Microbial attachment to surfaces including natural environments, water distribution systems, processing plant equipment, food contact surfaces, body implants, ...
SANITATION: SIGNIFICANCE OF BIOFll.MS*
Constantin Genigeorgis** . Department of Food Hygiene and Technology, Veterinary School Aristotle University, 54006, Thessaloniki, Greece and Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, 95616, USA
Microbial attachment to surfaces including natural environments, water distribution systems, processing plant equipment, food contact surfaces, body implants, foods of animal origin, and intestinal mucosa, have been studied extensively. Theories for the mechanism of attachment have been proposed and use of scanning electron and epifluorescent microscopy have improved our understanding of this mechanism. After a reversible non-specific absorption to the surface, microorganisms (MO) attach in a non-reversible specific way leading to the formation of mostly heterogeneous and complex colonies known as biofilms. These biofilms are difficult to remove. In addition to effects on the surfaces, biofilms--through cell sloughing--may become for the food industries, a product contamination problem: during and after processing there may be spoilage and pathogenic MO. Laboratory studies have shown the attachment of such MO to materials used for food processing equipment and food contact surfaces, and have identified parameters affecting attachment. Our knowledge is limited with respect to biofilm formation under field conditions in food processing plants. Cells in biofilms have an increased resistance to heating and to sanitizing agents. Use of detergents, followed by sanitizing agents, seems to control microbial attachment to food contact surfaces while non-food contact surfaces are not as cleanable.
Keywords: biofilms, attachment, adherence, cleaning and sanitizing
INTRODUCTION Microbial attachment and growth on surfaces and the implications of such growth, is of significant concern to water distribution agencies, municipal utilities, food production and food industries, and to medicine (Costerton and Lappin-Scott, 1989; Characklis and Marshall, 1990; Notermans et aI., 1991; Mattila-Sandholm and Wirtanen, 1992; Melo et aI., 1992; Marshall, 1992; Anwar and Costerton, 1992; Zottola and Sasahara, 1994). International statistics indicate (Mead et aI., 1990; Chaffey et aI., 1991; Farber and Peterkin., 1991; Chaffey et aI., 1991; Rocourt, 1994; Bryan and Doyle, 1995) that, with few exceptions (Ternstrom and Molin., 1987) for certain MO, there is an extensive level of contamination of raw meat, especially poultry meat, with important human pathogens like Salmonella, Campylobacter, Yersinia enterocolitica, Staphylococcus aureus and Listeria. This situation reflects the failure of the meat slaughtering industry to prevent environmental and product contamination or to use an effective and legal decontamination treatment.
*Proceedings, International Course on: Production of Processed Meats and Convenience Foods. Building a Predictable Safety. Athens, June 25-26, 1995. ECCEAMST, Utrecht. ** Professor Emeritus, University of California, Davis 1
Contaminated raw meats may pose an increased risk for foodborne disease either as a result of product under-cooking or through contamination of equipment, food contact surfaces and foods after processing. A better understanding of the mechanisms involved in the attachment and detachment of MO from food contact and non-food contact surfaces in processing plants and meat surfaces is of significance to the development of procedures which will minimize microbial presence in the plant environment and raw muscle tissues, thus enhancing product self-life and decreasing potential public health risks. In this review I will discuss some of the basic concepts concerning microbial attachment and colonization of surfaces in general; then I will focus on attachment and colonization of food contact surfaces and raw meats; I will discuss the parameters affecting these phenomena, and finally I will discuss the control of attachment and biofilm development. The subject of microbial attachment to the mucosa of the intestinal tract, which is so important to colonization, invasiveness, and control of enteric pathogens (Krogfelt, 1991; Hentges, 1992), is beyond the scope of this review and it will not be discussed. Also the subject of meat decontamination which has been evaluated extensively (Dickson and Anderson, 1992; Lillard, 1994; Kim et al., 1994; Jericho et al., 1995) will not be reviewed here. BACTERIAL ADHESION According to Marshall (1992) biofilms consist of microorganisms (MO) immobilized at substratum surface, typically embedded in an organic polymer matrix of bacterial origin. Such biofilms are ubiquitous in flowing aqueous environments, are not necessarily uniform in time and space and may trap inorganic substances within the polymer matrix. Mature biofilms in such environments require 2 to 4 weeks of growth to develop and have been termed by Zottola and Sasahara(1994) as classical biofilms. Most bacteria in natural habitats can exist in two distinct physical environments i) the planktonic state, whereby they function as individuals, and ii) the sessile state, whereby they attach to surfaces, form biofilms, and function as a closely integrated community. Low population densities are characteristic of most but not all, planktonic communities in natural microbial ecosystems. In these environments available nutrients are insufficient to support extensive microbial activity and most MO merely survive in a starved state. Often starvation is accompanied by a marked reduction in size and endogenous respiration and increased cell surface, hydrophobisity and an increase in adhesiveness. These factors make MO more prone to adhere to solid surfaces where they benefit from an enhanced nutrient status. In flowing systems attached MO gain access to a continual source of nutrients being carried past them by the flowing water. After attaching to a surface, MO grow to normal size and then begin cellular reproduction. Continued adhesion and growth lead to biofilm formation. Depending on their location within a biofilm individual MO can suffer intense competition for nutrients, oxygen and possibly space. On the other hand MO deep within a biofilm are protected from various antimicrobial substances (antibiotics, chlorine etc.,). In addition, some populations within a biofilm benefit from the metabolic by-products of other populations. A number of theories have been proposed to explain the mechanism of microbial attachment to surfaces. Attachment occurs in two steps (Marshall et al., 1971), three steps (Busscher and Weerkamp, 1987; Notermans et al., 1991), and five steps (Charachlis and Cooksey, 1983; Lawrence at al., 1987). The Marshall et al., (1971) two step theory for microbial attachment and biofilm formation assumes a first reversible stage of cell association near, but
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not in actual contact with, the substratum. If allowed to remain associated with the substratum, the cells eventually synthesize exopolymeric substances (EPS) that exude from the cell surface and directly bind the cell to the substratum in an irreversible way. The exact phenomena occurring between the cells and the substratum are described in further detail in the three-step theory of Busscher and Weerkamp (1987). When mostly negatively charged cells are at distances greater than 50nm from mostly negatively charged surfaces, attractive Van der Walls forces operate. At distances of 10 to 20nm in addition to Van der Walls forces repulsive electrostatic forces operate. Adhesion in this stage is envisioned to be reversible. At separation distances less than 1.5nm where the potential energy barrier has been overcome, specific interactions can take place provided that the organism is capable of extruding adhesion probes and hydrophobic groups are available. Physical parameters such as fluid flow rate, charge, hydrophobicity, and microtopography of substratum affect the degree to which cells are associated with the substratum. In order to get closer to the substratum, the cells must overcome a high energy repulsion barrier which is affected by the surface area of a cell (Van Loosdrecht et aI., 1989). Therefore a MO with surface protrusions such as pili or fimbriae or flagellae with very small diameters and presence of hydrophobic groups could conceivably overcome the repulsion barrier and assist the cell in adhering to the surface where microcolony and biofilm growth begins (Busscher and Weerkamp, 1987; Van Loosdrecht et aI., 1989; Zottola and Sasahara, 1994). Development of thread-like structures (protrusions) by bacteria in the process of attaching to surfaces such as stainless steel and the formation of biofilms have been demonstrated by electron microscopy (Zottola and Sasahara, 1994). Hydrophobic cells have been shown to adhere