Biofilms

1. Introduction
Microbial biofilms on surfaces cost the nation billions of dollars yearly in equipment damage, product contamination, energy losses and medical infections. Conventional methods of killing bacteria (such as antibiotics, and disinfection) are often ineffective with biofilm bacteria. The huge doses of antimicrobials required to rid systems of biofilm bacteria are environmentally undesirable (and perhaps not allowed by environmental regulations) and medically impractical (since what it would take to kill the biofilm bacteria would also kill the patient!). So new strategies based on a better understanding of how bacteria attach, grow and detach are urgently needed by many industries such as in dairy processing. Conversely, microbial processes at surfaces also offer opportunities for positive industrial and environmental effects, such as bioremediating hazardous waste sites, biofiltering industrial water, and forming biobarriers to protect soil and groundwater from contamination.

The ubiquity and significance of the biofilm phenomenon is confirmed by the ongoing, long term industrial interest in the work at the Montana State University Center for Biofilm Engineering, whose associates represent a wide range of industries, including petroleum, specialty chemicals, health, household products, drinking water, mining, and utilities Single-celled organisms generally exhibit two distinct modes of behavior. The first is the familiar free floating, or planktonic, form in which single cells float or swim independently in some liquid medium. The second is an attached state in which cells are closely packed and firmly attached to each other and usually form a solid surface. A change in behavior is triggered by many factors, including quorum sensing, as well as other mechanisms that vary between species. When a cell switches modes, it undergoes a phenotypic shift in behavior in which large suites of genes are up- and down- regulated.

2. Definitions biofilms
An aggregate of microbes with a distinct architecture. A biofilm is like a tiny city in which microbial cells, each only a micrometer or two long, form towers that can be hundreds of micrometers high. The "streets" between the towers are really fluid-filled channels that bring in nutrients, oxygen and other necessities for live biofilm communities. A microbial (bacterial, fungal, algae) community, enveloped by the extracellular biopolymer which these microbial cells produce, that adheres to the interface of a liquid and a surface. A complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased.

Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection. Dental plaque, the slimy coating that fouls pipes and tanks, and algae mats on bodies of water are examples of biofilms. While biofilms are generally pathogenic in the body, they can be used beneficially in treating sewage, industrial waste, and contaminated soil.

3. Formation biofilms
The formation of biofilms is in many aspects a most intriguing process. How biofilms are formed and behave is told by the Montana State University and the Science & Technical Encyclopedia. 3.1 Montana State University: Center for Biofilm Engineering Biofilm forms when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of material such as metals, plastics, soil particles, medical implant materials, and tissue. A biofilm can be formed by a single bacterial species, but more often biofilms consist of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products. Essentially, biofilm may form on any surface exposed to bacteria and some amount of water. Once anchored to a surface, biofilm microorganisms carry out a variety of detrimental or beneficial reactions (by human standards), depending on the surrounding environmental conditions.

Figure 1. Biofilm formation

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Place a clean sterile glass slide in a stream of water containing at least minimal nutrients, and over the course of days or weeks a microbial ecosystem will form consisting of a variety of microorganisms arranged in a complex relationship to one another and embedded in a mass of extracellular polysaccharides of their own making. The formation of this biofilm is far from a random process. To the contrary, the formation of a biofilm follows a course the nature of which can be predicted and recorded.

