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BioliSME II – Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes

Final Report Summary - BIOLISME II (BioliSME II – Demonstration, validation and preliminary promotion of a commercial prototype speedy system for sampling and detecting Listeria monocytogenes)

Executive Summary:
BIOLISME II is a collaborative Project for demonstration, validation and preliminary promotion of a commercial prototype Speedy system for sampling and detecting Listeria monocytogenes on surfaces. The project builds on the success of BIOLISME project, which resulted in the development of innovative sampling and detection technologies for rapid and selective detection of Listeria monocytogenes on surfaces, improving currently available methodologies of controlling the presence of this pathogen in food industries.
BIOLISME II involves 5 partners from 4 EU countries, combining their expertise in microbiology, optics, engineering, food safety and hygiene control to provide a commercially viable tool for enhanced control of Listeria monocytogenes.
During this project, functional prototypes have been constructed to validate the technology developed against conventional methods for controlling L. monocytogenes in food processing environments. These prototypes showcase the technology developed during BIOLISME project and display the latest state of development previously to their market introduction.
Construction of the prototypes has aimed at maximizing their technical performance and functionality to enhance their dissemination value. Prototypes produced have been used in dissemination activities and field trials to promote project results and to gather performance data that will support their future commercialization.
The technology developed and exemplified in the demonstration prototypes displays significant advantages over conventional methods currently used. Specifically, the novel technology allows obtaining results in under 2 hours from sample collection, against more than 24 hours with conventional methods. Operation of the equipment is semi-automatic, minimizing requirements for specialized personnel and facilities. Finally, cost of the equipment and consumables is contained and market competitive. In summary, the technology produced in BIOLISME II represents a valid alternative to current tools and methods and shows great commercial potential.
Dissemination activities have been carried out throughout the project to maximize awareness of project results among food industry and food safety professionals. Dissemination has employed varied tools available such as website and social networks, publications, press releases and participation in scientific and technical events. Additionally, a workshop was organized for public demonstration of the project results and the operation of the prototype.
Finally, actions towards exploitation of results have been carried out which include applying for patent protection for the sampling subsystem and for the integrated sampling and analysis device and a business plan has been produced setting the commercialization strategy for project results.
The results obtained have demonstrated to provide significant advantages to food industries for improved control of contamination by L. monocytogenes in terms of speed of analysis, costs and ease of operation. This is expected to result in more tools at the disposal of food industries for controlling microbial contamination in their facilities, enhancing prevention of food contamination and contributing to increase food safety across the EU.

Project Context and Objectives:
BIOLISME II project aims at supporting the commercialization of a novel technology for rapid detection of the food pathogen Listeria monocytogenes on hard surfaces in agri-food industries.
Listeria monocytogenes (L. monocytogenes) is one of the most important food pathogens due to its lethality and as such it is a source of major concern for food industries and regulatory agencies worldwide. This microorganism can cause the disease known as listeriosis. In humans, severe illness mainly occurs in developing fetuses, newborn infants, the elderly and those with weakened immune systems. Symptoms vary, ranging from mild flu-like symptoms and diarrhea, to life-threatening infections characterized by septicaemia and meningoencephalitis. In pregnant women, the infection can spread to the fetus, leading to severe illness at birth of death in the uterus, resulting in abortion. Illness is often severe with high hospitalization and mortality rates. Human infections are rare yet important, given the associated high mortality rate. These organisms are among the most important causes of death from food-borne infections in industrialized countries.
The bacterial genus Listeria currently comprises 10 species, but human cases of listeriosis are almost exclusively caused by the species Listeria monocytogenes. Listeria species are ubiquitous organisms that are widely distributed in the environment, especially in plant matter and soil. The principal reservoirs of Listeria are soil, forage and water. Other reservoirs include infected domestic and wild animals. The main route of transmission, to both humans and animals, is through consumption of contaminated food or feed. The bacterium can be found in raw foods and in processed foods which are contaminated after processing. Infection can also rarely be transmitted directly from infected animals to humans. Cooking at temperatures higher than 65 °C destroys Listeria, but the bacteria are able to multiply at temperatures as low as +2/+4 °C, which makes presence of Listeria in ready-to-eat (RTE) foods, with a relatively long shelf-life, of particular concern.
