Lysine Oxidase: An Enzyme to Control Biofilm, Survey, and Analysis

Effect of hydrodynamics on the formation and removal of microalgal biofilm in photobioreactors

ObjectiveTrichosporon asahii is the major causative fungus of disseminated or deep-seated trichosporonosis and forms a biofilm on medical devices. Biofilm formation leads to antifungal drug resistance, so biofilm-related infections are relatively difficult to treat and infected devices often require surgical removal. Therefore, prevention of biofilm formation is important in clinical settings. In this study, to identify metal cations that affect biofilm formation, we evaluated the effects of cation chelators on biofilm formation in T. asahii.ResultsWe evaluated the effect of cation chelators on biofilm formation, since microorganisms must assimilate essential nutrients from their hosts to form and maintain biofilms. The inhibition by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was greater than those by other cation chelators, such as deferoxamine, triethylenetetramine, and ethylenediaminetetraacetic acid. The inhibitory effect of TPEN was suppressed by the addition of zinc. TPEN also inhibited T. asahii hyphal formation, which is related to biofilm formation, and the inhibition was suppressed by the addition of zinc. These results suggest that zinc is essential for biofilm formation and hyphal formation. Thus, zinc chelators have the potential to be developed into a new treatment for biofilm-related infection caused by T. asahii.

Management of biofilms in irrigation lines is an important aspect to achieve Zero Liquid Discharge cultivation systems, as is the goal for Dutch greenhouse horticulture by 2027. A test system (in triplicate) and a protocol were developed and build to test the effectiveness of technologies and products for removal and prevention of formation of biofilms. Under greenhouse circumstances (temperature, water composition, irrigation line material, system length, etc.) this system and protocol have shown to be capable to produce a reproducible biofilm in three parallel test systems. Five technologies have been tested with this system: • (1) Oxyl-PRO S silver stabilised H2O2 has shown to be effective to prevent biofilm formation, but did not show a significant effect in biofilm removal. • (2) SureFlow ClO2 has shown to be effective in both removal of an existing biofilm and prevention of biofilm formation. • (3) Antibacterial pipelines, (4) AQUA4D® and (5) SonoPure Ultrasound did not show a significant effect in both prevention of biofilm formation and removal of an existing biofilm. None of the tested technologies had a selective effect on the composition of the microbial community in the biofilm.

Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a 'minimal' mimic, a well characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small angle x-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs.

Event Abstract Back to Event Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms Dalal Asker1, 2, Tarek Awad1, Perrin Baker3, Lynne Howell3, 4 and Benjamin Hatton1 1 University of Toronto, Materials Science and Engineering, Canada 2 Alexandria University, Food Science & Technology Department, Egypt 3 Hospital for Sick Children, Canada 4 University of Toronto, Dept. of Biochemistry, Canada Introduction: Implanted devices are at significant risk of developing bacterial biofilm-associated infections. Approximately 60% of all hospital-acquired infections (HAIs) are the result of biofilm formation on implanted medical devices and the bacterium Pseudomonas aeruginosa is one of the most prevalent isolated pathogens. Bacterial biofilms are communities of bacteria encased within a protective extracellular matrix. Exopolysaccharides (EPS) are the predominant component of biofilm matrix for many bacteria, contributing to initial adhesion, architecture, and resistance. Bacteria in biofilm are highly tolerant to antibiotics and can evade the host immune system. Recently we identified and produced several recombinant glycoside hydrolases that selectively target and hydrolyze biofilm EPS. Two of these enzymes - PslGh, and PelAh - hydrolyze the Psl and Pel polysaccharides from P. aeruginosa, respectively, and were found to be effective in solution at disrupting the biofilms from lab, clinical and environmental isolates. Current antibiotic therapy and antimicrobial surfaces are