Probing bacteria life stages and quantitative assays using material platforms
Date
2018
Authors
Neufeld, Bella Hirschl, author
Reynolds, Melissa M., advisor
Borch, Thomas, committee member
Fisher, Ellen, committee member
Popat, Ketul, committee member
Kota, Arun, committee member
Van Orden, Alan, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The growing concern associated with nosocomial infections has been attributed to both the increasing prevalence of superbugs that are resistant to one or more antibiotic and the decreased susceptibility of bacteria to therapeutics once in biofilm form. Nosocomial infections are a particular concern with implanted biomaterials as the material surface is ideal for bacterial attachment and biofilm formation. Thus, there remains a significant need to modify current biomaterials to either prevent or kill bacteria in the biofilm stage. The focus of this work is specific to polymer systems most commonly associated with applications such as extracorporeal circuitry, catheters, and wound dressings, all of which are known to undergo complications from bacterial infections. The ability to probe the different life stages of bacteria (planktonic, initial attachment, and mature biofilm) to elicit a desired biological effect requires discrete methodologies that are presented herein. Additionally, accurate measurements of complex biological systems using in vitro assays are utilized both for antibacterial studies as well as an in-depth analysis of potential interferences arising from these techniques. Specifically, the potent antibacterial agent nitric oxide (NO) is employed in two ways to determine efficacy against planktonic and mature biofilm bacteria (the former being utilized in a blended film and the latter by addition as a solution). These presented studies demonstrate the ability to target different bacteria life stages using the same therapeutic administered in different forms. They also provide quantification for the amount of NO necessary to impose antibacterial action, whether in planktonic or biofilm states. Another presented study explores the attachment stage of biofilm formation through the use of a water-stable metal composite material. A copper-based metal-organic framework is incorporated into a chitosan matrix and the materials demonstrate a remarkable ability to impede bacterial attachment. These composite materials are also shown to be reusable, highlighting their ability to be employed in an antibacterial surface fashion. Finally, two in vitro assays that exploit chemical spectroscopic transformations in the presence of cellular activity are analyzed for potential interferences. The assay starting reagents are placed in solution with 19 small molecules in the absence of cells and deviations from the control solutions are determined via absorbance measurements. This critical study (which ultimately revealed a high occurrence of false responses in the presence of multiple tested compounds) highlights the importance of implementing proper control studies when using these types of techniques. Overall, a combination of polymer-bacteria systems are presented using in vitro techniques to continue to mitigate the prevalence of clinical infections. As a result of the findings presented herein, there is a significantly deeper understanding of the susceptibility of bacteria both in their planktonic and biofilm states that will ultimately lead to an enhanced ability to target and effectively kill bacteria throughout their life stages.