Differences in colonisation of five marine bacteria on two types of glass surfaces

The retention patterns of five taxonomically different marine bacteria after attachment on two types of glass surfaces, as-received and chemically etched, have been investigated. Contact angle measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectrometry (XPS) were employed to investigate the impact of nanometer scale surface roughness on bacterial attachment. Chemical modification of glass surfaces resulted in a ～1 nm decrease in the average surface roughness (R _a) and the root-mean-squared roughness (R_q ) and in a ～8 nm decrease in the surface height and the peak-to-peak (R _max) and the 10-point average roughness (R_z ). The study revealed amplified bacterial attachment on the chemically etched, nano-smoother glass surfaces. This was a consistent response, notwithstanding the taxonomic affiliation of the selected bacteria. Enhanced bacterial attachment was accompanied by elevated levels of secreted extracellular polymeric substances (EPS). An expected correlation between cell surface wettability and the density of the bacterial attachment on both types of glass surfaces was also reported, while no correlation could be established between cell surface charge and the bacterial retention pattern.     

<Emphasis Type="Italic">Escherichia coli,Pseudomonas aeruginosa</Emphasis>, and<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> Attachment Patterns on Glass Surfaces with Nanoscale Roughness

Attachment tendencies of Escherichia coli K12, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus CIP 68.5 onto glass surfaces of different degrees of nanometer-scale roughness have been studied. Contact-angle and surface-charge measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) were employed to characterize substrata and bacterial surfaces. Modification of the glass surface resulted in nanometer-scale changes in the surface topography, whereas the physicochemical characteristics of the surfaces remained almost constant. AFM analysis indicated that the overall surface roughness parameters were reduced by 60–70%. SEM, CLSM, and AFM analysis clearly demonstrates that although E. coli, P. aeruginosa and S. aureus present significantly different patterns of attachment, all of the species exhibited a greater propensity for adhesion to the “nano-smooth” surface. The bacteria responded to the surface modification with a remarkable change in cellular metabolic activity, as shown by the characteristic cell morphologies, production of extracellular polymeric substances, and an increase in the number of bacterial cells undergoing attachment.     

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.     

Effect of Support Material on the Development of Microbial Fixed Films Converting Acetic Acid to Methane*

A study of the development of methanogenic fixed films on pieces of polyvinyl chloride plastic, etched glass and baked clay showed that support material markedly affected the rate of attachment and growth of bacteria converting acetic acid to methane. Film development, as indicated by the rate of acetate conversion to methane and carbon dioxide, was threefold faster on fired clay than on either PVC plastic or etched glass. Scanning electron micrographs showed that the film of bacteria attached to clay was thick and uniform, while the film attached to PVC plastic was thin although still uniform. Attachment to etched glass was spotty. The characteristics of clay which made it a superior support appeared to be its rough, porous surface which offered attachment sites to the micro-organisms and the presence of minerals in the clay, particularly iron which is known to stimulate methanogenesis and growth.     

Effects of surface topography and chemistry of Rumex obtusifolius leaves on the attachment of the beetle Gastrophysa viridula

Broad-leaved dock, Rumex obtusifolius L. (Polygonaceae), is the main host plant of the green dock beetle, Gastrophysa viridula Degeer (Coleoptera: Chrysomelidae). Adult beetles are able to attach and walk on leaves of this plant. Leaf surface is rather uneven, because of irregularly shaped prominent epidermal cells with a maximum height of about 9 µm. The surface is covered with a smooth epicuticular wax layer having relatively low free surface energy (FSE). The aim of this study was to measure beetle attachment force applying a 'centrifugal technique' on adult insects on the leaf surface and other substrates, in order to understand the effect of surface architecture and its physicochemical properties on insect attachment. We compared forces on an adaxial leaf surface with forces on a smooth silanized glass plate having low FSE, and on a polishing paper having slightly lower FSE and similar surface roughness (asperities' size = 9 µm). Smooth plate made of normal untreated clean glass with high FSE was used as a reference substrate. On the leaf surface and the polishing paper, attachment forces were lower compared to both glass samples. There was no significant difference between force values on leaves and the polishing paper, but on both glass surfaces they were significantly higher than on the other substrates. Hydrophobicity alone explains a decrease in attachment force of the beetle, but when combined with roughness the decrease in force is four times greater.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Influence of Substratum Characteristics on the Attachment of a Marine Pseudomonad to Solid Surfaces