ineffective against biofilm infections. Therefore, we aim to use surface-immobilized enzymes as a means of preventing biofilm formation on implanted medical devices such as endotracheal tubes. Materials and Methods: We tested the immobilization of PslGh onto polymer biomaterial surfaces as a means of preventing biofilm growth for P. aeruginosa in long term static and flow culture. The enzyme was covalently bound with glutaraldehyde cross-linking to glass and PDMS surfaces and its attachment confirmed by ATR-FTIR and contact angle measurements. To evaluate anti-biofilm efficacy, our biomaterial surfaces were incubated in P. aeruginosa culture that overexpresses the Psl polysaccharide for varying duration. Fluorescence microscopy and fluorescent dyes (SYTOX Green) were used and for quantitative analysis, we used turbidity and crystal violet staining methods. Results and Discussion: The covalently-bound PslGh inhibited both cell attachment and biofilm formation of the P. aeruginosa (Psl-dependent) cells (Fig 1a), compared with the control (i.e., no enzyme) or a catalytically inactive enzyme (Fig. 1b) over a 24 hour period. This indicates that the anti-biofilm activity of PslGh is directly related its catalytic activity. The covalently-immobilized PslGh enzyme was also effective at preventing biofilm formation for an 8 day static culture. Quantitative analysis using crystal violet straining indicated significant anti-biofilm activity for the immobilized PslG, as compared with the control sample. Conclusion: These results indicate the high specificity of PslGh in targeting the Psl exopolysacchride in P. aeruginosa biofilms, which appears to greatly inhibit bacterial colonization and biofilm growth, even when the enzyme is covalently-immobilized on the polymer surface. Figure 1. Inhibition of P. aeruginosa (Psl-dependent) biofilm attachment and formation by covalently-attached PslGh on PDMS samples. (a) untreated PDMS; (b) catalytically-inactive mutant PslGh (mPslGh) covalently immobilized on PDMS; and (c) PslGh covalently immobilized on PDMS. Images were observed with 20X dry objectives. Scale bars, 40 µm. Keywords: Bacteria, Implant, biofunctionalization, bioactive interface, enzyme Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Interfacial phenomena Citation: Asker D, Awad T, Baker P, Howell L and Hatton B (2016). Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00131 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Scholar Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton PubMed Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Immobilization of silver nanoparticles at varying concentrations on segments of polyvinyl chloride manufactured endotracheal tubes

Part 1 Biofilms in the food and beverage industries: Biofilms in the food and beverage industries: an introduction Molecular mechanisms involved in biofilm formation by food-associated bacteria Methods for imaging and quantifying the structure of biofilms in food processing and other environments Monitoring of biofilms in the food and beverage industries A centralized database for use in studying bacterial biofilms and quorum sensing in food processing and other environments: MicroBQs. Part 2 Microorganisms and their metabolites in biofilms: Biofilm formation by food spoilage microorganisms in food processing environments Biofilm formation by Listeria monocytogenes and transfer to foods Biofilm formation by Salmonella in food processing environments Biofilm formation by Gram-positive bacteria including Staphylococcus aureus, Mycobacterium avium and Enterococcus spp in food processing environments Biofilm formation by spore-forming bacteria in food processing environments. Part 3 Biofilm prevention, inactivation and removal and beneficial biofilms: Food contact surfaces, surface soiling and biofilm formation Cleaning and sanitation in food processing environments for the prevention of biofilm formation and biofilm removal Novel methods for biofilm control and removal from food processing equipment. Part 4 Biofilms in particular food industry sectors: Biofilms in red meat processing Biofilms in dairy processing Biofilms and brewing Biofilms in poultry processing Beneficial biofilms: wastewater and other industrial applications Biofilms in fish processing Biofilms in fresh fruit and vegetables. Part 5 Appendix: Sampling and quantification of biofilms in food processing and other environments.

Purpose:Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel.Materials and Methods:The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests.Results:GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group.Conclusion:AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

The author would like to thank the Independent Research Fund Denmark (Technology and Production) and the EU Horizon 2020 research programme for funding the research presented in this doctoral dissertation. In particular, I would like to thank Professor in Veterinary Pathology Henrik Elvang Jensen who participated in all parts of the experimental studies. Thanks for your inspiration, guidance and enthusiasm. My thanks also go to all the staff at the Pathobiological Sciences Section for being great colleagues and for the many happy times during work and social events. A special thanks to laboratory technicians Elizabeth Petersen and Betina Andersen for all your help in the histo-lab. I apologize for all the challenging and problematic issues regarding histological preparation of bones. Many thanks also to technicians Dennis Brok and Frederik Andersen for their skilful technical support and help producing images. I would also like to thank the animal technicians at the Department of Experimental Medicine for their excellent care of the pigs and help with anaesthesia, blood sampling and inoculation procedures. Thank you to all my research assistants during the past 5 years, including Kristine Dich-Jørgensen, Anne Sofie Boym Johansen, Nicole Lind Henriksen and Amalie Blirup-Plum. Thank you for your hard work, including endless hours spent evaluating and scoring histological slides, and thank you for bringing enthusiasm and fun into our daily routine. Thanks also to Expert in Experimental Surgery Janne Koch, Associate Professor in Veterinary Microbiology Bent Aalbæk and to Professor in Chronic Infections and Biofilm Thomas Bjarnsholt. You have all been indispensable for the research described in this dissertation. Thomas, I enjoyed our biofilm discussions and I am grateful for all your support. Thanks to everyone in Professor and Orthopedic Surgeon Kjeld Søballe's research group at Aarhus University Hospital for our collaboration on antimicrobial penetration into infected bone tissue. Mikkel Tøttrup, Pelle Hanberg and Mats Bue, it has been a pleasure to work with you. Finally, thanks to all my friends and family for their never ending support and trust, and thanks to my wonderful husband and daughters, Kim, Alberte and Frida, for your love and patience. Worldwide, there has been an ongoing increase in the number of bone infections that lead to amputations and lifelong disability, affecting millions of people every year. Therefore, research investigating the prevention, diagnosis and treatment of bone infections is vitally important. However, to develop effective new approaches and techniques for managing bone infections, preclinical testing and evaluations using reliable animal models are necessary. A novel porcine model of implant-associated osteomyelitis (IAO) in humans was recently developed. The model was based on female pigs, and osteomyelitis was induced by inoculation of Staphylococcus aureus bacteria into a predrilled tibial cavity (2 × 20 mm). Following inoculation, a steel implant (2 × 15 mm) was inserted into the cavity. The animals were euthanized 5 days after inoculation. Control pigs were exposed to the same surgical procedure and inoculated with sterile saline. The success rate of the model was 100%; that is, all the pigs inoculated with bacteria developed osteomyelitis. Bone lesions similar to those found in human patients with osteomyelitis developed in the porcine IAO model, and the inoculated bacteria were detected both within peri-implant bone tissue and on the surface of the implants. Thus, peri-implant bone tissue may serve as a reservoir for biofilm (bacterial aggregates surrounded by an extracellular matrix) shortly after surgical contamination. Biofilms are extremely tolerant to antibiotics and are reportedly the main reason for human bone infection treatment failure. This new porcine IAO model was used to develop a biofilm staining technique, which combined histochemistry (HC) and immunohistochemistry (IHC). The new staining technique allowed the bacterial cells and extracellular matrix to be visualized simultaneously: Alcian blue pH3 stained the carbohydrates of the extracellular matrix blue, and IHC treatment with an antibody specific for S. aureus coloured the bacterial cells red. The new staining technique could also be used reliably on human bone tissue with chronic staphylococcal osteomyelitis. Two microdialysis studies were performed using the porcine IAO model. These studies found that systemically administered antibiotics (cefuroxime and vancomycin) showed significantly less penetration into the tissue surrounding infected bone implants than into healthy bone tissue. Furthermore, this reduced antimicrobial penetration was correlated with the progression of peri-implant bone lesions. Bone lesions that extended to a depth of >3 mm from the implant cavity into the bone tissue showed almost no antimicrobial penetration. The reduced penetration was due to suppurative and necrotic bone inflammation and expansion of the implant cavity. Consequently, diffusion of antimicrobial agents from the capillary system into the implant cavity was hampered because it had to cover a greater distance. Bone inflammation also had a negative impact on the efficacy of a single dose of locally administered gentamicin. Different doses of gentamicin were added to the inoculum 1 min prior to inoculation into the porcine IAO model. Due to the development of acute inflammation including vasodilatation and increased vascular permeability, only high doses of gentamicin (>1000× minimum inhibitory concentration) were effective at the implant surface. This shows that the prophylactic concentration of locally administered antimicrobial agents cannot be evaluated solely by using in vitro assays. A systematic review of all contemporary large, non-rodent animal models of bone infection (i.e. goats, sheep, dogs, pigs and rabbits) was performed. Overall, it was found that experimental design was poorly reported, methods were of poor quality and the pathological parameters used varied significantly. Therefore, study template guidelines as a standard for reporting on animal models of bone infection was established. It was apparent that the animal species per se was one of the most important study design parameters. Based on a narrative review, we found that there were many advantages in using pigs for modelling bacterial biofilm infections in humans because comparable infections also occur spontaneously in pigs. Currently, there are many problems associated with treating patients who have chronic biofilm-based infections, including bone infections. Furthermore, the increasing prevalence of antimicrobial resistance means that new treatments for infectious diseases are urgently required. The novel porcine IAO model described here is a valuable and reliable tool for investigating new prophylactic strategies and treatment regimens for bone infections in humans. Flere og flere patienter får diagnosen knogleinfektion. En knogleinfektion fører til operation, indlæggelse, flere ugers antibiotikabehandling og i yderste konsekvens amputation. De fleste knogleinfektioner opstår, fordi der kommer bakterier ind i knoglen under en operation. Det kan være under indsættelsen af en ny kunstig hofte, eller ved korrektion af et benbrud hvor der anvendes osteosyntese. Knogleinfektioner er enormt svære at behandle, fordi bakterierne danner biofilm, dvs. de går i dvale, og danner en beskyttende matrix omkring sig. Biofilmdannelse beskytter bakterierne mod kroppens eget immunforsvar og det antibiotikum som gives i behandlingsøjemed. Den stigende udvikling af antibiotikaresistens udgør en alvorlig trussel mode vores evne til at bekæmpe infektioner, og derfor er forskning indenfor forebyggelse, diagnosticering og behandling af knogleinfektioner mere relevant end nogensinde før. For at udvikle nye tiltag og teknikker med klinisk relevans, er det helt afgørende, at disse testes og evalueres i velkarakteriserede dyremodeller, som efterligner den humane patologi. Denne afhandling beskriver udviklingen, karakteriseringen og anvendeligheden af en ny grisemodel for implantat-relateret osteomyelitis hos menneseker. Grisemodellen er baseret på, at der bores en lille kavitet i højre tibia hvori der inokuleres saltvand eller Stafylokokkus aureus bakterier. Efter inokuleringen indsættes et lille implantat (2x15mm). Grisene aflives efter 5 dage og alle dyr inokuleret med bakterier udvikler komparative knoglelæsioner. De inokulerede bakterier genfindes på både det indsatte implantat og et stykke inde (1 cm) i det omgivende væv. Modellen demonstrerer dermed, at det knoglevæv som omgiver et implantat kan udgøre et biofilmreservoir allerede korttid efter kirurgisk kontaminering. Den nye grisemodel er blevet brugt til, at udvikle en ny farvemetode der kan synliggøre biofilmdannelse i vævsnit ved brug af almindelig lysmikroskopi. Princippet i den nye metode er en kombination af almindelig histokemi og immunohistokemi. Ved at kombinere en protokol for Alcian Blue pH3 med immunhistokemi baseret på et S. aureus specifikt antistof, kunne både matrix og bakterier i biofilmaggregater visualiseres i to forskellige farver. Den nye farvemetode er også anvendt på humant væv med kronisk osteomyelitis. Knogleinfektioner kræver lang tids antibiotikabehandling og de eksisterende doseringsprotokoller bygger på farmakokinetiske studier af rask knoglevæv. Baseret på grisemodellen for implantat-relateret osteomyelitis blev det demonstreret, at penetrationen af systemisk indgivet antibiotika (cefuroxime og vancomycine) til inficerede knogleimplantater, er signifikant reduceret sammenlignet med normalt rask knoglevæv. Den reducerede antibiotika penetration var korreleret til udbredelsen af patologiske forandringer i knoglevævet omkring implantatet. I de tilfælde hvor læsionerne udbredte sig mere end 3 mm fra implantatet, var der næsten ingen penetration. Den reducerede penetration blev forårsaget af en purulent og nekrotisk inflammation, hvilket ødelage knoglevævet og skabte en kavitet omkring implantat. Derfor kan det antages, at diffusion af antibiotika fra kapillærerne og indtil implantatet er blevet nedsat delvist pga. en øget diffusionsafstand. Udover den store effekt på penetrationen af antibiotika, blev det også påvist, at det inflammatoriske respons har stor indflydelse på effekten af lokal indgivet antibiotika. Forskellige doser af gentamicin blev iblandet det bakterielle inokulum et minut før inokulering i grisemodellen. På grund af udviklingen af akut inflamamtion, med vasodilation og øget vaskulær permeabilitet, var det kun de meget høje gentamicin doser (>1000× MIC) som kunne fastholde en baktericid koncentration på implantatets overflade. Derfor kan det konkluderes, at det for knogleinfektioner, er utilstrækkeligt at basere effekten af antibiotika udelukkende på studier af raskt væv og på in vitro assays. I et stort systematisk review af alle ikke-gnaver modeller for knogleinfektioner blev det påvist, at der de sidste 10 år er sket en markant stigning i brugen af større forsøgsdyr som fx får og grise. Det blev også påvist, at afrapporteringen af studiedesign ofte var meget mangelfuld, og at den metodologiske kvalitet var meget lav. Dette førte til udviklingen af et set retningsliner for standard afrapportering ved udvikling og anvendelse af dyremodeller for knogleinfektioner. Det systematiske review viste, at dyrearten er en af de mest afgørende design parameter. Baseret på en gennemgang af alle grisemodeller for bakterielle infektioner hos mennesker, blev det konkluderet, at grisemodeller er særdeles fordelagtige som modeldyr for biofilm baseret infektioner hos mennesker, fordi grise (slagtesvin) udvikler komparative infektioner spontant. Der er store problemer med behandlingen af patienter som lider af kroniske infektioner inklusive knogleinfektioner. Det er tydeligt, at den nye grisemodel for implantat-relateret osteomyelitis er et brugbart og pålideligt redskab, til at studere nye profylaktiske strategier og behandlingsregimer. Worldwide, there has been an ongoing increase in the number of bone infections that lead to amputations and lifelong disability, affecting millions of people every year [1, 2]. Consequently, bone infections constitute a substantial economic burden in terms of patients, physicians, hospitals and healthcare systems [1, 2]. When applied to patients, the term 'bone infection' can include prosthetic joint infections (PJIs), fracture-related infections (FRIs), implant-associated osteomyelitis (IAO), chronic osteomyelitis (CO), osteomyelitis in children and diabetic foot osteomyelitis (DFO) [3]. Presently, DFO is the leading cause of lower extremity amputations, and it is estimated that a lower limb is lost every 30 s due to DFO [4, 5]. The increased number of bone infections is mainly due to an increase in the size of the elderly population, an increased prevalence of diabetes and an increase in the number of joint prostheses and bone fixation implants being used [1]. The persistence of the problem and the unsatisfactory proportion of positive treatment outcomes implies that the current prophylaxis and treatment strategies are incomplete despite best practice [2]. Therefore, future research must focus on prevention, diagnosis and treatment of bone infections. However, the development of effective new approaches and techniques will depend on preclinical testing and evaluations performed in reliable animal models. In this dissertation, a novel porcine IAO model is presented. The publications referred to (I–IX) describe the development, characterization and application of this model. Chronic bacterial infections are generally caused by biofilm-forming bacteria [6]. Therefore, it is surprising that most reports regarding bacterial biofilms are based on in vitro observations, because these laboratory findings cannot be extrapolated into clinical settings (VIII) [7]. Consequently, there are many problems associated with treating patients who have chronic biofilm-based infections, including bone infections. In addition, the increasing prevalence of antimicrobial resistance means that new treatments for chronic infectious diseases are urgently required [8]. The porcine IAO model has generated new relevant in vivo observations regarding biofilms (II, VI, VIII). In particular, studies have shown that biofilms do not simply involve artificial surface attachment; therefore, the old dogma used to describe biofilms in terms of a 'race for the surface' seems to be clinically inadequate (II, VI, VIII). Biofilm formation also affects tissues, and research into both tissue and implant biofilms is equally important for evaluating chronic bone infections. Promising new approaches to prevent biofilm formation in orthopaedic research include the following: 1) modification of implant surfaces to prevent bacterial adhesion, 2) coating of implants so that they can elute high concentrations of antibiotics locally (without causing systemic toxicity), 3) new drugs directed against bacterial adhesion molecules or quorum sensing and 4) the development of vaccines against biofilm-forming bacteria [9]. All of these new technologies for combating osteomyelitis may be tested using the porcine IAO model. During the past 5 years, the porcine IAO model has been applied in several studies [10-13]. In brief, the model was used as the basis of an EU HORIZON 2020 Research