Spatial heterogeneity in Pseudomonas aeruginosa biofilms and how it affects antibiotic tolerance
Richards, Lee Alexander
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This dissertation presents evidence of heterogeneity within Pseudomonas aeruginosa biofilms and the effects of said heterogeneity on antibiotic tolerance. The existence of oxygen concentration gradients within the biofilm was confirmed. There were in fact regions within the biofilm that were nearly anoxic, this was confirmed by use of dissolved oxygen microelectrodes. The size of the aerobic zone within the biofilm agreed with the size of the active zone indicated by the use of an inducible green fluorescent protein. We found that anoxia could explain some of the biofilm's recalcitrance to the antibiotics ciprofloxacin and tobramycin, but the effects of anoxia were not adequate to explain all of an intact biofilm's tolerance to antimicrobial treatment. It was also apparent that glucose limitation was not a factor in biofilm recalcitrance. In addition, dormancy within Pseudomonas aeruginosa biofilms was explored by use of a novel approach to labeling active and dormant cells within the biofilm using a strain of P. aeruginosa tagged with a stable, inducible, green fluorescent protein. Spatial patterns of activity were visualized by microscopy. Further, we found it possible to sort the active and dormant cells using a flow cytometer. It was thus possible to determine the relative viability of each population after treatment with the antibiotics tobramycin and ciprofloxacin. We found that dormant cells were much more tolerant to antibiotic treatment than were active cells within the same biofilm.