In 2012, 1,642 confirmed human cases of listeriosis were reported in the EU, a 10.5 % increase compared with 2011. The EU notification rate was 0.41 cases per 100,000 population with the highest country specific notification rates observed in Finland, Spain and Denmark (1.13 0.93 and 0.90 cases per 100,000 population, respectively).
A total of 198 deaths due to listeriosis were reported in the EU in 2012, which was the highest number of fatal cases reported since 2006. Fifteen countries reported one or more fatal cases with France reporting the highest number, 63 cases. The EU case fatality rate was 17.8 % among the 1,112 confirmed cases for which this information was reported (67.7 % of all confirmed cases).
EU legislation (Regulation (EC) No 2073/2005) lays down food safety criteria for L. monocytogenes in RTE foods. This regulation came into force in January 2006, and the criteria are described below:
• In RTE products intended for infants and for special medical purposes L. monocytogenes must not be present in 25 g.
• L. monocytogenes must not be present in levels exceeding 100 cfu/g during the shelf-life of other RTE products.
• In RTE foods that are able to support the growth of the bacterium, L. monocytogenes may not be present in 25 g at the time of leaving the production plant; however, if the producer can demonstrate, to the satisfaction of the competent authority, that the product will not exceed the limit of 100 cfu/g throughout its shelf-life, this criterion does not apply.
• In the case of RTE foods that are able to support the growth of L. monocytogenes, the microbiological criterion to be applied depends on the stage in the food chain and whether the producer has demonstrated that L. monocytogenes will not multiply to levels exceeding 100 cfu/g throughout the shelf-life.
Based on these criteria, 72 food alerts were issued in 2013 by the Rapid Alerts System for Foods and Feeds (RASFF) in the EU due to contamination by L. monocytogenes. The majority of these alerts corresponded to RTE fish products (27 cases), fresh cheeses (20) and RTE meat products (13). Most of these alerts resulted in product withdrawal from the market and its destruction, with subsequent costs for the manufacturer and impact on reputation.
Contamination of RTE products occurs primarily during post-lethality (during peeling, slicing, repackaging, etc.) exposure to the environment. Soil that persists on food processing equipment after use is inevitably contaminated with microorganisms which use its nutrients to form characteristic niches with the potential to attach and form biofilms, which upon detachment may cross-contaminate foods. For this reason, maintaining adequate levels of hygiene in food processing facilities is of paramount importance to prevent cross-contamination of food products and ensuring food safety. Food industries implement food safety procedures such as HACCP that involve routine control of microorganism presence in the food processing environment after cleaning and disinfection operations. Food contact surfaces are sampled to detect presence of undesired microorganisms and to assess results of cleaning and disinfection procedures.
Control of microbiological contamination is usually performed by conventional methods that involve collection of samples from surfaces using swabs or towels, transfer of samples to a culture growth medium, incubation in selective media and counting of grown colonies. This procedure requires availability of a microbiology laboratory and trained personnel and it is therefore not available to small sized companies which will normally subcontract these services. The main drawback of conventional methods is the long times required for obtaining results, due to the need to incubate samples for periods of 24 hours to 5 days and, in some cases, to perform a pre-enrichment stage for pathogens found in small numbers. In the case of L. monocytogenes, pre-enrichment of samples and incubation for 48 hours is required, as well as an additional confirmation stage for positive samples. The long processing times difficult strict control of hygiene conditions of surfaces as corrective measures can only be implemented after obtaining results.
Conventional methods in microbiological analysis are used despite their long turnover times because of their high selectivity and sensitivity. Rapid methods, in particular biosensors have the potential to shorten the time span between sample uptake and results, but their future lies in reaching selectivity and sensitivity comparable to established methods at a fraction of the cost. Although not so critical, issues such as ease of use, low maintenance and continuous operation also need to be considered.
