BIOFILMS ADD NEW DIMENSIONS TO FOOD MICROBIOLOGY

| June 24, 2008

If you are a food scientist or food technologist you are probably familiar with the term ‘biofilm’. You are probably aware that a biofilm consists of a layer of microorganisms embedded in a kind of microbial slime that forms on wet surfaces in food processing areas and makes them more difficult to clean properly. You will know that biofilms are a potential source of product contamination and that, in extreme cases, can cause serious fouling problems in equipment like plate heat exchangers. What you may not know is that recent research has shown that biofilms are much more that just a few microbes stuck to a surface. New microscopy techniques have revealed that they can be complex microbial communities – sometimes dubbed ‘slime cities’ – with their own architecture, organisation and communication systems. These discoveries could have profound implications for food microbiology and for food hygiene.

The science of microbiology has traditionally focused on microorganisms as single, self-contained entities living independent lives in whatever environment they happen to inhabit. Bacteria and other microbes are typically studied in pure cultures in broth or agar growth media. But in the last decade or so microbiologists have finally begun to understand that microbes don’t normally live like this. They much prefer to live together in communities, and it has been estimated that at least 99% of the world’s microbial biomass exists in biofilms, rather than in a free-living, or ‘planktonic’ state.

The existence of biofilms has been known for almost as long as the existence of microorganisms themselves. If a surface stays wet for long enough, a biofilm will eventually form on it, whether it is a stone in a pond, the hull of a ship, a contact lens, or the surfaces of food processing equipment. The typical food factory is full of wet surfaces that can support the development of biofilms and serve as a reservoir of contamination unless controlled. Not surprisingly, therefore, most research into biofilms has focused on their removal, a task that has sometimes proved to be far from simple.

But, over the last 15 years, the development of new technologies, especially advanced microscopy techniques, such as confocal scanning laser microscopy, has enabled researchers to look more closely at biofilms that form on all manner of surfaces. The advantage of this technique is that it allows the biofilm to be viewed without having to dry it first and without the use of destructive preparation methods that might alter its structure. Applying the technique to biofilms has revealed that, far from being simple slime layers, they can form complex three-dimensional structures.

Biofilm pic

Schematic of a complex three-dimensional biofilm

 

At the base is typically a thin adhesion layer, usually composed of a mixture of bacterial extracellular polymeric substances (EPS) secreted by cells that have adhered to the surface. Rising from this layer are many microcolonies, often produced by different bacterial species. These colonies also exist in an EPS matrix produced by the bacteria in the colony and are often mushroom shaped, with a narrow stalk and broader upper layer that may produce ‘streamers’ that break away from the colony. These ‘towers’ may rise to 100-200 ┬Ám above the surface. Between the microcolonies, a much less viscous gel matrix can be found, often riddled with water channels that allow the movement of nutrients, waste products and free-living cells around the film. Although films consist mainly of bacteria, microfungi, algae and protozoa may also be part of the community.

Such a complex structure means a complex environment allowing the development of redox potential gradients and chemical diffusion gradients that promote the movement of nutrients and metabolites around the biofilm and provide different habitats that can support the growth of a diverse microbial population. It is thought that the most metabolically active microbial cells are in the outer layers of the film, while growth is much slower deep within the EPS matrix. The microbes living in the biofilm inevitably interact with one another and may either compete, or cooperate.

The end result is a mixed microbial community that is much more resistant to environmental stress than free-living microbes or a single species biofilm. Importantly, these mixed species biofilms are also much more resistant to antimicrobials and sanitisers than free-living cells – one reason why they are so difficult to remove or inactivate.

Communication is the key

The question is – how do such apparently simple microbes manage to generate such complex and sophisticated communities? Microbiologists are now beginning to understand how the process operates.

The first stage is adhesion of bacterial cells onto the surface. This process can be passive, or active, depending on whether the cells are motile, and seems to be at least partly triggered by environmental factors, like nutrient levels. Firstly, a reversible attachment occurs involving electrostatic forces and other molecular interactions with the surface. The attachment becomes irreversible within a few hours, and happens through surface bonding of appendages on the cell, such as flagella or pili, and/or by EPS production. Once this has taken place, it is very difficult to remove the attached bacteria without strong mechanical or chemical action.

Although environmental factors seem to induce bacteria to switch from a planktonic to an attached state, the whole process is under genetic control. Many motile bacteria, such as pseudomonads, lose their flagella when they attach to a surface and immediately start to produce EPS as the result of the expression of genes that code for biofilm development. Bacteria attach more readily to some surfaces than to others, but recent research suggests that almost all materials used in food processing equipment will support a biofilm given time.

After attachment has taken place, the bacteria in the biofilm start to clump together and grow to produce microcolonies. Planktonic bacteria from the liquid medium can also be co-opted into the new biofilm at this stage and this seems to be controlled by a process known as ‘quorum sensing’. This phenomenon is best described as chemical communication between individual cells. Certain quorum sensing molecules, such as acyl-homoserine lactones, are produced by cells within the biofilm and diffuse into the medium. When the concentration in the medium is high enough it induces passing planktonic bacteria to switch to an attached state. This bacterial communication system may also be important in the initial attachment process and in regulating the development of the biofilm.

Microcolony development is accompanied by copious EPS production, and if conditions are suitable, the biofilm may start to develop an organised structure. During this ‘maturation’ process the characteristic three-dimensional structure of mushroom-shaped microcolonies in a heterogeneous EPS matrix cut through with many water channels can develop. But sometimes the mature biofilm remains as a single layer of cells embedded in EPS on the surface.

