Anti-adhesive properties of fish tropomyosins

Aims:  We have recently found that preconditioning of stainless steel surfaces with an aqueous fish muscle extract can significantly impede bacterial adhesion. The purpose of this study was to identify and characterize the primary components associated with this bacteria-repelling effect.
Methods and Results:  The anti-adhesive activity was assayed against Escherchia coli K-12, and bacterial adhesion was quantified by crystal violet staining and sonication methods. Proteolytic digestion, elution and fractionation experiments revealed that the anti-adhesive activity of the extract was linked to the formation of a proteinaceous conditioning film composed primarily of fish tropomyosins. These fibrous proteins formed a considerable anti-adhesive conditioning layer on and reduced bacterial adhesion to several different materials including polystyrene, vinyl plastic, stainless steel and glass. The protein adsorption profiles obtained from the various materials did not differ significantly, but elution was often incomplete making minor qualitative/quantitative differences indiscernible.
Conclusions:  The data highlights the significance of protein conditioning films on bacterial adhesion and emphasizes the importance of substratum's physiochemical properties and exposure time with regards to protein adsorption/elution efficiency and subsequent bacterial adhesion.
Significance and Impact of the Study:  Fish tropomyosin-coatings could potentially offer a nontoxic and relatively inexpensive measure of reducing bacterial colonization of inert surfaces.     

Conditioning film and initial biofilm formation on ceramics tiles in the marine environment

The formation of biofilm on surfaces in the marine environment is believed to be an important factor driving colonization and recruitment of some sessile invertebrate communities. The present study follows the process of biofilm buildup on unglazed ceramic tiles deployed into the marine environment in the northern Gulf of Eilat. PCR-DGGE of film eluted from the tile surface indicated the presence of bacteria as early as 2 h after deployment. The makeup of the biofilm bacterial community was dynamic. Bacterial presence was apparent microscopically 6 h after deployment, though a developed biofilm was not observed until 24 h following deployment. Total organic carbon (TOC) data suggest that a conditioning film was built within the first four hours following deployment. During this time period TOC reached the highest level possibly due to adhesion of organics (e.g., sugars, proteins and humic substances) from the water column. We suggest that the primary adhering bacteria, whilst still in the reversible stage of adhesion, utilize the conditioning film as food causing the decrease in TOC. Understanding the dynamics between these primary bacterial settlers is of importance, since they may play a role on the succession of invertebrate species settlement onto artificial surfaces.     

The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Bond Strengthening in Oral Bacterial Adhesion to Salivary Conditioning Films

Transition from reversible to irreversible bacterial adhesion is a highly relevant but poorly understood step in initial biofilm formation. We hypothesize that in oral biofilm formation, irreversible adhesion is caused by bond strengthening due to specific bacterial interactions with salivary conditioning films. Here, we compared the initial adhesion of six oral bacterial strains to salivary conditioning films with their adhesion to a bovine serum albumin (BSA) coating and related their adhesion to the strengthening of the binding forces measured with bacteria-coated atomic force microscopy cantilevers. All strains adhered in higher numbers to salivary conditioning films than to BSA coatings, and specific bacterial interactions with salivary conditioning films were accompanied by stronger initial adhesion forces. Bond strengthening occurred on a time scale of several tens of seconds and was slower for actinomyces than for streptococci. Nonspecific interactions between bacteria and BSA coatings strengthened twofold faster than their specific interactions with salivary conditioning films, likely because specific interactions require a closer approach of interacting surfaces with the removal of interfacial water and a more extensive rearrangement of surface structures. After bond strengthening, bacterial adhesion forces with a salivary conditioning film remained stronger than those with BSA coatings.     

Bacterial biofilm and clogging of biliary stents

Endoscopic biliary stenting has become a standard palliative treatment for obstructive jaundice due to malignancies of the pancreas and the hepatobiliary system. Despite the high initial success rate in achieving biliary drainage, durable endoscopic stenting has been limited by the clogging of biliary stents, usually after 4–5 months, due to formation of an adherent bacterial biofilm. Various methods have been investigated for the prevention of bacterial adhesion and prolongation of stent patency. These include: 1) prophylactic use of antimicrobial agents and bile salts; 2) testing of new stent material and new designs for these biliary stents; and 3) the recent introduction of self-expandable metal stents. Each method has its own merits as well as specific problems. This article reviews the pathogenesis of biofilm formation on the biliary stents and the latest status of research in avoiding the problem of stent occlusion.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Adhesion of monocytes to medical steel as used for vascular stents is mediated by the integrin receptor Mac-1 (CD11b/CD18; alphaM beta2) and can be inhibited by semiconductor coating

