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Cutibacterium acnes (previously known as Propionibacterium acnes) is frequently found on lipid-rich parts of the human skin. While C. acnes is most known for its role in the development and progression of the skin disease acne, it is also involved in many other types of infections, often involving implanted medical devices. C. acnes readily forms biofilms in vitro and there is growing evidence that biofilm formation by this Gram-positive, facultative anaerobic micro-organism plays an important role in vivo and is also involved in treatment failure. In this brief review we present an overview on what is known about C. acnes biofilms (including their role in pathogenesis and reduced susceptibility to antibiotics), discuss model systems that can be used to study these biofilms in vitro and in vivo and give an overview of interspecies interactions occurring in polymicrobial communities containing C. acnes.

Osseointegration is a well-established concept used in applications including the percutaneous Bone-Anchored Hearing System (BAHS) and auricular rehabilitation. To date, few retrieved implants have been described. A systematic review including cases where percutaneous bone-anchored implants inserted in the temporal bone were retrieved and analyzed was performed. We also present the case of a patient who received a BAHS for mixed hearing loss. After the initial surgery, several episodes of soft tissue inflammation accompanied by pain were observed, leading to elective abutment removal 14 months post-surgery. Two years post-implantation, the implant was removed due to pain and subjected to a multiscale and multimodal analysis: microbial DNA using molecular fingerprinting, gene expression using quantitative real-time polymerase chain reaction (qPCR), X-ray microcomputed tomography (micro-CT), histology, histomorphometry, backscattered scanning electron microscopy (BSE-SEM), Raman spectroscopy, and fluorescence in situ hybridization (FISH). Evidence of osseointegration was provided via micro-CT, histology, BSE-SEM, and Raman spectroscopy. Polymicrobial colonization in the periabutment area and on the implant, including that with Staphylococcus aureus and Staphylococcus epidermidis, was determined using a molecular analysis via a 16S-23S rDNA interspace [IS]-region-based profiling method (IS-Pro). The histology suggested bacterial colonization in the skin and in the peri-implant bone. FISH confirmed the localization of S. aureus and coagulase-negative staphylococci in the skin. Ten articles (54 implants, 47 patients) met the inclusion criteria for the literature search. The analyzed samples were either BAHS (35 implants) or bone-anchored aural epitheses (19 implants) in situ between 2 weeks and 8 years. The main reasons for elective removal were nonuse/changes in treatment, pain, or skin reactions. Most samples were evaluated using histology, demonstrating osseointegration, but with the absence of bone under the implants' proximal flange. Taken together, the literature and this case report show clear evidence of osseointegration, despite prominent complications. Nevertheless, despite implant osseointegration, chronic pain related to the BAHS may be associated with a chronic bacterial infection and raised inflammatory response in the absence of macroscopic signs of infection. It is suggested that a multimodal analysis of peri-implant health provides possibilities for device improvements and to guide diagnostic and therapeutic strategies to alleviate the impact of complications.

The bacterial biofilm constitutes a complex environment that endows the bacterial community within with an ability to cope with biotic and abiotic stresses. Considering the interaction with bacterial viruses, these biofilms contain intrinsic defense mechanisms that protect against phage predation; these mechanisms are driven by physical, structural, and metabolic properties or governed by environment-induced mutations and bacterial diversity. In this regard, horizontal gene transfer can also be a driver of biofilm diversity and some (pro)phages can function as temporary allies in biofilm development. Conversely, as bacterial predators, phages have developed counter mechanisms to overcome the biofilm barrier. We highlight how these natural systems have previously inspired new antibiofilm design strategies, e.g., by utilizing exopolysaccharide degrading enzymes and peptidoglycan hydrolases. Next, we propose new potential approaches including phage-encoded DNases to target extracellular DNA, as well as phage-mediated inhibitors of cellular communication; these examples illustrate the relevance and importance of research aiming to elucidate novel antibiofilm mechanisms contained within the vast set of unknown ORFs from phages. ispartof: VIRUSES-BASEL vol:14 issue:5 ispartof: location:Switzerland status: published

Bacterial adhesion and biofilm formation on surfaces are troublesome in many industrial processes. Here, nanoporous and nanopillared aluminum surfaces were engineered by anodizing and postetching processes and made hydrophilic (using the inherent oxide layer) or hydrophobic (applying a Teflon coating) with the aim of discouraging bacterial adhesion. Adhesion of Staphylococcus aureus ATCC 12600 (Gram-positive, spherically shaped) and Escherichia coli K-12 (Gram-negative, rod shaped) was evaluated to the nanoengineered surfaces under both static and flow conditions (fluid shear rate of 37 s(-1)). Compared to a nonstructured electropolished flat surface, the nanostructured surfaces significantly reduced the number of adhering colony forming units (CFUs) for both species, as measured using agar plating. For the hydrophilic surfaces, this was attributed to a decreased contact area, reducing bacterial adhesion forces on nanoporous and nanopillared surfaces to 4 and 2 nN, respectively, from 8 nN on flat surfaces. Reductions in the numbers of adhering CFUs were more marked on hydrophobic surfaces under flow, amounting to more than 99.9% and 99.4% for S. aureus and E. coli on nanopillared surfaces, respectively. Scanning electron microscopy revealed 4 few bacteria found on the hydrophobic nanopillared surfaces adhered predominantly to defective or damaged areas, whereas the intact area preserving the original nanopillared morphology was virtually devoid of adhering bacteria. The greater decrease in bacterial adhesion to hydrophobic nanopillared surfaces than to hydrophilic or nanoporous ones is attributed to effective air entrapment in the three-dimensional pillar morphology, rendering them superhydrophobic and slippery, in addition to providing a minimized contact area for bacteria to adhere to.