to a greater extent than hydrophilic cells to sulfate polystyrene (Van Loosdrecht et aI., 1987). Production of EPS, which are not subject to the same degree of repulsion as the cells, can form a bridge between the bacterium and the surface by various combinations of chemical bonding and assist in cell attachment to the surface. Substratum charge has a role in bacterial attachment. Maximum cell adhesion occurs on highly charged substrates like glass and less on lower charge substrata like polystyrene (Zottola and Sasahara, 1994). Furthermore "conditioning" of the substratum surface may affect the efficiency of bacterial attachment. Conditioning is a process which starts within minutes of substratum immersion into liquids and implies the adsorption onto the surface of organic macromolecules and other low-molecular-weight hydrophobic molecules. The conditioning films alter both the charges and free energy of the surface and thus may influence microbial attachment. In the food processing industry conditioning films may be proteins from milk and meat. Meat juice has been shown to reduce the negative charge of clean stainless steel surfaces. The reduced charge would improve the interaction with the negative charged bacterium and demonstrates the enhanced potential for microbial accumulation in food processing plants (Zottola and Sasahara, 1994). The role of cell hydrophobicity and surface charge to microbial attachment to surfaces has also been explored (Gilbert et aI., 1991; Mafu et aI., 1991a,b; Zottola and Sasahara, 1994). Cell hydrophobicity is considered to have a key role to non-specific microbial attachment. Yet hydrophobicity does not consistently predict the attachment of MO, nor does correlation with cell surface charge always apply (Vanhaecke et aI., 1990). Cell hydrophobicity and surface charge vary (Mafu et aI., 1991 a,b) with growth phase, ionic strength and pH. With respect to L. monocytogenes, Mafu et aI., (1991a) concluded that factors other than cell surface such as surface charge and presence of EPS may be of importance in the adhesion of this MO to stainless steel, glass, polypropylene and rubber surfaces. Overall MO adhere at
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different rates to a range of substrata. Some bacteria already possess necessary attachment structures (EPS, fimbriae, etc.) and immediately attach firmly to surfaces. In such instances, the adhesion is purely physiochemical and has been termed passive adhesion (Marshall, 1992). Other bacteria require prolonged exposure to attach firmly to surface, indicating some physiological response. This time-dependent process is termed active adhesion. During active adhesion, bacteria initially are attracted to the surface but do not firmly attach. In this stage of reversible adhesion, the bacteria still show Brownian motion and can be removed from the surface by a moderate shear force. Irreversible adhesion on the other hand is characterized by a lack of Brownian motion by the bacteria and an inability to remove them from the surface by a moderate shear force (Marshall, 1992). SURFACE COLONIZATION The colonization of surfaces depends on the mixtures of bacterial species present in the aqueous phase. Even when a single culture of bacteria is in contact with a uniform surface, adhesion introduces some heterogeneity, as the cells may adhere to uncolonized areas or may join colonized areas. When more than one species is present, colonization patterns may differ, as each species may react differently to the surface. Only one species may adhere or one species may act as a primary colonizer and attract cells of the same or different species. At the beginning adhesion sites are randomly located on a uniform surface. Once colonization commences, these sites begin to differ because of bacterial activity. Surface growth, replication microcolony formation, and production of the biofilm matrix (glycocalyx), all increase the heterogeneity of the attachment sites and this further increases as a confluent biofilm forms. The composition and properties of the biofilm matrix depend on the bacterial inhabitants. Production of EPS material by some species may bind metal ions and thus alter the biofilm nature. Metabolic products such as organic acids can become trapped within the biofilm, sometimes leading to marked differences in adjacent microcolonies or even affect helping microbial heterogeneity and even affecting profoundly the surface by corrosion or digestion. Biofilms capture inorganic and organic molecules from the bulk liquid, making these available as nutrients for the growth of MO within the biofilm. The glycocalyx holds cells within the biofilm together ensuring an exchange of metabolites to sustain growth, allowing the removal of toxins deadly to some of the inhabitants, and encouraging the combination of enzymatic capabilities from several bacteria in a consortium to degrade growth substrates. As multi-layers of bacteria are being formed and embedded in a polymer matrix, diffusion becomes a major factor in determining the microbial community structure within the biofilm. An oxygen gradient soon develops as a result of rapid utilization by aerobic bacteria at or near the water biofilm interface. The lack of oxygen diffusion at any significant depth of the biofilm leads to formation of an anoxic zone where anaerobic conditions prevail with fermentative bacteria particularly active. These bacteria form low-molecular-weight organic and fatty acids, as well as carbon dioxide and hydrogen products that encourage growth of sulfate-reducing-bacteria which may lead to metal corrosion problems (Marshall, 1992).
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CONSEQUENCES OF BIOFILM FORMATION
Biofilm formation on surfaces is of special interest to industry and to medicine. The consequences have been reviewed (Notennans et al., 1991; Mattila-Sandholm and Wirtanen., 1992; Zottola and Sasahara., 1994) and will be summarized here briefly. Biofilms can cause energy losses and blockages in power, municipal utilities, and chemical process industry because of their growth on condenser tubes, cooling tower materials, water and waste water circuits, and heat exchangers. Biofilm formation on ship hulls results in increased fuel consumption. Furthermore, due to processes in the lower layers, biofilms may accelerate material deterioration and metal corrosion in the above industrial settings. Biofilm formation by pathogens like Legionella in cooling towers and air-conditioning systems may present public health risks because of aerosol formation by detached cell aggregates. The adherence of microbial cells to surfaces in piping and water distribution systems is well documented (LeChevallier et al., 1987; Mattila-Sandholm and Wirtanen, 1992). Biofilm formation appears in lavatories, sinks, valves, and different joint areas throughout the piping system. Biofilms have been found also in swimming pools. Microbes such as Pseudomonas attach easily to surfaces of hydrophobic materials such as polystyrene. Hoses, tubes, filters, etc., containing polyvinyl-chloride increase the risks of contamination (Mattila- Sandholm and Wirtanen, 1992). Dental plaques are typical forms of biofilms. Biofilms of pathogenic MO (Staphylococcus or Pseudomonas) may be formed on foreign-body-instruments like cardiac pacemakers, catheters, artificial valves, etc. ATTACHMENT OF MICROORGANISMS TO FOOD CONTACT SURFACES
The potential attachment of MO to food contact surfaces and generally to food processing equipment and plant structures is of great interest to the food industry. This is due to the following reasons:) there is the creation of a reservoir of spoilage and pathogenic MO in the structure of the biofilm from where continuous contamination of the food through cell sloughing during and after processing may occur with unfortunate consequences; ii) studies have shown that MO in biofilms are by far more resistant to sanitizers and hard to clean (Schwach and Zottola, 1984; Frank and Koffi, 1990; Wirtanen and Mattila-Sandholm, 1992); iii) formation of biofilms in heat exchangers like those for product pasteurization may result in under-processing; iv) potential instrument and equipment corrosion. Scanning electron microscopy, epifluorescence microcsopy, interference reflection microscopy, and scanning laser confocal microscopy have permitted the accumulation of important information in recent years (Holah et al., 1988; Wirtanen and Mattila-Sandholm., 1993). Zoltai et al., (1981) were one of the first to show that P. fragi was able to attach to the surfaces of stainless steel chips and glass microscopic cover slips placed into actively growing cultures. They also demonstrated that as contact time with the surfaces increased, the degree of attachment, the number of cells and the size of the micro-colony increased. Attachment was assisted by appendages extending from the cells to the inert surfaces and to other cells.
