A biofilm is a collection of bacteria and/or fungi that exists in a multicellular or community form, encased in an extracellular polysaccharide matrix that they themselves synthesise. They require the presence of water, and form on a solid substrate, on soft tissue surfaces within living organisms and at liquid-air interfaces.

Formation of a biofilm starts with adherence of bacteria followed by production of the polysaccharide matrix. Other bacteria, other micro-organisms and debris become incorporated in the biofilm. Bacteria within a biofilm communicate by quorum sensing leading to phenotypic modifications and changes in gene expression. Bacteria within the biofilm are protected from disinfectants and have a fundamentally reduced antibacterial sensitivity compared with planktonic, free-living, bacteria. Biofilms are highly important in human medicine.

In the pig industry, they are important in reducing antibacterial and disinfectant efficacy, causing the persistence of infections in houses and systems, in blocking water systems and probably have a direct role in pig disease. Control of biofilms is difficult and addresses preventing initial formation and removing existing biofilms. Biofilms are best removed by oxidative disinfectants (Virkon® S or Hyperox® from DuPont Animal Health Solutions).

Jake Waddilove, MA, VetMB, MRCVS

Introduction

Microbes tend to be thought of as rapidly multiplying free floating simple cells. In the real world, this is a misconception. Most microbes live in mixed communities and in many circumstances, these are attached to an environmental surface encasing themselves in an extracellular polysaccharide matrix which they themselves have produced. These attached communities are known as biofilms.

Definition of a Biofilm

A collection of bacteria and/or fungi that exists in a multicellular or community form, encased in an extracellular polysaccharide matrix that they themselves synthesise.

Where are Biofilms Found?

They can occur on solid substratum's in contact with moisture, on soft tissue surfaces in living organisms and at liquid-air interfaces.

To give examples within a pig farm, they can occur in drinking water systems, on permanently damp surfaces in humid housing, within the intestines of pigs, and on dental plaque on teeth. They can also occur in many areas of the world in which we live. Examples include in mountain streams, on puddle surfaces, in plant roots and in heat exchangers in industry. They are highly important in human medicine. Globally, the cost of biofilms is estimated to be many billions of UK pounds.

How do Biofilms Form?

1. There is an initial organic monolayer formed on the slide surface. This is made up of polysaccharide and/or glycoprotein.
2. Free floating or planktonic bacteria encounter this monolayer and form a reversible, sometimes transient attachment to it. The mechanism of this attachment, or adsorption as it is often called, is unclear but appears to be influenced by electrical and electrostatic events.
3. If the attachment of the bacteria to the substrate occurs for long enough (probably a few minutes) other physical and chemical structures are produced which transform the attachment to an irreversible one.
4. Over the next few hours or days there is growth and division of the attached bacteria followed by the production of extracellular polymer substances (EPS). This gives the slimy nature of the biofilm, and contains many sugars such as fructose, glucose, mannose, rhamnose, galactose and N-acetylglucosamine. This stage provides the final part of the irreversible attachment.
5. The slimy EPS-bacterial combination traps particulate matter including organic debris, dead cells, other microbes and precipitated materials. Thus the biofilm becomes a complex structure. Its biological diversity will continue to increase as other microbes are entrapped or individually attach and grow.
6. The biofilm remains a dynamic structure. Bacteria can escape from the structure and regain their planktonic nature to spread elsewhere.

Large biofilms can be seen as pink jelly-like deposits, but they can vary in nature and size. Often they appear as an indistinct slime on a surface. The biofilm has a complex architectural structure with many channels through which water and nutrients can flow. The surface is very irregular and long streamer structures are formed from which bacteria detach. Within a biofilm there are surprising variations in environmental conditions. Large oxygen variations occur and there are significant diffusion gradients of nutrients and waste matter. Bacteria within a biofilm can be aerobic or anaerobic.

Biofilm formation is more than bacteria attaching to a solid surface. Bacteria aggregate with similar bacteria, and can form congregations with members of other species. They accomplish this through chemical mechanisms. When the local concentration of the chemicals indicates that the population has reached a certain minimum density, known as a quorum, the organisms undergo phenotypic changes. This can result in changes in cell metabolism. For example, Pseudomonas aeruginosa within biofilms has been shown to change from aerobic behaviour to anaerobic behaviour. The process of chemical communication is known as quorum sensing. Both gram-positive and gram-negative bacteria use quorum sensing but use different chemicals and systems.

Why are Biofilms Important in the Pig Industry?

