Search

For decades, Xanthomonas vasicola pv. musacearum (Xvm) has been an economically important bacterial pathogen on enset in Ethiopia. Since 2001, Xvm has also been responsible for significant losses to banana crops in several East and Central African countries, with devastating consequences for smallholder farmers. Understanding the genetic diversity within Xvm populations is essential for the smart design of transnationally reasoned, durable, and effective management practices. Previous studies have revealed limited genetic diversity in Xvm, with East African isolates from banana each falling into one of two closely related clades previously designated as sublineages SL 1 and SL 2, the former of which had also been detected on banana and enset in Ethiopia. Given the presumed origin of Xvm in Ethiopia, we hypothesized that both clades might be found in that country, along with additional genotypes not seen in Central and East African bananas. Genotyping of 97 isolates and whole‐genome sequencing of 15 isolates revealed not only the presence of SL 2 in Ethiopia, but additional diversity beyond SL 1 and SL 2 in four new clades. Moreover, SL 2 was detected in the Democratic Republic of Congo, where previously SL 1 was the only clade reported. These results demonstrate a greater range of genetic diversity among Xvm isolates than previously reported, especially in Ethiopia, and further support the hypothesis that the East/Central Africa xanthomonas wilt epidemic has been caused by a restricted set of genotypes drawn from a highly diverse pathogen pool in Ethiopia.\ud \ud

International audience; The bacterium Xylella fastidiosa (Xf) is a plant endophyte native to the Americas that causes diseases in many crops of economic importance (grapevine, Citrus, Olive trees etc). Xf has been recently detected in several regions outside of its native range including Europe where little is known about its potential geographical expansion. We collected data documenting the native and invaded ranges of the Xf subspecies fastidiosa, pauca and multiplex and fitted bioclimatic species distribution models (SDMs) to assess the potential climate suitability of European continent for those pathogens. According to model predictions, the currently reported distribution of Xf in Europe is small compared to the large extent of climatically suitable areas. The regions at high risk encompass the Mediterranean coastal areas of Spain, Greece, Italy and France, the Atlantic coastal areas of France, Portugal and Spain as well as the southwestern regions of Spain and lowlands in southern Italy. The extent of predicted climatically suitable conditions for the different subspecies are contrasted. The subspecies multiplex, and to a certain extent the subspecies fastidiosa, represent a threat to most of Europe while the climatically suitable areas for the subspecies pauca are mostly limited to the Mediterranean basin. These results provide crucial information for the design of a spatially informed European-scale integrated management strategy, including early detection surveys in plants and insect vectors and quarantine measures.

UMR BFP - Equipe Mollicutes; International audience; We report a disseminated infection caused by Spiroplasma apis, a honeybee pathogen, in a patient in France who had X-linked agammaglobulinemia. Identification was challenging because initial bacterial cultures and direct examination by Gram staining were negative. Unexplained sepsis in patients with agammaglobulinemia warrants specific investigation to identify fastidious bacteria such as Spiroplasma spp

