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BIOFILM FORMATION
BIOFILM FORMATION
FM Nattress, Agriculture and Agri-Food Canada, Lacombe, AB, Canada r 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by DR Korber, JR Lawrence, volume 1, pp 59–68, © 2004, Elsevier Ltd.
Glossary Brownian movement of bacteria A random movement of cells suspended in a fluid resulting from their collision as opposed to motility mediated by cellular appendages. Hydrophobic Lacking an affinity for water; insoluble in water; or repelling water. Planktonic cells Individual free-floating cells.
Quorum sensing A method of communication among bacterial cells by the release and sensing of small diffusible signal molecules. Spore-forming bacteria Bacteria that have the ability to develop spores, a resting stage of live bacteria encased in a ‘shell’ that is capable of protecting the cell under adverse conditions. Under appropriate growth conditions, the spores will germinate and cells will become active.
Introduction
Formation of Biofilms
The concept of microorganisms attaching to surfaces to form biofilms is not new. There are several different definitions of biofilms, but common to all is that bacteria form communities that are attached to solid surfaces. There is a general tendency for microorganisms to attach to wet surfaces upon which they multiply. They produce exopolymeric substances (EPS) in which they become embedded. EPS assist bacteria to adhere to surfaces and to each other, and protect the bacteria from adverse conditions. When these processes proceed without interruption, a biofilm is formed. A biofilm consists of a biologically active matrix of cells that is embedded in EPS produced by the cells and associated with or attached to a solid surface. The formation of biofilms by prokaryotic cells is a biologically unique developmental process in which the activities of the cells are coordinated to obtain effects of mutual benefit, such as the maintenance of open water channels. Such coordination of cellular activities requires there to be inter- and intraspecies communication. Various systems of cell-to-cell signaling have been identified and are currently being investigated. Many in vitro studies of biofilms have been undertaken using one or more bacterial species; however, the study of biofilms in situ remains challenging despite the availability of sophisticated molecular, electronic, and microscopic techniques. The application of in vitro findings for understanding of bacterial behavior in food processing environments is often not valid, so more study of natural biofilms is needed. Since bacteria living within biofilms can be very resistant to environmental stresses, such as drying and industrial sanitation processes, prevention of their development is an ongoing challenge for the meat industry. Of particular concern are nooks and crannies in commercial meat processing equipment that are hard to access and difficult to clean.
Stages of the Process
64
The formation and development of biofilms involves several stages. In the literature, there are different approaches to how these stages are defined and described (Table 1). Essentially, cells of microorganisms attach, first reversibly and then irreversibly, to a substratum that has been preconditioned with molecules in the environment around them. Once attached, and very early in biofilm development, the bacteria, and the microenvironment they create, become resistant to adverse or disruptive environmental conditions. If not disturbed, a mature biofilm will be formed. The formation of a mature biofilm can be achieved within a few hours but might instead take several weeks. Figures 1 and 2 are diagrammatic representations of the processes governing biofilm formation.
Surface Conditioning When a solid material is placed in a liquid, solutes from the liquid will concentrate on the surface of the solid material and form a conditioning film. The physicochemical properties of the surface, such as surface free energy, hydrophobicity, and electrostatic charges can change. In a food processing environment, the properties of the work surfaces might change depending on the type of conditioning film. For example, if surfaces in the facility are hydrophobic, they could show a high affinity for fat and the conditioning film on all surfaces could be high in fat content. Meat juice has been shown to reduce the negative charge on stainless steel, which would be expected to change the overall properties of the stainless steel.
Encyclopedia of Meat Sciences, Volume 1
doi:10.1016/B978-0-12-384731-7.00231-2
Table 1
Stages of biofilms formation as described by various authors
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Conditioning of a surface
Adhesion of cells
Formation of microcolony
Biofilm formation
Initial attachment of cells to the surface
Production of EPS resulting in more firmly adhered ‘irreversible’ attachment Transport of planktonic cells from the bulk liquid to the surface
Early development of biofilm architecture
Maturation of biofilm architecture
Detachment and dispersal of biofilms Dispersion of single cells from the biofilm
Adsorption of cells at the surface
Desorption of reversibly attached adsorbed cells
Some microbial cells remain after cleaning and sanitizing, and initiate growth
Larger biofilms formed with the help of expression of genes and quorum sensing
Preconditioning of the adhesion surface
Deposition of organic molecules (conditioning of a surface)
Biologically active molecules attracted to surface
Irreversible adsorption of bacterial cells at the surface
Stage 6
Stage 7
Stage 8
Stage 9
Reference Kumar and Anand (1998) Stoodley et al. (2002)
Production of cell–cell signaling molecules
Transport of substrates to and within the biofilm
Substrate metabolism by the biofilmbound cells and transport of products out of the biofilm
Biofilm removal by detachment or sloughing
Breyers and Ratner (2004)
Shi and Zhu (2009)
Biofilm Formation 65
66
Biofilm Formation
Pathogen Wet environment
Flow (milk, meat ...)
Sanitizer/detergents Organic material
Organic molecules
Persistent cell stress response
Attached monolayer
Cell proliferation
Microcolony
Gene expression
Biofilm
Quorum sensing
Figure 1 Sequence of events in biofilm formation on food contact surfaces. Firstly, organic molecules from food are deposited on the surface of equipment and form a conditioning film. Secondly, biologically active microorganisms are attracted to the organic molecules. Thirdly, persistent microbial cells remain after cleaning and sanitizing and initiate growth. Lastly, the biofilm forms with the expression cellular genes and quorum sensing. Reprinted from Shi, X., Zhu, X., 2009. Biofilm formation and food safety in food industries. Trends in Food Science and Technology 20, 407–413.
1. Substratum preconditioning by ambient molecules
2. Cell deposition
6. Convective and diffusive transport of O2 and nutrients
4. Desorption
3. Cell adsorption
5. Cell-to-cell signaling and onset of exopolymer production
9. Detachment, erosion, and sloughing 8. Secretion of polysaccharide matrix
7. Replication and growth
Substratum Figure 2 Distinct processes govern biofilm formation. Reprinted from Bryers, J.D., Ratner, J.P., 2004. Bioinspired implant material befuddle bacteria. American Society of Microbiology News 70, 232–237, with permission from ASM.
Transport of Planktonic Cells to Surfaces and Adhesion Factors that cause cells to undergo the transition from planktonic mode to biofilm mode or to become attached to or detached from surfaces are associated with a variety of environmental and physiological triggers that include quorum sensing, nutrient and oxygen availability, shear stress, microbial metabolic activity, microbial gene expression, and
cellular stress. Planktonic cells can move toward a conditioned surface actively, by means of flagella, or passively in response to forces such as gravity, diffusion, or fluid dynamics. Once cells are close to the surface, adhesion will be affected by factors like nutrient availability in the fluid, growth stage of the cells, and the microtopography of the contact surface. The first stage of adhesion is reversible and is the result of long-range interaction forces: electron acceptor–electron donor
Biofilm Formation
interactions and hydrophobic interactions that keep cells close to the conditioned surface. At this stage, the bacteria are not firmly attached and will continue to show Brownian movement. They can still be easily removed, if the microtopography of the surface does not keep cells trapped, by processes that produce shear forces, such as rinsing, or changes in charge and hydrophobicity. Until the bacterial surface appendages become involved, the bacteria usually cannot make direct contact with the surface due to repulsive forces between the bacterial cells and the surface. Bacterial cells produce surface appendages, including flagella, fimbriae, pili, and EPS fibrils that reduce the surface area that contacts the inert surface and by which they can overcome repulsive forces such as negative charges on both cell and surface to become attached. Flagella, composed of fine threads of flagellin protein, permit motility, which is hypothesized to overcome repulsive forces between cells and the surface so that a monolayer of cells can form on the surface. Flagella can also enable bacteria to move along the surface and allow growth and spread of the developing biofilm or form adhesive bonds between bacterial cells and surfaces. Fine, filamentous, hair-like proteinaceous appendages, including pili, and fimbriae present in both Gram-negative and Gram-positive bacteria are involved in various processes including conjugation, adherence, and twitching motility. All of these can affect biofilm formation and the characteristics of the bacteria in the biofilm. Although bacteria with fimbriae can adhere strongly to other bacterial cells and inorganic particles, and can probably overcome the initial electrostatic repulsion barrier between the cells and the surface, fimbriae are not always involved in the biofilm attachment process. They have, however, been shown to have a critical role in the initial stable cell-to-surface attachment for Salmonella spp. and numerous pathogenic and nonpathogenic Escherichia coli. A bacterial cell might be covered in fimbriae, giving the cell a hairy appearance. There are different types of pili, and cells can change the characteristics of the pili they produce to correspond to their environment. Cells may have one or several pili. The ends of pili are sticky and make cells adhesive so that bacteria with pili adhere strongly to other bacterial cells and inorganic particles. Pili are able to retract to bring the bacterial cell closer to the surface and Type IV pili enable cellular locomotion. Enterobacteriaceae produce proteinaceous fibers termed curli that are implicated in cell adhesion, cell aggregation, biofilm formation, and pathogenesis. Research into these fibers is relatively limited, but there is evidence that co-expression of curli and cellulose leads to the formation of networks of tightly packed cells that are aligned in parallel and that are highly hydrophobic. EPS also has a role in stabilizing the attachment of bacteria to surfaces.
Production of Exopolymeric Substances, Irreversible Attachment, and Development of Biofilm Architecture The production of EPS, irreversible attachment, and development of biofilm architecture are linked. An important characteristic of biofilms is that the cells are embedded in an EPS matrix. This matrix has a variety of functions, including cohesion and adhesion of cells and particulate matter; protection
67
from biocides and other chemicals; entrapment of molecules and nutrients; binding of cations, toxic metallic ions and other substances; and resistance to dessication. The polysaccharides and proteins form the structural elements of biofilms and determine their mechanical stability. The roles of the other components are not well established. At some time after attachment of cells to a surface, this attachment becomes irreversible. The cells grow and divide to form a microcolony. The microcolony enlarges, produces EPS, and coalesces so that it becomes anchored to the surface and stabilized. Microcolonies become protected from environmental stresses, and cells can show increased resistance to antimicrobial agents within a few hours of adhesion, even before they become embedded in the EPS matrix. Over time, cellular activity continues and the biofilm matures. Biofilms can be composed of different organisms or they can contain only one species. The organization of the cells ranges from a single layer to 3-D structures with the bacteria living within a large, complex, and organized ecosystem. There might be mechanisms whereby some species are encouraged to populate a biofilm, whereas attachment of other species is inhibited. In vitro studies show that multispecies biofilms are thicker and more stable than single species biofilms. The distribution of bacteria within biofilms is not even. Bacteria grow within the microcolonies that are surrounded by the EPS matrix and water channels are formed among them. These channels facilitate the transport of substrates, including oxygen, metabolites, and nutrients to the biofilm and within the biofilm; and also transport products, including waste products, out of the biofilm. The physiological state of biofilm bacteria appears to be different than that of planktonic bacteria. The combination of physiological modifications of biofilm-associated cells, including reduced growth rates and production of enzymes that can degrade antimicrobial substances, and physical protection provided by the biofilm matrix itself might be responsible for the extreme resistance to antimicrobials of biofilm bacteria. Part of the ‘life cycle’ of a biofilm is that damaged individuals are eliminated from the population. Programmed cell death and lysis can be a function of spatial orientation within the biofilm. Since deoxyribonucleic acid (DNA) is a component of the EPS and is thought to be related to the stability of the biofilm, the expectation is that cell lysis has a role in maintaining the stability of the biofilm structure. Biofilms are known to spread, but factors that control release of cells and detachment of sections of the biofilms are not well understood. They can release ‘pioneer’ cells or daughter cells individually, or there might be a sloughing or detachment of a relatively large part of the biofilm. When single cells or small clusters of cells detach, the effect on the biofilm is limited to the surface. When a large portion of a biofilm is sloughed off, the whole biofilm is impacted and may be lost.
Cellular Control of Biofilms – The Role of Cell-to-Cell Signaling Molecules and the Molecular Basis of Biofilm Formation The defining characteristic of biofilms is the formation of an integrated bacterial community. This requires self-organization
68
Biofilm Formation
and cooperation among cells, rather than the classical ‘competitive’ natural selection of individual microorganisms. Bacteria, which are colonial by nature, must be able to sense environmental changes and respond to them. They must also communicate with each other. Bacteria modulate gene expression and have systems of intercellular interactions and communications that are currently the focus of much study. The molecular changes that occur in a bacterium when it changes from its planktonic form to become sessile (permanently attached to the substrate) can be very complex and are only beginning to be understood. For example, in Listeria monocytogenes, expression of many proteins has been shown to be upregulated, including some related to stress response, protein synthesis, carbon metabolism, and regulation. These diverse molecular shifts show that the central metabolism of L. monocytogenes is affected during biofilm development. A major area of interest is the role of quorum sensing in development and maintenance of biofilms. Cell-to-cell signaling is mediated by small, diffusible molecules called auto-inducers that are produced and secreted during bacterial growth. As the bacterial population in the biofilm increases, the concentration of these small molecules increases too. When they reach a threshold or quorum level, the cells respond to them, with a variety of phenotypic responses being displayed as a result of the regulation of target genes that are quorum-sensing dependent. Quorum sensing seems to be involved in all stages of biofilm formation and maintenance. Some of the biofilm-related processes that can be controlled by quorum sensing are the establishment of bacteria in a mixed biofilm community, survival in food processing environments that are hostile to bacteria, the production of surface appendages, and motility. Since the discovery that bacterial cells communicate is relatively recent, much information is still required to fully explain the role of quorum sensing in the activities of biofilm bacteria. There is particular interest in the possibility of controlling the development of biofilms through disruption of the quorum sensing system.
