Final Report Summary - BIOLISME (Speedy system for sampling and detecting Listeria monocytogenes in agro-food and related European industries)
Executive summary:
The objective of BIOLISME was the development of new instruments that helped food companies improve the monitoring of Listeria monocytogenes on surfaces in production environments. The resulting instruments would allow the rapid, safe, on-site detection of the pathogen. In order to achieve this single objective, a double approach was adopted: working both at the sampling and at the detection stages.
Firstly, the contamination conditions on surfaces in contact with food and how food industries dealt with it were simulated. L. monocytogenes strains were isolated from different sources (food samples and reference culture collections), which included six different serotypes (1a, 1/2a, 1/2b, 1/2c, 3a, 4b). Methods were assessed for the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms. The influence of various parameters was considered, namely: substrata (stainless steel, polytetrafluoroethylene and ceramics), temperature (4, 22, 30 and 37 degrees of Celsius), biofilm age (4, 24 and 48 h), growth media (BHI, TSB, TSBYE, TW) and presence of other foodborne pathogens (Escherichia coli non VT O157 and Pseudomonas fluorescens ATCC 13525), among others.
Secondly, a sampling system was developed to detach and collect L. monocytogenes adhered to surfaces based on the use of compressed air technology. The sampling sequence took 2 min and the cleaning sequence between samplings, about 3 minutes. The recovery rates were 26 and 100 % for planktonic cells and for biofilms, respectively. These values were compared to the recovery rates of conventional techniques, such as swabs and Rodac plates, which were below 4 % in every case. This proved that the actual levels of contamination in industries might be underestimated with conventional sampling techniques. The BIOLISME sampling prototype could contribute to improve this situation.
Thirdly, a fluorescence-linked-immunosorbent assay (FLISA) was developed for the specific capture and detection of L. monocytogenes. It was based on a monoclonal antibody which was highly specific for the serotypes isolated at the beginning of the project and which did not react with related Listeria spp. Magnetic and fluorescent markers were used to enhance the capture and the detection, respectively. The incubation time was 10 min and the length of the whole assay, 40 minutes. The sensitivity of the sandwich FLISA was 1E3 cells / mL. These results were achieved for L. monocytogenes in different formats: planktonic cells, sessile cells and mix flora. Further investigations on the fluorescent labelling yielded a sandwich FLISA with a sensitivity of 1E2 cells / mL. Therefore, this was found to be an interesting technique for the rapid and specific capture and detection of L. monocytogenes in microbiology laboratories.
Fourthly, a detection system was developed to measure the concentration of L. monocytogenes in environmental samples. The design of the system was based on the FLISA. It comprised four hardware modules: microfluidics, magnetic separation, fluorescent measurement and control. Software applications running in the control unit implemented the semi-automatic execution of the immunoassay, the capture and processing of the resulting images and the estimation of the bacterial concentration. The instrument kept the main characteristics of the FLISA (length of the assay: 40 min; sensitivity: 1E3 L. monocytogenes cells / mL) but it was less labour-intensive.
Finally, a complete instrument was developed by interfacing the sampling and detection systems. As the detection stage was performed in liquid format, it was decided to recover the microorganisms after the sampling stage in liquid media too. In this way, both instruments could be connected directly so the detached microorganisms could be conveyed to the detection system without manipulation. In addition, this approach granted more flexibility to the complete system. Both instruments could be used separately and easily interfaced with minimum user intervention, just by plugging the recovery containers to the immunosensor.
Project context and objectives:
The Codex Alimentarius Commission (CAC) defined food hygiene as 'all conditions and measures necessary to ensure the safety and suitability of food at all stages of the food chain'. In addition, the European Union (EU)'s General Food Hygiene Directive defined food hygiene as 'all measures necessary to ensure the safety and wholesomeness of foodstuffs'.
An especially relevant safety issue for food industries has been food spoilage resulting from the presence of fastidious and pathogenic microorganisms. They have a tremendously negative impact, posing a high risk for consumers' health and provoking million losses for public and private sectors. One of the microorganisms that has caused great concern in the last decades has been Listeria monocytogenes. This bacterium is the origin of listeriosis, a rare but potentially lethal foodborne infection which can kill vulnerable people such as the elderly, pregnant women and people suffering from immunocompromising diseases such as cancer or human immunodeficiency virus (HIV).
In 2011, The European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) published the EU summary report on Trends and Sources of Zoonoses, Zoonotic Agents and Foodborne Outbreaks in 2009. According to the information submitted by 27 EU Member States, L. monocytogenes was seldom detected above the legal safety limit from ready-to-eat foods. Findings over this limit were most often reported from fishery products, cheeses, and meat products at levels of 0.3 - 1.1 %. However, the number of listeriosis cases in humans increased by 19.1 % compared to 2008, with 1 645 confirmed cases recorded in 2009. A high case fatality ratio of 16.6 % was reported amongst cases. Based on the reported fatality rates and the total numbers of reported confirmed cases, it was estimated that in 2009 there were approximately 270 human deaths due to listeriosis, compared to 90 deaths due to salmonellosis and 40 deaths due to campylobacteriosis in the EU.
In order to ensure food safety, business operators have to comply with microbiological criteria implemented in Europe according to the categories of ready-to-eat foods (e.g. foods intended for sensitive consumers and foods supporting or not supporting growth of L. monocytogenes). Application of microbiological criteria is only one of several management activities to ensure that ready-to-eat foods are of low risk for human. Microbiological criteria will assist in controlling the levels of L. monocytogenes e.g. absence in 25 g or 100 cfu / g at the point of consumption. A recent risk assessment concluded that most listeriosis cases were due to foods markedly above the latter limit.
Nevertheless and as stated in Regulation (EC) No 2073/2005, microbiological criteria give guidance not only on the acceptability of foodstuffs, but also on the manufacturing, handling and distribution processes. For that reason, they have been consistently applied in combination with good hygiene practices (GHP) and hazard analysis and critical control point (HACCP) systems in order to minimise the initial contamination at manufacturing level and/or reducing the potential for growth of L. monocytogenes. This has been supported by a number of surveys, which revealed associations of listerioris cases with food and food packaging type, some preparation practices such as the use of slicing machines for meat products, the lack of training of food handlers, the storage time and the storage temperature, among others.