and Innovation Programme project (Novel Marine Biomolecules Against Biofilm [NoMoRFilm] no. 634588) to identify new antibiotics in micro-algae. As a result, one promising anti-biofilm compound was isolated, synthesized, chemically characterized and evaluated for anti-infective properties in the porcine model. In addition, the NoMorFilm project also resulted in the development of a new surface-coating technique for orthopaedic implants. This technique provides a coating that can bind and release high concentrations of antibiotics. An EU patent application based on the new coating technique is currently under review and describes very successful test results obtained using the porcine IAO model. In addition to the prophylactic coating studies, the porcine model was also recently used to evaluate the therapeutic impact of a gentamicin-loaded biodegradable bone void filler, following limited or extensive debridement of osteomyelitis lesions [12]. Currently, the porcine IAO model is also being used to investigate the molecular orchestration of bacterial bone infections. Gene expression analyses of porcine and human infected bone biopsies have shown that bone tissue can mount and sustain a local acute phase response (i.e. an extra hepatic acute phase response) [11]. In addition, research has shown that the classical receptor activator of nuclear factor κB (RANK)–RANK-ligand (RANKL) pathway is not responsible for bone loss resulting from bacterial osteomyelitis [13]. In particular, the molecular studies demonstrated upregulation of specific inflammatory genes, which had no murine homologues; that is, the corresponding genes are not present in mice. This dissertation consists of six chapters. Chapter 1 provides background information regarding IAO in humans. Chapter 2 focuses on previously studied non-rodent animal models of bone infections and the advantages of using pigs for modelling osteomyelitis. In Chapter 3, the development of the new porcine IAO model is described. Chapter 4 focuses on a new staining technique for in situ visualization of biofilms and biofilm formation in spontaneous porcine bacterial infections. Chapter 5 describes the application of the porcine IAO model in studies investigating antimicrobial penetration into infected bone tissue and the prevention of biofilm formation. Finally, Chapter 6 summarizes the main conclusions from the publications on which this dissertation is based. In elective trauma surgery, bone infections occur at a rate of 1%–5% after closed fractures, and at a rate of 3%–50% after open fractures [14]. In 2004, it was estimated that two million fracture fixation procedures were performed each year in the United States leading to approximately 100,000 cases of IAO annually [15]. Risk factors for the development of infections include smoking and comorbidities such as diabetes, immunosuppression and chronic infections at other sites [16]. Fractures occur most frequently in the feet and hands, by the that is the tibia and 1) [16]. IAO after fracture fixation of a this may 1) or lead to loss or of the limb The term to inflammation of the bone and the term to inflammation of the bone including the [14]. osteomyelitis is the term and this is used for both bone infection in patients [14]. IAO may be based on the from to of the are within 2 after bone the and the and occur than 10 after can be acute they are caused by a they a chronic infection' or the of an infection' [16]. osteomyelitis is also frequently as acute or is as a bone infection with that for at 6 the of bone in an or infections that due to or In is associated with for implants Therefore, or IAO may also be as in the may also be using the which with treatments and The terms and are not used in this system and the are the and of the The system osteomyelitis into or osteomyelitis is to the cavity of the or osteomyelitis only the 3, or osteomyelitis both and In this the bone and the infection not involve the bone or osteomyelitis the of the with loss of The system a and the as or An is a with no systemic or local factors The is by systemic or local factors that the and local are patients who are so that they cannot have The of bacteria within bone tissue and on the surface of bone implants will lead to a suppurative inflammatory by (II, Bone will be induced by from the inflammatory cells and the of matrix Furthermore, the of will also to bone matrix the bone will increase the and cause of blood resulting in which to bone (i.e. the and within the and this will lead to a and blood and (i.e. of a bone a for bacterial an infectious bone will tissue and which may be surrounded by cells in the will into and a of an which the bone The is and is with which may into the surrounding and to the surface a The of the may increase and almost a new cases of IAO are caused by and Staphylococcus aureus and Staphylococcus 1) are the most that are frequently responsible for IAO include bacteria and infections are caused by such as S. A and bacteria The by which bacterial can bone tissue 1) 2) or 3) or infections are caused by bacterial of the bone tissue during trauma and or during the [16]. infections occur due to bacterial following and infections are a of progression from or tissue infections