Studying Biofilms The study of biofilms in situ is extremely difficult for many reasons. As a result, most knowledge about biofilms has been obtained from in vitro studies. Such results might not be relevant to circumstances in the food industry. Many disinfection processes are designed using bacteria that are planktonic, which are more sensitive to antimicrobials than bacteria embedded in biofilms. Enumerating or determining the types of bacteria residing within biofilms can be difficult. It has been estimated that for each planktonic bacterial cell detected, another 1000 cells could be present within a biofilm. In food processing environments, surfaces are usually treated with antimicrobial chemicals during cleaning, might not provide a lot of nutrients, can be hot or cold and dry, and might be otherwise hostile environments for bacteria causing them to be stressed and/or injured and nonculturable. The types of bacteria within a biofilm and the environment in which they develop will determine its characteristics. Researchers cannot as yet wholly duplicate naturally occurring biofilms.
A variety of microscopic techniques including scanning electron microscopy, transmission electron microscopy, and laser scanning microscopy are the tools used to observe biofilms and to monitor their development under controlled conditions. One instrument being studied as a way to monitor biofilm formation online is the mechatronic surface sensor. This type of sensor is based on the analysis of the vibration response of the surface and can detect biological and chemical characteristics of a variety of surfaces such as stainless steel and polyvinyl chloride.
Biofilms in the Food Industry The major focus of research on biofilms has been in relation to medical matters, but they are also very important in many industries, including the food industry. The bacteria in biofilms can cause food spoilage, compromise food safety and affect performance, and longevity of industrial equipment. Several pathogenic and spoilage bacteria including L. monocytogenes, Salmonella spp., Campylobacter spp., E. coli, Pseudomonas spp., and lactic acid bacteria have been associated with biofilm formation. In the meat and dairy industries, bacteria, including species of Pseudomonas, Staphylococcus, Enterobacter, Flavobacterium, yeasts, and Kluyvera were recovered from biofilms associated with clean floor materials. L. monocytogenes will form biofilms on floor drain surfaces, in storage tanks, on hand trucks, on conveyor belts, and on other food contact surfaces; and individual strains have been shown to persist in food plants for several years. Bacteria in biofilms can catalyze chemical and biological reactions to cause corrosion in pipelines and tanks, and if the biofilms are allowed to become thick, the heat transfer in heat exchangers and pipelines can be reduced. Equipment and processing plant design, cleaning, and disinfection regimens are the major tools available to the food industry to control the development of biofilms. Surfaces in meat processing facilities are frequently wet and biofilms can quickly form under wet conditions. Stainless steel, the material used for many food contact surfaces, is chemically and physiologically stable over a range of temperatures, and it is easy to clean. When the microtopographies of stainless steel surfaces are examined, they are seen to have many cracks and crevices that are good locations for bacteria to become attached. All areas of food processing environments including floors, walls, pipes, drains, conveyer belts, gaskets, and dead spaces are prone to biofilm formation. Similarly, materials that are commonly used in food processing facilities such as stainless steel, aluminum, nylon, Teflon, rubber, and plastic can become colonized by biofilms. The development and structure of biofilms depend on many factors, both intrinsic and extrinsic, including the species of bacteria in the consortium, the temperature, pH and nutrient status of the environment, and the flow conditions of fluids that contact surfaces. In the food industry, because cleaning and disinfection are frequent, mature biofilm structures or continuous bacterial films are not often observed on equipment surfaces; however, microcolony development with or without EPS is common. Whether these attached bacteria constitute a biofilm is debated. Although not showing the
Biofilm Formation
typical characteristics of biofilms, these microcolonies are important to the food industry because they can exhibit increased resistance to antimicrobial treatments within only a few hours of establishment; and cleaning and disinfection regimens must be designed to remove them completely. It is hypothesized that this resistance might, in part at least, result from a reduction in surface area exposed to antimicrobial treatment as a result of the attached surfaces not being in contact with the applied chemicals. Further research is required in this area, with particular emphasis on contamination of damp surfaces in food processing areas. In hard to access areas and areas that are infrequently cleaned, mature biofilms, with microbial cluster embedded in EPS and water channels, are observed. The best approach to control biofilm development is prevention of their establishment. Equipment should be designed so that areas that are hard to access and clean and areas that allow bacteria to accumulate, such as dead ends, corners, valves and joints, are avoided. Good water drainage should be assured and proper attention should be paid to the welding, material, and surface finishing on both exposed and nonexposed surfaces. All surfaces should be nonporous and smooth, without pits or crevices. Glass is a hard, smooth, and corrosion-resistant material but its applications are limited since it breaks easily. Stainless steel is easy to clean but the surface is easily damaged. Rubber surfaces deteriorate. Surfaces in food establishments must be cleaned, but regimens that cause corrosion and produce topographical defects should be avoided. Defects and roughness of surfaces have greater effect on the ease with which a surface can be cleaned than the type of finish. Since biofilm development occurs when moisture is present, maintenance of dry conditions will aid in prevention of their establishment. Even with proper equipment design and attention to surface characteristics and integrity, the likelihood of preventing biofilm development is slim. Cleaning and disinfection programs necessarily remain the main strategy for controlling surface contamination. Since bacterial adhesion is initiated soon after a surface is conditioned, regular cleaning and disinfection at short intervals are required to prevent firm bacterial attachment to surfaces and the formation of mixed species biofilms. Cleaning at short intervals will help to prevent sporulation by spore-forming bacteria, which is of particular importance to the dairy industry, but might become important to the meat industry as the involvement of psychrophilic (cold-loving) and pathogenic clostridia in meat spoilage becomes more common. One major factor militating against elimination of biofilms is that they can be reseeded with a very small number of cells. It is almost impossible to remove all cells associated with a biofilm. Pouring liquid onto a surface will erode a biofilm but a negligible number of the biofilm cells will be detached. Any cleaning and sanitation strategy must ensure that the biofilm bacteria are dead, not just dislodged. Surfaces should be cleaned prior to sanitation and cleaning processes must break up or dissolve the EPS so that sanitizers can make contact with the viable bacteria within the biofilm. Some materials used for cleaning are surfactants or alkaline products that suspend or dissolve food residues by decreasing surface tension, emulsification of fat, and denaturation of proteins. Acid products can
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also be used if the surfaces are coated with precipitated minerals or much food residue. If it is practicable, exposure of the biofilm to high temperature can reduce the requirements for physical cleaning. The type of cleaning process must not cause aerosols. For example, when mechanical force is applied using scrubbing and brushing, or pulsed laser beams are used, bacteria can become airborne. Such processes, including use of high pressure sprays, are being replaced by foam or gel cleaning. Bacterial cells within biofilms are highly resistant to antimicrobials. Even after a surface is cleaned, ridding the surface of viable cells is difficult. There are several mechanisms that have either been shown or are hypothesized to cause the elevated resistance of biofilm bacteria. Penetration of antimicrobials into the biofilm can be slow or the antimicrobial can be neutralized in the outer layers of the biofilm. The rate of diffusion might be slower than the rate at which the antimicrobial is inactivated, as is observed with resistance of biofilms to chlorine. The growth rate of bacteria deep in the biofilm is decreased and cells are in a ‘quasi-dormant’ state in which their resistance to biocides is increased. Older biofilms are more resistant to biocides than are younger ones. Genes controlling adaptive stress responses are expressed and biofilm cells are able to sense challenges from antimicrobials. Salmonella enteritidis isolated from biofilms had increased resistance to heat and chemicals when compared to planktonic cells. Repeated exposure of biofilm cells to antibiotics was shown to cause an increase in EPS synthesis in the biofilm. Cells, termed persister cells, that are phenotypically variant, can develop. These cells do not grow and do not die when challenged with antimicrobials. In the medical field, such cells are considered to be largely responsible for persistent infections resulting from biofilms. Their resistance is explained by their production of cellular toxins that block cellular processes such as translation and, so, render the cells resistant. Prevention and control of biofilms are topics of active research. At this time, there is no means known of preventing or controlling the development of biofilms without adverse side effects. Almost all materials will support the formation of biofilms. Some current efforts are focussed on the possibility of incorporating antimicrobial agents into either the construction materials themselves or surface coatings. Much work has been concerned with biomedical applications, but application in the food industry will likely follow and has been the focus of some studies. Laboratory studies in which fungicides are incorporated into flooring materials have been reported, but no results of long-term studies have been published. Covalent coupling of silicone rubber implants with quaternary ammonium coatings, coating surfaces with silver, preconditioning surfaces with surfactants and adsorption of nisin onto food contact surfaces are all in the experimental phase. In food processing, the importance of the ‘house microflora’ and the possibility that the presence of bacteriocin-producing bacteria and other endogenous strains might influence the establishment of biofilms are being studied. Novel approaches to prevention, removal, and inactivation of biofilm bacteria include the development of enzyme-based cleaners; various combinations of enzymes, detergents, surfactants, and phenolic antimicrobials; use of bacteriophages; interruption of quorum sensing; and manipulation of nutrient availability.
70
Biofilm Formation
The formation of biofilms is a unique, complex, and important biological phenomenon that enables many bacteria to survive and grow in interactive communities and in environments that are hostile to them. Biofilms can be advantageous, but in many parts of the food processing industry, they are considered to be biofouling agents responsible for problems with food spoilage, food safety, deterioration of equipment, and compromised efficiency of equipment. Our knowledge of the mechanisms that contribute to the formation, development and maintenance of biofilms is limited and improved control over biofilms will depend on significant advances in the field.
See also: Microbial Contamination: Decontamination of Processed Meat. Equipment Cleaning. Microbial Contamination: Decontamination of Fresh Meat; Microbial Contamination of Fresh Meat; Microbial Contamination of Processed Meat. Packaging: Technology and Films
Gandhi, M., Chikindas, M.L., 2007. Listeria: A foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15. Kumar, C.G., Anand, S.K., 1998. Significance of microbial biofilms in the food industry: A review. International Journal of Food Microbiology 42, 9–27. Marchand, S., De Block, J., De Jonghe, V., et al., 2012. Biofilm formation in milk production and processing environments; Influence on milk quality and safety. Comprehensive Reviews in Food Science and Food Safety 11, 133–147. Mettler, E., Carpentier, B., 1998. Variations over time of microbial load and physicochemical properties of floor materials after cleaning in food industry. Journal of Food Protection 61, 57–65. Shi, X., Zhu, X., 2009. Biofilm formation and food safety in food industries. Trends in Food Science and Technology 20, 407–413. Simões, M., Simões, L.C., Vieira, M.J., 2010. A review of current and emergent biofilm control strategies. LWT − Food Science and Technology 43, 573–583. Stoodley, P., Saur, K., Davies, D.G., Costerton, J.W., 2002. Biofilms as complex differentiated communities. Annual Reviews in Microbiology 56, 187–209. Van Houdt, R., Michiels, C.W., 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface. Journal of Applied Microbiology 109, 1117–1131. Zotolla, E.A., Sasahara, K.C., 1994. Microbial biofilms in the food processing industry − Should they be a concern? International Journal of Food Microbiology 23, 125–148.
Relevant Websites Further Reading Annous, B.A., Fratamico, P.M., Smith, J.L., 2009. Quorum sensing in biofilms: Why bacteria behave the way they do. Journal of Food Science 74, R24–R38. Bai, A.J., Rai, V.R., 2011. Bacterial quorum sensing and food industry. Comprehensive Reviews in Food Science and Food Safety 10, 184–194. Bryers, J.D., Ratner, J.P., 2004. Bioinspired implant material befuddle bacteria. American Society of Microbiology News 70, 232–237. Carpentier, B., Cerf, O., 1993. Biofilms and their consequences with particular references to hygiene in the food industry. Journal of Applied Bacteriology 75, 499–511.
http://www.ift.org/~/media/Knowledge%20Center/Science%20Reports/Scientific% 20Status%20Summaries/Editorial/editorial_0209_feat_biofilms.pdf Institute of Food Technologists. https://www.google.ca/search?q=biofilm+formation&oq=biofilm+formation &aqs=chrome..69i57j0l5.4979j0j8&sourceid=chrome&espv=210&es_sm= 122&ie=UTF-8#q=biofilm+formation+and+food+safety Manitoba Agriculture, Food and Rural Development.
