Research, Technology and Development III

RTD Leader: Luca Cocolin, UNIUD, Italy

 

WP8 Matrix Interactions

WP Leader: Nils Arneborg, LIFE, Denmark

This WP will determine the effect of pathogen-matrix interactions on adhesion, viability, resistance and virulence. The activities will be carried out on the basis of the tools developed in WP1, WP2, WP4 and WP5. The knowledge obtained will be used in WP11, WP13 and WP14.

The WP will concentrate on attachment and detachment of pathogens to food and food structures and to feed and food contact surfaces and environmental conditions representative for the food chain.

To study matrix interactions the techniques developed and adapted in WP1, the platform for bioimaging, will be applied. A stepwise procedure will be used with step one being based upon Fluorescence Radio Imaging Microscopy (FRIM) for time lapse studies of pHi-homeostasis as a measurement of the physiological status and resistance to various stresses for single cells of bacterial and fungal pathogens and protective and probiotic cultures attached to various matrices. The matrices will mimic feeds and foods and representative contact surfaces. The model for exposure to stresses will include acids, bases, ethanol, CO2 and a bacteriocin. Expression of virulence and adhesion will be monitored for populations of pathogens by the tools adapted in WP5. Expression of virulence and toxicity on solid matrices will include invasive growth of S. cerevisiae and biosynthesis of ochratoxin by P. nordicum. Special attention will be given to environmental factors affecting invasive growth of S. cerevisiae. Following intrinsic factors like C-source, N-source, O2/CO2 ratio, minerals, and extrinsic factors like temperature, pH and aw value will be considered. It is expected that they will affect invasive growth. It will include the relationship of the molecular heterogenicity of the yeast cell surface (i.e. chitins, mannans,  glucans, lectin type moieties), exposure to physico-chemical stresses relevant to food and virulence. Among environmental factors we will study intrinsic and extrinsic factors, which potentially effect yeast behaviour and its expression of genes and invasive growth. Invasive growth will be measured on model epithelial culture as well as with organic surfaces like agar, gelatine. For selected parameters single cell experiment will be conducted to elucidate respond of single cell to adhesion and to invasive growth. This will be measured via internal cell pH as universal marker of cell health status on one hand and as expression of oxidative stress respond via catalase, superoxid dismuatase, glutathione content and selected HSP markers as well. Specificity of yeast cell surface for attachment and invasive growth potential will be examined  via yeast cell surface protein profiles in 2D electrophoresis which will be marked for the purpose to compare invasive to non-invasive cell lines.

 

This WP involves the following tasks:

Task 8.1        Viability, adhesion and virulence expression of single bacterial cells attached to different solid matrices, the effect of physio-chemical stresses and selection of resistant cells for similar studies.

Task 8.2        Viability, adhesion and virulence expression (invasive growth) of S. cerevisiae as determined by physio-chemical stresses relevant to food

Task 8.3        The effect of growth on solid surfaces on the induction of mycotoxin (ochratoxin) biosynthetic genes in filamentous fungi

Task 8.4        Expression of functional genes of protective and probiotic cultures on solid matrices

 

Partner 1, Partner 5 and Partner 20 (UL-BF) will be involved in this WP.

Task 8.2 will be performed by colabaration with WP2

 

 

WP9 Host-Pathogen Interactions

WP Leader: Avrelija Cencic, UM, Slovenia

This WP will investigate the mechanisms of interactions between foodborne pathogens, farm animal hosts and the potential effect of protective and probiotic cultures on the host-pathogen interactions. For these purposes, the functional cell models developed in WP3, the pathogens and the protective and probiotic bacteria selected in WP10 will be used.

The work will concentrate on interactions between the pathogens (L. monocytogenes, M. avium subsp. paratuberculosis, C. jejuni, E.coli (STEC), invasive S.cerevisiae, hepatitis E virus (HEV) and tickborne encephalitis virus (TBEV) and the epithelial cells of pigs, chicken and ruminants. Gene and protein expression will be used to monitor the interaction in addition to the potential action of defined proteins from the immune system. The effect on the pathogens will be monitored by determination of viability and virulence. The results obtained will be used in WP14 for modelling the survival and virulence of pathogens originating from the intestinal tract of pig, poultry and ruminants.

The WP will include the following tasks:

Task 9.1        Interactions between the pathogens (L, monocytogenes, M. avium subsp. paratuberculosis, C. jejuni, E. coli (STEC), invasive S. cerevisiae, hepatitis E virus (HEV), tickbrne encephalitis virus (TBEV) and the epithelial cells of pigs, chicken and ruminants determined by gene and protein expression and potential action of defined proteins as well as determination of the relationship between gene expression in epithelial cells of pigs, chicken and ruminants and survival and presistance of the pathogens.

Task 9.2        ‘Cross-talk’ between probiotic cultures, pathogens and intestinal epithelial cells and cells of mucosal and blood immune system.

 

Partner 4, Partner 7, Partner 13 and Partner 20 (UL-MF) will be involved in this WP.

 

 

WP10 Protective and probiotic Cultures

WP Leader: Effie Tsakalidow, AUA, Greece

The aim of this WP is to find lactic acid bacteria and bifidobacteria strains able to inhibit, either at the level of farm animals or at the level of food products, the pathogens that are studied in PathogenCombat. Strains with this potential can then be considered as protective and probiotic cultures to be incorporated in the development of prevention strategies for foodborne pathogenic microorganisms throughout the food chain.

A wide range of strains already existing in the culture collections of the Partners involved in this WP will be examined. The aim is to screen as many strains as possible in order to increase the chances for indentifying strains with antimicrobial activity, especially against the Gram negative bacteria and the viruses. The strains will be screened for antagonistic activity against the food pathogens investigated in this project, and the ones exhibiting antimicrobial activity will be selected for further studies. High Throughput Screening (HTS) will be applied.

The selected strains will be examined with respect to parameters related with their survival in the gastorintestinal tract (e.g. low pH and presence of bile salts), their colonization in the gastrointestinal tract (e.g. adhesion) and their tolerance towards stress conditions prevailing in food processing, e.g. heat treatment, low pH, low water activity and starvation.

Further, the selected strains will be identified and typed by phenotypic and molecular techniques and their antibiotic resistance will be examined at phenotypic and molecular level. Finally, a kinetic analysis of the growth of strains and the production of their antimicrobial compounds will be conducted. The antimicrobial compound will be identified.

The selected strains will be used in for studies WP8 and WP9 to examine their ability (i) to adhere on the food matrix (Partner 1) and the intestinal tract (Partner 7) and (ii) to inhibit the adhesion of pathogenic bacteria (Partner 1 and Partner 7). In WP13 they will be used in actual trails in the food chain.

The WP will include following tasks:

Task 10.1      Screening of strains for antimicrobial activity against pathogens

Task 10.2         Screening of strains for survival under gastrointestinal tract and food processing conditions

Task 10.3      Identification of strains and determination of antibiotic resistance

Task 10.4      Characterization of the antimicrobial compounds

Task 10.5      Kinetic analysis of growth and production of antimicrobial compounds

 

Partner 5, Partner 6, Partner 18 and Partner 19 will all be involved in these activities. They will be being responsible for their own cultures, but benefit from the joint efforts.

 

 

WP11 Hygienic Processing Systems

WP Leader: Alan Friis, DTU, Denmak

The aim of this work package is to provide information on hygienic design, construction materials and cleaning and disinfection routines, which can be applied to minimise transfer of pathogenic agents to humans via inert (contact as well as non-contact) surfaces in food or feed processing plants. In addition, the WP will evaluate surface characteristics and techniques, which may prevent attachment or promote detachment of pathogens from the surface of relevant construction materials. Overall, this will prevent establishment and spreading of pathogenic micro-organisms through the food chain.

The work is based upon the following techniques:

Surface topography

·         Atomic Force Microscopy (AFM) involves a nanometre scale probe scanning a surface generating a 3D image of the surface. Differences in topographical features in the sub-microns can be determined i.e. surface roughness are measured in nanometres.

·         A surface tracing instrument is used to assess short scale surface irregularities with a stylus tracing a two-dimensional profile section

 

Non-microbial surface soiling

·         X-ray photoelectron spectroscopy (XPS) will be used to study organic soil composition on surfaces (to 10nm depth) and construction material composition.

·         Staining with fluorescence markers for protein, lipid and carbohydrates

 

Microbial surface soiling

·         Traditional microbial culture techniques

·         Fluorescence microscopic methods (DNA or rRNA staining)

·         The Optical Tweezer described in WP1

·         Impedance/conductance measuring equipment allows monitoring of electrical changes during bacterial growth and via calibration curves the level of micro-organisms may be estimated. The method has been adapted and used for quantification of bacteria on surfaces. Evaluation of the potential in real-time PCR for specific quantification of surface attached microorganisms

 

Hydrodynamic parameterrs

·         Computational Fluid Dynamics (CFD) can be used to visualise and predict flow patterns and parameters such as velocity, pressure, temperature, wall shear stress, turbulence fluctuations etc. in fluid flow. The method will be extended to predict flow properties near and at surfaces in closed system.

·         Flow visualisation will be carried out using transparent process equipment. Flow patterns will be studied qualitatively using tracers and specific light sources or quantitatively using Laser Doppler Anemometry (LDA) to measuring flow data (velocity, turbulence, etc.) in discrete points in the flow

 

This WP is divided into the following tasks:

Task 11.1      Development of laboratory scale test setup to study industrially relevant interactions between microbial pathogenic organisms, non-microbial surface films and inert food contact surfaces.

Task 11.2      Identification and establishment of methods for detection of biofilm and organic soil on inert food contact surfaces

Task 11.3      Determination of parameters affecting the presence of pathogen micro-organisms on inert surfaces in open processes in the food industry

Task 11.4      Establishment of hydrodynamic parameters controlling cleaning in closed food processes systems to prevent pathogen contamination and biofilm remains

Task 11.5      Development of improved cleaning agents and cleaning procedures for inert surfaces in the food and feed industry

 

Partner 4, Partner 21, Partner 22, Partner 23, Partner 24, Partner 25 and Partner 30 will be involved in this WP.

 

 

WP12 Novel Processing Techniques

WP Leader: Frank Devlieghere, UGENT, Belgium

This WP will identify appropriate inactivation techniques and inhibiting factors to control the microbial safety of food products through combination of new and currently available processing methods. It will create insight in the development of stress resistance and/or virulence in pathogenic bacteria due to minimal processing. The effect of the inactivation step in combination with growth inhibiting factors such as CO2 enriched atmosphere during packaging on the inactivation and sublethal damage of the pathogens will be quantified in a first step. Secondly, the risk on increased stress resistance and virulence will be investigated by studying the phenotypical and genotypical response of sublethal damage cells to elucidate the stress response mechanisms and to identify phenotypical or genetic indicators, which are linked to the above-mentioned phenomena. The research will be carried out with E. coli (STEC), C. jejuni, L. monocytogenes and M. a. paratuberculosis. For virus (HEV and TBEV) similar, but limited studies will be carried out. The data obtained will be applied in the modelling of microbial behaviour in the food chain and estimation of microbial safety risk (WP14).

This WP will include the following tasks

 

Task 12.1      Selection of relevant microbial strains

Task 12.2         Optimisation of novel and mild processing conditions to obtain a novel process that inactivates/inhibits foodborne pathogens. Methods to be used include high hydrostatic pressure, decontamination with organic acids or other decontamination agents (e.g. chlorine dioxide in the gas phase), and intense light pulses

Task 12.3         Quantification of cross resistance in the microbial population surviving novel processing.

Task 12.4         Effect of stress factors during minimal processing on virulence of bacterial food pathogens. 

 

Partner 9, Partner 10, Partner 13 and Partner 14 will be involved in this WP.