Insights into the Mechanisms of Metal Oxide and Cationic Antimicrobials

Abstract

Bacterial 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.