FM Nattress, Agriculture and Agri-Food Canada, Lacombe, AB, Canada r 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by DR Korber, JR Lawrence, volume 1, pp 59–68, © 2004, Elsevier Ltd.
Glossary Brownian movement of bacteria A random movement of cells suspended in a fluid resulting from their collision as opposed to motility mediated by cellular appendages. Hydrophobic Lacking an affinity for water; insoluble in water; or repelling water. Planktonic cells Individual free-floating cells.
Quorum sensing A method of communication among bacterial cells by the release and sensing of small diffusible signal molecules. Spore-forming bacteria Bacteria that have the ability to develop spores, a resting stage of live bacteria encased in a ‘shell’ that is capable of protecting the cell under adverse conditions. Under appropriate growth conditions, the spores will germinate and cells will become active.
Introduction
Formation of Biofilms
The concept of microorganisms attaching to surfaces to form biofilms is not new. There are several different definitions of biofilms, but common to all is that bacteria form communities that are attached to solid surfaces. There is a general tendency for microorganisms to attach to wet surfaces upon which they multiply. They produce exopolymeric substances (EPS) in which they become embedded. EPS assist bacteria to adhere to surfaces and to each other, and protect the bacteria from adverse conditions. When these processes proceed without interruption, a biofilm is formed. A biofilm consists of a biologically active matrix of cells that is embedded in EPS produced by the cells and associated with or attached to a solid surface. The formation of biofilms by prokaryotic cells is a biologically unique developmental process in which the activities of the cells are coordinated to obtain effects of mutual benefit, such as the maintenance of open water channels. Such coordination of cellular activities requires there to be inter- and intraspecies communication. Various systems of cell-to-cell signaling have been identified and are currently being investigated. Many in vitro studies of biofilms have been undertaken using one or more bacterial species; however, the study of biofilms in situ remains challenging despite the availability of sophisticated molecular, electronic, and microscopic techniques. The application of in vitro findings for understanding of bacterial behavior in food processing environments is often not valid, so more study of natural biofilms is needed. Since bacteria living within biofilms can be very resistant to environmental stresses, such as drying and industrial sanitation processes, prevention of their development is an ongoing challenge for the meat industry. Of particular concern are nooks and crannies in commercial meat processing equipment that are hard to access and difficult to clean.
Stages of the Process
64
The formation and development of biofilms involves several stages. In the literature, there are different approaches to how these stages are defined and described (Table 1). Essentially, cells of microorganisms attach, first reversibly and then irreversibly, to a substratum that has been preconditioned with molecules in the environment around them. Once attached, and very early in biofilm development, the bacteria, and the microenvironment they create, become resistant to adverse or disruptive environmental conditions. If not disturbed, a mature biofilm will be formed. The formation of a mature biofilm can be achieved within a few hours but might instead take several weeks. Figures 1 and 2 are diagrammatic representations of the processes governing biofilm formation.
Surface Conditioning When a solid material is placed in a liquid, solutes from the liquid will concentrate on the surface of the solid material and form a conditioning film. The physicochemical properties of the surface, such as surface free energy, hydrophobicity, and electrostatic charges can change. In a food processing environment, the properties of the work surfaces might change depending on the type of conditioning film. For example, if surfaces in the facility are hydrophobic, they could show a high affinity for fat and the conditioning film on all surfaces could be high in fat content. Meat juice has been shown to reduce the negative charge on stainless steel, which would be expected to change the overall properties of the stainless steel.
Encyclopedia of Meat Sciences, Volume 1
doi:10.1016/B978-0-12-384731-7.00231-2
Table 1
Stages of biofilms formation as described by various authors
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Conditioning of a surface
Adhesion of cells
Formation of microcolony
Biofilm formation
Initial attachment of cells to the surface
Production of EPS resulting in more firmly adhered ‘irreversible’ attachment Transport of planktonic cells from the bulk liquid to the surface
Early development of biofilm architecture
Maturation of biofilm architecture
Detachment and dispersal of biofilms Dispersion of single cells from the biofilm
Adsorption of cells at the surface
Desorption of reversibly attached adsorbed cells
Some microbial cells remain after cleaning and sanitizing, and initiate growth
Larger biofilms formed with the help of expression of genes and quorum sensing
Preconditioning of the adhesion surface
Deposition of organic molecules (conditioning of a surface)
Biologically active molecules attracted to surface
Irreversible adsorption of bacterial cells at the surface
Stage 6
Stage 7
Stage 8
Stage 9
Reference Kumar and Anand (1998) Stoodley et al. (2002)
Production of cell–cell signaling molecules
Transport of substrates to and within the biofilm
Substrate metabolism by the biofilmbound cells and transport of products out of the biofilm
Biofilm removal by detachment or sloughing
Breyers and Ratner (2004)
Shi and Zhu (2009)
Biofilm Formation 65
66
Biofilm Formation
Pathogen Wet environment
Flow (milk, meat ...)
Sanitizer/detergents Organic material
Organic molecules
Persistent cell stress response
Attached monolayer
Cell proliferation
Microcolony
Gene expression
Biofilm
Quorum sensing
Figure 1 Sequence of events in biofilm formation on food contact surfaces. Firstly, organic molecules from food are deposited on the surface of equipment and form a conditioning film. Secondly, biologically active microorganisms are attracted to the organic molecules. Thirdly, persistent microbial cells remain after cleaning and sanitizing and initiate growth. Lastly, the biofilm forms with the expression cellular genes and quorum sensing. Reprinted from Shi, X., Zhu, X., 2009. Biofilm formation and food safety in food industries. Trends in Food Science and Technology 20, 407–413.
1. Substratum preconditioning by ambient molecules
2. Cell deposition
6. Convective and diffusive transport of O2 and nutrients
4. Desorption
3. Cell adsorption
5. Cell-to-cell signaling and onset of exopolymer production
9. Detachment, erosion, and sloughing 8. Secretion of polysaccharide matrix
7. Replication and growth
Substratum Figure 2 Distinct processes govern biofilm formation. Reprinted from Bryers, J.D., Ratner, J.P., 2004. Bioinspired implant material befuddle bacteria. American Society of Microbiology News 70, 232–237, with permission from ASM.
