Date of Award:

8-2020

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Committee Chair(s)

Jeff R. Broadbent (Committee Co-Chair), Charles E. Carpenter (Committee Co-Chair)

Committee

Jeff R. Broadbent

Committee

Charles E. Carpenter

Committee

Marie K. Walsh

Committee

Brian A. Nummer

Committee

David J. Wilson

Abstract

Listeria monocytogenes is a gram-positive food-borne pathogen that is widely dispersed in the environment and can cause listeriosis with high fatality rates when consumed in contaminated food products. They are capable of growing over a wide range condition. Listeria is also able to tolerate adverse conditions which allows the bacterium to survive in unfavorable environments. The ubiquitous nature of L. monocytogenes makes it difficult to eliminate from food systems. One major problem in the food industry is the survival of L. monocytogenes under sublethal low pH-environment since organic acids are widely used as food decontaminants. Prior research has suggested that the use of organic acids in the food industry may unintentionally enhance pathogenicity of L. monocytogenes. This study examined the stress response of two strains of L. monocytogenes, N1-227 and R2-499, after lactic acid or acetic acid habituation.

The first phase of the study investigated the impact of habituation to lactic acid and acetic acid on expression of transcription factors and genes related to acid resistance, bile resistance and virulence in L. monocytogenes strains N1-227 and R2-499 by qRT-PCR. Listeria cells were treated with a sublethal organic acid concentration and RNA samples were collected for transcriptome analysis after 20 minutes. Statistical analysis was performed to identify genes that increased or decreased in expression during organic acid habituation compared to cells without organic acid habituation. Results showed that organic acid habituation significantly induced expression of the acid and bile stress response genes in both strains, while expression of virulence genes was strain dependent.

The second phase of this study investigated the in vivo virulence of habituated L. monocytogenes using the Galleria mellonella infection model. Virulence was determined by injecting the cells into G. mellonella larvae. After injection, the survival of G. mellonella and the L. monocytogenes growth kinetics in insects were evaluated and the median lethal time (LT50) was determined. Results showed that habituation in organic acid increased virulence of both strains as evidenced by decreased LT50 of G. mellonellalarvae. The growth of L. monocytogenes growth kinetics in insects between treatments for either strain showed no significant difference, indicating that the enhanced virulence observed in organic acid habituated cells is not due to enhanced survival or growth in the larvae.

The third phase of this study investigated comprehensive transcriptional profile of L. monocytogenes strains N1-227 and R2-499 by RNA-seq in the presence or absence of organic acid. Results revealed detailed information about the mechanisms of L. monocytogenes responses to organic acid. As compared to L. monocytogenes grown in standard media, more differentially expressed genes (DEGs) were identified when cells were habituated with organic acid compared to cells habituated with inorganic acid. Induced expression of acid and bile stress response genes and virulence genes profiled using RT-qPCR technique in phase one was also validated by RNA-seq results. RNA-seq data were strongly correlated with the gene expression values obtained for those genes shared in the parallel qRT-PCR analysis (R2 = 0.74 for strain N1-227 and R2 = 0.79 for strain R2-499). Other DEGs included genes involved in cell motility, membrane transport and carbohydrate, amino acid metabolism and quorum sensing.

Results from this project have increased the understanding of organic acid stress response in L. monocytogenes and may provide new leads for research and help to develop better strategies to prevent L. monocytogenes contamination in food.

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