Theses and Dissertations at Montana State University (MSU)
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Item Role of transport limitation in the resistance of Pseudomonas aerunginosa biofilms to ciprofloxacin and levofloxacin(Montana State University - Bozeman, College of Engineering, 1996) Vrany, Julia DawnBacterial biofilm infections are often far less susceptible to antibiotic therapy than their planktonic, or freely suspended counterparts. Transport limitation of the antibiotic within the biofilm has been proposed as one explanation for this resistance. To explore the possibility that transport limitation contributes to a decreased antibiotic efficacy, a Comparison was made between the efficacies of two fluoroquinolone antibiotics, ciprofloxacin and levofloxacin, against freely suspended and biofilm bacteria. Transport of the antibiotics to the biofilm-substratum interface was monitored using ATR-FTIR spectroscopy techniques. Both biofilms and planktonic organisms were treated with antibiotic concentrations of 100, 250, and 500 μg ml-1 for 30 min and rinsed for 1 h with fresh media. Antibiotic efficacy was determined by the ratio of culturable bacteria to total cell counts. The experimental ATR-FTIR transport data was then simulated with a mathematical computer model containing the processes of molecular diffusion, adsorption, and desorption to provide possible explanations for differences in antibiotic delivery. Levofloxacin was found to be more efficacious against planktonic organisms than biofilm cells. However, no difference in efficacy was seen when planktonic and biofilm bacteria were treated with ciprofloxacin. ATR-FTIR results showed that the biofilm provided very little transport limitation for each antibiotic, thus suggesting that the reduced susceptibility of a biofilm to antimicrobial treatment may be due to factors other than transport.Item Spatial heterogeneity in Pseudomonas aeruginosa biofilms and how it affects antibiotic tolerance(Montana State University - Bozeman, College of Engineering, 2006) Richards, Lee Alexander; Chairperson, Graduate Committee: Philip S. StewartThis 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.