Transport of Planktonic Cells to Surfaces and Adhesion Factors that cause cells to undergo the transition from planktonic mode to biofilm mode or to become attached to or detached from surfaces are associated with a variety of environmental and physiological triggers that include quorum sensing, nutrient and oxygen availability, shear stress, microbial metabolic activity, microbial gene expression, and
cellular stress. Planktonic cells can move toward a conditioned surface actively, by means of flagella, or passively in response to forces such as gravity, diffusion, or fluid dynamics. Once cells are close to the surface, adhesion will be affected by factors like nutrient availability in the fluid, growth stage of the cells, and the microtopography of the contact surface. The first stage of adhesion is reversible and is the result of long-range interaction forces: electron acceptor–electron donor
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interactions and hydrophobic interactions that keep cells close to the conditioned surface. At this stage, the bacteria are not firmly attached and will continue to show Brownian movement. They can still be easily removed, if the microtopography of the surface does not keep cells trapped, by processes that produce shear forces, such as rinsing, or changes in charge and hydrophobicity. Until the bacterial surface appendages become involved, the bacteria usually cannot make direct contact with the surface due to repulsive forces between the bacterial cells and the surface. Bacterial cells produce surface appendages, including flagella, fimbriae, pili, and EPS fibrils that reduce the surface area that contacts the inert surface and by which they can overcome repulsive forces such as negative charges on both cell and surface to become attached. Flagella, composed of fine threads of flagellin protein, permit motility, which is hypothesized to overcome repulsive forces between cells and the surface so that a monolayer of cells can form on the surface. Flagella can also enable bacteria to move along the surface and allow growth and spread of the developing biofilm or form adhesive bonds between bacterial cells and surfaces. Fine, filamentous, hair-like proteinaceous appendages, including pili, and fimbriae present in both Gram-negative and Gram-positive bacteria are involved in various processes including conjugation, adherence, and twitching motility. All of these can affect biofilm formation and the characteristics of the bacteria in the biofilm. Although bacteria with fimbriae can adhere strongly to other bacterial cells and inorganic particles, and can probably overcome the initial electrostatic repulsion barrier between the cells and the surface, fimbriae are not always involved in the biofilm attachment process. They have, however, been shown to have a critical role in the initial stable cell-to-surface attachment for Salmonella spp. and numerous pathogenic and nonpathogenic Escherichia coli. A bacterial cell might be covered in fimbriae, giving the cell a hairy appearance. There are different types of pili, and cells can change the characteristics of the pili they produce to correspond to their environment. Cells may have one or several pili. The ends of pili are sticky and make cells adhesive so that bacteria with pili adhere strongly to other bacterial cells and inorganic particles. Pili are able to retract to bring the bacterial cell closer to the surface and Type IV pili enable cellular locomotion. Enterobacteriaceae produce proteinaceous fibers termed curli that are implicated in cell adhesion, cell aggregation, biofilm formation, and pathogenesis. Research into these fibers is relatively limited, but there is evidence that co-expression of curli and cellulose leads to the formation of networks of tightly packed cells that are aligned in parallel and that are highly hydrophobic. EPS also has a role in stabilizing the attachment of bacteria to surfaces.
Production of Exopolymeric Substances, Irreversible Attachment, and Development of Biofilm Architecture The production of EPS, irreversible attachment, and development of biofilm architecture are linked. An important characteristic of biofilms is that the cells are embedded in an EPS matrix. This matrix has a variety of functions, including cohesion and adhesion of cells and particulate matter; protection
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from biocides and other chemicals; entrapment of molecules and nutrients; binding of cations, toxic metallic ions and other substances; and resistance to dessication. The polysaccharides and proteins form the structural elements of biofilms and determine their mechanical stability. The roles of the other components are not well established. At some time after attachment of cells to a surface, this attachment becomes irreversible. The cells grow and divide to form a microcolony. The microcolony enlarges, produces EPS, and coalesces so that it becomes anchored to the surface and stabilized. Microcolonies become protected from environmental stresses, and cells can show increased resistance to antimicrobial agents within a few hours of adhesion, even before they become embedded in the EPS matrix. Over time, cellular activity continues and the biofilm matures. Biofilms can be composed of different organisms or they can contain only one species. The organization of the cells ranges from a single layer to 3-D structures with the bacteria living within a large, complex, and organized ecosystem. There might be mechanisms whereby some species are encouraged to populate a biofilm, whereas attachment of other species is inhibited. In vitro studies show that multispecies biofilms are thicker and more stable than single species biofilms. The distribution of bacteria within biofilms is not even. Bacteria grow within the microcolonies that are surrounded by the EPS matrix and water channels are formed among them. These channels facilitate the transport of substrates, including oxygen, metabolites, and nutrients to the biofilm and within the biofilm; and also transport products, including waste products, out of the biofilm. The physiological state of biofilm bacteria appears to be different than that of planktonic bacteria. The combination of physiological modifications of biofilm-associated cells, including reduced growth rates and production of enzymes that can degrade antimicrobial substances, and physical protection provided by the biofilm matrix itself might be responsible for the extreme resistance to antimicrobials of biofilm bacteria. Part of the ‘life cycle’ of a biofilm is that damaged individuals are eliminated from the population. Programmed cell death and lysis can be a function of spatial orientation within the biofilm. Since deoxyribonucleic acid (DNA) is a component of the EPS and is thought to be related to the stability of the biofilm, the expectation is that cell lysis has a role in maintaining the stability of the biofilm structure. Biofilms are known to spread, but factors that control release of cells and detachment of sections of the biofilms are not well understood. They can release ‘pioneer’ cells or daughter cells individually, or there might be a sloughing or detachment of a relatively large part of the biofilm. When single cells or small clusters of cells detach, the effect on the biofilm is limited to the surface. When a large portion of a biofilm is sloughed off, the whole biofilm is impacted and may be lost.
Cellular Control of Biofilms – The Role of Cell-to-Cell Signaling Molecules and the Molecular Basis of Biofilm Formation The defining characteristic of biofilms is the formation of an integrated bacterial community. This requires self-organization
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Biofilm Formation
and cooperation among cells, rather than the classical ‘competitive’ natural selection of individual microorganisms. Bacteria, which are colonial by nature, must be able to sense environmental changes and respond to them. They must also communicate with each other. Bacteria modulate gene expression and have systems of intercellular interactions and communications that are currently the focus of much study. The molecular changes that occur in a bacterium when it changes from its planktonic form to become sessile (permanently attached to the substrate) can be very complex and are only beginning to be understood. For example, in Listeria monocytogenes, expression of many proteins has been shown to be upregulated, including some related to stress response, protein synthesis, carbon metabolism, and regulation. These diverse molecular shifts show that the central metabolism of L. monocytogenes is affected during biofilm development. A major area of interest is the role of quorum sensing in development and maintenance of biofilms. Cell-to-cell signaling is mediated by small, diffusible molecules called auto-inducers that are produced and secreted during bacterial growth. As the bacterial population in the biofilm increases, the concentration of these small molecules increases too. When they reach a threshold or quorum level, the cells respond to them, with a variety of phenotypic responses being displayed as a result of the regulation of target genes that are quorum-sensing dependent. Quorum sensing seems to be involved in all stages of biofilm formation and maintenance. Some of the biofilm-related processes that can be controlled by quorum sensing are the establishment of bacteria in a mixed biofilm community, survival in food processing environments that are hostile to bacteria, the production of surface appendages, and motility. Since the discovery that bacterial cells communicate is relatively recent, much information is still required to fully explain the role of quorum sensing in the activities of biofilm bacteria. There is particular interest in the possibility of controlling the development of biofilms through disruption of the quorum sensing system.
Studying Biofilms The study of biofilms in situ is extremely difficult for many reasons. As a result, most knowledge about biofilms has been obtained from in vitro studies. Such results might not be relevant to circumstances in the food industry. Many disinfection processes are designed using bacteria that are planktonic, which are more sensitive to antimicrobials than bacteria embedded in biofilms. Enumerating or determining the types of bacteria residing within biofilms can be difficult. It has been estimated that for each planktonic bacterial cell detected, another 1000 cells could be present within a biofilm. In food processing environments, surfaces are usually treated with antimicrobial chemicals during cleaning, might not provide a lot of nutrients, can be hot or cold and dry, and might be otherwise hostile environments for bacteria causing them to be stressed and/or injured and nonculturable. The types of bacteria within a biofilm and the environment in which they develop will determine its characteristics. Researchers cannot as yet wholly duplicate naturally occurring biofilms.
A variety of microscopic techniques including scanning electron microscopy, transmission electron microscopy, and laser scanning microscopy are the tools used to observe biofilms and to monitor their development under controlled conditions. One instrument being studied as a way to monitor biofilm formation online is the mechatronic surface sensor. This type of sensor is based on the analysis of the vibration response of the surface and can detect biological and chemical characteristics of a variety of surfaces such as stainless steel and polyvinyl chloride.
Biofilms in the Food Industry The major focus of research on biofilms has been in relation to medical matters, but they are also very important in many industries, including the food industry. The bacteria in biofilms can cause food spoilage, compromise food safety and affect performance, and longevity of industrial equipment. Several pathogenic and spoilage bacteria including L. monocytogenes, Salmonella spp., Campylobacter spp., E. coli, Pseudomonas spp., and lactic acid bacteria have been associated with biofilm formation. In the meat and dairy industries, bacteria, including species of Pseudomonas, Staphylococcus, Enterobacter, Flavobacterium, yeasts, and Kluyvera were recovered from biofilms associated with clean floor materials. L. monocytogenes will form biofilms on floor drain surfaces, in storage tanks, on hand trucks, on conveyor belts, and on other food contact surfaces; and individual strains have been shown to persist in food plants for several years. Bacteria in biofilms can catalyze chemical and biological reactions to cause corrosion in pipelines and tanks, and if the biofilms are allowed to become thick, the heat transfer in heat exchangers and pipelines can be reduced. Equipment and processing plant design, cleaning, and disinfection regimens are the major tools available to the food industry to control the development of biofilms. Surfaces in meat processing facilities are frequently wet and biofilms can quickly form under wet conditions. Stainless steel, the material used for many food contact surfaces, is chemically and physiologically stable over a range of temperatures, and it is easy to clean. When the microtopographies of stainless steel surfaces are examined, they are seen to have many cracks and crevices that are good locations for bacteria to become attached. All areas of food processing environments including floors, walls, pipes, drains, conveyer belts, gaskets, and dead spaces are prone to biofilm formation. Similarly, materials that are commonly used in food processing facilities such as stainless steel, aluminum, nylon, Teflon, rubber, and plastic can become colonized by biofilms. The development and structure of biofilms depend on many factors, both intrinsic and extrinsic, including the species of bacteria in the consortium, the temperature, pH and nutrient status of the environment, and the flow conditions of fluids that contact surfaces. In the food industry, because cleaning and disinfection are frequent, mature biofilm structures or continuous bacterial films are not often observed on equipment surfaces; however, microcolony development with or without EPS is common. Whether these attached bacteria constitute a biofilm is debated. Although not showing the
Biofilm Formation
typical characteristics of biofilms, these microcolonies are important to the food industry because they can exhibit increased resistance to antimicrobial treatments within only a few hours of establishment; and cleaning and disinfection regimens must be designed to remove them completely. It is hypothesized that this resistance might, in part at least, result from a reduction in surface area exposed to antimicrobial treatment as a result of the attached surfaces not being in contact with the applied chemicals. Further research is required in this area, with particular emphasis on contamination of damp surfaces in food processing areas. In hard to access areas and areas that are infrequently cleaned, mature biofilms, with microbial cluster embedded in EPS and water channels, are observed. The best approach to control biofilm development is prevention of their establishment. Equipment should be designed so that areas that are hard to access and clean and areas that allow bacteria to accumulate, such as dead ends, corners, valves and joints, are avoided. Good water drainage should be assured and proper attention should be paid to the welding, material, and surface finishing on both exposed and nonexposed surfaces. All surfaces should be nonporous and smooth, without pits or crevices. Glass is a hard, smooth, and corrosion-resistant material but its applications are limited since it breaks easily. Stainless steel is easy to clean but the surface is easily damaged. Rubber surfaces deteriorate. Surfaces in food establishments must be cleaned, but regimens that cause corrosion and produce topographical defects should be avoided. Defects and roughness of surfaces have greater effect on the ease with which a surface can be cleaned than the type of finish. Since biofilm development occurs when moisture is present, maintenance of dry conditions will aid in prevention of their establishment. Even with proper equipment design and attention to surface characteristics and integrity, the likelihood of preventing biofilm development is slim. Cleaning and disinfection programs necessarily remain the main strategy for controlling surface contamination. Since bacterial adhesion is initiated soon after a surface is conditioned, regular cleaning and disinfection at short intervals are required to prevent firm bacterial attachment to surfaces and the formation of mixed species biofilms. Cleaning at short intervals will help to prevent sporulation by spore-forming bacteria, which is of particular importance to the dairy industry, but might become important to the meat industry as the involvement of psychrophilic (cold-loving) and pathogenic clostridia in meat spoilage becomes more common. One major factor militating against elimination of biofilms is that they can be reseeded with a very small number of cells. It is almost impossible to remove all cells associated with a biofilm. Pouring liquid onto a surface will erode a biofilm but a negligible number of the biofilm cells will be detached. Any cleaning and sanitation strategy must ensure that the biofilm bacteria are dead, not just dislodged. Surfaces should be cleaned prior to sanitation and cleaning processes must break up or dissolve the EPS so that sanitizers can make contact with the viable bacteria within the biofilm. Some materials used for cleaning are surfactants or alkaline products that suspend or dissolve food residues by decreasing surface tension, emulsification of fat, and denaturation of proteins. Acid products can