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Attachment of P. aeruginosa to stainless steel was also shown by Stanley (1983). Stone and Zottola(1985) demonstrated the attachment of P. fragi to stainless steel in a constantly moving milk flow. P. fragi produced fimbriae within 30 minutes at 25°C and 2 hr. at 4°C. Herald and Zottola (1988a,b) demonstrated that and Yersinia enterocolitica attached to stainless steel under a variety of pH conditions, but most efficiently at pH 8. Under the same conditions greater numbers of P. fragi attached than the two pathogens. Production of EPS exuding from the surfaces of P. fragi cells during attachment was shown (Herald and Zottola, 1988c).Herald and Zottola (1988b,c) demonstrated the effects of pH and temperature of the attachment of L. monocytogenes and Y. enterocolitica. Mafu et al., (1990) demonstrated the attachment of L. monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces within 20 minutes of contact. Attachment to cast iron (Spurlock and Zottola, 1991) was not accomplished as readily as was attachment to stainless steel. Work done by Krysinski et al., (1992) also demonstrated the attachment of L. monocytogenes to stainless steel and polypropylene. Hood and Zottola (1992) sampled for biofilms throughout different meat processing plants by placing stainless steel and cast iron chips in several locations where biofilm formation may occur. Biofilms were formed on chips placed on floor drains but were not formed on chips placed on food contact surfaces. Gram positive and gram negative MO were isolated from the floor drains and food contact surfaces. Sasahara and Zotttola (1993) reported that when P. fragi and L. monocytogenes grow together they form a more complex biofilm on glass and stainless steel than when either is growing independently. P. fragi appeared to be the predominate glycocalyx former and was present in greater numbers in the biofilm. Jeong and Frank (1993) demonstrated the ability of L. monocytogenes to grow in multi species biofilms formed on stainless steel at 10°C in the presence of bacteria isolated from dairy and meat plant environments. Abrishami et al., (1994) evaluated the adherence and viability of E. coli inoculated onto the surfaces of plastic cutting boards and new and old wooden boards. The studies corroborated the anecdotal concept that bacteria are better retained on wood than plastic cutting board surfaces. A higher percentage of cells were retained by dry wood than water conditioned wood surface and from new woods than used ones. Bacteria adhering to wood surfaces resided within the structural and vegetative elements of the wood's xylem tissues and were alive. Bacteria that adhere to plastic surfaces were more easily removed by lowtemperature washing than were cells that adhered to wood surfaces. The attachment of MO to milk processing equipment has been evaluated. Bouman et al., (1982) found more thermos-resistant S. thermophilus cells adhering to heat exchangers on the pasteurized milk effluent side than where raw milk entered. S. thermophilus remained attached in high numbers even when milk flow rate was 100kglh. In contrast, Czechowski (1990) noted that more MO were observed attached to the raw milk side of milk processing equipment. Suarez et al., (1992) investigated the attachment of MO, isolated from milk, to stainless steel, glass and rubber. They found that the bacteria showing the greatest degree of attachment to substratum were inhibited from attaching in the presence of milk. Their results caution against the extrapolation of data from bacteriological media to food systems (Zottola and Sasahara, 1994).
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PARAMETERS AFFECTING MICROBIAL ATTACHMENT TO INERT SURFACES A better understanding of what enhances or discourages MO from forming biofilms is of significance to any effort to minimize the problem for the food industry. Numerous reports addressed this subject. Generally the consolidation (Notermans et al., 1991) of the attachment to a surface is dependent on a number of parameters. Attachment is a time-dependent process and the number of cells becoming attached is proportional to the initial concentration (Chung et al., 1989 in Notermans et al., 1991). Other factors include pH (Herald and Zottola 1988b), temperature (Lewis et al., 1987; Herald and Zottola, 1988a,b), cell surface charge (Fletcher and Loeb, 1979), cell hydrophobicity (Dahlback et al., 1981), bacterial motility (Stanley, 1983), bacterial structures, including the production of extracellular polysaccharides and flagella (Notermans and Kampelmacher., 1974), the ionic concentration at the interface (Stanley, 1983; Roller, 1991), the growth phase of the organism (Stone and Zottola, 1985; Rolle, 1991), the nature of the medium where the planktonic cells originate including nutrient content (Marshal, 1992; Zottola and Sasahara, 1994; Kim and Frank, 1995), the nature of the collecting surface (Fletcher and Loeb, 1979; Marshall, 1992; Helke et al., 1993), and the nature of compounds which might be on the collecting surface (Al-Makhlafi et al., 1994). Evidence suggests that the conditions of culture influence the attachment process (Notermans et al., 1980). When, for example, the feces of chickens given certain bacterial strains were used experimentally, higher rates of attachment were observed for these organisms than with the same strains grown in a laboratory medium. There is disagreement over the role of surface structures, since non-fimbriated and nonflagellated cells have been reported to attach at rates similar to those of cells which possess these structures. However, other reports indicated that motile bacteria attach to surfaces more rapidly than non-motile strains (Dickson and Koohmaraie, 1989). Stanley (1983) demonstrated that the attachment of P. aeruginosa to stainless steel depended on the pH, ionic strength and dynamic environment. Herald and Zottola (1988a,b) found that the formation of fibers or fibrils during the attachment process of L. monocytogenes and Y. enterocolitica was affected by temperature and pH. Roller (1991) demonstrated the significance of sodium chloride concentration and phase of growth on the attachment of P. fragi to polystyrene. The interactive effect of L. monocytogenes and P. fragi on their concurrent attachment to glass and stainless steel (Sasahara and Zottola, 1993) has been mentioned before. Cell hydrophobicity has been considered as a major determining factor for the attachment of cells to negatively charged polystyrene (Van Loosdrecht et al., 1987). However as the relative hydrophobicity decreased, electrophoretic mobility had more influence on attachment. Immediately upon immersion of a solid substratum into an aquatic environment, macromolecules and low molecular weight hydrophobic molecules from the aqueous phase begin to adsorb to the surfaces to form conditioning films. These films alter both the charge and the free energy of the surface which in turn modify the efficiency of bacterial adhesion to the surface in question (Marshall, 1992). Research in water distribution systems showed that biofilm formation is enhanced under low nutrient conditions. The reason for the enhanced attachment is attributed to the increased concentration of nutrients at the substratum surface rather than in the bulk fluid and bacteria sense this concentration gradient and migrate toward the surface.(Zottola and Sasahara, 1994). Often starvation of cells is marked by cell size reduction, an increase in cell hydrophobicity, and an increase in adhesiveness (Marshall, 1992; Zottola and Sasahara, 1994). Not all biofilm formation occurs under such conditions. Wolfaardt