The preventive approach to the problem involves examining every stage of the process where contamination can occur in order to assure that the final product is safe. In this line, the analysis of the production and processing environments is one of the most effective ways to identify and prevent the presence of pathogenic microorganisms in foodstuffs, in particular of L. monocytogenes. This is even more crucial if we bear in mind their ability to attach to surfaces and form biofilms.
By definition, biofilms are structures which are constituted by one or several species of microorganisms (that can include bacteria, viruses and / or fungi) embedded in a complex matrix composed by exopolymeric substance (EPS) and water. Biofilms are ubiquitous in nature and have been recognised for their beneficial properties, such as wastewater treatment. On the contrary, the presence of biofilms in different systems, such as drinking water pipelines, heat exchangers or surfaces in contact with food, is of great concern for many industries. In food processing environments, uncontaminated food such as salads, raw meat or smoked fish, may become cross-contaminated by biofilms growing on rich-nutrient manufacturing facilities. Therefore, disinfection and removal of food industry biofilms is mandatory for an efficient control of food safety, even because it has been shown that sessile cells (cells in biofilms) are much more resistant to disinfectants and biocides than in the planktonic phase.
However, the analysis of environments presents some restrictions coming from the limitations of the associated sampling and testing methods. Some sampling methods commonly used nowadays in food industries are sponges, swabs, wipes and agar contact plates, among others. Their main limitations are their low recovery rates (lower than 10 %, in some cases) and the restrictions as for the type of surfaces that can be sampled (limited size and, in some cases, only flat surfaces). With regard to current detection methods for routine monitoring of contamination in production plants, they comprise culture-, molecular- and immunoassay-based techniques, amongst others. The main limitations of these techniques result from the need of sample enrichment (because the limits of detection are not low enough) and of specialised equipment and personnel (which usually implies outsourcing the analyses).
These limitations, together with the cost of the analyses and the time to get the results, have a great influence on companies when establishing sampling frequencies and dispatching times. Nevertheless, they are allowed to use analytical methods other than the reference ones, in particular more rapid methods, as long as their use can provide equivalent results. The overall objective of BIOLISME was to take advantage of this possibility and develop new instruments allowing food business operators to improve the analysis of the production environments and the assessment of the cleaning processes, with a particular focus on Listeria monocytogenes.
For that purpose, the first specific objective was to simulate the contamination conditions on surfaces in contact with food and how food industries deal with it. It would be studied the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms as well as their recovery, in particular of strains isolated from real food samples. It would be also considered the influence of various parameters, such as materials, temperatures and presence of other foodborne pathogens. This would result in useful information not only for the design of the BIOLISME instruments, but also for the evaluation of the reference methods.
The second specific objective was the construction of a sampling and recovery system based on compressed air technology. This technology was extensively used in almost all industries (papers, plastics, textiles, etc.), even in food industry for dehydration, bottling and vacuum packing, among others. Moreover, it had also proven its efficacy for removing bacteria in other areas, such as odontology. Knowledge on the state of the art of the technology and knowledge on contamination conditions would therefore be combined in order to develop a more flexible and efficient system to detach and collect L. monoctyogenes cells from surfaces in food processing environments.
The third specific objective was the construction of a detection system based on biosensor technology. This technology had been applied for the detection of different substances in several fields, such as in health (e.g. for glucose in blood), environment (e.g. for pesticides in water) and food (e.g. for antibiotics in meat). The main advantages of biosensors were their specificity, sensitivity and response time. Hence, it was decided to integrate recent progress on immunotechnology, labelling strategies and optics in the development of a more sensitive and rapid system for the detection L. monoctyogenes.
The instrument developed in BIOLISME was eventually aimed at providing user companies with several advantages. Firstly, the easy interfacing of both the sampling and measuring stages. This would allow companies carrying out their L. monocytogenes monitoring programmes in a more quick, efficient and safe manner than with current tools. Nevertheless and despite that, the sampling and measuring systems would be developed independently. This would enable their separate use and would result in greater flexibility of the system. Moreover, the approach to the design and the operation of the system would be set up from a general perspective to ensure an easy application to the detection of other important pathogens as well as the detection of Listeria monocytogenes in other sectors, such as clinical and environmental.
Project results:
The CAC defined food hygiene as 'all conditions and measures necessary to ensure the safety and suitability of food at all stages of the food chain'. In addition, the EU's General Food Hygiene Directive defined food hygiene as 'all measures necessary to ensure the safety and wholesomeness of foodstuffs'.
An especially relevant safety issue for food industries has been food spoilage resulting from the presence of fastidious and pathogenic microorganisms. They have a tremendously negative impact, posing a high risk for consumers' health and provoking million losses for public and private sectors. One of the microorganisms that has caused great concern in the last decades has been Listeria monocytogenes. This bacterium is the origin of listeriosis, a rare but potentially lethal foodborne infection which can kill vulnerable people such as the elderly, pregnant women and people suffering from immunocompromising diseases such as cancer or HIV.
In 2011, The EFSA and the ECDC published the EU summary report on Trends and Sources of Zoonoses, Zoonotic Agents and Foodborne Outbreaks in 2009. According to the information submitted by 27 EU Member States, L. monocytogenes was seldom detected above the legal safety limit from ready-to-eat foods. Findings over this limit were most often reported from fishery products, cheeses, and meat products at levels of 0.3 - 1.1 %. However, the number of listeriosis cases in humans increased by 19.1 % compared to 2008, with 1 645 confirmed cases recorded in 2009. A high case fatality ratio of 16.6 % was reported amongst cases. Based on the reported fatality rates and the total numbers of reported confirmed cases, it was estimated that in 2009 there were approximately 270 human deaths due to listeriosis, compared to 90 deaths due to salmonellosis and 40 deaths due to campylobacteriosis in the EU.
In order to ensure food safety, business operators have to comply with microbiological criteria implemented in Europe according to the categories of ready-to-eat foods (e.g. foods intended for sensitive consumers and foods supporting or not supporting growth of L. monocytogenes). Application of microbiological criteria is only one of several management activities to ensure that ready-to-eat foods are of low risk for human. Microbiological criteria will assist in controlling the levels of L. monocytogenes e.g. absence in 25 g or <=100 cfu / g at the point of consumption. A recent risk assessment concluded that most listeriosis cases were due to foods markedly above the latter limit.
