Initial Adhesion Rate Research Articles

Probing the Affinity of Coronavirus with Contact Surfaces in Simulated Body Fluids.

Transmission of viral pathogens has raised serious public health concerns, but the affinity and strength of viruses adhering to high-touch surfaces are not clear. We systematically investigated the propensities of a coronavirus, Murine hepatitis virus A59 (MHV), adhering onto and releasing from four representative contact surfaces, silica, stainless steel, cellulose, and polystyrene, in simulated saliva and urine using quartz crystal microbalance with dissipation monitoring (QCM-D). We also quantified the interactions between MHV and contact surfaces using atomic force microscopy (AFM). Both initial adhesion rates and saturated adhesion mass of MHV were higher in urine buffer than in saliva buffer, which is attributed to the higher repulsions between the virus and surfaces in the presence of mucin. The maximum adhesion mass of MHV follows the order of stainless steel > silica > cellulose ≈ polystyrene in both urine and saliva buffers. Stainless steel and silica are surfaces with likely higher risks of virus contamination due to their highest maximum adhesion mass in both urine and saliva buffers and lower virus release percentages upon water rinse. The results of this study will provide insights into risk assessment and control of pathogens associated with contact surfaces.

Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Characterization of stratified EPS and their role in the initial adhesion of anammox consortia

Anammox bacteria tend to self-aggregate, and biofilm-based anammox processes are widely used as sustainable alternative methods for nitrogen removal from wastewater. However, the mechanism for the initial adhesion of anammox bacteria remains unclear. In this work, extracellular polymeric substances (EPS) were extracted separately from anammox granules and then characterized by multi-methods. The adhesion properties of anammox consortia to biotic and abiotic surfaces were examined separately before and after the extraction of three stratified EPS, using aggregation assays and a quartz crystal microbalance technique with dissipation monitoring, respectively. The extraction of each of the three stratified EPS gradually increased the initial aggregation of anammox consortia from 77.6 ± 3.0% to 85.2 ± 2.6%. Fourier transform infrared spectrometry confirmed that the aggregation of anammox consortia depended largely on the interactions between functional groups on the cell surfaces. All three stratified EPS had positive effects on the initial adhesion rate and mass of anammox consortia to abiotic surfaces. More importantly, the structure of the adhered layer was more compact before the extraction of each of the three stratified EPS. We therefore hypothesized that the initial adhesion among anammox consortia was due to the ability of the anammox bacteria to express adhesion molecules on the bacterial surfaces and that the three stratified EPS were excreted to adhere inert particulates and form a compact biofilm structure. This study clarifies the role of stratified EPS on the initial formation of anammox biofilms and provides a theoretical basis for accelerating the formation of anammox biofilms.

Effects of denture adhesives on growth and morphological transformation of Candida albicans

PurposeWe investigated the effects of denture adhesives (cream (Cr), powder (Po), and cushion (Cu)) on growth and adhesive-related morphological transformation of Candida albicans. For this purpose, the numbers of adherent C. albicans, hyphae-specific gene expressions, and the SEM images were examined. MethodsAcrylic resin blocks were prepared as controls (Co). Cr, Po, and Cu were thinly spread on the surface of the resin block.C. albicans suspension was seeded on the specimens and incubated at 4 °C for 2 h. The numbers of C. albicans adhering to each specimen at each incubation time period (1, 2, 3, 6, 12, and 24 h) were quantified using real-time RT-PCR. The hyphae-specific genes expressions were examined. The surface of each specimen was observed under the SEM to detect the transformation to the hyphal form. ResultsThe initial adhesion rates in all groups were not statistically significant. The numbers of C. albicans adhering increased with time in all groups, and those adhering to the Cr, Po, and Cu were significantly greater than that adhering to the Co. In the Cr and Po, the hyphal-specific genes expressions were higher after incubation for 6 h. The transformation to the hyphal form was identified in the Cr and Po after incubation for 6 and 12 h. ConclusionsThe denture adhesives used in this study accelerated the growth of C. albicans. Moreover, the early transformation to the hyphal form on the Cr- and Po-type adhesives was observed, suggesting that we should carefully use Cr- and Po-type adhesives.

Biofilm Formation Potential of Heat-Resistant Escherichia coli Dairy Isolates and the Complete Genome of Multidrug-Resistant, Heat-Resistant Strain FAM21845.

ABSTRACTWe tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays.IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption.

Influence of flow direction and flow rate on the initial adhesion of seven Listeria monocytogenes strains to fine polished stainless steel

The effects of flow direction and shear stress on the adhesion of different strains of Listeria monocytogenes to fine polished stainless steel under liquid flow conditions were investigated. Furthermore, the relationship between cell surface properties and cell size and the initial adhesion rate (IAR) was studied. A method, including fluorescence microscopy and a flow perfusion system, was developed and used to examine the real-time initial cell adhesion of different L. monocytogenes species in situ to opaque surfaces under flow conditions.The results demonstrated that shear stress was the determining factor for the initial adhesion of L. monocytogenes under flow conditions. The flow direction in relation to the orientation of surface features (the scratches) could be disregarded. IARs were dependent on the shear stress and strain type. The strain EGDe, which had the lowest IAR, had the largest cell size, was the least hydrophobic and possessed the most electron-donating cell surface. Except for the L. monocytogenes strain EGDe, no clear correlations were found between the IAR and cell surface properties, or cell size.In conclusion, many factors may be involved in determining the initial adhesion of L. monocytogenes to stainless steel under flow conditions. Two of the main factors are flow rate/shear stress and strain specificity.

Initial adhesion of Listeria monocytogenes to solid surfaces under liquid flow

Some strains of the food borne pathogen Listeria monocytogenes persist in food processing environments. The exact reason behind this phenomenon is not known, but strain differences in the ability to adhere to solid surfaces could offer an explanation. In the present work, initial adhesion of nine strains of L. monocytogenes was investigated under liquid flow at two levels of shear stress on six different surfaces using a flow chamber set-up with microscopy measurements. The surfaces tested were glass and PVC, and glass coated with beef extract, casein, and homogenised and unhomogenised milk. In addition, the effect of prior environmental stress (5% NaCl, low nutrient availability) on initial adhesion was investigated. The hydrophobicity of the investigated surfaces was determined by contact angle measurements and the surface properties of the investigated L. monocytogenes strains were determined using Microbial Adhesion To Solvents (MATS). All surfaces with the exception of PVC were found to be hydrophilic. Strain differences were found to significantly influence the initial adhesion rate (IAR) of all nine strains to all the surfaces (p<0.05) at both low and high shear stress. Furthermore, there was a significant effect of the surfaces tested (p<0.05) in the adhesion ability of almost all strains. The IAR was affected by flow rate (shear stress) as seen by a decrease in adhesion at high shear stress for most strains. A significant effect of interactions between strain-surface and strain-shear stress (p<0.001) was observed but not of interactions between surface-shear stress. No correlation between surface hydrophobicity and IAR was observed. Addition of 5% NaCl during propagation resulted in a decrease in IAR whilst propagation in low nutrient media caused an increase indicating a general change in surface characteristics under these conditions. Known persisting strains did not display general better adherence.

Reducing Milk Protein Adhesion Rates: A Transient Surface Treatment of Stainless Steel

The primary factor in developing a surface that diminishes fouling during food processing is reducing the rate of formation of the initial foulant layer. Modifying the properties of plant processing surfaces, which are typically 316 L or 304 L stainless steel, is one way of achieving a reduction in initial adhesion rate. We have developed a transient treatment that modifies the surface properties of the metal oxide surface, that is shown to reduce the interaction potential between the stainless steel processing surface and phosphate anions. The phosphate anions are involved in the conditioning layer associated with foulant formation during milk processing. The transient nature of the treatment is that it is present during the processing cycle, but is removed at high pH, which is an environment that exists during the cleaning cycle. The transient surface treatment was utilized in fouling trials, conducted in a pilot plant milk pasteurizer, a model system to emulate milk fouling in industrial plant. Evaluation of the result shows significant reductions in fouling rates compared to the typical controls, as indicated by the pressure drop across the plate heat exchanger and the observed reduction in the fouling of the heat exchanger plate.

Effect of Membrane Surface Properties During the Fast Evaluation of Cell Attachment

The biofouling potential is one of the important factors to design and to select membranes for water and wastewater treatment. In this investigation, the effect of membrane surface properties during the attachment of S. cerevisiae cells was examined using a laboratory‐scale membrane filtration cell enabling direct microscopic observation of microbial cell deposition. The experimental results from 6 commercially available membranes showed that the initial adhesion rate, k d , was affected by the zeta potentials, hydrophobicity, and roughness of membrane surfaces. The k d value was significantly lower at the membrane which had more negative, hydrophilic, and smooth surfaces. The results will be helpful to minimize the time for selecting membranes in different situations, and for testing the performance of newly designed membranes.

Adhesion and biofilm formation of Candida albicans on native and Pluronic-treated polystyrene

Candida albicans forms part of the normal human flora whose growth is usually restricted by the normal flora bacteria and the host's immune system. It is an opportunistic fungal pathogen that causes infections in immunocompromised individuals, mechanical trauma victims and iatrogenic patients. Candida albicans can ingress the human host by adhering to a plastic surface (i.e. prosthetic devices, catheters, artificial organs, etc.) that is subsequently implanted, and forms a protective biofilm that provides a continuous reservoir of yeast to be hematogenously dispersed. In order for the medical profession to battle device-related infections, initial adhesion and biofilm formation of C. albicans needs to be better understood. There has been some skepticism as to whether the initial adhesion events bear any relationship to subsequent biofilm formation. Thus, to better comprehend the relationship between the initial adhesion rates and growth rate and biofilm formation, these events were studied on two different, well-defined culture surfaces, native polystyrene and Pluronic F127-conditioned polystyrene. The adhesion studies determined that Pluronic F127 adsorption dramatically reduced the adhesion of C. albicans to polystyrene. The biofilm growth studies, analyzed by confocal scanning laser microscopy, revealed that Pluronic F127 decreased the biofilm surface coverage, cluster group size, thickness and the presence of hyphal elements over the untreated polystyrene. These findings indicate that the effect of a material's surface chemistry on the initial adhesion process has a direct influence on subsequent biofilm formation.

Enhancement of biofilm formation onto surface-modified hollow-fiber membranes and its application to a membrane-aerated biofilm reactor

Surface-modified hollow-fiber membranes were prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidylmethacrylate (GMA), onto a polyethylene-based fiber (PE-fiber). The epoxy ring of GMA was opened by introduction of diethylamine (DEA). The bacterial adhesivity to this material (DEA-fiber) was tested by immersion into a nitrifying bacterial suspension. The initial adhesion rates and the amount of attached bacteria of the DEA-fiber were 6-10-fold and 3-fold greater than those of the PE fiber, respectively. A membrane-aerated biofilm reactor (MABR) composed of DEA fibers was developed for partial nitrification with nitrite accumulation. Prior to the nitrification test, it was confirmed that the oxygen supply rate (OSR) was proportional to air pressure up to 100 kPa, allowing easy control of oxygen supply. Stable nitrite accumulation was observed in the partial nitrification test at a fixed oxygen supply throughout the operation period, indicating that oxygen was consumed only by ammonia oxidizers. Furthermore, it was demonstrated that oxygen utilization efficiency (OUE) in the ammonia oxidation process was nearly 100% after 300 h incubation.

Protein and bacterial fouling characteristics of peptide and antibody decorated surfaces of PEG-poly(acrylic acid) co-polymers

