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

Due to concerns over bacterial tolerance to sanitizers, FDA and FSIS recommend rotating sanitizers in RTE food processing facilities to better control foodborne pathogens, in particular, Listeria monocytogenes (Lm). These recommendations are nonbinding; whether Lm develops tolerance to common sanitizers remains inconclusive and debated. Even if Lm develops tolerance through sub-lethal exposure to sanitizers, how long and how strong the tolerance can last should be considered in determining whether sanitizer rotation is needed and how often it should be applied. Lack of consensus and quantitative data on possibility and duration of sanitizer tolerance creates confusions and dilemmas, especially when sanitizer rotation presents considerable challenges in training, compliance, and cost control to the industry. This proposal describes studies to help settle the debate and fill critical knowledge gaps regarding Lm tolerance to chlorine and quaternary ammonium compounds. We will measure residual sanitizer levels in produce processing facilities. We will perform laboratory assays to investigate tolerance development and persistence. We will explore machine-learning-aided tolerance prediction and identify evolutionary signals (or lack thereof) of tolerance development from whole genome sequencing data. Our results will provide the industry and regulators with scientific evidence for substantiating, better implementing, or justifiably shelving sanitizer rotational programs.

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.

1. Survey of residual sanitizer concentrations in selected locations in two different produce processing facilities between sanitation shifts. 

2. Measurement of intrinsic tolerance to sodium hypochlorite and benzalkonium chloride in 200–300 strategically selected Listeria monocytogenes (Lm) strains. 

3. Evaluation of how different levels of sanitizers and lengths of sanitizer exposure affect the degree and duration of acquired sanitizer tolerance in selected Lm strains. 

4. Characterization of transcriptomic shifts that accompany the waning of acquired sanitizer tolerance. 

5. Whole genome sequencing (WGS) analyses of Lm to (1) develop machine-learning classifiers for intrinsic sanitizer tolerance prediction, and (2) search for evolutionary evidence for intrinsic tolerance development.

Benzalkonium chloride (BC) resistance of L. monocytogenes in the US is mostly mediated by the bcrABC gene cassette and can be reliably predicted by its presence/absence in the isolate genome. Chlorine resistance is uniformly present in L. monocytogenes, with little variation among isolates. 

-General selection for BC-tolerant strains of L. monocytogenes are associated with food processing environments. Compared with other source categories, including various food animals, such selection appears to be relatively moderate among isolates from fruits and vegetables. 

-Under a growth promoting condition (nutrient culture and 30°C), the upper bound of adaptive BC tolerance was 25 µg/ml in the BC-susceptible (BCs) strain and 50 µg/ml in the BCtolerant strain. To reach the upper bounds, a two-phase process of tolerance development was required under sublethal exposure to BC, which took 21 h for the BC-susceptible strain and 72 h for the BC-tolerant strain. Once developed, the tolerance remained stable, not dependent on external BC, and hereditary in the BC-susceptible strain, but waned within an hour in the BC-tolerant strain after the removal of BC exposure. 

-Recognition of the distinct temporal patterns of adaptive BC tolerance between BC-tolerant and BC-sensitive strains allows a differential assessment of their respective likelihood and risk regarding the tolerance. The long exposure requirement for full tolerance (50 µg/ml) development in BC-tolerant strains (72 h) makes it unlikely to occur under the frequency of daily sanitation. Even fully developed, the rapid loss of the tolerance after BC exposure makes a lingering concern unlikely. In comparison, the finding that BC-susceptible strains could develop full, stable and hereditary tolerance (25 µg/ml) within the interval of consecutive sanitation shifts (24 h) may warrant further mechanistic understanding of the tolerance. 

-Under growth limiting conditions (0.1% buffered peptone water at 4 and 25°C) that more realistically mimicked growth constraints on L. monocytogenes in produce processing environments, neither the BC-susceptible nor the BC-tolerant strain developed any adaptive tolerance.

-No adaptive tolerance to chlorine was observed in L. monocytogenes. 

-External constraints make the development of adaptive BC tolerance in L. monocytogenes unlikely in produce processing environments. A daily regime of pre-production sanitation by QAC (400 µg/ml BC) and post-production water rinse left no detectable level of BC residuals for L. monocytogenes to be exposed to in multiple sampled locations in a produce processing facility. Development of adaptive tolerance also requires optimal growth of the bacteria, which is unlikely to occur in produce processing environments. 

For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes.