Dual pulsed laser deposition of Ag nanoparticles on calcium phosphate coatings for biomedical applications.

Pulsed laser deposition (PLD) represents a promising bottom-up methodology for the synthesis and transference of nanoparticles to the surface of a biomedical device. Silver (Ag) nanoparticles directly incorporated on the metallic implant emerge as an alternative strategy for local action against prosthetic joint-associated infections. In the present research, a dual sequential PLD process is proposed to obtain a bilayer coating with (1) a bio-derived calcium phosphate (CaP) layer, to provide osteointegrative properties and (2) the controlled growth of the Ag nanoparticles over it, ranging the number of laser pulses from 100 to 500. The characterization by SEM, EDS, TEM, XPS and AFM revealed the uniform deposit of Ag rounded nanoparticles, with a narrow mean size distribution, in the original non-oxidized metallic state. Moreover, given the evidences from XPS and AFM techniques, the occurrence of a coalescence phenomenon from 400 pulses onwards was proposed together with the expected positive linear relation between the number of pulses and Ag contribution with a deposition rate of 0.05 at. % of Ag per pulse. Conversely, the decrease in roughness as the Ag content increased was also verified. Finally, the expected bacteriostatic activity for these PLD deposited metallic state Ag nanoparticles against the bacterial strainStaphylococcus aureuswas confirmed. Moreover, the evaluation of the osteoblast-like MG-63 cells viability on the Ag(100-500)-CaP coatings revealed a significant increased proliferation (p > 0.05) on the Ag100-CaP coating compared to the control (Ag0-CaP). When same coating was evaluated againstS. aureusthe effect was not significant. The possibility of modulating the amount of nanoparticles in the bilayer coating to obtain a greater or lesser effect in combination with CaP was revealed.

Quantitative proteomic analysis of marine biofilms formed by filamentous cyanobacterium.

Cyanobacterial molecular biology can identify pathways that affect the adhesion and settlement of biofouling organisms and, consequently, obtain novel antifouling strategies for marine applications. Proteomic analyses can provide an essential understanding of how cyanobacteria adapt to different environmental settings. However, only a few qualitative studies have been performed in some cyanobacterial strains. Considering the limited knowledge about protein expression in cyanobacteria in different growing conditions, a quantitative proteomic analysis by LC-MS/MS of biofilm cells from a filamentous strain was performed. Biofilms were also analysed through standard methodologies for following cyanobacterial biofilm development. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). Biofilm development was higher at 4 s-1 and no significant differences were found between surfaces. Proteomic analysis identified 546 proteins and 41 were differentially expressed. Differences in protein expression were more noticeable between biofilms formed on glass and perspex at 4 s-1. When comparing biofilms formed on different surfaces, results suggest that biofilm development may be related to the expression of several proteins like a beta-propeller domain-containing protein, chaperone DnaK, SLH domain-containing proteins, an OMF family outer membrane protein, and/or additional uncharacterized proteins. Regarding the hydrodynamic effect, biofilm development can be related to SOD enzyme expression, to proteins related to photosynthetic processes and to a set of uncharacterized proteins with calcium binding domains, disordered proteins, and others involved in electron transfer activity. Studies that combine distinct approaches are essential for finding new targets for antibiofilm agents. The characterisation performed in this work provides new insights into how shear rate and surface affect cyanobacterial biofilm development and how cyanobacteria adapt to these different environmental settings from a macroscopic standpoint to a proteomics context.

Cell adhesion in microchannel multiple constrictions - Evidence of mass transport limitations.

Biofilm growth (fouling) in microdevices is a critical concern in several industrial, engineering and health applications, particularly in novel high-performance microdevices often designed with complex geometries, narrow regions and multiple headers. Unfortunately, on these devices, the regions with local high wall shear stresses (WSS) also show high local fouling rates. Several explanations have been put forward by the scientific community, including the effect of cell transport by Brownian motion on the adhesion rate. In this work, for the first time, both WSS and convection and Brownian diffusion effects on cell adhesion were evaluated along a microchannel with intercalate constriction and expansion zones designed to mimic the hydrodynamics of the human body and biomedical devices. Convection and Brownian diffusion effects were numerically studied using a steady-state convective-diffusion model (convection, diffusion and sedimentation). According to the numerical results, the convection and Brownian diffusion effects on cell adhesion are effectively more significant in regions with high WSS. Furthermore, a good agreement was observed between experimental and predicted local Sherwood numbers, particularly at the entrance and within the multiple constrictions. However, further mechanisms should be considered to accurately predict cell adhesion in the expansion zones. The described numerical approach can be used as a way to identify possible clogging zones in microchannels, and defining solutions, even before the construction of the prototype.

