubiquitous across microbiota of human and pig populations, they probably relied on different genetic elements for their dissemination within each population.IMPORTANCE There is growing concern that antibiotic resistance genes could spread from the husbandry environment to human pathogens through dissemination mediated by mobile genetic elements. In this study, we investigated the contribution of mobile genetic elements to the abundance of highly prevalent antibiotic resistance genes found in commensal bacteria of both human and pig intestinal microbiota originating from the same region. Our results reveal that for most of these antibiotic resistance genes, the abundance is not explained by the same mobile genetic element in each host, suggesting that the human and pig microbial communities promoted a different set of mobile genetic carriers for the same antibiotic resistance genes. These results deepen our understanding of the dissemination of antibiotic resistance genes among and between human and pig gut microbiota.

We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south.

International audience; Flavobacterium psychrophilum affects salmonid health worldwide and causes economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored, and apparently healthy individuals were sampled over 1 year. A total of 288 isolates were recovered from fish organs (gills and spleen) and eggs. Pulsed field gel electrophoresis revealed high genetic diversity. Multilocus sequence typing performed on a selection of 31 isolates provided congruent results, as follows: 18 sequence types (STs) were found, of which 13 were novel. The mean gene diversity (H = 0.8413) was much higher than that previously reported for this host species, although the sampling was restricted to a single watershed and 1 year. Seven isolates out of 31 were assigned to clonal complex ST10 (CC-ST10), which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilum.IMPORTANCE The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolates from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to clonal complex sequence type 10 (CC-ST10), the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harbored by rainbow trout.

International audience; Viral tropism and transmission of herpesviruses are best studied in their natural host for maximal biological relevance. In the case of alphaherpesviruses, few reports have focused on those aspects, primarily because of the few animal models available as natural hosts that are compatible with such studies. Here, using Marek's disease virus (MDV), a highly contagious and deadly alphaherpesvirus of chickens, we analyze the role of tegument proteins pUL47 and pUL48 in the whole life cycle of the virus. We report that a virus lacking the UL48 gene (vΔUL48) is impaired in growth in cell culture and has diminished virulence in vivo In contrast, a virus lacking UL47 (vΔUL47) is unaffected in its growth in vitro and is as virulent in vivo as the wild-type (WT) virus. Surprisingly, we observed that vΔUL47 was unable to be horizontally transmitted to naive chickens, in contrast to the WT virus. In addition, we show that pUL47 is important for the splicing of UL44 transcripts encoding glycoprotein gC, a protein known as being essential for horizontal transmission of MDV. Importantly, we observed that the levels of gC are lower in the absence of pUL47. Notably, this phenotype is similar to that of another transmission-incompetent mutant ΔUL54, which also affects the splicing of UL44 transcripts. This is the first study describing the role of pUL47 in both viral transmission and the splicing and expression of gC.IMPORTANCE Host-to-host transmission of viruses is ideally studied in vivo in the natural host. Veterinary viruses such as Marek's disease virus (MDV) are, therefore, models of choice to explore these aspects. The natural host of MDV, the chicken, is small, inexpensive, and economically important. MDV is a deadly and contagious herpesvirus that can kill infected animals in less than 4 weeks. The virus naturally infects epithelial cells of the feather follicle epithelium from where it is shed into the environment. In this study, we demonstrate that the viral protein pUL47 is an essential factor for bird-to-bird transmission of the virus. We provide some molecular basis to this function by showing that pUL47 enhances the splicing and the expression of another viral gene, UL44, which is essential for viral transmission. pUL47 may have a similar function in human herpesviruses such as varicella-zoster virus or herpes simplex viruses.

Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease., Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.

The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances., Bacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. The results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and, more surprisingly, a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth, and the main effect involved in the return to the basal level was identified by flux calculation as growth dilution. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology. IMPORTANCE The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependence on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaechei clinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaechei strains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured. Single nucleotide polymorphisms (SNPs) were determined on the sequenced genomes. The expression of genes involved in antibiotic resistance among Enterobacter cloacae complex strains were determined by reverse transcription-quantitative PCR (qRT-PCR). All the strains belonged to sequence type 66 and were distant by a maximum of nine SNPs. After 3 months of hospitalization, three strains presented a significant increase in MICs for ceftazidime, cefepime, temocillin, ertapenem, tigecycline, ciprofloxacin, and chloramphenicol. Those resistant strains did not acquire additional antibiotic resistance genes but harbored a 16-bp deletion in the ramR gene. This deletion led to upregulated expression of RamA, AcrA, AcrB, and TolC and downregulated expression of OmpF. The ΔramR mutant harbored the same phenotype as the resistant clinical strains regarding tigecycline, chloramphenicol, and ciprofloxacin. The increased expression of RamA due to partial deletion in the ramR gene led to a cross-resistance phenotype by an increase of antibiotic efflux through the AcrAB-TolC pump and a decrease of antibiotic permeability by porin OmpF. ramR appears to be an important adaptative trait for E. hormaechei strains.

Pectobacterium punjabense is a newly described species causing blackleg disease in potato plants. Therefore, by the combination of long (Oxford Nanopore Technologies, MinION) and short (Illumina MiSeq) reads, we sequenced the complete genome of P. punjabense SS95T, which contains a circular chromosome of 4.793 Mb with a GC content of 50.7%.

Chemical-only fertilization is ubiquitous in contemporary conventional agriculture despite the fact that sustainability of this agricultural practice is increasingly being questioned because of the current observed soil degradation. We explored how long-term chemical-only versus organic-only fertilizations impacted the soil microbiota reservoir in terms of both diversity and induced assembly processes. The results showed that long-term chemical-only fertilization resulted in deep selection pressure on the soil microbial community reservoir, with both a higher proportion of specialists and a stronger signature of deterministic processes. The soil microbiota has clearly changed as a consequence of the fertilization regime. The diagnoses of the functional consequences of these ecoevolutionary changes in relation to agricultural practices are key to imagining agriculture in the time ahead and especially regarding future efforts for the conservation, restoration, and management of the soil microbiota reservoir which is key to the fertility of the ecosystem., Fertilization regimes are known to drive succession of the soil microbial community, whereas the assembly rules involved remain elusive. Moreover, the ecological roles of microbial “generalists” and “specialists” in soils with contrasting fertilization regimes have not been characterized. We explored how long-term fertilization regimes (i.e., treatments conducted for at least 30 years) impact the soil bacteria by modifying species richness, diversity, bacterial assembly, and niche breadth. Compared with long-term organic fertilizer input, the soils having undergone chemical-only fertilization contained smaller amounts of carbon resources and had a more acidic environment. This strong environmental constraint lowered the soil bacterial reservoir and resulted in a detectable ecoevolutionary transformation, with both a higher proportion of specialists and a stronger signature of deterministic processes. Overall, this study provided a new comprehensive understanding of the assembly rules of bacterial generalists and specialists under long-term fertilization regimes. This study also highlighted that chemical-only fertilization, a ubiquitous agricultural practice of current conventional agriculture, induced a strong and similar environmental force that transformed the soil microbiota from 28°N to 46°N included in this study. IMPORTANCE Chemical-only fertilization is ubiquitous in contemporary conventional agriculture despite the fact that sustainability of this agricultural practice is increasingly being questioned because of the current observed soil degradation. We explored how long-term chemical-only versus organic-only fertilizations impacted the soil microbiota reservoir in terms of both diversity and induced assembly processes. The results showed that long-term chemical-only fertilization resulted in deep selection pressure on the soil microbial community reservoir, with both a higher proportion of specialists and a stronger signature of deterministic processes. The soil microbiota has clearly changed as a consequence of the fertilization regime. The diagnoses of the functional consequences of these ecoevolutionary changes in relation to agricultural practices are key to imagining agriculture in the time ahead and especially regarding future efforts for the conservation, restoration, and management of the soil microbiota reservoir which is key to the fertility of the ecosystem.

The human gut harbors a complex bacterial community that plays important roles in host health and disease, including nutrient acquisition, maturation of the immune system, and resistance to infections. The capacity to adhere to surfaces and form communities called biofilms is believed to be important for niche colonization and maintenance of gut bacteria. However, little is known about the adhesion capacity of most gut bacteria. In this study, we investigated biofilm formation in Bacteroides thetaiotaomicron, one of the most abundant bacteria of the normal mammalian intestine. We identified that B. thetaiotaomicron capsules, a group of eight surface-exposed polysaccharidic layers mediating important interactions with the gut environment, are also major determinants of biofilm formation that mask or unmask adhesion factors. Studying how B. thetaiotaomicron regulates its adhesion properties will allow us to better understand the physiology and specific properties of this important gut symbiont within anaerobic biofilms., Bacteroides thetaiotaomicron is one of the most abundant gut symbiont species, whose contribution to host health through its ability to degrade dietary polysaccharides and mature the immune system is under intense scrutiny. In contrast, adhesion and biofilm formation, which are potentially involved in gut colonization and microbiota structure and stability, have hardly been investigated in this intestinal bacterium. To uncover B. thetaiotaomicron biofilm-related functions, we performed a transposon mutagenesis in the poorly biofilm-forming reference strain VPI-5482 and showed that capsule 4, one of the eight B. thetaiotaomicron capsules, hinders biofilm formation. We then showed that the production of capsules 1, 2, 3, 5, and 6 also inhibits biofilm formation and that decreased capsulation of the population correlated with increased biofilm formation, suggesting that capsules could be masking adhesive surface structures. In contrast, we showed that capsule 8 displayed intrinsic adhesive properties. Finally, we demonstrated that BT2934, the wzx homolog of the B. thetaiotaomicron glycosylation locus, competes with capsule production and impacts its adhesion capacity. This study therefore establishes B. thetaiotaomicron capsule regulation as a major determinant of B. thetaiotaomicron biofilm formation, providing new insights into how modulation of different B. thetaiotaomicron surface structures affects in vitro biofilm formation.

sCJD occurrence is currently assumed to result from spontaneous and stochastic formation of a misfolded PrP nucleus in the brains of affected patients. This original nucleus then recruits and converts nascent PrPC into PrPSc, leading to the propagation of prions in the patient’s brain. Our study demonstrates the coexistence of two prion strains in the brains of a majority of the 23 sCJD patients investigated. The relative proportion of these sCJD strains varied both between patients and between brain areas in a single patient. These findings strongly support the view that the replication of an sCJD prion strain in the brain of a patient can result in the propagation of different prion strain subpopulations. Beyond its conceptual importance for our understanding of prion strain properties and evolution, the sCJD strain mixture phenomenon and its frequency among patients have important implications for the development of therapeutic strategies for prion diseases., Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently classified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K-digested abnormal prion protein (PrPres) isoform identified by Western blotting (type 1 or type 2). Converging evidence led to the view that MM/MV1, VV/MV2, and VV1 and MM2 sCJD cases are caused by distinct prion strains. However, in a significant proportion of sCJD patients, both type 1 and type 2 PrPres were reported to accumulate in the brain, which raised questions about the diversity of sCJD prion strains and the coexistence of two prion strains in the same patient. In this study, a panel of sCJD brain isolates (n = 29) that displayed either a single or mixed type 1/type 2 PrPres were transmitted into human-PrP-expressing mice (tgHu). These bioassays demonstrated that two distinct prion strains (M1CJD and V2CJD) were associated with the development of sCJD in MM1/MV1 and VV2/MV2 patients. However, in about 35% of the investigated VV and MV cases, transmission results were consistent with the presence of both M1CJD and V2CJD strains, including in patients who displayed a “pure” type 1 or type 2 PrPres. The use of a highly sensitive prion in vitro amplification technique that specifically probes the V2CJD strain revealed the presence of the V2CJD prion in more than 80% of the investigated isolates, including isolates that propagated as a pure M1CJD strain in tgHu. These results demonstrate that at least two sCJD prion strains can be present in a single patient.

International audience; Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE: Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