The attachment of a marine Pseudomonas sp. to a variety of surfaces was investigated, and the number of bacteria which became attached was related to the surface charge and degree of hydrophobicity of the substratum. Large numbers of bacteria attached to hydrophobic plastics with little or no surface charge [Teflon, polyethylene, polystyrene, poly(ethylene terephthalate)]; moderate numbers attached to hydrophilic metals with a positive (platinum) or neutral (germanium) surface charge; and very few attached to hydrophilic, negatively charged substrata (glass, mica, oxidized plastics). The results suggest that both electrostatic and hydrophobic interactions are involved in bacterial attachment.     

Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Proving the antimicrobial spectrum of an amphoteric surfactant-sol-gel coating: a food-borne pathogen study

An antimicrobial coating was evaluated in this work for its antimicrobial efficacy against common food-borne pathogens. Dodecyl-di(aminoethyl)-glycine, an organic disinfectant, was immobilized in a silicon oxide matrix to generate thin films over surfaces by means of the sol-gel process. Tetraethoxysilane was used as the polymeric precursor. No alteration of optical transparency on the covered surfaces was observed. Topographic images obtained with atomic force microscopy showed a homogeneous film with no additional roughness added by the polymer to the surface. The attenuated total reflectance-Fourier transform infrared spectral data showed the presence of dodecyl-di(aminoethyl)-glycine in the silicon oxide network after a normal cleaning procedure. The antimicrobial efficacy test was performed by exposing coated slides to suspensions of common food-borne pathogens: Escherichia coli, Staphyloccocus aureus, E. coli O157:H7, Salmonella typhi, S. cholerasuiss, Listeria innocua and L. monocytogenes. The coating activity was not only bacteriostatic but also bactericidal. The percent reduction of viable microorganism exposure over 24 h to the coated surface ranged between 99.5%, for the more resistant gram-positive bacteria, and over 99.999%, for most gram-negative bacteria. The silicon matrix itself did not account for any reduction of viable microbial, even more an increase was observed.     

The role of exopolymers in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Green mussel Perna viridis L.: attachment behaviour and preparation of antifouling surfaces

The green mussel Perna viridis LINNE can be kept in simulated seawater for more than 6 months in good condition. The mussel forms many threads by secreting an adhesive protein from the foot, and attaches with more than 50 byssal threads, which makes most mussels clump together. In order to investigate the preparation of the antifouling surfaces toward green mussels, the attachment of mussels was tested using glass surfaces modified with silane coupling agents, together with non-treated material surfaces such as glass and silicone. The correlation between the attachment percentage and the mean number of the secreted byssus was highly significant, indicating that the mussel selects a favorable surface prior to the secretion of byssus. The relationships between the mussel attachment and the surface chemical parameters (surface free energy (sfe) and its dispersion and polar components) were examined based on a working hypothesis, which we have previously reported. The result of statistical regression test indicated that a certain correlation was found between the dispersion component and the mussel attachment, while the polar component did not correlate to the mussel attachment. The present surface chemical approach provided an additional clue for the preparation of ecologically clean antifouling materials that takes into account the combination of the wettability of both the marine adhesive proteins (MAP) and the modified surfaces.     

Comparison of fluorinated polymers against stainless steel, glass and polypropylene in microbial biofilm adherence and removal

Biofilm formation is a long-standing problem in ultrapure water and bioprocess fluid transport lines. The standard materials used in these applications (316L stainless steel, polypropylene and glass) have long been known to be good surfaces for the attachment of bacteria and other biological materials. To compare the relative tenacity of biofilms grown on materials used in manufacturing processes, a model system for biofilm attachment was constructed that approximates the conditions in industrial process systems. New fluorinated polymers were compared to the above materials by evaluating the surface area coverage of bacterial populations on materials before and after mild chemical treatment. In addition, contact angle studies compared the relative hydrophobicity of surfaces to suspensions of bacteria in growth media, and scanning electron microscopy and atomic force microscopy studies were used to characterize surface smoothness and surface defects. Biofilm adherence to polymer-based substrata was determined to be a function of both surface finish and surface chemistry. Specifically, materials that are less chemically reactive, as indicated by higher contact angle, can have rougher surface finishes and still be amenable to biofilm removal. Received 20 March 1997/ Accepted in revised form 15 July 1997     

Immobilization of the purple non-sulfur bacterium Rhodobacter sphaeroides on glass surfaces

Summary A method for improving the adsorption of bacteria on glass surfaces was developed. The modification of a glass surface by LS-2480 greatly increased the number of bacteria that were immobilized. The conditions for bacteria immobilization on the modified glass surface were optimized.     

Effects of surfactant adsorption and biodegradability on the distribution of bacteria between sediments and water in a freshwater microcosm.

A microcosm containing resuspended river sediment was used to investigate the effect of anionic surfactants on the distribution of bacteria between planktonic and attached populations. Freshwater river sediment containing viable bacteria was preequilibrated in the microcosm, which was subsequently supplemented with biodegradable or recalcitrant surfactants and a non-surface-active carbon and energy source. Population dynamics of both free-living and attached bacteria were measured by epifluorescence microscopy with simultaneous analysis of the residual solution concentration of the xenobiotic carbon source. The addition of the readily biodegradable anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate in separate experiments caused an increase in the number of attached bacteria and a concomitant decrease in the number of free-living bacteria. As biodegradation of the surfactants progressed, these trends reversed and the bacterial populations had returned to their preaddition values by the time when biodegradation was completed. In contrast, sodium tetradecyl sulfate or sodium dodecane sulfonate did not stimulate bacterial association with sediment, nor were they biodegraded in the microcosm. Sodium pyruvate, a non-surface-active carbon and energy source, was readily utilized but caused no bacterial attachment to the sediment. These results indicate that for an anionic surfactant to induce bacterial attachment to river sediment, it must be biodegradable. The bacterial attachment to the sediment appears to be reversible and may be dependent on the accumulation of the surfactant at the surface or as a result of alteration of the surface free energies.     

Effects of surfactant adsorption and biodegradability on the distribution of bacteria between sediments and water in a freshwater microcosm.

A microcosm containing resuspended river sediment was used to investigate the effect of anionic surfactants on the distribution of bacteria between planktonic and attached populations. Freshwater river sediment containing viable bacteria was preequilibrated in the microcosm, which was subsequently supplemented with biodegradable or recalcitrant surfactants and a non-surface-active carbon and energy source. Population dynamics of both free-living and attached bacteria were measured by epifluorescence microscopy with simultaneous analysis of the residual solution concentration of the xenobiotic carbon source. The addition of the readily biodegradable anionic surfactants sodium decyl sulfate and sodium dodecyl sulfate in separate experiments caused an increase in the number of attached bacteria and a concomitant decrease in the number of free-living bacteria. As biodegradation of the surfactants progressed, these trends reversed and the bacterial populations had returned to their preaddition values by the time when biodegradation was completed. In contrast, sodium tetradecyl sulfate or sodium dodecane sulfonate did not stimulate bacterial association with sediment, nor were they biodegraded in the microcosm. Sodium pyruvate, a non-surface-active carbon and energy source, was readily utilized but caused no bacterial attachment to the sediment. These results indicate that for an anionic surfactant to induce bacterial attachment to river sediment, it must be biodegradable. The bacterial attachment to the sediment appears to be reversible and may be dependent on the accumulation of the surfactant at the surface or as a result of alteration of the surface free energies.     

