Biofilm



A biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium.

Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.

Formation
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 adhesion via 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.

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 using such products as AHL. 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. The development of a biofilm may allow for an aggregate cell colony (or colonies) to be increasingly antibiotic resistant.

Development
There are five stages of biofilm development (see illustration at right):
 * 1) Initial attachment:
 * 2) Irreversible attachment:
 * 3) Maturation I:
 * 4) Maturation II:
 * 5) Dispersion:

Dispersal
Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle. 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. Biofilm matrix degrading enzymes may be useful as anti-biofilm agents. Recent evidence has shown that a fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces cyclo hetermorphic cells in several species of bacteria and the yeast Candida albicans

Properties
Biofilms are usually found on solid substrates submerged in or exposed to an 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 (visible to the naked eye). Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performs specialized metabolic functions. However, some organisms will form single-species films under certain conditions.

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 a thousandfold. Lateral gene transfer is greatly facilitated in biofilms and leads to a more stable biofilm structure.

However, biofilms are not always less susceptible to antibiotics. For instance, the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials than do stationary-phase planktonic cells, although when the biofilm is compared to logarithmic phase planktonic cells, the biofilm does have greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.

Examples
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 will form on virtually every non-shedding surface in a non-sterile aqueous (or very humid) environment.
 * 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 can grow in the most extreme environments: from, for example, the extremely hot, briny waters of hot springs ranging from very acidic to very alkaline, to frozen glaciers.
 * In the human environment, biofilms can grow in showers very easily since they provide a moist and warm environment for the biofilm to thrive. Biofilms can form inside water and sewage pipes and cause clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas.
 * Biofilms in cooling- or heating-water systems are known to reduce heat transfer.
 * Biofilms in marine engineering systems, such as pipelines of the offshore oil and gas industry, can lead to substantial corrosion problems. Corrosion is mainly due to abiotic factors; however, at least 20% of corrosion is caused by microorganisms that are attached to the metal subsurface (i.e., microbially-influenced corrosion).
 * 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 reduce maximum vessel speed by up to 20%, prolonging voyages and consuming 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), while 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).
 * Stromatolites are layered accretionary structures formed in shallow water by the trapping, binding and cementation of sedimentary grains by microbial biofilms, especially of cyanobacteria. Stromatolites include some of the most ancient records of life on Earth, and are still forming today.
 * Biofilms are present on the teeth of most animals as dental plaque, where they may cause tooth decay and gum disease.
 * Biofilms are found on the surface of and inside plants. They can either 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 used in microbial fuel cells (MFCs) to generate electricity from a variety of starting materials, including complex organic waste and renewable biomass.

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 efficiency in healing or treating 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.

Biofilms can also be formed on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices.

New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergy-inflammations.

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 planktonic 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 sanguinis), 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. The bacterial strains identification of the biofilms isolated from the dental plaque or from the biofilms attached to the surfaces of some dental alloys, impression materials, dental implants, restorative and cement materials play an essential role concerning the biofilms establishment dynamics towards the physical-chemical properties of the materials which biofilms are attached to.

Legionellosis
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.

Neisseria gonorrhoeae biofilms
Neisseria gonorrhoeae is an exclusive human pathogen. Recent studies have demonstrated that it utilizes two distinct mechanisms for entry into human urethral and cervical epithelial cells involving different bacterial surface ligands and host receptors. In addition it has been demonstrated that the gonococcus can form biofilms on glass surfaces and over human cells. There is evidence for formation of gonococcal biofilms on human cervical epithelial cells during natural disease and that outer membrane blebbing by the gonococcus is crucial in biofilm formation over human cervical epithelial cells.

Molecular genetics
Technological progress in microscopy, molecular genetics and genome analysis has significantly advanced our understanding of the structural and molecular aspects of biofilms, especially of extensively studied model organisms such as Pseudomonas aeruginosa. Biofilm development can be divided into several key steps including attachment, microcolony formation, biofilm maturation and dispersion; and in each step bacteria may recruit different components and molecules including flagellae, type IV pili, DNA and exopolysaccharides. The rapid progress in biofilm research has also unveiled several genetic regulation mechanisms implicated in biofilm regulation such as quorum sensing and the novel secondary messenger cyclic-di-GMP. Understanding the molecular mechanisms of biofilm formation has facilitated the exploration of novel strategies to control bacterial biofilms.