Polymethylmethacrylate (PMMA) also referred as (acrylic) bone cement is a non-degradable biomaterial that has been used in clinical orthopedic practice for several decades. PMMA can be used in a plain formulation, but is often used in an antibiotic-loaded formulation in (primary and revision) arthroplasty and in treatment of orthopedic infections as prosthetic joint infections (PJI) and chronic osteomyelitis. In treatment of PJIs antibiotic-loaded PMMA is often used as a carrier material for local antibiotic delivery in addition to treatment with systemic antibiotics. In this case, the antibiotic-loaded PMMA is often used as a spacer or as a bead chain. Since the introduction of PMMA as an antibiotic carrier there is a tremendous amount of scientific and clinical papers published, which studied numerous different aspects of antibiotic-loaded PMMA. This paper will review the research regarding basic principles of antibiotic-loaded PMMA as mechanism of action, antibiotic-release capacities, choice of antibiotics and influences on mechanical properties of PMMA. Subsequently, concerns regarding the application of antibiotic-loaded PMMA, biofilm formation, antibiotic resistance and local or systemic toxicity will be discussed. In addition to these subjects, the role of antibiotic loaded PMMA in clinical treatment of PJIs and chronic osteomyelitis is discussed in the final part of this paper

Polymethylmethacrylate (PMMA) also referred as (acrylic) bone cement is a non-degradable biomaterial that has been used in clinical orthopedic practice for several decades. PMMA can be used in a plain formulation, but is often used in an antibiotic-loaded formulation in (primary and revision) arthroplasty and in treatment of orthopedic infections as prosthetic joint infections (PJI) and chronic osteomyelitis. In treatment of PJIs antibiotic-loaded PMMA is often used as a carrier material for local antibiotic delivery in addition to treatment with systemic antibiotics. In this case, the antibiotic-loaded PMMA is often used as a spacer or as a bead chain. Since the introduction of PMMA as an antibiotic carrier there is a tremendous amount of scientific and clinical papers published, which studied numerous different aspects of antibiotic-loaded PMMA. This paper will review the research regarding basic principles of antibiotic-loaded PMMA as mechanism of action, antibiotic-release capacities, choice of antibiotics and influences on mechanical properties of PMMA. Subsequently, concerns regarding the application of antibiotic-loaded PMMA, biofilm formation, antibiotic resistance and local or systemic toxicity will be discussed. In addition to these subjects, the role of antibiotic loaded PMMA in clinical treatment of PJIs and chronic osteomyelitis is discussed in the final part of this paper.

Background: The use of drains in patients undergoing abdominal surgery has been a subject of debate for several decades. In this paper, the usefulness of quantitative cultures of sonicated abdominal drains for diagnosing surgical site of infection (SSI) and the association between culture results with patient outcome is evaluated. Methods: Forty-five abdominal drainage tubes from 35 patients who underwent abdominal surgery were studied. Samples were sonicated for 5 min, the sonicate was centrifuged, and the sediment was cultured on different media. Total bacterial counts were adjusted to the actual surface of the drainage tubing. Clinical information of the patients was reviewed retrospectively. Results: A relation was observed between SSI and the use of drains for more than 3 d (p = 0.0216). The presence of a suspected pathogen was related to the prevalence of SSI (p = 0.035), complications (p = 0.013), and greater leukocyte count (p = 0.048 Mann Whitney test), as well as to the use of drains for more than 3 d (p = 0.0386) and to the serous appearance of the exudates at the point of insertion of the drain (p = 0.0399). The sonication procedure showed a sensitivity of 50%, specificity of 84.2%, positive predictive value of 72.72%, and negative predictive value of 66.67% in the diagnosis of SSI. The most commonly isolated group of organisms was coagulase-negative staphylococci, being present in 18 patients (51.43%) who, however, were not associated with SSI. One or two organisms considered as pathogens were detected in 11 patients (31.43%). The more common pathogens detected were Enterobacteriae spp. (nine patients): Enterobacter aerogenes (2), Enterobacter cloacae (1), Escherichia coli (4), Klebsiella pneumoniae (1), Morganella morganii (1); and Pseudomonas aeruginosa (five patients). Candida spp. and Enterococcus spp. were detected in one patient each one. Conclusions: The detection and quantification of organisms not present in skin microbiota after drain sonication is helpful in the diagnosis of SSI and it is associated with a worse outcome in patients. Duration of use of drainage tubes is an independent risk factor for the development of SSI.

The incidence of mesh-related infection after abdominal wall hernia repair is low, generally between 1 and 4%; however, worldwide, this corresponds to tens of thousands of difficult cases to treat annually. Adopting best practices in prevention is one of the keys to reduce the incidence of mesh-related infection. Once the infection is established, however, only a limited number of options are available that provides an efficient and successful treatment outcome. Over the past few years, there has been a tremendous amount of research dedicated to the functionalization of prosthetic meshes with antimicrobial properties, with some receiving regulatory approval and are currently available for clinical use. In this context, it is important to review the clinical importance of mesh infection, its risk factors, prophylaxis and pathogenicity. In addition, we give an overview of the main functionalization approaches that have been applied on meshes to confer anti-bacterial protection, the respective benefits and limitations, and finally some relevant future directions. (C) 2018 Elsevier Ltd. All rights reserved.

Polyetheretherketone (PEEK) films were oxygen plasma treated to increase surface free energy and characterized by X-ray photoelectron microscopy, atomic force microscopy, and water contact angles. A parallel plate flow chamber was used to measure Staphylococcus epidermidis, Staphylococcus aureus, and U-2 OS osteosarcomal cell-line adhesion to the PEEK films in separate monocultures. In addition, bacteria and U-2 OS cells were cocultured to model competition between osteoblasts and contaminating bacteria for the test surfaces. Plasma treatment of the surfaces increased surface oxygen content and decreased the hydrophobicity of the materials, but did not lead to a significant difference in bacterial or U-2 OS cell adhesion in the monocultures. In the S. epidermidis coculture experiments, the U-2 OS cells adhered in greater numbers on the treated surfaces compared to the untreated PEEK and spread to a similar extent. However, in the presence of S. aureus, cell death of the U-2 OS occurred within 10 h on all surfaces. The results of this study suggest that oxygen plasma treatment of PEEK may maintain the ability of osteoblast-like cells to adhere and spread, even in the presence of S. epidermidis contamination, without increasing the risk of preoperative bacterial adhesion. Therefore, oxygen plasma-treated PEEK remains a promising method to improve implant surface free energy for osseointegration. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4427-4434, 2014.

The acetogen Clostridium ljungdahlii is capable of syngas fermentation and microbial electrosynthesis. Biofilm formation could benefit both these applications, but was not yet reported for C. ljungdahlii. Biofilm formation does not occur under standard growth conditions, but attachment or aggregation could be induced by different stresses. The strongest biofilm formation was observed with the addition of sodium chloride. After 3 days of incubation, the biomass volume attached to a plastic surface was 20 times higher with than without the addition of 200 mM NaCl to the medium. The addition of NaCl also resulted in biofilm formation on glass, graphite and glassy carbon, the latter two being often used electrode materials for microbial electrosynthesis. Biofilms were composed of extracellular proteins, polysaccharides, as well as DNA, while pilus-like appendages were observed with, but not without, the addition of NaCl. A transcriptome analysis comparing planktonic (no NaCl) and biofilm (NaCl addition) cells showed that C. ljungdahlii coped with the salt stress by the upregulation of the general stress response, Na+ export and osmoprotectant accumulation. A potential role for poly-N-acetylglucosamines and D-alanine in biofilm formation was found. Flagellar motility was downregulated, while putative type IV pili biosynthesis genes were not expressed. Moreover, the gene expression analysis suggested the involvement of the transcriptional regulators LexA, Spo0A and CcpA in stress response and biofilm formation. This study showed that NaCl addition might be a valuable strategy to induce biofilm formation by C. ljungdahlii, which can improve the efficacy of syngas fermentation and microbial electrosynthesis applications.

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 μM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 μM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analogs when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria.

Here we report on the viscous nature of the bond between adhering bacteria and a substratum surface. A tailor-made script was written for an atomic force microscope, that enabled a constant loading force of 1 or 5 nN to act for 30 s upon a bacterium compressed between a cantilever and a glass surface, while measuring its deformation. Time-dependent deformation was fitted to a one element Kelvin-Voigt analogue of the bond to yield a characteristic relaxation time and viscosity of the bond. Viscosities of streptococcal bonds were smaller (20 kPa s) than those of staphylococcal bonds (31 kPa s). Since staphylococci are relatively rich in extracellular polymeric substances, it can be inferred that the presence of extracellular polymeric substances yields the major contribution to the viscous response. The viscous nature of the bond between adhering bacteria and substratum surfaces provides the bacteria with more time to respond and protect themselves against external stresses.

Biostable fiber-reinforced composite (FRC) implants prepared from bisphenol-A-dimethacrylate and triethyleneglycoldimethacrylate resin reinforced with E-glass fibers have been successfully used in cranial reconstructions in 15 patients. Recently, porous FRC structures were suggested as potential implant materials. Compared with smooth surface, porous surface allows implant incorporation via bone ingrowth, but is also a subject to bacterial attachment. Non-cytotoxic silver-polysaccharide nanocomposite coatings may provide a way to decrease the risk of bacterial contamination of porous FRC structures. This study is focused on the in vitro characterization of the effect porosity on the antimicrobial efficiency of the coatings against Staphylococcus aureus and Pseudomonas aeruginosa by a series of microbiological tests (initial adhesion, antimicrobial efficacy, and biofilm formation). Characterization included confocal laser scanning microscopy and scanning electron microscopy. The effect of porosity on the initial attachment of S. aureus was pronounced, but in the case of P. aeruginosa the effect was negligible. There were no significant effects of the coatings on the initial bacterial attachment. In the antimicrobial efficacy test, the coatings were potent against both strains regardless of the sample morphology. In the biofilm tests, there were no clear effects either of morphology or of the coating. Further coating development is foreseen to achieve a longer-term antimicrobial effect to inhibiting bacterial implant colonization.

The presence of bacteria in human milk has been acknowledged since the seventies. For a long time, microbiological analysis of human milk was only performed in case of infections and therefore the presence of non-pathogenic bacteria was yet unknown. During the last decades, the use of more sophisticated culture-dependent and -independent techniques, and the steady development of the -omic approaches are opening up the new concept of the ‘milk microbiome’, a complex ecosystem with a greater diversity than previously anticipated. In this review, possible mechanisms by which bacteria can reach the mammary gland (contamination versus active migration) are discussed. In addition, the potential roles of human milk for both infant and maternal health are summarised. A better understanding of the link between the milk microbiome and health benefit, the potential factors influencing this relationship and whether or not it can be influenced by nutrition is required to open new avenues in the field of pregnancy and lactation.

Biofilms are detrimental in many industrial and biomedical applications and prevention of biofilm formation has been a prime challenge for decades. Biofilms consist of communities of adhering bacteria, supported and protected by extracellular-polymeric-substances (EPS), the so-called house of biofilm organisms. EPS consists of water, proteins, polysaccharides and extracellular-DNA (eDNA). eDNA, being the longest molecule in EPS, connects the different EPS components and therewith holds an adhering biofilm together. eDNA is associated with bacterial cell surfaces by specific and non-specific mechanisms, mediating binding of other biopolymers in EPS. eDNA therewith assists in facilitating adhesion, aggregation and maintenance of biofilm structure. Here, a new method is described to prevent biofilm formation on surfaces by applying a DNase I enzyme coating to polymethylmethacrylate, using dopamine as an intermediate. The intermediate coupling layer and final DNase I coating are characterized by water-contact-angle measurements and X-ray photoelectron-spectroscopy. The DNase I coating strongly reduces adhesion of Staphylococcus aureus (95%) and Pseudomonas aeruginosa (99%) and prevents biofilm formation up to 14 h, without affecting mammalian cell adhesion and proliferation. Also agarose-gel-electrophoresis indicates loss of enzyme activity between 8 and 24 h. This duration however, is similar to many local antibiotic-delivery devices, which makes it an ideal coating for biomaterial implants and devices, known to fail due to biofilm formation with disastrous consequences for patients and high costs to the healthcare system. With threatening increases in antibiotic resistance, the DNase I coating may provide a timely, potent new approach to biofilm prevention on biomaterial implants and devices.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms., The authors would like to acknowledge the support from the EU COST Action BacFoodNet FA1202.