In search of the microbe/mineral interface: quantitative analysis of bacteria on metal surfaces using vertical scanning interferometry

To understand the development of biofilms on metal surfaces, analysis of initial bacterial attachment to surfaces is crucial. Here we present the results of a study, using Shewanella oneidensis MR-1 as a model organism, in which vertical scanning interferometry (VSI) was used to investigate the initial stages of cell attachment to glass, steel and aluminium surfaces. It was found that while VSI gave unambiguous results with opaque surfaces, when reflective surfaces were used, an artifact sometimes appeared, with the bacteria appearing as rod-shaped pits rather than as cells on the surface. When the bacteria were altered to increase opacity, this artifact disappeared, and upon further investigation, it was found that the observational artifact was the result of a conflict between light reflected from the bacteria and the light reflected from the bacteria–metal interface. These results suggest that not only can bacteria be measured on surfaces using VSI, but with some modifications to the analytical software, there may be a unique window for studying the bacterial/substrate interface that can be used for quantitative observations. Imaging and characterization of the bacteria–substrate interface in vivo (previously invisible) will provide new insights into the interactions that occur at this important juncture.     

Genetic diversity of gamma-hexachlorocyclohexane-degrading sphingomonads isolated from a single experimental field

Aim:  To determine the effect of the surface roughness of denture acrylic on the attachment of Streptococcus oralis .
Methods and Results:  Roughened denture acrylic samples were assessed for bacterial attachment, over time, using microscopy. The area of the image covered by bacteria was calculated and converted into a percentage of the total area sampled. The results showed an increasing bacterial coverage with time of incubation and increasing roughness. Differences were seen between heat cured acrylic and cold cured acrylic.
Conclusion:  This study successfully demonstrated a system for the assessment of the amount of attached bacteria on denture acrylic varying roughness. The system was able to discern the difference in surface area coverage by attached bacteria over a roughness range relevant to brushing dentures with dentifrices.
Significance and Impact of Study:  This study provides strong support for the scratches caused by brushing dentures with dentifrice encouraging bacterial attachment. This is likely to have a significant effect on efficacy of denture cleaning, general hygiene and biofilm re-formation between cleaning regimens and may indicate that alternative low abrasive cleaners, such as antimicrobial denture-cleaning tablets, offer a more appropriate regimen.     

Adhesive performance of the stick-capture apparatus of rove beetles of the genus Stenus (Coleoptera, Staphylinidae) toward various surfaces

Rove beetles of the genus Stenus possess a unique adhesive prey-capture apparatus that enables them to catch elusive prey such as springtails over a distance of several millimeters. The prey-capture device combines the hierarchically organized morphology of dry adhesive systems with the properties of wet ones, since an adhesive secretion is released into the contact zone. We hypothesize that this combination enables Stenus species successfully to capture prey possessing a wide range of surface structures and chemistries. We have investigated the influence of both surface energy and roughness of the substrate on the adhesive performance of the prey-capture apparatus in two Stenus species. Force transducers have been used to measure both the compressive and adhesive forces generated during the predatory strike of the beetles on (1) epoxy resin surfaces with defined roughness values (smooth versus rough with asperity diameters ranging from 0.3 to 12 μm) and (2) hydrophobic versus hydrophilic glass surfaces. Our experiments show that neither the surface roughness nor the surface energy significantly influences the attachment ability of the prey-capture apparatus. Thus, in contrast to the performance of locomotory adhesive systems in geckos, beetles, and flies, no critical surface roughness exists that might impede adhesion of the prey-capture apparatus of Stenus beetles. The prey-capture apparatus of Stenus beetles is therefore well adapted to adhere to the various unpredictable surfaces with diverse roughness and surface energy occurring in a wide range of potential prey.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.