Project description:The bactericidal effect of biomimetic nanostructured surfaces has been known for a long time, with recent data suggesting an enhanced efficiency of the nanostructured surfaces under fluid shear. While some of the influential factors on the bactericidal effect of nanostructured surfaces under fluid shear are understood, there are numerous important factors yet to be studied, which is essential for the successful implementation of this technology in industrial applications. Among those influential factors, the orientation of the nanostructured surface can play an important role in bacterial cell adhesion onto surfaces. Gravitational effects can become dominant under low flow velocities, making the diffusive transport of bacterial cells more prominent than the advective transport. However, the role of nanostructure orientation in determining its bactericidal efficiency under flow conditions is still not clear. In this study, we analysed the effect of surface orientation of nanostructured surfaces, along with bacterial cell concentration, fluid flow rate, and the duration of time which the surface is exposed to flow, on bacterial adhesion and viability on these surfaces. Two surface orientations, with one on the top and the other on the bottom of a flow channel, were studied. Under flow conditions, the bactericidal efficacy of the nanostructured surface is both orientation and bacterial species dependent. The effects of cell concentration, fluid flow rate, and exposure time on cell adhesion are independent of the nanostructured surface orientation. Fluid shear showed a species-dependent effect on bacterial adhesion, while the effects of concentration and exposure time on bacterial cell adhesion are independent of the bacterial species. Moreover, bacterial cells demonstrate preferential adhesion onto surfaces based on the surface orientation, and these effects are species dependent. These results outline the capabilities and limitations of nanostructures under flow conditions. This provides valuable insights into the applications of nanostructures in medical or industrial sectors, which are associated with overlaying fluid flow.
Project description:Bacteria often thrive in surface-attached communities, where they can form biofilms affording them multiple advantages. In this sessile form, fluid flow is a key component of their environments, renewing nutrients and transporting metabolic products and signaling molecules. It also controls colonization patterns and growth rates on surfaces, through bacteria transport, attachment and detachment. However, the current understanding of bacterial growth on surfaces neglects the possibility that bacteria may modulate their division behavior as a response to flow. Here, we employed single-cell imaging in microfluidic experiments to demonstrate that attached Escherichia coli cells can enter a growth arrest state while simultaneously enhancing their adhesion underflow. Despite utilizing clonal populations, we observed a non-uniform response characterized by bistable dynamics, with co-existing subpopulations of non-dividing and actively dividing bacteria. As the proportion of non-dividing bacteria increased with the applied flow rate, it resulted in a reduction in the average growth rate of bacterial populations on flow-exposed surfaces. Dividing bacteria exhibited asymmetric attachment, whereas non-dividing counterparts adhered to the surface via both cell poles. Hence, this phenotypic diversity allows bacterial colonies to combine enhanced attachment with sustained growth, although at a reduced rate, which may be a significant advantage in fluctuating flow conditions.
Project description:In medicine and food industry, bacterial colonisation on surfaces is a common cause of infections and severe illnesses. However, the detailed quantitative information about the dynamics and the mechanisms involved in bacterial proliferation on solid substrates is still lacking. In this study we investigated the adhesion and detachment, the individual growth and colonisation, and the cell size control of Escherichia coli (E. coli) MG1655 on polyethylene terephthalate (PET) surfaces. The results show that the bacterial growth curve on PET exhibits the distinct lag and log phases, but the generation time is more than twice longer than in bulk medium. Single cells in the lag phase are more likely to detach than clustered ones in the log phase; clustered bacteria in micro-colonies have stronger adhesive bonds with surfaces and their neighbours with the progressing colonisation. We show that the cell size is under the density-dependent pathway control: when the adherent cells are at low density, the culture medium is responsible for coordinating cell division and cell size; when the clustered cells are at high population density, we demonstrate that the effect of quorum sensing causes the cell size decrease as the cell density on surfaces increases.
Project description:Different nanostructured surfaces have bactericidal properties that arise from the interaction between the bacteria and the nanostructured surface. In this study, we focused on the relationship between bacterial motility and bactericidal properties. The motility of Escherichia coli (E. coli) was tuned by genetic engineering, and four types of E. coli (wild type (WT), lacking flagella, and flagellated with deficient motility or deficient chemotaxis) were used to evaluate the adhesion and bactericidal properties of nanostructured surfaces. Cicada (Cryptotympana facialis) wings and Si nano-pillar array substrates were used as natural and artificial nanostructured surfaces, respectively. Differences in motility and chemotaxis strongly influenced the adhesion behavior and to some extent, the damage to the cell membrane. These results suggest that the bactericidal properties of nanostructured surfaces depend on bacterial motility.
Project description:A novel biofilm model is described which systemically couples bacteria, extracellular polymeric substances (EPS) and solvent phases in biofilm. This enables the study of contributions of rheology of individual phases to deformation of biofilm in response to fluid flow as well as interactions between different phases. The model, which is based on first and second laws of thermodynamics, is derived using an energetic variational approach and phase-field method. Phase-field coupling is used to model structural changes of a biofilm. A newly developed unconditionally energy-stable numerical splitting scheme is implemented for computing the numerical solution of the model efficiently. Model simulations predict biofilm cohesive failure for the flow velocity between [Formula: see text] and [Formula: see text] m s(-1) which is consistent with experiments. Simulations predict biofilm deformation resulting in the formation of streamers for EPS exhibiting a viscous-dominated mechanical response and the viscosity of EPS being less than [Formula: see text]. Higher EPS viscosity provides biofilm with greater resistance to deformation and to removal by the flow. Moreover, simulations show that higher EPS elasticity yields the formation of streamers with complex geometries that are more prone to detachment. These model predictions are shown to be in qualitative agreement with experimental observations.