to the bone fixation [16]. bone tissue exposed to and the inserted bone implants are to infections the bacteria are not they will to the bone tissue the surface of the implant (II, VI, This of bacterial is by for surface and or specific adhesion to such as and healthy bone tissue is to Thus, the increased to infection is a of orthopaedic procedures. to implant or fractures or IAO will cause tissue and of exposed bone and the bone tissue and inserted implant will be by 2 and the coating a important in bacterial surface adhesion to implants than the implant steel or the bacteria are after to the bone tissue or they will into a biofilm [6]. However, aggregates of biofilm from the may also the infection A biofilm is as a of bacterial cells in an extracellular which is tolerant antimicrobial agents and of the than bacteria in a biofilm will bacteria will be to the antibiotics used to (VIII) [6]. This increased of biofilm bacteria is mainly due to their lower and their extracellular which can bind and antimicrobial agents [6]. Biofilm bacteria are to tolerant of antibiotics than their A bone infection biofilm will also increasing antimicrobial of this biofilm the of treating a bone infection from to treatment is than after infection research has shown that biofilms is extremely is in treating these infections, due to the high of antimicrobial by and implant and debridement of necrotic bone tissue may be 1) after of the infected implant and extensive systemic treatment several may be to the bone negative [16]. the main reason for of IAO is bacterial biofilm in the bone tissue animals such as sheep, goats, dogs, pigs and are being used frequently in preclinical orthopaedic research due to their which are for the of many orthopaedic than those of and This is also being in the study of bone infections. Therefore, a systematic review was to an of studies on non-rodent (i.e. sheep, goats, dogs, pigs and rabbits) animal models of bone infections The was to identify in study design for bacterial inoculation dose or infection the animal The review also the quality of the studies and and of and The review was based on a systematic of two and of This resulted in a of publications that the (i.e. experimental bacterial inoculation of animals in to model of bacterial bone infection in pigs and have been used frequently as experimental animals to study bone infections that patients the of and porcine models is an to previously models developed in dogs, the increasing of models a for and in vivo studies in a animal than a a were in the study design parameters the animal species inoculation dose or from inoculation to However, porcine including the model described in this dissertation, were regarding a number of specific In particular, the bacterial inoculation dose was lower and the infection inoculation to was in pigs than in all other species VIII). of the animal species used or the of bone infection the bacteria used most frequently for inoculation was S. most of the studies not a specific for the inoculated In studies on models that were based on inoculation into a specific bone using a significantly lower inoculation dose an implant was inserted at the same as the Therefore, results from preclinical bone infection studies are with those obtained in by et who demonstrated that a can the number of bacteria to an infection et × of S. aureus into the of human resulting in only of the infected and of the cases being However, et inserted an implant and of S. all infected and of the infections were

The prevention of biofilm formation on orthopedic implants is essential, as biofilms are the main challenge in the effective treatment of periprosthetic joint infection (PJI). A silver multilayer (SML) coating was developed to prevent biofilm formation on the implant surface. Previous studies have already demonstrated its antibacterial properties without cytotoxic effects. However, the coating has not been previously tested when applied to common titanium surfaces used in total joint arthroplasty implants. These surfaces often have increased roughness and porosity in the case of cementless implants, which can alter the antibacterial effect of the coating. In this study, we assessed the antibacterial and anti-biofilm properties of the SML coating on corundum-blasted and plasma-sprayed microporous-coated titanium alloy surfaces, using S. aureus, S. epidermidis, and E. coli. An antibacterial activity test following the principles of ISO 22196, ASTM E2180-18, and JIS Z 2801 standards was performed, as well as a biofilm proliferation assay investigating bacterial adhesion and biofilm formation. The SML coating exhibited strong antibacterial effects for all bacterial strains. After 24 h biofilm culture, a >4-log reduction in CFU was induced by the SML coating for S. epidermidis and E. coli on the corundum-blasted and plasma-sprayed microporous-coated titanium surfaces, respectively, when compared to the uncoated surfaces. The coating showed bactericidal properties against Gram-positive bacteria on the corundum-blasted discs. The SML coating on two common titanium surfaces demonstrates significant potential as an effective strategy in combating PJI across a wide range of orthopedic implants.

