Date of Award

5-14-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Dacheng Ren

Keywords

Biofilm, Efflux Pump, Eravacycline, Persister

Abstract

Eradicating persistent bacterial infections is an ever-growing problem in the medical field. Conventional antibiotics are becoming less effective against persistent infections in tuberculosis, cystic fibrosis, Lyme disease, device-associated, urinary tract, gastric and pneumonia still remains a problem. Left untreated, the persistent infections have the potential to increase mortality, morbidity, and the development of antibiotic resistant bacteria. One major mechanism that contributes to the failure of current antibiotic treatment is the formation of persister cells. Persisters are formed when bacterial cells undergo phenotypic changes and enter dormancy which enables their survival under lethal doses of antibiotics. As phenotypic variants, persister formation is transient and reversible. During antibiotic treatment, persisters remain growth arrested and repopulate after the antibiotic is withdrawn, causing the failure of treatment and relapse of many infections. Because most, if not all, antibiotics target growth-associated activities, eradication of persister cells has been challenging.Here, we report new findings that challenge the conventional view that bacterial persister cells are tolerant to all antibiotics and provide evidence that it is possible to achieve persister killing by leveraging dormancy-associated reduction of antibiotic efflux. We demonstrate that antibiotics capable of penetrating bacterial cells by energy- independent diffusion and binding to their target strongly can kill persister cells during wake-up. This was demonstrated using minocycline. At the concentration of 100 μg/mL, minocycline killed Escherichia coli persister cells by 70.8 ± 5.9% while it did not have significant killing of normal cells. Consistently, the results showed that persister cells had a higher intracellular minocycline concentration (~2.6 times more minocycline per cell) compared to normal cells. The results were corroborated with tests using efflux pump mutants and efflux pump inhibitors. Specifically, treatment with carbonyl cyanide m- chlorophenylhydrazone (CCCP), an efflux pump inhibitor, at 10μM led to 94.7 ± 2.5% killing of E. coli normal cells by minocycline. This is a 4-time increase compared to treatment without membrane depotentiation, which only led to 22.0 ± 3.3% killing. We demonstrated here that E. coli persister cells have reduced efflux and thus more accumulation of minocycline than normal cells, leading to an effective killing of this dormant population. Encouraged by these findings, we then sought out to determine if persister killing can be further enhanced by increasing the target binding affinity of antibiotics. Through literature research, we found eravacycline, which also targets the ribosome but has a stronger binding than minocycline. Our results demonstrated eravacycline can kill E. coli persisters by 3 logs when treated at 100 μg/mL. In addition, it has a strong synergy with ampicillin, eradicating both normal and persister cells of E. coli. With our proposed mechanism, we have proven thus far, that the increased in sensitivity of persister cells is the result of at least three factors which are low membrane potential, reduced efflux pump activity and the tight binding affinity of this antibiotic with the ribosome. To validate this new strategy, it is necessary to test if these antibiotics are also effective against persister cells of other pathogenic bacteria. This motivated us to test eravacycline on uropathogenic E. coli (UPEC) and Pseudomonas. aeruginosa. Our results demonstrated that eravacycline is effective in killing both UPEC normal and persister cells; by 97.8 ± 0.7% and 99.9 ± 0.1%, respectively at a concentration of 100 μg/mL. In addition, we tested the effects of eravacycline on P. aeruginosa PAO1 (wild-type) and PDO300 (mucoid mutant) persisters cells. Our results demonstrate that CCCP-isolated PAO1(wildtype) and PDO300 (mucoid) persisters can be

effectively killed by eravacycline (by 99.7 ± 0.0% and 99.0 ± 0.1%, respectively) with a concentration of 100 μg/mL. Lastly, we tested the effects of eravacycline on biofilm associated persister cells given that biofilms play a major role in chronic infections. At the concentration of 100 μg/mL eravacycline reduced the number of viable biofilm cells of UPEC, P. aeruginosa PAO1, and P. aeruginosa PDO300 by 99.2 ± 0.5%, 99.9 ± 0.8%, and 99.6 ± 0.4%, respectively, compared to untreated controlFurthermore, the results from E. coli persister data were used to develop a set of criteria for persister control. The set of criteria outline that effective control agents need to (1) be positively charged under physiological condition to interact with the negatively charged lipopolysaccharides on bacterial outer membrane, (2) be able to penetrate persister cells via energy-independent diffusion, (3) be amphiphilic to have membrane activity for penetration, and (4) have strong binding to an intracellular target. Using this set of criteria, we optimized a chemoinformatic clustering algorithm to design and select persister control agents. To validate these criteria, we first extracted structural and physiochemical parameters of persister killing agents identified in this study (minocycline, rifamycin SV, and eravacycline) and those from a small chemical library (80 chemical compounds). Next, we used k-means clustering to assess the logP (octanol-water partition) and the number of halogen atoms that appear on the compounds. We selected the top ten compounds based on the clustering result to furhter test for persister killing.

Among the tested top leads, we discovered four persister control agents that showed potent activity against E. coli persister cells. While most of the compounds had similar moderate effects on normal (~20% killing) and persister cells (~30% killing) when treated at 100 μg/mL, four compounds (161,171, 173 and 175) exhibited stronger effects against persister cells than normal cells. At a concentration of 100 μg/mL, they showed ~ 30% killing of normal cells; however, compounds 161, 171, 173, and 175 killed 95.5 ± 1.7% (p<0.0001), 85.2 ± 2.6% (p=0.0003), 94.2 ± 1.4% (p<0.0001), and 99.6 ± 0.1% (p<0.0001) respectively of the persister cells. To further understand the stronger killing efficacy of persister cells than normal cells by these compounds, we quantified the intracellular concentrations and found persister cells accumulated more compared to normal cells. Since, we demonstrated that persister cells have reduced efflux activities, we further evaluated if both compounds are substrate of the multidrug AcrAB-TolC efflux pump. To test this, E. coli JW4364 (ΔacrA mutant), JW5536 (ΔacrB mutant), and JW5503 (ΔtolC mutant) were compared with their wild-type strain E. coli BW25113 for susceptibility to compounds. Increased killing of all three efflux mutants compared to the wild-type strain was observed for all three of the four compounds. This finding further demonstrates a correlation between the lack of efflux and increase in persister killing.While it is commonly stated that persister cells are tolerant to conventional antibiotics, our study reveals that these dormant cells can be killed by selecting the right antibiotics with appropriate treatment conditions. Specifically, we demonstrate that antimicrobial capable of penetrating bacterial cells by energy-independent diffusion and binding to their target strongly can kill persister cells during wake-up. These results can help develop better strategies to combat persistent infections.

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