BGPI : Equipe 2 : Interactions Virus Insecte Plante (VIP); International audience; Vector transmission plays a primary role in the life cycle of viruses, and insects are the most common vectors. An important mode of vector transmission, reported only for plant viruses, is circulative nonpropagative transmission whereby the virus cycles within the body of its insect vector, from gut to salivary glands and saliva, without replicating. This mode of transmission has been extensively studied in the viral families Luteoviridae and Geminiviridae and is also reported for Nanoviridae. The biology of viruses within these three families is different, and whether the viruses have evolved similar molecular/cellular virus-vector interactions is unclear. In particular, nanoviruses have a multipartite genome organization, and how the distinct genome segments encapsidated individually transit through the insect body is unknown. Here, using a combination of fluorescent in situ hybridization and immunofluorescence, we monitor distinct proteins and genome segments of the nanovirus Faba bean necrotic stunt virus (FBNSV) during transcytosis through the gut and salivary gland cells of its aphid vector Acyrthosiphon pisum. FBNSV specifically transits through cells of the anterior midgut and principal salivary gland cells, a route similar to that of geminiviruses but distinct from that of luteoviruses. Our results further demonstrate that a large number of virus particles enter every single susceptible cell so that distinct genome segments always remain together. Finally, we confirm that the success of nanovirus-vector interaction depends on a nonstructural helper component, the viral protein nuclear shuttle protein (NSP), which is shown to be mandatory for viral accumulation within gut cells.IMPORTANCE An intriguing mode of vector transmission described only for plant viruses is circulative nonpropagative transmission, whereby the virus passes through the gut and salivary glands of the insect vector without replicating. Three plant virus families are transmitted this way, but details of the molecular/cellular mechanisms of the virus-vector interaction are missing. This is striking for nanoviruses that are believed to interact with aphid vectors in ways similar to those of luteoviruses or geminiviruses but for which empirical evidence is scarce. We here confirm that nanoviruses follow a within-vector route similar to that of geminiviruses but distinct from that of luteoviruses. We show that they produce a nonstructural protein mandatory for viral entry into gut cells, a unique phenomenon for this mode of transmission. Finally, noting that nanoviruses are multipartite viruses, we demonstrate that a large number of viral particles penetrate susceptible cells of the vector, allowing distinct genome segments to remain together.

International audience; Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-Pseudomonas aeruginosa, where IMPs are most frequently detected.

Proliferation of microcystin (MC)-producing Microcystis aeruginosa in brackish waters has been described in several locations and represents a new concern for public and environmental health. While the impact of a sudden salinity increase on M. aeruginosa physiology has been studied, less is known about the mechanisms involved in salt tolerance after acclimation. This study aims to compare the physiological responses of two strains of M. aeruginosa (PCC 7820 and PCC 7806), which were isolated from contrasted environments, to increasing salinities. After acclimation, growth and MC production rates were determined and metabolomic analyses were conducted. For both strains, salinity decreased the biovolume, growth, and MC production rates and induced the accumulation of polyunsaturated lipids identified as monogalactosyldiacylglycerol. The distinct salt tolerances (7.5 and 16.9) obtained between the freshwater (PCC 7820) and the brackish-water (PCC 7806) strains suggested different strategies to cope with the osmotic pressure, as revealed by targeted and untargeted metabolomic analyses. An accumulation of trehalose as the main compatible solute was obtained in the freshwater strain, while sucrose was mainly accumulated in the brackish one. Moreover, distinct levels of glycine betaine and proline accumulation were noted. Altogether, metabolomic analysis illustrated a strain-specific response to salt tolerance, involving compatible solute production. IMPORTANCE Blooms of Microcystis aeruginosa and the production of microcystins are major issues in eutrophic freshwater bodies. Recently, an increasing number of proliferations of M. aeruginosa in brackish water has been documented. The occurrence of both M. aeruginosa and microcystins in coastal areas represents a new threat for human and environmental health. In order to better describe the mechanisms involved in Microcystis sp. proliferation in brackish water, this study used two M. aeruginosa strains isolated from fresh and brackish waters. High salinity reduced the growth rate and microcystin production rate of M. aeruginosa. In order to cope with higher salinities, the strains accumulated different cyanobacterial compatible solutes, as well as unsaturated lipids, explaining their distinct salt tolerance.