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also be used if the surfaces are coated with precipitated minerals or much food residue. If it is practicable, exposure of the biofilm to high temperature can reduce the requirements for physical cleaning. The type of cleaning process must not cause aerosols. For example, when mechanical force is applied using scrubbing and brushing, or pulsed laser beams are used, bacteria can become airborne. Such processes, including use of high pressure sprays, are being replaced by foam or gel cleaning. Bacterial cells within biofilms are highly resistant to antimicrobials. Even after a surface is cleaned, ridding the surface of viable cells is difficult. There are several mechanisms that have either been shown or are hypothesized to cause the elevated resistance of biofilm bacteria. Penetration of antimicrobials into the biofilm can be slow or the antimicrobial can be neutralized in the outer layers of the biofilm. The rate of diffusion might be slower than the rate at which the antimicrobial is inactivated, as is observed with resistance of biofilms to chlorine. The growth rate of bacteria deep in the biofilm is decreased and cells are in a ‘quasi-dormant’ state in which their resistance to biocides is increased. Older biofilms are more resistant to biocides than are younger ones. Genes controlling adaptive stress responses are expressed and biofilm cells are able to sense challenges from antimicrobials. Salmonella enteritidis isolated from biofilms had increased resistance to heat and chemicals when compared to planktonic cells. Repeated exposure of biofilm cells to antibiotics was shown to cause an increase in EPS synthesis in the biofilm. Cells, termed persister cells, that are phenotypically variant, can develop. These cells do not grow and do not die when challenged with antimicrobials. In the medical field, such cells are considered to be largely responsible for persistent infections resulting from biofilms. Their resistance is explained by their production of cellular toxins that block cellular processes such as translation and, so, render the cells resistant. Prevention and control of biofilms are topics of active research. At this time, there is no means known of preventing or controlling the development of biofilms without adverse side effects. Almost all materials will support the formation of biofilms. Some current efforts are focussed on the possibility of incorporating antimicrobial agents into either the construction materials themselves or surface coatings. Much work has been concerned with biomedical applications, but application in the food industry will likely follow and has been the focus of some studies. Laboratory studies in which fungicides are incorporated into flooring materials have been reported, but no results of long-term studies have been published. Covalent coupling of silicone rubber implants with quaternary ammonium coatings, coating surfaces with silver, preconditioning surfaces with surfactants and adsorption of nisin onto food contact surfaces are all in the experimental phase. In food processing, the importance of the ‘house microflora’ and the possibility that the presence of bacteriocin-producing bacteria and other endogenous strains might influence the establishment of biofilms are being studied. Novel approaches to prevention, removal, and inactivation of biofilm bacteria include the development of enzyme-based cleaners; various combinations of enzymes, detergents, surfactants, and phenolic antimicrobials; use of bacteriophages; interruption of quorum sensing; and manipulation of nutrient availability.
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Biofilm Formation
The formation of biofilms is a unique, complex, and important biological phenomenon that enables many bacteria to survive and grow in interactive communities and in environments that are hostile to them. Biofilms can be advantageous, but in many parts of the food processing industry, they are considered to be biofouling agents responsible for problems with food spoilage, food safety, deterioration of equipment, and compromised efficiency of equipment. Our knowledge of the mechanisms that contribute to the formation, development and maintenance of biofilms is limited and improved control over biofilms will depend on significant advances in the field.
See also: Microbial Contamination: Decontamination of Processed Meat. Equipment Cleaning. Microbial Contamination: Decontamination of Fresh Meat; Microbial Contamination of Fresh Meat; Microbial Contamination of Processed Meat. Packaging: Technology and Films
Gandhi, M., Chikindas, M.L., 2007. Listeria: A foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15. Kumar, C.G., Anand, S.K., 1998. Significance of microbial biofilms in the food industry: A review. International Journal of Food Microbiology 42, 9–27. Marchand, S., De Block, J., De Jonghe, V., et al., 2012. Biofilm formation in milk production and processing environments; Influence on milk quality and safety. Comprehensive Reviews in Food Science and Food Safety 11, 133–147. Mettler, E., Carpentier, B., 1998. Variations over time of microbial load and physicochemical properties of floor materials after cleaning in food industry. Journal of Food Protection 61, 57–65. Shi, X., Zhu, X., 2009. Biofilm formation and food safety in food industries. Trends in Food Science and Technology 20, 407–413. Simões, M., Simões, L.C., Vieira, M.J., 2010. A review of current and emergent biofilm control strategies. LWT − Food Science and Technology 43, 573–583. Stoodley, P., Saur, K., Davies, D.G., Costerton, J.W., 2002. Biofilms as complex differentiated communities. Annual Reviews in Microbiology 56, 187–209. Van Houdt, R., Michiels, C.W., 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface. Journal of Applied Microbiology 109, 1117–1131. Zotolla, E.A., Sasahara, K.C., 1994. Microbial biofilms in the food processing industry − Should they be a concern? International Journal of Food Microbiology 23, 125–148.
Relevant Websites Further Reading Annous, B.A., Fratamico, P.M., Smith, J.L., 2009. Quorum sensing in biofilms: Why bacteria behave the way they do. Journal of Food Science 74, R24–R38. Bai, A.J., Rai, V.R., 2011. Bacterial quorum sensing and food industry. Comprehensive Reviews in Food Science and Food Safety 10, 184–194. Bryers, J.D., Ratner, J.P., 2004. Bioinspired implant material befuddle bacteria. American Society of Microbiology News 70, 232–237. Carpentier, B., Cerf, O., 1993. Biofilms and their consequences with particular references to hygiene in the food industry. Journal of Applied Bacteriology 75, 499–511.
http://www.ift.org/~/media/Knowledge%20Center/Science%20Reports/Scientific% 20Status%20Summaries/Editorial/editorial_0209_feat_biofilms.pdf Institute of Food Technologists. https://www.google.ca/search?q=biofilm+formation&oq=biofilm+formation &aqs=chrome..69i57j0l5.4979j0j8&sourceid=chrome&espv=210&es_sm= 122&ie=UTF-8#q=biofilm+formation+and+food+safety Manitoba Agriculture, Food and Rural Development.