Serratiopeptidase reduces the invasion of osteoblasts by Staphylococcus aureus

Finding new strategies to counteract periprosthetic infection and implant failure is a main target in orthopedics. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter and kill osteoblasts, to stimulate pro-inflammatory chemokine secretion, to recruit osteoclasts, and to cause inflammatory osteolysis. Moreover, by entering eukaryotic cells, staphylococci hide from the host immune defenses and shelter from the extracellular antibiotics. Thus, infection persists, inflammation thrives, and a highly destructive osteomyelitis occurs around the implant. The ability of serratiopeptidase (SPEP), a metalloprotease by Serratia marcescens, to control S. aureus invasion of osteoblastic MG-63 cells and pro-inflammatory chemokine MCP-1 secretion was evaluated. Human osteoblast cells were infected with staphylococcal strains in the presence and in the absence of SPEP. Cell proliferation and cell viability were also evaluated. The release of pro-inflammatory chemokine MCP-1 was evaluated after the exposure of the osteoblast cells to staphylococcal strains. The significance of the differences in the results of each test and the relative control values was determined with Student’s t-test. SPEP impairs their invasiveness into osteoblasts, without affecting the viability and proliferation of bone cells, and tones down their production of MCP-1. We recognize SPEP as a potential tool against S. aureus bone infection and destruction

Drug loaded homogeneous electrospun PCL/gelatin hybrid nanofiber structures for anti-infective tissue regeneration membranes

YesInfection is the major reason for guided tissue regeneration/guided bone regeneration (GTR/GBR) membrane failure in clinical application. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection by electrospinning of poly(ε-caprolactone) (PCL) and gelatin blended with metronidazole (MNA). Acetic acid (HAc) was introduced to improve the miscibility of PCL and gelatin to fabricate homogeneous hybrid nanofiber membranes. The effects of the addition of HAc and the MNA content (0, 1, 5, 10, 20, 30, and 40 wt.% of polymer) on the properties of the membranes were investigated. The membranes showed good mechanical properties, appropriate biodegradation rate and barrier function. The controlled and sustained release of MNA from the membranes significantly prevented the colonization of anaerobic bacteria. Cells could adhere to and proliferate on the membranes without cytotoxicity until the MNA content reached 30%. Subcutaneous implantation in rabbits for 8 months demonstrated that MNA-loaded membranes evoked a less severe inflammatory response depending on the dose of MNA than bare membranes. The biodegradation time of the membranes was appropriate for tissue regeneration. These results indicated the potential for using MNA-loaded PCL/gelatin electrospun membranes as anti-infective GTR/GBR membranes to optimize clinical application of GTR/GBR strategies

Biological evaluation of alginate-based hydrogels, with antimicrobial features by Ce(III) incorporation, as vehicles for a bone substitute

In this work three different hydrogels were developed to associate, as vehicles, with the synthetic bone substitute GR-HA. One based on an alginate matrix (Alg); a second on a mixture of alginate and chitosan (Alg/Ch); and a third on alginate and hyaluronate (Alg/HA), using Ca2+ ions as cross-linking agents. The hydrogels, as well as the respective injectable bone substitutes (IBSs), were fully characterized from the physical-chemical point of view. Weight change studies proved that all hydrogels were able to swell and degrade within 72 hours at pH 7.4 and 4.0, being Alg/HA the hydrogel with the highest degradation rate (80%). Rheology studies demonstrated that all hydrogels are non-Newtonian viscoelastic fluids, and injectability tests showed that IBSs presented low maximum extrusion forces, as well as quite stable average forces. In conclusion, the studied hydrogels present the necessary features to be successfully used as vehicles of GR-HA, particularly the hydrogel Alg/HA.The authors would like to acknowledge the financial support from FCT (Fundacao para a Ciencia e a Tecnologia) through the grant SFRH/BD/76237/2011 and project ENMED/0002/2010, from FEDER funds through the program COMPETE-Programa Operacional Factores de Competitividade-under the project PEst-C/EME/UI0285/2011, as well as to the project I&DT BIOMAT&CELL n. 1372

Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7

Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4 degrees C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors

Solar Energy: Incentives to Promote PV in EU27

The growth in the use of renewable energies in the EU has been remarkable. Among these energies is PV. The average annual growth rate for the EU-27 countries in installed PV capacity in the period 2005-2012 was 41.2%. While the installed capacity of PV has reached almost 82 % of National Renewable Energy Action Plan (NREAP) targets for the EU-27 countries for 2020, it is still far from being used at its full potential. Over recent years, several measures have been adopted in the EU to enhance and promote PV. This paper undertakes a complete review of the state of PV power in Europe and the measures taken to date to promote it in EU-27. 25 countries have adopted measures to promote PV. The most widespread measure to promote PV use is Feed- in Tariffs. Tariffs are normally adjusted, in a decreasing manner, annually. Nevertheless, currently, seven countries have decided to accelerate this decrease rate in view of cost reduction of the installations and of higher efficiencies. The second instrument used to promote PV in the EU-27 countries is the concession of subsidies. Nevertheless, subsidies have the disadvantage of being closely linked to budgetary resources and therefore to budgetary constraints. In most EU countries, subsidies for renewable energy for PV are being lowered. Twelve EU-27 countries adopted tax measures. Low-interest loans and green certificate systems were only sparingly used

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

A Mouse Model of Post-Arthroplasty Staphylococcus aureus Joint Infection to Evaluate In Vivo the Efficacy of Antimicrobial Implant Coatings

Post-arthroplasty infections represent a devastating complication of total joint replacement surgery, resulting in multiple reoperations, prolonged antibiotic use, extended disability and worse clinical outcomes. As the number of arthroplasties in the U.S. will exceed 3.8 million surgeries per year by 2030, the number of post-arthroplasty infections is projected to increase to over 266,000 infections annually. The treatment of these infections will exhaust healthcare resources and dramatically increase medical costs.To evaluate novel preventative therapeutic strategies against post-arthroplasty infections, a mouse model was developed in which a bioluminescent Staphylococcus aureus strain was inoculated into a knee joint containing an orthopaedic implant and advanced in vivo imaging was used to measure the bacterial burden in real-time. Mice inoculated with 5x10(3) and 5x10(4) CFUs developed increased bacterial counts with marked swelling of the affected leg, consistent with an acute joint infection. In contrast, mice inoculated with 5x10(2) CFUs developed a low-grade infection, resembling a more chronic infection. Ex vivo bacterial counts highly correlated with in vivo bioluminescence signals and EGFP-neutrophil fluorescence of LysEGFP mice was used to measure the infection-induced inflammation. Furthermore, biofilm formation on the implants was visualized at 7 and 14 postoperative days by variable-pressure scanning electron microscopy (VP-SEM). Using this model, a minocycline/rifampin-impregnated bioresorbable polymer implant coating was effective in reducing the infection, decreasing inflammation and preventing biofilm formation.Taken together, this mouse model may represent an alternative pre-clinical screening tool to evaluate novel in vivo therapeutic strategies before studies in larger animals and in human subjects. Furthermore, the antibiotic-polymer implant coating evaluated in this study was clinically effective, suggesting the potential for this strategy as a therapeutic intervention to combat post-arthroplasty infections