In this context, the BIOLISME project (No. 232037) was launched in 2009, supported by the 7th Framework Programme under the “Research for the benefit of SMEs” theme. The aim of the project was developing a technology and methodology for enabling rapid detection of L. monocytogenes on surfaces in a selective, easy and cost-efficient manner. The main result of this project was a prototype device that integrated the functions of sample collection, sample processing and measurement of levels of L. monocytogenes in sample. The device was based on a novel biosensor that comprised an immunoassay for selective isolation and marking of Listeria monocytogenes cells and a detector that measured fluorescence light emitted by the sample after illumination. The main technological achievements of the technology developed during BIOLISME project (2009 – 2011) were:
• Reduced processing time or around 3 hours from sample collection to obtaining results.
• Highly efficient microorganism recovery rate from sampled surfaces (up to 90 %).
• Selective identification of L. monocytogenes cells
While the base technology that enabled the above performance was developed during BIOLISME project, this was still far from market requirements in terms of usability and functionality for demonstration actions. Based on the success of the project and the interest of the partners involved in pursuing commercialization of the technology developed, BIOLISME II project (No. 286713) was launched in 2012 by the same Consortium, under the Demonstration Action work programme of the EU’s 7th Framework Programme.
BIOLISME II project aims at disseminating BIOLISME project results among food industries and food safety actors, through dissemination actions and demonstration activities, as well as providing the means to support commercialization of the technology across the EU territory. The specific objectives of BIOLISME II project are:
• Construction of demonstration prototypes that display the technical achievements resulting from BIOLISME project.
• Documenting the performance and construction costs of prototypes for supporting commercial viability of the technology.
• Obtaining IP protection for foreground developed.
• To create awareness of the BIOLISME technology among food industries, food safety specialists, regulatory agencies and laboratory equipment manufacturers.
Project Results:
As a result of BIOLISME II project, the following scientific and technologic foregrounds achieved are the following:
A. A pre-commercial prototype mobile laboratory for detection of Listeria monocytogenes on hard surfaces.
B. Increased knowledge on behavior of L. monocytogenes cells on surfaces in different growth medium and biofilm forming conditions.
C. Optimized immunoassay for rapid and selective isolation and identification of Listeria monocytogenes cells.
A. The pre-commercial prototype integrates the technology developed during BIOLISME project and enables its routine use in demonstration activities and field trials to display the technical features of the technology.
The prototype consists of two differentiated subsystems: a sampling subsystem and a detection subsystem. The sampling subsystem allows collection of liquid samples from hard surfaces containing microorganisms. Sampling is based on a sampling tip of cylindrical form which presents a cavity to be placed on the surface to be examined. Sample collection occurs by injection of pressurized air and water into the cavity. Microorganisms are detached by impact with the pressurized jet and the mixture containing water and retrieved microorganisms is collected through and outlet into a sample collection chamber. Sample collection is aided by an additional cavity that surrounds the main cavity where vacuum is created. This enables strong attachment of the sampling tip to the surface to prevent the pressurized jet escaping the sampling area and its use on vertical surfaces or even upside down.
This method of sample collection presents several advantages over conventional sampling methods. For example, microorganism collection efficiency is very high, with recovery rates ranging from 90 to 99 % of microorganisms present on the surface, even when they are grown inside a biofilm. By comparison, conventional sampling methods such as swabs or towels only achieve recovery rates lower that 15 %. Additionally, the sampling subsystem delivers samples in liquid format, with an approximate volume of 40 mL. Samples collected in this form can then be processed for detection in the BIOLISME device or even analyzed in a standard microbiology laboratory using conventional procedures (incubation and colony counting).
The sampling tip is also connected to a cleaning circuit that allows circulation of a disinfecting solution to remove microbial contamination between each sample collection. The sampling subsystem comprises three deposits for water, disinfecting solution and waste collection, connected to the sampling tip through plastic, flexible pipes. Fluid transportation is achieved through peristaltic pumps and electrovalves. These are actioned through a programmable logic controller (PLC) which can be operated on a touch screen. The PLC includes options for manually operating the valves and pumps in the sampling subsystems and for automatic sampling, based on a pre-established sequence of operations.
