Activity of and resistance to cationic antimicrobial agents in Pseudomonas aeruginosa

This thesis investigates mechanisms of adaptive resistance to the cationic antimicrobial agents tobramycin and polymyxin B in the opportunistic pathogen Pseudomonas aeruginosa. Using a comprehensive mutant library of P. aeruginosa to screen for genes capable of affecting tobramycin susceptibility, 135 genes were identified that caused increased resistance when insertionally inactivated. Transcriptional profiling studies demonstrated downregulation of 53 of these genes in response to tobramycin and significant up-regulation of a number of heat shock genes including an alternative lon protease, AsrA. Induced expression of asrA in trans demonstrated its ability to induce heat shock genes in the absence of tobramycin and also provided protection against tobramycin in the first hour after exposure to a lethal dose of 4 μg/ml. Upregulation of the known efflux pump MexXY was observed after prolonged exposure to sub-inhibitory concentrations of tobramycin but induction of this operon was not observed as part of the immediate response to lethal concentrations of tobramycin. When investigating susceptibility testing methods for polymyxins, 24 P. aeruginosa clinical isolates were observed to have a distinct, reproducible phenotype in which skipped wells were observed during microbroth dilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated in two of these isolates and one isolate demonstrating a constitutive resistance phenotype. The effects of varying concentrations of polymyxin B on growth, on expression of the resistance genes phoQ, arnB and PA4773 (pmrAB operon), and on outer membrane permeability were assessed. The isolates presenting the skipped well phenotype demonstrated adaptations in growth, gene expression and membrane permeabilization in response to specific concentrations of polymyxin B consistent with the involvement of Lipid A modifications in the adaptive resistance phenotype. The results of this thesis highlight the complexity of the bacterial response to cationic antimicrobial agents, as we have demonstrated that adaptation conferring immediate protection (induction of heat shock) differs from that providing long term protection (induction of efflux and Lipid A modifications). Furthermore, the regulatory systems involved in conferring resistance through Lipid A modifications, PhoPQ and PmrAB, are complex and may vary between strains as they adapt further to the pressures imposed by antimicrobial treatment.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat