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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Food Science

Year Degree Awarded

2016

Month Degree Awarded

February

First Advisor

Dr. Sam R. Nugen

Second Advisor

Dr. David A. Sela

Third Advisor

Dr. Eric A. Decker

Fourth Advisor

Dr. Steven J. Sandler

Subject Categories

Biotechnology | Food Microbiology | Molecular Genetics

Abstract

Bacteria are ubiquitous and vital constituents of our environment, our foods, and our bodies. A small percentage of this vast, microbial population is pathogenic to humans, but represents a significant burden on public health. There is a current public health focus on two subgroups: foodborne pathogenic bacteria and antibiotic resistance bacteria. A key challenge for public health is the rapid identification of these bacteria to prevent their consumption and to ensure proper treatment for infections. This challenge calls for the development of novel, low-cost diagnostics that combine sensitivity and accuracy with speed and ease-of-use.

Bacteriophages represent rapid, readily targeted, and easily produced probes for the detection of bacterial pathogens. Furthermore, modern molecular tools have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Our work here demonstrates the successful bioengineering of bacteriophage to create platforms that: (i) enable multiplex detection of bacterial pathogens; (ii) allow for the rapid detection of antibiotic resistant bacteria; (iii) and can be used synergistically with other diagnostic platforms, like paper-fluidics, plate readers, and MALDI-TOF.

In particular we demonstrate: (i) the modification of T7 bacteriophage to carry TEV protease enabling proof-of-principle detection of E. coli in 3 hours after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for MALDI-TOF MS analysis; (ii) the development and use of a phage amplification-based lateral flow assay for the detection of low levels of E. coli in less than 7 hours in both broth and water; and (iii) the modification of T7 phage to carry phoA; enabling the rapid detect of low levels of bacteria and their antibiotic susceptibility in under 6 hours. These phage-based platforms could be readily adopted in many labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection. Further research into the bioengineering of phage-based reporters and demonstrating their incorporation into common and novel diagnostics platforms, will highlight their potential and ideally result in the creation of diagnostics that impact public health.

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