Possibility, duration, and molecular predictors of sanitizer tolerance in Listeria monocytogenes

There is still no scientific consensus on whether Listeria monocytogenes (Lm) develops sanitizer tolerance. We hypothesize that development of two types of sanitizer tolerance may occur in Lm. First, short-term adaptation to sub-lethal levels of sanitizers induces acquired tolerance, which is transient and not hereditary. Second, long- term selection by sanitizers causes intrinsic tolerance, which is established in Lm populations by evolutionary changes to Lm genomes. To help settle the debate, we will test our hypothesis by distinguishing and investigating both types of tolerance in Lm using chlorine and a quaternary ammonium compound as example sanitizers. In this study, we will survey residual sanitizer levels in a leafy green and a tomato processing facilities to evaluate if laboratory-derived sanitizer levels optimal for tolerance development are relevant to produce processors. We will assess the possibility of acquired tolerance by measuring the difference in minimum inhibition concentrations (MIC) before and after sanitizer adaptation. We will study how different sanitizer levels and exposure time affect the development of acquired tolerance, including how long the tolerance can last after exposure to sanitizers. We will explore the mechanisms behind the development of acquired sanitizer tolerance by characterizing temporal shifts in Lm transcriptome throughout the duration of the tolerance. We will assess intrinsic tolerance in a collection of 200-300 strategically selected Lm strains using high-throughput growth kinetics assays. We will search for evolutionary evidence that suggests the development of intrinsic tolerance in recent history by analyzing whole genome sequencing (WGS) data of these strains. We
will build a machine-learning classifier to predict tolerance levels and identify key tolerance predictors from WGS. This research will provide valuable prerequisite information for determining if sanitizer rotation is necessary for preventing the development of Lm tolerance to sanitizers. Scientific data from the project will also help optimize sanitation practices to mitigate tolerance development and determine frequency for sanitizer rotation if rotation is needed.