Implications for the food industry

The complexity of biofilms is very interesting, but what does it mean for the food industry? It has to be said that most of the recent research on the structure and development of biofilms has been done in the water supply and water treatment sectors. Biofilms in water may behave very differently from those in a food-processing environment. For example, complex biofilms seem to develop most readily in low nutrient conditions, which are less common in food industry environments. Furthermore, microbial diversity is likely to be much less in some food processing situations than in natural ecosystems, especially where any kind of heat process is employed.

So do these ‘cities of slime’ exist in food processing environments? Unfortunately, there really hasn’t been enough research carried out to be sure. While environments like drains and process water supply pipework certainly have the potential to support these complex communities, it seems less likely that they can develop elsewhere in food factories.

What is known is that some bacterial species that are very important in food microbiology are known to have a propensity to form biofilms. First among these is the pathogen Listeria monocytogenes, a species that is known to colonise wet environments and one which is notoriously difficult to eradicate from food processing environments once established. Listeria has been shown to survive and grow in multi-species biofilms and can form biofilms on many of the materials used in food factories. Persistent strains of Listeria may well be those that form biofilms most readily. Although the role of biofilms in foodborne outbreaks of listeriosis is unknown, it would be surprising if it were never a factor.

Biofilm pic 2

Photomicrograph of a multi-species biofilm on a stainless steel surface

 

Pseudomonas spp. too are often found in biofilms and produce large amounts of EPS. These important food spoilage bacteria have also been shown to form films on stainless steel and other food contact materials and to survive in multi-species biofilms with foodborne pathogens like Listeria. Heat resistant Bacillus spp. and Salmonella are other important food-related bacteria known to attach to surfaces and to form biofilms.

Once bacteria are protected by a biofilm they become remarkably resistant to sanitisers and other chemicals. For example, one study showed that 150 ppm of chlorine was not enough to completely remove a Salmonella biofilm from stainless steel, and other species have been able to survive much higher levels of various sanitisers when protected within a biofilm than when free-living.

A further feature of biofilms that is often overlooked is the difficulty of detecting them when they are present. Most environmental monitoring techniques depend on using a swab, or sponge, to remove bacteria and soil from a surface before using conventional culture methods, or ATP assay, to determine the degree of contamination. Unfortunately, swabbing doesn’t necessarily remove an established biofilm completely, especially if it has formed on a rough surface. This means that the sample could be unrepresentative and give a misleadingly satisfactory result, when, in fact, contamination levels are unacceptably high.

Getting rid of biofilms

Surely an effective cleaning schedule removes any concerns about biofilms? Unfortunately, what is known about biofilms suggests that such a view would be complacent. Once a biofilm is established on any surface, it is very difficult to remove. The presence of EPS is the key to this. It not only attaches very strongly to surfaces and protects bacterial cells from sanitisers and cleaning chemicals, but it also supplies a surface to which more bacteria can readily attach when production resumes. Food particles and other debris can also stick to the EPS layer if it is not removed.

Nevertheless, there are steps that can be taken to prevent the development of biofilms and to remove them when they occur. The best way to approach this is to use HACCP principals and look for steps in a process where biofilm formation is likely, then build in the appropriate controls.

Hygienic design is an important first step. Designing plant that is easy to clean and is constructed from materials that are resistant to biofilm formation is an obvious precaution, but still no guarantee. In areas where biofilm development is possible, cleaning chemicals and sanitisers should be chosen accordingly. For example, in dairy processing, peracetic acid has been shown to be much more effective in dealing with biofilms than chlorine-based sanitisers.

In the future, it may be possible to do much more to prevent the formation of biofilms on food processing surfaces. Although claims have been made in the past for the biofilm resistance of various materials, trials under real processing conditions have often proved disappointing. But one novel approach that is currently being investigated is a nanotechnology solution using very small silver particles to coat surfaces. Silver is known to inhibit bacterial growth and applying a coating at a molecular level could prevent bacterial attachment and biofilm development. Clearly, biofilm-resistant surfaces offer a more effective control solution than improved biofilm removal.

Conclusions

The comparative lack of information about biofilms in food processing environments means that it is difficult to judge their importance. But it seems safe to assume that biofilms do occur, and when they become established in such a situation, will remain as a potential source of product contamination with foodborne pathogens and spoilage bacteria until eradicated. Many persistent contamination problems that defy thorough plant cleaning may well be explained by the presence of a biofilm in the plant. This situation can be improved by the development of plant construction materials that resist biofilm development and by better cleaning methods

While this is a failsafe view to take, it doesn’t address the issue of how recent findings about biofilms affect other, more general assumptions about the behaviour of bacteria in food. Most of what is known about food microbiology is based on pure culture experiments using laboratory strains of free-living cells. But as we have seen, some important food bacteria will form biofilms given the chance and may behave differently as a result, especially if the biofilm contains a mixture of species. We are only just beginning to uncover the complexities of these communities and how they influence product contamination, spoilage and foodborne disease transmission. The investigation of biofilms may reveal some surprises in the future and could even change food microbiology at a fundamental level.

Reference

Biofilm formation and control in food processing facilities
Chmielewski, R.A.N. & Frank, J.F.
Comprehensive Reviews in Food Science and Food Safety, 2(1), 22-32.

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Category: Features

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