Impact of nano-topography on bacterial attachment

The adhesion of bacteria to surfaces is an important biological process, but one that has resisted simple categorization due to the number and complexity of parameters involved. The roughness of the substrate is known to play a significant role in the attachment process, particularly when the surface irregularities are comparable to the size of the bacteria and can provide shelter from unfavorable environmental factors. According to this scenario, roughness on a scale much smaller than the bacteria would not be expected to influence the initial attachment. To test this hypothesis, the impact of nanometer-scale roughness on bacterial attachment has been investigated using as-received and chemically etched glass surfaces. The surface modification by etching resulted in a 70% reduction in the nanoscale roughness of the glass surface with no significant alteration of its chemical composition or charge. Nevertheless, the number of bacteria adhering to the etched surface was observed to increase by a factor of three. The increase in attachment was also associated with an alteration in cellular metabolic activity as demonstrated by changes in characteristic cell morphologies and increased production of extracellular polymeric substances. The results indicate that bacteria may be more sensitive to nanoscale surface roughness than was previously believed.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.     

Bacterial attachment to stainless steel welds: Significance of substratum microstructure

AISI Type 304 L stainless steel (SS) is a widely used material in industry due to its strength and resistance to corrosion. However, corrosion on SS is reported largely at welds or adjacent areas. Bacteria were observed to colonize preferentially near welds as a result of surface roughness. In the present study, the influence of another important metal surface condition on bacterial adhesion has been evaluated, i.e. substratum microstructure. Type 304 L SS weld samples were prepared and machined to separate weld metal, the heat affected zone (HAZ) and base metal regions. The coupons were molded in resin so that only the surfaces polished to a 3 p.m finish were exposed to the experimental medium with Pseudomonas sp. isolated from a corrosive environment in Japan. The coupons were exposed for varying durations. The area of bacterial attachment showed significant differences with time of exposure and; the type of coupons. Generally, the weld metal samples showed more attachment whilst the base metal showed the least. The area of attachment was inversely proportional to the average grain size of the three samples. As the bacteria started colonizing, attachment mainly occurred on the grain boundaries of the base metal (after 8h, 84.62% and 15.38% of the total number of bacteria attached in the field of view (FOV) at the grain boundary and matrix, respectively) and on the austenite‐ferrite interface in the weld metal (after 8h, 88.33% and 11.77% of the total number of bacteria attached in the FOV at the boundary and matrix, respectively). The weld area had more grains and hence more grain boundary/ unit area than the base metal, resulting in more bacterial attachment. SEM observations showed this increased attachment of Pseudomonas sp. resulted in the initiation of microbiologically influenced corrosion (MIC) on the weld coupons by 16 d. Therefore, the results provide data to support the fact that substratum microstructure influences bacterial attachment, which in turn leads to corrosion.     

EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

The conditioning role of saliva in streptococcal attachment to hydroxyapatite surfaces

The solution properties of saliva and its role in conditioning both the substrate and the bacterial surface have been investigated with regard to the attachment of oral streptococci to hydroxyapatite surfaces. Saliva from eight subjects was used and the attachment of three organisms, Streptococcus mutans strains FA-1 (serotype b) and KPSK2 (serotype c) and S. sanguis T175-1, was studied. An adsorbed salivary layer on a hydroxyapatite surface substantially reduced the affinity of the organisms for the surface. Adsorbed saliva on the bacterial surfaces, however, tended to increase the organisms' affinity for saliva-coated apatite. The source of saliva was important in determining the extent of inhibition of attachment. The data indicated that the negatively charged and hydrophilic nature of salivary conditioning films was important in controlling bacterial adsorption to hydroxyapatite. The results also suggested that hydrophobic salivas could promote binding of the more hydrophobic bacteria known to be early colonizers of the teeth.     

Cathodic protection and microbially induced corrosion

Cathodic protection, using sacrificial anodes or impressed current, has been recognized for a long time as an effective way to prevent marine corrosion. Cathodic polarization leads to the formation of a protective calcareous layer on the surfaces. It is well documented that the attachment of bacteria to metal surfaces and subsequent biofilm formation changes some physical and chemical parameters at the interface and influences the corrosion process. The objective of this study was to determine whether there is a relationship between cathodic polarization and development of biofilms on surfaces exposed to both synthetic and natural seawater. Experiments were conducted on clean surfaces, biofilmed surfaces, in natural or synthetic seawater using bacterial monocultures and cocultures. In marine sediments, cathodically produced hydrogen encouraged growth of hydrogenase-containing sulphate-reducing bacteria while in aerated seawater biofilms competed with the magnesium and calcium deposition. Both low pH induced by bacterial metabolism and exopolymers affect the deposition process and the stability of the calcareous layer.     