Pesticides are key stressors of soil microorganisms with reciprocal effects on ecosystem functioning. These effects have been mainly attributed to the parent compounds, while the impact of their transformation products (TPs) has been largely overlooked. We assessed in a meadow soil (soil A) the transformation of iprodione and its toxicity in relation to (i) the abundance of functional microbial groups, (ii) the activity of key microbial enzymes, and (iii) the diversity of bacteria, fungi, and ammonia-oxidizing microorganisms (AOM) using amplicon sequencing. 3,5-Dichloroaniline (3,5-DCA), the main iprodione TP, was identified as a key explanatory factor for the persistent reduction in enzymatic activities and potential nitrification (PN) and for the observed structural changes in the bacterial and fungal communities. The abundances of certain bacterial (Actinobacteria, Hyphomicrobiaceae, Ilumatobacter, and Solirubrobacter) and fungal (Pichiaceae) groups were negatively correlated with 3,5-DCA. A subsequent study in a fallow agricultural soil (soil B) showed limited formation of 3,5-DCA, which concurred with the lack of effects on nitrification. Direct 3,5-DCA application in soil B induced a dose-dependent reduction of PN and NO3−-N, which recovered with time. In vitro assays with terrestrial AOM verified the greater toxicity of 3,5-DCA over iprodione. “Candidatus Nitrosotalea sinensis” Nd2 was the most sensitive AOM to both compounds. Our findings build on previous evidence on the sensitivity of AOM to pesticides, reinforcing their potential utilization as indicators of the soil microbial toxicity of pesticides in pesticide environmental risk analysis and stressing the need to consider the contribution of TPs in the toxicity of pesticides on the soil microbial community. IMPORTANCE Pesticide toxicity on soil microorganisms is an emerging issue in pesticide risk assessment, dictated by the pivotal role of soil microorganisms in ecosystem services. However, the focus has traditionally been on parent compounds, while transformation products (TPs) are largely overlooked. We tested the hypothesis that TPs can be major contributors to the soil microbial toxicity of pesticides using iprodione and its main TP, 3,5-dichloroaniline, as model compounds. We demonstrated, by measuring functional and structural endpoints, that 3,5-dichloroaniline and not iprodione was associated with adverse effects on soil microorganisms, with nitrification being mostly affected. Pioneering in vitro assays with relevant ammonia-oxidizing bacteria and archaea verified the greater toxicity of 3,5-dichloroaniline. Our findings are expected to advance environmental risk assessment, highlighting the potential of ammonia-oxidizing microorganisms as indicators of the soil microbial toxicity of pesticides and stressing the need to consider the contribution of TPs to pesticide soil microbial toxicity.

International audience; No gold standard exists for histopathological diagnosis of a prosthetic joint infection (PJI). The historical criterion considers the presence of neutrophil infiltration upon examination of periprosthetic tissue. Morawietz et al. proposed a classification of periprosthetic membranes (Morawietz et al., Clin Pathol 59:591-597, 2006, https://doi.org/10.1136/jcp.2005.027458) and a more recently described classification with a new cutoff value of 23 neutrophils in 10 high-power fields (Morawietz et al., Histopathology 54:847-853, 2009. https://doi.org/10.1111/j.1365-2559.2009.03313.x). We performed a multicenter prospective study, which compared both methods for the diagnosis of PJI. All suspicions of PJI ( = 264) between December 2010 and March 2012 in seven centers were prospectively included. Five perioperative specimens were collected per patient for cultures, and one was collected for histology. Diagnosis of PJI was made according to the Infectious Diseases Society of America (IDSA) guidelines. Histopathological analysis classified the patients according to the threshold of 23 neutrophils and according to the classification of Morawietz. Performances of both methods were compared by using clinical and/or bacteriological criteria as the gold standard. Among 264 patients with suspected PJI, a diagnosis of infection was confirmed in 215 and unconfirmed in 49 patients. Histopathological analysis was available for 150 confirmed PJI and 40 unconfirmed PJI cases. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.7%, 90.0%, 96.7%, 52.9%, and 81.1%, respectively, for the Morawietz classification, and 82.0%, 90.0%, 96.9%, 57.1%, and 83.7%, respectively, for the 23-neutrophil threshold. The new algorithm using a threshold of 23 neutrophils can be proposed as a new gold standard for the histopathological diagnosis of PJI.

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions., IMPORTANCE Clostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

Bacterial communities associated with plant roots play an important role in the suppression of soil-borne pathogens, and multispecies probiotic consortia may enhance disease suppression efficacy. Here we introduced defined Pseudomonas species consortia into naturally complex microbial communities and measured the importance of Pseudomonas community diversity for their survival and the suppression of the bacterial plant pathogen Ralstonia solanacearum in the tomato rhizosphere microbiome. The survival of introduced Pseudomonas consortia increased with increasing diversity. Further, high Pseudomonas diversity reduced pathogen density in the rhizosphere and decreased the disease incidence due to both intensified resource competition and interference with the pathogen. These results provide novel mechanistic insights into elevated pathogen suppression by diverse probiotic consortia in naturally diverse plant rhizospheres. Ecologically based community assembly rules could thus play a key role in engineering functionally reliable microbiome applications., IMPORTANCE The increasing demand for food supply requires more-efficient control of plant diseases. The use of probiotics, i.e., naturally occurring bacterial antagonists and competitors that suppress pathogens, has recently reemerged as a promising alternative to agrochemical use. It is, however, still unclear how many and which strains we should choose for constructing effective probiotic consortia. Here we present a general ecological framework for assembling effective probiotic communities based on in vitro characterization of community functioning. Specifically, we show that increasing the diversity of probiotic consortia enhances community survival in the naturally diverse rhizosphere microbiome, leading to increased pathogen suppression via intensified resource competition and interference with the pathogen. We propose that these ecological guidelines can be put to the test in microbiome engineering more widely in the future.

Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses., The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs. IMPORTANCE Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

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

International audience; Beetles thriving on tyrosine-poor diet sources often develop mutualistic associations with endosymbionts able to synthesize aromatic amino acids. This surplus of aromatic amino acids is used to reinforce the insect's protective cuticle.Nutritional symbioses between insects and intracellular bacteria (endosymbionts) are a major force of adaptation, allowing animals to colonize nutrient-poor ecological niches. Many beetles feeding on tyrosine-poor substrates rely on a surplus of aromatic amino acids produced by bacterial endosymbionts. This surplus of aromatic amino acids is crucial for the biosynthesis of a thick exoskeleton, the cuticle, which is made of a matrix of chitin with proteins and pigments built from tyrosine-derived molecules, providing an important defensive barrier against biotic and abiotic stress. Other endosymbiont-related advantages for beetles include faster development and improved fecundity. The association between Sitophilus oryzae and the Sodalis pierantonius endosymbiont represents a unique case study among beetles: endosymbionts undergo an exponential proliferation in young adults concomitant with the cuticle tanning, and then they are fully eliminated. While endosymbiont clearance, as well as total endosymbiont titer, are host-controlled processes, the mechanism triggering endosymbiont exponential proliferation remains poorly understood. Here, we show that endosymbiont exponential proliferation relies on host carbohydrate intake, unlike the total endosymbiont titer or the endosymbiont clearance, which are under host genetic control. Remarkably, insect fecundity was preserved, and the cuticle tanning was achieved, even when endosymbiont exponential proliferation was experimentally blocked, except in the context of a severely unbalanced diet. Moreover, a high endosymbiont titer coupled with nutrient shortage dramatically impacted host survival, revealing possible environment-dependent disadvantages for the host, likely due to the high energy cost of exponentially proliferating endosymbionts.IMPORTANCE Beetles thriving on tyrosine-poor diet sources often develop mutualistic associations with endosymbionts able to synthesize aromatic amino acids. This surplus of aromatic amino acids is used to reinforce the insect's protective cuticle. An exceptional feature of the Sitophilus oryzae/Sodalis pierantonius interaction is the exponential increase in endosymbiotic titer observed in young adult insects, in concomitance with cuticle biosynthesis. Here, we show that host carbohydrate intake triggers endosymbiont exponential proliferation, even in conditions that lead to the detriment of the host survival. In addition, when hosts thrive on a balanced diet, endosymbiont proliferation is dispensable for several host fitness traits. The endosymbiont exponential proliferation is therefore dependent on the nutritional status of the host, and its consequences on host cuticle biosynthesis and survival depend on food quality and availability.

International audience; The human T-cell leukemia virus (HTLV)-1 is responsible for an aggressive neurodegenerative disease (HAM/TSP) and multiple neurological alterations. The capacity of HTLV-1 to infect central nervous system (CNS) resident cells, together with the neuroimmune-driven response, has not been well-established. Here, we combined the use of human induced pluripotent stem cells (hiPSC) and of naturally STLV-1-infected nonhuman primates (NHP) as models with which to investigate HTLV-1 neurotropism. Hence, neuronal cells obtained after hiPSC differentiation in neural polycultures were the main cell population infected by HTLV-1. Further, we report the infection of neurons with STLV-1 in spinal cord regions as well as in brain cortical and cerebellar sections of postmortem NHP. Additionally, reactive microglial cells were found in infected areas, suggesting an immune antiviral response. These results emphasize the need to develop new efficient models by which to understand HTLV-1 neuroinfection and suggest an alternative mechanism that leads to HAM/TSP.

The response of bacteria in bulk soil and earthworm casts to carbon enrichment was studied by an RNA stable-isotope probing/terminal restriction fragment length polymorphism strategy with 13 C-labeled glucose and acetate. Both the soil microsite status and the carbon enrichment selected rapidly for different active bacterial communities, which resulted in different degradation kinetics. Our study clearly illustrates the biases that are generated by adding C substrates to detect metabolically active bacteria in soil.

