Air-flow resistances of silicone rubber voice prostheses after formation of bacterial and fungal biofilms.

Laryngectomized patients use silicone rubber voice prostheses to rehabilitate their voice. However, biofilm formation limits the lifetime of voice prostheses by causing leakage or an increased air-flow resistance and the prosthesis has to be replaced. To determine which bacterial or yeast strains, isolated from explanted voice prostheses, contribute most to increases in air-flow resistance of silicone rubber voice prostheses, biofilms consisting of either a bacterial or a yeast strain were grown on voice prostheses in the artificial throat model. The effects of these biofilms on air-flow resistances were determined by calculating the difference in air-flow resistance of the individual voice prosthesis as covered with a 7-day-old biofilm with the situation prior to biofilm formation. Conspicuously, voice prosthetic biofilms formed by the bacterial strains Staphylococcus aureus GB 2/1 and Rothia dentocariosa GBJ 41/25B and their excreted organic matter showed larger increases in air-flow resistance (more then 30 cm H(2)O.s/L) than biofilms formed by Candida species. This is contrary to the literature, where there seems to be agreement that Candida species are mainly responsible for clinical failure of silicone rubber voice prostheses.     

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-beta-(1-->6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.     

Extracellular DNA in biofilms

Extracellular DNA (eDNA) is an important biofilm component that was recently discovered. Its presence has been initially observed in biofilms of Pseudomonas aeruginosa, Streptococcus intermedius, Streptococcus mutans, then Enterococcus faecalis and staphylococci. Autolysis is the common mechanism by which eDNA is released. In P. aeruginosa eDNA is generated by lysis of a bacterial subpopulation, under control of quorum sensing system. In E. faecalis autolysis proceeds in a fratricide mode, resulting from a process similar to necrosis of eukaryotic cells. In Staphylococcus aureus autolysis originates by an altruistic suicide, i.e., a programmed cell death similar to apoptosis of eukaryotic cells. In S. aureus autolysis is mediated by murein hydrolase, while in S. epidermidis by the autolysin protein AtlE. In P. aeruginosa eDNA is located primarily in the stalks of mushroom-shaped multicellular structures. In S. aureus the crucial role of eDNA in stabilizing biofilm is highlighted by the disgregating effect of DNase I. eDNA represents an important mechanism for horizontal gene transfer in bacteria. eDNA and other microbial structural motifs are recognized by the innate immune system via the TLR family of pattern recognition receptors (PRRs).     

In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber

Biomaterial-centered infection is a dreaded complication associated with the use of biomedical implants. In this paper, the antimicrobial activity of silicone rubber with a covalently coupled 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) coating was studied in vitro and in vivo. Gram-positive Staphylococcus aureus ATCC 12600, Staphylococcus epidermidis HBH, 102, and Gram-negative Esherichia coli O2K2 and Pseudomonas aeruginos AK1 were seeded on silicone rubber with and without QAS-coating, in the absence or presence of adsorbed human plasma proteins. The viability of the adherent bacteria was determined using a live/dead fluorescent stain and a confocal laser scanning microscope. The coating reduced the viability of adherent staphylococci from 90% to 0%), and of Gram-negative bacteria from 90% to 25% while the presencc of adsorbed plasma proteins had little influence. The biomaterials were also subcutaneously implanted in rats for 3 or 7 days, while pre- or postoperatively seeded with S. aureus ATCC 12600. Preoperative seeding resulted in infection of 7 out of 8 silicone rubber implants against 1 out of 8 QAS-coated silicone rubber implants. Postoperative seeding resulted in similar infection incidences on both implant types, but the numbers of adhering bacteria were 70% lower on QAS-coated silicone rubber. In conclusion, QAS-coated silicone rubber shows antimicrobial properties against adhering bacteria, both in vitro and in vivo.     

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.     

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.     

Anti-biofilm properties of chitosan-coated surfaces

Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 (10)log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 (10)log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.     