Implantation of stents into stenosed arteries helps to restore normal blood flow in ischemic organs. However, limited biocompatibility of the applied medical steel can cause acute thrombosis and long-term restenosis. Adhesion of monocytes to stent metal may participate in those acute and long-term complications of stent placement. Based on described prominent electrochemical properties of the interaction between the monocyte integrin receptor Mac-1 and its various ligands, we hypothesized, that this receptor is a central mediator of monocyte adhesion to stent metal and that semiconductor coating of medical steel reduces monocyte adhesion. Adhesion of monocytes on L-316 stainless steel was directly evaluated by light microscopy. Mac-1 could be identified as mediator of monocyte adhesion, since cell adhesion could be blocked by anti-Mac-1-antibodies, including the cross-reacting anti-GPIIb/IIIa antibody fragment abciximab. To further prove the central role of Mac-1, two CHO cell lines were generated expressing recombinant Mac-1 either as wild type, resulting in a low affinity receptor, or mutant with a GFFKR deletion of the alpha(M) subunit, resulting in a high affinity receptor. Indeed, adhesion was specific for Mac-1 and dependent on the affinity state of this integrin. Finally, we could demonstrate that Mac-1-mediated adhesion of monocytes to stents can be significantly inhibited by silicon carbide coating of the stent metal. In conclusion, the integrin Mac-1 and its affinity state could be identified as major mediators of monocyte adhesion on medical steel. As therapeutic strategies, the blockade of Mac-1 by antibodies or silicon carbide coating of steel inhibits monocyte adhesion on stents.     

A new stent with streamlined cross-section can suppress monocyte cell adhesion in the flow disturbance zones of the endovascular stent

We proposed a new stent with streamlined cross-sectional wires, which is different from the clinical coronary stents with square or round cross-sections. We believe the new stent might have better hemodynamic performance than the clinical metal stents. To test the hypothesis, we designed an experimental study to compare the performance of the new stent with the clinical stents in terms of monocyte (U-937 cells) adhesion. The results showed that when compared with the clinical stents, the adhesion of U-937 cells were much less in the new stent. The results also showed that, when Reynolds number increased from 180 (the rest condition for the coronary arteries) to 360 (the strenuous exercise condition for the coronary arteries), the flow disturbance zones in the clinical stents became larger, while they became smaller with the new stent. The present experimental study therefore suggests that the optimization of the cross-sectional shape of stent wires ought to be taken into consideration in the design of endovascular stents.     

Fabrication of drug-eluting covered stents with micropores and differential coating of heparin and FK506

To reduce in-stent restenosis rates, we developed a novel drug-eluting covered stent with a microporous elastometric covered film, in which its luminal surface was flat and immobilized with heparin for anticoagulation and its outer surface immobilized with FK506 to prevent neointimal hyperplasia. One month after implantation into the bilateral common carotid arteries, all stented arteries were patent and the luminal surfaces were fully covered with a confluent of endothelial cells irrespective of the drug immobilization. In the control group, which consisted of covered stents without drug immobilization, intensive inflammatory cells adjacent to the stents and neointimal hyperplasia, indicating vascular injury, were observed. In contrast, in the developed drug-eluting stents, only a few inflammatory cells around the stent strut and covered film were observed, and there was no significant neointimal thickening.     

Traversal of a polarized epithelium by pathogenic Neisseriae: facilitation by type IV pili and maintenance of epithelial barrier function.

BACKGROUND: Gonococci (GC) and meningococci (MC) are gram-negative bacterial pathogens that infect human mucosal epithelia. We would like to understand the functions of specific bacterial components at each stage of mucosal colonization: adhesion, cell invasion, and traversal into subepithelial tissues. As no animal model of mucosal colonization by GC or MC is available, increasingly sophisticated in vitro approaches have been used to address these issues. MATERIALS AND METHODS: We adapted the polarized T84 human epithelial cell system to study GC and MC colonization. Epithelial barrier function was monitored by permeability to soluble tracers and with electrical resistance measurements. Polarized cells were used to assay bacterial traversal of the monolayers, and cells grown on plastic were used to assay adhesion and cell invasion. RESULTS: All pathogenic Neisseriae examined traversed the monolayers. The traversal times were species specific and identical to times established previously in organ culture studies. In contrast to experiments with some enteric pathogens, transmigration by GC and MC was not accompanied by disruption of the epithelial barrier. GC mutants lacking type IV pili were compromised in adhesion, invasion, and traversal of T84 cells. CONCLUSIONS: Experiments with polarized T84 cells mimic key features of organ culture infections and reveal additional aspects of neisserial infection. Epithelial barrier function can be retained during bacterial traversal. Experiments with a nonpiliated GC mutant and its wild-type parent indicated an unexpected role for pili in cell invasion. Our results are consistent with the hypothesis that bacterial adhesion, invasion, or both are rate-limiting for traversal across the epithelium.     