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection., IMPORTANCE SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4+ T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.

International audience; Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. IMPORTANCE Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by S. Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

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.

International audience; Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria, such as Gardnerella vaginalis, and low levels of "healthy" lactobacilli leads to bacterial vaginosis (BV), usually associated with a low-grade inflammatory process. Despite appropriate antibiotic treatment, G. vaginalis-associated BV is characterized by significant recurrence. The use of probiotics could be an interesting alternative therapy due to their ability to rebalance vaginal microbiota. In this study, we investigated the effects of a wellcharacterized probiotic strain, Lacticaseibadllus rhamnosus Lcr35, on epithelial vaginal and dendritic cell (DC) immune responses after G. vaginalis infection. In an in vitro coculture model with human monocyte-derived dendritic cells and a vaginal epithelial cell NEC) monolayer, the Lcr35 strain induced DC activation, as evidenced by the induction of maturation and synthesis of interleukin-8 (IL-8) and CCL-20 chemokines upon apical challenge of the VECs by G. vaginas. Analysis of the vaginal epithelial response showed that the presence of Lcr35 significantly increased the production of the proinflammatory cytokines IL-8 and IL-1 beta and human beta-defensin 2 (HBD-2), whereas the concentration of secretory leukocyte protease inhibitor (SLPI) was decreased in G. vagina/is-infected vaginal epithelial cells. Treatment with recombinant SLPI was associated with upregulation of Lcr35-stimulated IL-8 and HBD-2 production. These results suggest that inhibition of SLPI by Lcr35 in vaginal epithelial cells contributes to the host defense response against G. vaginalis infection.

Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes.

International audience; Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multi locus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors.IMPORTANCE Nonharmful bacteria are the primary microbial symbionts that inhabit the human gastrointestinal tract. These bacteria play many beneficial roles and in some cases can modify disease states, making it important to understand which nutrients sustain specific lineages. This knowledge will in turn lead to strategies to intentionally manipulate the gut microbial ecosystem. We designed a scalable, high throughput platform for measuring the ability of gut bacteria to utilize polysaccharides, of which many are derived from dietary fiber sources that can be manipulated easily. Our results provide paths to expand phenotypic surveys of more diverse gut bacteria to understand their functions and also to leverage dietary fibers to alter the physiology of the gut microbial community.

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (N(Nuc)), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to N(Nuc) and L, allowing the attachment of the L polymerase to the N(Nuc) template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and N(Nuc), which has not been demonstrated yet. Here, we finely characterized the binding P-N(Nuc) interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to N(Nuc) and that P binds to the N-terminal domain of N (N(NTD)), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-N(Nuc) interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

International audience; Here, we report the complete genome sequence of the race 4 strain Xanthomonas campestris pv. campestris SB80, which was isolated from a symptomatic white head cabbage leaf in Samsun Province, Turkey, in 2019. The genome consists of a circular chromosome (5,129,762 bp) with a G+C content of 64.98%, for which 4,159 putative protein-coding genes, 2 rRNA operons, 54 tRNAs, and 86 noncoding RNAs (ncRNAs) were predicted.

International audience

We report here the complete genome sequences of three Bacillus cereus group strains isolated from blood cultures from premature and immunocompromised infants hospitalized in intensive care units in three French hospitals. These complete genome sequences were obtained from a combination of Illumina HiSeq X Ten short reads and Oxford Nanopore MinION long reads.

International audience; Necrotizing enterocolitis (NEC) is among the most relevant gastrointestinal diseases affecting mostly prematurely born infants with low birth weight. While intestinal dysbiosis has been proposed as one of the possible factors involved in NEC pathogenesis, the role of the gut microbiota remains poorly understood. In this study, the gut microbiota of preterm infants was explored to highlight differences in the composition between infants affected by NEC and infants prior to NEC development. A large-scale gut microbiome analysis was performed, including 47 shotgun sequencing data sets generated in the framework of this study, along with 124 retrieved from publicly available repositories. Meta-analysis led to the identification of preterm community state types (PT- CSTs), which recur in healthy controls and NEC infants. Such analyses revealed an overgrowth of a range of opportunistic microbial species accompanying the loss of gut microbial biodiversity in NEC subjects. Moreover, longitudinal insights into preterm infants prior to NEC development indicated Clostridium neonatale and Clostridium perfringens species as potential biomarkers for predictive early diagnosis of this disease. Furthermore, functional investigation of the enzymatic reaction profiles associated with pre-NEC condition suggested DL-lactate as a putative metabolic biomarker for early detection of NEC onset.IMPORTANCE Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease occurring predominantly in premature infants whose etiology is still not fully understood. In this study, the analysis of infant fecal samples through shotgun metagenomics approaches revealed a marked reduction of the intestinal (bio)diversity and an overgrowth of (opportunistic) pathogens associated with the NEC development. In particular, dissection of the infant's gut microbiome before NEC diagnosis high-lighted the potential involvement of Clostridium genus members in the progression of NEC. Remarkably, our analyses highlighted a gastrointestinal DL-lactate accumulation among NEC patients that might represent a novel potential functional biomarker for the early diagnosis of NEC.