Bacterial biofilm formation on synthetic polymers plays an important role in industry and in modern medicine, leading, for example, to difficult-to-treat infections caused by colonized foreign bodies. Prevention of biofilm formation is a necessary step in the successful prophylaxis of such infections. One approach is to inhibit bacterial adherence by polymer surface modification. We have investigated polymer modification by glow discharge treatment in order to study the influence of the modified surface on bacterial adherence. Surface roughness, surface charge density and contact angles of the modified polymers were determined and related to the adherence of Staphylococcus epidermidis KH6. Although no influence of surface roughness and charge density on bacterial adherence was noticed, a correlation between the free enthalpy of adhesion (estimated from contact angle measurements) and adherence was observed. There seems to exist a certain minimum bacterial adherence, independent of the nature of the polymer surface. Modified polymers with negative surface charge allow for bacterial adherence close to the adherence minimum. These polymers could be improved further by the ionic bonding of silver ions to the surface. Such antimicrobial polymers are able to prevent bacterial colonization, which is a prerequisite for biofilm formation. It is suggested that modification of polymers and subsequent surface coupling of antimicrobials might be an effective approach for the prevention of bacterial biofilm formation.

3 - Biofilm risks

ABSTRACTThere is currently an urgent need to find new strategies to tackle antimicrobial resistance and biofilm-related infections. This study has two aims. First, we evaluated the in vitro efficacy of hyperthermia in preventing biofilm formation on the surfaces of polyvinyl chloride discs. Second, we assessed the in vivo efficacy of hyperthermia in preventing biofilm formation in endotracheal tubes (ETTs) of a rabbit model. For the in vitro studies, nine clinical extensively drug-resistant/multidrug-resistant Gram-negative isolates of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa and three clinical methicillin-resistant Staphylococcus aureus strains were studied. For biofilm formation, an adhesion step of 30 or 90 min followed by a growth step of 24 h were performed with application of one, two, and three pulses at 42°C for 15 min each pulse after the adhesion step. For the in vivo studies, New Zealand rabbits were intubated with ETTs previously colonized with K. pneumoniae or P. aeruginosa strains, and three pulses at 42°C for 15 min were applied after the adhesion step. The application of three pulses at 42°C for 15 min each pulse was needed to achieve the prevention of the in vitro biofilm formation of 100% of the tested strains. The application of heat pulses in a rabbit intubation model led to biofilm prevention of 85% against two K. pneumoniae strains and 80% against two P. aeruginosa strains compared to the control group. Hyperthermia application through pulses at 42°C could be a new nonantibiotic strategy to prevent biofilm formation in ETTs.IMPORTANCE Biofilm-producing microorganisms are considered medically crucial since they cause 80% of the infections that occur in the human body. Medical devices such as endotracheal tubes (ETTs) can act as a reservoir for pathogens providing the surface to which microorganisms can adhere and cause biofilm-associated infections in critically ill patients. This biofilm has been related with the development of ventilator-associated pneumonia (VAP), with an incidence of 8 to 28%, a mortality rate up to 17% and its associated high extra costs. Although some VAP-preventive measures have been reported, they have not demonstrated a significant reduction of VAP incidence. Therefore, we present a new nonantibiotic strategy based on hyperthermia application to prevent biofilm formation inside ETTs. This technology could reduce VAP incidence, intubation duration, hospital and intensive care unit (ICU) length stays, and mortality rates. Consequently, this could decrease the antibiotics administered and influence the impact of antibiotic resistance in the ICU.

Biofilm Formation on Clinically Noninfected Penile Prostheses

SummaryBiofilms are immobile communities of micro‐organisms attached to any surface, such as stainless steel or a food matrix surface or on packaging material. They may be composed of a single species, but more generally, in the natural environment, they consist of mixed species together with an extracellular matrix. Biofilms provide a common mechanism of persistence for a number of bacterial species especially in food processing environments, and therefore, prevention of biofilm formation and the removal of preformed biofilms are an important issue for the food industry. This article reviews the current understanding on the formation of biofilms and recent developments in biological and chemical methods for prevention and removal.