Shakerley, Nicole Theses Advised
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Item Investigating minocycline’s anti-inflammatory role in alveolar macrophages(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Scholl, Erica '23Minocycline has been FDA-approved for 50 years and is utilized to treat a wide range of infections. More recently, minocycline has been used to treat several sterile inflammatory conditions due to its non-antimicrobial anti-inflammatory properties. While minocycline is a unique and beneficial therapeutic, its immunomodulatory impact on inflammation during an active infection is not well understood. Elucidating the molecular mechanism of minocycline’s ability to modulate the inflammatory response can lead to the development of novel therapeutic strategies that take advantage of the antibiotic capabilities of minocycline while also protecting the host against post-infection inflammatory tissue damage. We hypothesized that minocycline exerts anti-inflammatory properties that suppress the cytokine production after macrophage activation, altering the host immune response to pathogens and limiting tissue damage. Our data indicates that minocycline is nontoxic to alveolar macrophages even at doses far beyond therapeutic potential. Additionally, pretreatment with minocycline significantly decreased the activation of nuclear factor kappa beta (NF-κB), as well as the secretion of several inflammatory cytokines following treatment with ligands to activate specific toll-like receptors. Total levels of key signaling molecules MyD88 and NF-κB were not altered following minocycline pretreatment, suggesting that minocycline may be altering the activation status within the cascade. Lastly, the pretreatment of macrophages with minocycline appears to have no significant effect on the production of oxidative stress following ligand treatment at 24 hours. Our long-term goal is to identify immunomodulatory mechanisms exerted by minocycline in the presence of an infection, as it could be the key to managing infections by not only eliminating the pathogen but also minimizing host inflammatory tissue damage, which is a driver of poor patient outcomes.Item Alternative Treatment Options against Carbapenem-Resistant Acinetobacter baumannii Infections(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Putra, Vibert '22Carbapenem-resistant Acinetobacter baumanii (CRAB) is a multidrug-resistant nosocomial gram-negative pathogen that has become increasingly prevalent in the United States. CRAB has been labelled by the U.S. Centre for Disease Control and World Health Organisation as an “urgent threat” and “critical priority”, respectively. Due to its plethora of resistance arsenals, current treatment options against CRAB are extremely limited and combination treatments have become the norm. The purpose of this study to explore alternative combination treatments by evaluating clinically used antibiotic combinations and identifying a novel non-antibiotic combination strategy. Polymyxin B plus meropenem and polymyxin B plus minocycline were evaluated by in vitro hollow fibre infection models against minocycline-susceptible clinical CRAB strains. Our results showed polymyxin B combination with meropenem have greater bacterial killing despite minocycline monotherapy susceptibility in vitro. Previous studies showed that a USFDA-approved rheumatoid arthritis drug, auranofin, inhibits bacterial thioredoxin reductase within the antioxidant system that restores protein structures damaged by oxidative stress and regulates downstream effector proteins. Thioredoxin reductase activates thioredoxin A (TrxA), which is the effector protein in the thioredoxin system. Our 24-hour static concentration time kill assays showed that auranofin and meropenem combinations were beneficial against clinical CRAB isolates but not meropenem-susceptible clinical isolate. Furthermore, disc diffusion assay showed that auranofin has additive effects with minocycline, colistin, and chloramphenicol. Our total antioxidant assay showed that the TrxA-deleted mutant showed has significantly increased total antioxidant activity under meropenem stress, suggesting that the pathogen can compensate for the lack of antioxidant activity from the thioredoxin system. Hence, we propose that the increased antibiotic susceptibility observed under auranofin treatment was due to thioredoxin modulation of resistance mechanisms. Our study showed that TrxA modulates tetracycline efflux pump activities, while TrxA only modulates baseline total β-lactamase activities and not its induction overtime under meropenem stress. Therefore, our study showed the two potential avenues of therapies against CRAB infections that can either be applied immediately in the clinics or further characterised in vitro. Further evaluation of polymyxin B plus meropenem combination dose ranges and determination of thioredoxin role in modulation of resistance are needed to develop optimal treatments.Item Thioredoxin-mediated modulation of Acinetobacter baumannii virulence and antibiotic resistance(Albany College of Pharmacy and Health Sciences Theses, 7/15/2020) Phelps, Catherine '20Bacterial antioxidant enzymes have been shown to aid intracellular survival and antibiotic resistance pathways in many microbes. These enzymes represent a largely unexamined pool of potential drug targets in Acinetobacter baumannii. Due to rapidly increasing drug resistance and limited knowledge of specific contributing mechanisms, A. baumannii has been classified as a critical pathogen for the development of new therapeutics by both the World Health Organization and CDC. Thioredoxin A (trxA) is an oxidoreductase produced by A. baumannii which maintains bacterial redox homeostasis by recycling electrons. In this study, we examine a trxA deficient strain of Acinetobacter (\316\224trxA) alongside a wild type (Ci-79) and complement strain (\316\224trxA+ptrxA) to better understand the role of trxA in antibiotic resistance and virulence. \316\224trxA was shown to be more sensitive to oxidizing compounds pyrogallol, hydrogen peroxide, and diamide in disc diffusion assay, growth curve generation, and CFU recovery assays. Using a combination of in vitro and in vivo infection models, we demonstrate that \316\224trxA exhibits reduced survival in J774 macrophages and attenuated virulence in Galleria mellonella. Susceptibility to multiple classes of antibiotics was determined by disc diffusions and minimum inhibitory concentration (MIC) determinations. \316\224trxA demonstrated significantly greater sensitivity than Ci-79 or \316\224trxA+ptrxA to all antibiotic classes tested, suggesting that thioredoxin may be a novel treatment target. To reproduce this heightened sensitivity in the WT strain, Ci-79 was exposed to thiol stress-inducing agent diamide alongside a conventional antibiotic, meropenem. Treatment with both agents demonstrated synergistic activity in Ci-79 as well as other clinical isolate strains of A. baumannii. Further testing with FDA approved thioredoxin inhibitors utilized in combination with front line antibiotics also showed synergistic activity against Ci-79, providing proof-of-principle for a novel treatment protocol against multidrug-resistant Acinetobacter. This finding supports further investigation of additional antioxidant enzymes of A. baumannii as potential therapeutic targets.