Date of Graduation

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Poultry Science (PhD)

Degree Level

Graduate

Department

Poultry Science

Advisor/Mentor

Kwon, Young Min

Committee Member

Barabote, Ravi D.

Second Committee Member

Lessner, Daniel J.

Third Committee Member

Kong, Byung-Whi

Keywords

Biological sciences; Campylobacter; Desiccation; Essential genome; Host stressors; Salmonella; Transposon sequencing

Abstract

Non-typhoidal Salmonella (NTS) and Campylobacter play a major role in foodborne illness caused by the consumption of food contaminated by pathogens worldwide. A comprehensive understanding of the genetic factors that increase the survival fitness of these foodborne pathogens will effectively help us formulate mitigation strategies without affecting the nutrition ecology. The objective of this study was to identify the genetic determinants of Salmonella and Campylobacter that are required for fitness under various in vitro conditions. For the purpose, we used a high throughput Transposon sequencing (Tn-seq) that utilizes next generation sequencing (NGS) to screen hundreds of thousands of mutants simultaneously. In Chapter 1, we reviewed the technical aspects of different Tn-seq methods along with their pros and cons and compressive summary of recently published studies using Tn-seq methods. In Chapter 2, we exposed complex Tn5 library of Salmonella Typhimurium 14028S (S. Typhimurium) to the mimicked host stressors in vitro conditions. Such as low acidic pH (pH 3) found in the stomach, osmotic (3% NaCl) and short chain fatty acid (SCFAs, 100 mM Propionate) found in intestine, and oxidation (1mM H2O2) and starvation (12-day survival in PBS) found in macrophage. There was an overlapping set of 339 conditionally essential genes (CEGs) required by S. Typhimurium to overcome these host stressors. In Chapter 3, we screened of S. Typhimurium Tn5 library for desiccation survival. Salmonella spp. is the most notable and frequent cause of contamination in low-water activity foods. We identified 61 genes and 6 intergenic regions required for fitness during desiccation stress. In Chapter 4, the essential genome of Campylobacter jejuni (C. jejuni) NCTC 11168 and C. jejuni 81-176 was investigated using Tn-seq. We identified 166 essential protein-coding genes and 20 essential transfer RNA (tRNA) in C. jejuni NCTC 11168 which were intolerant to Tn5 insertions during in vitro growth. The reconstructed library C. jejuni 81-176 had 384 protein coding genes with zero Tn5 insertions. The genetic determinants Salmonella and Campylobacter identified in this study have high potential to be explored as food safety intervention, therapeutic and vaccine target to curb the spread of the foodborne pathogens making world a safer place.

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