Photodynamic action of merocyanine 540 on Staphylococcus epidermidis biofilms

Photodynamic treatment (PDT) has been proposed as a new approach for inactivation of biofilms associated with medical devices that are resistant to chemical additives or biocides. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. Effect of the combined photodynamic action of MC 540 and 532 nm laser was investigated on the viability and structure of biofilms of two Staphylococcus epidermidis strains, RP62A and 1457. Significant inactivation of cells was observed when biofilms were exposed to MC 540 and laser simultaneously. The effect was found to be light dose-dependent but S. epidermidis 1457 biofilm proved to be slightly more susceptible than S. epidermidis RP62A biofilm. Furthermore, significant killing of both types of cells was attained even when a fixed light dose was delivered to the biofilms. Confocal laser scanning microscope (CLSM) analysis indicated damage to bacterial cell membranes in photodynamically treated biofilms, while disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).     

Effect of alkaline pH on staphylococcal biofilm formation

Biofilms are a serious problem, cause of severe inconvenience in the biomedical, food and industrial environment. Staphylococcus aureus and S. epidermidis are important pathogenic bacteria able to form thick and resistant biofilms on various surfaces. Therefore, strategies aimed at preventing or at least interfering with the initial adhesion and subsequent biofilm formation are a considerable achievement. The aim of this study was to evaluate the effect of alkaline pH on bacterial adhesion and further biofilm formation of S. aureus and S. epidermidis strains by biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis. The results demonstrated that the amount of biofilm biomass formed and the surface hydrophobicity were significantly less than what were observed at higher levels of pH. SEM and CLSM images revealed a poorly structured and very thin biofilm (2.5-3 times thinner than that of the controls). The inhibiting effect of the alkaline pH on the bacterial attachment impaired the normal development of biofilm that arrested at the microcolony stage. Alkaline formulations could be promising towards the control of bacterial colonization and therefore the reduction of the biofilm-related hazard. In the clinical setting, alkaline solutions or cleaners could be promising to prevent the bacterial colonization, by treating surfaces such as catheters or indwelling medical devices, reducing the risk of biofilm related infections.     

The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines

The comparison of copolymers containing sulfobetaine or phosphobetaine moieties for use as potential biocompatible coatings has been investigated. Two statistical copolymers were produced by a free radical polymerisation technique, one based on a sulfobetaine and the other on a phosphobetaine, both with a silyl group component to allow thermal crosslinking after coating. PMMA and glass discs were dip-coated with the polymers and their properties were compared to the uncoated controls. Bacterial adhesion to these coated materials was assessed using Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa. Human macrophages and granulocytes were used to assess the adhesion and activation of inflammatory cells whilst mouse 3T3 fibroblast cells were used to assess the propensity for the materials to support fibroblast cell adhesion. In all cases the polymer coatings reduced cell adhesion with respect to the base materials. The phosphobetaine-based copolymer coatings were shown to be markedly superior to the sulfobetaine-based copolymer coatings.     

Nitric oxide-releasing sol-gels as antibacterial coatings for orthopedic implants

To assess the benefits of nitric oxide (NO)-releasing sol-gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol-gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol-gel-coated stainless steel is evaluated at both ambient and physiological temperature. Sol-gel films incubated at 25 degrees C have a lower NO flux over the first 24 h compared to those at 37 degrees C, but release more than five times longer. The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol-gel, and NO-releasing sol-gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25 degrees C and 37 degrees C. Cell adhesion to bare and sol-gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.     

Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces

In this work, we report a study of long-chain zwitterionic poly(sulfobetaine methacrylate) (pSBMA) surfaces grafted via atom transfer radical polymerization (ATRP) for their resistance to bacterial adhesion and biofilm formation. Previously, we demonstrated that p(SBMA) is highly resistant to nonspecific protein adsorption. Poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) grafted surfaces were also studied for comparison. Furthermore, we quantify how surface grafting methods will affect the long-term biological performance of the surface coatings. Thus, self-assembled monolayers (SAMs) of alkanethiols with shorter-chain oligo(ethylene glycol) (OEG) and mixed SO3-/N+(CH3)3 terminated groups were prepared on gold surfaces. The short-term adhesion (3 h) and the long-term accumulation (24 or 48 h) of two bacterial species (Gram-positive Staphylococcus epidermidis and Gram-negative Pseudomonas aeruginosa) on these surfaces were studied using a laminar flow chamber. Methyl-terminated (CH3) SAM on gold and a bare glass were chosen as references. p(SBMA) reduced short-term adhesion of S. epidermidis and P. aeruginosa relative to glass by 92% and 96%, respectively. For long-term biofilm formation, qualitative images showed that p(SBMA) dramatically reduced biofilm formation of S. epidermidis and P. aeruginosa as compared to glass.     

Staphylococcus quorum sensing in biofilm formation and infection

Cell population density-dependent regulation of gene expression is an important determinant of bacterial pathogenesis. Staphylococci have two quorum-sensing (QS) systems. The accessory gene regulator (agr) is genus specific and uses a post-translationally modified peptide as an autoinducing signal. In the pathogens Staphylococcus aureus and Staphylococcus epidermidis, agr controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. However, the role of agr during infection is controversial. A possible second QS system of staphylococci, luxS, is found in a variety of Gram-positive and Gram-negative bacteria. Importantly, unlike many QS systems described in Gram-negative bacteria, agr and luxS of staphylococci reduce rather than induce biofilm formation and virulence during biofilm-associated infection. agr enhances biofilm detachment by up-regulation of the expression of detergent-like peptides, whereas luxS reduces cell-to-cell adhesion by down-regulating expression of biofilm exopolysaccharide. Significant QS activity in staphylococci is observed for actively growing cells at a high cell density, such as during the initial stages of an infection and under optimal environmental conditions. In contrast, the metabolically quiescent biofilm mode of growth appears to be characterized by an overall low activity of the staphylococcal QS systems. It remains to be shown whether QS control in staphylococci represents a promising target for the development of novel antibacterial agents.     

Comparative antibody-mediated phagocytosis of Staphylococcus epidermidis cells grown in a biofilm or in the planktonic state

Staphylococcus epidermidis is an important cause of nosocomial infections. Virulence is attributable to elaboration of biofilms on medical surfaces that protect the organisms from immune system clearance. Even though leukocytes can penetrate biofilms, they fail to phagocytose and kill bacteria. The properties that make biofilm bacteria resistant to the immune system are not well characterized. In order to better understand the mechanisms of resistance of bacteria in biofilms to the immune system, we evaluated antibody penetration throughout the biofilm and antibody-mediated phagocytic killing of planktonic versus biofilm cells of S. epidermidis by using a rabbit antibody to poly-N-acetylglucosamine (PNAG). These antibodies are opsonic and protect against infection with planktonic cells of PNAG-positive Staphylococcus aureus and S. epidermidis. Antibody to PNAG readily penetrated the biofilm and bound to the same areas in the biofilm as did wheat germ agglutinin, a lectin known to bind to components of staphylococcal biofilms. However, biofilm cells were more resistant to opsonic killing than their planktonic counterparts in spite of producing more PNAG per cell than planktonic cells. Biofilm extracts inhibited opsonic killing mediated by antibody to PNAG, suggesting that the PNAG antigen within the biofilm matrix prevents antibody binding close to the bacterial cell surface, which is needed for efficient opsonic killing. Increased resistance of biofilm cells to opsonic killing mediated by an otherwise protective antibody was due not to a biofilm-specific phenotype but rather to high levels of antigen within the biofilm that prevented bacterial opsonization by the antibody.     

Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis

Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear beta-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent in S. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression in ica- and polysaccharide-positive strains of both S. aureus and S. epidermidis. These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.     

Antibacterial and antifouling catheter coatings using surface grafted PEG-b-cationic polycarbonate diblock copolymers

Intravascular catheter-associated infections (CAIs), which are normally induced by microbial adhesion and subsequent biofilm formation, are a major cause of morbidity and mortality. Therefore, strategies to prevent CAIs are in great demand. In this study, a series of diblock copolymers of PEG and cationic polycarbonate with various compositions were synthesized by metal-free organocatalytic ring-opening polymerization, and coated onto silicone rubber (a commonly used catheter material) at different concentrations via a reactive polydopamine coating. Static contact angle and X-ray photoelectron spectroscopy measurements proved the successful coating, and quartz crystal microbalance results showed that the coating thickness increased as polymer concentration increased. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates - leading causes of intravascular CAIs - were employed to evaluate the antibacterial and antifouling activities of the polymer coatings. Polymer coatings with a hydrophobic component effectively killed planktonic MSSA and MRSA in solution and prevented their fouling on silicone rubber surface. Live/dead cell staining experiments revealed that polymer coatings with the optimal polymer composition possessed significantly higher antifouling activity than PEG coating. In addition, scanning electron microscopic studies showed that the polymer coating inhibited S.?aureus biofilm formation over a period of 7 days. Furthermore, the polymer coating caused no significant hemolysis, and there was no blood protein adsorption or platelet adhesion observed. Therefore, PEG-b-cationic polycarbonates with optimal compositions are effective antifouling and antibacterial coatings for the prevention of intravascular CAIs.     