Nevertheless and as stated in Regulation (EC) No 2073/2005, microbiological criteria give guidance not only on the acceptability of foodstuffs, but also on the manufacturing, handling and distribution processes. For that reason, they have been consistently applied in combination with GHP and HACCP systems in order to minimise the initial contamination at manufacturing level and / or reducing the potential for growth of L. monocytogenes. This has been supported by a number of surveys, which revealed associations of listerioris cases with food and food packaging type, some preparation practices such as the use of slicing machines for meat products, the lack of training of food handlers, the storage time and the storage temperature, amongst others.
The preventive approach to the problem involves examining every stage of the process where contamination can occur in order to assure that the final product is safe. In this line, the analysis of the production and processing environments is one of the most effective ways to identify and prevent the presence of pathogenic microorganisms in foodstuffs, in particular of L. monocytogenes. This is even more crucial if we bear in mind their ability to attach to surfaces and form biofilms.
By definition, biofilms are structures which are constituted by one or several species of microorganisms (that can include bacteria, viruses and/or fungi) embedded in a complex matrix composed by EPS and water. Biofilms are ubiquitous in nature and have been recognised for their beneficial properties, such as wastewater treatment. On the contrary, the presence of biofilms in different systems, such as drinking water pipelines, heat exchangers or surfaces in contact with food, is of great concern for many industries. In food processing environments, uncontaminated food such as salads, raw meat or smoked fish, may become cross-contaminated by biofilms growing on rich-nutrient manufacturing facilities. Therefore, disinfection and removal of food industry biofilms is mandatory for an efficient control of food safety, even because it has been shown that sessile cells (cells in biofilms) are much more resistant to disinfectants and biocides than in the planktonic phase.
However, the analysis of environments presents some restrictions coming from the limitations of the associated sampling and testing methods. Some sampling methods commonly used nowadays in food industries are sponges, swabs, wipes and agar contact plates, amongst others. Their main limitations are their low recovery rates (lower than 10 %, in some cases) and the restrictions as for the type of surfaces that can be sampled (limited size and, in some cases, only flat surfaces). With regard to current detection methods for routine monitoring of contamination in production plants, they comprise culture-, molecular- and immunoassay-based techniques, amongst others. The main limitations of these techniques result from the need of sample enrichment (because the limits of detection are not low enough) and of specialised equipment and personnel (which usually implies outsourcing the analyses).
These limitations, together with the cost of the analyses and the time to get the results, have a great influence on companies when establishing sampling frequencies and dispatching times. Nevertheless, they are allowed to use analytical methods other than the reference ones, in particular more rapid methods, as long as their use can provide equivalent results. The overall objective of BIOLISME was to take advantage of this possibility and develop new instruments allowing food business operators to improve the analysis of the production environments and the assessment of the cleaning processes, with a particular focus on Listeria monocytogenes.
For that purpose, the first specific objective was to simulate the contamination conditions on surfaces in contact with food and how food industries deal with it. It would be studied the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms as well as their recovery, in particular of strains isolated from real food samples. It would also be considered the influence of various parameters, such as materials, temperatures and presence of other foodborne pathogens. This would result in useful information not only for the design of the BIOLISME instruments, but also for the evaluation of the reference methods.
The second specific objective was the construction of a sampling and recovery system based on compressed air technology. This technology was extensively used in almost all industries (papers, plastics, textiles, etc.), even in food industry for dehydration, bottling and vacuum packing, among others. Moreover, it had also proven its efficacy for removing bacteria in other areas, such as odontology. Knowledge on the state of the art of the technology and knowledge on contamination conditions would therefore be combined in order to develop a more flexible and efficient system to detach and collect L. monoctyogenes cells from surfaces in food processing environments.
The third specific objective was the construction of a detection system based on biosensor technology. This technology had been applied for the detection of different substances in several fields, such as in health (e.g. for glucose in blood), environment (e.g. for pesticides in water) and food (e.g. for antibiotics in meat). The main advantages of biosensors were their specificity, sensitivity and response time. Hence, it was decided to integrate recent progress on immunotechnology, labelling strategies and optics in the development of a more sensitive and rapid system for the detection L. monoctyogenes.
The instrument developed in BIOLISME was eventually aimed at providing user companies with several advantages. Firstly, the easy interfacing of both the sampling and measuring stages. This would allow companies carrying out their L. monocytogenes monitoring programmes in a more quick, efficient and safe manner than with current tools. Nevertheless and despite that, the sampling and measuring systems would be developed independently. This would enable their separate use and would result in greater flexibility of the system. Moreover, the approach to the design and the operation of the system would be set up from a general perspective to ensure an easy application to the detection of other important pathogens as well as the detection of Listeria monocytogenes in other sectors, such as clinical and environmental.
Potential impact:
At the beginning of the project, the official public access website was launched at http://www.biolisme.eu. It was periodically updated with news about the consortium and its members, about information generated in the project and considered as public and about events of the project itself as well as connected to it. Moreover, the private area of the website, only accessible to project partners and to the REA via the project officer, granted access to internal documents (deliverables, dissemination documents, meetings minutes, official reports and working documents) and a private agenda (for meetings and participation in dissemination events).
Another dissemination material to publicise was the project leaflet, which was specially addressed to the industry. It introduced the project and presented with high simplicity and graphically the present and the future situation of the analysis of L. monocytogenes in food industries. 5 000 copies were printed in four different languages (English, Spanish, French and Czech). This allowed reaching more industries more easily.
A remarkable effort was made by all the partners in order to contribute to the dissemination of BIOLISME. The activities carried out in this area as well as the scientific (peer reviewed) publications originated from the project are listed next:
(1) Media briefings:
- 'Betelgeux participa en el desarrollo de un sistema de detección de Listeria en superficies'. Betelgeux noticias, 2009.
- 'Photek Limited joins the European Seventh Framework Programme BIOLISME'. Photek website, 2009.
(2) Press releases:
- 'Photek joins the European Seventh Framework Programme for fluorescence detection of food pathogens'. OptoIQ, 2009.
- 'Betelgeux participa en el proyecto BIOLISME de detección de Listeria' Investigación, Tecnología y Seguridad Alimentaria, 2009.
- 'Novo sistema permitirá detector Listeria em superfícies em contacto com alimentos'. Qualidad & Segurança Alimentar, 2009.
- 'Evitar la listeriosis en las personas más vulnerables'. Website of the Asociación Industrial de Laboratorios Farmacéuticos AG, 2009.
- 'Detección de Listeria en superfícies en contacto con alimentos'. Agroinformación, 2010.
- 'Ainia lidera un proyecto para un sistema de detección rápida de Listeria'. Diario Crítico de la Comunidad Valenciana, 2010.
- 'Ainia lidera un proyecto para un sistema de detección rápida de Listeria'. La Información Civil, 2010.
- 'Ainia lidera un proyecto para desarrollar un sistema de detección rápida de Listeria'. Europa Press, 2010.
- 'Ainia lidera un proyecto europeo para el desarrollo de un sistema de detección rápida de Listeria'. Eurocarne Digital, 2010.
- 'Ainia lidera un proyecto que desarrolla un sistema para la detección rápida de Listeria'. Asociación Española de Comunicación Científica, 2010.
- 'Ainia lidera un proyecto que desarrolla un sistema para la detección rápida de Listeria'. Bitácoras, 2010.
- 'Betelgeux participa en una reunión del proyecto BIOLISME'. Eurocarne Digital, 2011.
(3) Articles published in the popular press:
- 'Evitar la listeriosis en las personas más débiles'. Consumer Eroski, 2009.
- Belenguer, J. 'Nuevas técnicas para el muestreo y la detección de Listeria monocytogenes'.
- Tecnifood. La revista de la tecnología alimentaria, 2010.
- Belenguer, J. 'Hacia la reducción de tiempos en el control de Listeria monocytogenes en entornos productivos'. Alimentación, Equipos y Tecnología, 2011.
(4) Scientific (peer reviewed) publications:
- Byrne, B. 'Antibody-based sensors: Principles, problems and potential for detection of pathogens and associated toxins'. Sensors 9, Multidisciplinary Digital Publishing Institute, 2009, pp.
- Lynam, C. 'Carbon Nanotube based transducers for Immunoassays'. Carbon 47, Elsevier, 2009, pp. 4407-4445.
- Lahiff, E. 'The Increasing Importance of Nanostructured Surfaces in Biosensor Fabrication'. Analytical and Bioanalytical Chemistry 398, Elsevier, 2010, pp. 1575-1589.
- Crean, C. 'Polyaniline nanofibres as templates for the covalent immobilisation of biomolecules'. Synthetic metals 161, Elsevier, 2011, pp. 285-292.
- Gilmartin, N. 'Bionanotechnologies for the detection and reduction of pathogens'. Enzyme and Microbial Technology 50, Elsevier, 2012, pp. 87-95.
- Keevil, B. 'The biofilm safe haven and Listeria prevention'. UK Science & Technology 5, publicservice.co.uk Ltd.
(5) Posters:
- Gião, M. S. 'Episcopic differential interference contrast/epifluorescence microscopy to characterise Listeria'. International symposium on problems of listeriosis (ISOPOL XVII), 2010.
- Gilmartin, N. 'Characterisation of antibodies for use in an immunosensor for on-site detection of L. monocytogenes'. International symposium on problems of listeriosis (ISOPOL XVII), 2010.
- Gião, M. S. 'Hydrodynamic forces to detach Listeria monocytogenes biofilms from stainless steel surfaces'. Biofilms4. The International Biofilms Conference, 2010.
- Porta, S. 'New techniques for sampling Listeria monocytogenes from food industry surfaces'. Foodinnova, 2010.
- Lorenzo, F. 'Detección rápida de Listeria monocytogenes en superficies' Química Fusión, 2011.
(6) Presentations:
- Gilmartin, N. 'A rapid on-site immunosensor for the detection of L. monocytogenes'. ASSET 2011. The Food Integrity and Traceability Conference.
- Lorenzo, F. 'Innovative biosensors for effectively and easy detecting Listeria monocytogenes'. Reunión Anual de Consorcios Españoles de la RED Enterprise Europe Network, 2011.
(7) Conferences:
- Foley, C. 'Rapid simple immunoassay for the detection of Listeria monocytogenes' DRHEA undergraduate symposium, 2011.
(8) Workshops:
- Verdú, L. 'Controles analíticos en fábrica'. Prevención y control de Listeria monocytogenes, 2010.
- Porta, S. 'Monitorización de la limpieza y desinfección. El proyecto BIOLISME'. Seguridad Alimentaria e Higiene. Importancia de la Limpieza y Desinfección, 2010.
As a bridge towards exploitation activities, the participants integrated the exploitable results of the project as technology offers in the Enterprise Europe Network (EEN) database. The EEN brings together more than 580 business support organisations from across 49 countries. Some of the services that the network offers to the small and medium-sized enterprises (SMEs) are: international cooperation, technology transfer, access to EU projects and funding and information and assessment on EU legislation. The SMEs of the BIOLISME consortium submitted a technology offer for testing of new applications. The purpose of the offer was to have the opportunity of increasing the number of tests in industrial environments at international level and, in turn, making the project known to a higher range of companies.
A workshop was also organised to publicise the project results among potential end users. The event took place in Gandia (Valencia, Spain) and consisted of oral presentations describing the project concept, its development and the results obtained in comparison with current methods for control of Listeria. Afterwards, the prototype resulting from the project was presented, describing its components and mode of work. Finally, a practical demonstration was performed, simulating the operation of the prototype in real conditions.
Finally, the BIOLISME consortium submitted the proposal entitled: 'BIOLISME II - Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes'. It was approved by the REA in the framework of the 'Research for the benefit of SMEs' programme, within the call for demonstration activities. The objective of these activities is to prove the viability of new technologies that offer a potential economic advantage but which cannot be commercialised directly (e.g. testing of product-like prototypes). This is the last development stage before products or processes enter production.
Contact details: Mr José Belenguer Ballester (Project Coordinator)
Ainia - Centro Tecnológico
Email: jbelenguer@ainia.es
Telephone.: +34-961-366090
Ms Michaela Bitsakis (Project Officer)
Research Executive Agency
Email: Michaela.BITSAKIS@ec.europa.eu
Tel: +32-229-80279
List of websites: http://www.biolisme.eu.
