Biofilms are common in natural environments and the water treatment industry and are also a major problem in the medical sector. Food technologists too are familiar with biofilms as layers of micro-organisms embedded in a kind of microbial slime that form on wet surfaces in food processing areas and make them more difficult to clean properly. Biofilms are a potential source of product contamination and, in extreme cases, can cause serious fouling problems in equipment like plate heat exchangers. But 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 testing and for food hygiene.
The science of microbiology has traditionally focused on micro-organisms 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 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.
Looking inside biofilms
Over the last 15 years, the development of new techniques, especially advanced microscopy techniques, such as confocal laser scanning microscopy (CLSM), has enabled researchers to look more closely at biofilms that form on all manner of surfaces. CLSM was developed in the late 1980s and has become an important technique in medical microscopy. It works by passing a beam of laser light through a narrow aperture that allows it to be focused onto a very small volume of the sample being observed. The sample is treated with dyes that fluoresce when illuminated by the laser. This creates a mix of reflected laser light and emitted fluorescent light that is collected by the objective lens. The collected light then passes through a beam splitter that only allows the laser light to pass through and reflects the fluorescent light through another small aperture and into a photodetection instrument, such as a photomultiplier tube. The signal is then recorded as a piece of digital data. The effect of the detector aperture is to block all the light from the specimen that is not coming from the focal point, and which would therefore cause blurring of the image. The result is a very sharp and discrete image of a tiny portion of the focal plane. The system then scans across the entire focal plane and builds up a complete digital image of it. As the laser light only illuminates a small focal volume for each individual part of the image, CLSM is capable of taking optical sections through a relatively thick specimen. By raising and lowering the microscope stage, multiple sections can be scanned, allowing a three-dimensional image of the specimen to be built up by a computer. The advantage of this technique is that it allows the specimen to be viewed without having to dry it first and without the use of destructive preparation methods that might alter its structure. It can even be used to
examine living biological systems.
Applying this technique to biofilms has revealed that, far from being simple slime layers, they can form complex three-dimensional structures. 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 µm 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 natural 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.
How biofilms are built
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 eventually support a biofilm.
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 carried out 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 pipes 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 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.
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. Even relatively delicate bacteria like Campylobacter may be able to survive in biofilms. It has long been a mystery how these fragile pathogens seem able to persistently recontaminate poultry products in the harsh environment of the poultry processing plant. Recent research from the University of Arkansas has shown that Campylobacter jejuni is able to survive inside established Pseudomonas biofilms on processing machinery, even though it is unable to form a biofilm itself.
There is evidence that biofilms may form on the inside surfaces of packaging, especially in relatively-long shelf life products like cheese and cooked meats. This could have an effect on product shelf life. Biofilm-like communities may form on the surface of foods or even within them, especially in complex products assembled from more than one component and where boundary layers exist. This could have important implications for microbiological sampling and testing methods.
Detecting and controlling biofilms
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. Other methods, such as agar contact plates, are even less effective in this respect. This means that the sample could be unrepresentative and give a misleadingly satisfactory result, when, in fact, contamination levels are unacceptably high. At present there is simply no reliable method for quantifying microorganisms existing in biofilms on food processing surfaces.
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. Food particles and other debris can also stick to the EPS layer if it is not removed. Some degree of control can be accomplished by a combination of hygienic design and careful choice of cleaning and sanitising chemicals. In the future, it may be possible to design surfaces that are resistant to biofilm development, perhaps by applying a coating of nanosilver particles to prevent bacterial attachment.
Wider implications for food
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.
But are biofilms anything more than just a nuisance? There is reason to suspect that they may be much more important than we had previously thought. 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 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. Nearly everything we know about the behaviour of micro-organisms in foods, such as their resistance to high salt concentrations or low pH values, is based on experiments carried out in culture and we design food safety controls based on the results. But do these properties change when microbes exist in biofilms? The answer is that we simply do not know at present. We can see inside biofilms with CLSM, but what is needed now is a set of tools to study the characteristics of microbes, not in a test tube, but in a biofilm community.
Molecular biology could provide some of the answers. As we learn more about the genes that control microbial characteristics important in food safety, it may be possible to determine if and when these genes are expressed in the planktonic and biofilm environments. This could open up a whole new field within food microbiology and help in the design of safer and longer lasting