Samples are collected in a 60 mL, sterile plastic syringe. This is a cheap, easily available and functional consumable which can be discarded after use.
The detection subsystem comprises two additional subsystems: a sample processing unit and a detector unit. The sample processing unit contains a holder for attaching the syringe containing the sample. This holder connects the syringe to the detector unit from the syringe tip and to deposits containing the immunoassay reagents and waste collection from the syringe upper opening. Isolation, concentration and fluorescent marking of Listeria monocytogenes cells is performed in the sampling processing unit through an immunoassay reaction. This allows separating L. monocytogenes cells from other microorganisms present in the sample and their semi-quantitative detection in the detector unit.
Sample processing through immunoassay reaction comprises the following steps:
• Addition of L. monocytogenes-specific antibodies bound to magnetic nanoparticles.
• Incubation
• Activation of magnet so that L. monocytogenes cells conjugated to antibodies are attracted to magnets and retained.
• Emptying of syringe to leave 2 mL.
• Washing through injection of water and emptying to waste.
• Addition of water up to 50 mL
• Addition of biotnylated antibodies bound to fluorescent markers
• Incubation
• Magnet activation and emptying
• Washing
All operations are controlled through the same PLC screen, which allows manual activation of pumps and electrovalves or automatic operation based on pre-programmed parameters. The immunoassay process for isolating and identifying L. monocytogenes cells constitutes one of the main technical achievements of the BIOLISME technology, since the reaction takes less than 1 hour to be completed, enabling fast obtaining of results and excellent specificity.
Sample processed as described above is moved by means or a peristaltic pump to the detector unit. The detector unit contains a sample holding chamber with a volume of 1 mL through which the sample fluid passes. Listeria monocytogenes cells are retained in the holding chamber by means of a magnet placed below. A dichroic mirror cube is placed over the holding chamber to allow illumination of sample and separation of the fluorescence signal emitted. Sample is thus excited by illumination with a LED source emitting at 405 nm. The fluorescent markers attached to antibodies bound to L. monocytogenes cells absorb incoming light and emit fluorescence of 650 nm wavelength. The detector sensor is placed above the dichroic mirror cube and collects fluorescence signal emitted by the sample. This sensor consists of a silicon photomultipliers that transform incoming light into an electrical current whose intensity is proportional to the amount of light collected. The detector contains an LCD screen which displays a numerical value based on measured signal along a pass/fail indication based on comparison of the measured signal against a reference value. Therefore, samples displaying a measured fluorescence above a reference threshold value will be classed as ‘fail’, representing a positive Listeria monocytogenes detection.
Sample handling through the detection unit is also controlled by the PLC screen, wich allows automatic and manual operation. The detection unit comprises an additional detector in the prototypes constructed. This detector is based on a CCD camera that obtains an optical image of the sample holding chamber under excitation. The image captured displays illuminated areas corresponding to clusters of cells emitting fluorescence. The image is analyzed through software that measures the illuminated areas, with the value of illuminated area proportional to the concentration of L. monocytogenes cells in the sample. This secondary detector is intended to complement the much easier to operate silicon photomultiplier detector and to be used for diagnostic purposes.
The detection subsystem allows detection of levels of Listeria monocytogenes over 100 cells/mL and results are obtained almost instantly after sampling processing.
All components and subsystems are contained in a mobile cart that allows transportation of the device and its use in demonstration activities and field trials. The equipment is fully functional and operation is semi-automatic as minimum personnel intervention is required. In summary, the technical features of this foreground that represent an advantage over current methodologies for control of Listeria monocytogenes on surfaces are:
• Rapid processing time. Results are obtained in under 2 hours from sampling to expression of results. Conventional microbiology techniques require at least 24 hours.
• Semi-automatic operation, requiring basic training for personnel involved in handling the equipment, in contrast with specialized facilities and personnel required in conventional microbiology analysis.
• High recovery rates in sampling of over 90 %, providing more reliable sampling.
• Low limit of detection of 10 cells/mL, corresponding to 9 cells/cm2 of surface sampled.
• Possibility of expansion of technical capabilities through further development to support parallel processing of multiple samples or detection of other pathogens of interest by modification of the immunoassay reaction.
B. As a result of tests performed to assess the performance of the sampling subsystem, enhanced knowledge on the behavior of Listeria monocytogenes cells when attached to hard surfaces was obtained. In particular, knowledge was gained regarding survival capacity of L. monocytogenes cells under different conditions, strength of attachment to surfaces of different materials and ability to form biofilms. The findings in this regard have implications on design and control of cleaning operations as appropriate water pressure and medium composition must be applied for effective removal of Listeria monocytogenes cells.
Biofilm growth experiments using tap water representative of wash procedures in kitchens and food industries showed that L. monocytogenes can form biofilms on stainless steel surfaces at any temperature between 4 °C and 37 °C. It was found that part of the inoculated cells could remain viable and cultivable for at least 48 hours on surfaces and that the rest of them could enter the viable but not cultivable state. This adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.
C. While the immunoassay reaction was developed during BIOLISME project, the optimized sequence of reaction steps was optimized during BIOLISME II as a result of tests performed on the prototypes constructed. This reaction allows isolating L. monocytogenes cells from other microorganisms which may be present in the sample and their identification by marking with a fluorescent attachment enabling their detection by fluorescence measurement.
The reaction relies on the use of antibodies that are specific of Listeria monocytogenes cells. These are Anti-InlA monoclonal antibodies 2B3 that attach to Internalin A surface protein found in Listeria monocytogenes. Antibodies are either conjugated to nanomagnetic particles or fluorescent labelled and a sandwich structure is formed during incubation where a Listeria monocytogenes cell in bound to a magnetic antibody and a fluorescent antibody. Proper performance of the immunoassay reaction is aided by the use of magnets to separate and retain L. monocytogenes cells coupled to magnetic antibodies.
The main features of the immunoassay reaction are its specificity, as only L. monocytogenes cells are coupled to the antibodies, sensitivity, with a limit of detection of 100 cells/mL, and speed of process, with the whole reaction taking place in around 1 hour.

Potential Impact:
The socio-economic impact of BIOLISME II project must be considered in the framework of food safety and control of microbiological contamination in the food industry.
As described above, Listeria monocytogenes is one of the pathogens causing major concern in the food industry, due to its wide distribution, survival at refrigeration temperatures and high rates of lethality. In 2012, 1642 confirmed cases of listeriosis in humans were reported in the EU territory, with 198 deaths as a result.
Foodborne illnesses have a significant impact on the society and the economy. Some studies have tried to evaluate this impact. Hoffmann et al. revised studied the 14 most frequent food pathogens in USA, which are involved in 95 % of intoxications and hospital admissions. The estimated annual cost due to medical expenses, lost productivity and premature mortality was $14,000 million in USA. Economic impact on the EU, while not directly comparable, must certainly be in the same order of magnitude.
Additionally, food contamination by pathogens have an impact of the food industry, due to alerts and recalls that will impact on the industry’s reputation and involve costs associated to destruction of products affected, declining sales, fines and compensation, etc.
In this context, the technology made available by BIOLISME project provides a tool for improving control of microbial contamination in food processing environments. One of the main disadvantages of conventional control methodologies is the long processing times required to obtain results (at least 24 hours). As a result, the hygienic condition of food processing environments is only assessed indirectly and not immediately, which may possess a risk for the safety of the foods produced. Industries demand faster and cheaper methods for performing controls of presence of microorganism in their facilities, being time and cost the main reasons for not increasing internal controls and thus enhancing food safety.
The results obtained constitute the basis of a promising technology for performing in-situ controls of microorganism presence after cleaning and disinfection operations, with Listeria monocytogenes being the first pathogen that can be tackled this way. Availability of these tools will encourage food industries to carry out more frequent and extensive controls in their facilities, thus minimizing the risks of food contamination and enhancing food safety.
The final result in terms of societal impact will be increasing safety of foods produced and consumed within the EU, reducing the number of illnesses associated to foodborne intoxications and therefore reducing the economic impact that these illnesses convey.
