Heterogeneity in single-cell outgrowth of Listeria monocytogenes in half Fraser enrichment broth is affected by strain variability and physiological state

Jasper W Bannenberg,Marcel H Tempelaars,Marcel H Zwietering,Tjakko Abee,Heidy M.W Den Besten

doi:10.1016/j.foodres.2021.110783

Jasper W Bannenberg, Marcel H Tempelaars + Show 3 more

Open Access

https://doi.org/10.1016/j.foodres.2021.110783

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Abstract

The behaviour of pathogens at the single-cell level can be highly variable and can thus affect the detection efficacy of enrichment-based detection methods. The outgrowth of single cells of three Listeria monocytogenes strains was monitored after fluorescence-activated single-cell sorting in non-selective brain heart infusion (BHI) broth and selective half Fraser enrichment broth (HFB) to quantify outgrowth heterogeneity and its effect on the detection probability. Single-cell heterogeneity was higher in HFB compared to non-selective BHI and heterogeneity increased further when cells were heat-stressed. The increase in heterogeneity was also strain-dependent because the fast-recovering strain Scott A showed less outgrowth heterogeneity than the slower-recovering strains EGDe and H7962. Modelling of the outgrowth kinetics during the primary enrichment demonstrated that starting at low cell concentrations could fail detection of L. monocytogenes at least partly due to cell heterogeneity. This highlights that it is important to take single-cell heterogeneity into account when optimizing enrichment formulations and procedures when L. monocytogenes contamination levels are low.

Highlights

Listeria monocytogenes is ubiquitous in nature and can be introduced to food processing environments where it may contaminate products
Outgrowth in brain heart infusion (BHI) seemed to be more homogeneous than the outgrowth in half Fraser enrichment broth (HFB) (Fig. 1), which suggests that outgrowth medium can impact the heterogeneity
The three strains of L. monocytogenes showed a clear dif ference in outgrowth kinetics, which is in line with earlier research where outgrowth kinetics of reference cells and heat-stressed cells were quantified at the population level for 23 strains (Bannenberg et al, 2020)

Summary

Introduction

Listeria monocytogenes is ubiquitous in nature and can be introduced to food processing environments where it may contaminate products. The heterogeneity in growth initiation is increased even more when cells are recovering from (sub-lethal) injury due to prior food processing (Guillier, Pardon, & Augustin, 2005; Koutsouma nis & Sofos, 2005) Stresses such as freezing, heating, and exposure to low pH, which L. monocytogenes may encounter during food production can have a profound effect on single-cell lag durations (Dupont & Augustin, 2009). When starting cell concentrations are low, the differences in single-cell outgrowth potential can lead to failure to reach the necessary detection threshold for detection Such cases would lead to false-negative results where the presence of L. monocytogenes is not detected and thereby posing a risk for food safety. The aim of this study was to quantify the impact of outgrowth heterogeneity of L. monocytogenes at single-cell level during primary enrichment following the ISO 11290–1 protocol in half Fraser broth using a selection of slow, medium, and fastrecovering strains of L. monocytogenes

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