Surface immobilization of kanamycin-chitosan nanoparticles on polyurethane ureteral stents to prevent bacterial adhesion

Bacterial adhesion is a major problem that can lead to the infection of implanted urological stents. In this study, kanamycin-chitosan nanoparticles (KMCSNPs) were immobilized on the surface of a polyurethane ureteral stent (PUS) to prevent urinary bacterial infection. KMCSNPs were synthesized using the ionic gelation method. The synthesized KMCSNPs appeared spherical with a ζ-average particle size of 225 nm. KMCSNPs were immobilized on the PUS surface by covalent immobilization techniques. The surface-modified PUS was characterized using attenuated total reflectance Fourier transform infrared spectroscopy, field emission scanning electron microscopy, and energy dispersive X-ray spectroscopy. The surface-modified PUS showed significantly increased antibacterial activity against Escherichia coli MTCC 729 and Proteus mirabilis MTCC 425 relative to the surface of an unmodified PUS. These findings suggest that the KMCSNP-immobilized PUS has the potential to prevent bacterial infection in the human urinary tract.     

Leitbakteria of microbial biofilm communities causing occlusion of biliary stents

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.     

烫伤创面绿脓杆菌定植动态的实验研究

本文从细菌以多细胞生理活动观点出发,以认识定植稳定过程为目的。进行了实验大白鼠烫伤创面绿脓杆菌定植与抗定植动态观察。通过用铁浸染色法对细菌群体结构定量化研究,用糖包被负染法对群体结构内部结构观察,对粘附在组织表面细菌数量的测定,反映结构与粘附力的关系。进一步结合电镜观察及细菌生长状态的分析,证明了烫伤创面上绿脓杆菌群体结构的形成是细菌分裂繁殖所致。通过群体结构,糖包被,粘附力及生长状态的动态观察,表现出与定植的稳定程度呈平行关系,显示其重要性。联系抗定植力研究,表明定植与抗定植的一致性。最后分析了定植三个主要条件,和稳定性定植的三要素。     

Specific and non-specific interactions in bacterial adhesion to solid substrata

Abstract Based on a literature review, a hypothesis is forwarded on the mechanism of initial bacterial adhesion to solid substrata, which accounts both for the role of specific microscopic surface components as well as for the role of non-specific macroscopic surface properties (surface free energy, zeta potential or hydrophobicity). Three distinct regions in the adhesion process are suggested in which at large and intermediate separation distances adhesion is mediated by the macroscopic surface properties as surface free energy and surface charge, respectively. At small separation distances specific short-range interactions can occur, leading to a strong and irreversible bonding, provided the water film present in between the interaction surfaces can be removed. A major role of hydrophobic groups, supposed to be associated with bacterial surface appendages is suggested to be its dehydrating capacity, enabling the removal of the vicinal water film yielding small areas of direct contact between protruberant parts of the cell surface and the substratum.     

冠状动脉内重叠支架置入术的临床研究进展

重叠支架置入术是临床上用来治疗冠状动脉弥漫性长病变的常用方法,以往曾采取重叠裸金属支架置入术,但其临床预后不佳,目前较为常用的重叠药物洗脱支架置入术被认为相对安全、有效,但仍存在许多潜在问题。本文介绍了适合使用重叠支架置入术进行治疗的冠状动脉病变的特点,回顾了重叠裸金属支架的临床应用情况,阐述了近年来药物洗脱支架的发展以及重叠药物洗脱支架置入术的优势,比较了四种负载不同药物的药物洗脱支架同种重叠置入后的临床疗效,观察了异种药物洗脱支架混合重叠置入后的临床特点和支架重叠段对临床预后的影响,分析了重叠支架置入术与几种其它治疗冠状动脉弥漫性长病变方法的应用区别,并对新一代药物支架的重叠应用进行了展望。     

