A short artificial antimicrobial peptide shows potential to prevent or treat bone infections.

Infection of bone is a severe complication due to the variety of bacteria causing it, their resistance against classical antibiotics, the formation of a biofilm and the difficulty to eradicate it. Antimicrobial peptides (AMPs) are naturally occurring peptides and promising candidates for treatment of joint infections. This study aimed to analyze the effect of short artificial peptides derived from an optimized library regarding (1) antimicrobial effect on different bacterial species, (2) efficacy on biofilms, and (3) effect on osteoblast‑like cells. Culturing the AMP-modifications with Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus (including clinical isolates of MRSA and MSSA) and Staphylococcus epidermidis identified one candidate that was most effective against all bacteria. This AMP was also able to reduce biofilm as demonstrated by FISH and microcalorimetry. Osteoblast viability and differentiation were not negatively affected by the AMP. A cation concentration comparable to that physiologically occurring in blood had almost no negative effect on AMP activity and even with 10% serum bacterial growth was inhibited. Bacteria internalized into osteoblasts were reduced by the AMP. Taken together the results demonstrate a high antimicrobial activity of the AMP even against bacteria incorporated in a biofilm or internalized into cells without harming human osteoblasts

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

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 antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells

Fluorescence in situ Hybridization (FISH) in the Microbiological Diagnostic of Deep Sternal Wound Infection (DSWI)

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 [email protected]: 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, as well as individual risk factors and the microbiologic results were documented.Results: Tissue samples were taken from 12 patients suffering from DSWI. Classical microbiological culture resulted in the growth of microorganisms in the specimens of five patients (42%), including bacteria and in one case Candida. FISHseq gave additional diagnostic information in five cases (41%) and confirmed culture results in seven cases (59%).Conclusion: Microbial biofilms are not always present in DSWI wounds, but microorganisms are distributed in a “patchy” pattern in the tissue. Therefore, a deep excision of the wound has to be performed to control the infection. We recommend to analyze at least two wound samples from different locations by culture and in difficult to interpret cases, additional molecular biological analysis by FISHseq.Keywords: biofilm, FISH, mediastinitis, microorganisms, DSW

Is Ureaplasma spp. the leading causative agent of acute chorioamnionitis in women with preterm birth?

Objectives: Aim of this study was to detect microorganisms in fetal membranes and placental tissue in preterm chorioamnionitis by combining fluorescence in situ hybridization (FISH) with broad range PCR. The combination of the two molecular techniques enables identification and localization of the microorganisms within the tissue, confirming their clinical relevance. Methods: In a prospective cohort study, we compared 31 women with preterm premature rupture of membranes or preterm labour and preterm delivery by caesarean section with a control group of 26 women undergoing elective caesarean section at term. Fetal membranes and placental tissue were analysed by FISH and broad range 16S rRNA- gene PCR and sequencing. Results: For 20 women in the preterm group, caesarean section was performed because of a clinical diagnosis of chorioamnionitis. Microorganisms were detected in the tissues by both molecular techniques in 11 out of 20 women. Among those, Ureaplasma spp. was most abundant, with five cases that remained culture-negative and would have been missed by routine diagnostic procedures. Other infections were caused by Staphylococcus aureus, Streptococcus mitis or Escherichia coli. FISH and PCR were negative for all women without suspected chorioamnionitis and for the control group. Conclusions: Combination of FISH with broad- range PCR and sequencing permitted unambiguous identification of the causative microorganisms in chorioamnionitis. The high prevalence of Ureaplasma spp. should lead to a re-evaluation of its clinical significance and possible therapeutic consequences. (C) 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms

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 antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells.Keywords: antibiotic release, Staphylococcus aureus, Staphylococcus epidermidis, fluorescence in situ hybridization, isothermal microcalorimetr

Neuartiges Modell zur Untersuchung der Insulintherapie auf die Ausbildung Biofilm-assoziierter Wunden in diabetischen Mäusen

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7m +/+ Leprdb/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with Staphylococcus aureus ATCC 49230 (106 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence in situ hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19).Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and S. aureus -induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.Einleitung: Patienten mit Diabetes mellitus leiden häufiger unter Biofilm-assoziierten Infekten als normoglykämische Patienten. Das vermehrte zeitgleiche Auftreten erhöhter Blutzuckerwerte und Biofilm-assoziierter Wundinfekte ist gut beschrieben; der dieser erhöhten Infektvulnerabilität zugrundeliegende Pathomechanismus ist allerdings bisher nicht abschließend erforscht. Um dieses Forschungsfeld künftig tiefergehend zu studieren, haben wir in unserem Labor ein neues Tiermodel zur Untersuchung der Biofilmbildung in diabetischen Mäusen unter einer kontrollierten Insulinbehandlung etabliert. Material und Methoden: 16 Wochen alten BKS.Cg-Dock7m +/+ Leprdb/J Mäusen wurde in einem Rückenhautkammermodell eine dermale Wunde gesetzt. Diese Wunden wurden mit Staphylococcus aureus ATCC 49230 (106 Zellen/mL) infiziert. Zeitgleich wurden den Tieren je nach Versuchsgruppe (je N=5) entweder Insulin verabreichende subkutane Implantate (LinShin Canada Inc., Toronto, Canada) oder wirkstofffreie Implantate appliziert. Die Tiere lebten insgesamt 6 Tage. Täglich wurden das Gewicht, die Temperatur und Blutzuckerwerte der Tiere bestimmt. Nach 6 Tagen wurden Gewebeproben jeweils für Fluoreszenz-in-situ -Hybridisierung, CFU und immunhistochemische Färbungen gewonnen. Die Versuche sind entsprechend der Tierversuchsordnung des Landes Sachsen durchgeführt worden (TVV05/19).Ergebnisse: Wir konnten ein neuartiges zuverlässiges Modell zur Untersuchung der Insulintherapie auf die Ausbildung Biofilm-assoziierter Wunden in diabetischen Mäusen etablieren. Die Insulinpumpentherapie konnte eine Reduktion der Blutzuckerwerte erreichen. Die Kombination des Rückenhautkammermodells, welches kontinuierliche In-vivo-Untersuchungen der Infektion erlaubt, mit Insulinimplantaten ist ein für die diabetischen Mäuse gut tolerierbares Modell.Diskussion: Wir haben ein neuartiges Modell zur Untersuchung des Zusammenhangs zwischen Insulin- und Blutzuckerwerten auf die Bildung von Biofilm-assoziierten Wundinfekten am diabetischen Tiermodell entwickelt. Dieses Modell eröffnet ein weites Spektrum an künftigen Forschungsfeldern

Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (S alpha ThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of S alpha ThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues

Acrylic microparticles increase daptomycin intracellular and in vivo anti-biofilm activity against Staphylococcus aureus

© 2018 Published by Elsevier B.V.Daptomycin (DAP) is a cyclic lipopeptide antibiotic with potential clinical application in orthopedic infections caused by staphylococci. However, it failed to eradicate Staphylococcus aureus in vitro, in intracellular infection studies, as well as in vivo in an experimental model of implant-associated biofilm infections. In this study, the antimicrobial effect of DAP encapsulated in poly(methyl methacrylate)-Eudragit (PMMA-EUD) microparticles (DAP-MPs) on intracellular S. aureus was evaluated in human osteoblast cells using fluorescence in situ hybridization (FISH) analysis. Encapsulated DAP was able to reduce the amount of intracellular S. aureus by 73% compared to blank microparticles (MPs). Then, the advantage of treating with DAP-MPs versus free DAP was evaluated in a murine model of implant-associated biofilm infection. Free DAP showed a >3 log10 decrease in planktonic and adherent bacteria but failed to eradicate adherent methicillin-resistant S. aureus (MRSA), whereas DAP-MPs showed a clearance of planktonic MRSA, significantly reduced adherent MRSA by more than 3 log10 and cured the infection in 60%. This was linked to the prolonged higher DAP concentration within the tissue cage fluid compared to free DAP. To our knowledge, this study provides the first evidence for the high intracellular and in vivo anti-biofilm efficacy of DAP-MPs to target staphylococcal infections.Portuguese government, Fundação para a Ciência e Tecnologia (FCT), (Projects: EXCL/CTM-NAN/0166/2012; Pest-UID/DTP/04138/2014). The paper is based upon work from COST TD1305 (Improved Protection of Medical Devices against infection).info:eu-repo/semantics/publishedVersio

Determining Rg of IDPs from SAXS Data

There is a great interest within the research community to understand the structure-function relationship for intrinsically disordered proteins (IDPs); however, the heterogeneous distribution of conformations that IDPs can adopt limits the applicability of conventional structural biology methods. Here, scattering techniques, such as small-angle X-ray scattering, can contribute. In this chapter, we will describe how to make a model-free determination of the radius of gyration by using two different approaches, the Guinier analysis and the pair distance distribution function. The ATSAS package (Franke et al., J Appl Crystallogr 50:1212-1225, 2017) has been used for the evaluation, and throughout the chapter, different examples will be given to illustrate the discussed phenomena, as well as the pros and cons of using the different approaches