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Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis. Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed. PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (p

Background: L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis. Methods: L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) differentmixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis. Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Although arable landscapes are positively valued by the society, intensified cropping and livestock systems continue to predominate in western EU rural territories. The increase of farm size, conversion/loss of non-crop features and simplification of crop rotations should maintain the trend toward a reduction in landscape diversity associated to an impoverishment of plant and animal communities, a soil and water quality loss due to pollutants transfer, an increase of greenhouse gases and potentially a reduction of the agro-ecosystem’s capacity for carbon sequestration (Stoate et al., 2001). To overcome these issues, in 2012 France decided to set up a strategic programme for the generalisation of “agro-ecology”. All farms are proposed to reach economic, social and environmental performances all in one (MAAF, 2016). Innovative cropping/livestock systems are thus expected and special attention is granted to agroforestry which is more than ever widely considered, being even sometimes proposed as the unique solution enable to limit most of the externalities from intensified agriculture. However, the ecosystem services from agroforestry are not well known. When some are studied, the use of references produced is limited to the sole cropping system, region and tree species of the study area. That is why the appropriation of agroforestry usually remains theoretical (practical use being possible but not without precautions and risks). References on the potential advantages and limits of agroforestry are very frequently expected and questioned within rural territories, by farmers or students. This is particularly the case of water operators which have the duties and the financial capacities to propose to farmers the improvement of the ecological status of their agricultural fields in order to locally contribute to the protection and/or the recovering of the water quality. Very often, [1] a range of agro-ecological infrastructures such as agricultural parcel (re)forestation (APF), peripheral hedge (PHD), living slope fascine (LSF), mechanical slope fascine (MSF), Grass-covering conversion (GCC), intercalated agroforestry (AGF), short rotation coppice (SRC), in-parcel grass strip (GST), in-parcel slope or ditch (SLO), pond (PON) and [2] a range of agri-technical alternatives such as no-tillage technics (NOT), direct (undercover) seeding (DIS) or even tillage direction change (TDC) are proposed to farmers to compensate/replace their intensified practices. But alternatives’ adoption remains limited because farmers ignore or are skeptical of such alternatives and the local farm advisory services often lack expertise. The PREVALTERA project conduced in the Nord - Pas-de-Calais region (Grandgirard et al., 2011) aimed to fill the needs of knowledge and expertise. Experts’ perception and knowledge about the potential ecosystem services from agro-ecological and agro-technical alternatives have been surveyed and analysed. Objectives are three: (i) to identify the main ecosystem services to be related to the alternatives assessed, (ii) to propose a first cross-ranking of these alternatives and (iii) to identify and rank the ecosystem potentials of the two main agroforestry types questioned in France which are PHD and AGF.

National audience; Le génie végétal, ou génie biologique, désigne la mise en oeuvre des techniques utilisant les végétaux et leurs propriétés mécaniques et/ou biologiques pour le contrôle de l’érosion des sols, la restauration de milieux dégradés et la dépollution des sols et des eaux. L’ingénierie végétale désigne la conception des projets d’application du génie végétal ou génie biologique. Aujourd’hui, la filière de ces deux domaines est en plein essor, en particulier en France et en Europe. Les professionnels expriment tous leurs besoins de reconnaissance et de développement des compétences et du savoir-faire, de consolidation et d’évolution des réglementations et directives en vigueur, ainsi que d’une meilleure identification des bénéfices issus du génie végétal. Ainsi, depuis peu, les acteurs de cette filière se structurent pour mieux se connaître et mettre leurs compétences en synergie. Les spécialistes de l’ingénierie végétale et du génie végétal prônent une éthique et une réglementation renforcées pour l’exercice de leur métier. Enfin, les professionnels reconnaissent la nécessité aujourd’hui de mieux identifier les bénéfices que les milieux naturels et les populations retirent des actions par et/ou pour le vivant. Pour cela, l’accroissement des connaissances et des données scientifiques, ainsi que la formalisation de formations tant initiales que continues, peuvent avantageusement contribuer à ce développement actuel de l’ingénierie végétale et du génie végétal.

Presentation From mid 2000, in order to address global challenges, European member states’ strategies for agriculture have progressively identified and given priority to the set up of a multi-functional farm models. In France, to achieve both food security and socio-environmental sustainability, the design, test and deployment of new Productive and Efficient Cropping Systems (PECS) was then engaged. Therefore, those of the particular PECS targeting the re-conquest of water quality often work at reducing drastically, or even renouncing to chemical pesticides. For that, within the 2012 EXPE Dephy Ecophyto program, a 6-years SCA0PEST project (pesticide-free agroforestry cropping system) has been funded. Considering the experimental farm objectives and local sectors' potentials, SCA0PEST objectives are (i) the review of the current conventional agroforestry cropping system and its experts' opinions-based re-design by respecting principles of integrated pest & weed management, (ii) the set up of 6 over 8 of the crops of the crop rotation each year, (iii) the ex-ante and continuous economic, environmental and social assessments of its performances. Depending on raw products sale prices, energy and fertilizers costs, preliminary SCA0PEST ex-ante assessment provided limited economic prevision, as direct margin would decrease from 140 up to 250€/ha mainly due to yield losses and additional costs for seeds. Conversely, environmental performance would be improved as annual GHG emissions from the cultivated compartment fall down to 1140 kg eq.CO2/ha (48% of the reference cropping system with pesticides); additionally, when simulated, annualized carbon sequestration by trees should reach 650 kg eq.CO2/ha. Finally, inclusion of a 2-years alfalfa production within the rotation would provide a supplementary 230€/ha indirect gain when incorporated at 40% within the dairy cows ration. If this first SCA0PEST assessment points an acceptable economic balance and an improved environmental footprint, continuous assessments would have to confirm the potential of this innovative free-pesticide cropping system.

Poster Facing new challenges of agriculture, the research for more sustainable agrosystems includes a growing interest in agroforestry practices. Over the past years, a large number of experimental and demonstration plots have been established all around France. However, coordination between research and extension partners is lacking. In order to support innovations in agriculture, the French government encourages partnership between research, development and education stakeholders, through the creation of Mixed Technological Networks (Réseaux Mixtes Technologiques, RMT). These RMTs benefit from national recognition and receive a grant for network coordination and communication. In 2014, a new network was created: the RMT “AgroforesterieS” brings together about fifty members involved in agroforestry, from research, semi-public and associative farmers’ organizations, technical institutes, engineering offices, and agricultural schools. By sharing expertise, databases and demonstration sites, the network aims at (i) promoting collective dynamics for the development of sustainable and innovative agroforestry systems adapted to the French territorial constraints, and (ii) developing technical and methodological tools for the setting up and management of agroforestry systems. It will particularly achieve these by: 1. Creating an observatory of agroforestry practices, including both experimental and commercial sites, in order to provide technical references for farmers; 2. Carrying out a multicriteria assessment of agroforestry systems, in terms of economical, technical, agronomical and environmental performances, as well as adoption factors; 3. Ensuring the coordination of scientific and technical stakeholders in order to initiate new collectively-thought projects of research and development in agroforestry; 4. Developing and/or improve decision support tools for farmers and land managers for the design and management of agroforestry systems; 5. Drawing up a strategy for knowledge diffusion towards future advisors, learners, and users.