Ehrlichia ruminantium est une alpha-protéobactérie à Gram-négatif de la famille des Anaplasmataceae, responsable d'une maladie tropicale mortelle chez les ruminants domestiques et sauvages, la cowdriose. E. ruminantium est une bactérie intracellulaire obligatoire, transmise par les tiques du genre Amblyomma. En raison de son fort impact sur l'élevage en zone tropicale, il est crucial de proposer des méthodes de lutte efficaces contre cette maladie. La question centrale de cette thèse était de mieux comprendre et appréhender la pathogenèse d'Ehrlichia ruminantium, notamment en identifiant et caractérisant les déterminants moléculaires qui permettent à la bactérie de contrôler la cellule hôte et de s'y répliquer. Un premier volet de cette étude nous a permis d'identifier Erip1 comme un effecteur de type IV essentiel pour l'infection par E. ruminantium en inactivant l'expression du gène par un peptide nucleic acid (PNA) ciblant erip1. Nous avons ainsi montré qu'une répression ciblée d'un gène est maintenant possible chez E. ruminantium. Par des approches d'immunoprécipitation de la chromatine (ChIP-seq) nous avons montré qu'Erip1 ciblerait plusieurs régions du génome bovin. Cette protéine se fixe notamment sur six promoteurs dont celui de MRP14 et de deux ARN non codants (ARNsno et ARNlnc). Le second volet de cette thèse s'est attaché à étudier cinq effecteurs de type IV prédits et conservés chez les Anaplasmataceae. L'étude de la sécrétion type IV-dépendante par un système rapporteur hétérologue chez Legionella pneumophila n'a pas permis de confirmer que ces protéines sont sécrétées par le système de sécrétion de type IV. Cette partie du projet devra donc être poursuivie dans un système plus proche de notre modèle d'étude. Le dernier volet de ces travaux de recherche a montré que l'autophagie est un mécanisme physiologique essentiel pour le développement de la bactérie dans sa cellule hôte. En effet, l'utilisation de la Spautin- 1, inhibiteur de la Beclin-1, dans une culture de cellules endothéliales bovines réprime E. ruminantium. Pour terminer, une analyse du transcriptome par une approche RNA-seq, nous a permis d'identifier que la voie de biosynthèse du cholestérol endogène est induite au cours de l'infection. Nous avons aussi montré que la Spautin-1 induit une répression de 448 protéines à 48h post-infection. Parmi ces gènes réprimés, nous retrouvons ceux de la famille des map1, des virB/virD4 mais également le super régulateur erxR. L'ensemble des résultats de cette thèse permettent de mieux comprendre la prise de contrôle de la cellule hôte par Ehrlichia ruminantium et constitue un pas de plus dans la lutte contre ce pathogène.

International audience; Intragenesis is an important alternative to transgenesis to produce modified plants 27 containing native DNA only. A key point to develop such a strategy is the availability of 28 regulatory sequences controlling the expression of the gene of interest. With the aim of 29 finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora 30 (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes 31 encode oxidative enzymes involved in many physiological processes and have been 32 previously shown to be up-regulated during the Ea infection process. We found ten PPO and 33 two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 34 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-35 constitutive regulatory sequences respectively. Expression levels of reporter genes fused to 36 these promoters and transiently or stably expressed in apple were quantified after various 37 treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and 38 strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System 39 dependent manner. Altogether our results indicate that pKFDV02 did not keep its promises as 40 a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned 41 from apple, can be a useful component of intragenic strategies to create fire blight resistant 42 apple genotypes.

MIB and MIP bind to antibodies and disrupt the antigen binding site to promote the dissociation of antibody-antigen interactions., Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

International audience; Bacillus cereus is a ubiquitous endospore‐forming bacterium, which mainly affects humans as a food‐borne pathogen. B. cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR‐F48, was isolated and characterized. VAR‐F48 can survive in sterilized groundwater over a long period in a vegetative form, and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR‐F48 compared to its parental strain. The results revealed a significant rerouting of the metabolism in the absence of Spo0A. We concluded that VAR‐F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A‐mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.

TheRalstonia solanacearumspecies complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identifiedR.pseudosolanacearum(phylotypes I and III) andR.solanacearum(phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

UMR BFP - Equipe Mollicutes; International audience; CRISPR/Cas systems provide adaptive defense mechanisms against invading nucleic acids in prokaryotes. Because of its interest as a genetic tool, the Type II CRISPR/Cas9 system from Streptococcus pyogenes has been extensively studied. It includes the Cas9 endonuclease that is dependent on a dual-guide RNA made of a tracrRNA and a crRNA. Target recognition relies on crRNA annealing and the presence of a protospacer adjacent motif (PAM). Mollicutes are currently the bacteria with the smallest genome in which CRISPR/Cas systems have been reported. Many of them are pathogenic to humans and animals (mycoplasmas and ureaplasmas) or plants (phytoplasmas and some spiroplasmas). A global survey was conducted to identify and compare CRISPR/Cas systems found in the genome of these minimal bacteria. Complete or degraded systems classified as Type II-A and less frequently as Type II-C were found in the genome of 21 out of 52 representative mollicutes species. Phylogenetic reconstructions predicted a common origin of all CRISPR/Cas systems of mycoplasmas and at least two origins were suggested for spiroplasmas systems. Cas9 in mollicutes were structurally related to the S. aureus Cas9 except the PI domain involved in the interaction with the PAM, suggesting various PAM might be recognized by Cas9 of different mollicutes. Structure of the predicted crRNA/tracrRNA hybrids was conserved and showed typical stem-loop structures pairing the Direct Repeat part of crRNAs with the 5′ region of tracrRNAs. Most mollicutes crRNA/tracrRNAs showed G + C% significantly higher than the genome, suggesting a selective pressure for maintaining stability of these secondary structures. Examples of CRISPR spacers matching with mollicutes phages were found, including the textbook case of Mycoplasma cynos strain C142 having no prophage sequence but a CRISPR/Cas system with spacers targeting prophage sequences that were found in the genome of another M. cynos strain that is devoid of a CRISPR system. Despite their small genome size, mollicutes have maintained protective means against invading DNAs, including restriction/modification and CRISPR/Cas systems. The apparent lack of CRISPR/Cas systems in several groups of species including main pathogens of humans, ruminants, and plants suggests different evolutionary routes or a lower risk of phage infection in specific ecological niches.

Mycoplasma hominis is an opportunistic human pathogen associated with genital and neonatal infections. Until this study, the lack of a reliable transformation method for the genetic manipulation of M. hominis hindered the investigation of the pathogenicity and the peculiar arginine-based metabolism of this bacterium. A genomic analysis of 20 different M. hominis strains revealed a number of putative restriction-modification systems in this species. Despite the presence of these systems, a reproducible polyethylene glycol (PEG)-mediated transformation protocol was successfully developed in this study for three different strains: two clinical isolates and the M132 reference strain. Transformants were generated by transposon mutagenesis with an efficiency of approximately 10−9 transformants/cell/µg plasmid and were shown to carry single or multiple mini-transposons randomly inserted within their genomes. One M132-mutant was observed to carry a single-copy transposon inserted within the gene encoding P75, a protein potentially involved in adhesion. However, no difference in adhesion was observed in cell-assays between this mutant and the M132 parent strain. Whole genome sequencing of mutants carrying multiple copies of the transposon further revealed the occurrence of genomic rearrangements. Overall, this is the first time that genetically modified strains of M. hominis have been obtained by random mutagenesis using a mini-transposon conferring resistance to tetracycline.

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

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects and plants. Here, we demonstrate that highly virulentMycoplasma mycoidessubspeciescapri(Mmc) can be fully attenuatedviatargeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately ten percent of the originalMmcgenome, were successively deleted usingSaccharomyces cerevisiaeas an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually nonessential in the JCVI-Syn3.0 minimal cell, were excised from the genome.In vitrocharacterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though ten percent of the genome content were removed. A novelin vivochallenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reaching endpoint criteria within seven days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of aMycoplasmastrain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequentin vitroorin vivotrials employing theMycoplasmachassis will allow us to dissect the role of individual candidateMycoplasmavirulence factors and lead the way for the development of an attenuated designer vaccine.IMPORTANCEMembers of theMycoplasma mycoidescluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools toMycoplasma mycoidesin order to design and create a fully attenuatedMycoplasmastrain that was subsequently confirmedin vivousing a novel caprine infection model. This is the first time that aMycoplasmamutant developed by applying synthetic biology tools has been testedin vivoto pin point candidate virulence traits. The mutant strain is similar to “apathogenicE. coliK12” strains that boosted the research on host-pathogen interactions for the genusEscherichiaand other bacterial genera.