Figure 2. Stages in biofilm formation Typically, within minutes, an organic monolayer adsorbs to the surface of the slide substrate. This changes the chemical and physical properties of the glass slide or other substrate. These organic compounds are found to be polysaccharides or glycoproteins. These adsorbed materials condition the surface of the slide and appear to increase the probability of the attachment of planktonic bacteria. Free floating or planktonic bacteria encounter the conditioned surface and form a reversible, sometimes transient attachment often within minutes. This attachment called adsorption is influenced by electrical charges carried on the bacteria, by Van der Waals forces and by electrostatic attraction although the precise nature of the interaction is still a matter of intense debate. In some instances, as for example, in the association between a pathogen and the receptor sites of cells of its host there may be a stereospecificity which though still reversible is stronger than that achieved strictly by ionic or electrostatic forces. If the association between the bacterium and its substrate persists long enough, other types of chemical and physical structures may form which transform the reversible adsorption to a permanent and essentially irreversible attachment. The final stage in the irreversible adhesion of a cell to an environmental surface is associated with the production of extracellular polymer substances or EPS. Most of the EPS of biofilms are polymers containing sugars such as glucose, galactose, mannose, fructose, rhamnose, N-acetylglucosamine and others. This layer of EPS and bacteria can now entrap particulate materials such as clay, organic materials, dead cells and precipitated minerals adding to the bulk and diversity of the biofilm habitat. This growing biofilm can now serve as the focus for the attachment and growth of other organisms increasing the biological diversity of the community.
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Figure 3. A full grown biofilm The microbial inhabitants with in biofilms and microbial mats in a significant sense behave as multicellular assemblages. Far from being the homogeneous populations usually assumed in planktonic pure cultures, biofilms as simple as colonies on agar surfaces and as complex as pathogen interactions with host cells and the bacterial populations inhabiting the surface of mud flats behave in many respects like the tissues of a multicellular organism. The accompanying illustration presented here (figure 3)courtesy of the Center for Biofilm Engineering gives some concept of the structure and complexity of a typical biofilm. At one time, most scientists thought that biofilms consisted of bacteria randomly distributed within a uniform slimy matrix. However, once scientists began using new imaging tools like the Confocal Scanning Laser Microscope, which allows viewing of hydrated living biofilms, they found that biofilm structures take a wide variety of forms depending on their age and growing conditions. This structural complexity allows bacteria deep within the biofilm to have access to nutrients carried by the convective flow of water. Some of the features typically seen in biofilms are labeled in figure 3. 3. 2 Answers.com Sci Tech Encyclopedia - Biofilms Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. Pili is a fine, filamentous hairlike structure found on the surface of many gram-negative bacteria, shorter, thinner and straighter than flagella. The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Some species are not able to attach to a surface on their own but are often able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. This development of biofilm allows for the cells to become more antibiotic resistant.

3.2.1 Development stages biofilm

During biofilm development, a large number of phenomena occur simultaneously and interact over a large range of length and time scales. As a result of nutrient conversions, the biofilm expands based on bacterial growth and production of extracellular polymers. Chemical species need to be continuously transported to and from the biofilm system by physical processes such as molecular diffusion and convection. Fluid flow drives biofilm growth by regulating the concentrations of available substrates and products. On the other hand, the flow also shears the biofilm surface, and determines biofilm detachment processes. All these linked phenomena create a dynamic picture of the biofilm three-dimensional structure.

In the development of biofilms five stages of development can be recognized. Each stage of development in the diagram is paired with a photomicrograph of a developing Pseudomonas aeruginosa biofilm. All photomicrographs are shown to same scale. The five stages of biofilm development are the following: 1. initial attachment 2. irreversible attachment 3. maturation I 4. maturation II 5. dispersion

3.2.2 Biofilm dispersal

Dispersal of cells from the biofilm colony is an essential stage of the biofilm lifecycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. [2] [3] Biofilm matrix degrading enzymes may be useful as anti-biofilm agents.

3.2.3 Properties
Biofilms are usually found on solid substrates submerged in or exposed to some aqueous solution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performing specialized metabolic functions. However, some organisms will form monospecies films under certain conditions.

Figure 4. Biofilm of Staphylococcus aureus on a catheter Researchers from the Helmholtz Center for Infection Research have investigated the strategies used by biofilms. They discovered that biofilm bacteria apply chemical weapons in order to defend themselves against disinfectants and antibiotics, phagocytes and our immune system. The lead researcher, Dr. Carsten Matz, began a serious investigation in order to find why phagocytes cannot annihilate the biofilm bacteria. He analyzed the marine bacteria, which defend themselves against the amoebae, the behavior of which copies the behavior of phagocytes. The amoebae behave in the sea just like the immune cells in human body: they search for and feed on the bacteria. When bacteria are alone and separated in the water, they become an easy catch for the attackers. However, when they attach to a surface and join other bacteria, the amoebae cannot assault them. The researcher stated that biofilms may be seen as a source of new bioactive agents. When bacteria are organized in biofilms, they produce effective substances which individual bacteria are unable to produce alone. 3.2.4 Extracellular matrix The biofilm is held together and protected by a matrix of excreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signalling molecules. This matrix is strong enough that under certain conditions, biofilms can become fossilized. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000 fold.

