Date of Award

Spring 2020

Document Type

Open Access Dissertation

Department

Environmental Health Sciences

First Advisor

Jamie R. Lead

Abstract

Silver nanoparticles (AgNPs) have attracted tremendous attention as a potential broadspectrum antimicrobial agent to overcome multidrug resistant (MDR) infections. However, a comprehensive understanding to AgNPs bactericidal mechanism of action and the relative role of particulate versus ionic Ag in AgNPs antibacterial activity is lacking but essential for their optimization for potential medical applications. Therefore, a novel method to separate and quantify Ag internalization in P. aeruginosa was developed and validated through multimethod approach. The methods used were optical density at 600 nm (OD600), LIVE/DEAD staining, transmission electron microscopy (TEM), and sorbed Ag liberation assays. After optimizing the method, it was applied to quantify Ag content in different bacterial fractions. To better understand the dynamic transformations of AgNPs upon exposure and the relative bioavailability/biouptake of different Ag species, bimetallic Au@Ag NPs was used and Ag:Au ratios were calculated in different bacterial compartments. Results showed a nearly constant OD600 within the first two hours of 50 mM EDTA treatment indicating complete bacterial growth cessation. However, bacterial density declined beyond this treatment condition indicating cell lysis. LIVE/DEAD staining images indicated intact bacterial membrane before- and after- EDTA treatment for six hours. Cell counts observed in untreated controls were statistically similar to cell counts after two hours exposure to EDTA. Whereas significantly (p <0.0001) fewer cells were observed in images after six hours EDTA exposure when compared to untreated controls. This suggested that complete disruption and lyses of cells may have occurred upon EDTA

exposures exceeding two hours rather than the simple cell membrane damage or/and increased permeability. TEM results indicated intact, smooth, and dense cell boundaries for untreated controls and cells exposed to EDTA for two hours. No membrane disintegrations were detected even after six hours EDTA treatment, but cells featured very light and thin surfaces near the cell edges compared to control. However, 50 mM EDTA treatment is a very high concentration and is expected to remove most of the periplasm. The plateau obtained in the Ag liberation curve, from the operationally defined sorbed fraction, within the first two hours of EDTA treatment suggested complete removal of sorbed Ag. Whereas the statistically significant (p <0.05) increase in Ag mass content after six hours EDTA treatment was likely as a result of cells lysis and/or increased membrane permeability. The weight of evidence across the multimethod approach suggested that two hours of EDTA treatment at a concentration of 50 mM provided a reproducible estimation of Ag uptake in the operationally defined internalized fraction approximating the accurate value in the Gram-negative bacterium P. aeruginosa. Therefore, this method was applied to quantify metallic distribution upon exposing bacteria to Au@Ag NPs. Ag distribution data indicated that the total Ag accumulated in the abiotic phase was mostly in dissolved form and was 45.5% and 54.8 % for 0.2 mg L-1 and 2.0 mg L-1 Au@Ag NPs exposure, respectively. The external cellular fractions after periplasm removal were 42.2% for 0.2 mg L-1 Au@Ag NPs exposure whereas for 2.0 mg L-1 Au@Ag NPs exposure it was 44.7%. The internalized fraction accumulated 16.2% ± 1.8 in 2.0 mg L-1 Au@Ag NPs exposure that was significantly greater (p = 0.0066) than 9.9% ± 1.1 in 0.2 mg L-1 Au@Ag NPs exposure. On the other hand, molar ratios were significantly greater consistently in all biotic phase fractions at lower exposure concentration except for internalized fraction.

Molar ratios detected in both abiotic phase (p >0.9999) and internalized fractions (p =0.9999) were not significantly different from pristine NPs. Ag:Au ratios indicated a significant concentration-dependent effect on Ag transformations and bioavailability. At higher exposure concentration, Ag was less bioavailable (in both external and internalized cellular fractions), and aggregation appeared to be the dominant process. In contrast, Ag was more bioavailable, and dissolution seemed to be the dominant process at lower exposure concentration. The weight of evidence suggests that Ag+ preferentially adsorbed to the external cellular surface, whereas a complex mixture of depleted original Au@Ag NPs and dissolved Ag in either ionic or secondary NPs forms accumulated in the internalized fraction. However, these fractions were operationally defined and might not perfectly match their corresponding theoretical definitions. Consistent with previous studies, our results suggest that both AgNPs and dissolved Ag are bioavailable to bacterial cells and may interact with multiple cellular targets simultaneously.

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