Poster In order to provide potable water to the Parisian users, the Régie des Eaux de Paris (EDP) has set up since 2000 an upstream water protection strategy across territories housing its groundwater catchments. Despite the financial incentives granted for the (re)conversion to organic farming or to the grass covering of very vulnerable areas, water quality at the Vanne catchment is continuously and punctually exceeding potability thresholds. Therefore, in 2012, EDP has decided to conduct an experimental project for the development of agroforestry systems onto the Vanne catchments basin in order to limit nitrate, suspended matters and pesticides' transfers to surface and groundwater. For that, a previously-developed multicriteria and GIS-based methodology (PREVALTERA) has been adapted to provide a decision support system (DSS) enable to recommend for each parcel, when needed, efficient and matching agroforestry and/or agrotechnical alternatives. The methodology is facing both (i) the nature and the level of intrinsic vulnerability of the parcels to erosion, runoff, infiltration risks and again the potential benefit for the local ecological connectivity of its land improvement, (ii) the morphological and hydrological characteristics of the parcels and its close environment. It results by allocating different agroforestry and/or agrotechnical alternatives to individual parcels or to groups of parcels when local particularities (e.g. ponor) asked for a collective management plan. Results and recommendations obtained from the DSS are used as references by the territorial engineers together with the farmers to (1) localize hot spots and conduct in situ vulnerability diagnosis at parcel/farm scales to precise risks and assess agricultural pressures, (2) envisage the technical and economic feasibility of agrotechnical and/or agroforestry alternatives in their parcels. Later, as tested in other zones, the methodology makes possible the integration of farmers' preferences about agroecological alternatives and their estimated efficiency to decide of adapted, efficient but also accepted solutions.

130 ref. *INRA Dijon, Documentation, 17 rue Sully, BP 86510, 21065 Dijon cedex Diffusion du document : INRA Dijon, Documentation, 17 rue Sully, BP 86510, 21065 Dijon cedex Diplôme : Dr. d'Université

International audience

International audience

Background Approximately 7% of survivors from meningococcal meningitis (MM) suffer from neurological sequelae due to brain damage in the course of meningitis. The present study focuses on the role of matrix metalloproteinases (MMPs) in a novel mouse model of MM-induced brain damage. Methods The model is based on intracisternal infection of BALB/c mice with a serogroup C Neisseria meningitidis strain. Mice were infected with meningococci and randomised for treatment with the MMP inhibitor batimastat (BB-94) or vehicle. Animal survival, brain injury and host-response biomarkers were assessed 48 h after meningococcal challenge. Results Mice that received BB-94 presented significantly diminished MMP-9 levels (p

Background Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10–20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. Methods BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. Results Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. Conclusions Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM.

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-alpha-D-glucosamine into N-acetyl-D-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (alpha 2 -> 9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.

Background: Biocides and antibiotics are used to eradicate or prevent the growth of microbial species on surfaces (occasionally on catheters), or infected sites, either in combination or sequentially, raising concerns about the development of co-resistance to both antimicrobial types. The effect of such compounds on Salmonella enterica, a major food-borne and zoonotic pathogen, has been analysed in different studies, but only few works evaluated its biological cost, and the overall effects at the genomic and transcriptomic levels associated with diverse phenotypes resulting from biocide exposure, which was the aim of this work. Results: Exposure to triclosan, clorhexidine, benzalkonium, (but not to hypochlorite) resulted in mutants with different phenotypes to a wide range of antimicrobials even unrelated to the selective agent. Most biocide-resistant mutants showed increased susceptibility to compounds acting on the cell wall (β-lactams) or the cell membranes (poly-L-lysine, polymyxin B, colistin or toxic anions). Mutations (SNPs) were found in three intergenic regions and nine genes, which have a role in energy production, amino acids, carbohydrates or lipids metabolism, some of them involved in membrane transport and pathogenicity. Comparative transcriptomics of biocide-resistant mutants showed over-expression of genes encoding efflux pumps (sugE), ribosomal and transcription-related proteins, cold-shock response (cpeE) and enzymes of microaerobic metabolism including those of the phosphotransferase system. Mainly ribosomal, metabolic and pathogenicity-related genes had affected expression in both in vitro-selected biocide mutants and field Salmonella isolates with reduced biocide susceptibility. Conclusions: Multiple pathways can be involved in the adaptation of Salmonella to biocides, mainly related with global stress, or involving metabolic and membrane alterations, and eventually causing “collateral sensitivity” to other antimicrobials. These changes might impact the bacterial-environment interaction, imposing significant bacterial fitness costs which may reduce the chances of fixation and spread of biocide resistant mutants.

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1544-y) contains supplementary material, which is available to authorized users.