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Nick Spindler,1 Annette Moter,2,3 Alexandra Wiessner,2,3 Tanja Gradistanac,4 Michael Borger,5 Arne C Rodloff,6 Stefan Langer,1 Judith Kikhney2,3 1Department of Orthopedic Surgery, Traumatology and Plastic Surgery, University Hospital Leipzig, Leipzig, Germany; 2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health Institute of Microbiology, Infectious Diseases and Immunology, Biofilmcenter, Berlin, Germany; 3MoKi Analytics GmbH, Berlin, Germany; 4Department of Pathology, University Clinic Leipzig, Leipzig, Germany; 5Department of Cardiac Surgery, Leipzig Heart Center, University of Leipzig, Leipzig, Germany; 6Institute of Microbiology and Epidemiology of Infectious Diseases, University Hospital Leipzig, Leipzig, GermanyCorrespondence: Nick SpindlerDepartment of Orthopedic Surgery, Traumatology and Plastic Surgery, University Hospital Leipzig, Leipzig, GermanyTel +49-341 - 9717140Fax +49-341 - 9717139Email nick.spindler@medizin.uni-leipzig.dePurpose: Postoperative mediastinitis after cardiac surgery is still a devastating complication. Insufficient microbiological specimens obtained by superficial swabbing may only detect bacteria on the surface, but pathogens that are localized in the deep tissue may be missed. The aim of this study was to analyze deep sternal wound infection (DSWI) samples by conventional microbiological procedures and fluorescence in situ hybridization (FISH) in order to discuss a diagnostic benefit of the culture-independent methods and to map spatial organization of pathogens and microbial biofilms in the wounds.Methods: Samples from 12 patients were collected and analyzed using classic microbiological culture and FISH in combination with molecular nucleic acid amplification techniques (FISHseq). Frequency of and the time to occurrence of a DSWI was recorded, previous operative interventions, complications

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient‐days. Of these cases, catheter‐associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health‐care costs of £1–2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary‐tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device‐associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1–2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.

Inês Santos Ferreira,1 Ana F Bettencourt,1 Lídia MD Gonçalves,1 Stefanie Kasper,2 Bertrand Bétrisey,3 Judith Kikhney,2 Annette Moter,2 Andrej Trampuz,4 António J Almeida1 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; 2Biofilmcenter, German Heart Institute Berlin, Berlin, Germany; 3Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 4Center for Musculoskeletal Surgery, Charité – University Medicine Berlin, Berlin, Germany Abstract: The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micro­meter size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antiba

Inês Santos Ferreira,1 Ana F Bettencourt,1 Lídia MD Gonçalves,1 Stefanie Kasper,2 Bertrand Bétrisey,3 Judith Kikhney,2 Annette Moter,2 Andrej Trampuz,4 António J Almeida1 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; 2Biofilmcenter, German Heart Institute Berlin, Berlin, Germany; 3Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 4Center for Musculoskeletal Surgery, Charité – University Medicine Berlin, Berlin, Germany Abstract: The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micro­meter size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antiba

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phyl

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phyl