Deciphering the cell heterogeneity and the spatial distribution of pathogens in food matrices in interaction with microbial communities – PathoFood

The cellular heterogeneity of the foodborne pathogenic bacteria E. coli O157: H7 and L. monocytogenes is investigated by single-cell microbiological approaches based on the most probable number technique (MPN) by applying experimental designs to assess the effect of different environmental conditions, such as pH, temperature or organic acid concentration. The adaptive capacity, growth and survival of these pathogens are thus evaluated at the populational and cellular levels under different micro-environment conditions encountered in the food. Furthermore, the differential spatial distribution and expression of genes of interest are followed by fluorescence microscopy approaches based on the use of fluorescent reporter genes. In addition to the behaviour of these pathogens in response to different conditions representative of the food biotopes, interactions with the microbial communities (microbiota) in food are also studied, in particular by approaches based on luminescence as a marker of the physiological state of pathogenic bacteria. Finally, the data generated are used to develop predictive microbiological models, in particular stochastic mathematical models aiming at integrating the notion of heterogeneity in the behaviour of foodborne pathogens.

We have determined and modelled the impact of abiotic factors (temperature, pH, organic acids, and structure of the environment) on the probability of cell growth of E. coli O157:H7 and L. monocytogenes. Temperature and pH as well as the structure of the medium showed the most important effects. Through different cultural approaches, we were able to demonstrate that these two pathogens could exhibit better tolerance to certain stresses when they are cultivated in jellified model matrices rather than in liquid culture media. The colonisation of these matrices takes place in the form of isolated sessile or motile cells or of micro-colonies of variable sizes and shapes depending on the environmental conditions. The use of advanced fluorescence techniques has allowed us to visualise and quantify cellular heterogeneity within these structures at the single cell level. The molecular determinants involved in the colonisation process of the food matrix are also studied. The growth of both pathogens was also investigated in the presence of microbial consortia representative of the food matrices studied. The results for the cheese consortia (lactic acid bacteria, ripening bacteria and yeasts) show that their presence strongly modulates the maximum concentration reached (Nmax) by the pathogens. The impact on the pathogen depends on the ecological niche of the microorganism composing the food community. The models developed for cell growth and competition between species were validated on food matrices.

The fitness, the heterogeneity of the cell physiological profiles, the spatial distribution and the molecular determinants involved in the interaction with food matrix components will be characterised for L. monocytogenes and E. coli O157:H7. Furthermore, the impact of the microbial communities commonly identified in ground beef and soft cheeses on the physiology and localisation of the two pathogens will be considered. Finally, the impact of these different interactions will be integrated into a predictive modelling approach and validated in ground beef and soft cheese. The better understanding of the bacterial pathogen interactions in food ecosystems, i.e. considering both the biotope and the biocenosis, represents a challenging and crucial issue for food safety improvement. This project represents a first and major step towards a more advanced understanding and integration of cell heterogeneity in the ecophysiology and predictive microbiology of the foodborne pathogens L. monocytogenes and EHEC.

The spatial organisation of bacterial pathogens in food matrices and the heterogeneity of expression have mainly resulted in the following publications and communications:
-Saint Martin et al. 2022 Genetic, physiological and cellular heterogeneities of bacterial pathogens in food matrices: consequences on food safety. Comprehensive Reviews in Food Science and Food Safety. idHAL:hal-03762779
-Dubois-Brissonnet et al. 2022. Genetic, physiological and cellular heterogeneities of bacterial pathogens in food matrices: consequences on food safety. Iseki-food e-conference «Current innovation trends« 23 November 2022. idHAL:hal-03867169
-Saint Martin et al. 2022. Spatial organisation of L. monocytogenes and E. coli O157:H7 cultivated in gel matrices. Food Microbiology. idHAL:hal-03480607
-Saint Martin et al. 2023. Spatially localised expression of the glutamate decarboxylase gadB in E. coli O157:H7 microcolonies in hydrogel matrices. npj Science of Food. idHAL:hal-04242126
The molecular determinants of bacterial pathogens involved in food matrix colonization have led to the following publications:
-Ageorges et al. 2020. Molecular determinants of surface colonisation in diarrhoeagenic E. coli: From bacterial adhesion to biofilm formation. FEMS Microbiology Reviews. idHAL:hal-02532777
-Monteiro et al. 2020. The secretome landscape of E. coli O157:H7: Deciphering the cell-surface, outer membrane vesicle and extracellular subproteomes. Journal of Proteomics. idHAL:hal-03103302
-Chafsey et al. 2022. Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of L. monocytogenes. Journal of Proteomics. idHAL:hal-03413889
-Ageorges et al. 2023. Genome-Wide Analysis of Antigen 43 (Ag43) Variants: New Insights in Their Diversity, Distribution and Prevalence in Bacteria. International Journal of Molecular Sciences. idHAL:hal-04064345
-Monteiro et al. 2023. Specific proteomic identification of collagen-binding proteins in E. coli O157:H7: Characterisation of OmpA as a potent vaccine antigen. Cells. idHAL:hal-04138700
Environmental factors and food microbial communities on the cell heterogeneity of foodborne bacterial pathogens have primarily resulted in the following communications:
-Stahl et al. 2021. Quels sont les impacts de conditions abiotiques sur la croissance cellulaire de deux pathogènes alimentaires L. monocytogenes et E. coli 0157:H7 ? SFM Microbes, Nantes, France, 22-24 septembre 2021. idHAL:hal-03735731
-Stahl et al. 2022. E. coli O157:H7 exposée à des consortia microbiens alimentaires : quels impacts sur sa croissance cellulaire ? SFM Microbes, Montpellier, France, 3-5 octobre 2022. idHAL:hal-04193006
-Mangavel et al. 2023. Microbiome engineering as a biopreservation approach to reduce L. monocytogenes in food. BIC 2023, Lille, France 19-21 juin 2023. idHAL:hal-04193008

For microbial risk management, current regulatory affairs provide food safety recommendations and rules essentially based on a threshold. However, this limit does not take into account the dissimilar bacterial population of a foodborne pathogen in a heterogeneous environment. Indeed, within a given food matrix diverse microenvironments co-exist respective to their physico-chemical properties, biochemical composition and food microbial communities. Phenotypic heterogeneity at single-cell level now appears as an intrinsic feature of foodborne pathogens to be considered for controlling food safety. Thus, relevant quantitative microbial risk assessment must encompass the bacterial variability. The PathoFood project aims at deciphering cell heterogeneity and spatial distribution of pathogens in interactions with the dominant food microbial communities by characterising their local physiology in their microenvironment and modelling bacterial behaviour in food system. It addresses the issue of food contamination at low levels focusing on Listeria monocytogenes and Escherichia coli O157:H7, two foodborne pathogens representing a few infection cases per year but responsible of serious sequelae for patients. Meat and dairy products, in particular ground beef and soft cheese, are main exposure vehicles of these foodborne pathogens. In these complex matrices, the pathogens will experience multiple heterogeneous micro-environments and interact with their microbial communities. The fitness, the heterogeneity of the cell physiological profiles, the spatial distribution and the molecular determinants involved in the interaction with food matrix components will be characterised for Listeria monocytogenes and Escherichia coli O157:H7. Furthermore, the impact of the microbial communities commonly identified in ground beef and soft cheeses on the physiology and localisation of the two pathogens will be considered. Finally, the impact of these different interactions will be integrated into a predictive modelling approach and validated in ground beef and soft cheese. The better understanding of the bacterial pathogen interactions in food ecosystems, i.e. considering both the biotope and the biocenosis, represents a challenging and crucial issue for food safety improvement.