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and Cloete (1992) reported increased microbial attachment to stainless steel, aluminum and glass in flowing systems with increased dissolved organic carbon content. After adhesion to the surface, starved bacteria grow to normal size and then begin cellular reproduction (Marshall, 1992). Organic molecules in conditioning films represent a potential source of energy for the bacteria attaching to the substratum. Sasahara and Zottola (1993) found that L. monocytogenes in a continuous flowing system did not attach to glass cover slips when cells were cultured in both nutrient -rich and nutrient-limited conditions. This was an interesting observation since glass is a highly charged substratum that readily adsorbs dissolved solutes, which then act as conditioning film upon which a bacterium should attach (Fletcher, 1992). In the food industry, and especially the meat industry the high level of organic material remaining on food contact surfaces could result in a conditioning film onto which MO become entrapped. The adsorption of organic materials to a surface can alter the characteristics of the surface and subsequently promote or inhibit microbial attachment. Speers and Gilmour (1985) documented differences in attachment of several common milk-borne MO to stainless steel and rubber surfaces in the presence of milk and milk components. Helke et al., (1993) reported that the attachment of L. monocytogenes and S. typhimurium to stainless steel and Buna-n rubber in the presence of milk and individual milk components was significantly inhibited. In contrast Al-Makhlafi et al., (1994) found that attachment of L. monocytogenes to hydrophobic and hydrophilic silica surfaces varied with the nature of pre-adsorbed milk proteins. Production of EPS, which is important to attachment, depends on the organism, the nature of the collecting surface, and the interface (Fletcher and Loeb, 1979). Scanning electron photography has revealed the micro-topography of many of the materials used in the construction of equipment for the food industry and food contact surfaces including stainless steel, Teflon, rubber, aluminum, tile and concrete flooring. Teflon and tile surfaces were shown (Notermans et al., 1991) to be smooth while stainless steel surface had channels and crevices the width of an E. coli cell, whereas the aluminum surface had large channels and a sponge like appearance. Such surfaces reveal the potential entrapment and the difficulty in removing MO. Organisms held in this fashion have an opportunity to become firmly attached to the collecting surface and produce extracellular polymers. By being protected from routine cleaning and disinfection of the processing plant, the organisms may become indigenous (Notermans et al., 1991; Mead and Dodds, 1990). ATTACHMENT OF MICROORGANISMS TO MEAT AND POULTRY The attachment of MO to meat and poultry subject has attracted scientific interest and has been reviewed (Firtenberg-Eden, 1981; Benedict, 1988; Lillard, 1989; Selgas et al., 1994). Brief comments will be made in this review. The theories about the mechanism of microbial attachment to surfaces and the parameters which may affect the attachment and biofilm formation have already been discussed. With respect to the attachment of MO to meat surfaces the picture is not very clear. As early as 1974 Notermans and Kampelmacher evaluated the variables affecting the attachment of MO to the skin of broiler chickens. The literature on the attachment of bacteria to meat surfaces was reviewed by Firstenberg-Eden in 1981. The issue of whether motility ofMO, and pH and temperature of the attaching bath played an important role to the attachment was not clarified. Most authors agreed that a direct relationship existed between numbers of MO in the
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attachment medium and the extend of bacterial attachment and that higher populations enhanced attachment. Schwach and Zottola (1982) demonstrated the attachment of P. fragi to beef. They showed that the MO appeared to be entrapped within the collagen fibers of the raw meat and could be transferred to a clean stainless steel surface. The finding verified how easily food contact surfaces can become contaminated. Me Meekin et al., (1984) and Campbell et al., (1987) confirmed the adhesion of Salmonella, fimbriated strains of E. coli and a strain of Campylobacter to collagen fibers of chicken muscle fascia when the tissues and the cells were suspended in media of low ionic strength. Saline suspension fluids inhibited attachment and adherent cells were removed by rinsing the tissue in saline media. Attachment occurred only when the cells were suspended in distilled water. Farber and Idziak (1984) found minimal competition for attachment when two different MO out of seven tested were in contact with longissimus dorsi muscle and faster rates of attachment by motile MO. Lillard (1985) reported a linear increase in the rate of attachment of MO with time following exposure of poultry skin to suspending medium and that non-flagellated MO attach as readily as flagellated. In a later communication (Lillard, 1988) she reported that ionic strength had little effect on Salmonella attachment to poultry skin or muscle and assumed that electrostatic attraction between tissue and bacterial cells was not the main mechanism of attachment. The potential of Salmonella firmly attached to poultry skin to detach and transfer to stainless steel surfaces was shown (Carson et al., 1987). Lillard (1989) review her overall findings with respect to the factors affecting the persistence of Salmonella during processing of poultry and compared them to findings reported by others. She reported that neither fimbriae, flagella nor electrostatic attraction seem to playa significant role in the mechanism of attachment. Electron microscopy demonstrated the entrapment of MO to ridges and crevices which became more pronounced in the skin and muscle following water immersion. It was also shown that MO are firmly attached to poultry skin before the broilers arrived at the plant and that high numbers were still recovered after 40 consecutive whole carcass rinses of a single carcass. Chung et al., (1989) evaluated the attachment of six species of bacteria including L. monocytogenes to beef. They reported that the number of cells attached was proportional to the initial cell concentration. Nearly all bacteria attached better to lean meat than to fat at ambient temperature for 72h. No significant competition for attachment between any two species of bacteria was seen. Dickson and Koohmaraie (1989) reported a linear correlation between the negative charge on bacterial cell surface and the initial attachment to beef lean muscle and fat tissue. The relative hydrophobicity of each bacterium tested was dependent on the specific method of determination, with wide variations noted among the methods. Transfer of L. monocytogenes and S. typhimurium between beef tissue surfaces was shown by Dickson (1990). Sanderson et al., (1991) explored the molecular basis of Salmonella attachment to chicken muscle fascia. Their findings suggested that Salmonella attached to fascia through an interaction with hyalunan a component of the glycosamine-glycans matrix of the chicken skin. The interaction was not due to ionic or hydrophobic bonding but probably to a specific ligand receptor reaction. The possibility of cell outer proteins participating in the attachment was explored and suspected. Based on changes of skin tissue micro-topography induced by water take up, Thomas and Mc Meekin (1991) concluded that retention of Salmonella is probably a function of the amount of water bound by the connective tissue of the skin poultry processing. The adhesion of Staphylococcus aureus and its importance poultry processing was reviewed by Mead and Dodd (1990) and Chaffey et al., (1991). The authors emphasized the significance of certain strains to produce extracellular slime rich in glucosamine
