Date of Graduation

7-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Poultry Science (PhD)

Degree Level

Graduate

Department

Poultry Science

Advisor/Mentor

Kwon,Young

Committee Member

Adams, Rich

Second Committee Member

Sun, Xiaolun

Third Committee Member

Huang, Yan

Keywords

cecal extract; chicken cecum; conditional essential genes; genome-wide fitness; Salmonella Typhimurium; transposon insertion sequencing

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) remains a major foodborne pathogen associated with chicken, where it can persist in the intestinal tract without overt disease and contribute to zoonotic transmission. The chicken cecum represents a complex ecological niche shaped by microbial competition, host-derived substrates, short-chain fatty acids, oxygen limitation, extracellular components, and diverse metabolic gradients. Although transposon insertion sequencing (Tn-seq) has been widely used to define bacterial fitness determinants, limited work has examined genome-wide adaptation of S. Typhimurium in chicken ceca-derived ex vivo systems that preserve key features of the avian cecal environment. This dissertation investigated conditionally essential genes required for S. Typhimurium adaptation to chicken cecal extract (CE) using an ex vivo model and high-density Tn-seq. The ecological relevance of CE was first supported through characterization of extracellular DNA in whole cecal suspension, washed cell pellet, and cell-free supernatant fractions. Microbiota profiling and confocal fluorescence microscopy confirmed extracellular DNA structures and distinct microbial features within cecal fractions, supporting CE as a physiologically structured cecal model. Genome-wide mutant fitness was evaluated under CE aerobic (CE+O₂), CE anaerobic (CE−O₂), and LB anaerobic (LB−O₂) conditions. Fitness was assessed using normalized insertion counts, log₂-transformed fitness indices, false discovery rate correction, and linear mixed-effects modeling. Across experimental conditions, 588 genes were significantly depleted using combined statistical and biological thresholds (FDR ≤ 0.05; log₂ fold change ≤ −1). Among these, 62 genes were uniquely associated with CE conditions, including 34 specific to CE aerobic, 17 specific to CE anaerobic, and 11 shared across both CE environments. Mixed-effects modeling demonstrated that environmental condition significantly influenced mutant fitness, with anaerobic CE imposing stronger selective pressure than aerobic CE. No overlap was observed between CE-specific genes and essential genes in LB, indicating that CE-associated determinants represent condition-dependent requirements specific to the chicken-derived environment. Functional interpretation revealed enrichment in stress adaptation, metabolic flexibility, membrane transport, RNA regulation, and virulence-associated processes. To independently validate Tn-seq predictions, defined single-gene deletion mutants were evaluated through monoculture growth kinetics and structured direct competition assays in unfiltered CE under both oxygen conditions. While most CE-associated mutants did not exhibit severe intrinsic growth defects in monoculture, a subset including pnp, rfbP, srmB, and STM14_3319 demonstrated reproducible growth impairment consistent with Tn-seq. Competitive fitness assays showed strong quantitative concordance with Tn-seq-derived fitness estimates (r = 0.62–0.68; R² up to 0.46), with 65% directional agreement across platforms. Under aerobic CE, the highest severity tier included STM14_0346, sipB, csrB, idnK, rfbP, pagO, gsp, yohD, and STM14_4653. Under anaerobic CE, a partially restructured but overlapping severity tier included csrB, gsp, kduI, pagO, rfbP, sicA, sipB, STM14_0346, STM14_2638, STM14_4653, araA, and zntA. Several loci including STM14_0346, sipB, csrB, rfbP, pagO, gsp, and yohD remained within the highest severity tier under both oxygen conditions, indicating preservation of core CE survival determinants independent of oxygen availability. Mixed-effects variance partitioning confirmed that gene-level variance exceeded replicate-level variance, supporting the intrinsic biological basis of competitive phenotypes. Collectively, this dissertation demonstrates that chicken CE imposes structured, oxygen-sensitive selective pressures distinct from conventional laboratory media and that CE-based Tn-seq reliably identifies biologically authentic cecal fitness determinants. These findings advance understanding of the genetic architecture underlying S. Typhimurium persistence in a chicken -relevant intestinal environment and establish ex vivo cecal modeling as a robust platform for identifying pathogen adaptation mechanisms that provide potential targets for future Salmonella Typhimurium intervention strategies.

Download

Included in

Microbiology Commons

Share

COinS