Periodic Reporting for period 1 - Multispeciesbiofilm (Understanding of multispecies biofilms in the food industry is indispensible to control biofilm related food safety and quality issues)
Periodo di rendicontazione: 2022-01-01 al 2023-12-31
The prevention of contamination with certain microbes in food processing industries is important for the safety and quality of food products. Cleaning and disinfection practices are mostly appropriately applied n food industries, but nevertheless, it seems that food contact surfaces like stainless steel are often laden with surface-adhered microbial communities termed biofilms. This is because not all food contact surfaces are in ideal condition or some places in the equipment, like dead ends, are difficult to clean. From time to time, this biofilm can disintegrate and release bacterial cells or enzymes and toxins which can contaminate the food product flowing past in the pipelines or on conveyor belts. As a result, biofilms in food industries can be responsible for food spoilage, process downtime, and reduced system efficiency of heat exchangers as a result of biofouling and corrosion of pipelines and tanks. It is important for the food industry to be able to prevent the formation of these biofilms or to effectively remove them during cleaning and disinfection. But also for society it is important to have a food system in which risks for deterioration of food quality or safety are still better managed.
In these biofilms, a large diversity of bacterial species are present and they are protected from the environment with a special matrix which is produced by some of these bacteria. Bacterial species belonging to Pseudomonas, Microbacterium, Bacillus, Stenotrophomonas, Staphylococcus, and Streptococcus have been observed to be widely present as contaminants across diverse food processing industries after the cleaning and disinfection practices. Pathogens like Listeria monocytogenes, Salmonella and Campylobacter can be present on these food contact surfaces too. It's the hypothesis that several of these bacteria work together in this biofilm through synergistic interactions making them more resilient and thus difficult to remove following cleaning and disinfection regimes.
The overall objective of the Marie Skłodowska-Curie action Multispeciesbiofilm was to understand interspecies interactions in the biofilms formed by the dominant bacterial isolates of dairy, meat and egg processing industries, with more emphasis on dairy processing. Starting from a large number of possible combinations, we wanted to know which combinations of species (with a maximum of 4 species together) are most detrimental to form biofilms on stainless steel under conditions mimicking real life processing conditions. We wanted to explore how these specific multispecies combinations respond to routine industrial cleaning and disinfection using a lab model with stainless steel coupons. We used a combination of cutting edge technologies like metatranscriptome analysis to explore which genes are expressed, and confocal laser scanning electron microscopy to visualize the biofilm at structural levels. In addition, we investigated the possibility to implement a biosensing device for real-time monitoring of the formation of biofilms.
In these biofilms, a large diversity of bacterial species are present and they are protected from the environment with a special matrix which is produced by some of these bacteria. Bacterial species belonging to Pseudomonas, Microbacterium, Bacillus, Stenotrophomonas, Staphylococcus, and Streptococcus have been observed to be widely present as contaminants across diverse food processing industries after the cleaning and disinfection practices. Pathogens like Listeria monocytogenes, Salmonella and Campylobacter can be present on these food contact surfaces too. It's the hypothesis that several of these bacteria work together in this biofilm through synergistic interactions making them more resilient and thus difficult to remove following cleaning and disinfection regimes.
The overall objective of the Marie Skłodowska-Curie action Multispeciesbiofilm was to understand interspecies interactions in the biofilms formed by the dominant bacterial isolates of dairy, meat and egg processing industries, with more emphasis on dairy processing. Starting from a large number of possible combinations, we wanted to know which combinations of species (with a maximum of 4 species together) are most detrimental to form biofilms on stainless steel under conditions mimicking real life processing conditions. We wanted to explore how these specific multispecies combinations respond to routine industrial cleaning and disinfection using a lab model with stainless steel coupons. We used a combination of cutting edge technologies like metatranscriptome analysis to explore which genes are expressed, and confocal laser scanning electron microscopy to visualize the biofilm at structural levels. In addition, we investigated the possibility to implement a biosensing device for real-time monitoring of the formation of biofilms.
