Spatial growth patterns of Pseudomonas aeruginosa biofilms
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.