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International audience ; AbstractSalmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. © 2014, American Society for Microbiology. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants ERANET Pathogenomic (GEN2006-27792-C2-1-E/PAT), BIO2011-30503- C02-02, and AGL2011-23954 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation Department of the Government of Navarra. We thank Enrique Calvo from the Proteomic Service of CNIC for his expert help in protein identification and Victor Segura from the Genomics, Proteomics and Bioinformatics Unit at the Center for Applied Medical Research, University of Navarra, for microarray data analysis. ; Peer Reviewed

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Modification of the biomaterial surface topography is a promising strategy to prevent bacterial adhesion and biofilm formation. In this study, we use direct laser interference patterning (DLIP) to modify polystyrene surface topography at sub-micrometer scale. The results revealed that three-dimensional micrometer structures have a profound impact on bacterial adhesion. Thus, line- and pillar-like patterns enhanced S. aureus adhesion, whereas complex lamella microtopography reduced S. aureus adhesion in static and continuous flow culture conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus adhesion both when the surface is coated with human serum proteins and when the material is implanted subcutaneously in a foreign-body associated infection model. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants AGL2011-23954 and BIO2011-30503-C02-02 from the Spanish Ministry of Economy and Competitivity and IIQ14066. RI1 from Innovation Department of the Government of Navarra. A. Lasagni, D. Langhenirich, and R. Helbig thank the Deutsche Forschungsgemeinschaft (DFG) for the financial support of the project "Mechanically stable anti-adhesive polymer surfaces" (LA-2513 4-1). ; Peer Reviewed

In the past 15 years, many efforts were made to create functionalized artificial surfaces showing special anti-bacterial and anti-biofouling properties. Thereby, the topography of medical relevant materials plays an important role. However, the targeted fabrication of promising surface structures like hole-, lamella- and pyramid-like patterns with feature sizes in the sub-micrometer range in a one-step process is still a challenge. Optical and e-beam lithography, molding and selfassembly layers show a great potential to design topographies for this purpose. At the same time, most of these techniques are based on sequential processes, require masks or molds and thus are very device relevant and time consuming. In this work, we present the Direct Laser Interference Patterning (DLIP) technology as a capable method for the fast, flexible and direct fabrication of periodic micrometer- and submicrometer structures. This method offers the possibility to equip large plain areas and curved devices with 1D, 2D and 3D patterns. Simple 1D (e.g. lines) and complex 3D (e.g. lamella, pillars) patterns with periodic distances from 0.5 μm to 5 μm were fabricated on polymeric materials (polyimide, polystyrene). Subsequently, we characterized the adhesion behavior of Staphylococcus epidermidis and S. aureus bacteria under in vitro and in vivo conditions. The results revealed that the topographies have a significant impact on bacteria adhesion. On the one side, one-dimensional line-like structures especially with dimensions of the bacteria enhanced microbe attachment. While on the other hand, complex three-dimensional patterns prevented biofilm formation even after implantation and contamination in living organisms. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants AGL2011-23954 and BIO2011-30503-C02-02 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation Department of the Government of Navarra. A. Lasagni, D. Günther, and R. Helbig thank the Deutsche Forschungsgemeinschaft for the financial support of the project "Mechanically stable anti-adhesive polymer surfaces" (LA-2513 4-1, WE 2539/17-1). ; Peer reviewed

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi- GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine. ; SB is a predoctoral fellow from the Public University of Navarra. CG and BG are recipients of a postdoctoral contract under Grants IIM 13329.RI1 and BIO2011-30503-C02-02, respectively. This work was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2011-30503-C02-02 and BIO2014-53530-R from the Spanish Ministry of Economy and Competitiveness. ; Peer Reviewed