BackgroundProsthetic meshes are increasingly popular in abdominal wall reconstructive surgery owing to a reduction in hernia recurrence rate. Individual meshes have been evaluated with respect to the formation of infectious biofilms, but no comprehensive comparison yet exists. The aim of this study was to evaluate the effect of the material and morphology of surgical meshes on biofilm growth.MethodsBiofilms of a coagulase-negative staphylococcus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were grown in vitro on different meshes. Intact biofilms were stained fluorescently and evaluated with confocal laser scanning microscopy to distinguish dead from live bacteria and slime.ResultsNumbers of adhering bacteria and the amount of slime in the biofilm were dependent on the morphology of the mesh. Hydrophobicity and the presence of niches in multifilament meshes contributed most to increased biofilm growth.ConclusionThe morphological properties of surgical meshes as well as their hydrophobicity influence bacterial growth and slime production. Differences in slime production may explain why the effectiveness of antibiotic treatment varies for different types of mesh.

Item does not contain fulltext The fate of secondary biomaterial implants was determined by bio-optical imaging and plate counting, after antibiotic treatment of biomaterials-associated-infection (BAI) and surgical removal of an experimentally infected, primary implant. All primary implants and tissue samples from control mice showed bioluminescence and were culture-positive. In an antibiotic treated group, no bioluminescence was detected and only 20% of all primary implants and no tissue samples were culture-positive. After revision surgery, bioluminescence was detected in all control mice. All the implants and 80% of all tissue samples were culture-positive. In contrast, in the antibiotic treated group, 17% of all secondary implants and 33% of all tissue samples were culture-positive, despite antibiotic treatment. The study illustrates that due to the BAI of a primary implant, the infection risk of biomaterial implants is higher in revision surgery than in primary surgery, emphasizing the need for full clearance of the infection, as well as from surrounding tissues prior to implantation of a secondary implant. 01 oktober 2010

Oral biofilm can never be fully removed by oral hygiene measures. Biofilm left behind after brushing is often left behind on the same sites and exposed multiple times to antimicrobials from toothpastes and mouthrinses, after which removal becomes increasingly difficult. On the basis of this observation, we hypothesize that oral bacteria adhering to salivary conditioning films become more difficult to remove after adsorption of antimicrobials due to stiffening of their adhesive bond. To verify this hypothesis, bacteria adhering to bare and saliva-coated glass were exposed to 3 different mouthrinses, containing chlorhexidine-digluconate, cetylpyridinium-chloride, or amine-fluoride, after which bacterial vibration spectroscopy was carried out or a liquid-air interface was passed over the adhering bacteria to stimulate their detachment. Brownian motion–induced nanoscopic vibration amplitudes of 4 oral streptococcal strains, reflecting their bond stiffness, decreased after exposure to mouthrinses. Concurrently, the percentage detachment of adhering bacteria upon the passage of a liquid-air interface decreased after exposure to mouthrinses. A buffer control left both vibration amplitudes and detachment percentages unaffected. Exposure to either of the selected mouthrinses yielded more positively charged bacteria by particulate microelectrophoresis, suggesting antimicrobial adsorption to bacterial cell surface components. To rule out that exposure of adhering bacteria to the mouthrinses stimulated polysaccharide production with an impact on their detachment, Fourier transform infrared spectroscopy was carried out on bacteria adhering to an internal reflection element, prior to and after exposure to the mouthrinses. Infrared absorption band areas indicated no significant change in amount of polysaccharides after exposure of adhering bacteria to mouthrinses, but wave number shifts demonstrated stiffening of polysaccharides in the bond, as a result of antimicrobial adsorption to the bacterial cell surface and in line with changes in surface charge. Clinically, these findings suggest that accumulation of oral biofilm exposed to antimicrobials should be prevented (interdental cleaning aids, floss use), as removal becomes progressively more difficult upon multiple exposures.

Background: Staphylococcal species are the most common organisms causing prosthetic mesh infections, however, infections due to rapidly growing mycobacteria are increasing. This study evaluates the resistance of biomaterial for abdominal wall prostheses against the development of postoperative infection in a rat model. Material and methods: In 75 rats, we intramuscularly implanted three different types of prostheses: (1) low-density polypropylene monofilament mesh (PMM), (2) high-density PMM, and (3) a composite prosthesis composed of low-density PMM and a nonporous hydrophilic film. Meshes were inoculated with a suspension containing 108 colony-forming units of Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium fortuitum, or Mycobacterium abscessus before wound closure. Animals were sacrificed on the eighth day postoperatively for clinical evaluation, and the implants were removed for bacteriologic analyses. Results: Prostheses infected with S aureus showed a higher bacterial viability, worse integration, and clinical outcome compared with infection by other bacteria. Composite prostheses showed a higher number of viable colonies of both M fortuitum and Staphylococcus spp., with poorer integration in host tissue. However, when the composite prosthesis was infected with M abscessus, a lower number of viable bacteria were isolated and a better integration was observed compared with infection by other bacteria. Conclusions: Considering M abscessus, a smaller collagen-free contact surface shows better resistance to infection, however, depending on the type of bacteria, prostheses with a large surface, and covered with collagen shows reduced resistance to infection, worse integration, and worse clinical outcome. (C) 2016 Elsevier Inc. All rights reserved.

Biofilm has been recognized as a well-protected form of living for bacteria, contributing to bacterial pathogenicity, particularly for opportunistic species. Biofilm-associated infections are marked by their persistence. Extensive research has been devoted to the formation and composition of biofilms. The immune response against biofilms remains rather unexplored, but there is the notion that bacteria within a biofilm are protected from host defences. Here we glance at the mechanisms by which neutrophils recognize and face biofilms in implant infections and discuss the implications of this interplay, as well as speculate on its significance.

Objectives The purpose of this study was to assess the efficacy of different cleansing agents in killing mixed species biofilms on silicone facial prostheses. Materials and methods Two bacterial and three yeast strains, isolated from silicone facial prostheses, were selected for the mixed species biofilms. A variety of agents used to clean facial prostheses were employed, viz., antibacterial soap, essential-oil-containing mouth rinse, ethanol 27 %, chlorhexidine mouth rinse, and buttermilk. Colony forming units (CFUs) and live/dead staining were analyzed to assess the efficacy of these cleansing agents against 24-h and 2-week biofilms and regrown biofilms on silicone samples. Results Chlorhexidine was the most effective cleansing agent. Chlorhexidine killed 8 log unit CFUs (>99.99 % killing) in a 24-h biofilm and 5 log unit CFUs (>99.99 % killing) in 2-week biofilms. Also, after regrowth and repeated treatment of the biofilm, chlorhexidine was the most effective cleansing agent showing no detectable CFUs. The essential-oil-containing mouth rinse (containing 26.9 % ethanol) showed a similar efficacy as ethanol (27 %) alone. Antibacterial soap and buttermilk were the least effective agents tested. Conclusions Chlorhexidine showed the highest reduction in CFUs in 24-h, 2-week, and regrown mixed species biofilm of microorganisms isolated from silicone facial prostheses. Clinical relevance Chlorhexidine mouth rinse (easy obtainable and relatively cheap) is very effective in killing bacteria and yeast present in biofilms on silicone facial prostheses. When applied on a regular basis, cleansing a facial prosthesis with chlorhexidine will presumably increase its lifetime and reduce skin irritations.

