Romain BRIANDET, Research Director, INRA Micalis (FR)
Microbial life is teeming on most surfaces of our planet. A support, water and some nutrients to microbes are sufficient to initiate the construction of real fortresses called microbial biofilms. The organic matrix, which ensures the cohesion of the biological structure, is essentially constituted by a sticky polymeric mixture. These microscopic architectures impact our daily life in several ways since they can form in natural and man-made surfaces. For the microbial roommates of the matrix, there are many benefits to community lifestyle, including on industrial equipment tolerance to antimicrobial, resistance to hydrodynamic stresses and cleaning operations The variety of survival strategies developed by these surface ecosystems is just beginning to be decrypted in the case of rudimentary model systems. Far from being mere three-dimensional assemblies of identical cells, biofilms are composed of subgroups with heterogeneous traits that are largely involved in their ecological success.
Philip S. STEWART, Center for Biofilm Engineering, Montana State University (USA)
This presentation will discuss fundamental physical, chemical, and biological concepts important to understanding control of detrimental biofilms. Four phenomena that are important in the action of antimicrobial agents against a biofilm will be examined: diffusion, hydrodynamics, physiology, and the genetic basis of biofilm tolerance. Direct microscopic observation of facile diffusion of a fluorophore-tagged antibiotic into biofilm is contrasted with failure of hydrogen peroxide, a much smaller molecule, to penetrate. These seemingly contradictory observations can be reconciled by recognizing that penetration of antimicrobials into biofilms is governed by the balance of reaction and diffusion. Time-lapse imaging of biofilms subjected to antimicrobial treatments reveals that in many cases these treatments do not remove the biofilm. In instances where removal is observed it is clear that forces applied by the flowing fluid are an important component of the removal process. Oxygen and nutrient concentration gradients within biofilms lead to stratified patterns of anabolic activity. Staining techniques reveal that the same biofilm can harbor, in distinct spatial niches, growing and dormant cells. Variation in the physiological activity is accompanied by alterations in susceptibility to antimicrobials. Patterns of gene expression within biofilms in response to local environmental chemistry are hypothesized to contribute to protection from antimicrobial agents. An illustrative transcriptomic and mutant susceptibility analysis is presented. The biofilm transcriptome is found to be enriched for genes associated with oxygen limitation and stationary phase growth. Mutants in regulatory genes associated with the response to growth arrest or hypoxia exhibit diminished tolerance to an antibiotic when grown as biofilms. These analyses suggest a model in which overlapping starvation and stress responses control the expression of multiple genes that are activated in mature biofilms and contribute to biofilm antimicrobial tolerance. The biofilm defense against biocides and antibiotic is multifactorial and so requires integrated and interdisciplinary science.
Prof. Dr. Hans-Curt FLEMMING, Professor emeritus, University of Duisburg-Essen, Biofilm Centre, Universitätsstr., Essen (DE)
In biofilms, the organisms live within a complex matrix of extracellular polymeric substances (EPS) which consists of polysaccharides, proteins, nucleic acids and humic substances. These provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional, functional polymer network that interconnects and transiently immobilizes biofilm cells. They can develop long-term interactions synergistic consortia, which can orchestrate the degradation of recalcitrant substrates, not available to single species. The matrix retains extracellular enzymes, resulting in a vast and versatile extracellular digestion system. All the characteristic features of biofilms — such as social cooperation, resource capture, extended communication and gene exchange, as well as enhanced survival of exposure to antimicrobials — rely on the structural and functional properties of the matrix. In biofilms, bacteria exhibit a set of ‘emergent properties’ that differ substantially from free-living bacterial cells. This includes fierce competition, resulting in constant adaptation to population shifts and environmental conditions. This makes the biofilm life the most successful and widespread mode of life on Earth.
Dr Alex Verplaetse and ing Thijs Vackier KULeuven (BE)
Formation of biofilms on food contact surfaces is a potential source of contamination and can impair food quality or stimulate growth of pathogens in food products. It is well known that microorganisms can attach and grow on different materials used in the food industry and can cause recurrent microbial problems lowering shelf life and endangering food safety. In a recent study performed in six food companies (meat, dairy and ready-meals) different biofilm forming microorganisms were isolated and identified. More than one third of the isolated biofilm forming organisms was considered to be strong biofilm forming bacteria meaning that they were irreversibly attached to stainless steel coupons. In all food companies a similar group of biofilm forming microorganisms was detected which mainly consisted of four families of microorganisms: Pseudomonas spp., Stenotrophomonas spp., Acinetobacter spp. and Microbacterium spp.
To study the development of biofilms of the food related microorganisms a Biofilm Reactor Model was validated for stainless steel coupons. The growth of biofilms in this model system was reproducible for the different food isolates. In this model the efficiency to remove biofilms was tested with classical cleaning and disinfection protocols used in the food industry. Although most industrial products could reduce the biofilm, a high regrowth was noticed within 24 hours indicating that biofilms could be kept under control with these classical protocols but could not be eliminated from the installation. Hence, novel strategies are needed for the removal of biofilms in the food industry because known protocols are not efficient enough.
As a case study, natural antimicrobial compounds were tested during cleaning and disinfection to inhibit biofilm growth or disrupt existing biofilms of Campylobacter spp. The potential of a series of natural antimicrobial compounds will be demonstrated for the inhibition and disruption of these pathogenic biofilms.
Dr. M.N. (Masja) NIEROP GROOT, Senior scientist Sustainable Food Processing/Microbiology (NL)
Bacillus cereus is a spore forming food pathogen that can persist in factory environments in the form of biofilms. Biofilms can be difficult to eradicate from surfaces including stainless steel used in the food industry.Microbes embedded in biofilms often display increased resistance to antimicrobial agents due to the self-produced matrix of extracellular polymeric substances functioning as a protective barrier.
B. cereus forms biofilms preferentially at the air-liquid interface typically present in semi-filled pipe lines or dead-ends of poorly designed processing equipment. The biofilm formation capacity of B. cereus is influenced by different parameters including those present in the factory environment, such as nutrient availability and type of surface but also varies highly between different B. cereus strains. Within the biofilm, heat resistant spores can be formed, a process that can be accelerated by drying of the biofilm or exposure to air. Under favourable conditions, a B. cereus biofilm can be formed and (partly) released within 24h.Consequently, bacterial cells and spores will be released and potentially lead to contamination of food products.
Novel insights in B. cereus biofilm formation and underlying mechanisms will be presented and translated to possibilities in design of novel strategies to prevent or eradicate B. cereus biofilms from food processing environments.
Even HEIR, Ph.D. Research Scientist, NOFIMA AS (NO)
Listeria monocytogenes is regarded the most serious food safety challenge for many food industry processors. Concerns are linked to the ability of Listeria to establish and grow in food processing environments where subsequent consumption of contaminated foods can cause listeriosis. Control strategies of L. monocytogenes in processing environments are key elements to improve the Listeria situation in the food industry.
At Nofima, we have coordinated several projects in collaboration with food industry producing Listeria risk products (cheese, meat, fish). These projects have documented the Listeria situation in the food processing environments and identified production practices and challenges linked to the Listeria situation in processing plants.
Factors influencing the elimination of Listeria include processing/environmental conditions as well as L. monocytogenes characteristics enabling their survival, growth and persistence in food industry conditions. The studies identified priority areas for enhanced Listeria control and determined the effects of selected interventions to combat Listeria in food processing facilities. Persistent house strains were observed in nearly all facilities. Laboratory studies showed that
persistent strains were not more tolerant to sanitation routines than non-persistent isolates. However, a possible role of resistance genes towards disinfectants in the ability of L. monocytogenes to grow in food processing environments was indicated. Growth competition and sanitation at recommended user concentrations did not eliminate L. monocytogenes in mixed microbiota biofilms. Follow-up studies have demonstrated whole genome sequencing to be suited to answer questions regarding persistence and epidemiology of L. monocytogenes in food processing facilities.
The results of these and previous studies indicate the complex challenges in controlling this pathogen in food industry. Possible strategies for enhanced Listeria prevention and elimination will be discussed.
Dr Steve FLINT, Professor in Food Safety and Microbiology Massey University (NZ)
Microbiological contamination is the main constraint in the manufacture of dairy products. This contamination originates from biofilms that grow on the internal surfaces of manufacturing plant, releasing bacteria, spores and enzymes into milk resulting in poor product quality. Blockage and loss of efficiency in heat transfer are also attributed to biofilm development. Biofilms can develop many stages of dairy manufacture stainless steel surfaces where raw milk is handled through to the waste treatment plant. The type of biofilm varies depending on the stage of dairy product manufacture. Psycrotrophic bacteria dominate in the raw milk handling zones while thermophilic bacteria dominate in the zones such as plate heat exchanges and evaporators where milk is heat treated. We now have evidence from comparative genomic sequencing that some of these bacteria, such as G. stearothermophilus have become adapted to the dairy environment, acquiring the ability to use lactose as a carbon source while others, including Bacillus licheniformis appear to thrive without this capability. Factors contributing to biofilm development include the need for ions, especially calcium ions. Clean in place systems are used to maintain the hygiene of dairy manufacturing plant but they are often not completely successful in eliminating the biofilm, allowing rapid re-colonisation of the manufacturing plant. Smart engineering solutions to disrupt the conditions that favour microbial growth or reducing the surface area available for biofilm growth, have helped in controlling biofilm development. In products where the ions are removed some thermophilic bacteria will not grow and manufacturing run lengths can be increased. Attempts to develop surfaces that prevent microbial colonisation have had limited success. Research is now focussed on understanding the composition of the biofilm matrix in a dairy system with an aim to improve the cleaning systems used in dairy manufacture.
Christophe DUFOUR, Scientific Director Microbiology Europe, Mérieux NutriSciences (FR)
Protecting the production environment from transient residues and pathogens has a direct effect on the quality of food products, specific concerns are related to the development of Biofilm in the food. The actual regulations specifically requests environmental monitoring of production, especially for infant formulas or ready-to-eat products, to chase the source of contamination.
After a specific risk assessment including the identification of microbiological hazards (Listeria, Salmonella, Biofilms ...), but also chemical, allergens, foreign bodies... it is essential to set up an effective environmental monitoring program including sampling plans, analyzes and corrective actions.
This presentation aims to highlight the key elements to build but also manage an effective environmental monitoring program. In particular, it will highlight the multiple approaches for identifying bacterial contaminations, including Biofilms presence. Building and operating a monitoring plan is a real force to anticipate or react as soon as possible in the beginning of environmental factory contamination. This presentation will also present the effectiveness of new environmental management tools, which is particularly effective in managing and planning environmental sample.
Prof. J. Azeredo, Universidade do Minho
Bacteriophages (phages) are viruses that specifically infect bacteria and present a great potential to be used as antibacterial agents. Strictly lytic phages recognize their hosts through specific cell surface receptors, replicate inside the bacteria and burst, killing the bacteria in a few minutes to release new phage particles capable of infecting new bacterial cells. We have demonstrated that certain bacteriophages can effectively control bacterial biofilms causing a biomass reduction of up to 97%, our studies proved that bacteriophages are able to penetrate the dense biofilm structures, through the water channels and because of their ability to replicate in low metabolic state cells they are able to tackle bacterial cells present in the inner layers causing biofilm dispersion. This process of killing is assisted by phages enzymes (endolysins and depolymerases) encoded in phage genomes and produced during the phage lytic cycle or displayed at phage tails or spikes. We have performed an in silico analysis of fully sequenced ds DNA phages and identified 723 phage endolysins (peptidoglycan degrading enzymes) and 160 putative depolymerases (including sialidases, levanases, xylosidases, dextranases, rhamnosidases, hyaluronidases, peptidases as well as alginate and pectate/pectin lyases). We further cloned and expressed endolysins and depolymerases active against gram negative and gram positive pathogens (namely S. aureus, Acinetobacter spp. and P. aeruginosa) and characterized their activity as biofilm preventing and controlling agents.
In this talk I will show how bacteriophages interact with biofilms and why certain bacteriophages have good anti-biofilm properties. I will also present the classes of endolysins and depolymerases encoded in phage genomes and how these enzymes can be used to prevent and control biofilm formation.
Raphaël RAYMOND and Florence MOIMEAUX, SODEXO (FR)
As the leading provider of food and facilities management services, Sodexo is committed to providing high quality and safe food service. For our customers and clients, food safety is mandatory and is included in the quality of the meal.
For a long time, Sodexo has understood the consequences of a food crisis :
loss of confidence of our consumers and clients
degradation of the company’s branding
loss of time and money to manage the crisis…
This is why Sodexo puts significant resources toward food safety prevention and training, and also relies on innovations.
