Acquisition of an Antibiotic Tolerant Phenotype in Enterobacter cloacae and Klebsiella pneumoniae through Antibiotic-Induced Metabolic Regulation
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Author
Harrison, Lucas B.
Date
2019-04-30
Degree
PhD (Doctor of Philosophy), Medical Microbiology and Immunology
Copyright: Thesis/Dissertation © Lucas B. Harrison, 2019
2019-04-30
Degree
PhD (Doctor of Philosophy), Medical Microbiology and Immunology
Copyright: Thesis/Dissertation © Lucas B. Harrison, 2019
Metadata
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Abstract
While Enterobacter cloacae and Klebsiella pneumoniae are part of the normal human microbiota, they are also potential human pathogens and have been associated with the spread of antibiotic resistance. These organisms can enter a physiological state that allows a fraction of each population to survive antibiotic exposure at concentrations above what is necessary to treat most of the population. Because most of the population is inhibited by the antibiotic, the subpopulation that can tolerate the antibiotic exposure is not detected by traditional phenotypic susceptibility testing. Multiple mechanisms for acquiring this antibiotic tolerant state have been proposed, and the downstream effects of many of these mechanisms govern global cellular functions like growth rate or metabolic activity. What is less clear is whether the tolerant organisms are preexisting or if they are induced into an antibiotic tolerant state. I hypothesized that antibiotic exposure can induce an antibiotic tolerant state in Enterobacter cloacae and Klebsiella pneumoniae through metabolic downregulation via decreasing intracellular concentrations of ATP. To address this hypothesis, I challenged a panel of clinical isolates to identify bacteria that exhibited the greatest degree of modification by evaluating the regulation of the antibiotic resistance gene blaKPC. Representative isolates were sequenced and a transcriptomic analysis was performed on ertapenem and tigecycline treated organisms to identify metabolic pathways that were differentially expressed between treated and untreated isolates. I identified the methylglyoxal pathway as a candidate pathway that bacteria can use to acquire antibiotic tolerance due to its ability to decrease ATP production in the cell, leading to metabolic downregulation. A panel of clinical isolates of Enterobacter cloacae and Klebsiella pneumoniae was evaluated for their ability to 1) express an antibiotic tolerant phenotype in response to antibiotic exposure, 2) regulate intracellular concentrations of ATP in response to antibiotic exposure and 3) induce expression of methylglyoxal pathway genes in response to antibiotic exposure. I found that the antibiotic tolerant phenotype was present in most of the clinical isolates tested. However, I also found that neither gene expression of the methylglyoxal pathway nor decreasing intracellular concentrations of ATP alone were sufficient to provide a tolerant phenotype for all isolates examined. In a glpT knockout of a clinical isolate that activated the methylglyoxal pathway through a GlpT-mediated feeder pathway, the knockout strain produced fewer tolerant cells than the parent strain. The knockout also showed higher intracellular concentration of ATP compared to the parent strain when both were exposed to ertapenem. From these results, I concluded that GlpT can contribute to antibiotic tolerance by providing a carbon substrate for the methylglyoxal pathway. While fewer tolerant bacteria were present in the knockout strain, this also meant that knocking out glpT alone is insufficient to eliminate the tolerant population of bacteria. From these findings, I conclude that multiple mechanisms of antibiotic tolerance are present in these organisms. Further, this suggests that different subpopulations within a bacterial population employ their own unique mechanism of antibiotic tolerance. If multiple mechanisms for acquiring an antibiotic tolerant phenotype in different bacteria have the same downstream effect, then it may be possible to identify a therapeutic target that affects multiple subpopulations of antibiotic tolerant bacteria at once.