Understanding the flow behavior around marine biofilms.

In vitro platforms capable of mimicking the hydrodynamic conditions prevailing in natural aquatic environments have been previously validated and used to predict the fouling behavior on different surfaces. Computational Fluid Dynamics (CFD) has been used to predict the shear forces occurring in these platforms. In general, these predictions are made for the initial stages of biofilm formation, where the amount of biofilm does not affect the flow behavior, enabling the estimation of the shear forces that initial adhering organisms have to withstand. In this work, we go a step further in understanding the flow behavior when a mature biofilm is present in such platforms to better understand the shear rate distribution affecting marine biofilms. Using 3D images obtained by Optical Coherence Tomography, a mesh was produced and used in CFD simulations. Biofilms of two different marine cyanobacteria were developed in agitated microtiter plates incubated at two different shaking frequencies for 7 weeks. The biofilm-flow interactions were characterized in terms of the velocity field and shear rate distribution. Results show that global hydrodynamics imposed by the different shaking frequencies affect biofilm architecture and also that this architecture affects local hydrodynamics, causing a large heterogeneity in the shear rate field. Biofilm cells located in the streamers of the biofilm are subjected to much higher shear values than those located on the bottom of the streamers and this dispersion in shear rate values increases at lower bulk fluid velocities. This heterogeneity in the shear force field may be a contributing factor for the heterogeneous behavior in metabolic activity, growth status, gene expression pattern, and antibiotic resistance often associated with nutrient availability within the biofilm.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

The Use of 3D Optical Coherence Tomography to Analyze the Architecture of Cyanobacterial Biofilms Formed on a Carbon Nanotube Composite.

The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.

Visualization of Bacterial Colonization and Cellular Layers in a Gut-on-a-Chip System Using Optical Coherence Tomography.

Imaging of cellular layers in a gut-on-a-chip system has been confined to two-dimensional (2D)-imaging through conventional light microscopy and confocal laser scanning microscopy (CLSM) yielding three-dimensional- and 2D-cross-sectional reconstructions. However, CLSM requires staining and is unsuitable for longitudinal visualization. Here, we compare merits of optical coherence tomography (OCT) with those of CLSM and light microscopy for visualization of intestinal epithelial layers during protection by a probiotic Bifidobacterium breve strain and a simultaneous pathogen challenge by an Escherichia coli strain. OCT cross-sectional images yielded film thicknesses that coincided with end-point thicknesses derived from cross-sectional CLSM images. Light microscopy on histological sections of epithelial layers at the end-point yielded smaller layer thicknesses than OCT and CLSM. Protective effects of B. breve adhering to an epithelial layer against an E. coli challenge included the preservation of layer thickness and membrane surface coverage by epithelial cells. OCT does not require staining or sectioning, making OCT suitable for longitudinal visualization of biological films, but as a drawback, OCT does not allow an epithelial layer to be distinguished from bacterial biofilms adhering to it. Thus, OCT is ideal to longitudinally evaluate epithelial layers under probiotic protection and pathogen challenges, but proper image interpretation requires the application of a second method at the end-point to distinguish bacterial and epithelial films.

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions.

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s-1 ) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.

Bacterial Density and Biofilm Structure Determined by Optical Coherence Tomography.

Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.

Role of Viscoelasticity in Bacterial Killing by Antimicrobials in Differently Grown Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa colonizes the sputum of most adult cystic fibrosis patients, forming difficult-to-eradicate biofilms in which bacteria are protected in their self-produced extracellular polymeric substance (EPS) matrices. EPS provide biofilms with viscoelastic properties, causing time-dependent relaxation after stress-induced deformation, according to multiple characteristic time constants. These time constants reflect different biofilm (matrix) components. Since the viscoelasticity of biofilms has been related to antimicrobial penetration but not yet bacterial killing, this study aims to relate killing of P. aeruginosa, in its biofilm mode of growth, by three antimicrobials to biofilm viscoelasticity. P. aeruginosa biofilms were grown for 18 h in a constant-depth film fermenter, with mucin-containing artificial sputum medium (ASM+), artificial sputum medium without mucin (ASM-), or Luria-Bertani (LB) broth; this yielded 100-µm-thick biofilms that differed in their amounts of matrix environmental DNA (eDNA) and polysaccharides. Low-load compression testing, followed by three-element Maxwell analyses, showed that the fastest relaxation component, associated with unbound water, was most important in LB-medium-grown biofilms. Slower components due to water with dissolved polysaccharides, insoluble polysaccharides, and eDNA were most important in the relaxation of ASM+-grown biofilms. ASM--grown biofilms showed intermediate stress relaxation. P. aeruginosa in LB-medium-grown biofilms was killed most by exposure to tobramycin, colistin, or an antimicrobial peptide, while ASM+ provided the most protective matrix, with less water and most insoluble polysaccharides and eDNA. In conclusion, stress relaxation of P. aeruginosa biofilms grown in different media revealed differences in matrix composition that, within the constraints of the antimicrobials and growth media applied, correlated with the matrix protection offered against different antimicrobials.

