| Surfaces of facilities, devices and materials in the fields of food and medicine are prone to infections caused by the attached microbes, and eventually-biofilms, leading to food spoilage and tissue contamination, which is a severe threat to the health of consumers and patients. Biofilms would be very difficult to be cleaned after the maturation, therefore in order to reduce the contamination of biofilms on surfaces, the early stages before biofilms mature(i.e., bacterial adhesion and growth) need to be widely investigated.Nowadays, a host of chemical strategies have been employed to reduce initial adhesion on surfaces, which generally involves a chemical surface modification with covalently bonding disinfectant agents and antibiotics. But the above strategies have risks, e.g. the issues of cytotoxicity of the disinfectant agents and antibiotic resistance in bacterial strains, which limit the application of chemical strategies in food and medicine industry. Thus the physical approach i.e., utilizing three-dimensional surface topographies with regularly shaped patterns, has been increasingly highlighted. However, the mechanism of bacterial adhesion on these surface topographies has not been understood yet; the studies on bacterial growth after adhesion are also still lacking, and the dynamics of proliferation and bacterial behavior growing on surfaces have not been reported.In this dissertation, polyethylene terephthalate(PET) films that are widely applied in food and medicine industry were used as substrates and Escherichia coli(E.coli) as test strain. Firstly, a proper method was developed to fabricate surface topographies with multiple features and different dimensions on PET substrates. The effect of surface topographies on bacterial adhesion was then studied under different incubating conditions, in order to clarify the mechanism of bacterial response to surface topographies with defined configurations. In terms of bacterial growth after adhesion, detailed investigations from the view of biological physics were conducted on the dynamics of bacterial proliferation, the mechanism of cell division and cell size control, bacterial behavior over different growing phases, and the interaction between bacteria and PET substrates. Furthermore, the role of fimbriae in bacterial adhesion and proliferation were also specified. The main research contents of this dissertation are shown as follows:(1) Fabrication of PET surface topography with regularly shaped patterns and the effect of the topography on the adhesion of E.coli on PET surfacesUV lithography was combined with hot embossing: The two-dimensional patterns designed by AutoCAD software were transferred onto the surface of the master mold made of silicon wafer by UV lithography, resulting in the formation of three-dimensional topography on the master mold. Then polydimethylsiloxane(PDMS) soft stamp and the epoxy were used to transfer the topography twice in order to produce the mold for hot embossing. Subsequently, the topography with regularly shaped patterns was fabricated on PET substrates using hot embossing. The surface topography on surfaces was characterized using scanning electron microscope(SEM) and atomic force microscope(AFM). The results showed that three-dimensional topography with defined 1-8 ?m configurations was well fabricated on PET surfaces. Infrared spectroscopy(IR) and contact angle measurements of PET films before and after hot embossing were conducted to respectively determine the changes of molecular structure and hydrophobic property. The results demonstrated that molecular structure and hydrophobic property of PET surfaces have not been significantly changed after hot embossing.Topographic features simultaneously including curved and straight edges, flat plateaus(top of pillars), and flat surfaces between pillars were fabricated on patterned PET surfaces using the above method, and the features displayed six dimensions. Confocal Laser Scanning Microscope(CLSM) and SEM were used to investigate the amount and distribution of adherent E.coli on all topographic features, under the incubation with oligotrophic/rich culture medium, respectively in static/dynamic conditions. The results showed that bacterial cell could sense the surface topography: E.coli cells exhibited obvious preference to attach on the edges with less curvature; The cells were hard to get in regions with features of large curvature, however, after longer incubation, these cells could mask this disadvantageous surface topography; The less preferential attachment which bacteria displayed, the less amount of cells would be attached on the surface topography. Moreover, bacterial sensing ability was affected by environmental conditions. When the nutrition was little and fluid shear stress existed, bacterial cells would lose the ability in sensing surface topography.(2) The growth and cell size control of E.coli on PET surfacesCLSM was conducted to observe the division and proliferation after E.coli cells were attached on the surface. The results showed that the growth curve of adherent cells exhibited the distinct lag and log phases as traditional growth curve did. The generation time of sessile daughter cells was 38 min, while that of planktonic cells was 16 min under the same incubation condition. Meanwhile, it was the first time to find and verify that the morphology of micro-colonies and cell size were all controlled by quorum sensing during bacterial growth on surfaces.(3) The role of type I fimbriae in the adhesion and the proliferation of E.coli on PET surfacesThe E.coli mutant that lack type I fimbriae was constructed. CLSM was applied to study the effect of type I fimbriae on bacterial reversible adhesion, irreversible adhesion and proliferation. The results showed that type I fimbriae were not required for reversible adhesion, but critical for irreversible adhesion. The adherent non-fimbriated mutants could not become irreversibly attached, thus they would not progress into the next stage to grow on the surface.(4) The response of E.coli adhered and growing on surfaces to shear stressThe fluid shear stress produced by the device of reciprocating linear vibration was applied to E.coli which was in the stages of adhesion or proliferation on PET surfaces. CLSM was used to determine the fraction of total cells and viable/dead status of cells remaining on PET surfaces after shear stress applied, thus the interactions between bacteria and substrates during adhesion and growth were understood. The effect of quorum sensing on bacterial response to fluid shear stress was also studied. The results showed that the fraction of total cells remaining and the bacterial adhesive force were both followed this order of living status increasingly: lag phase, single layer micro-colony phase and multi-layered micro-colony phase. The fraction of total cells remaining was affected by bacterial living status, not by incubation time nor the size of micro-colonies; The fraction of viable cells was found to be related with the size of micro-colonies, not with the living status; When the quorum sensing was inhibited, bacterial resistance to the fluid shear stress in the culture environment were weaken. |
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