Bile acid resistance by Lactococcus lactis depends on the ABC-type multidrug transporter LmrCD. Upon deletion of the lmrCD genes, cells can reacquire bile acid resistance upon prolonged exposure to cholate, yielding the Δ lmrCD r strain. The resistance mechanism in this strain is non-transporter based. Instead, cells show a high tendency to flocculate, suggesting cell surface alterations. Contact angle measurements demonstrate that the Δ lmrCD r cells are equipped with an increased cell surface hydrophilicity compared to those of the parental and wild-type strains, while the surface hydrophilicity is reduced in the presence of cholate. Δ lmrCD r cells are poor in biofilm formation on a hydrophobic polystyrene surface, but in the presence of subinhibitory concentrations of cholate, biofilm formation is strongly stimulated. Biofilm cells show an enhanced extracellular polymeric substance production and are highly resistant to bile acids. These data suggest that non-transporter-based cholate resistance in L. lactis is due to alterations in the cell surface that stimulate cells to form resistant biofilms.

Many next-generation biomaterials will need the ability to not only promote healthy tissue integration but to simultaneously resist bacterial colonization and resulting biomaterials-associated infection. For this purpose, antimicrobial nanofibers of polycaprolactone (PCL) were fabricated by incorporating calcium peroxide. PCL nanofibers containing different ratios of calcium peroxide (1%, 5% and 10% (w/w)) with or without ascorbic acid were fabricated using an electrospinning technique. Antimicrobial evaluations confirmed the inhibitory properties of the nanofibers on the growth of E. coli and S. epidemidis because of a significant burst release of calcium peroxide from the nanofibers. Analysis of tissue cell response showed that despite an initial toxic effect over the first 24 h, after 4 days of culture, osteoblast viability and morphology were both healthy. These results demonstrate that oxygen-generating nanofibers can be designed and developed to provide a short-term peroxide-based antimicrobial response while still maintaining attractive tissue-integration properties.

Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol) (PEG) brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid) (bioactive "PEG-RGD") were compared to monofunctional PEG brush-coatings (biopassive "PEG") and bare titanium oxide (TiO(2)) surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s(-1) for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s(-1)). After 2 h, staphylococcal adhesion was reduced to 3.6 +/- 1.8 x 10(3) and 6.0 +/- 3.9 x 10(3) cm(-2) on PEG and PEG-RGD coatings respectively, compared to 1.3 +/- 0.4 x 10(5) cm(-2) for the TiO(2) surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s(-1)). U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO(2). The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the "race for the surface" between bacteria and mammalian cells.

This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings. (C) 2009 Elsevier Ltd. All rights reserved.

This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings. (C) 2009 Elsevier Ltd. All rights reserved.

Upon initial microbial adhesion to a surface multiple events occur that include interfacial re-arrangements in the region between an adhering organism and a surface Application of physico-chemical mechanisms to explain microbial adhesion to surfaces requires better knowledge of the interfacial re arrangement occurring immediately after adhesion than hitherto available (C) 2010 Elsevier Ltd All rights reserved

Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol) (PEG) brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid) (bioactive "PEG-RGD") were compared to monofunctional PEG brush-coatings (biopassive "PEG") and bare titanium oxide (TiO(2)) surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s(-1) for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s(-1)). After 2 h, staphylococcal adhesion was reduced to 3.6 +/- 1.8 x 10(3) and 6.0 +/- 3.9 x 10(3) cm(-2) on PEG and PEG-RGD coatings respectively, compared to 1.3 +/- 0.4 x 10(5) cm(-2) for the TiO(2) surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s(-1)). U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO(2). The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the "race for the surface" between bacteria and mammalian cells.

Recent studies suggest that diamond-like carbon (DLC) coatings are suitable candidates for application on biomedical devices and implants, due to their high hardness, low friction, high wear and corrosion resistance, chemical inertness, smoothness, and tissue and blood compatibility. However, most studies have neglected the potential susceptibility of DLC coatings to bacterial adhesion, which is the first step in the development of implant-related infections. This study compares adhesion of seven bacterial strains, commonly implicated in implant-related infections, to tetrahedral amorphous carbon, with their adhesion to AISI 316L surgical steel. The results show that bacterial adhesion to DLC was similar to the adhesion to commonly used stainless steel. This suggests that DLC coating can be advantageously used on implants made of AISI 316L or other materials without increasing the risk to implant-related infections. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 90B: 882-885, 2009

Using a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion (F(prev)) and to detach adhering bacteria (F(det)) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air-liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air-liquid interface. F(prev) varied from 0.03 to 0.70 pN, while F(det) varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DIDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory (F(DLVO)) towards the secondary interaction minimum were higher on glass than on DIDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air-liquid interface passages) to the substratum surface.

Polymer brushes of poly(ethylene glycol) have long been considered the gold standard for antifouling surfaces that resist adsorption of biomolecules and attachment of microorganisms. However, despite displaying excellent resistance to protein adsorption, the polymer brush coatings cannot entirely avoid colonization by bacteria. Here we investigate and identify which non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings and how these challenges might be overcome. We quantified biofilm formation on a well-known polymer brush coating of poly(L-lysine)graft-poly(ethylene glycol) (PLL-g-PEG) on titanium. The coating successfully resisted colonization by Staphylococcus aureus and Pseudomonas aeruginosa, but not Staphylococcus epidermidis. This colonization pattern was also reflected on the adhesion forces measured on single bacterial cells. The biofilm produced from S. epidermidis on PLL-g-PEG were found to be rich in polysaccharides and extracellular DNA, and quantification of DNA, polysaccharides and proteins on PLL-g-PEG surfaces revealed that although the coating almost fully resisted protein adsorption, polysaccharides could adsorb, and exposure to DNA led to desorption of the polymer from the titanium surface. We hypothesized that this problem could be overcome by increasing the polymer brush density to better resist the penetration of DNA and polysaccharides into the polymer layer. Indeed, high density PLL-g-PEG brushes prepared by the recently discovered temperature-induced polyelectrolyte (TIP) grafting method resisted the interaction with DNA and polysaccharides, and therefore also the colonization by S. epidermidis. The TIP grafting is a simple improvement of PLL-g-PEG brush formation, and our results suggest that it provides an important advancement to the bacterial resistance by polymer brush coatings.Statement of SignificanceThe antifouling properties of poly(ethylene glycol) brush coatings against protein adsorption are well documented, but it is not well understood why these coatings do not perform as well against bacterial colonization when tested against a wide range of species and over periods of days. Here we investigated bacterial colonization on poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) grafted on Ti, and revealed that bacteria relying mostly on polysaccharides and extracellular DNA for adhesion and biofilm formation could successfully colonize PLL-g-PEG coated surfaces. The coatings could not resist adsorption of polysaccharides, and DNA could even desorb the coatings from the Ti surface. Fortunately, the shortcomings of conventional PLL-g-PEG could be overcome by increasing the graft density, using the recently discovered and very simple grafting method, 'temperature-induced polyelectrolyte (TIP) grafting'. Our study highlights that it is of utmost importance to develop coatings which resist adsoprtion of non-proteinaceous bacterial adhesins such as polysaccharides and DNA, and we demonstrated that TIP grafted high density PLL-g-PEG coatings are promising materials to achieve diverse bacterial resistance. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Infectious diseases are everywhere around us, and we need to keep improving our knowledge about our defence mechanisms and the evasion strategies of the pathogens. The work in this thesis describes multiple techniques that can contribute to fast screening methods in order to come up with new strategies against infectious diseases. The use of automated micro-injectors in combination with large flow cytometers and automated microscopy has shown added value (chapters 2, 3 & 4) for research questions about (opportunistic) pathogens. The collaboration between scientists of different research areas has proved to be very successful in the development of an easy to use analysis platform for the analysis of RNAseq data (chapter 5). This has led to very detailed description of host molecular expression patterns following infection by these pathogens. This could be used to gain more insight in how biomaterials behave in a host environment in the presence or absence of infection (chapter 6). All together this has led this to a variety of research methods that can be used for studies of infections caused by many bacteria such as S. epidermidis and M. marinum described in this thesis, but, also by other microbes, such as fungi. zebrafish, Staphylococcus-epidermidis, Mycobacterium-marinum, high-throughput,screening, method-development, transcriptome-analysis, molecular-cell-biology, immunology, host-pathogen-interaction; disease-models

Polyetheretherketone (PEEK) films were oxygen plasma treated to increase surface free energy and characterized by X-ray photoelectron microscopy, atomic force microscopy, and water contact angles. A parallel plate flow chamber was used to measure Staphylococcus epidermidis, Staphylococcus aureus, and U-2 OS osteosarcomal cell-line adhesion to the PEEK films in separate monocultures. In addition, bacteria and U-2 OS cells were cocultured to model competition between osteoblasts and contaminating bacteria for the test surfaces. Plasma treatment of the surfaces increased surface oxygen content and decreased the hydrophobicity of the materials, but did not lead to a significant difference in bacterial or U-2 OS cell adhesion in the monocultures. In the S. epidermidis coculture experiments, the U-2 OS cells adhered in greater numbers on the treated surfaces compared to the untreated PEEK and spread to a similar extent. However, in the presence of S. aureus, cell death of the U-2 OS occurred within 10 h on all surfaces. The results of this study suggest that oxygen plasma treatment of PEEK may maintain the ability of osteoblast-like cells to adhere and spread, even in the presence of S. epidermidis contamination, without increasing the risk of preoperative bacterial adhesion. Therefore, oxygen plasma-treated PEEK remains a promising method to improve implant surface free energy for osseointegration. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4427-4434, 2014.

Bacterial cell surfaces play a crucial role in their adhesion to surfaces. In the present study, physico-chemical cell surface properties of Pseudomonas aeruginosa, isolated from a case of contact lens associated keratitis, are determined for mid-exponential and early stationary phase cells and for cells after exposure to a lens care solution or after mechanical damage by sonication. Exposure to a lens care solution and mechanical cell surface damage reduced the cell surface hydrophobicity and water contact angles decreased from 129 degrees to 96 degrees and 83 degrees, respectively. Zeta potentials in saline (-9 mV) were hardly affected after mechanical damage, but tri-modal zeta potential distributions, with subpopulation zeta potentials at -11, -28 and -41 mV, were observed after exposure of bacteria to a lens care solution. X-ray photoelectron spectroscopy indicated changes in the amounts of oxygen-, nitrogen- and phosphorus-rich cell surface components. Mid-exponential phase cells had more nitrogen-rich cell surface components than early stationary phase cells, but water contact angles and zeta potentials were not very different. In addition, mid-exponential phase cells adhered better than early stationary phase cells to hydrophobic and hydrophilic substrate in a parallel plate flow chamber. The capacity of P. aeruginosa to adhere was decreased after inflicting cell surface damage. Exposure to a lens care solution yielded a larger reduction in adhesion capacity than sonication, likely because sonication left most of the cells in a viable state, in contrast to exposure to a lens care solution. It is argued that for clinically relevant experiments. it may be preferable to work; with surface damaged cells rather than with gently harvested organisms. (C) 2001 Elsevier Science B.V. All rights reserved.

The biofilm mode of growth protects the plaque microorganisms against environmental attacks, such as from antimicrobials or detergents. Detergents have a demonstrated ability to detach initially adhering bacteria from enamel surfaces, but the ability of detergent components to detach plaque bacteria is not always obvious from in vivo experiments and reports on their clinical efficacy are inconsistent. It is likely that antimicrobials or detergents are unable to penetrate the plaque and reach the bacteria that actually link the plaque mass to the substratum surface. Attenuated total reflectance/Fourier transform infrared spectroscopy was used to measure the transport of sodium lauryl sulphate (SLS) through Streptococcus mutans HG 985 biofilms. The transport of SLS to the base of the S. mutans biofilms was not hindered, while moreover an accumulation of SLS near the base of the biofilms was found, suggesting that SLS was adsorbed to biofilm components. X-ray photoelectron spectroscopy confirmed the ability of S. mutans, grown on sucrose supplemented medium, to adsorb SLS, and simultaneously indicated that exposure of cells to SLS might lead to a loss of surface proteins. Furthermore, experiments in a parallel-plate flow chamber demonstrated that initially adhering S. mutans HG 985 could be stimulated to detach by SLS, but that, depending on the growth stage of the biofilm, only maximally 27% of biofilm bacteria could be stimulated to detach by a 4% (w/v) SLS solution.

Curing biomaterial-associated infection (BAI) frequently includes antibiotic treatment, implant removal and re-implantation. However, revision implants are at a greater risk of infection as they may attract bacteria from their infected surroundings. Polymer brush-coatings attract low numbers of bacteria, but the virtue of polymer brush-coatings in vivo has seldom been investigated. Here, we determine the possible benefits of polymer brush-coated versus pristine silicone rubber in revision surgery, using a murine model.BAI was induced in 26 mice by subcutaneous implantation of silicone rubber discs with a biofilm of Staphylococcus aureus Xen29. During the development of BAI, half of the mice received rifampicin/vancomycin treatment. After 5 days, the infected discs were removed from all mice, and either a polymer brush-coated or pristine silicone rubber disc was re-implanted. Revision discs were explanted after 5 days, and the number of cfu cultured from the discs and the surrounding tissue was determined.None of the polymer brush-coated discs after antibiotic treatment appeared colonized by staphylococci, whereas 83% of the pristine silicone rubber discs were re-infected. Polymer brush-coated discs also showed reduced colonization rates in the absence of antibiotic treatment when compared with pristine silicone rubber discs. Tissue surrounding the discs was culture-positive in all cases.Polymer brush-coatings are less prone to re-infection than pristine silicone rubber when used in revision surgery, i.e. when implanted in a subcutaneous pocket infected by a staphylococcal BAI. Antibiotic pre-treatment during the development of BAI hardly had any effect in preventing the colonization of pristine silicone rubber.

11 p.-6 fig., Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system., This work was supported by grants SAF2009-10824 and SAF2012-39444-C02-01/02 from Dirección General de Investigación Científica y Técnica and MINECO, respectively.

International audience; Aims The purpose of this study was to quantify the extracellular matrix of Listeria monocytogenes biofilm. A preliminary study was carried out to establish a relationship between phylogenetic lineage of 27 strains and their ability to form biofilm in various conditions. Methods and Results Biofilm formation on microtitre plates of 27 strains of L.monocytogenes belonging to lineages I or II was evaluated in different conditions [two temperatures (37 and 22 degrees C) and two media (tryptone soy broth yeast extract medium (TSBYE) and MCDB 202 defined medium)] using crystal violet assay. Lineage II strains produced significantly more biofilm than lineage I strains. In microtitre plates assay, biofilm quantities were greater in MCDB 202 vs TSBYE medium [confirmed by scanning electron microscopy (SEM) analysis] and at 37 vs 22 degrees C. Cultivable bacteria from biofilm population on Petri dishes were enumerated in greater quantities in TSBYE than in MCDB 202 medium. The SEM investigation established that L.monocytogenes biofilms produce extracellular matrix in both media at 37 degrees C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain that presented similar pH values and exopolymer contents in both media. Proteins were the most abundant exopolymer components, followed by DNA and polysaccharides. Conclusions The interpretation of results of biofilm quantification was depending on the growth conditions, the viability of the bacteria and the analysis method. The quantities of proteins, DNA and polysaccharides were different according to the strains and the medium. Significance and Impact of the Study This study screened the potential of a wide panel of L.monocytogenes strains to synthesize exopolymers in biofilm growing condition. The characterization of L.monocytogenes biofilm composition may help to develop new strategies to prevent the formation of biofilms and to remove the biofilms.

To evaluate the effect of total bowel decontamination (TD) and selective bowel decontamination (SD) in a non-protective environment clinical and laboratory data of children treated for acute leukaemia between 1983 and 1991 were analysed retrospectively. From 1983 until 1989 34 patients [18 acute non-lymphoblastic leukaemia (ANLL) patients, 16 acute lymphoblastic leukaemia (ALL) patients] received TD and 31 patients (8 ANLL patients, 23 ALL patients) received SD from 1987 until 1991. TD consisted of colistin sulphate, neomycin, cephaloridine and amphotericin B orally as well as Orabase and sterilized food, while the patients were nursed in a single room. SD consisted of oral colistin sulphate, neomycin and amphotericin B. Those patients with ANLL were nursed in a single roomy patients with ALL were nursed in a single room during remission induction therapy only. All patients except those with ANLL receiving TD received Pneumocystis carinii pneumonia prophylaxis with cotrimoxazole. Because the two groups were heterogeneous for diagnosis and chemotherapy the occurrence of fever (central body temperature at least 38.5 degrees C) and major infections (septicaemia or infections of the deep tissues or organs) were registered during periods of neutropenia (neutrophilic granulocytes less than or equal to 500/mm(3) for at least 8 days). Patients on TD had 55 periods of neutropenia, patients on SD 80, Patients on TD had 89.1 periods of fever/100 periods of neutropenia whereas patients on SD had 56.3. Also patients on TD had 27.3 major infections/100 periods of neutropenia whereas patients on SD had 11.3, Major infections predominantly consisted of septicaemia caused by gram-positive bacteria. We conclude that, in this study, TD in a non-protective environment does not offer better protection against major infections than SD in patients with ALL or ANLL.