The objective of BIOLISME was the development of new instruments that helped food companies improve the monitoring of Listeria monocytogenes on surfaces in production environments. The resulting instruments would allow the rapid, safe, on-site detection of the pathogen. In order to achieve this single objective, a double approach was adopted: working both at the sampling and at the detection stages.
Firstly, the contamination conditions on surfaces in contact with food and how food industries dealt with it were simulated. L. monocytogenes strains were isolated from different sources (food samples and reference culture collections), which included six different serotypes (1a, 1/2a, 1/2b, 1/2c, 3a, 4b). Methods were assessed for the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms. The influence of various parameters was considered, namely: substrata (stainless steel, polytetrafluoroethylene and ceramics), temperature (4, 22, 30 and 37 degrees of Celsius), biofilm age (4, 24 and 48 h), growth media (BHI, TSB, TSBYE, TW) and presence of other foodborne pathogens (Escherichia coli non VT O157 and Pseudomonas fluorescens ATCC 13525), among others.
Secondly, a sampling system was developed to detach and collect L. monocytogenes adhered to surfaces based on the use of compressed air technology. The sampling sequence took 2 min and the cleaning sequence between samplings, about 3 minutes. The recovery rates were 26 and 100 % for planktonic cells and for biofilms, respectively. These values were compared to the recovery rates of conventional techniques, such as swabs and Rodac plates, which were below 4 % in every case. This proved that the actual levels of contamination in industries might be underestimated with conventional sampling techniques. The BIOLISME sampling prototype could contribute to improve this situation.
Thirdly, a fluorescence-linked-immunosorbent assay (FLISA) was developed for the specific capture and detection of L. monocytogenes. It was based on a monoclonal antibody which was highly specific for the serotypes isolated at the beginning of the project and which did not react with related Listeria spp. Magnetic and fluorescent markers were used to enhance the capture and the detection, respectively. The incubation time was 10 min and the length of the whole assay, 40 minutes. The sensitivity of the sandwich FLISA was 1E3 cells / mL. These results were achieved for L. monocytogenes in different formats: planktonic cells, sessile cells and mix flora. Further investigations on the fluorescent labelling yielded a sandwich FLISA with a sensitivity of 1E2 cells / mL. Therefore, this was found to be an interesting technique for the rapid and specific capture and detection of L. monocytogenes in microbiology laboratories.
Fourthly, a detection system was developed to measure the concentration of L. monocytogenes in environmental samples. The design of the system was based on the FLISA. It comprised four hardware modules: microfluidics, magnetic separation, fluorescent measurement and control. Software applications running in the control unit implemented the semi-automatic execution of the immunoassay, the capture and processing of the resulting images and the estimation of the bacterial concentration. The instrument kept the main characteristics of the FLISA (length of the assay: 40 min; sensitivity: 1E3 L. monocytogenes cells / mL) but it was less labour-intensive.
Finally, a complete instrument was developed by interfacing the sampling and detection systems. As the detection stage was performed in liquid format, it was decided to recover the microorganisms after the sampling stage in liquid media too. In this way, both instruments could be connected directly so the detached microorganisms could be conveyed to the detection system without manipulation. In addition, this approach granted more flexibility to the complete system. Both instruments could be used separately and easily interfaced with minimum user intervention, just by plugging the recovery containers to the immunosensor.
Project context and objectives:
The Codex Alimentarius Commission (CAC) defined food hygiene as 'all conditions and measures necessary to ensure the safety and suitability of food at all stages of the food chain'. In addition, the European Union (EU)'s General Food Hygiene Directive defined food hygiene as 'all measures necessary to ensure the safety and wholesomeness of foodstuffs'.
An especially relevant safety issue for food industries has been food spoilage resulting from the presence of fastidious and pathogenic microorganisms. They have a tremendously negative impact, posing a high risk for consumers' health and provoking million losses for public and private sectors. One of the microorganisms that has caused great concern in the last decades has been Listeria monocytogenes. This bacterium is the origin of listeriosis, a rare but potentially lethal foodborne infection which can kill vulnerable people such as the elderly, pregnant women and people suffering from immunocompromising diseases such as cancer or human immunodeficiency virus (HIV).
In 2011, The European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) published the EU summary report on Trends and Sources of Zoonoses, Zoonotic Agents and Foodborne Outbreaks in 2009. According to the information submitted by 27 EU Member States, L. monocytogenes was seldom detected above the legal safety limit from ready-to-eat foods. Findings over this limit were most often reported from fishery products, cheeses, and meat products at levels of 0.3 - 1.1 %. However, the number of listeriosis cases in humans increased by 19.1 % compared to 2008, with 1 645 confirmed cases recorded in 2009. A high case fatality ratio of 16.6 % was reported amongst cases. Based on the reported fatality rates and the total numbers of reported confirmed cases, it was estimated that in 2009 there were approximately 270 human deaths due to listeriosis, compared to 90 deaths due to salmonellosis and 40 deaths due to campylobacteriosis in the EU.
In order to ensure food safety, business operators have to comply with microbiological criteria implemented in Europe according to the categories of ready-to-eat foods (e.g. foods intended for sensitive consumers and foods supporting or not supporting growth of L. monocytogenes). Application of microbiological criteria is only one of several management activities to ensure that ready-to-eat foods are of low risk for human. Microbiological criteria will assist in controlling the levels of L. monocytogenes e.g. absence in 25 g or 100 cfu / g at the point of consumption. A recent risk assessment concluded that most listeriosis cases were due to foods markedly above the latter limit.
Nevertheless and as stated in Regulation (EC) No 2073/2005, microbiological criteria give guidance not only on the acceptability of foodstuffs, but also on the manufacturing, handling and distribution processes. For that reason, they have been consistently applied in combination with good hygiene practices (GHP) and hazard analysis and critical control point (HACCP) systems in order to minimise the initial contamination at manufacturing level and/or reducing the potential for growth of L. monocytogenes. This has been supported by a number of surveys, which revealed associations of listerioris cases with food and food packaging type, some preparation practices such as the use of slicing machines for meat products, the lack of training of food handlers, the storage time and the storage temperature, among others.
The preventive approach to the problem involves examining every stage of the process where contamination can occur in order to assure that the final product is safe. In this line, the analysis of the production and processing environments is one of the most effective ways to identify and prevent the presence of pathogenic microorganisms in foodstuffs, in particular of L. monocytogenes. This is even more crucial if we bear in mind their ability to attach to surfaces and form biofilms.
By definition, biofilms are structures which are constituted by one or several species of microorganisms (that can include bacteria, viruses and / or fungi) embedded in a complex matrix composed by exopolymeric substance (EPS) and water. Biofilms are ubiquitous in nature and have been recognised for their beneficial properties, such as wastewater treatment. On the contrary, the presence of biofilms in different systems, such as drinking water pipelines, heat exchangers or surfaces in contact with food, is of great concern for many industries. In food processing environments, uncontaminated food such as salads, raw meat or smoked fish, may become cross-contaminated by biofilms growing on rich-nutrient manufacturing facilities. Therefore, disinfection and removal of food industry biofilms is mandatory for an efficient control of food safety, even because it has been shown that sessile cells (cells in biofilms) are much more resistant to disinfectants and biocides than in the planktonic phase.
However, the analysis of environments presents some restrictions coming from the limitations of the associated sampling and testing methods. Some sampling methods commonly used nowadays in food industries are sponges, swabs, wipes and agar contact plates, among others. Their main limitations are their low recovery rates (lower than 10 %, in some cases) and the restrictions as for the type of surfaces that can be sampled (limited size and, in some cases, only flat surfaces). With regard to current detection methods for routine monitoring of contamination in production plants, they comprise culture-, molecular- and immunoassay-based techniques, amongst others. The main limitations of these techniques result from the need of sample enrichment (because the limits of detection are not low enough) and of specialised equipment and personnel (which usually implies outsourcing the analyses).
These limitations, together with the cost of the analyses and the time to get the results, have a great influence on companies when establishing sampling frequencies and dispatching times. Nevertheless, they are allowed to use analytical methods other than the reference ones, in particular more rapid methods, as long as their use can provide equivalent results. The overall objective of BIOLISME was to take advantage of this possibility and develop new instruments allowing food business operators to improve the analysis of the production environments and the assessment of the cleaning processes, with a particular focus on Listeria monocytogenes.
For that purpose, the first specific objective was to simulate the contamination conditions on surfaces in contact with food and how food industries deal with it. It would be studied the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms as well as their recovery, in particular of strains isolated from real food samples. It would be also considered the influence of various parameters, such as materials, temperatures and presence of other foodborne pathogens. This would result in useful information not only for the design of the BIOLISME instruments, but also for the evaluation of the reference methods.
The second specific objective was the construction of a sampling and recovery system based on compressed air technology. This technology was extensively used in almost all industries (papers, plastics, textiles, etc.), even in food industry for dehydration, bottling and vacuum packing, among others. Moreover, it had also proven its efficacy for removing bacteria in other areas, such as odontology. Knowledge on the state of the art of the technology and knowledge on contamination conditions would therefore be combined in order to develop a more flexible and efficient system to detach and collect L. monoctyogenes cells from surfaces in food processing environments.
The third specific objective was the construction of a detection system based on biosensor technology. This technology had been applied for the detection of different substances in several fields, such as in health (e.g. for glucose in blood), environment (e.g. for pesticides in water) and food (e.g. for antibiotics in meat). The main advantages of biosensors were their specificity, sensitivity and response time. Hence, it was decided to integrate recent progress on immunotechnology, labelling strategies and optics in the development of a more sensitive and rapid system for the detection L. monoctyogenes.
The instrument developed in BIOLISME was eventually aimed at providing user companies with several advantages. Firstly, the easy interfacing of both the sampling and measuring stages. This would allow companies carrying out their L. monocytogenes monitoring programmes in a more quick, efficient and safe manner than with current tools. Nevertheless and despite that, the sampling and measuring systems would be developed independently. This would enable their separate use and would result in greater flexibility of the system. Moreover, the approach to the design and the operation of the system would be set up from a general perspective to ensure an easy application to the detection of other important pathogens as well as the detection of Listeria monocytogenes in other sectors, such as clinical and environmental.
Project results:
The CAC defined food hygiene as 'all conditions and measures necessary to ensure the safety and suitability of food at all stages of the food chain'. In addition, the EU's General Food Hygiene Directive defined food hygiene as 'all measures necessary to ensure the safety and wholesomeness of foodstuffs'.
An especially relevant safety issue for food industries has been food spoilage resulting from the presence of fastidious and pathogenic microorganisms. They have a tremendously negative impact, posing a high risk for consumers' health and provoking million losses for public and private sectors. One of the microorganisms that has caused great concern in the last decades has been Listeria monocytogenes. This bacterium is the origin of listeriosis, a rare but potentially lethal foodborne infection which can kill vulnerable people such as the elderly, pregnant women and people suffering from immunocompromising diseases such as cancer or HIV.
In 2011, The EFSA and the ECDC published the EU summary report on Trends and Sources of Zoonoses, Zoonotic Agents and Foodborne Outbreaks in 2009. According to the information submitted by 27 EU Member States, L. monocytogenes was seldom detected above the legal safety limit from ready-to-eat foods. Findings over this limit were most often reported from fishery products, cheeses, and meat products at levels of 0.3 - 1.1 %. However, the number of listeriosis cases in humans increased by 19.1 % compared to 2008, with 1 645 confirmed cases recorded in 2009. A high case fatality ratio of 16.6 % was reported amongst cases. Based on the reported fatality rates and the total numbers of reported confirmed cases, it was estimated that in 2009 there were approximately 270 human deaths due to listeriosis, compared to 90 deaths due to salmonellosis and 40 deaths due to campylobacteriosis in the EU.
In order to ensure food safety, business operators have to comply with microbiological criteria implemented in Europe according to the categories of ready-to-eat foods (e.g. foods intended for sensitive consumers and foods supporting or not supporting growth of L. monocytogenes). Application of microbiological criteria is only one of several management activities to ensure that ready-to-eat foods are of low risk for human. Microbiological criteria will assist in controlling the levels of L. monocytogenes e.g. absence in 25 g or <=100 cfu / g at the point of consumption. A recent risk assessment concluded that most listeriosis cases were due to foods markedly above the latter limit.