血糖对树鼩感染表皮葡萄球菌清除能力及植入性材料细菌黏附性的影响

背景：高浓度葡萄糖培养条件下,细菌在生物材料表面有较强的生物膜形成能力。 目的：观察血糖升高对表皮葡萄球菌在动物体内清除能力及植入性生物材料上细菌生物膜形成的影响。 方法：注射链脲佐菌素建立高血糖树鼩模型(血糖≥11.1 mmol/L),采用生物膜形成阳性与阴性表皮葡萄球菌感染高血糖与正常对照树鼩,并同时在动物股静脉内植入PVC导管。 结果与结论：感染生物膜形成阳性的表皮葡萄球菌株后,血糖≥11.1 mmol/L树鼩血液、心脏、肝脏、肾脏、胰腺的细菌感染率及菌落计数较正常对照组高(P < 0.05);扫描电镜观察血糖≥11.1 mmol/L组植入生物材料上有明显的生物膜形成。感染生物膜形成阴性表皮葡萄球菌株后无论血糖高低,均未观察到生物膜形成。表明血糖升高不仅使植入生物材料树鼩的细菌清除能力下降,同时可诱导细菌在植入生物材料表面形成明显的生物膜。     

人工心脏瓣膜材料细菌粘附的初步研究

在体外采用平板计数方法测定了金黄色葡萄球菌，表皮样葡萄球菌，大肠杆菌，绿脓杆菌及白色念珠菌等５种细菌对医用涤纶及热解碳的粘附。结果表明：实验所用细菌对两种人工心脏瓣膜材料均有粘附性，其对涤纶的粘附均明显强于热解碳。     

In vitro and in vivo comparisons of staphylococcal biofilm formation on a cross-linked poly(ethylene glycol)-based polymer coating

Poly(ethylene glycol) (PEG) coatings are known to reduce microbial adhesion in terms of numbers and binding strength. However, bacterial adhesion remains of the order of 10⁴ cm^?2. It is unknown whether this density of bacteria will eventually grow into a biofilm. This study investigates the kinetics of staphylococcal biofilm formation on a commercially produced, robust, cross-linked PEG-based polymer coating (OptiChem^®) in vitro and in vivo. OptiChem^® inhibits biofilm formation in vitro, and although adsorption of plasma proteins encourages biofilm formation, microbial growth kinetics are still strongly delayed compared to uncoated glass. In vivo, OptiChem^®-coated and bare silicone rubber samples were inserted into an infected murine subcutaneous pocket model. In contrast to bare silicone rubber, OptiChem^® samples did not become colonized upon reimplantation despite the fact that surrounding tissues were always culture-positive. We conclude that the commercial OptiChem^® coating considerably slows down bacterial biofilm formation both in vitro and in vivo, making it an attractive candidate for biomaterials implant coating.     

Effect of plastic catheter material on bacterial adherence and viability

The kinetics of adherence of single isolates of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Escherichia coli to catheters made of polyvinyl chloride (PVC), Teflon, siliconised latex, polyurethane and Vialon was evaluated by a radiometric assay. Radiolabelled bacteria (10(8) cfu/ml) were incubated in vials containing 1-cm lengths of catheter for up to 3 days. The peak of maximal adherence to each biomaterial was reached after 24 h for P. aeruginosa and after 72 h for the other strains. Bacterial adherence to PVC and siliconised latex was significantly higher (2-6 times; p less than 0.05) than to the other biomaterials for all the strains. The lowest values of adherence were observed with polyurethane and Vialon for the staphylococci but with Teflon for E. coli and P. aeruginosa. Bacterial viability and growth was evaluated in eluates obtained from incubation of segments of each catheter in buffer for 24 h. None of the eluates affected the viability of the staphylococci. However, all of them, significantly increased the growth of E. coli and P. aeruginosa with the exception of the eluate from siliconised latex, in which the inoculum count was reduced to an undetectable level for E. coli. We conclude that bacterial adherence to catheters may depend in part on the nature of the biomaterial and that certain substances eluted from the catheters may affect the viability and growth of different micro-organisms.