Abstract
Pathogens that produce the Klebsiella pneumoniae carbapenemase (KPC) have become a major threat to the global health care system. Patients infected with KPC-producing pathogens have a higher incidence of morbidity and mortality, as these infections have been associated with high levels of β- lactam resistance, in addition to other antibiotic drug classes. This, in addition to the lack of novel antimicrobials with activity against Gram-negative bacteria, forces physicians to treat infections caused by KPC-producing pathogens with potentially toxic antibiotics, such as the aminoglycosides and polymyxins. The rapid spread of blaKPC into multiple genera of Gram-negative bacteria, the lack of effective treatment regimens, and the difficulty of identifying these pathogens using current detection methods in the clinical laboratories are the biggest challenges the KPC-producing organisms pose.|Although in vitro testing indicates that the KPC β-lactamase can efficiently hydrolyze the carbapenems, multiple studies have reported variability in susceptibility testing of KPC-producing clinical isolates. The first hypothesis tested in this dissertation was that the variation observed during susceptibility testing was due to variations in gene expression and/or gene copy number. Variation in KPC enzyme production could contribute to the variability observed during susceptibility testing. However, the individual influence of the KPC β-lactamase cannot be determined in clinical isolates since all of the organisms evaluated to date produce multiple β-lactam resistance mechanisms. Little is known about the role gene expression plays in KPC-mediated resistance or its affect on KPC production during susceptibility testing. Therefore, I sought to elucidate the molecular mechanisms that contribute to the variation in susceptibility of Gram-negative bacteria harboring blaKPC.|To address this problem, we created a panel of four different genera of Gram-negative bacteria that expressed the blaKPC gene without the influence of other resistance mechanisms. Using this panel, we evaluated the relationship between blaKPC gene expression to β-lactam MICs, gene copy number, and protein production in comparison to KPC-producing clinical isolates. These studies identified variation in blaKPC gene copy number, expression and protein production in the clinical isolates compared to their|genus-specific control. These variations in expression and protein production did not always correlate with the β-lactam MICs observed during susceptibility testing. Evaluation of RNA expression identified two novel transcriptional start sites and promoter elements for blaKPC. Subsequent analysis of the|upstream promoter regions using constructed promoter deletion clones demonstrated that the proximal promoter elements were responsible for the β-lactam resistant phenotype.|As the expression and protein data do not elucidate the problems associated with susceptibility testing and the rapid spread of this gene among numerous genera, a second hypothesis was tested: Exposure to sub- inhibitory levels of antibiotics stimulates blaKPC expression and/or contributes to the mobilization of Tn4401 carrying blaKPC. I evaluated the effect of “collateral damage,” or the unintended effects of exposure to antibiotics on transmission and expression of KPC–mediated antimicrobial resistance. Five KPC-producing clinical isolates were exposed to antibiotics from five different drug classes to determine whether they would stimulate blaKPC expression or mobilization of the transposon Tn4401 carrying blaKPC. While no changes in Tn4401 mobility were observed, there were subtle increases in blaKPC gene|expression with gentamicin and tigecycline, both of which are used in the treatment of KPC-producing pathogens. Of note, non-β-lactam drugs influenced blaKPC gene expression more often than the β-lactam drugs, but strain to strain variation occurred.|The third hypothesis was that a high resolution melt assay would be able to detect and differentiate between blaKPC alleles. To circumvent the|difficulties that conventional phenotypic testing can pose an alternative testing algorithm was designed to decrease the turn around time required to identify the presence of the KPC carbapenemase in Gram-negative clinical isolates. To do so, a single tube, PCR-based assay was designed to detect and differentiate between blaKPC gene alleles. Validation of this study involved testing 166 Gram-negative clinical isolates, 66 of which were known to be KPC-positive. This assay demonstrated 100% diagnostic sensitivity and specificity for both blaKPC gene detection and differentiation into blaKPC-2-like and blaKPC-3like allele groups.|Taken together, these data demonstrate the level of complexity by which blaKPC is regulated and the preliminary findings may represent a means of differential expression under various selective pressures. Although the fold differences in blaKPC gene expression are subtle, these differences cannot be ignored. This variation may represent differences in metabolic processes or other compensatory mechanisms that may have been selected for in the patients they infect, where they can be exposed to a variety of different physiological conditions and stressors. It is imperative that we continue to evaluate the complexity of this resistance mechanism in order to better understand how it spreads to susceptible patient populations. As it is apparent that there may be several, strain dependent mechanisms involved in the susceptibility testing variation, the development of molecular assays in the clinical microbiology laboratory may be a superior testing method for the identification of KPCs.