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which allows them to attach strongly to equipment, to become endemic in the plant and later contaminate poultry carcasses. The ability of the strains for cell to cell adhesion to produce a clump is considered an important physical barrier to penetration by disinfectants increasing the resistance of these strains and making their elimination in the plant very difficult. The establishment of endemic strains in the de-feathering equipment and their transferring to carcasses was especially emphasized. The adhesion of spoilage bacteria to fat and tendon slices and to glass was evaluated by Piette and Idziak (1991). According to their findings adhesion was influenced by the physiological age of the cells but was greatly influenced by the composition of the adhesion medium, in particular in sodium content. Irrespective of the surface or the adhesion medium, no correlation was found between adhesion of the various MO and their hydrophobicity and surface charge. In a later model study the same authors (1992) explored the adhesion of P. fluorescens to meat. The authors demonstrated attachment by dead cells, indicating that physiological activity was not required. Adhesion also increased with increasing ionic strength up to 10-1 OOmM, suggesting that at low ionic strength electrostatic interaction were involved in the adhesion. At higher ionic strengths adhesion was sharply reduced. Based on their findings they also suggested that if the adhesion was specific, the attachment sites on the tendon surface used must be located within the collagen or the proteoglycan molecules. In a series of papers Kim and Doores (1993a,b) and Kim et al., (1993) evaluated the effect of defeathering methods on the penetration of S.typhymurium into turkey skin. Penetration increased with incubation time and was affected by the method of de-feathering which caused different skin micro-topographies, and resulted in different amounts of bacterial adhesion. The high number of attached cells and the greatest amount of fibril formation on the surface of steam spray skin suggested the positive relationship between bacterial attachment and fibril formation. Contrary to a previous report (Dickson and Koohmaraie, 1989) that there is a relationship between cell surface charge and a bacterium's ability to contaminate beef. Dickson and Siragusa reported recently (1994) their inability to associate attachment of L .monocytogenes, exhibiting rough colony appearance, to beef tissue surfaces. CONTROL OF BIOFILM DEVELOPMENT As mentioned earlier microbial attachment to food contact surfaces and to processing equipment are of great significance to the food industry. To what extend classical biofilm formation takes place in a food processing plant as in aquatic environments is not well defined. Formation of the latter may take several days to several weeks (Zottola and Sasahara, 1994). The frequent cleaning taking place in a food processing plant probably is not conducive to biofilm formation. Yet in places where cleaning may not be easy and ineffective cleaning may create problems (Holah and Kearney, 1992; Holah and Thorpe, 1990). Environmental studies done in meat, dairy and other types of food processing plants have demonstrated the extensive prevalence of L. monocytogenes and other pathogens (Cox et al., 1989; Charlton et al., 1990; Klausner and Donnelly, 1991) L.monocytogenes has been detected in drains, condensed or stagnant water, floors process equipment and may survive in aerosols (Rocourt, 1994). Our own studies in Greece not only demonstrated the extensive environmental contamination of meat and dairy plants but also demonstrated that the products with the highest Listeria prevalence were those produced in the plants with the most frequent isolations (Genigeorgis et al., 1991b).
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The effect of sanitizers and detergents on planktonic (free-living) bacterial cells and especially Listeria cell has been evaluated extensively (Lopes, 1986; Mustapha and Liewen, 1989; Genigeorgis et al., 1991a; Van de Weyer et al., 1993 ) in recent years and it will not be reviewed here. While Listeria and other pathogens are easily destroyed by commercial sanitizers in water, the presence of organic matter may affect significantly the function of sanitizers (Genigeorgis et al., 1991; Van de Weyer et al., 1993). In our studies we evaluated twelve disinfectants and three soap preparations against L. monocytogenes, L. ivanovii, L. seeligeri, E. coli, P. aeruginosa and S. typhimurium. When the disinfectants in deionized water were tested at the manufacturers' recommended level, they were all effective against all organisms. However in the presence of 10% reconstituted dry skim milk in the deionized water only one iodophore and a phenolic preparation were effective at the recommended level and exposure time against all organisms. One of the quaternary ammonium compounds (QAC) was effective against 417 organisms and one of the acids and a combined QAC+acid preparation were effective against 217 organisms. All soap preparations at 1: 1 dilution caused 5 decimal reductions to the organisms within 0.5 minutes. Ren and Frank (1993) reported that starved planktonic cells were 390 times more resistant to QAC than non starving cells. They associated starved cells with processing plant environments where cleaning programs remove food residues from surfaces. In the past fifteen years several papers have been published that dealt with the resistance of adhered or biofilm bacteria to sanitizers. Scwach and Zottola (1984) using scanning electron microscopy demonstrated that the use of water rinse followed by sodium hypochloride rinse was not sufficient to remove the P. fragi, S . montevideo and B. cereus and debris attached to stainless steel chips. Stone and Zottola (1985) evaluated the effect of two clean-in-place cleaning systems on P. fragi attached to stainless steel in a pilot sized milk transfer system. They showed that the use of adequate cleaning and sanitizing, i.e., proper concentration, temperature and flow rates removed adherent bacteria and inactivated any remaining organisms. LeChevalier et al., (1987) evaluated several treatment procedures for the control of biofilm composed of coliform bacteria in water distribution system. Their finding indicated recontamination of water with coliforms within days after treating the pipeline with up to 4.3 ppm of free residual chlorine, flushing with high flow rates and mechanically cleaning with polyfoam swabs. Use of mono-chloramine was more effective in killing the biofilm cells than free chlorine because mono-chloramine does not react with organic compounds which are known to decrease the efficacy of chlorine before penetrating the outermost layer of the biofilm (LeChevalier et al., 1988). Frank and Koffi (1990) and Lee and Frank (1991) demonstrated the increased resistance of attached L. monocytogenes to glass or to stainless steel to QAC an anionic acid sanitizer, hypochloride and to heating at 55 and 70°C as compared to planktonic cells. Mafu et al., (1990) examined the efficiency of four commercial sanitizing agents against L. monocytogenes attached on four types of surfaces. The sanitizers were more efficient when the pathogen was attached to non-porous surfaces than to porous surfaces and at 20 that 4°C. The reports of Dhaliway et al., (1992), Wirtanen and Mattila-Sandholm (1992a,b), Mosteller and Bishop (1993) further demonstrated the fact that sanitizers alone are unable to completely inactivate MO attachment to surfaces. Czechowski and Banner (1992) investigated the efficacy of cleaning and sanitizing procedures in reducing the viability of bacteria attached to stainless steel, Teflon and Buna-n gaskets in pipeline systems. Their results showed that a combination of chlorinated alkaline and alkaline detergents with sanitizers were effective in reducing the viable cell populations to greater than 98%. By
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increasing the sanitizer contact time, a further reduction of the viable counts was achieved. The study suggested that frequent cleaning over a 24 hour processing period and thorough drying of processing equipment after cleanup would provide another hurdle in biofilm-forming MO trying to establish a niche. The study of Krysinski et aI., (1992)--dealing with the removal and inactivation of L. monocytogenes which attached to various surfaces--supported the conventional wisdom that cleaning must precede sanitizing in order to remove and inactivate MO. They also reported that the type of surface (stainless steel, polyester, or polyester backed with polyurethane) had little effect on the rate of cell attachment but affected the efficacy of various sanitizers and cleaners. After developing biofilms on stainless steel and Buna-n rubber by seven L. monocytogenes and one Salmonella strain, Ronner and Wong (1993) challenged them with four types of detergent and non-detergent sanitizers. Resistance to sanitizers was strongly influenced by the type of surface. Biofilms on Buna-n were more resistant than biofilms on stainless steel. Chlorine and anionic acid sanitizers removed extracellular materials from biofilms better than iodine and quaternary ammonium detergent sanitizers. SEM demonstrated that biofilm cells and extracellular matrices could remain on surfaces from which nonviable cells were removed. Trisodium phosphate (TSP) has been approved by the U.S. Department of Agriculture as a post-chill antimicrobial treatment for raw poultry. Recently, Somers et aI., (1994) evaluated its effectiveness on biofilm and planktonic cells of Campylobacter jejuni, E. coli 0157:H7, L. monocytogenes and S. typhimurium at room temperature and at l0°C. As shown before, planktonic cells were more sensitive then biofilm cells attached to stainless steel and Buna-n rubber. Listeria was the most resistant bacterium requiring exposure to 8% of TSP for 10 min. at room temperature to reduce biofilm bacteria by at least one log. At 1 % concentration, and the same temperature TSP reduced the biofilms of the other bacteria by 2.4- 5 logs after 0.5 min. of exposure. The attachment of bacteria to meat surfaces has been discussed before. Attachment might be due to specific mechanisms and also to involve entrapment of the cells within collagen fibers, in ridges, crevices and channels brought about by immersion of carcasses in water and enhanced by de-feathering procedures at least in poultry. Removal and destruction of bacteria under such condition by approved sanitizers and detergents is not an easy task. This subject has been evaluated extensively (Dickson and Anderson, 1992; Lillard, 1994; Kim et aI., 1994; Jericho et al, 1995) but it will not be reviewed here. CONCLUSIONS What has been achieved? In the last ten years there has been a continuously increasing number of reported studies and interest on the general subject of biofilms. As a result there is a better understanding on the mechanism of their formation, factors affecting the formation, the consequences in specific environments and means for their prevention and removal. It is encouraging that in the last five years special attention by researchers has been given to the significance of biofilms to the food industry, including the mechanism and nature of microbial attachment to food contact surfaces and muscle foods the increased microbial resistance to heat and sanitizing agents, the identification of critical areas in the processing line where biofilm formation will have a major impact on product shelf-life and safety, as well as the development of appropriate cleaning and sanitizing procedures.
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What has been neglected? The continuing high prevalence of important pathogens like Salmonella, Campylobacter, Listeria, Yersinia, and S. aureus in raw meats and poultry is well documented. As a result one may assume that the current practices of animal processing have not maximized available scientific knowledge on microbial attachment and detachment from surfaces and means for control. Another implication might be the progress made in automation in food processing and the development of complex equipment often creating new niches for microbial entrapment and attachment (Notermans, 1994). What needs to be done? Considering the frequent cleaning of surfaces in food processing plants, the potential for biofilm formation should be further explored. This should be done under field conditions since it is well known that microbial behavior in laboratory media and plant environments may differ extensively. Advances are needed in the design of new detergents and sanitizers which take into account the production of extracellular polymeric substances by attached MO, known to enhance microbial resistance to sanitizers and heating. The phenomenon of potential microbial adaptation to sanitizers needs to be explored further. The mechanism of attachment of pathogens like Listeria which are shown to be consistent environmental contaminants needs further clarification. Additional future research needs have been proposed recently in a scientific status summary on the significance of biofilms to the food industry (Zottola and Sasahara, 1994)
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Characklis, W.G. and Marshall, K.C., (Eds.), 1990. Biofilms. John Wiley & Sons Inc. New York Charlton, B.R, Kinde, H and Jensen, L.H., 1990. Environmental survey for Listeria species in California milk processing plants. J. Food Prot. 53, 198-201 Chung, K.T., Dickson, lS. and Crouse, lD., 1989. Attachment and proliferation of bacteria on meat. l Food Prot. 52, 173-177 Costerton, J.W. and Lappin-Scott, HM., 1989. Behavior of bacteria in biofilms. ASM News. 55, 650-654 Costerton, lW., and Lappin-Scott, 1989. Behavior of bacteria in biofilms. ASM News. 55, 650654 Cox, L.l, Kleis, T., Cordier, lL. et al., 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6, 49-61 Czechowski, M.H and Banner, M., 1992. Control ofbiofilms in breweries through cleaning and sanitizing. Master Brew. Assoc. Am. Tech. Q. 29, 86-88 Dahlback, B., Hermansson, M., Kjelleberg, S. and Nokrans, B., 1981. The hydrophobicity of bacteria-an important factor in their initial adhesion at the air-water interfaces. Arch. Microbiol. 128,267-270. Dhaliwal, D.S., Cordier, lL. and Cox, L.l, 1992. Impedimetric evaluation of the efficiency of disinfectants against biofilms. Letters Appl, Microbiol. 15,217-221 Dickson, lS. and Anderson, M.E., 1994. Microbiological decontamination of food animal carcasses by washing and sanitizing systems: a review. J. Food Prot. 55, 133-140 Dickson, lS. and Koohmaraie, 1989. Cell surface charge characteristics and their relationship to bacterial attachment to meat surfaces. Appl. Envir. Microbiol. 55, 832-836 Dickson, lS. and Siragusa, G.R, 1994. Cell surface charge and initial attachment characteristics of rough strains of Listeria monocytogenes. Letters Appl. Microbiol. 19, 192-199 Dickson, lS., 1990. Transfer of Listeria monocytogenes and S.typhimurium between beef tissue surfaces. J. FoodProt. 53,51-55 Farber, lM. and Idziak, E.S., 1984. Attachment of psychrotrophic meat spoilage bacteria to muscle surfaces. J. Food Prot. 47,92-95 Farber, lM. and Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55,476-511 Firstenberg-Eden, R, 1981. Attachment of bacteria to meat surface: a review. J. Food Prot. 44, 602-607 Fletcher, M. and Loeb, G.I., 1979. Influence of substrate characteristics on the attachment of a marine pseudomona to solid surfaces. Appl. Environ. Microbiol. 37, 67-72 Fletcher, M., 1992. The measurement of bacterial attachment to surfaces in static systems. In: L. Melo., T.R Bott., M.Fletcher, and B. Capdeville (Eds.). Biofilms-Science and Technology. Kluwer, Dordrecht, The Netherlands, pp. 603-614 Frank, IF. and Koffi, RA., 1990. Surface-adherent growth of Listeria monocytogenes is associated with increased resistance to surfactant sanitizers and heat. J. Food Prot. 53, 550-55 Genigeorgis, c., Panoulis ,C., Theodoridis, A et al., 1991. Prevalence of Listeria spp in raw and processed foods of animal origin and the environment of processing plants. Proc Intern. Conf.: Listeria and Food Safety. pp. 213. ASEPT, Laval, France
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Genigeorgis, C., Rosales, J. and Verder-Elepano, M., 1991. Effect of selected sanitizer cleaners and soaps on Listeria spp, S.typhimurium, E.coli, and P.aeruginosa in the presence or absence of organic matter. Proc. Inter. Conf. Listeria and food Safety, pp.186, ASEPT, Laval, France Gilbert, P., Evans, D.J., Evans, E,., Duguid, I.G. and Brown, M.RW., 1991. Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis. J. Appl. Bacteriol. 71, 72-77 Helke, D.M., Somers, AB. and Wong, AC.L., 1993. Attachment of Listeria monocytogenes and Salmonella typhimurium to stainless steel and Buna-n in the presence of milk and individual milk components. J. Food Prot. 56,479-484 Hentges, D.J. 1992. Gut flora and disease resistance. In: Fuller, R(Ed) Probiotics-the scientific basis. Chapman & Hall, London. pp. 87-110 Herald, P.J. and Zottola, E.A, 1988. Attachment of Listeria monocytogenes to stainless steel surfaces at various temperatures and pH values. J. Food Sci. 53, 1549-1552,1562 Herald, PJ. and Zottola, E.A, 1988. Scanning electron microscopic examination of Yesinia enterocolitica attached to stainless steel at selected temperatures and pH values. J. Food Prot. 51,445-448 Herald, P.J. Zottola, E.A, 1988c. The use of transmission electron microscopy to study composition Pseudomonas fragi attachment material. Food Microstructure. 7, 53-57 Holah, J.T and Kearney, L.R, 1992. Introduction ofbiofilms in the food industry. In: L.F. Melo, TR Bott, M.Fletcher and B. Capdeville (Eds.) Biofilms-science and Technology. Kluwer, Dordrecht, the Netherlands, pp. 35-41 Holah, J.T and Thorpe, RH., 1990. Cleanability in relation to bacterial retention on unused and abraded domestic sink materials. J. Appl. Bacteriol. 69, 599-608 Holah, J.T .. , Betts, RP. and Thorpe, RH., 1988. The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces. J. Appl. Bacteriol. 65,215-221 Jeong, D.K, and Frank, J.F., 1994. Growth of Listeria monocytogenes at lOOC in biofilms with microorganisms isolated from meat and dairy processing environments. J. Food Prot. 57, 576-586 Jericho, W., Bradley, J.A and Kozub, G.c., 1995. Microbiologic evaluation of carcasses before and after washing in a slaughter plant. JA VMA. 206,452-455 Kim, J-W, and Doores, S., 1993. Attachment of salmonella typhimurium to skins of turkey that had been defeathered through three different systems: scanning electron microscopic examination. J. Food Prot. 56, 395-400 Kim, J-W, Slavic, M.F. and Bender, F.G., 1994. Removal of attached to chicken skin by rinsing with Trisodium phosphate solution: scanning electron microscopic examination. J. Food Safety. 14, 77-84 Kim, J-W and Doores, S., 1993. Influence of three defeathering systems on microphotography of turkey skin and adhesion Salmonella typhimurium. J. Food Prot. 56,286-291,305 Kim, J-W., Knabel, S.J. and Doores, S., 1993. Penetration of Salmonella typhymurium into turkey skin. J. Food Prot. 56, 292-296 Kim, K.Y, and Frank, J.F., 1994. Effect of growth nutrients on attachment of Listeria monocytogenes to stainless steel. J. Food Prot. 57, 720-726 Kim, K.Y. and Frank, J.F., 1995. Genes on stainless steel. J. Food Prot. 58,24-28
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Klausner, RB. and Donnelly, C.W., 1991. Environmental sources of Listeria and Yersinia in Vermont dairy plants. J. Food Prot. 54,607-611 Klausner, RB. and Donnelly, C.W., 1991. Environmental sources of Listeria and Yersinia in Vermont dairy plants. J. Food Prot. 54,607-611 Krogfelt, KA, 1991. Bacterial adhesion: genetics, biogenesis and role in pathogenesis of fimbrial adhesions of Escherichia coli. Rev. Infectious Dis. 13, 721-735 Krysinski, E.P., Brown, L.l and Marchiselo, Tl, 1992. Effect of cleaners and sanitizers on Listeria monocytogenes attached to product contact surfaces. J. Food Prot. 55,246-251 Lawrence, J.R, Delaquis, P.l, Korber, D.R and Caldwell, 1987. Boundary layers of surface microenvironments. Microb. ecol. 14, 1-14 LeChevalier, M.W., Cawthon, C.D. and Lee, RG., 1988. Inactivation ofbiofilm bacteria. Appl. Envir. Microbiol. 54, 2492-2499 LeChevallier, M.W., Babcock, TM., and Lee, RG., 1987. Examination and characterization of distribution system biofilms. Appl. Environ. Microbiol. 53,2714-2724 Lee, S-H, and Frank, J.F., 1991. Inactivation of surface-adherent Listeria monocytogenes. Hypochlorite and heat. J. Food prot. 54,4-6 Lewis, S.l and Gilmour, A, 1987. Microflora associated with the internal surface of rubber and stainless steel transfer pipeline. J. Appl. Bacteriol. 62, 327-333 Lillard, HS., 1985. Bacterial cell characteristics and conditions influencing their adhesion to poultry skin. J. Food Prot. 48, 803-807 Lillard, HS., 1988. Effect of surfactant or changes in ionic strength on the attachment of Salmonella thyphimurium to poultry skin and muscle. J. Food Sci. 53, 727-730 Lillard, HS., 1989. Factors affecting the persistence of Salmonella during the processing of poultry. l Food Prot. 52, 829-832 Lillard, HS., 1994. Decontamination of poultry skin by sonic action. Food Tech. 48(12), 72-73 Loosdrecht, M.C.M., Lyklema, L, Norde, W., Schraa, G. and Zenhder, AlB., 1987. The role of bacterial cell wall hydrophobicity in adhesion. Appl. Environ. Microbiol. 53, 1893-1897 Lopes, J.A, 1986. Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeria monocytogenes. J. Dairy Sci. 69,2791-2796 Mafu, AA, Roy, D., Goulet, J et al., 1990. Efficiency of sanitizing agents for destroying Listeria monocytogenes on contaminated surfaces. J. Dairy Sci. 73, 3428-3432 Mafu, AA, Roy, D., Goulet, J. and Savoie, D., 1991b. Characteristics of physicochemical forces involved in adhesion of Listeria monocytogenes to surfaces. Appl. Environ. Microbiol. 57, 1969-1973 Mafu, AA, Roy, D., Savoie, L. and Goulet, L, 1991a. Bioluminescence assay for estimating the hydrophobic properties of bacteria as revealed by hydrophobic interactive chromatography. Appl. Environ. Microbiol. 57, 1640-1643 Marshall, KC., 1992. Biofilms: An overview of bacterial adhesion, activity and control at surfaces. ASM News. 58, 202-207. Marshall, KC., Stout, R and Mitchell, R, 1971. Mechanism of the initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68,337-348 Mattila-Sandholm, T, and Wirtanen, G., 1992. Biofilm formation in the industry: a review. Food Reviews International. 8(4),573-603 McGuire, L, and Krisdhasima, v., 1991. Surface chemical influences on protein adsorption kinetics. Food Technol. 45(12),92-96
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McMeekin, TA, Thomas, c.J. and Pennington, P.I., 1984. Contamination and decontamination of poultry carcass neck tissue. J. Food Safety. 6, 79-88 Mead, G.c. and Dodd, C.E.R, 1990. Incidence, origin and significance of staphylococci on processed poultry. J. Appl. Bacteriol. Symp. Suppl. S81-S91 Mosteller, TM. and Bishop, lR, 1993. Sanitizer efficacy against attached bacteria in a milk biofilm. J. Food Prot. 56, 34-41 Mustapha, A and Liewen, M.B., 1989. Destruction of Listeria monocytogenes by sodium hypochlorite and quaternary ammonium sanitizers. J. Food Prot. 52, 306-311 Notermans, S., 1994. The significance of biofouling to the food industry. Food Technol. 48(7), 13-14. Notermans, S. and Kampelmacher, E.H., 1974. Attachment of some bacterial strains to the skin of broiler chickens. Br. Poult Sci. 15,573-585 Notermans, S., Terbijhe, Rl and Van Schothorst, M., 1980. Removing fecal contamination of broilers by spray-cleaning during evisceration. Br. Poult. Sci. 21, 115-121 Notermans, S., Dormans, lAM.A, and Mead, G.C., 1991. Contribution of surface attachment to the establishment of micro-organisms in food processing plants: a review. Biofouling. 5,21-36. Piette, G.J-P, and Idziak, E.S., 1991. Adhesion of meat spoilage bacteria to fat and tendon slices and to glass. Biofouling. 5,3-20 Piette, G.J-P, and Idziak, E.S. 1992. A model study of factors involved in adhesion of Pseudomonasfluorescens to meat. Appl. Envir. Microbiol. 58,2783-2791. Pontefract, RD., 1991. Bacterial adherence: its consequences in food processing. Can. Inst. Sci. Technol. l24(3/4),113-117 Ren, T-J and Frank, J.F., 1993. Susceptibility of starved planktonic and biofilm L.monocytogenes to quaternary ammonium sanitizer as determined by direct viable and agar plate counts. l Food Prot. 56, 573-576 Rocourt, l,1994. Listeria monocytogenes: the state of the science. Dairy, Food Envir. Sanit. 14, 70-82 Roller, S.D., 1991. Effect of sodium chloride on the adhesion of Pseudomonas fragi to polystyrene. Biofouling. 5,57-63 Ronner, AB. and Wong, AL., 1993. Biofilm development and sanitizer inactivation of Listeria monocytogenes and Salmonella typhimurium on stainless steel and Buna-n rubber. J. Food Prot. 56, 750-758 Sanderson, K., Thomas, C.l, and McMeeking T, A, 1991. Molecular basis of Salmonella serotypes to chicken musscle fascia. Biofouling. 5,89-101. Sasahara, K.c. and Zottola, E.A, 1993. Biofilm formation by Listeria monocytogenes utilizes a primary colonizing microorganism in flowing systems. J. Food Prot. 56, 1022-1028 Scwach, TS. and Zottola, E.A, 1984. Scanning electron microscopic study on some effects of sodium hypo chloride on attachment of bacteria to stainless steel. J. Food prot. 47, 656759. Selgas, D., Marin, M.L., Pin, c., and Casas, c., 1993. Attachment of bacteria to meat surfaces: a review. Meat Science. 34,265-273 Somers, E.B., Schoni, J.L., and Wong, AC.L., 1994. Effect of trisodium phosphate on biofilm and planktonic cells of Campylobacter jejuni, Escherichia coli, 0157:H7, Listeria monocytogenes and Salmonella typhimurium. Int. J. Food Microbiol. 22, 269-276
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Speers, JG.S. and Gilmour, A, 1985. The influence of milk and milk components on the attachment of bacteria to farm dairy equipment surfaces. J Appl. Bacteriol. 59,325-332 Spurlock, AT and Zottola, E.A, 1991. Growth and attachment of Listeria monocytogenes to cast iron. J Food Prot. 54, 925-929 Stanley, P.M., 1983. Factors affecting the irreversible attachment of Pseudomonas aeruginosa to stainless steel. Can. J Microbiol. 29, 1493-1499 Stone, L.S. and Zottola, EA, 1985. Effect of cleaning and sanitizing on the attachment of Pseudomonas fragi to stainless steel. J. Food Sci. 50,951-956 Suarez, B., Rerreiros, C.M. and Criado, M.-T, 1992. Adherence of psychrotrophic bacteria to dairy equipment surfaces. J Dairy. Res. 59,381-388 Ternstrom, A and Molin, G., 1987. Incidence of potential pathogens on raw pork, beef and chicken in Sweden, with special reference to Erysipelothrix rhusiopathiae. J Food Prot. 50., 141-146. Thomas, c.J and McMeekin, TA, 1991. Factors which affect retention of salmonella by chicken muscle fascia. Biofouling. 5, 75-87 Van de Weyer, A, Devleeschouwer, M.J and Dony, J., 1993. Bactericidal activity of disinfectants on Listeria. J Appl. Bacteriol. 74, 480-483 Van Loosdrecht, M.C.M., Lyklema, J, Norde, W. and Zenhder, AJB., 1989. Bacterial adhesion: a physicochemical approach. Microb. Ecol. 17, 1-15 Van Loosdrecht, M.C.M., Lyklema, J, Norde, W., Schraa, G and Zehnder, AJB., 1987. The role of bacterial cell wall hydrophobocity in adhesion. Appl. Environ. Microbiol. 53, 18931897. Vanhaecke, E, Remon, J-P., Moors, M. et al., 1990. Kinetics of Pseudomonas aeruginosa adhesion to 304 and 316-L stainless steel: role of cell surface hydrophobicity. Appl. Environ. Microbiol. 56, 788-795 Wirtanen, G., and Mattila-Sandholm, T, 1992. Removal of foodborne biofilms-Comparison of surface and suspension tests. Part 1. Lebensm.-Wiss.u.-Technol.25, 43-49 Wirtanen, G., and Mattila-Sandholm, T, 1992. Effect of growth phase of foodborne biofilms on their resistance to a chlorine sanitizer. Part II. Lebensm.-Wiss.u.-Technol.25, 50-54 Wirtanen, G. and Mattila-Sandholm, T, 1993. Epifluorescence image analysis and cultivation of foodborne biofilm bacteria grown on stainless steel surfaces. J. Food Prot. 56, 678-683 Wolfaardt, G.M. and Cloete, TA, 1992. The effect of some environmental parameters on surface colonization of microorganisms. Water Res. 26, 527-537 Zoltai, P.T, Zottola, E.A and McKay, L.L., 1981. Scanning electronmicroscopy of microbial attachment to milk contact surfaces. J Food Prot. 44,204-208 Zottola, AE, and Sasahara, 1994. Microbial biofilms in the food processing industry-Should they be a concern? Inter. J Food Microbiol. 23, 125-148 Zottola, EA, 1994. Scientific Status Summary. Microbial attachment and biofilm formation: A new problem for the food industry? Food Technol. 48(7), 107-114
Organizers of the course: ECCEAMST/Greece with the technical support of AGRO-UETP, the COMET program of the European Commission and the additional support from the Greek food companies Goodies and Nikas S.A.
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