A quantitative description at multiple scales of observation of accumulation and displacement patterns in single and dual-species biofilms
Klayman, Benjamin Joseph.
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This research represents a novel approach for describing biofilm accumulation at multiple scales of observation in both single and dual-species biofilms. Pseudomonas aeruginosa PAO1 and Escherichia coli O157:H7 were grown as single and dual-species biofilms in 1 mm glass capillary flow cells and monitored over time using confocal microscopy. Colonization and biofilm development patterns were associated with the fluid flow regime as evaluated using the finite volume analysis program CFX (ANSYS Europe, Ltd). The shear stress was shown to vary along the surface from a minimum near the edges to a maximum in the center of the flow path. Initial colonization by both species occurred at the outer edges of the flow path (low shear). P. aeruginosa was subsequently observed to migrate perpendicular to the flow direction towards the center of the flow path (high shear), but E. coli was never observed outside of the 200 micron outer edge. E. coli was unable to persist in the flow cell unless P. aeruginosa was present as a colonizing partner. Bio-volumes of each species were calculated using the Metamorph (Molecular Devices) image analysis program and are reported over time.P. aeruginosa reached a much higher final cell density when examining the entire surface (>99% total bio-volume), while an analysis of the outer 200 microns of the flow path revealed that in this microenvironment E. coli was observed to out-compete P. aeruginosa. (>50% total bio-volume). Additionally, significant advancement was achieved in describing accumulation and displacement at the single cluster level in developing (non steady-state) singlespecies, dual-labeled P. aeruginosa biofilms. User script was written in Metamorph software to allow for volume and centroid measurements of single clusters as well as pockets of cells within clusters. From measurements made over time the accumulation rates and displacement vectors were calculated. The distribution of cluster accumulation rates was observed to be upper bound by the planktonic growth rate for small cluster sizes, and was frequently observed to be negative (indicating net decrease in bio-volume) for larger cluster sizes. Expanding larger clusters were observed to physically displace neighboring cells and smaller cell clusters.