The potential advantages of using a poly(HPMA) brush in urinary catheters: effects on biofilm cells and architecture.

Infections related to bacterial colonization of medical devices are a growing concern given the socio-economical impacts in healthcare systems. Colonization of a device surface with bacteria usually triggers the development of a biofilm, which is more difficult to eradicate than free-floating or adhered bacteria and can act as a reservoir for subsequent infections. Biofilms often harbor Viable but nonculturable (VBNC) cells that are likely to be more resistant to antibiotic treatment and that can become active in more favorable conditions causing infection. Biofilm formation is dependent on different factors, chiefly the properties of the surface and of the surrounding medium, and the hydrodynamic conditions. In this work, the antifouling performance of a poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brush was evaluated in vitro in conditions that mimic a urinary catheter using Escherichia coli as a model organism. The results obtained with the brush were compared to those obtained with two control surfaces, polydimethylsiloxane (PDMS) (the most common material for catheters) and glass. A decrease in initial adhesion and surface coverage was observed on the brush. This antifouling behavior was maintained during biofilm maturation and even in a simulated post-bladder infection period when the reduction in total cell number reached 87 %. Biofilms were shown to adapt their architecture during that period and VBNC cells adsorbed weakly on the brushes and were completely washed away. Taken together, these results suggest that the use of the poly(HPMA) brush in urinary tract devices such as catheters and stents may reduce biofilm formation and possibly render the formed biofilms more susceptible to antibiotic treatment and with reduced infectivity potential.

Fabrication and Hydrodynamic Characterization of a Microfluidic Device for Cell Adhesion Tests in Polymeric Surfaces.

A fabrication method is developed to produce a microfluidic device to test cell adhesion to polymeric materials. The process is able to produce channels with walls of any spin coatable polymer. The method is a modification of the existing poly-dimethylsiloxane soft lithography method and, therefore, it is compatible with sealing methods and equipment of most microfluidic laboratories. The molds are produced by xurography, simplifying the fabrication in laboratories without sophisticated equipment for photolithography. The fabrication method is tested by determining the effective differences in bacterial adhesion in five different materials. These materials have different surface hydrophobicities and charges. The major drawback of the method is the location of the region of interest in a lowered surface. It is demonstrated by bacterial adhesion experiments that this drawback has a negligible effect on adhesion. The flow in the device was characterized by computational fluid dynamics and it was shown that shear stress in the region of interest can be calculated by numerical methods and by an analytical equation for rectangular channels. The device is therefore validated for adhesion tests.

Impact of modified diamond-like carbon coatings on the spatial organization and disinfection of mixed-biofilms composed of Escherichia coli and Pantoea agglomerans industrial isolates.

This work investigated the effects of diamond-like carbon (DLC) coatings on the architecture and biocide reactivity of dual-species biofilms mimicking food processing contaminants. Biofilms were grown using industrial isolates of Escherichia coli and Pantoea agglomerans on bare stainless steel (SST) and on two DLC surface coatings (a-C:H:Si:O designated by SICON® and a-C:H:Si designated by SICAN) in order to evaluate their antifouling activities. Quantification and spatial organization in single- and dual-species biofilms were examined by confocal laser scanning microscopy (CLSM) using a strain specific labelling procedure. Those assays revealed that the E. coli isolate exhibited a higher adhesion to the modified surfaces and a decreased susceptibility to disinfectant in presence of P. agglomerans than alone in axenic culture. While SICON® reduced the short-term growth of E. coli in axenic conditions, both DLC surfaces increased the E. coli colonization in presence of P. agglomerans. However, both modified surfaces triggered a significantly higher log reduction of E. coli cells within mixed-species biofilms, thus the use of SICON® and SICAN surfaces may be a good approach to facilitate the disinfection process in critical areas of food processing plants. This study presents a new illustration of the importance of interspecies interactions in surface-associated community functions, and of the need to evaluate the effectiveness of hygienic strategies with relevant multi-species consortia.