So we investigate to understand how Listeria crisis occurred in order to avoid it. This work enabled us to understand the path towards Listeria crisis. The use of traditional chemicals products could lead to micro fouling of surfaces which could contribute to the settlement of biofilms. According to these facts, we worked with REALCO on a preventive cleaning program with Enzymes in order to break the biofilm development. This program is managed by using the biofilm detection kit. The results of the preventive cleaning with enzyme lead to less micro fouling and less Listeria detection.
Thierry BENEZECH, INRA (FR)
Food contact surfaces in processing equipment are considered to be major factors in the risk of food contamination in the food and beverage industries. Joint responsibility of equipment manufacturers and food producers could be suggested. Hence surface hygiene concerns could be tackled both by looking at the machinery design (geometry and material) and at the cleaning and disinfection operation conditions and frequencies.
Over recent years, apart from numerous studies of surface disinfections, great interest has been shown in both the mechanisms of (i) surface contamination, including the potential roles of materials and environmental conditions and of (ii) surface cleaning, largely governed by the machinery design impacting the mechanical action of the detergent flow under cleaning-in-place (CIP) conditions.
Hygienic design of food processing equipment is nowadays considered to be mandatory in the reduction of the risk microbial food contamination. The presentation will focus on two aspects, (i) how the design could play a role in biofilm installations and (ii) in its persistence in spite of cleaning operations. Better knowledge of surface contamination and cleaning mechanisms would positively impact hygienic design principles, thereby mitigating any environmental impact of the cleaning operations in the food and beverage industries.
Delhalle L., Blavier J., Louis C. and Vandersmissen I.
For the past 15 years, many efforts have been made to improve microbiological quality and security in the food chain. However many foodborne outbreaks are still reported each year in the European Union. Common foodborne pathogens have readily been found to produce biofilms on surfaces as Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Clostridium perfringens, Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, and Yersinia enterocolitica. Most of the time, it is difficult for industry to find the contamination origin especially from biofilm. One way to solve and to prevent microbial contamination from biofilm is to use microbial risk assessment (MRA) tools.
MRA has emerged as a comprehensive and systematic approach for addressing the risk of pathogens and spoilage bacteria in specific food and/or processes. The formalized structure of an MRA is well appropriate to government health agency decision-making tasks. It also has relevance to a number of industrial situations such as troubleshooting, shelf-life determination, thermal process setting, ingredient selection, assessment of innovative non-thermal processes and new product development. However, although much knowledge has been gained from a number of published risk assessments these past ten years, it are not usually used by food industry in hygiene management.
The overall objective of the presentation is to provide an overview of the tools available for the MRA with considerations for different approaches that can be used to analyze and to collect the relevant information (e.g.: metagenomics analysis, predictive microbiology, and modelling). Some examples of MRA in industry will be provided with a focus on the interpretation of results and the outputs from the use of various tools.
Dirk Nikoleiski - Mondelez International, Associate Director Product Protection & Hygienic Design
The application of hygienic design principles to buildings, infrastructure and equipment enable food manufacturers to clean all assets in a food manufacturing efficiently and effectively and it will minimise contamination risks. Directives and Regulations make hygienic design a legal obligation in the European Union and describe the expected outcomes. The available norms, standards and guidelines as regards food manufacturing equipment that are referring to the Directive 2006/42/EC (“Machinery Directive”) give details of what hygienic design means and how good design looks like. However, there is little information available on what stakeholders should do for a successful integration and implementation of hygienic entities.
Hygienic design should not only be understood as a technical foundation for delivering good design solutions, but also as an integrated approach. This implies the following:
The food manufacturer as the user of hygienically designed entities should determine the needs specific to the intended use, which includes the development of a user requirement specification as the basis for further discussions with vendors.
Very often underestimated, though critically important for success, is the iterative qualification process during the concept development and design stages before hygienic systems are being build.
During fabrication and commissioning, the design of hygienic entities should be verified at all stages to ensure that the requirements of qualified specifications are constantly and consistently met.
Typically, at the end of this process, a cleaning validation is conducted before hygienic entities can be released for normal operations to produce safe food products.
It should be noted, that project team members need to be familiar with hygienic design requirements for having the same understanding and making the right decisions. This requires training for developing knowledge paired with experience. In addition, project teams should be capable of applying powerful methods and tools, such as the Failure Mode and Effects Analysis (FMEA) and other.
Hygienic Design is more than a long list of technical criteria. It should be also recognized as a well-structured collaborative way of working embedded into a company’s business process.