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces.

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 µm-3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 µm-3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.

Influence of biofilm lubricity on shear-induced transmission of staphylococcal biofilms from stainless steel to silicone rubber.

In real-life situations, bacteria are often transmitted from biofilms growing on donor surfaces to receiver ones. Bacterial transmission is more complex than adhesion, involving bacterial detachment from donor and subsequent adhesion to receiver surfaces. Here, we describe a new device to study shear-induced bacterial transmission from a (stainless steel) pipe to a (silicone rubber) tube and compare transmission of EPS-producing and non-EPS-producing staphylococci. Transmission of an entire biofilm from the donor to the receiver tube did not occur, indicative of cohesive failure in the biofilm rather than of adhesive failure at the donor-biofilm interface. Biofilm was gradually transmitted over an increasing length of receiver tube, occurring mostly to the first 50 cm of the receiver tube. Under high-shearing velocity, transmission of non-EPS-producing bacteria to the second half decreased non-linearly, likely due to rapid thinning of the lowly lubricious biofilm. Oppositely, transmission of EPS-producing strains to the second tube half was not affected by higher shearing velocity due to the high lubricity and stress relaxation of the EPS-rich biofilms, ensuring continued contact with the receiver. The non-linear decrease of ongoing bacterial transmission under high-shearing velocity is new and of relevance in for instance, high-speed food slicers and food packaging.

Detachment and successive re-attachment of multiple, reversibly-binding tethers result in irreversible bacterial adhesion to surfaces.

Bacterial adhesion to surfaces occurs ubiquitously and is initially reversible, though becoming more irreversible within minutes after first contact with a surface. We here demonstrate for eight bacterial strains comprising four species, that bacteria adhere irreversibly to surfaces through multiple, reversibly-binding tethers that detach and successively re-attach, but not collectively detach to cause detachment of an entire bacterium. Arguments build on combining analyses of confined Brownian-motion of bacteria adhering to glass and their AFM force-distance curves and include the following observations: (1) force-distance curves showed detachment events indicative of multiple binding tethers, (2) vibration amplitudes of adhering bacteria parallel to a surface decreased with increasing adhesion-forces acting perpendicular to the surface, (3) nanoscopic displacements of bacteria with relatively long autocorrelation times up to several seconds, in absence of microscopic displacement, (4) increases in Mean-Squared-Displacement over prolonged time periods according to tα with 0 < α ≪ 1, indicative of confined displacement. Analysis of simulated position-maps of adhering particles using a new, in silico model confirmed that adhesion to surfaces is irreversible through detachment and successive re-attachment of reversibly-binding tethers. This makes bacterial adhesion mechanistically comparable with the irreversible adsorption of high-molecular-weight proteins to surfaces, mediated by multiple, reversibly-binding molecular segments.

Normalization of vasomotion in laser Doppler perfusion monitoring.

Laser Doppler flux signals show temporal fluctuations caused by physiological phenomena like heartbeat, respiration, and local variation of vascular tonus, vasomotion. This study investigates the influence of fiber arrangement, equipment and two probe locations on the variations in laser Doppler flux signals in five frequency bands in the absence of provocations. Two probes with detecting optical fibers at several distances from the illuminating source were used, as well as instruments from two manufacturers. The results show that normalization of the filtered flux signals with the mean flux leads to an enormous decrease of the influence of fiber distance. The difference between instruments is small after normalization. Some influence of probe location remains after normalization. Development of a standard method for normalization of the variations in laser Doppler signals is recommended.

Low-load compression testing: a novel way of measuring biofilm thickness.

Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.