Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Thesis - MUSC Only

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

David A. Schofield

Second Advisor

Michael G. Schmidt

Third Advisor

Laura M. Kasman

Fourth Advisor

Caroline Westwater

Abstract

Anthrax is a rapidly progressive disease caused by cutaneous, inhalational or gastrointestinal contact with the spores of Bacillus anthracis. Deliberate release of spores in a bioterror event could potentially lead to massive anthrax outbreaks and public panic. Spores are extremely recalcitrant within the environment and may maintain their virulence for more than 200 years, leaving the affected area uninhabitable until remediation. To ensure public health preparedness for such an event, an efficient environmental detection system for B. anthracis is essential. B. anthracis can be identified using microbiological methods such as the FDA-approved gamma phage lysis assay, or molecular methods such as polymerase chain reaction (PCR). However, these assays may not be suitable for complex environmental samples due their lengthy processing time (lysis assay) or inability to discriminate between viable and non-viable cells (PCR). To address this need, a ‘light-tagged’ B. anthracis reporter phage was generated by integrating genes encoding luciferase into the genome of the temperate phage, Wβ. The resulting engineered phage, Wβ::luxAB-2, was able to rapidly transduce bioluminescence to viable B. anthracis cells within 20 minutes. The ability of the system to transition from detecting viable B. anthracis in pure cultures to complex environmental samples was assessed. Using Wβ::luxAB-2, detection of B. anthracis in spore-contaminated water was 101, 102 and 102 CFU/mL within 12h from pond, brackish and lake water, respectively. However, when the phage were used to evaluate soil samples, spore detection was more challenging with a limit of detection of 105 CFU/g in 6h. In environmental samples, the presence of endogenous microbial flora, high salt content and poor vegetative cell viability inhibited spore detection. Upon further examination of these biological limitations, it was concluded that detection was significantly dependent on bacterial growth phase and the presence of competitive species naturally residing in soil such as B. thuringiensis. Collectively, these results demonstrate that Wβ::luxAB-2 displays potential for rapid detection of viable spores from contaminated environmental samples.

Rights

All rights reserved. Copyright is held by the author.

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