Degree Type

Thesis

Date of Award

2020

Degree Name

Doctor of Philosophy

Department

Animal Science

Major

Microbiology

First Advisor

Stephan Schmitz-Esser

Abstract

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis and is commonly isolated from food products and production facilities. Some of these isolates are known to contain plasmids, though previous plasmid studies were conducted with a limited data set of 14 sequenced strains. To determine the presence of plasmids in a variety of L. monocytogenes strains and from various sequence types (ST), a large-scale analysis of 1924 L. monocytogenes genome sequences was conducted to determine the presence of plasmids in different STs, years, countries, and isolation sources. On average, 53% of all L. monocytogenes strains, and up to 92% of the strains within a ST (i.e. ST121), analyzed contained a plasmid. These plasmids ranged in size between 3 kb to 140 kb, with the majority being around 58 kb (± 1kb) and the other around 77 kb (± 1 kb). Plasmid conservation was a minimum of 91.9% nucleotide identity across all plasmids among all STs and the average similarity was 92.3% similarity. Examination of six previously identified plasmid-encoded stress-related genes indicated the genes present in L. monocytogenes plasmids are highly conserved, but their presence in plasmids is highly variable. Analysis of the dataset also identified a novel mercury-resistance operon containing seven genes involved in increasing tolerance in the presence of mercury.

Understanding that plasmids often play a role in stress survival, it was necessary to determine the contribution of L. monocytogenes plasmids to stress tolerance when under such conditions. For this aim, we studied three isogenic pairs of L. monocytogenes ST5, ST8, and ST121 strains, isolated from dairy foods and production facilities, and that either contained or were cured of plasmids harboring putative stress response genes. Wildtype (wt) and plasmid-cured (pc) strains were exposed to sublethal concentrations of oxidative stress (0.01% H2O2), salinity (15% wt/vol NaCl, pH 5), acidity (1% v/v lactic acid, pH 3.4), heat stress, or disinfectant (benzalkonium chloride, BC). When exposed to H2O2, NaCl, or lactic acid, CFU counts for all wt strains were significantly higher (P < 0.05) than those of the pc strains. Our data revealed that L. monocytogenes ST5, ST8, and ST121 plasmids confer increased tolerance against elevated temperature, salinity, acidic environments, oxidative stress and disinfectants.

Further examination of the phenotypic and functional characteristics of plasmids was required to better understand the mechanisms used by L. monocytogenes plasmids to confer tolerance to stress conditions. Therefore, a transcriptomics survey of plasmid-carrying L. monocytogenes strains 6179 and R479a was conducted under acidic and oxidative stress conditions, using the acidic and oxidative conditions of functional characterization, with an exposure time of 30 minutes at 20°C. The data showed no significant gene expression in the oxidative stress conditions for the R479a strain, and a single differentially expressed (DE) gene in the 6179 strain, a protein of unknown function.

For the lactic acid conditions, pLM6179 had 35 DE genes, 16 of which were upregulated, including clpL, a gene predicted to be involved in general stress response. For pLMR479a, the most upregulated DE gene under lactic acid stress was a putative zinc riboswitch, found upstream of a zinc-transporting ATPase. Seventy percent of the transcripts of pLMR479a during lactic acid stress were mapped to this putative zinc riboswitch, indicating heavy metal riboswitches and their potentially corresponding transporters may be important for L. monocytogenes stress response.

DOI

https://doi.org/10.31274/etd-20200624-38

Copyright Owner

Annabel Naditz

Language

en

File Format

application/pdf

File Size

231 pages

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