The potential for base poly(ethylene glycol) graft poly(acrylic acid) PEG-g-PA copolymers and surface-modified PEG-g-PA materials to inhibit random protein fouling and bacterial adhesion are investigated. PEG-g-PA co-polymers were synthesized that inhibited non-specific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV) or fragments of antibodies to monocyte/macrophage integrin receptors (Anti-VLA4, Anti- β 1, Anti- β 2, and Anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Materials produced in this work were characterized using: hydrophobicity by contact angle; angle-resolved X-ray Photoelectron Spectroscopy to confirm the presence of PEG in the bulk material and the surface; degree of hydration; differential scanning calorimetry; and thermal gravimetric analysis. To evaluate the non-fouling efficacy of the various modified surfaces, three proteins, human serum albumin, human fibronectin (Fraction I) and human immunoglobulin were 125I labeled. Samples of base PEG-g-PA and PEG-g-PA, modified with various peptides, were exposed to solutions containing either 2 or 200 μg/ml of one of the labeled proteins at 37°C for 24 h. PEG-g-PA substrata modified with directly bound peptides exhibited protein adsorption that varied depending upon the surface bounded peptide. PEG-g-PA modified with peptides linked by linear PEG tethers reduced protein adsorption at 24 h by ∼45% in comparison to PEG-g-PA. Peptides linked by way of StarPEO and StarlikePEO tethers further decreased protein adsorption in comparison to PEG-g-PA. The ability of peptide:PEOtethers to inhibit protein adsorption appeared to be a function of type and surface coverage of the PEO tether and not influenced by the amount or molecular structure the tethered peptide. Peptides directly coupled to the PEG-g-PA increased the amount of protein fouling relative to controls and there appeared to be some dependency of the amount of protein adsorption on which peptide was tethered. Two 14C-labeled pathogens, Staphylococcus epidermidis and Pseudomonas aeruginosa, were used to quantify the degree of bacterial adhesion using two types of laminar flow cell chambers; one that provided invasive sampling of the target substrata and one that provided non-invasive microscopic surveillance of adhering bacterial cells. Attachment of both species to PEG-g-PA and peptide-modified PEG-g-PA was reduced compared to the basic poly(acrylic acid). Presence of peptides on the surface, whether directly bound or bound by the PEO tether did not influence adhesion of P. aeruginosa relative to controls. S. epidermidis adhesion rates increased slightly for those materials where peptides were directly bound to the surface but were reduced relative to base PEG-g-PA when peptides were bound by PEO tethers. All PEG-g-PA surfaces modified with fragments of monoclonal antibodies dramatically enhanced bacterial initial adhesion rates and maximum extent of attachment.

The effects of urine and temperature on the physicochemical surface properties and adhesion behaviour of uropathogenic bacteria

Bacterial adhesion in relation to urinary-tract infections has gained importance in the last years because of the increasing catheterization in hospitals to assist post-surgery flow of urine. Since the initial adhesion of bacteria to biomaterials is governed by physicochemical forces emerging from the physicochemical properties of both interacting phases, we have investigated the physicochemical surface changes of uropathogen Enterococcus faecalis ATCC29212 bacteria due to the presence of urine in its growth medium and to the differences in the environmental temperature. Urine-grown cells were found to be less hydrophobic based on water contact angles at 22°C, while no changes were detected at 37°C. In addition, they exhibited higher acid-base surface energy component than urine-free cultured cells. These changes in surface properties were also reflected in thermodynamic predictions of the adhesion to glass and silicone, which were experimentally compared with the in vitro adhesion curves obtained in a parallel plate flow chamber. The shapes of the adhesion graphs indicated that interaction free energies should be used to describe only the initial adhesion stages. Adhesion to silicone was always enhanced by urine-grown cells, while the adhesion to glass did not seem to be affected by the urine constituents. Despite the fact that the interaction free energies were not able to explain the adhesion process in some cases, changes in the electron-donor and electron-acceptor parameters of their surface free energy due to urine addition seemed to have a relation with initial adhesion rates.

Cellular Responses on a Wettability Gradient Surface with Continuous Variations in Surface Compositions of Carbonate and Hydroxyl Groups

This article reports adhesion, spreading, migration, and proliferation responses of bovine endothelial cells (ECs) on the surface with a unidirectional wettability gradient. The surface chemical gradient, in which molar fractions of cyclic carbonate and hydroxyl groups were inversely varied, was prepared by continuous immersion of a poly(vinylene carbonate) (PVCa) film into an aqueous solution of sodium hydroxide at a constant speed. Advancing water contact angles of the surface, which ranged from 60° for an untreated PVCa surface to around 20° for a well-treated surface, gradually decreased with the distance from the untreated end of the film. Quantitative analyses of cellular characteristics of adhered cells on the gradient surface, which included the initial adhesion rate and the morphological indices of adhered cells such as spreading area, peripheral length, and circular coefficient, showed that these cells gave the highest values on the untreated PVCa surface and rapidly decreased on the surface regions with shorter periods of hydrolysis, followed by a slower decrease on surface regions with longer periods ofhydrolysis with increasing distance from the untreated end. Little adhesion occurred in a well-hydrolyzed, very hydrophilic surface region. The highest migration rate of adhered cells occurred on the slightly hydrophobic surface region (advancing contact angle, 35°) where adhered cells tended to delaminate as tissue formation proceeded. These results indicate that each cellular potential of ECs is dependent on the wettability of the gradient surface, producing polymorphorous states of adhered cells. Thus, the wettability gradient surface served as an excellent substrate to simultaneously study cellular response dependence on surface chemical composition and wettability.

Effects of cellobiose on cellulose colonization by a mesophilic, cellulolytic Clostridium (strain C401)

When cultured on a mixture of cellobiose and cellulose, Clostridium C401 did not initially attach to cellulose but remained in the liquid phase. After cellobiose exhaustion, bacterial cells grew in association with the insoluble cellulose. Carboxymethylcellulase (CMCase) production on Avicel cellulose was four- to fivefold greater than on cellobiose, and cellulose-grown cells adhered to filter paper with an initial adhesion rate about four- to fivefold greater than did cellobiose-grown cells. Using tritiated thymidine incorporation as a measure of growth, it appeared that transfer of strain C401 from cellobiose to cellulose required an adaptation phase. An extracellular cellulase complex was isolated by affinity chromatography. This enzyme system is a multicomponent aggregate (molecular mass above 5 MDa), and yielded two major polypeptide bands by SDS-PAGE having molecular masses of 130 and 70 kDa. Cellobiose strongly inhibited Avicelase activity and slightly inhibited p-nitrophenylcellobiose hydrolysis (pNPCbase), but had no effect on the CMCase activity of the cellulase complex. In addition, polyclonal antibodies, raised against the purified 130 kDa protein inhibited Avicelase activity, but not CMCase and pNPCbase activities.

Modulation of Trypanosoma cruzi adhesion to host muscle cell membranes by ligands of muscarinic cholinergic and beta adrenergic receptors

Plasma membrane vesicles (PMVs) of Trypanosoma cruzi adhered to L6 myoblast host cells as a function of time and concentration in saturation phenomena in a similar fashion to that reported in a previous publication [1]. The initial adhesion rate (A 0) of T. cruzi PMVs to L6 myoblasts in tissue culture was inhibited by acetylcholine (10 -5 M), isoproterenol (10 -5 M) and norepinephrine (10 -8 M) (range 29.1–50.3% of control). Atropine, the antagonist of muscarinic cholinergic receptors (10 -5 M), and propranolol or pindolol, the antagonists of β-adrenergic receptors (10 -5 M), were also equal inhibitors of the T. cruzi PMV to L6 myoblast adhesion rate (range 26.1 to 55.5% of control). The α-adrenergic receptor ligands yohimbine and phentolamine (10 -5 M) showed no A 0 inhibitory activity in similar assays. The interaction of T. cruzi PMVs with type I host muscle sarcolemma receptors was clearly defined in assays which used porcine heart atrial membranes (PAMs) immobilized on cationic polyacrylamide beads. In this parasite membrane-host cell membrane assay system, 10 -10 M atropine and 5 × 10 -9 M propanolol produced a shift of an S-shaped T. cruzi PMV to PAM saturation isotherm to the right, suggesting that a negative cooperative interaction was produced between the intramembrane ligand binding site and another, surface heterotropic T. cruzi PMV adhesion site. Atropine and propranolol were equieffective inhibitors of the T. cruzi striated muscle sarcolemma recognition process, raising the possibility that T. cruzi attachment molecules annexed pairs of muscarinic cholinergic and β-adrenergic receptors to effect adhesion of the two membrane surfaces.

Complementary surface epitopes, myotropic adhesion and active grip in Trypanosoma cruzi-host cell recognition

Plasma membranes with complementary surface epitopes and with essentially the same orientation as tissue infective metacyclic trypomastigotes and epimastigotes of Trypanosoma cruzi adhere to L6 myoblast host cells preferentially to smooth muscle and epithelial cells as a function of time, surface area and concentration in saturation phenomena at 4 and 37°C. The initial adhesion rates are partially calcium ion dependent and, at saturation, they are also dependent on high energy phosphorylated intermediates, exerting an active grip on adherent parasite membranes. These phenomena are consistent with the existence of parasite attachment molecules on the external surface of the plasma membrane and complementary host cell receptor structures with the capacity to bind them.