3.2.5 Examples of biofilms

Biofilm in Yellowstone National Park. Longest raised mat area is about half a meter long. Biofilms are ubiquitous. Nearly every species of microorganism, not only bacteria and archaea, have mechanisms by which they can adhere to surfaces and to each other.

Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often form on the surface of stagnant pools of water. In fact, biofilms are important components of food chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed. Biofilms grow in hot, acidic pools in Yellowstone National Park (USA) and on glaciers in Antarctica. Biofilms can grow in showers very easily since they provide a moist and warm environment for the biofilm to thrive. Biofilms can develop on the interiors of pipes leading to clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas. Biofilms in cooling water systems are known to reduce heat transfer. Bacterial adhesion to boat hulls serves as the foundation for biofouling of seagoing vessels. Once a film of bacteria forms, it is easier for other marine organisms such as barnacles to attach. Such fouling can inhibit vessel speed by up to 20%, making voyages longer and requiring additional fuel. Time in dry dock for refitting and repainting reduces the productivity of shipping assets, and the useful life of ships is also reduced due to corrosion and mechanical removal (scraping) of marine organisms from ships hulls. Biofilms can also be harnessed for constructive purposes. For example, many sewage treatment plants include a treatment stage in which waste water passes over biofilms grown on filters, which extract and digest organic compounds. In such biofilms, bacteria are mainly responsible for removal of organic matter (BOD); whilst protozoa and rotifers are mainly responsible for removal of suspended solids (SS), including pathogens and other microorganisms. Slow sand filters rely on biofilm development in the same way to filter surface water from lake, spring or river sources for drinking purposes. What we regard as clean water is a waste material to these microcellular organisms since they are unable to extract any further nutrition from the purified water. Biofilms can help eliminate petroleum oil from contaminated oceans or marine systems. The oil is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB). Biofilms are also present on the teeth of most animals as dental plaque, where they may become responsible for tooth decay and gum disease.
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Biofilms are found on the surface of and inside plants. They can both contribute to crop disease or, as in the case of nitrogen fixing Rhizobium on roots, exist symbiotically with the plant. Examples of crop diseases related to biofilms include Citrus Canker, Pierce's Disease of grapes, and Bacterial Spot of plants such as peppers and tomatoes.

Biofilms are found in food processing plants such as in heaters and in evaporators and may cause biofilm recontamination in passing bulk fluid.

3.2.6. Biofilms and infectious diseases

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves. More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficacy in infected skin wounds. It has recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology. Biofilms were also found on samples from two of 10 healthy controls mentioned. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present. New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergy-inflammations. According to the researchers from the Helmholtz Center for Infection Research, they were able to discover the strategies that biofilm bacteria use. In most cases biofilm bacteria develop in crowds and squat on areas where they create a community with other bacteria. They can develop on any surface to which bacteria can affix to. It is interesting to note that these biofilms cannot be destroyed by any disinfectants and antibiotics, not even phagocytes and our immune system are able to annihilate the biofilm bacteria. This is one of the major problems in hospitals if biofilms create a community a catheter or implant, surfaces where bacteria may cause a dangerous infection. Researchers working at the Helmholtz Centre for Infection Research, located in Braunschweig, claim that they have spotted one of the main mechanisms that biofilms use in order to defend themselves against the attack of phagocytes. Teamed up with colleagues from Australia, Great Britain and the United States, scientists are now publishing their discoveries in the popular specialist publication PLoS ONE. The finding states that biofilm bacteria apply chemical weapons to protect themselves. Until recent findings, researchers could not understand the core of the biofilm problem - why phagocytes cannot destroy the biofilm bacteria. Dr. Carsten Matz was the one to start a serious investigation of the problem. The model for his research was the marine bacteria, which face continuous threats in their environment. The threat comes from the amoebae,
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whose behavior resembles the behavior of phagocytes. The amoebae acts the same way in the sea as the immune cells in human body, namely they look for and feed on the bacteria. As long as bacteria are free and separated in the water, they turn into an easy target for the attackers. But when they become affixed to a surface and form a community with other bacteria, the amoebae is unable to assault them.
"The surprising thing was that the amoebae attacking the biofilms were de-activated or even killed.