In order to pursue the impact described above as a result of commercialization of the results of the project, a series of dissemination activities have been performed during the project to create awareness of the technology developed. Additionally, exploitation of results has been initiated by project partners to support future commercialization of results.
Main dissemination activities are described below:
A. Demonstration workshop. A workshop was held in Gandia (Valencia, Spain) towards the end of the project (15th July 2014). The main objective of this workshop was to show for the first time the BIOLISME prototype to an audience composed of food industry professionals, academics and journalists. The workshop included talks from each partner participating in the project, followed by a live demonstration of prototype #3. Talks included information on each partner, their contribution to the project and the technical features they were responsible for. The workshop was attended by around 100 people, from different food industries, universities, journalists and manufacturers of laboratory equipment. The workshop was largely covered by specialized online media in Spain
B. Project website. A website is available in www.projectbiolisme.eu to be used as the main vehicle for spreading and interaction with the public seeking information about the BIOLISME II project. Besides providing information about the project, the website is also used as a vehicle to make available to all visitor, documents, articles and reports on the project. In addition to the website, a profile was created on the social network Twitter (@BIOLISME), which helped disseminate news and events related to the project. This was particularly useful when providing quasi-real time information on events such as meetings and the presentation workshop held at the end of the project. Followers include food safety professionals, food industries and food industry associations.
C. Participation in scientific and technical events. All partners participated in varied conferences and congresses in different European countries aimed at either scientific public or industry public. This participation helped disseminate results and obtain feedback from attendees. BIOLISME II results were presented at a total of 8 events.
D. Publications. Scientific and technological foreground resulting from the project was made available to specialized and general public by publications in different media: scientific journals, specialized media or books. A total of 6 publications were produced in English, French and English throughout the project.
E. Press releases. Periodic information on project progress and achievements was disseminated through press releases either on the project websites or on websites belonging to the project partners. Usually, these press releases were replicated by online specialized media with wide audience across the food industry.
F. Media appearances. Dissemination of project results was further supported by media appearances of some of the project partners. These consisted on a radio interview by BBC Radio to SOTON and a short piece on national Spanish TV by BETELGEUX and AINIA (the latter occurred after the end of the project).
In general, the project partners have pursued dissemination of project results using all channels within their reach and taking advantage of online possibilities. As a result, significant awareness of the project, its objectives and results has been created among food industry and food safety professionals. Dissemination impact was confirmed by feedback received from partners usually involving queries for further details on technical performance and availability of the BIOLISME technology. Dissemination of results has helped to create expectation within the food industry for the novel technology, thus supporting future commercialization efforts.
Finally, exploitation of results has been the initial aim of the industrial partners involved in the project. In order to set the path to future commercialization of project results, the following actions have been performed within the scope of BIOLISME II project:
A. A Joint Dissemination and Exploitation Plan was produced at the early stages of the project. This Plan set the strategy for dissemination of results and the guidelines to exploit the results obtained, based on the expectations of the project partners. The Plan aimed at maximizing impact of dissemination of results and potential of exploitation of the technology.
B. IP protection of the foreground resulting for the project was acquired in the form of patent applications. Two patent applications were submitted during the project: a French national patent covering the sampling subsystem and a European patent application covering the method and the device developed and improved during BIOLISME and BIOLISME II projects.
C. A Business Plan was produced by the industrial partners where the strategy for commercialization of project results was defined. The business model takes in consideration the limitations of the project partners to industrialize the production of equipment at large scale, and thus it relies on licensing the technology to a specialist manufacturer of laboratory equipment for final refinement of the technology and commercialization.
D. Contact with potential external manufacturers has been initiated in the aftermath of the project and as a result of dissemination actions, in order to assess potential interest in licensing and commercialization of results. Initial feedback is very encouraging, supporting the proposed business model.

List of Websites:
Project website address: www.projectbiolisme.eu.
Contact details (coordinator):
Dr Fernando Lorenzo
Betelgeux, S.L.
Ador (Valencia), Spain
f.lorenzo@betelgeux.es
www.betelgeux.es