Nevertheless and as stated in Regulation (EC) No 2073/2005, microbiological criteria give guidance not only on the acceptability of foodstuffs, but also on the manufacturing, handling and distribution processes. For that reason, they have been consistently applied in combination with GHP and HACCP systems in order to minimise the initial contamination at manufacturing level and / or reducing the potential for growth of L. monocytogenes. This has been supported by a number of surveys, which revealed associations of listerioris cases with food and food packaging type, some preparation practices such as the use of slicing machines for meat products, the lack of training of food handlers, the storage time and the storage temperature, amongst others.
The preventive approach to the problem involves examining every stage of the process where contamination can occur in order to assure that the final product is safe. In this line, the analysis of the production and processing environments is one of the most effective ways to identify and prevent the presence of pathogenic microorganisms in foodstuffs, in particular of L. monocytogenes. This is even more crucial if we bear in mind their ability to attach to surfaces and form biofilms.
By definition, biofilms are structures which are constituted by one or several species of microorganisms (that can include bacteria, viruses and/or fungi) embedded in a complex matrix composed by EPS and water. Biofilms are ubiquitous in nature and have been recognised for their beneficial properties, such as wastewater treatment. On the contrary, the presence of biofilms in different systems, such as drinking water pipelines, heat exchangers or surfaces in contact with food, is of great concern for many industries. In food processing environments, uncontaminated food such as salads, raw meat or smoked fish, may become cross-contaminated by biofilms growing on rich-nutrient manufacturing facilities. Therefore, disinfection and removal of food industry biofilms is mandatory for an efficient control of food safety, even because it has been shown that sessile cells (cells in biofilms) are much more resistant to disinfectants and biocides than in the planktonic phase.
However, the analysis of environments presents some restrictions coming from the limitations of the associated sampling and testing methods. Some sampling methods commonly used nowadays in food industries are sponges, swabs, wipes and agar contact plates, amongst others. Their main limitations are their low recovery rates (lower than 10 %, in some cases) and the restrictions as for the type of surfaces that can be sampled (limited size and, in some cases, only flat surfaces). With regard to current detection methods for routine monitoring of contamination in production plants, they comprise culture-, molecular- and immunoassay-based techniques, amongst others. The main limitations of these techniques result from the need of sample enrichment (because the limits of detection are not low enough) and of specialised equipment and personnel (which usually implies outsourcing the analyses).
These limitations, together with the cost of the analyses and the time to get the results, have a great influence on companies when establishing sampling frequencies and dispatching times. Nevertheless, they are allowed to use analytical methods other than the reference ones, in particular more rapid methods, as long as their use can provide equivalent results. The overall objective of BIOLISME was to take advantage of this possibility and develop new instruments allowing food business operators to improve the analysis of the production environments and the assessment of the cleaning processes, with a particular focus on Listeria monocytogenes.
For that purpose, the first specific objective was to simulate the contamination conditions on surfaces in contact with food and how food industries deal with it. It would be studied the inoculation of L. monocytogenes planktonic cells and for the formation of L. monocytogenes biofilms as well as their recovery, in particular of strains isolated from real food samples. It would also be considered the influence of various parameters, such as materials, temperatures and presence of other foodborne pathogens. This would result in useful information not only for the design of the BIOLISME instruments, but also for the evaluation of the reference methods.
The second specific objective was the construction of a sampling and recovery system based on compressed air technology. This technology was extensively used in almost all industries (papers, plastics, textiles, etc.), even in food industry for dehydration, bottling and vacuum packing, among others. Moreover, it had also proven its efficacy for removing bacteria in other areas, such as odontology. Knowledge on the state of the art of the technology and knowledge on contamination conditions would therefore be combined in order to develop a more flexible and efficient system to detach and collect L. monoctyogenes cells from surfaces in food processing environments.
The third specific objective was the construction of a detection system based on biosensor technology. This technology had been applied for the detection of different substances in several fields, such as in health (e.g. for glucose in blood), environment (e.g. for pesticides in water) and food (e.g. for antibiotics in meat). The main advantages of biosensors were their specificity, sensitivity and response time. Hence, it was decided to integrate recent progress on immunotechnology, labelling strategies and optics in the development of a more sensitive and rapid system for the detection L. monoctyogenes.
The instrument developed in BIOLISME was eventually aimed at providing user companies with several advantages. Firstly, the easy interfacing of both the sampling and measuring stages. This would allow companies carrying out their L. monocytogenes monitoring programmes in a more quick, efficient and safe manner than with current tools. Nevertheless and despite that, the sampling and measuring systems would be developed independently. This would enable their separate use and would result in greater flexibility of the system. Moreover, the approach to the design and the operation of the system would be set up from a general perspective to ensure an easy application to the detection of other important pathogens as well as the detection of Listeria monocytogenes in other sectors, such as clinical and environmental.
Potential impact:
At the beginning of the project, the official public access website was launched at http://www.biolisme.eu. It was periodically updated with news about the consortium and its members, about information generated in the project and considered as public and about events of the project itself as well as connected to it. Moreover, the private area of the website, only accessible to project partners and to the REA via the project officer, granted access to internal documents (deliverables, dissemination documents, meetings minutes, official reports and working documents) and a private agenda (for meetings and participation in dissemination events).
Another dissemination material to publicise was the project leaflet, which was specially addressed to the industry. It introduced the project and presented with high simplicity and graphically the present and the future situation of the analysis of L. monocytogenes in food industries. 5 000 copies were printed in four different languages (English, Spanish, French and Czech). This allowed reaching more industries more easily.
A remarkable effort was made by all the partners in order to contribute to the dissemination of BIOLISME. The activities carried out in this area as well as the scientific (peer reviewed) publications originated from the project are listed next:
(1) Media briefings:
- 'Betelgeux participa en el desarrollo de un sistema de detección de Listeria en superficies'. Betelgeux noticias, 2009.
- 'Photek Limited joins the European Seventh Framework Programme BIOLISME'. Photek website, 2009.
(2) Press releases:
- 'Photek joins the European Seventh Framework Programme for fluorescence detection of food pathogens'. OptoIQ, 2009.
- 'Betelgeux participa en el proyecto BIOLISME de detección de Listeria' Investigación, Tecnología y Seguridad Alimentaria, 2009.
