Karin I. Metselaar

Isolation and quantification of highly acid resistant variants of Listeria monocytogenes.

Heterogeneity in stress response of bacteria is one of the biggest challenges posed by minimal processing, which aims at finding the balance between microbiologically stable foods while maintaining the characteristics of fresh products. In this study, exposure of Listeria monocytogenes LO28 to acid stress, which can be encountered in the food processing environment as well as in the human body upon ingestion, led to inactivation kinetics showing considerable tailing, which was described by a biphasic inactivation model. Stable acid resistant variants of L. monocytogenes LO28 were isolated after exposure of late-exponential phase cells to pH 3.5 for 90 min. The resulting 23 stable resistant isolates could be divided in three groups: (a) highly increased acid resistance (<1 log₁₀ reduction, n=16), (b) slightly increased acid resistance (1-3 log₁₀ reduction, n=6), and (c) one isolate showing a variable acid stress response. The highly acid resistant group showed increased resistance to the tested pH range of 2.5 to 3.5 in both late-exponential and stationary phase. Increased acid resistance showed to be significantly correlated to reduced growth rate. The Weibull model was reparameterized, resulting in improved parameter estimation, and was used to estimate the inactivation kinetics at mild pH. Studying the growth boundaries of the wild type 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. This study shows that acid exposure of late-exponential phase cells reveals the presence of acid resistant subpopulations and that there is a phenotypic diversity amongst them. The occurrence of heterogeneity and stress resistant subpopulations may lead to a higher number of surviving microorganisms than expected. Also, stress resistant subpopulations can become part of the domestic flora in a food production line. The currently isolated acid resistant variants are a new group of stress resistant variants and underline the importance of gaining more insight in the mechanisms underlying this heterogeneity and increased resistance.

Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers

ABSTRACT One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Impact of Pathogen Population Heterogeneity and Stress-Resistant Variants on Food Safety

This review elucidates the state-of-the-art knowledge about pathogen population heterogeneity and describes the genotypic and phenotypic analyses of persister subpopulations and stress-resistant variants. The molecular mechanisms underlying the generation of persister phenotypes and genetic variants are identified. Zooming in on Listeria monocytogenes, a comparative whole-genome sequence analysis of wild types and variants that enabled the identification of mutations in variants obtained after a single exposure to lethal food-relevant stresses is described. Genotypic and phenotypic features are compared to those for persistent strains isolated from food processing environments. Inactivation kinetics, models used for fitting, and the concept of kinetic modeling-based schemes for detection of variants are presented. Furthermore, robustness and fitness parameters of L. monocytogenes wild type and variants are used to model their performance in food chains. Finally, the impact of stress-resistant variants and persistence in food processing environments on food safety is discussed.

Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU.

The dynamic response of microorganisms to environmental conditions depends on the behavior of individual cells within the population. Adverse environments can select for stable stress resistant subpopulations. In this study, we aimed to get more insight in the diversity within Listeria monocytogenes LO28 populations, and the genetic basis for the increased resistance of stable resistant fractions isolated after acid exposure. Phenotypic cluster analysis of 23 variants 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 activity correlated with increased acid resistance. Whole genome sequencing revealed mutations in rpsU, encoding ribosomal protein S21 in the largest phenotypic cluster, while mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and high hydrostatic pressure 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. The finding that acid stress selects for rpsU variants provides potential insights in the mechanisms underlying population diversity of L. monocytogenes.

Microbial variability in growth and heat resistance of a pathogen and a spoiler: All variabilities are equal but some are more equal than others.

Quantitative microbiology is used in risk assessment studies, microbial shelf life studies, product development, and experimental design. Realistic prediction is, however, complicated by different sources of variability. The final concentration of microorganisms at the moment of consumption is affected by different sources of variability: variability in the storage times and temperatures, variability in product characteristics, variability in process characteristics, variability in the initial contamination of the raw materials, and last but not least, microbiological variability. This article compares different sources of microbiological variability in growth and inactivation kinetics of a pathogen and a spoiler, namely experimental variability, reproduction variability (within strain variability), strain variability (between strain variability) and variability between individual cells within a population (population heterogeneity). Comparison of the different sources of microbiological variability also allows to prioritize their importance. In addition, the microbiological variability is compared to other variability factors encountered in a model food chain to evaluate the impact of different variability factors on the variability in microbial levels encountered in the final product.

Performance of stress resistant variants of Listeria monocytogenes in mixed species biofilms with Lactobacillus plantarum.

Population diversity and the ability to adapt to changing environments allow Listeria monocytogenes to grow and survive under a wide range of environmental conditions. In this study, we aimed to evaluate the performance of a set of acid resistant L. monocytogenes variants in mixed-species biofilms with Lactobacillus plantarum as well as their benzalkonium chloride (BAC) resistance in these biofilms. L. monocytogenes LO28 wild type and 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. Homolactic fermentation of glucose by L. plantarum created an acidic environment with pH values below the growth boundary of L. monocytogenes. 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, indicating differences in adaptive behaviour between the variants. Two variants that showed a high level of acid adaptation when grown in liquid cultures, showed also better performance in mixed species biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. It can be concluded that acid resistant variants of L. monocytogenes show diversity in their adaptation to acidic conditions and their capacity to survive in mixed cultures and biofilms with L. plantarum.

Modeling and Validation of the Ecological Behavior of Wild-Type Listeria monocytogenes and Stress-Resistant Variants.

ABSTRACT Listeria monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress-resistant variants. This study aimed to evaluate the impact of the presence of stress-resistant variants of Listeria monocytogenes and their corresponding trade-offs on population composition under different environmental conditions. A set of stress robustness and growth parameters of the wild type (WT) and an rpsU deletion variant was obtained and used to model their growth behavior under combined mild stress conditions and to model their kinetics under single- and mixed-strain conditions in a simulated food chain. Growth predictions for the WT and the rpsU deletion variant matched the experimental data generally well, although some deviations from the predictions were observed. The data highlighted the influence of the environmental conditions on the ratio between the WT and variant. Prediction of performance in the simulated food chain proved to be challenging. The trend of faster growth and lower stress robustness for the WT than for the rpsU variant in the different steps of the chain was confirmed, but especially for the inactivation steps and the time needed to resume growth after an inactivation step, the experimental data deviated from the model predictions. This report provides insights into the conditions which can select for stress-resistant variants in industrial settings and discusses their potential persistence in food processing environments. IMPORTANCE Listeria monocytogenes exhibits a heterogeneous stress response which can partially be attributed to the presence of genetic variants. These stress-resistant variants survive better under severe conditions but have, on the other hand, a reduced growth rate. To date, the ecological behavior and potential impact of the presence of stress-resistant variants is not fully understood. In this study, we quantitatively assessed growth and inactivation behavior of wild-type L. monocytogenes and its stress-resistant variants. Predictions were validated under different conditions, as well as along a model food chain. This work illustrates the effects of environmental factors on population dynamics of L. monocytogenes and is a first step in evaluating the impact of population diversity on food safety.

Gene profiling-based phenotyping for identification of cellular parameters that contribute to fitness, stress-tolerance and virulence of Listeria monocytogenes variants.

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed that 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of the two rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants.