Carabelli, Alessandro M.
(2019)
Exploring bacteria-surface interactions.
PhD thesis, University of Nottingham.
Abstract
Bacteria adhere to almost any surface. Medical-device biofilm-centred infections pose an enormous threat, particularly from multi-antibiotic resistant pathogens. Biofilm formation is largely understood and encompasses an initial,reversible bacterial cell surface-attachment phase followed by irreversible surface-attachment, leading to the formation of biofilms that can be up to 1,000 times more resistant to antibiotics and refractory to host immune defences. A better understanding of bacterial-surface interactions should aid human intervention into this process to reduce biofilm formation on implanted medical devices.
Previously, Hook et al. screened an (meth)acrylatepolymer microarray in high-throughput for polymers that prevent biofilm formation by a selection of bacterial pathogens including Pseudomonas aeruginosa. This resulted in the discovery of poly(ethylene glycol dicyclopentenyl ether acrylate)(pEGdPEA) which has recently been approved for human use as a coating (BACTIGON®) for urinary tract catheters. However, the mechanism(s) by which pEGdPEA prevents biofilm formation is not known beyond the observation that it does not inhibit bacterial growth. This thesis describes the response of bacteria to pEGdPEA in comparison with that of a biofilm-promoting polymer, poly(neopentyl glycol propoxylate diacrylate polymer) (pNGPDA). Fluorescence and electron microscopy after 24 h of incubation with P. aeruginosa showed that little extracellular matrix formed on pEGdPEA in contrast with pNGPDA where a robust biofilm was observed.
A method for spatio-temporal characterisation of bacterial motility at and above surfaces was developed using a custom designed microscope that allowed the interaction of Pseudomonas on different materials to be followed over time. This multimode 2D-3D microscope utilised several optical techniques simultaneously with the ability to record stable video data of individual bacterial cells: namely digital holography, differential interference contrast (DIC), TIRM (total internal reflection microscopy),TIRF (total internal reflection fluorescent microscopy) and widefield epifluorescence microscopy. The behaviour of large numbers of bacterial cells was characterised simultaneously from videos using high-throughput motion analysis algorithms. To study bacteria surface adhesion strength on different chemistries a method was developed using microfluidics and xurography or razor writing to form channels. P. aeruginosa cells showed weaker surface adhesion strength, moved faster and with shorter residence times on pEGdPEA compared with pNGPDA. P. aeruginosa cells were also observed, post cell-division, to leave the pEGdPEA surface with a higher frequency than from the pNGPDA surface. By using a cdrA::gfp fusion as a cyclic diguanylate (c-di-GMP) biosensor, the inability of P. aeruginosa cells interacting with pEGdPEA to increase c-di-GMP levels was observed and contrasted markedly with the rapid induction of high levels of fluorescence in cells on pNGPDA.
Biofilm-forming bacterial cells are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on the extracellular polymeric substance (EPS) which is known to affect surface adhesion. Here, by combining EPS staining and bacterial tracking, P. aeruginosa was shown not to deposit exopolysaccharides on pEGdPEA, in contrast to that observed on pro-biofilm pNGPDA. These data, together with weaker adhesion strength, higher frequency of detachment events and lower c-di-GMP intracellular levels, suggest that on pEGdPEA, P. aeruginosa is unable to switch from the reversible to irreversible attachment stage. The key signalling and sensing pathways used by P. aeruginosa to respond to surfaces were investigated and the data obtained suggest that bacteria actively “decide” whether to attach to a particular surface and that the decision not to form a biofilm on pEGdPEA is likely to involve the sadB pathway. SadB is a cytoplasmic protein with an, as yet, unknown function but its upstream regulatory pathway involves c-di-GMP signalling. How this protein is involved in the ability of bacteria to maintain a surface-associated state which leads to an irreversible attachment is described.
Bacteria have been shown to clump as multicellular aggregates during the process of biofilm formation either in bulk (as planktonic aggregates) or at the surface. Here differential planktonic aggregation with respect to pEGdPEA and pNGPDA is described where greater aggregation was observed on the latter. The formation of aggregates in the bulk liquid phase above these polymers was characterised using bright-field microscopy and laser diffraction analysis. The differential aggregation observed was also examined for ‘aggregation inducing’ signals by analysis of the cell-free spent media using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparative metabolomics enabled the identification of the aggregation-inducing signal N-(3-oxododecanoyl)-L-homoserine lactone, which was at a significantly lower concentration in spent medium from cells previously exposed to pEGdPEA compared with the pro-biofilm pNGPDA. These data provide new insights into the understanding of how bacteria, after being in contact with different surface chemistries, are able to alter their bulk liquid phase behaviour. Additionally, this highlights the potential of novel use of these polymer surfaces to investigate bacterial biofilm development and to develop new solutions for the prevention of bacterial biofilm-centred infections.
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