The bacteria are clearly not just building a fortress, they are also fighting back," says Carsten Matz. In order to protect themselves, bacteria use chemical weapons. Marine bacteria, for instance, uses a very effective molecule called pigment violacein. After the protection system is prepared, the biofilm bacteria turn color soft purple. In case the invaders consume only one cell of the biofilm, together with the pigment included, they are instantly paralyzed and the chemical weapon activates their suicide mechanism. "I feel that these results could offer a change of perspective. Biofilms may no longer be seen just as a problem; they may also be a source of new bioactive agents. When organized in biofilms, bacteria produce highly effective substances which individual bacteria alone cannot produce," says Carsten Matz. Researchers look forward to apply these molecules in order to deal with a certain group of pathogens: parasites living in human body and causing serious infections, including sleeping illness and malaria. It is worth mentioning that amoeba represent olden relatives of these pathogens, which is why weapons obtained by the biofilm bacteria may offer a great basis for the development of new parasiticidal drugs.

Pseudomonas aeruginosa biofilms

The achievements of medical care in industrialised societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the aging population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planctonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies.

Dental plaque
Dental plaque is the material that adheres to the teeth and consists of bacterial cells (mainly Streptococcus mutans and Streptococcus sanguis), salivary polymers and bacterial extracellular products. Plaque is a biofilm on the surfaces of the teeth. This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease.

Legionellosis
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Legionella bacteria are known to grow under certain conditions in biofilms, in which they are protected against disinfectants. Workers in cooling towers, persons working in air conditioned rooms and people taking a shower are exposed to Legionella by inhalation when the systems are not well designed, constructed, or maintained.

4. Biofilms: development and control A novel approach to control biofilms

A pilot plant-scale cheese-milk pasteurisation plant was designed and constructed to study the development of biofilms of Streptococcus thermophilus during pasteurisation of milk, and to investigate methods for preventing this growth from occurring. Under base run conditions, S. thermophilus grew on surfaces in the cool-down sections of the pilot plant, between 50 and 35 C (bulk milk temperature), and could be detected in the product stream after 810 h production, reaching levels of 106 CFU ml1 after 16 h. Thermoduric bacteria also grew in the heating sections of the pilot plant, although to a lesser extent, as did non-thermoduric bacteria that originated in the raw milk. The novel application of temperature step changes, implemented periodically to the growth region of S. thermophilus, successfully controlled the development of biofilms of these organisms. The growth of bacteria on the heating side of the pilot plant was also prevented by the implementation of these same step changes. The optimum step change conditions required to achieve a 20-h production run without detectable growth of S. thermophilus comprised a step change to 55 C, applied for 10 min, with a 60-min interval between step changes.

References:
1. Montana State University: Center for biofilm engineering 2. Answers.com Sci Tech Encyclopedia - Biofilms 3. Temperature step changes: a novel approach to control biofilms of Streptococcus thermophilus in a pilot plant-scale cheese-milk pasteurisation plant . G. C. Knight, R. S. Nicol and T. A. McMeekin . a Centre for Food Safety and Quality, School of Agricultural Science and Tasmanian Institute of Agricultural Research, University of Tasmania, Hobart, Tasmania, Australia b Birubi Innovation Pty. Ltd., 1 Bevan St., Albert Park, Victoria 3206, Australia.

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