- 'Novo sistema permitirá detector Listeria em superfícies em contacto com alimentos'. Qualidad & Segurança Alimentar, 2009.
- 'Evitar la listeriosis en las personas más vulnerables'. Website of the Asociación Industrial de Laboratorios Farmacéuticos AG, 2009.
- 'Detección de Listeria en superfícies en contacto con alimentos'. Agroinformación, 2010.
- 'Ainia lidera un proyecto para un sistema de detección rápida de Listeria'. Diario Crítico de la Comunidad Valenciana, 2010.
- 'Ainia lidera un proyecto para un sistema de detección rápida de Listeria'. La Información Civil, 2010.
- 'Ainia lidera un proyecto para desarrollar un sistema de detección rápida de Listeria'. Europa Press, 2010.
- 'Ainia lidera un proyecto europeo para el desarrollo de un sistema de detección rápida de Listeria'. Eurocarne Digital, 2010.
- 'Ainia lidera un proyecto que desarrolla un sistema para la detección rápida de Listeria'. Asociación Española de Comunicación Científica, 2010.
- 'Ainia lidera un proyecto que desarrolla un sistema para la detección rápida de Listeria'. Bitácoras, 2010.
- 'Betelgeux participa en una reunión del proyecto BIOLISME'. Eurocarne Digital, 2011.
(3) Articles published in the popular press:
- 'Evitar la listeriosis en las personas más débiles'. Consumer Eroski, 2009.
- Belenguer, J. 'Nuevas técnicas para el muestreo y la detección de Listeria monocytogenes'.
- Tecnifood. La revista de la tecnología alimentaria, 2010.
- Belenguer, J. 'Hacia la reducción de tiempos en el control de Listeria monocytogenes en entornos productivos'. Alimentación, Equipos y Tecnología, 2011.
(4) Scientific (peer reviewed) publications:
- Byrne, B. 'Antibody-based sensors: Principles, problems and potential for detection of pathogens and associated toxins'. Sensors 9, Multidisciplinary Digital Publishing Institute, 2009, pp.
- Lynam, C. 'Carbon Nanotube based transducers for Immunoassays'. Carbon 47, Elsevier, 2009, pp. 4407-4445.
- Lahiff, E. 'The Increasing Importance of Nanostructured Surfaces in Biosensor Fabrication'. Analytical and Bioanalytical Chemistry 398, Elsevier, 2010, pp. 1575-1589.
- Crean, C. 'Polyaniline nanofibres as templates for the covalent immobilisation of biomolecules'. Synthetic metals 161, Elsevier, 2011, pp. 285-292.
- Gilmartin, N. 'Bionanotechnologies for the detection and reduction of pathogens'. Enzyme and Microbial Technology 50, Elsevier, 2012, pp. 87-95.
- Keevil, B. 'The biofilm safe haven and Listeria prevention'. UK Science & Technology 5, publicservice.co.uk Ltd.
(5) Posters:
- Gião, M. S. 'Episcopic differential interference contrast/epifluorescence microscopy to characterise Listeria'. International symposium on problems of listeriosis (ISOPOL XVII), 2010.
- Gilmartin, N. 'Characterisation of antibodies for use in an immunosensor for on-site detection of L. monocytogenes'. International symposium on problems of listeriosis (ISOPOL XVII), 2010.
- Gião, M. S. 'Hydrodynamic forces to detach Listeria monocytogenes biofilms from stainless steel surfaces'. Biofilms4. The International Biofilms Conference, 2010.
- Porta, S. 'New techniques for sampling Listeria monocytogenes from food industry surfaces'. Foodinnova, 2010.
- Lorenzo, F. 'Detección rápida de Listeria monocytogenes en superficies' Química Fusión, 2011.
(6) Presentations:
- Gilmartin, N. 'A rapid on-site immunosensor for the detection of L. monocytogenes'. ASSET 2011. The Food Integrity and Traceability Conference.
- Lorenzo, F. 'Innovative biosensors for effectively and easy detecting Listeria monocytogenes'. Reunión Anual de Consorcios Españoles de la RED Enterprise Europe Network, 2011.
(7) Conferences:
- Foley, C. 'Rapid simple immunoassay for the detection of Listeria monocytogenes' DRHEA undergraduate symposium, 2011.
(8) Workshops:
- Verdú, L. 'Controles analíticos en fábrica'. Prevención y control de Listeria monocytogenes, 2010.
- Porta, S. 'Monitorización de la limpieza y desinfección. El proyecto BIOLISME'. Seguridad Alimentaria e Higiene. Importancia de la Limpieza y Desinfección, 2010.
As a bridge towards exploitation activities, the participants integrated the exploitable results of the project as technology offers in the Enterprise Europe Network (EEN) database. The EEN brings together more than 580 business support organisations from across 49 countries. Some of the services that the network offers to the small and medium-sized enterprises (SMEs) are: international cooperation, technology transfer, access to EU projects and funding and information and assessment on EU legislation. The SMEs of the BIOLISME consortium submitted a technology offer for testing of new applications. The purpose of the offer was to have the opportunity of increasing the number of tests in industrial environments at international level and, in turn, making the project known to a higher range of companies.
A workshop was also organised to publicise the project results among potential end users. The event took place in Gandia (Valencia, Spain) and consisted of oral presentations describing the project concept, its development and the results obtained in comparison with current methods for control of Listeria. Afterwards, the prototype resulting from the project was presented, describing its components and mode of work. Finally, a practical demonstration was performed, simulating the operation of the prototype in real conditions.
Finally, the BIOLISME consortium submitted the proposal entitled: 'BIOLISME II - Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes'. It was approved by the REA in the framework of the 'Research for the benefit of SMEs' programme, within the call for demonstration activities. The objective of these activities is to prove the viability of new technologies that offer a potential economic advantage but which cannot be commercialised directly (e.g. testing of product-like prototypes). This is the last development stage before products or processes enter production.
Contact details: Mr José Belenguer Ballester (Project Coordinator)
Ainia - Centro Tecnológico
Email: jbelenguer@ainia.es
Telephone.: +34-961-366090
Ms Michaela Bitsakis (Project Officer)
Research Executive Agency
Email: Michaela.BITSAKIS@ec.europa.eu
Tel: +32-229-80279
List of websites: http://www.biolisme.eu.