The development of synthetic genomics (SG) allowed the emergence of several groundbreaking techniques including the synthesis, assembly and engineering of whole bacterial genomes. The successful implantation of those methods, which culminated in the creation of JCVI-syn3.0 the first nearly minimal bacterium with a synthetic genome, mainly results from the use of the yeast Saccharomyces cerevisiae as a transient host for bacterial genome replication and modification. Another method played a key role in the resounding success of this project: bacterial genome transplantation (GT). GT consists in the transfer of bacterial genomes cloned in yeast, back into a cellular environment suitable for the expression of their genetic content. While successful using many mycoplasma species, a complete understanding of the factors governing GT will most certainly help unleash the power of the entire SG pipeline to other genetically intractable bacteria.

Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety. IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri. Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

UMR BFP - Equipe Mollicutes; International audience; Spiroplasmas are bacteria that do not possess flagella and their motility is linked to kink propagation coupled to changes in the cell body helicity. While the motility of bacteria with flagellar motion has been studied extensively, less work has been devoted to the motility of spiroplasmas. We first show that the motility of such bacteria has large variability from individual to individual as well as large fluctuations in time. The Brownian motion of such bacteria both in orientation and translation is also highlighted. We propose a simple model to disentangle the different components of this motility by examining trajectories of single bacteria in different viscosity solvents. The mean velocity of the bacteria turns out to depend on the viscosity of the medium as it increases with viscosity. Further, the temporal fluctuations of the bacteria motility turn out to be very strong with a direct link to tumbling events particular to this bacteria.

This paper presents the impact on antimicrobial resistance (AMR) in poultry and pig bacteria of the French EcoAntibio plan, a public policy to reduce antimicrobial use in animals. The analysis was performed using sales data of veterinary antimicrobials and AMR data from bacteria obtained at slaughterhouse and from diseased animals. From 2011-2018, fluoroquinolones exposure decreased by 71.5 % for poultry and 89.7 % for pigs. For Campylobacter jejuni isolated from broilers at slaughterhouses, ciprofloxacin resistance increased from 51 % in 2010 to 63 % in 2018, whereas for turkeys the percentages varied from 56 % in 2014 to 63 % in 2018. For commensal E. coli isolated from the caecal content of broilers at slaughterhouses, the resistance to ciprofloxacin - assessed using an epidemiological cut-off value - increased in broiler isolates from 30.7 % in 2010 to 38.1 % in 2018. In turkeys, the percentage of resistant E. coli isolates decreased from 21.3 % in 2014 to 15.2 % in 2018, whereas in pigs, it increased from 1.9 % in 2009 to 5.5 % in 2017. However, for E. coli isolated from diseased animals, when the breakpoints of 2018 were applied, resistance to fluoroquinolones significantly decreased between 2010 and 2018 from 9.0%-5.4% for broilers/hens, from 7.4 % to 3.4 % for turkeys and from 9.4 % to 3.6 % for pigs. These data show that the major, rapid decrease in the exposition to fluoroquinolones had contrasting effects on resistance in the diverse bacterial collections. Co-selection or fitness of resistant strains may explain why changes in AMR do not always closely mirror changes in use.

Première réunion du groupe REID - «Hétérogénéité clonale chez les procaryotes et interactions micro-organismes -hôte »; DNA Methylation in the entomopathogenic bacterium [i]Photorhabdus luminescens[/i]. Hétérogénéité clonale chez les procaryotes et interactions micro-organismes -hôte

UMR BFP - Equipe Mollicutes; International audience; Background: Mycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extragenital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants. Results: A 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions. A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain. Conclusion: For the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.

Little is known about biogenically synthesized Zinc oxide nanoparticles (ZnONPs) from Isodon rugosus. Synthesis of metal oxide NPs from essential oil producing medicinal plants results in less harmful side effects to the human population as compared to chemically synthesized NPs. In this article, we report biogenic synthesis of ZnONPs from in vitro derived plantlets and thidiazuron (TDZ) induced callus culture of Isodon rugosus. Synthesized NPs were characterized using UV-spectra, XRD, FTIR, SEM and EDX. Furthermore, the NPs were evaluated for their potential cytotoxic (against HepG2 cell line) and antimicrobial (against drug resistant Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa) activities. Pure crystalline ZnONPs with hexagonal and triangular shapes were obtained as a result of callus extract (CE) and whole plant extract (WPE), respectively. ZnONPs showed potent cytotoxic and antimicrobial potential. The antimicrobial and cytotoxic activities of ZnONPs were found to be shape and surface bound phytochemicals dependent. CE mediated hexagonal ZnONPs showed superior anti-cancer and antimicrobial activities as compared to WPE mediated triangular shaped ZnONPs. It is concluded that biogenic ZnONPs have incredible potential as theranostic agents and can be adopted as useful drug delivery system in next generation treatment strategies., EXCLI Journal; 17:Doc671; ISSN 1611-2156

Statement of the Problem: Bacterial cyclomodulins are a growing family of microbial virulence factors that not only alter host cell cycle progression, but that also interfere with host cell activity, thus favoring the hijacking of host cell protective functions for their own benefit. Staphylococcus aureus (S. aureus), a highly versatile Gram-positive pathogen can cause life-threatening infections. The implications of S. aureus in the alteration of the eukaryotic cell cycle and the biological significance of such an alteration has not been fully investigated. The purpose of the study is to explore the mechanism and to identify staphylococcal compounds that caused host cell cycle arrest and to evaluate the benefit provided by cyclomodulins to bacteria. Methodology & Theoretical Orientation: Flow Cytometry analysis, size exclusion chromatography, mass spectroscopy analysis, Western blotting and immunofluorescence methods were used to identify staphylococcal cyclomodulins and characterize the mechanism. Findings: We demonstrated that S. aureus-induced G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3. Phenol-soluble modulin a (PSMa) peptides were found responsible for this effect. The use of S. aureus mutants confirmed the findings. We showed that the G2 phase was preferential for bacterial proliferation and found that PSMa-induced G2/M transition delay correlated with a decrease in the defensins genes expression. We demonstrated that additionally to secreted staphylococcal cyclomodulins the membrane-anchored lipoprotein-like proteins exert cyclomodulin activity.

Statement of the Problem: Bacterial cyclomodulins are a growing family of microbial virulence factors that not only alter host cell cycle progression, but that also interfere with host cell activity, thus favoring the hijacking of host cell protective functions for their own benefit. Staphylococcus aureus (S. aureus), a highly versatile Gram-positive pathogen can cause life-threatening infections. The implications of S. aureus in the alteration of the eukaryotic cell cycle and the biological significance of such an alteration has not been fully investigated. The purpose of the study is to explore the mechanism and to identify staphylococcal compounds that caused host cell cycle arrest and to evaluate the benefit provided by cyclomodulins to bacteria. Methodology & Theoretical Orientation: Flow Cytometry analysis, size exclusion chromatography, mass spectroscopy analysis, Western blotting and immunofluorescence methods were used to identify staphylococcal cyclomodulins and characterize the mechanism. Findings: We demonstrated that S. aureus-induced G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3. Phenol-soluble modulin a (PSMa) peptides were found responsible for this effect. The use of S. aureus mutants confirmed the findings. We showed that the G2 phase was preferential for bacterial proliferation and found that PSMa-induced G2/M transition delay correlated with a decrease in the defensins genes expression. We demonstrated that additionally to secreted staphylococcal cyclomodulins the membrane-anchored lipoprotein-like proteins exert cyclomodulin activity.

International audience; Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii, One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109 Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 109 Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.

Background It has been suggested that prophages in the ST398 S. aureus clone are responsible for expanding ST398's spectrum of action and increasing its ability to cause human infections. We carried out the first characterization of the various prophages carried by 76 ST398 bloodstream infection (BSI) isolates obtained over 9 years of observation. Results Whole-genome sequencing of 22 representative isolates showed (1) the presence of the φ3-prophage and diverse genetic features typical of animal-associated isolates (i.e., SCCmec XI element, Tn916 transposon and non φ3-prophages) in a majority of BSI isolates, (2) one BSI isolate devoid of the φ3-prophage but otherwise similar to an animal-infecting isolate, (3) 35 prophages carrying numerous genes previously associated with virulence or immune evasion in animal models of staphylococcal infections. The analysis of prophage content in all 76 BSI isolates showed an increasing prevalence of polylysogeny over time. Overall, over the course of the last 10 years, the BSI isolates appear to have acquired increasing numbers of genetic features previously shown to contribute to bacterial adaptation and virulence in animal models of staphylococcal infections. Conclusions We hypothesize that lysogeny has played a significant role in increasing the ability of the ST398 clone to cause infections in humans. Our findings highlight the risk that the ST398 lineage will increase its threat to public health by continuing to acquire virulence and/or multiple antibiotic-resistance genes from hospital-associated clones of Staphylococcus aureus. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3516-x) contains supplementary material, which is available to authorized users.

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes., Author summary The level of interest in intrinsic resistance genes has increased recently, and one of reasons is that their mobilization could lead to emergence of resistant pathogens. Insertion sequences (ISs) or plasmids can capture intrinsic resistance genes and disseminate them in bacterial populations. In this study, we identified a novel subclass of aminoglycoside nucleotidyltransferases which are intrinsic in A. baumannii and other Acinetobacter species. The genes encoding the aminoglycoside nucleotidyltransferase were frequently horizontally transferred between different Acinetobacter species by homologous recombination. This work reports a novel mechanism of natural resistance in Acinetobacter and an overlooked pathway for the dissemination of resistance among species in this genus.

Salmonella enterica is a prominent bacterial pathogen with implications on human and animal health. Salmonella serovars could be classified as gastro-intestinal or extra-intestinal. Genome-wide comparisons revealed that extra-intestinal strains are closer relatives of gastro-intestinal strains than to each other indicating a parallel evolution of this trait. Given the complexity of the differences, a systems-level comparison could reveal key mechanisms enabling extra-intestinal serovars to cause systemic infections. Accordingly, in this work, we introduce a unique resource, SalmoNet, which combines manual curation, high-throughput data and computational predictions to provide an integrated network for Salmonella at the metabolic, transcriptional regulatory and protein-protein interaction levels. SalmoNet provides the networks separately for five gastro-intestinal and five extra-intestinal strains. As a multi-layered, multi-strain database containing experimental data, SalmoNet is the first dedicated network resource for Salmonella. It comprehensively contains interactions between proteins encoded in Salmonella pathogenicity islands, as well as regulatory mechanisms of metabolic processes with the option to zoom-in and analyze the interactions at specific loci in more detail. Application of SalmoNet is not limited to strain comparisons as it also provides a Salmonella resource for biochemical network modeling, host-pathogen interaction studies, drug discovery, experimental validation of novel interactions, uncovering new pathological mechanisms from emergent properties and epidemiological studies. SalmoNet is available at http://salmonet.org.

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response., The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of coincubation of hDCs with M. homini s, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs. IMPORTANCE Mycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.

During treatment of organic waste by anaerobic digestion, composting or in wastewater treatment plants, pathogenic bacteria are submitted to various biotic and abiotic stress. In treated wastes (digestates, composts…) increase of bacterial counts obtained by culture for indicators (E. coli) and pathogenic bacteria (Salmonella sp., Shigella sp., Listeria monocytogenes) are sometimes recorded, but the underlying mechanisms remain unknown. Some recent studies indicate that this may be due to the induction of Viable but Non-Culturable (VBNC) state during treatment, followed by ‘resuscitation’ during storage of treated products. Here, a microcosm approach was used to evaluate the persistence of three pathogenic bacteria (Salmonella Derby, Campylobacter coli and Listeria monocytogenes) in digestates from agricultural wastes, stored for later land spreading. Nine samples, including raw digestates, liquid fractions of digestate and composted digestates, were inoculated with each pathogen and maintained for 40 days at 24°C. Concentrations of pathogens were monitored using culture and qPCR methods. In some digestates, the concentration of the pathogens by qPCR assay was several orders of magnitude higher than the concentration of culturable cells, suggesting a potential loss of culturability and induction of VBNC state. The potential VBNC state which was generally not observed in the same digestate for the three pathogens, occurred more frequently for C. coli and L. monocytogenes than for Salmonella Derby. The effect of NH4+/NH3 on the culturability of C. coli and Salmonella Derby was also shown. Our results underline the importance of considering VBNC cells when evaluating the sanitary effect of an anaerobic digestion process and the persistence of pathogens during the storage of digestates and subsequent land spreading.

Bacteria of the genus Xanthomonas are a major group of plant pathogens. They are hazardous to important crops and closely related to human pathogens. Being collectively a major focus of molecular phytopathology, an increasing number of diverse and intricate mechanisms are emerging by which they communicate, interfere with host signalling and keep competition at bay. Interestingly, they are also biotechnologically relevant polysaccharide producers. Systems biotechnology techniques have revealed their central metabolism and a growing number of remarkable features. Traditional analyses of Xanthomonas metabolism missed the Embden-Meyerhof-Parnas pathway (glycolysis) as being a route by which energy and molecular building blocks are derived from glucose. As a consequence of the emerging full picture of their metabolism process, xanthomonads were discovered to have three alternative catabolic pathways and they use an unusual and reversible phosphofructokinase as a key enzyme. In this review, we summarize the synthetic and systems biology methods and the bioinformatics tools applied to reconstruct their metabolic network and reveal the dynamic fluxes within their complex carbohydrate metabolism. This is based on insights from omics disciplines; in particular, genomics, transcriptomics, proteomics and metabolomics. Analysis of high-throughput omics data facilitates the reconstruction of organism-specific large- and genome-scale metabolic networks. Reconstructed metabolic networks are fundamental to the formulation of metabolic models that facilitate the simulation of actual metabolic activities under specific environmental conditions.

BFP1332-Equipe Mollicutes; Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent cloning and subsequent engineering of other mycoplasma genomes in yeast opens new possibilities for studies of the genomes of genetically intractable organisms. Here, we report the successful one-step cloning of the M. hominis PG21 genome in yeast using the transformation-associated recombination (TAR) cloning method. At low passages, the M. hominis genome cloned into yeast displayed a conserved size. However, after ~60 generations in selective media, this stability was affected, and large degradation events were detected, raising questions regarding the stability of large heterologous DNA molecules cloned in yeast and the need to minimize host propagation. Taking these results into account, we selected early passage yeast clones and successfully modified the M. hominis PG21 genome using the CRISPR/Cas9 editing tool, available in Saccharomyces cerevisiae. Complete M. hominis PG21 genomes lacking the adhesion-related vaa gene were efficiently obtained.

Un corrigendum de cet article a été publié dans le vol 7, article 120 avec avec la clé UT (WOS) 000398420900001 et le DOI 10.3389/fcimb.2017.00120; During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets for the design of alternative therapeutics against intracellular pathogens. The collection of putative effectors of E. chaffeensis described in our paper could serve as a roadmap for future studies of the function and evolution of effectors.

SGI1, BLSE et AmpC chez des Proteus spp d’origine animale. 36. Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse (RICAI 2016)

