Microbial biofilms – a concern for industry?

Many bacteria are able to attach to and colonise environmental surfaces by producing a biofilm, which allows the organisms to persist in the environment and resist desiccation, UV light and treatment with antimicrobials and sanitising agents. Biofilms are formed when microbes attach to a solid support and to each other by extracellular polymeric substances (EPS), and on a wide variety of surfaces, including metal, plastic, rock and living or dead tissue. Once in a biofilm, bacteria can be several orders of magnitude more resistant to antimicrobials than their planktonic counterparts1.

Biofilms are of particular concern in the process and food industries as well as in potable and wastewater distribution systems. Biofilms formed on the inside of pipes can reduce flow rates while increased fouling can lead to decreases in heat transmission and thus ineffective processing, product contamination and pipe corrosion due to acid production in the biofilm. Biofilm formation in drinking water distribution systems can lead to a decrease in water velocity and carrying capacity, clogging and pipe corrosion, increase in energy utilisation and decreased operational efficiency. In marine and other aquatic environments, submerged surfaces attract organisms such as algae, diatoms and bacteria that are able to attach and form biofilms on ships’ hulls and become resistant to the different antifouling paints that have been developed to prevent the initial colonisation. This results in increased fluid frictional resistance and fuel consumption with financial implications to the marine and naval transport and also food industries. In the food industry, contamination of food processing and/or food contact equipment often leads to post-process contamination and reduced shelf life of products since when biofilms detach from the surfaces, spoilage and hazardous microorganisms can easily be spread2.

Incidences of foodborne illness linked to pathogen contaminated fresh produce have significantly increased over the years. Human pathogens such as E. coli and Salmonella have all been found capable of attaching to and colonising the surfaces of growing plants forming biofilms on plant tissues. It is this biofilm formation that has been recognised as one of the main factors that affects the efficiency of standard washing treatments. Lindow and Brandl3 reported that between 30 per cent and 80 per cent of bacteria on plant surfaces exist within biofilms as a mechanism for the cells to withstand the harsh environmental conditions (temperature change, oxidative stress, etc.) of the plant surface.

In the meat industry, contamination of raw meat products has been detected mainly as a result of inappropriate cleaning practices. Organisms such as Salmonella, Campylobacter, Yersinia, Staphylococcus and Listeria have been isolated from poultry surfaces. They have not only been shown to be associated with slaughtering processes but are also responsible for cross contamination of uncontaminated carcasses. In natural and industrial water systems, biofilm formation is a common phenomenon despite the lack of nutrients, with organisms such as Pseudomonas, Acinetobacter, Flavobacterium, Moraxella and others prevailing. Streptococcus thermophilus and Bacillus cereus spores, part of the natural flora of milk, have been found attached to stainless steel surfaces and heat exchangers in milk processing plants. Milk pasteurisation is not sufficient to kill the spores of B. cereus while standard cleaning practices have no effect on the spores of the organism which are able to then germinate in the product during storage at ambient conditions and produce spoilage and enterotoxins. Listeria monocytogenes has been isolated from a number of different food processing environments, in the meat, fish, dairy and fresh produce environments, including drains, floors, stagnant water, processing equipment and food contact surfaces such as conveyor belts.

Contamination of food by L. monocytogenes is thought to occur most often in foodprocessing environments where cells are able to persist through their ability to attach to surfaces. Once attached, biofilms may be produced that are resistant to disinfection procedures; cells may then become detached from the biofilm resulting in contamination of food products. The ability of L. monocytogenes to adsorb to inert surfaces, such as those found in the processing environment, has been well documented4,5. It has been observed that there are differences in both the rate and extent of adsorption depending on the type of surface selected, surface pre-treatment, environmental and growth conditions, pH and temperature6. Studies of large numbers of strains have indicated significant differences between strains and serotypes in their ability to adhere to various surfaces7.

Numerous studies on the factors that are important in microbial attachment and biofilm formation have been published; however evidence exists to indicate that such factors are mainly species specific and can also vary amongst the different surfaces studied. Nutrient availability and temperature of the environment are two of the main factors affecting the ability of different microorganisms to attach to different surfaces and form biofilms.

In water distribution systems where nutrient availability is quite limited, enhanced biofilm formation occurs. Some of the early research in the area reported that available nutrients are usually concentrated at the substratum surface rather than in the bulk fluid and since bacteria are able to sense this concentration gradient, they then migrate towards the surface. Nutrients saturate surface cell receptors when present in higher levels in the bulk fluid phase and are thus unavailable for specific binding at the substratum surface8. However, since then, numerous studies have shown that not all systems work in this way, highlighting the importance of nutrient availability on microbial attachment and formation of biofilms on different food contact and other surfaces and that attachment potential and biofilm formation is a result of a combination of factors including the microbial species / strains implicated, the type of surface used and environmental temperature.

In the work of Kopiec et al.9, the ability of different E. coli strains to attach onto different food contact surfaces, including stainless steel (SS) and conveyor belt material (polyvinyl chloride (PVC), polyurethane (PU) and polystyrene (PS)), was evaluated as affected by nutrient availability and temperature. Results indicated that E. coli was successfully recovered from all contact surfaces, however recovery was seen to be both temperature and surface dependent. E. coli was not recovered when attachment trials were set up at 37°C and 42°C. Cells were recovered from all surfaces at set up temperatures between 15 – 30°C. As the trial set up temperatures were lowered to 5 – 10°C, recovery was low and variable from PS, PU and PVC, but with no recovery from SS. Maximum cell recovery was observed between 15 – 25°C for PU and PVC, 10 – 30°C for PS and 25°C for SS. The results further indicated that nutrient availability, as this was provided through the use of nutrient-rich laboratory broth media, was an important additional factor in the attachment and maintenance of cells on the different surfaces, and their subsequent detachment by swabbing. Use of carbon sources in the form of glucose, glycerol and succinic acid favoured cell attachment on all contact surfaces apart from stainless steel with glucose giving the broadest recovery across PS, PU and PVC surfaces.