Project description:Quantifying bacterial cell numbers is crucial for experimental assessment and reproducibility, but the current technologies have limitations. The commonly used colony forming units (CFU) method causes a time delay in determining the actual numbers. Manual microscope counts are often error-prone for submicron bacteria. Automated systems are costly, require specialized knowledge, and are erroneous when counting smaller bacteria. In this study, we took a different approach by constructing three sequential generations (G1, G2, and G3) of counter-on-chip that accurately and timely count small particles and/or bacterial cells. We employed 2-photon polymerization (2PP) fabrication technology; and optimized the printing and molding process to produce high-quality, reproducible, accurate, and efficient counters. Our straightforward and refined methodology has shown itself to be highly effective in fabricating structures, allowing for the rapid construction of polydimethylsiloxane (PDMS)-based microfluidic devices. The G1 comprises three counting chambers with a depth of 20 µm, which showed accurate counting of 1 µm and 5 µm microbeads. G2 and G3 have eight counting chambers with depths of 20 µm and 5 µm, respectively, and can quickly and precisely count Escherichia coli cells. These systems are reusable, accurate, and easy to use (compared to CFU/ml). The G3 device can give (1) accurate bacterial counts, (2) serve as a growth chamber for bacteria, and (3) allow for live/dead bacterial cell estimates using staining kits or growth assay activities (live imaging, cell tracking, and counting). We made these devices out of necessity; we know no device on the market that encompasses all these features.
Project description:Mouse serum raised against killed antigen preparations of Mycobacterium tuberculosis failed to recognize most of the recombinant antigens of M. tuberculosis that were originally identified by reactivity to tuberculosis (TB) patient sera. Similar results were obtained with serum from guinea pigs immunized with live and killed mycobacteria. Antibodies raised against seven random TB patient serum-reactive antigens detected each of these antigens in the sonicate preparation. The nucleotide sequences of the genes for these seven antigens revealed that all represented hitherto unreported genes of M. tuberculosis. Our results suggest differential presentation to the host immune system of the same antigens derived from live and killed mycobacteria.
Project description:There has been a growing interest in understanding the ways in which bacteria interact with nano-structured surfaces. As a result, there is a need for innovative approaches to enable researchers to visualize the biological processes taking place, despite the fact that it is not possible to directly observe these processes. We present a novel approach for the three-dimensional visualization of bacterial interactions with nano-structured surfaces using the software package Autodesk Maya. Our approach comprises a semi-automated stage, where actual surface topographic parameters, obtained using an atomic force microscope, are imported into Maya via a custom Python script, followed by a 'creative stage', where the bacterial cells and their interactions with the surfaces are visualized using available experimental data. The 'Dynamics' and 'nDynamics' capabilities of the Maya software allowed the construction and visualization of plausible interaction scenarios. This capability provides a practical aid to knowledge discovery, assists in the dissemination of research results, and provides an opportunity for an improved public understanding. We validated our approach by graphically depicting the interactions between the two bacteria being used for modeling purposes, Staphylococcus aureus and Pseudomonas aeruginosa, with different titanium substrate surfaces that are routinely used in the production of biomedical devices.
Project description:Tissue integration is an important property when inducing transplant tolerance, however, the hemocompatibility of the biomaterial surface also plays an important role in the ultimate success of the implant. Therefore, in order to induce transplant tolerance, it is critical to understand the interaction of blood components with the material surfaces. In this study, we have investigated the adsorption of key blood serum proteins, in vitro adhesion and activation of platelets and clotting kinetics of whole blood on flat polycaprolactone (PCL) surfaces, nanowire (NW) surfaces and nanofiber (NF) surfaces. Previous studies have shown that polymeric nanostructured surfaces improve cell adhesion, proliferation and viability; however it is unclear how these polymeric nanostructured surfaces interact with the blood and its components. Protein adsorption results indicate that while there were no significant differences in total albumin (ALB) adsorption on PCL, NW and NF surfaces, NW surfaces had higher total fibrinogen (FIB) and immunoglobulin-G (IgG) adsorption compared to NF and PCL surfaces. In contrast, NF surfaces had higher surface FIB and IgG adsorption compared to PCL and NW surfaces. Platelet adhesion and viability studies show more adhesion and clustering of platelets on the NF surfaces as compared to PCL and NW surfaces. Platelet activation studies reveal that NW surfaces have the highest percentage of unactivated platelets, whereas NF surfaces have the highest percentage of fully activated platelets. Whole blood clotting results indicate that NW surfaces maintain an increased amount of free hemoglobin during the clotting process compared to PCL and NF surface, indicating less clotting and slower rate of clotting on their surfaces.
Project description:Quorum sensing (QS) is a population-density dependent chemical process that enables bacteria to communicate based on the production, secretion and sensing of small inducer molecules. While recombinant constructs have been widely used to decipher the molecular details of QS, how those findings translate to natural QS systems has remained an open question. Here, we compare the activation of natural and synthetic Pseudomonas aeruginosa LasI/R QS systems in bacteria exposed to quiescent conditions and controlled flows. Quantification of QS-dependent GFP expression in suspended cultures and in surface-attached microcolonies revealed that QS onset in both systems was similar under quiescent conditions but markedly differed under flow. Moderate flow (Pe > 25) was sufficient to suppress LasI/R QS recombinantly expressed in Escherichia coli, whereas only high flow (Pe > 102) suppressed QS in wild-type P. aeruginosa. We suggest that this difference stems from the differential production of extracellular matrix and that the matrix confers resilience against moderate flow to QS in wild-type organisms. These results suggest that the expression of a biofilm matrix extends the environmental conditions under which QS-based cell-cell communication is effective and that findings from synthetic QS circuits cannot be directly translated to natural systems.