Browsing by Author "Xu, Ruifang"

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Reaction-diffusion theory explains hypoxia and heterogeneous growth within microbial biofilms associated with chronic infections
(2016-06) Stewart, Philip S.; Zhang, Tian-Yu; Xu, Ruifang; Pitts, Betsey; Walters, Marshall C., III; Roe, Frank L.; Kikhney, Judith; Moter, Annette
Reaction–diffusion models were applied to gain insight into the aspects of biofilm infection and persistence by comparing mathematical simulations with the experimental data from varied bacterial biofilms. These comparisons, including three in vitro systems and two clinical investigations of specimens examined ex vivo, underscored the central importance of concentration gradients of metabolic substrates and the resulting physiological heterogeneity of the microorganisms. Relatively simple one-dimensional and two-dimensional (2D) models captured the: (1) experimentally determined distribution of specific growth rates measured in Pseudomonas aeruginosa cells within sputum from cystic fibrosis patients; (2) pattern of relative growth rate within aggregates of streptococcal biofilm harboured in an endocarditis vegetation; (3) incomplete penetration of oxygen into a Pseudomonas aeruginosa biofilm under conditions of exposure to ambient air and also pure oxygen; (4) localisation of anabolic activity around the periphery of P. aeruginosa cell clusters formed in a flow cell and attribution of this pattern to iron limitation; (5) very low specific growth rates, as small as 0.025 h−1, in the interior of cell clusters within a Klebsiella pneumoniae biofilm in a complex 2D domain of variable cell density.
Spatial growth patterns of Pseudomonas aeruginosa biofilms
(Montana State University - Bozeman, College of Engineering, 2004) Xu, Ruifang; Chairperson, Graduate Committee: Philip S. Stewart
Biofilms are less susceptible to antimicrobial action compared to their planktonic counterparts. The protective mechanisms are not fully understood. Physiological heterogeneity within biofilms is thought to contribute to the low susceptibility and was therefore studied. Expression of green fluorescent protein (GFP), induction of alkaline phosphatase (APase) by phosphate starvation, and the cell viability assay using the LIVE/DEAD BacLight bacterial viability stain were performed to visualize the spatial patterns of growth and viability within 5-d-old Pseudomonas aeruginosa biofilms. The capillary reactor and the drip-flow reactor were employed to obtain biofilms of a range of thickness. Biofilms cultivated in the capillary reactor were usually thinner than those grown in the low-shear drip-flow reactor. The former were examined by in situ confocal scanning laser microscopy (CSLM) whereas the latter were cryoembedded and cryosectioned prior to conventional fluorescence microscopic observation. P. aeruginosa PAO1 with the plasmid pAB1 carrying an inducible, stable gfp was used to identify zones of active protein synthesis. The induction of gfp proved suitable for the visualization of spatial growth patterns within biofilms. Greater GFP activity was evident at the surface of clusters and was not as bright in their centers after induction. Activity appeared more uniform in smaller clusters and less uniform in larger clusters. The APase activity induced by phosphate starvation showed a sharply delineated band of active APase synthesizing cells close to the biofilm-bulk fluid interface and some local APase synthetic activity in the depth of the biofilm. The results of biofilm viability staining using the LIVE/DEAD BacLight bacterial viability kit turned out to be puzzling and cast doubt on the methodological validity of applying the LIVE/DEAD BacLight bacterial viability staining method to P. aeruginosa biofilms. The findings of the spatial growth patterns illustrate the physiological heterogeneity that is present in these biofilms. Such variation in the metabolic activity probably contributes to the reduced susceptibility of these biofilms to antimicrobial agents.