AbstractListeria monocytogenes is a food-borne pathogen and the causative agent of listeriosis, a severe infection resulting in septicaemia, meningitis and still birth. Infection typically arises through the consumption of contaminated foodstuffs. L. monocytogenes is a hardy organism which can survive several food control measures. Psychotrophic and facultatively anaerobic properties permit growth under refrigeration conditions and within modified atmosphere packaging. Through transcriptional and translational changes L. monocytogenes is able to mount adaptive responses against stressors. Such responses typically cross protect against subsequent stresses, including effectors of the human immune system. The aim of this study was to assess the ability of L. monocytogenes to adaptively respond to stressors, and to assess the phenotypic and molecular responses that such exposures have on resistance and virulence potential.
Using adaptation and repeated exposure assays L. monocytogenes cells were assessed for their ability to develop resistance and to adaptively respond to stressors. Using qRT-PCR and insertional mutagenesis the roles of several candidate genes in stress response were assessed. Using a simulated gastro-intestinal transit model the effects of refrigeration and oxygen limitation on virulence potential were investigated, while microarray analysis allowed elucidation of the molecular mechanisms accounting for altered resistance properties and virulence potential.
Stationary phase L. monocytogenes cells were not found to adapt to sub lethal exposure to citric acid, TSP, NaCIO and H2O2. Susceptibility to stressors was increased or unchanged following sub-lethal pre-exposure.
Sub-lethal exposure to NaCIO increased expression of Imo0669 (oxidoreductase) by 4.6-fold, while a 2-fold increase in gadA was observed during TSP exposure. These responses permit survival under NaCIO and TSP stress, and may have implications in subsequent stress exposure and/or virulence potential. Inactivation of ctsR, hfq, lisR and lisK by site-directed mutagenesis gave rise to mutant cells with increased sensitivity to H2O2. Citric acid resistance was impaired by ctsR and hfq disruption. Pre-conditioning under oxygen limiting conditions significantly increased acid tolerance in L. monocytogenes FSL R2-499.
When assessing the effects of pre-conditioning on gastro-intestinal transit L. monocytogenes FSL R2-499 displayed growth phase, pre-conditioning and pH dependant resistance profiles. Cells grown under oxygen limiting conditions typically demonstrated increased resistance towards simulated gastric juice (pH 2.5), however, only stationary phase cells were able to survive bile salt exposure. L. monocytogenes cells grown under oxygen limiting conditions displayed increased acid tolerance but decreased H2O2 resistance. Transcriptional analysis revealed significant up-regulation of the acid response gene, gadA, under oxygen limiting conditions, while catalase was significantly down-regulated.
The findings of this study provide important information for food manufacturers who can use this data to "intelligently apply" food control measures, or hurdles, to improve food safety.
|Date of Award||2014|
|Supervisor||Carol A Phillips (Supervisor) & Stuart C H Allen (Supervisor)|