1. Antibacterial testing is based on pure cultures and planktonic free floating bacteria. Bacteria within biofilms are protected from antibacterial action. Once in a biofilm they can have several orders of magnitude more resistance to antibiotics than their planktonic counterparts.
2. The resistance of microbes within a biofilm to disinfectants is significantly increased. The resistance of E.coli within a biofilm to chloride within the water has been shown to increase 3000 times. This relates to physical and chemical protection and other possible mechanisms.
3. Water systems can therefore contain pathogens which survive the presence of antimicrobials and disinfectants. Once treatment stops and planktonic organisms start to be shed they can re-infect pigs, or infect the next batch of pigs.
4. The presence of biofilms can make the monitoring of the bacterial status of water inaccurate. If the amount of shedding of planktonic bacteria is low when a sample is taken a false impression of the water quality may be obtained.
5. The increased use of water medication on sugar bases will lead to increased biofilm formation in the water system. Similar effects can be seen with other additives including organic acids. The removal of antimicrobial growth promoters from pig feeds is likely to increase the use of this type of product and hence the incidence of biofilms on farm.
6. Biofilms forming on the roof of a humid house can drip on to underlying structures leading to infection of the pigs below.
7. Biofilms formed on fans or cooling systems could act as seeding points of infection via aerial spread.
8. Thick biofilms can block drinkers, water filters or even water pipes.
9. The undiscovered role of biofilms in some diseases. Apart from other effects, it is known that bacteria within a biofilm are protected from phagocytes.

Biofilms in Human Medicine

1. Resistance of antimicrobials
2. Resistance to disinfectants
3. Biofilms forming on prostheses, implants and catheters leading to infections, e.g. urinary, catheters, heart valves
4. Biofilms leading to infection of humans from the environment, e.g. Legionnaire's disease
5. Biofilms leading to non-healing wounds.
6. Biofilms causing clinical signs, e.g. Pseudomonas aeruginosa biofilms occurring in the lungs of patients with cystic fibrosis leading to pneumonia which is difficult to treat and organisms which are shielded from host defence mechanisms.
7. In dental plaque leading to tooth decay.

The incidence and importance of biofilms in human medicine can be shown by the fact that in the US the Centre for Disease Control and Prevention estimates that biofilms account for two-thirds of the bacterial infections that physicians encounter.

Antibiotic Resistance in Biofilms

It has been stated that bacteria within a biofilm have an increased antimicrobial resistance when compared with planktonic bacteria, and that this is profound.

The exact mechanism for this is unclear, but three hypotheses have been made:

1. The drug fails to penetrate beyond the biofilm surface layer
2. Some bacteria differentiate into a protective phenotypic state
3. Antibiotic action is antagonised within the regions of nutrient depletion or waste production

There now appears to be a fourth mechanism, persistence, where increased expression of certain regulatory genes renders the bacteria less sensitive to a wide range of different antibiotics.

How Can we Control Biofilms?

With their huge cost to industry and health control of biofilms is obviously a major area for research. Control can be divided into several areas.

1. Prevent formation of the biofilm
   1. Obviously the best method is dryness as biofilms are unable to form or survive without water. In pig buildings, decreasing humidity, reducing leaks from pipes and improving poor drainage would be significant controls.
   2. By preventing the initial adhesion of bacteria. Practically this is very difficult. In one example, a special silver coating was developed to stop endocarditis associated with biofilm forming on replacement heart valves. It failed because the coating promoted rather than prevented biofilm formation. If a coating could be found for water pipes, it would help.
   3. By interfering with quorum sensing mechanisms, and thus the structure of the biofilm. Possibly a very important approach in human medicine.
   4. By sensitising biofilm bound bacteria to existing antibiotics.
   5. Passing an electrical current through a biofilm can affect this.
   
   
   
2. By removing biofilms once they have formed. This is difficult to achieve but it is the only current practical approach in pig housing.
   1. By allowing the surface on which the biofilm is formed to dry. This is effective where it can be done.
   2. By proper cleaning and disinfection. For a biofilm to be removed from a surface the surface must first be cleaned of all other organic contamination. This involves thorough washing using a heavy-duty detergent, e.g. Biosolve® Plus (DuPont Animal Health Solutions). The surface then needs to be disinfected using an oxidising disinfectant, e.g. Virkon® S and Hyperox® (DuPont Animal Health Solutions). In severe situations, combinations of detergents and oxidising disinfectants are needed.
   3. Within drinking water systems a degree of control can be achieved once the system is cleared of biofilms by frequent sanitation of the system with a disinfectant which prevents biofilms developing but can be drunk by animals, e.g. Virkon® S (DuPont Animal Health Solutions) at a dilution of 1:1000.

Biofilms Can be Good

It is worth being aware that biofilms do have a positive side. They have shown promise in cleaning up ground water and helping rid over-fertilised land of excess nitrogen. They can be used to contain spills by forming biobarriers, and have been used in microbial enhanced recovery from old oil fields and spills, and similarly to clear up jet fuel, volatile organic compounds and chlorinated solvents. They have uses in scavenging certain metals from water.

They are used in water treatment plants and private septic systems to remove pathogens and reduce organic matter in the waste water. Finally they can be used to help produce biochemicals which may be of use as medicines, food additives or similarly.

This paper was presented at the Meeting of the British Pig Veterinary Society and to the Association Française de Médecine Vétérinaire Porcine, Paris, and was originally published in The Pig Journal.

Further Reading

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