A study of the transcriptomic responses elicited by Salmonella to humectants used in food production – do bacteria have a memory?

OVERVIEW

Humectants are used by the food industry for numerous reasons, such as the use of salt as flavour enhancing ingredient. They are most commonly used to reduce the moisture content of a food matrix, leading to a more stable product with a longer shelf life. Ultimately the aim is to reduce the incidence of foodborne illness. However, as consumer concern over the increased risk of detrimental health effects resulting from the over-intake of NaCl, food producers need to explore alternative humectants that can be utilised. Two of these are KCl and glycerol. Salmonella have been shown to survive for extended periods of time in numerous low-moisture products such as chocolate and peanut butter. Previous work has identified the minimum bactericidal concentrations of NaCl, KCl and glycerol towards a panel of Salmonella isolates. Other work has examined the effect of heat resistance post habituation within these compounds, and identified several genes up-regulated in response to NaCl shock. However, information is lacking regarding the transcriptomic responses towards KCl and glycerol humectants.

The aim of this study is to compare the response of the model organism Salmonella Typhimurium ST4/74 to sub-inhibitory concentrations of NaCl, KCl and glycerol to understand the mechanisms utilised to sustain viability. Using deep-level RNA sequencing (RNA-seq) the experiment will also allow direct comparison of the responses observed within all three conditions. It is hypothesised that genes involved in sustaining viability at sub-inhibitory levels will also prove important to survival within high concentrations of the humectants. To test this hypothesis, deletion mutants will be created based on genes identified by transcriptomic studies, and viability measured in comparison to the wild-type under a range of humectant concentrations.

As recovery of viable cells from high levels of humectants has been shown to be difficult using traditional culture techniques, we propose the use of flow cytometry for the enumeration of cells exposed to humectant stress. The BacLight kit would also allow accurate enumeration of cells. Cells with intact membranes (live) will be stained by the cell permeable SYTO9, while compromised (dead) will stain with the impermeable propidium iodide. It will also allow the identification intermediate populations of cells with slightly compromised membrane, which may then have the potential to repair and replicate should favourable conditions be restored. These will also be characterised by WGS.

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Introduction
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