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Bacterial stress response and heterogeneity therein is one of the biggest challenges posed by minimal processing. Heterogeneity and resulting tailing representing a more resistant fraction of the population, can have several causes and can be transient or stably in nature. Stable increased stress resistance is caused by alterations in the genome and therefore inheritable and is referred to as stable stress resistant variants. Also L. monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress resistant variants. Adverse environments were shown to select for stable stress resistant variants. The objective of the research described in this thesis was to evaluate if L. monocytogenes population diversity and the presence of stable resistant variants is a general phenomenon that is observed upon different types of stress exposure, to get more insight in the mechanisms leading to increased resistance and to evaluate the ecological behaviour and potential impact on food safety of these stable resistant variants. Acid stress was chosen as it is an important hurdle both in food preservation, as well as in stomach survival. First, the non-linear inactivation kinetics of L. monocytogenes upon acid exposure were quantitatively described. A commonly used biphasic inactivation model was reparameterized, which improved the statistical performance of the model and resulted in more accurate estimation of the resistant fraction within L. monocytogenes WT populations. The observed tailing suggested that stable stress resistant variants might also be found upon acid exposure. Indeed, 23 stable acid resistant variants of L. monocytogenes LO28 were isolated from the tail after exposure of late-exponential phase cells to pH 3.5 for 90 min, with different degrees of acid resistance amongst them. Increased acid resistance showed to be significantly correlated to reduced growth rate. Studying the growth boundaries of the WT and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress. A set of variants were further characterized phenotypically and cluster analysis was performed. This resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation and virulence indicators. A higher glutamate decarboxylase (GAD) activity correlated with increased acid resistance. Whole genome sequencing of a set of variants was performed and revealed mutations in rpsU, encoding ribosomal protein S21. This rpsU mutation was found in all 11 variants comprising the largest phenotypic cluster, indicating a potential role of this ribosomal protein in stress resistance. Mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and HHP resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants. Next, the performance in mixed species biofilms with Lactobacillus plantarum was evaluated, as well as their benzalkonium chloride (BAC) resistance in these biofilms. It was hypothesized that the acid resistant variants might also show better survival in biofilms with L. plantarum, which provide an acidic environment by lactose fermentation with pH values below the growth boundary of L. monocytogenes when biofilms mature. L. monocytogenes LO28 WT and eight acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, which could be correlated to increased survival in the mixed biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. Lastly, a set of robustness and fitness parameters of WT and variants was obtained and used to model their growth behaviour under combined mild stress conditions and to model their performance in a simulated food chain. This gave more insight in the trade-off between increased stress resistance and growth capacity. Predictions of performance were validated in single and mixed cultures by plate counts and by qPCR in which WT and an rpsU deletion variant were distinguished by specific primers. Growth predictions for WT and rpsU deletion variant were matching the experimental data generally well. Globally, the variants are more robust than the WT but the WT grows faster than most variants. Validation of performance in a simulated food chain consisting of subsequent growth and inactivation steps, confirmed the trend of higher growth fitness and lower stress robustness for the WT compared to the rpsU variant. This quantitative data set provides insights into the conditions which can select for stress resistant variants in industrial settings and their potential persistence in food processing environments. In conclusion, the work presented in this thesis highlights the population diversity of L. monocytogenes and the impact of environmental conditions on the population composition, which is of great importance for minimal processing. The work of this thesis resulted in more insight in the mechanisms underlying increased resistance of stress resistant variants and quantitative data on the behaviour of stress resistant variants which can be implemented in predictive microbiology and quantitative risk assessments aiming at finding the balance between food safety and food quality.

Quantitative and ecological aspects of Listeria monocytogenes population heterogeneity