The effect of metal microstructure on the initial attachment of Escherichia coli to 1010 carbon steel

Metallurgical features have been shown to play an important role in the attachment of microorganisms to metal surfaces. In the present study, the influence of the microstructure of as-received (AR) and heat-treated (HT) 1010 carbon steel on the initial attachment of bacteria was investigated. Heat treatment was carried out with the aim of increasing the grain size of the carbon steel coupons. Mirror-polished carbon steel coupons were immersed in a minimal medium inoculated with Escherichia coli (ATCC 25922) to investigate the early (15, 30 and 60?min) and relatively longer-term (4?h) stages of bacterial attachment. The results showed preferential colonisation of bacteria on the grain boundaries of the steel coupons. The bacterial attachment to AR steel coupons was relatively uniform compared to the HT steel coupons where an increased number of localised aggregates of bacteria were found. Quantitative analysis showed that the ratio of the total number of isolated (ie single) bacteria to the number of bacteria in aggregates was significantly higher on the AR coupons than the HT coupons. Longer-term immersion studies showed production of extracellular polymeric substances by the bacteria and corrosion at the grain boundaries on both types of steel coupon tested.     

Ellipsometric Measurement of Bacterial Films at Metal-Electrolyte Interfaces

Ellipsometric measurements were used to monitor the formation of a bacterial cell film on polarized metal surfaces (Al-brass and Ti). Under cathodic polarization bacterial attachment was measured from changes in the ellipsometric angles. These were fitted to an effective medium model for a nonabsorbing bacterial film with an effective refractive index (n_f) of 1.38 and a thickness (d_f) of 160 ± 10 nm. From the optical measurements a surface coverage of 17% was estimated, in agreement with direct microscopic observations. The influence of bacteria on the formation of oxide films was monitored by ellipsometry following the film growth in situ. A strong inhibition of metal oxide film formation was observed, which was assigned to the decrease in oxygen concentration due to the presence of bacteria. It is shown that the irreversible adhesion of bacteria to the surface can be monitored ellipsometrically. Electrophoretic mobility is proposed as one of the factors determining bacterial attachment. The high sensitivity of ellipsometry and its usefulness for the determination of growth of interfacial bacterial films is demonstrated.     

Bacterial cell attachment, the beginning of a biofilm

The ability of bacteria to attach to surfaces and develop into a biofilm has been of considerable interest to many groups in numerous industries, including the medical and food industry. However, little is understood in the critical initial step seen in all biofilm development, the initial bacterial cell attachment to a surface. This initial attachment is critical for the formation of a bacterial biofilm, as all other cells within a biofilm structure rely on the interaction between surface and bacterial cell for their survival. This review examines what are believed to be some of the most important aspects involved in bacterial attachment to a surface.     

Acidithiobacillus ferrooxidans fixation on mercuric surfaces and its application in stripping voltammetry

The adsorption (fixation) of bacteria Acidithiobacillus ferrooxidans on Hg and Cu metallic surfaces was qualitatively studied owing to two independent methods: frequency measurement using a quartz crystal microbalance and light absorption measuring at the Hg/bacterial culture interface. A method using a dropping mercury electrode (DME) allowed quantifying this bacterial fixation. Determining the superficial tension at Hg/bacterial culture interface led to determine bacteria adsorption on Hg through the Gibbs isotherm. A modified stripping voltammetry was proposed taking benefit of both bacterial adsorption on Hg surface and metal fixation capacity on biomass. Metal preconcentration on the biologically modified Hg electrode appeared to improve the measurement sensitivity of differential pulse anodic stripping voltammetry (DPASV). The height of the detected peaks was thus increased of 17.6% for copper, 48.4% for lead, and 132% for cadmium determinations compared to those obtained with an unmodified mercury electrode. Such augmentation depended on bulk bacteria concentration and bacteria preconcentration.     

Time-resolved fluorimetry in the study of attachment of staphylococci and streptococci on substrate-bound fibronectin

Abstract The application of time-resolved fluorimetry was evaluated in the study of staphylococcal and streptococcal attachment to fibronectin-coated coverslips. The test system allowed the use of low bacterial concentrations (2 × 10⁵−10⁷ bacteria per ml), in contrast to the much higher concentrations of bacteria used in earlier assays. The bacteria attached much better to fibronectin-coated plastic surfaces than to albumin-coated ones, but there were differences between the individual strains. Soluble fibronectin inhibited the adsorption of staphylococci but enhanced streptococcal attachment to fibronectin-coated surfaces. Purified antibodies to fibronectin inhibited both staphylococcal and streptococcal adhesion in a dose-dependent way. Our results show that time-resolved fluorimetry is a very sensitive method for quantitating bacterial attachment.     

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 substrate wettability on the attachment of marine bacteria to various surfaces

The effect of the initial substrate surface condition, as indicated by the critical surface tension for wetting, on the rate of attachment of marine bacteria to a variety of solid surfaces has been measured. The techniques used to determine the number of bacteria attached per unit surface area were a lipopolysaccharide test utilizing Limulus lysate and direct examination of the surface by scanning electron microscopy. The results obtained by the two techniques are compared and their significance to the control of microbiological slime film formation (microfouling) is discussed.     

