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Responses of Pseudomonas aeruginosa to sub-inhibitory antibiotics Brazas, Michelle Denise

Abstract

Emergence of resistance is never far behind the introduction of new antimicrobials. In response to antimicrobial challenge, bacteria have the capacity to alter their genomes in order to survive. Antibiotic environments play an important but poorly defined role in mediating such changes. The primary focus of this research was to further define the role of sub-inhibitory antimicrobials in the bacterial response to antimicrobial challenge, with particular attention to adaptive resistance responses. A greater understanding of this biological signature will help in evaluating dosing regimes, understanding mechanism of action and in designing new antimicrobials. The effect of sub-inhibitory ciprofloxacin was studied here, beginning with a global analysis using custom DNA microarray technology. Before conducting this analysis, a custom DNA microarray to 5,378 of the 5,570 open reading frames in the Pseudomonas aeruginosa genome was designed, constructed and validated, along with the accompanying set of RNA isolation, cDNA preparation and labelling, and microarray hybridization protocols. Analysis of the P. aeruginosa transcriptome following exposure to sub-inhibitory ciprofloxacin found expression changes in numerous genes. Prominent among these changes was up-regulation of the SOS DNA-repair response and the R2/F2 pyocin region. These changes were confirmed at both the transcription and protein level. Mutants in the R2/F2 pyocin region were found to be resistant to quinolones as well as other DNA damaging agents like mitomycin C, highlighting a role for this region in mediating susceptibility to DNA damage. I also found that sub-inhibitory ciprofloxacin, particularly 0.3μg/ml ciprofloxacin, induced adaptive resistance responses in P. aeruginosa. Similar responses were not observed in a R2/F2 pyocin mutant strain, although the extent of the role of the R2/F2 pyocin region in mediating adaptive resistance to ciprofloxacin awaits further characterization. Overall, this study indicated that sub-inhibitory concentrations of ciprofloxacin antimicrobial do play an important role in the development of resistance in P. aeruginosa, and highlight the clinical importance of better understanding bacterial-antimicrobial interactions.

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