The project's objective was to enhance understanding of bacterial interspecies interactions in multispecies biofilms within the food industry, while preparing the researcher for future professional challenges through comprehensive training. The researcher underwent extensive training at the home institute ILVO in various transferable skills (e.g. presenting, grant writing, food regulations) and on food processing operations in the Food Pilot, such as milk powder production, plant protein processing, and fish feed manufacturing. During secondment periods, training through research was performed at KU Leuven (biosensor) and the University of Copenhagen (confocal microscopy). The research involved examining the biofilm-forming capabilities in vitro of approximately 100 bacterial strains isolated from biofilm samples in dairy, meat, and egg processing industries. The study then focused on interspecies interactions in biofilm models composed of various combinations of up to four species isolated from a dairy pasteurizer. From this, two 4-species biofilm models were selected based on their synergistic bacterial interactions for further studies.
It was discovered that various social interactions within these dairy communities are crucial for species coexistence within the biofilm, going from cooperation, over competition to even exploitation. The stability and population dynamics of each species were found to be influenced by these interactions. The concept of 'keystone species' (e.g. the dairy species Microbacterium lacticum) was introduced, emphasizing their critical role in biofilm stability. Targeting these keystone species could be pivotal in developing strategies for biofilm eradication. At KU Leuven, the researcher innovatively utilized biosensors to study interspecies interactions on metallic coated surfaces, revealing for the first time the possibility to follow in real time the attachment and release of bacterial cells in a biofilm. Confocal microscopy at the University of Copenhagen was successfully employed to understand the spatial organization of each bacterial species in biofilms using specific FISH (fluorescent in situ hybridization) probes. This study revealed how bacteria benefit from their spatial organization under abiotic stress conditions such as cleaning and disinfection. Furthermore, new metabolic pathways and expressed genes related to bacterial adaptation in various environments were identified, offering insights into controlling biofilms and understanding interspecies interactions at an unprecedented level.
The findings have been disseminated through general articles in popular and trade magazines, a school activity and social media, enhancing public and industry understanding. In the academic sphere, four scientific articles have already been published, and more are underway, as well as oral and poster presentations at four international conferences. Several food or supplier companies have expressed their interest in the findings which may lead to follow-up research or industrial innovation.
It was discovered that various social interactions within these dairy communities are crucial for species coexistence within the biofilm, going from cooperation, over competition to even exploitation. The stability and population dynamics of each species were found to be influenced by these interactions. The concept of 'keystone species' (e.g. the dairy species Microbacterium lacticum) was introduced, emphasizing their critical role in biofilm stability. Targeting these keystone species could be pivotal in developing strategies for biofilm eradication. At KU Leuven, the researcher innovatively utilized biosensors to study interspecies interactions on metallic coated surfaces, revealing for the first time the possibility to follow in real time the attachment and release of bacterial cells in a biofilm. Confocal microscopy at the University of Copenhagen was successfully employed to understand the spatial organization of each bacterial species in biofilms using specific FISH (fluorescent in situ hybridization) probes. This study revealed how bacteria benefit from their spatial organization under abiotic stress conditions such as cleaning and disinfection. Furthermore, new metabolic pathways and expressed genes related to bacterial adaptation in various environments were identified, offering insights into controlling biofilms and understanding interspecies interactions at an unprecedented level.
The findings have been disseminated through general articles in popular and trade magazines, a school activity and social media, enhancing public and industry understanding. In the academic sphere, four scientific articles have already been published, and more are underway, as well as oral and poster presentations at four international conferences. Several food or supplier companies have expressed their interest in the findings which may lead to follow-up research or industrial innovation.
This project represents a significant advancement beyond the current state of the art in understanding bacterial interactions in biofilms, especially in food processing environments. By utilizing advanced techniques, we have uncovered intricate interspecies dynamics and spatial organization within multispecies biofilms which further explains their tolerance to current cleaning and disinfection practices. By introducing an innovative biofilm model consisting of a panel of carefully selected and interacting microbial species which represent a realistic worst case scenario, new possibilities are made to evaluate and improve cleaning and disinfection practices in industry. This creates new market opportunities for companies providing such products or services. The socio-economic impact of this project is substantial, as it offers strategies for more effective biofilm management in the food industry, potentially improving food safety and reducing waste. The wider societal implications include enhancing public health and contributing to sustainable food processing practices. We anticipate that our findings will not only contribute to scientific knowledge but also inform industry standards and policy like Food 2030, the Research and Innovation policy of the EU to transform food systems.