Microbial growth in meat from traditional handled Arius heudelotti fish during fermentation at 25-30°C in Senegal has been determined. Microorganisms involved in fermentation and pathogen microorganisms were analyzed in function of salt and millet addition [1/1 (w/v)]. Total viable microorganisms, lactic acid bacteria, H2S-producing Enterobacteriaceae, staphylococci, fungi and spore-forming bacteria counts in the crude meat fish reached 6.34 ± 0.28, 4.10 ± 0.61, 4.33 ± 0.45, 3.71 ± 0.69, 1.50 ± 0.3 and 1.33 ± 0.58 Log10 CFU/g. H2S-producing bacteria predominated (8.3 ± 0.25 Log10 CFU/g) after 24 h incubation at 25-30°C of untreated meat fish or that immersed in saline [14% NaCl (w/v)]. The pH of raw meat fish was 6.32 ± 0.1. It increased during unsalted fermentation, while it slightly decreased for the saline procedure. Meat fish fermentation in salty water added with NaCl at 80% (w/v), widespread in Senegal, allowed weak acidification in addition to growth inhibition of H2S producing Enterobacteriaceae which dropped to 3.53 ± 0.45 Log10 CFU/g after 24 h of fermentation. The fish fermentation in water added with malted millet flour at 15% (w/v) enabled significant growth of lactic acid bacteria and pH dropping to 4.9 ± 0.19. SH2-producing Enterobacteriaceae, Staphyloccoci and spore-forming bacteria showed a weak growth in the meat fish significantly acidified in millet solution, indicating preservative factor improvement when compared to the abusive salting traditional procedure which is a dietetic concern.

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.

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

International audience; Despite the development of on-farm anaerobic digestion as a process for making profitable use of animal by-products, factors leading to the inactivation of pathogenic bacteria during storage of digestates remain poorly described. Here, a microcosm approach was used to evaluate the persistence of three pathogenic bacteria (Salmonella enterica Derby, Campylobacter coli and Listeria monocytogenes) in digestates from farms, stored for later land spreading. Nine samples, including raw digestates, liquid fractions of digestate and composted digestates, were inoculated with each pathogen and maintained for 40 days at 24∘C. Concentrations of pathogens were monitored using culture and qPCR methods. The persistence of L. monocytogenes, detected up to 20 days after inoculation, was higher than that of Salmonella Derby, detected for 7-20 days, and of C. coli (not detected after 7 days). In some digestates, the concentration of the pathogens by qPCR assay was several orders of magnitude higher than the concentration of culturable cells, suggesting a potential loss of culturability and induction of Viable but Non-Culturable (VBNC) state. The potential VBNC state which was generally not observed in the same digestate for the three pathogens, occurred more frequently for C. coli and L. monocytogenes than for Salmonella Derby. Composting a digestate reduced the persistence of seeded L. monocytogenes but promoted the maintenance of Salmonella Derby. The effect of NH+44+/NH3 on the culturability of C. coli and Salmonella Derby was also shown. The loss of culturability may be the underlying mechanism for the regrowth of pathogens. We have also demonstrated the importance of using molecular tools to monitor pathogens in environmental samples since culture methods may underestimate cell concentration. Our results underline the importance of considering VBNC cells when evaluating the sanitary effect of an anaerobic digestion process and the persistence of pathogens during the storage of digestates and subsequent land spreading.

The mechanisms of interaction between Lactococcus lactis and the food pathogen Staphylococcus aureus are of crucial importance, as one major role of lactic acid bacteria (LAB) in fermented foods is to inhibit undesirable and pathogenic flora. It was never questioned if the presence of a pathogen can actively modify the gene expression patterns of LAB in a shared environment. In this study, transcriptome and biochemical analyses were combined to assess the dynamic response of L. lactis in a mixed culture with S. aureus . The presence of S. aureus hardly affected the growth of L. lactis but dramatically modified its gene expression profile. The main effect was related to earlier carbon limitation and a concomitantly lower growth rate in the mixed culture due to the consumption of glucose by both species. More specific responses involved diverse cellular functions. Genes associated with amino acid metabolism, ion transport, oxygen response, menaquinone metabolism, and cell surface and phage expression were differentially expressed in the mixed culture. This study led to new insights into possible mechanisms of interaction between L. lactis and S. aureus . Moreover, new and unexpected effects of L. lactis on the virulence of S. aureus were discovered, as described elsewhere (S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubière, and Y. Le Loir, Appl. Environ. Microbiol. 75:4459-4472, 2009).