International audience; The emergence of multidrug-resistant Escherichia coli ST131 is a major worldwide public health problem in humans. According to the "one health" approach, this study investigated animal reservoirs of ST131, their relationships with human strains, and the genetic features associated with host colonization. High-quality genomes originating from human, avian, and canine hosts were classified on the basis of their accessory gene content using pangenomic. Pangenomic clusters and subclusters were specifi- cally and significantly associated with hosts. The functions of clustering accessory genes were mainly enriched in functions involved in DNA acquisition, interactions, and virulence (e.g., pathogenesis, response to biotic stimulus and interaction between organisms). Accordingly, networks of cooccurrent host interaction factors were significantly associated with the pangenomic clusters and the originating hosts. The avian strains exhibited a specific content in virulence factors. Rarely found in humans, they corresponded to pathovars responsible for severe human infections. An emerging subcluster significantly associated with both human and canine hosts was evidenced. This ability to significantly colonize canine hosts in addition to humans was associated with a specific content in virulence factors (VFs) and metabolic functions encoded by a new pathogenicity island in ST131 and an improved fitness that is probably involved in its emergence. Overall, VF content, unlike the determinants of antimicrobial resistance, appeared as a key actor of bacterial host adaptation. The host dimension emerges as a major driver of genetic evolution that shapes ST131 genome, enhances its diversity, and favors its dissemination.IMPORTANCE Until now, there has been no indication that the evolutionary dynamics of Escherichia coli ST131 may reflect independent and host-specific adaptation of this lineage outside humans. In contrast, the limited number of ST131 reports in animals supported the common view that it rather reflects a spillover of the human sector. This study uncovered a link between host, ST131 population structure, and virulence factor content which appeared to reflect adaptation to hosts. This study helps to better understand the reservoir of ST131, the putative transmission flux, associated risks and the evolutionary dynamics of this bacterial population and highlights a paradigm in which host colonization stands as a key ecological force of the ST131 evolution.

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5′ end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m(7)GTP is complexed to the enzyme to form an m(7)GMP-nsP1 covalent intermediate. The m(7)GMP is then transferred onto the 5′-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m(7)GMP to yield m(7)GpppA, m(7)GpppC, or m(7)GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.

Aerobic bacteria are frequent primocolonizers of the human naive intestine. Their generally accepted role is to eliminate oxygen, which would allow colonization by anaerobes that subsequently dominate bacterial gut populations., Aerobic bacteria are frequent primocolonizers of the human naive intestine. Their generally accepted role is to eliminate oxygen, which would allow colonization by anaerobes that subsequently dominate bacterial gut populations. In this hypothesis-based study, we revisited this dogma experimentally in a germfree mouse model as a mimic of the germfree newborn. We varied conditions leading to the establishment of the dominant intestinal anaerobe Bacteroides thetaiotaomicron. Two variables were introduced: Bacteroides inoculum size and preestablishment by bacteria capable or not of consuming oxygen. High Bacteroides inoculum size enabled its primocolonization. At low inocula, we show that bacterial preestablishment was decisive for subsequent Bacteroides colonization. However, even non-oxygen-respiring bacteria, a hemA Escherichia coli mutant and the intestinal obligate anaerobe Clostridium scindens, facilitated Bacteroides establishment. These findings, which are supported by recent reports, revise the long-held assumption that oxygen scavenging is the main role for aerobic primocolonizing bacteria. Instead, we suggest that better survival of aerobic bacteria ex vivo during vectorization between hosts could be a reason for their frequent primocolonization.