Insights into the Mechanisms of Metal Oxide and Cationic Antimicrobials

dc.contributor.authorBenmamoun, Zachary Wangen
dc.contributor.committeechairDucker, William A.en
dc.contributor.committeememberBortner, Michael J.en
dc.contributor.committeememberMartin, Stephen Michaelen
dc.contributor.committeememberFalkinham, Joseph O.en
dc.contributor.departmentChemical Engineeringen
dc.date.accessioned2025-03-07T09:00:51Zen
dc.date.available2025-03-07T09:00:51Zen
dc.date.issued2025-03-06en
dc.description.abstractBacterial infections are a leading cause of death worldwide with over 7 million bacterial infection-related deaths reported in 2019 alone. Metal oxide- and polyelectrolyte-based antimicrobials are widely studied and used, but their mechanisms of action and interactions with bacterial cells and other substances found in nature are incomplete. The overall goal of this work is to gain insight on the mechanisms of metal oxide and polycationic disinfectants. The first part of this work presents the development and study of antimicrobial facemasks that kill 99.9% of pathogenic bacteria responsible for hospital acquired infections, Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae within 30 minutes. These facemasks were made by adhering cuprous oxide (Cu2O) microparticles to polypropylene textiles. Furthermore, we investigated whether surface contact was necessary to kill bacteria in drying droplets on the facemask surface and found that surface contact produced better killing than exposure to Cu+ ions that dissolve into the droplet. The second part of this work describes the mechanism of action of polycationic antimicrobials. The goals of this section were twofold. Our first aim was to develop a method of studying both polycation adsorption on bacterial cells and antimicrobial activity with single-cell and time-resolved measurements. Our second aim was to determine whether the surface density of antimicrobial required for kill is a better metric of effectiveness than solution concentration required for kill. We adsorbed bacteria to the surface of glass coverslips inside a flow cell. We then flowed through the cell a mixture of fluorescently tagged polycation, polydiallyldimethyl ammonium chloride (PDADMAC) tagged with Cy3, and a fluorescent dye, Sytox Blue, that indicated membrane permeability. This allowed us to image the density of PDADMAC on the cell surface, as well as the time taken for individual cells to permeabilize using fluorescence microscopy. We found a time lag of 5–10 minutes between adsorption and death and found that the time-to-die of an individual cell was well correlated with the rate of adsorption. We also found that the time-to-die and equilibrium adsorption differed among species but followed a trend of more adsorption onto bacterial species with a more negative zeta potential. Most importantly, we found that there was a wide range of cell responses, highlighting the usefulness of single-cell measurements in addition to ensemble-average measurements. Polycationic disinfectants need to be deployed in a wide range of environments, ranging from almost pure water to hypertonic salt. Owing to their cationic charge, one would expect the salt content of the medium to affect the antimicrobial action. So, we investigated the effect of ionic strength on polycation antimicrobial activity. We used our previous method to measure the time-course of adsorption and kill of our labelled cationic polymer in NaCl solution. We found that addition of NaCl decreased the density of polymer adsorption and diminished efficacy of PDADMAC. At salt concentrations at or above 0.15 M, which is similar to normal saline, PDADMAC was no longer bactericidal but instead bacteriostatic (stops growth). Fluorescence depolarization measurements showed that PDADMAC rigidified model bacterial membranes, but salt reduced this rigidity. We also found that the bacteriostatic effect is reversible, and cell growth resumed once PDADMAC was removed. The third section of this work focused on the effect of capillary geometry on the height of capillary rise. Angled capillaries are common in natural and engineered systems such as porous media. However, this system has not been studied by experiment or modelling. The goal of this section is to examine the equilibrium height of a meniscus in a trapezoidal capillary as an example of an angled capillary. To do this, we constructed glass capillaries from hydrophilic borosilicate glass microscope slides and used pure water or ethanol-water solutions as the liquid. This system was modelled by numerical solution to the Laplace equation to obtain the shape of the vapor-liquid interface. Both experiment and theory showed that there is less capillary rise for greater wall angles of the trapezoid, and the rise is more sensitive to the wall angle (α) than the contact angle (θ) at angles close to vertical.en
dc.description.abstractgeneralBacterial infections are a leading cause of death, with over 7 million deaths from bacterial infections reported in 2019. Many disinfectants use metal oxide particles or polymers as the active ingredient. While these are very effective at cleaning surfaces, the exact mechanism of how they work is still unknown. The overall goal of our research is to understand how metal oxide and polymer-based antimicrobials work so we can design better disinfectants and deploy them in environments where they are effective. The first part of my work focused on making a facemask that could both filter microbes out of the air like a regular facemask but then kill microbes that have landed on the mask. To make the facemasks, we adhered antimicrobial solid (cuprous oxide) particles to material (polypropylene) that is used to make facemasks. We found that our treated facemasks killed 99.9% of adsorbed bacteria within 30 minutes, and we found that bacteria die faster when they were forced to contact the surface. The second section of this work focused on understanding how polymer-based disinfectants work. Polycations are polymer molecules that have positive charges on one or more of the repeat units. Polycations are widely studied disinfectants, but their mechanism is not completely understood. The goals of our research were to 1) find a method of determining the amount of polymer on a bacterium and whether or not the bacterium was alive on a single-cell level, and 2) determine which was more important in determining bacterial kill: the amount of antimicrobial on the cell surface or the amount in solution. We found that cells died about 5 – 10 minutes after the polymer adhered to the cell. We also found that the time-to-die and maximum amount of polymer that can adhere to the cell differed among species but followed a trend of more adherence on more negatively charged species. Most importantly, we found that individual bacteria of the same species had a very large range of response, which highlights the usefulness of measuring the response of individual cells. We continued our polymer research to study how salt affects polymer antimicrobial efficacy. Antimicrobials need to be deployed in a wide range of environments ranging from pure water to saltwater. As they are charged, they will interact with other charged species in the sample, such as salts. Once the salt concentration rose to ~1% - which is the same concentration as found in blood serum – the polycation no longer killed bacterial cells, but instead, halted their growth. Additionally, polycations make bacterial cell membranes more rigid, and this effect disappears at high salt concentrations. Last, we found that the halted cell growth is reversible, and cells will regrow once the polymer is removed. The third section of this work focused on capillary rise in angled pores. Capillary rise is a well-studied effect where surface tension pulls liquid confined in a thin capillary against gravity. Capillaries with angled walls occur in nature and in engineered systems. However, there is no work that describes how the capillary rise is affected by an increase in capillary width with height. Our goal was to use experiment and theory to examine the liquid rise in trapezoidal capillaries. The test liquids were pure water or water-ethanol mixtures. Both theory and experiment showed that capillary rise decreased as the angle of the capillary increased, and that the rise was more sensitive to the wall angle (α) than the contact angle (θ).en
dc.description.degreeDoctor of Philosophyen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:42589en
dc.identifier.urihttps://hdl.handle.net/10919/124821en
dc.language.isoenen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectDisinfectanten
dc.subjectmetal oxideen
dc.subjectpolyelectrolyteen
dc.subjectbacteriaen
dc.subjectfluorescent microscopyen
dc.subjectcapillary riseen
dc.titleInsights into the Mechanisms of Metal Oxide and Cationic Antimicrobialsen
dc.typeDissertationen
thesis.degree.disciplineChemical Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.nameDoctor of Philosophyen

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