Direct observation of microbial inhibition of calcite dissolution

Vertical scanning interferometry (VSI) provides a method for quantification of surface topography at the angstrom to nanometer level. Time-dependent VSI measurements can be used to study the surface-normal retreat across crystal and other solid surfaces during dissolution or corrosion processes. Therefore, VSI can be used to directly and nondestructively measure mineral dissolution rates with high precision. We have used this method to compare the abiotic dissolution behavior of a representative calcite (CaCO(3)) cleavage face with that observed upon addition of an environmental microbe, Shewanella oneidensis MR-1, to the crystal surface. From our direct observations, we have concluded that the presence of the microbes results in a significant inhibition of the rate of calcite dissolution. This inhibition appears to be a 2nd-order effect that is related to the formation of etch pits. The opening of etch pits was greatly inhibited in the presence of added bacteria, suggesting that the bacterial cells exert their effect by inhibiting the formation of etch pits at high-energy sites at the crystal surface caused by lattice defects, e.g., screw or point dislocations. The experimental methodology thus provides a nondestructive, directly quantifiable, and easily visualized view of the interactions of microbes and minerals during weathering (or corrosion) processes or during mineral precipitation.     

Direct Observation of Microbial Inhibition of Calcite Dissolution

Vertical scanning interferometry (VSI) provides a method for quantification of surface topography at the angstrom to nanometer level. Time-dependent VSI measurements can be used to study the surface-normal retreat across crystal and other solid surfaces during dissolution or corrosion processes. Therefore, VSI can be used to directly and nondestructively measure mineral dissolution rates with high precision. We have used this method to compare the abiotic dissolution behavior of a representative calcite (CaCO₃) cleavage face with that observed upon addition of an environmental microbe, Shewanella oneidensis MR-1, to the crystal surface. From our direct observations, we have concluded that the presence of the microbes results in a significant inhibition of the rate of calcite dissolution. This inhibition appears to be a 2nd-order effect that is related to the formation of etch pits. The opening of etch pits was greatly inhibited in the presence of added bacteria, suggesting that the bacterial cells exert their effect by inhibiting the formation of etch pits at high-energy sites at the crystal surface caused by lattice defects, e.g., screw or point dislocations. The experimental methodology thus provides a nondestructive, directly quantifiable, and easily visualized view of the interactions of microbes and minerals during weathering (or corrosion) processes or during mineral precipitation.     

An improved spectrophotometric method to study the transport, attachment, and breakthrough of bacteria through porous media

This study reports an improved spectrophotometric method for studying bacterial (Pseudomonas fluorescens UPER-1) transport and attachment in saturated porous media (silica sand). While studying the effect of ionic strength by the traditional packed-column spectrophotometric method, we encountered an artifact. The absorbance of a well-stirred bacterial suspension was found to decrease with time in the presence of high concentrations of sodium and potassium phosphate salts (> or = 10(-2) M) as the cells continued to age in a resting stage. Our results show that collision efficiency and a bed ripening index will be in error by as much as 20% if breakthrough is measured by the traditional spectrophotometric technique. We present an improved experimental technique that will minimize the artifact and should substantially advance the understanding of bacteria transport in porous media.     

Impedimetric approach for quantifying low bacteria concentrations based on the changes produced in the electrode-solution interface during the pre-attachment stage

This paper describes an approach for quantifying low concentrations of bacteria, particularly Escherichia coli, based on the measurement of the initial attachment of bacteria to platinum surfaces, using impedance spectroscopy. The value of the interface capacitance in the pre-attachment stage (before 1min of attachment) showed correlation with suspended concentration of bacteria from 10(1) to 10(7)CFUmL(-1) (colony forming units per mL). This method was found to be sensitive to the attachment time, to the applied potential and to the size of the counter electrode. The sensor lifetime was also evaluated.     

Comparison of cell-specific activity between free-living and attached bacteria using isolates and natural assemblages

Marine snow aggregates are microbial hotspots that support high bacterial abundance and activities. We conducted laboratory experiments to compare cell-specific bacterial protein production (BPP) and protease activity between free-living and attached bacteria. Natural bacterial assemblages attached to model aggregates (agar spheres) had threefold higher BPP and two orders of magnitude higher protease activity than their free-living counterpart. These observations could be explained by preferential colonization of the agar spheres by bacteria with inherently higher metabolic activity and/or individual bacteria increasing their metabolism upon attachment to surfaces. In subsequent experiments, we used four strains of marine snow bacteria isolates to test the hypothesis that bacteria could up- and down-regulate their metabolism while on and off an aggregate. The protease activity of attached bacteria was 10-20 times higher than that of free-living bacteria, indicating that the individual strains could increase their protease activity within a short time (2 h) upon attachment to surfaces. Agar spheres with embedded diatom cells were colonized faster than plain agar spheres and the attached bacteria were clustered around the agar-embedded diatom cells, indicating a chemosensing response. Increased protease activity and BPP allow attached bacteria to quickly exploit aggregate resources upon attachment, which may accelerate remineralization of marine snow and reduce the downward carbon fluxes.     

Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.     

Surface contact stimulates the just-in-time deployment of bacterial adhesins

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.