Quantitative Assessments of Staphylococcal Phage and Biofilm Properties for Evaluating Phage as Antibiofilm Therapeutics

Mori, Kim

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Quantitative Assessments of Staphylococcal Phage and Biofilm Properties for Evaluating Phage as Antibiofilm Therapeutics

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Due to the difficulty of treating Staphylococcus epidermidis biofilm infections with traditional antibiotics, lytic phage are emerging as potential antibiofilm therapeutics. S. epidermidis frequently causes biofilm infections on medical devices and is estimated to be responsible for 50-70% of infections associated with surgical implants. This thesis studies the effect of phage Andhra, a lytic staphylococcal phage, on S. epidermidis biofilms to assess how different ratios of the concentrations of phage to bacterial cells in the biofilm affect biofilm height, porosity, and biomass. Phage Andhra was found to have a size of 121.36 ± 1.47 nm in diameter and a zeta potential of -12 ± 0.58 mV. A quantitative method to assess the bacterial cell concentration in biofilms prior to phage addition was developed where the local number density of bacterial cells and the total biofilm biomass extracted from image analysis of confocal laser scanning microscopy image volumes collected at two different length scales were used to estimate the number of bacterial cells in the biofilm. The development of this quantitative method for determining the ratios of phage to bacterial cells in the biofilm upon the addition of phage to biofilms allows for a more controlled method for studying phage treatments. With the known concentrations of phage and bacterial cells in the biofilm, the ratio of these concentrations—the multiplicity of infection (MOI)—was varied to study how the gross structure of the biofilm changes with MOIs of 0.07, 0.67, and 1.16. Overall, there is not a linear relationship between the increasing value of the MOI and the height, porosity, and biomass of the biofilm. The height and biomass decrease from MOIs 0 to 0.07 and 0.07 to 0.67 and then increase from an MOI of 0.67 to an MOI of 1.16. The porosity decreases at an MOI of 0.67 and increases at an MOI of 1.16. Although there were some significant changes in the biofilm properties of these experiments, most changes in properties were modest across MOI values used. Characterization of the size and zeta potential of phage Andhra, the development of a controlled method to calculate MOI prior to phage addition to biofilms, and studying the effects different MOIs have on biofilm structure are critical steps toward evaluating lytic phage Andhra as a biofilm therapeutic. Moving forward, additional factors that must be considered when evaluating phage treatments include the viability of bacterial cells within the biofilm, the penetration of the phage within the biofilm, the interactions of the phage with the biofilm matrix, and the different time points and dosages of phage treatments. This M.S. thesis made important steps to provide new insights into the potential of lytic staphylococcal phage as therapeutics for biofilm infections.

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