Escherichia coli adhesion, biofilm development and antibiotic susceptibility on biomedical materials.

The aim of this work was to test materials typically used in the construction of medical devices regarding their influence in the initial adhesion, biofilm development and antibiotic susceptibility of Escherichia coli biofilms. Adhesion and biofilm development was monitored in 12-well microtiter plates containing coupons of different biomedical materials--silicone (SIL), stainless steel (SS) and polyvinyl chloride (PVC)--and glass (GLA) as control. The susceptibility of biofilms to ciprofloxacin and ampicillin was assessed, and the antibiotic effect in cell morphology was observed by scanning electron microscopy. The surface hydrophobicity of the bacterial strain and materials was also evaluated from contact angle measurements. Surface hydrophobicity was related with initial E. coli adhesion and subsequent biofilm development. Hydrophobic materials, such as SIL, SS, and PVC, showed higher bacterial colonization than the hydrophilic GLA. Silicone was the surface with the greatest number of adhered cells and the biofilms formed on this material were also less susceptible to both antibiotics. It was found that different antibiotics induced different levels of elongation on E. coli sessile cells. Results revealed that, by affecting the initial adhesion, the surface properties of a given material can modulate biofilm buildup and interfere with the outcome of antimicrobial therapy. These findings raise the possibility of fine-tuning surface properties as a strategy to reach higher therapeutic efficacy.

The effects of surface properties on Escherichia coli adhesion are modulated by shear stress.

The adhesion of Escherichia coli to glass and polydimethylsiloxane (PDMS) at different flow rates (between 1 and 10 ml s(-1)) was monitored in a parallel plate flow chamber in order to understand the effect of surface properties and hydrodynamic conditions on adhesion. Computational fluid dynamics was used to assess the applicability of this flow chamber in the simulation of the hydrodynamics of relevant biomedical systems. Wall shear stresses between 0.005 and 0.07 Pa were obtained and these are similar to those found in the circulatory, reproductive and urinary systems. Results demonstrate that E. coli adhesion to hydrophobic PDMS and hydrophilic glass surfaces is modulated by shear stress with surface properties having a stronger effect at the lower and highest flow rates tested and with negligible effects at intermediate flow rates. These findings suggest that when expensive materials or coatings are selected to produce biomedical devices, this choice should take into account the physiological hydrodynamic conditions that will occur during the utilization of those devices.

96-well microtiter plates for biofouling simulation in biomedical settings.

Microtiter plates with 96 wells are routinely used in biofilm research mainly because they enable high-throughput assays. These platforms are used in a variety of conditions ranging from static to dynamic operation using different shaking frequencies and orbital diameters. The main goals of this work were to assess the influence of nutrient concentration and flow conditions on biofilm formation by Escherichia coli in microtiter plates and to define the operational conditions to be used in order to simulate relevant biomedical scenarios. Assays were performed in static mode and in incubators with distinct orbital diameters using different concentrations of glucose, peptone and yeast extract. Computational fluid dynamics (CFD) was used to simulate the flow inside the wells for shaking frequencies ranging from 50 to 200 rpm and orbital diameters from 25 to 100 mm. Higher glucose concentrations enhanced adhesion of E. coli in the first 24 h, but variation in peptone and yeast extract concentration had no significant impact on biofilm formation. Numerical simulations indicate that 96-well microtiter plates can be used to simulate a variety of biomedical scenarios if the operating conditions are carefully set.

Macroscale versus microscale methods for physiological analysis of biofilms formed in 96-well microtiter plates.

Microtiter plates with 96 wells have become one of the preferred platforms for biofilm studies mainly because they enable high-throughput assays. In this work, macroscale and microscale methods were used to study the impact of hydrodynamic conditions on the physiology and location of Escherichia coli JM109(DE3) biofilms formed in microtiter plates. Biofilms were formed in shaking and static conditions, and two macroscale parameters were assayed: the total amount of biofilm was measured by the crystal violet assay and the metabolic activity was determined by the resazurin assay. From the macroscale point of view, there were no statistically significant differences between the biofilms formed in static and shaking conditions. However, at a microscale level, the differences between both conditions were revealed using scanning electron microscopy (SEM). It was observed that biofilm morphology and spatial distribution along the wall were different in these conditions. Simulation of the hydrodynamic conditions inside the wells at a microscale was performed by computational fluid dynamics (CFD). These simulations showed that the shear strain rate was unevenly distributed on the walls during shaking conditions and that regions of higher shear strain rate were obtained closer to the air/liquid interface. Additionally, it was shown that wall regions subjected to higher shear strain rates were associated with the formation of biofilms containing cells of smaller size. Conversely, regions with lower shear strain rate were prone to have a more uniform spatial distribution of adhered cells of larger size. The results presented on this work highlight the wealth of information that may be gathered by complementing macroscale approaches with a microscale analysis of the experiments.

Influence of flow rate variation on the development of Escherichia coli biofilms.

This work investigates the effect of flow rate variation on mass transfer and on the development of Escherichia coli biofilms on a flow cell reactor under turbulent flow conditions. Computational fluid dynamics (CFD) was used to assess the applicability of this reactor for the simulation of industrial and biomedical biofilms and the numerical results were validated by streak photography. Two flow rates of 374 and 242 L h(-1) (corresponding to Reynolds numbers of 6,720 and 4,350) were tested and wall shear stresses between 0.183 and 0.511 Pa were predicted in the flow cell reactor. External mass transfer coefficients of 1.38 × 10(-5) and 9.64 × 10(-6) m s(-1) were obtained for the higher and lower flow rates, respectively. Biofilm formation was favored at the lowest flow rate because shear stress effects were more important than mass transfer limitations. This flow cell reactor generates wall shear stresses that are similar to those found in some industrial and biomedical settings, thus it is likely that the results obtained on this work can be used in the development of biofilm control strategies in both scenarios.

The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance.

AIMS: This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions. METHODS AND RESULTS: The pET28 and pUC8 plasmids were separately used to transform E. coli JM109(DE3). Biofilm formation, removal and antimicrobial susceptibility to the cationic biocide benzyldimethyldodecylammonium chloride (BDMDAC) were assessed. Transformed cells formed thicker biofilms with higher cell densities, and the metabolic activity was higher whereas nontransformed cells had higher viabilities. Biocide treatment was not efficient for biofilm removal but was effective for cell killing. Biofilms formed by nontransformed cells were less affected by the treatment. CONCLUSIONS: Cell transformation with the tested plasmids has significant impacts on biofilm formation, cell viability, metabolic activity and resistance to biocide treatment. Our results show that in biofilm studies involving deletion/complementation experiments, a control with the strain carrying a plasmid devoid of the gene under investigation must be included so that the real effects of the genetic manipulation are not biased by the presence of the plasmid backbone. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report where the presence of nonconjugative plasmids is assessed in flow conditions analysing biofilm formation, removal and antimicrobial susceptibility of high cell-density biofilms.

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow.

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l⁻¹) and a low (150 mg glucose l⁻¹) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s⁻¹ was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.

Recombinant protein secretion in Escherichia coli.

The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.

Evaluation of bottlenecks in proinsulin secretion by Escherichia coli.

This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g(-1) dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17-24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle.

Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB.

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system.

A sandwich-type enzyme-linked immunosorbent assay (ELISA) was established for monitoring the secretion of ZZ-fusion proteins. Two antibodies, a monoclonal mouse anti-human proinsulin and a rabbit anti-bovine IgG (strongly binding to the ZZ-domain), were used to quantify the secretion of recombinant human ZZ-proinsulin to the growth medium of Escherichia coli cultures. The method here reported conjugates the advantages of sandwich-type ELISA assays, namely, high sensitivity, specificity, and throughput, with the possibility of quantifying small protein molecules (e.g., peptides). A further advantage of gene fusion techniques integrating both downstream processing and product detection and quantitation is highlighted. The method is capable of detecting levels of 0.05 ng of ZZ-proinsulin.

Troubleshooting in gene splicing by overlap extension: a step-wise method.

A step-wise method for cloning intron-containing genes from genomic DNA is described. The two exons of the human proinsulin gene were separately amplified in two steps using, in the first step, completely homologous primers. This reduces unwanted interactions between mismatched primers and a complex DNA template such as genomic DNA. The fragments were amplified in a second step polymerase chain reaction (PCR) using mismatched primers that incorporated additional bases complementary to the other exon, and these products were spliced together in a third step PCR.