Biomimetic surfaces and their effect on bacterial attachment adhesion and retention

Spall, Joshua (2019) Biomimetic surfaces and their effect on bacterial attachment adhesion and retention. Masters by Research thesis (MSc), Manchester Metropolitan University for the degree of Master of Science (by Research).

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Abstract

Biofouling in the dairy industry accounts for billions of dollars in lost product each year. Surface properties, such as macro, micro and nano topography and hydrophobicity were analysed with bacteria in monoculture and co-culture to determine the surface characteristics that prevented biofilm formation. Replica biomimetic surfaces were made using dental wax from five different types of plant leaves (White Cabbage (Brassica oleracea capitate), Leek (Allium ampeloprasu), Tender Heart (Brassica oleracea), Cauliflower (Brassica oleracea var. botrytis), and Gladioli (Gladiolus); this included a flat surface wax control. Surface physicochemistry was determined using contact angle measurements and surface topography (Sa, Sq, and Spv) using optical profilometry. Monoculture and co-culture bacterial attachment, adhesion and retention assays were carried out using Escherichia coli and Listeria monocytogenes and determined using colony-forming units/mL. Scanning Electron Microscopy provided quantitative cell counts (CFU/cm2). The results demonstrated that the Tenderheart leaf surface was the most hydrophobic with the highest surface free energy, highest γsAB, most electron-donating and most electron-accepting surface. The Leek surface demonstrated the lowest surface free energy. The White cabbage surface was the most non-polar surface, with the least γsAB properties, the least electron-accepting and least electron-donating surface. However, it had the highest Sa and Sq values. The Cauliflower leaf surface was the least hydrophobic and least nonpolar surface whilst the Gladioli surface was found to have the highest Spv values. Finally, the flat surface showed the lowest Sa, Sq and Spv values. Following the attachment, adhesion and retention assays, E. coli in monoculture did not show any trends between the surface properties and the number of cells retained. However, for L. monocytogenes in monoculture, following the attachment and retention assays the Flat surface showed the least number of cells (6 Log10 CFU/cm2 and 4.5 Log10 CFU/cm2 respectively). Following the adhesion and retention assays, the Gladioli surface (highest Spv values) displayed the lowest numbers of L. monocytogenes cells (6 Log10 CFU/cm2 and 3.7 Log10 CFU/cm2 respectively). Use of the bacteria in co-cultures demonstrated that for both the attachment and retention assays, the Tenderheart surface (most hydrophobic) displayed the lowest number of cells (4.5 Log10 CFU/cm2 and 3.4 Log10 CFU/cm2 for E. coli, 5.1 Log10 CFU/cm2 and 3.9 Log10CFU/cm2 for L.monocytogenes respectively). SEM analysis did not correlate with the CFU/mL assays. However, with L. monocytogenes the flat surfaces (lowest roughness) retained the lowest numbers of cells (4.7 Log10 cells /cm2) and regarding the co-culture, the White cabbage surface (most hydrophilic) displayed the lowest number of cells when tested for bacterial attachment, adhesion and retention (4.1 Log10 cells /cm2, 4.5 Log10 cells /cm2 and 0 Log10 cells /cm2 respectively. These results demonstrate that when more topographically complex surfaces are analysed, the conclusions drawn between the effect of the surface properties on bacterial attachment, adhesion and retention from more uniform surfaces do not apply. Further, the processes of bacterial attachment, adhesion and retention are different and hence differentiation between these classifications needs to be clarified. It became apparent that the varying methods used produced a wide range of results and that the use of different bacteria in monoculture and co-culture affected the microbial assays. Hence, a new approach needs to be taken to understand the cell: surface interactions on complex surfaces.