Bacterial patterning at the three-phase line of contact with microtextured alkanes

Aliphatic crystallites, characteristic of the eicosane and docosane components of naturally occurring lipids, were found to form microtextures that were structured by specific interactions with ordered graphite (HOPG) used as the underlying substratum, as confirmed by scanning electron microscopy (SEM) and fast Fourier transform (FFT) analysis. Confocal scanning laser microscopy (CLSM) showed highly directed bacterial alignment for two bacterial species (spherical and rod-shaped), reflecting the preferential orientation of the crystallite–air–water interfaces to give linear and triangular bacterial patterning. The mechanisms of bacterial attachment are demonstrated in terms of the balance between effective radial adhesional forces and the capillary forces resulting from the water contact angle of the bacteria at the three-phase line (TPL) of the lipid surface. It is suggested that these microtextured surfaces, which exhibit the ability to limit bacterial adhesion to a precise patterning at the lipid TPL, could be used as a means of controlling bacterial colonization.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization.     

The effect of dissolved organic carbon on bacterial adhesion to conditioning films adsorbed on glass from natural seawater collected during different seasons

Adhesion of three marine bacterial strains, i.e. Marinobacter hydrocarbonoclasticus, Psychrobacter sp. and Halomonas pacifica with different cell surface hydrophobicities was measured on glass in a stagnation point flow chamber. Prior to bacterial adhesion, the glass surface was conditioned for 1 h with natural seawater collected at different seasons in order to determine the effect of seawater composition on the conditioning film and bacterial adhesion to it. The presence of a conditioning film was demonstrated by an increase in water contact angle from 15 degrees on bare glass to 50 degrees on the conditioned glass, concurrent with an increase in the amount of adsorbed organic carbon and nitrogen, as measured by X-ray photoelectron spectroscopy. Multiple linear regression analysis on initial deposition rates, with as explanatory variables the temperature, salinity, pH and concentration of dissolved organic carbon (DOC) of the seawater at the time of collection, showed that the concentration of DOC was most strongly associated with the initial deposition rates of the three strains. Initial deposition rates of the two most hydrophilic strains to a conditioning film, increased with the concentration of DOC in the seawater, whereas the initial deposition rate of the most hydrophobic strain decreased with an increasing concentration of DOC.     

Biliary Endoprosthesis: A Prospective Analysis of Bacterial Colonization and Risk Factors for Sludge Formation

Bacterial colonization of biliary stents is one of the driving forces behind sludge formation which may result in stent occlusion. Major focus of the study was to analyze the spectrum and number of microorganisms in relation to the indwelling time of stents and the risk factors for sludge formation. 343 stents were sonicated to optimize the bacterial release from the biofilm and identified by matrix-associated laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF). 2283 bacteria were analyzed in total. The most prevalent microorganisms were Enterococcus species (spp.) (504;22%), followed by Klebsiella spp. (218;10%) and Candida spp. (188;8%). Colonization of the stents mainly began with aerobic gram-positive bacteria (43/49;88%) and Candida spp. (25/49;51%), whereas stents with an indwelling time>60 days(d) showed an almost equal colonization rate by aerobic gram-negative (176/184;96%) and aerobic gram-positive bacteria (183/184;99%) and a high proportion of anaerobes (127/184;69%). Compared to stents without sludge, more Clostridium spp. [(P = 0.02; Odds Ratio (OR): 2.4; 95% confidence interval (95%CI): (1.1–4.9)]) and Staphylococcus spp. [(P = 0.03; OR (95%CI): 4.3 (1.1–16.5)] were cultured from stents with sludge. Multivariate analysis revealed a significant relationship between the number of microorganisms [P<0.01; OR (95%CI): 1.3(1.1–1.5)], the indwelling time [P<0.01; 1–15 d vs. 20–59 d: OR (95%CI): 5.6(1.4–22), 1–15 d vs. 60–3087 d: OR (95% CI): 9.5(2.5–35.7)], the presence of sideholes [P<0.01; OR (95%CI): 3.5(1.6–7.9)] and the occurrence of sludge. Stent occlusion was found in 70/343(20%) stents. In 35% of cases, stent occlusion resulted in a cholangitis or cholestasis. In conclusion, microbial colonization of the stents changed with the indwelling time. Sludge was associated with an altered spectrum and an increasing number of microorganisms, a long indwelling time and the presence of sideholes. Interestingly, stent occlusion did not necessarily lead to a symptomatic biliary obstruction.