Browsing by Subject "Antibiotic resistance"
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Item A molecular biological model describing silver nanoparticle mechanism of toxicity and associated antibiotic resistance(2018-05-04) Chambers, Bryant Allson; Kirisits, Mary Jo; Katz, Lynn E; Saleh, Navid; Hofmann, Hans; Parsek, MatthewControl of microbial growth is key to proper function of engineered systems and human health. Combating biological contamination in engineered processes is complicated due to the limited number of materials that are both able to impede microbial growth and are benign with respect to human and environmental health. Silver nanoparticles (AgNPs) have emerged as a novel biocide, reducing biological fouling in consumer goods and health care materials. Their almost ubiquitous usage is primarily due to their microbial cytotoxicity, limited human toxicity, and their ability to be incorporated into a wide variety of materials. The use of AgNPs is not without challenges; microbial toxicity varies by exposure methodology, and studies have shown that AgNPs have the potential to disrupt engineered biological processes either as nanoparticles or through the dissolution of aqueous silver (Ag([subscript aq])). The use of AgNPs is further complicated by their mechanisms of action; there is significant overlap of their biological targets with the targets of antibiotics. Thus, antibiotic resistance might result from AgNP exposure through the processes of co- and cross-resistance, in which one chemical selects for microbial resistance to a second (unrelated) chemical. In this work, the impact of AgNP aggregation and dissolution on toxicity to Escherichia coli was examined. Data indicate that conditions promoting high fractal dimension promote greater toxicity and induce an oxidative stress response. Subsequent studies on the opportunistic human pathogen Pseudomonas aeruginosa were directed at elucidating the mechanisms of action of AgNPs and the microbial response. Transcriptomic and proteomic studies focused on defining a model of bacterial AgNP interaction and isolated mechanisms of toxicity of AgNPs. Further these data provided the first evidence of AgNP exposure resulting in antibiotic resistance through the expression of multidrug efflux pumps. Transcriptomic data indicated that the stress response systems activated as a result of AgNP exposure were localized to the periplasm while the stress response systems activated as a result of Ag([subscript aq]) exposure were localized to the cytoplasm, which supports a surface attachment model of bacterial AgNP interaction distinct from that of Ag([subscript aq]). Transcriptomic studies revealed that key antibiotic resistance systems, including mexGHI and mexPQ, were stimulated by AgNP exposure. P. aeruginosa cells that were pre-exposed to a sublethal concentration of AgNPs demonstrated increased resistance in subsequent antibiotic challenges, demonstrating that antibiotic resistance can be induced by AgNPs. The findings of this study are an important contribution to our understanding of the impacts of co- and cross-resistance induced by AgNP exposure and will ultimately help inform decisions related to human and environmental healthItem An investigation of New Delhi metallo-[beta]-lactamase : clinical variants, lipid modification & inhibition(2017-05-12) Stewart, Alesha; Fast, Walter L.; Whitman, Christian; Kerwin, Sean; Lee, Seongmin; Hoffman, DavidMany β-lactam drugs, including penicillins, monobactams cephalosporins, and carbapenems, are used in clinical treatment of bacterial infections. However, resistant bacterial strains have surfaced, in part due to expression of β-lactam hydrolyzing enzymes called β-lactamases. One class of β-lactamases, metallo-β-lactamases (MBL), confers resistance to almost all clinically used bicyclic β-lactams through hydrolysis facilitated by active-site metal ions. The non-covalent mechanism of these enzymes enables them to escape the action of clinically used β-lactamase inhibitors, and there are currently no approved drugs that counteract their activity. One MBL that has caused particular alarm is New Delhi metallo-β-lactamase (NDM-1), that can be contracted through both hospital and communal settings. Clinically isolated bacteria strains that host NDM-1 have been reported to carry mutations in their bla[subscript NDM-1] gene sequence. The NDM-4, NDM-9, NDM-10*, and NDM-12 variants were characterized for zinc-binding and catalytic activity against a panel of structurally-diverse substrates. Unlike NDM-1, NDM-4 and NDM-12 (which share a M154L mutation) maintained high hydrolytic activity at low zinc concentrations for penam and carbapenem substrates. Furthermore, NDM-4 and NDM-12 were shown to have increased zinc affinity that may help maintain activity at low zinc concentrations similar to those found at infection sites. Full length NDM-1 has an N-terminal sequence that targets the transcribed enzyme to be transported to the periplasm where it binds Zn(II). The mature protein is lipid modified at the cysteine 26 position and embedded into the inner leaflet of the outer membrane. Transwell culture plates were used to examine if lipidation sequesters the enzyme and only provides resistance to the host bacterium (a "private good") in contrast to a β-lactamase lacking lipidation. Despite similar expression levels for enzyme variants, full-length NDM-1 does not protect neighboring cells against β-lactam treatment. However, the N-terminal deletion construct, which lacks the lipidation site, can effectively provide resistance to bystander cells (a "public good"). Finally, high-throughput screening assays were developed to analyze compounds and provide feedback that aided in the development and derivation of prospective NDM-1 inhibitors. There are no clinically-effective NDM-1 targeting drugs in use, so understanding the specificity and ligand-binding properties of this enzyme and its variants is important for the design of new drugs to counteract this global threatItem The MAR1 transporter of Arabidopsis thaliana has roles in aminoglycoside antibiotic transport and iron homeostasis(2009-08) Conte, Sarah Schorr; Lloyd, Alan M.Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics, however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here we report that mutations in the Arabidopsis locus Multiple Antibiotic Resistance (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Resistance is highly specific for aminoglycosides and does not extend to antibiotics of other classes, including the aminocyclitol, spectinomycin. Yeast expressing MAR1 are hypersensitive to the aminoglycoside, G418, but not to chloramphenicol or cycloheximide. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:YFP fusion protein localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside, gentamicin, while mar1-1 mutant chloroplasts accumulate less than wild type. MAR1 overexpression lines are slightly chlorotic, and this chlorosis is rescued by application of exogenous iron. MAR1 expression is also downregulated by low iron. Taken together, these data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis, and allows opportunistic entry of multiple antibiotics into the chloroplast. mar1 mutants represent an interesting example of plant antibiotic resistance that is based on the restriction of antibiotic entry into a subcellular compartment. Knowledge about this process – and other processes of antibiotic entry – could enable the production of crop plants that are incapable of antibiotic accumulation, aid in development of phytoremediation strategies for decontamination of water and soils polluted with antibiotics, and further the development of new plant-based molecular markers. The work described here also contributes to our understanding of how plants interact with the antibiotics they encounter, both in the laboratory (where aminoglycosides such as kanamycin are used heavily to select for transgenics) and in the natural environment.Item Microfabricated environments for the study of bacterial group behavior(2015-12) Fitzpatrick, Mignon Denise; Shear, Jason B.; Whiteley, MarvinThis thesis describes the application of micro three dimensional printing (µ3DP) techniques to create protein microstructures for the study of bacterial group behavior in small populations. Studies involving aggregates of ~10^1 to ~10^5 cells have shown extensive and complex communication and spatial organization. Multiphoton lithography (MPL) provides a means to quickly design and execute the fabrication of microscale structures with submicron resolution from a variety of biocompatible polymers. Using this technique, intricate spatial arrangements of bacteria can be achieved while maintaining small population sizes at high cell density (≥10^8 cells/mL), providing in vitro culture conditions which better simulate in vivo settings. As a result, valuable information can be obtained about bacterial social interactions through the coupling of additional analytical techniques to detect the presence or absence of extracellular signaling molecules. While quorum sensing (QS) remains the most extensively studied means of bacterial communication, it is becoming increasingly apparent that additional factors are necessary to effect certain changes in population-wide genetic expression which can lead to increased virulence, pathogenicity, and the development of antibiotic resistance. The work presented in this thesis addresses the influences of cell density, chemical heterogeneity of the environment within cell aggregates, and level of cell surface attachment as mechanisms to induce or influence the development of antibiotic resistance. Building upon previous work presented by members of the Shear lab, BSA-gelatin protein microstructures were used to study the behavior and response of the opportunistic pathogen Pseudomonas aeruginosa under these conditions. Antibiotic resistance was observed in low cell number/high density populations in agreement with previous work presented by the Shear lab. In addition, it was found that localized regions of oxygen depletion do not correlate directly with antibiotic resistance development, as the population size required for depletion far exceeded that for development of resistance. Finally, a new technique directed at simultaneous biofilm inhibition and cell removal from solution was explored.Item Overcoming antibiotic resistance in microbial populations : an interdisciplinary perspective(2015-08) Kaushik, Karishma Surendra; Gordon, Vernita DianeAntibiotic resistance is a major public health problem. The increase in antibiotic-resistant bacteria and decline in the approval of newer antibiotics has prompted the need for novel therapeutic approaches. In the environment and in the human body, microbes are exposed to varying spatial landscapes. Further, bacteria assemble into multicellular assemblies called biofilms, which possess intricate spatial structure. Inherent to this spatial structure, microbial communities also possess population structure, characterized by cell density, spatial organization, and different cell types. This dissertation has three main goals: i) to study the effect of microbial population structure on the survival of antibiotic resistant mutants, using P. aeruginosa, an opportunistic human pathogen as a model organism, ii) to evaluate the therapeutic potential of using bicarbonate to enhance the efficacy of aminoglycoside antibiotics, first-line agents for P. aeruginosa infections, and develop an improved method of analysis of drug interactions iii) to develop a low-cost, hands-on, educational module to characterize antimicrobial compounds using an interdisciplinary, biophysical approach.