Abstract
The expression of multi-drug efflux pumps belonging to the Resistance-Nodulation- Division family of exporters has been shown to contribute to both intrinsic and acquired resistance among Pseudomonas aeruginosa. These pumps are able to recognize and export diverse compounds including several classes of antibiotics. The overproduction of RND pumps can lead to high-level, multi-drug resistant phenotypes. The goal of this research was to understand how the expression of these pumps are governed in wild-type susceptible and drug resistant mutants and how these pumps interact with other cell factors to generate unique resistance phenotypes. In addition, a drug combination of fluoroquinolone-imipenem was explored to prevent the emergence of resistance during therapy by efflux and other chromosomal mechanisms.|Contrary to previous reports in the literature, four of the well characterized efflux pumps, mexAB-oprM, mexCD-opri, mexEF-oprN, and mexXY, were shown to be expressed in a wild-type susceptible strain of P. aeruginosa. The effect of growth-phase on expression of these pumps had varying results depending upon which RNA isolation method and endogenous controls w'ere utilized. When RNA was isolated by the hot phenol method and analyzed by semi-quantitative RT-PCR, similar conclusions were reached with other investigators as data demonstrated a growth-phase regulation of mexA expression with increasing levels of expression being observed from the earliest stages of growth through late stationary phase growth. Furthermore, we confirmed that expression of oprM is driven by its own promoter and independent of mexAB expression. In contrast to mex A, the highest levels of mexC and mex E expression were observed earlier in the growth phase, and mexX expression remained similar through the growth-phases. Surprisingly, when RNA was isolated by a modified Trizol method and analyzed by Real Time RT-PCR, P. aeruginosa failed to show any growth-phase regulation of mex A expression in contradiction to results obtained from semi-quantitative RT-PCR. Expression of the other operons decreased over the course of the growth cycle.|When the mexCD-oprJ efflux system is hyperexpressed, P. aeruginosa becomes resistant to certain antibiotics (i.e. fluoroquinolones) but simultaneously exhibits hypersusceptibility to imipenem and other drugs. In order to explore the relationship between levels of pump expression and susceptibility, phenotypic reveilants were generated by exposing imipenem-hypersusceptible, MexCD-Opr.) overproducing mutants to imipenem. Full phenotypic reversion was observed in some but not all revertants and did not coincide with a loss of pump overexpression. These data suggest that the hypersusceptibility phenotype associated with mexCD-opri hyperexpression is more complex than anticipated and involves multiple factors. Hypersusceptibility to the carbapenem, imipenem, in mexCD-opri overproducing mutants did not involve AmpC as previously suggested in the literature, nor the outer membrane porin OprD. Therefore, hypersusceptibility to imipenem may be linked to downregulation of a yet unidentified pump, changes in target, or increased permeability of the drug through a mechanism unrelated to OprD. Our studies demonstrated that hypersusceptibility to the aminoglycoside, kanamycin, involved a decrease in mexXY induction in mexCD-opri hyperexpressing mutants. The involvement of all four efflux pumps as well as other mechanisms in drug- resistant clinical isolates was examined in two separate studies. In the first study, fluoroquinolone resistance among a collection of fluoroquinolone-carbapenem dual resistant isolates from a Texas hospital was shown to involve the hyperexpression of the mexXY efflux pump, as well as changes with the intracellular drug targets. In contrast, carbapenem resistance was shown to involve the decreased expression of oprD. These data suggested that the dual resistance to fluoroquinolones and carbapenerns in these isolates emerged by independent mutational processes and not by single selection of the mexEF-oprN efflux pump by the fluoroquinolones. In the second study, the emergence of fluoroquinolone resistance was detected in P. aeruginosa isolated from a patient with Cystic Fibrosis. Resistance was associated with fluoroquinolone usage in the patient and involved the overexpression of the mexEF-oprN efflux system. However, contrary to what is reported in the literature, dual resistance to the carbapenerns was not associated with the mexEF-oprX hvperexpression. This provides yet another example of the complexity of the P. aeruginosa efflux pumps and how they interact with other resistance mechanisms.|Finally, a fluoroquinolone-imipenem drug combination was explored to prevent the emergence of resistance during therapy. It was hypothesized that fluoroquinolones would be active against imipenem-resistant mutants while imipenem would be active against fluoroquinolone resistant mutants. This combination was tested in an In vitro Pharmacokinetic Mode! against three susceptible clinical isolates and one isolate intermediately resistant to both drugs. With either drug alone, rapid regrowth was observed due to the selection of resistant subpopulations. In contrast, the fluoroquinolone-imipenem combination rapidly eradicated all three P. aeruginosa clinical isolates and provided effective killing against the intermediately resistant strain. The outgrowth of a resistant subpopulation did not occur with the combination. These data suggest that fluoroquinolone-imipenem may be an effective combination for preventing the emergence of resistance among P. aeruginosa, even when subpopulations resistant to both drugs are present. Further studies are warranted to evaluate this combination against strains with established resistance to either or both drugs.|Taken together, results presented in this dissertation show the contribution of efflux to antimicrobial resistance and the complexity of phenotypes generated as a result of pump overexpression. Future studies are needed to unravel the interaction of these pumps with other factors in the cell leading to these complex phenotypes. In the meantime, clinical studies with combinations, such as the fluoroquinolone-imipenem combination, are needed to identify a method to curve the growing trend towards multi-drug resistant P. aeruginosa.