In the work of Komitopoulou et al.10, seven L. monocytogenes strains were used in different trials to assess attachment potential, survival under different temperature and nutrient availability conditions, cell recovery and cell transfer onto foods that come into contact with contaminated surfaces over time. The results indicated that attachment and survival of cells on food contact surfaces was a straindependent phenomenon that was affected by the environmental temperature. Preliminary cell attachment and recovery was better at lower temperatures (5°C and 8°C), than at the optimum growth temperatures (25 – 37°C). However, attachment over time in nutrient rich environments was favoured at temperatures closer to the optimum for the growth of the organism. Of the three food contact surfaces tested (stainless steel, polyurethane and conveyor belt material), polyurethane was shown to better support initial attachment, survival and recovery of L. monocytogenes over time at the different exposure temperatures. Cell transfer onto cheese slices (in contact with contaminated surfaces) indicated that cell transfer was higher from the polyurethane surface to the cheese slices.

It has also been demonstrated that L. monocytogenes secretes a functional autoinducer 2 (AI-2)-like signal (quorum sensing signaling molecule) and that mutation in the luxS gene, which encodes the pre-cursor to AI-2, results in a luxS-deficient mutant able to produce a denser biofilm and attach to a surface 19 times better than the parent strain11. The expression of a range of physiological functions in many bacteria is modulated by quorum sensing, a population-dependent signalling mechanism that involves the production and detection of extracellular signalling molecules. In a study carried out by Komitopoulou & Leverington12, it was demonstrated that Listeria monocytogenes was able to produce functional autoinducer-2 (AI-2) activity which could accumulate gradually during the exponentialgrowth phase and reached its maximum levels during stationary-phase. A significant drop in the oxidation-reduction (redox) potential during the growth of L. monocytogenes occurred at the onset of the stationary-phase. The relationship between AI-2 activity and growth phase in L. monocytogenes confirmed the role of redox potential on AI-2 production. In an oxidised (high redox potential) environment, L. monocytogenes was shown unable to produce AI-2-like activity. When the redox potential was normally reduced during growth, AI-2 activity was re-established. The levels of AI-2 activity produced by L. monocytogenes were directly proportional to the degree of oxidation of the growth medium. The higher the redox potential of the growth medium, the lower the AI-2 activity produced by L. monocytogenes was. Although the mechanism of redox potentialregulated AI-2 activity has not yet been clearly defined, the study indicated the importance of such regulation in the ability of L. mono – cytogenes to form biofilms on plastic surfaces. Interfering with AI-2 activity levels by oxidation in L. monocytogenes resulted in the formation of biofilms of different density. The lower the levels of AI-2 activity produced by L. monocytogenes, the weaker the biofilm formed.

Even though a lot of work has been carried out looking at the role of quorum sensing promoting bacterial defence mechanisms, little is currently known about its role in biofilm formation, bacterial virulence and food spoilage. Some preliminary work carried out by the Food Safety Team at Leatherhead Food Research indicated that food spoilage is associated with a high level of AI-2 in a meat and fruit juice product in what seemed to be a co-ordinated response at high levels of background flora. However, there are still a number of questions that need to be answered, mainly looking at the ways that food components and food processing environments affect quorum sensing and the resultant microbial cross-talk and biofilm production.

References

1. Schachter, B. (2003) Slimy business – the biotechnology of biofilms. Nature Biotechnology, 21, 361

2. Kumar, C.G., and Anand, S.K. (1998) Significance of microbial biofilms in food industry: a review, International Journal of Food Microbiology, 42, 9-27

3. Lindow, S.E., and Brandl, M.T. (2003) Microbiology of the phyllosphere, Applied and Environmental Microbiology, 69,1875–1883

4. Hood, S.K., and Zottola, E.A. (1997) Adherence to stainless steel by food-borne micro-organisms during growth in food model systems. International Journal of Food Microbiology, 37,145

5. Takhistov, P., and George, B. (2004) Linearized kinetic model of Listeria monocytogenes biofilm growth. Bioprocess and Biosystems Engineering, 26 (4), 259

6. Kalmokoff, M.L., Austin, J.W., Wan, X.-D., Sanders, G., Banerjee, S., and Farber, J.M. (2001) Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions. Journal of Applied Microbiology, 91, 725

7. Lunden, J.M., Miettinen, M.K., Autio, T.J., and Korkeala, H.J. (2000) Persistent Listeria mono – cytogenes strains show enhanced adherence to food contact surfaces after short contact times. Journal of Food Protection, 63, 1204

8. Brown, C.M., Ellwood, D.C. and Hunter, J.R. (1977) Growth of bacteria at surfaces: influence of nutrient limitation, FEMS Microbiology Letters, 1, 163-166

9. Kopiek, J. Haines, J., and Komitopoulou, E. (2007) The potential of Escherichia coli O157 to form biofilms on food contact surfaces, Leatherhead Food Research, Forum Report No. 927

10. Komitopoulou, E. Gibbs, P.A. and Harward, C (January 2009) Virulence and cell attachment in Listeria monocytogenes, Leatherhead Food Research, Forum Report No.933

11. Sela, S., Frank, S., Belausov, E., and Pinto, R. (2006) A mutation in the luxS gene influences Listeria monocytogenes biofilm formation, Applied and Environmental Microbiology, 72, 5653–5658

12. Komitopoulou, E. and Leverington, C. (2005) Oxidation affects AI-2 activity and biofilm formation in Listeria monocytogenes, Leatherhead Food Research, Forum Report No. 864