Purpose Biomaterial-associated infections (BAI) remain a major concern in modern health care. BAI is difficult to treat and often results in implant replacement or removal. Pathogens can be introduced on implant surfaces during surgery and compete with host cells attempting to integrate the implant. Here we studied the influence of prophylactically given cephatholin in the competition between highly virulent Staphylococcus aureus and human osteoblast-like cells (U-2 OS, ATCC HTB-94) for a poly(methyl methacrylate) surface in vitro using a peri-operative contamination model. Method S. aureus was seeded on the acrylic surface in a parallel plate flow chamber prior to adhesion of U-2 OS cells. Next, S. aureus and U-2 OS cells were allowed to grow simultaneously under shear (0.14 1/s) in a modified culture medium containing cephatholin for 8 h, the time period this drug is supposed to be active in situ. Subsequently, the flow was continued with modified culture medium for another 64 h. Results In the absence of cephatholin, highly virulent S. aureus caused U-2 OS cell death within 18 h. In contrast, the presence of cephatholin for 8 h resulted in survival of U-2 OS cell up to 72 h during simultaneous growth of U-2 OS cells and bacteria. Not all adhering bacteria were killed however, but they showed a delayed growth. Conclusions These findings are in line with the recalcitrance of biofilms against antibiotic treatment observed clinically, and represent another support for the use of in vitro co-culture models in mimicking the clinical situation.

Biomaterial-associated infections occur on both permanent implants and temporary devices for restoration or support of human functions. Despite increasing use of biomaterials in an aging society, comparatively few biomaterials have been designed that effectively reduce the incidence of biomaterial-associated infections. This review provides design guidelines for infection-reducing strategies based on the concept that the fate of biomaterial implants or devices is a competition between host tissue cell integration and bacterial colonization at their surfaces.

Biomaterial-associated infections occur on both permanent implants and temporary devices for restoration or support of human functions. Despite increasing use of biomaterials in an aging society, comparatively few biomaterials have been designed that effectively reduce the incidence of biomaterial-associated infections. This review provides design guidelines for infection-reducing strategies based on the concept that the fate of biomaterial implants or devices is a competition between host tissue cell integration and bacterial colonization at their surfaces.

Bacteria adhere to virtually all natural and synthetic surfaces [1,2]. Although there are a number of different reasons as to why bacteria adhere to a surface, the summarizing answer is brief: ‘‘Adhesion to a surface is a survival mechanism for bacteria’’. Nutrients in aqueous environments have the tendency to accumulate at surfaces [1,3], giving adhering bacteria a benefit over free floating, so-called planktonic ones. This is why mountain creeks may contain crystal clear, drinkable water, while stepping stones underneath the water may be covered with a slippery film of adhering microbes. In the oral cavity, adhesion to dental hard and soft tissues is life-saving to the organisms, because microbes that do not manage to adhere and remain planktonic in saliva are swallowed with an almost certain death in the gastrointestinal tract. Bacterial adhesion is generally recognized as the first step in biofilm formation, and for the human host, the ability of a bacterium to adhere is a definite virulence factor, especially in immunocompromised patients and in the growing number of elderly patients relying on biomaterials implants and devices for the restoration of function after (oncological) intervention surgery, trauma, or wear [4]. Well-known examples of biomaterials implants are dental implants, vascular grafts, and prosthetic hips and knee joints. Bacterial adhesion is a virulence factor, because it stimulates the organism to produce extracellular polymeric substances (EPSs), such as polysaccharides, proteins, nucleic acids, and lipids [5], through which they embed themselves in a protective matrix. This protective matrix provides mechanical stability to a biofilm and constitutes the main difference between planktonic bacteria and bacteria adhering to a surface in a socalled biofilm mode of growth. Bacteria organized in biofilms are at least ten to 1,000 times more resistant to antibiotics [6] than bacteria in a planktonic state and can cope much better with unfavorable external conditions in the host immune system than their planktonic counterparts. Not surprisingly, the fate of an infection associated with a biomaterials implant is mostly removal and replacement of the implant [7], at high costs to the health care system and great inconvenience to the patient.

The aim of this study was to investigate the role of extracellular DNA (eDNA) on the adhesion strength of Streptococcus mutans LT11 on substrata with different hydrophobicities at high and low ionic strengths. AFM adhesion forces to a hydrophilic and hydrophobic substratum increased with increasing surface-delay times and ionic strength and were stronger on a hydrophobic than on a hydrophilic substratum. The presence of eDNA on the streptococcal cell surface enhanced its adhesion force to a hydrophobic substratum significantly more than to a hydrophilic substratum, especially after bond maturation. Bond maturation on a hydrophilic substratum was accompanied by an increasing number of minor adhesion peaks, indicating the involvement of acid base interactions, whereas on the hydrophobic substratum surface the number of minor adhesion peaks remained low. More minor adhesion peaks developed on the hydrophilic substratum at low ionic strength than at high ionic strength. The final rupture distance in retraction force distance curves was independent of ionic strength on a hydrophilic substratum and increased with increasing surface delay time. On the hydrophobic surface, the final rupture distance did not increase with surface delay time but was significantly smaller at low than at high ionic strength. Final rupture distances were different in presence and absence of eDNA, and the lower values of this difference coincided with the decrease in hydrodynamic radius of the streptococci upon increasing ionic strength, measured using dynamic light scattering. AFM also yielded higher values for the ionic strength induced difference in final rupture distance because in AFM rupture is forced, while in dynamic light scattering differences in radius are only induced by ionic strength differences.

The fluctuation-induced, Casimir-like interaction between two parallel rods of length L adsorbed on a fluid membrane is calculated analytically at short separations d<, Comment: 6 pages, 3 figures

Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a newin vitromodel for the post-operative bacterial contamination of implant surfaces and investigate the effects of contamination on the balance between mammalian cell growth and bacterial biofilm formation. U2OS osteosarcoma cells were seeded on poly(methyl methacrylate) in different densities and allowed to grow for 24 h in a parallel-plate flow chamber at a low shear rate (0.14 s−1), followed by contamination withStaphylococcus epidermidisATCC 35983 at a shear rate of 11 s−1. The U2OS cells and staphylococci were allowed to grow simultaneously for another 24 h under low-shear conditions (0.14 s−1). Mammalian cell growth was severely impaired when the bacteria were introduced to surfaces with a low initial cell density (2.5×104cells cm−2), but in the presence of higher initial cell densities (8.2×104cells cm−2and 17×104cells cm−2), contaminating staphylococci did not affect cell growth. This study is believed to be the first to show that a critical coverage by mammalian cells is needed to effectively protect a biomaterial implant against contaminating bacteria.