Biofilm

Formation

Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. While still not fully understood, it is thought that the first colonists of a biofilm adhere to the surface initially through weak, reversible adhesion via van der Waals forces and hydrophobic effects. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. Hydrophobicity also plays an important role in determining the ability of bacteria to form biofilms, as those with increased hydrophobicity have reduced repulsion between the extracellular matrix and the bacterium.

Some species are not able to attach to a surface on their own but are instead 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 (QS) using products such as N-acyl homoserine lactone (AHL). Some bacteria are unable to form biofilms as successfully due to their limited motility. Non-motile bacteria cannot recognize the surface or aggregate together as easily as motile bacteria. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. Polysaccharide matrices typically enclose bacterial biofilms. In addition to the polysaccharides, these matrices may also contain material from the surrounding environment, including but not limited to minerals, soil particles, and blood components, such as erythrocytes and fibrin. The final stage of biofilm formation is known as dispersion, 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 resistant to antibiotics. Cell-cell communication or quorum sensing has been shown to be involved in the formation of biofilm in several bacterial species.

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 heteromorphic cells in several species of bacteria and the yeast Candida albicans. Nitric oxide has also been shown to trigger the dispersal of biofilms of several bacteria species at sub-toxic concentrations. Nitric oxide has the potential for the treatment of patients that suffer from chronic infections caused by biofilms.

It is generally assumed that cells dispersed from biofilms immediately go into the planktonic growth phase. However, recent studies have shown that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. Hence, the dispersal process is a unique stage during the transition from biofilm to planktonic lifestyle in bacteria. Dispersed cells are found to be highly virulent against macrophages and Caenorhabditis elegans, but highly sensitive towards iron stress, as compared with planktonic cells.

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. The social structure (cooperation/competition) within a biofilm depends highly on the different species present.

Extracellular matrix

The biofilm is held together and protected by a matrix of secreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide, although the latter one only refers to the polysaccharide moiety of EPS. In fact, the EPS matrix consists not only of polysaccharides but also of proteins (which may be the major component in environmental and waste water biofilms) and nucleic acids. A large proportion of the EPS is more or less strongly hydrated, however, hydrophobic EPS also occur; one example is cellulose which is produced by a range of microorganisms. This matrix encases the cells within it and facilitates communication among them through biochemical signals as well as gene exchange. The EPS matrix is an important key to the evolutionary success of biofilms. One reason is that it traps extracellular enzymes and keeps them in close proximity to the cells. Thus, the matrix represents an external digestion system and allows for stable synergistic microconsortia of different species (Wingender and Flemming, Nat. Rev. Microbiol. 8, 623-633). 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 (Stromatolites).

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. Extracellular DNA is a major structural component of many different microbial biofilms. Enzymatic degradation of extracellular DNA can weaken the biofilm structure and release microbial cells from the surface.

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.

Habitat

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.

Taxonomic diversity

Many different bacteria form biofilms, including gram-positive (e.g. Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and Lactococcus lactis) and gram-negative species (e.g. Escherichia coli, or Pseudomonas aeruginosa). Cyanobacteria also form biofilms in aquatic environments.

Biofilms are formed by bacteria that colonize plants, e.g. Pseudomonas putida, Pseudomonas fluorescens, and related pseudomonads which are common plant-associated bacteria found on leaves, roots, and in the soil, and the majority of their natural isolates form biofilms. Several nitrogen-fixing symbionts of legumes such as Rhizobium leguminosarum and Sinorhizobium meliloti form biofilms on legume roots and other inert surfaces.

Along with bacteria, biofilms are also generated by a range of eukaryotic organisms, including fungi e.g. Cryptococcus laurentii and microalgae. Among microalgae, one of the main progenitors of biofilms are diatoms, which colonise both fresh and marine environments worldwide.

For other species in disease-associated biofilms and biofilms arising from eukaryotes see below.

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 bacterial vaginosis, 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. Early detection of biofilms in wounds is crucial to successful chronic wound management. Although many techniques have developed to identify planktonic bacteria in viable wounds, few have been able to quickly and accurately identify bacterial biofilms. Future studies are needed to find means of identifying and monitoring biofilm colonization at the bedside to permit timely initiation of treatment.

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.

Research has shown that sub-therapeutic levels of β-lactam antibiotics induce biofilm formation in Staphylococcus aureus. This sub-therapeutic level of antibiotic may result from the use of antibiotics as growth promoters in agriculture, or during the normal course of antibiotic therapy. The biofilm formation induced by low-level methicillin was inhibited by DNase, suggesting that the sub-therapeutic levels of antibiotic also induce extracellular DNA release. Moreover, from an evolutionary point of view, the creation of the tragedy of the commons in pathogenic microbes may provide advanced therapeutic ways for chronic infections caused by biofilms via genetically engineered invasive cheaters who can invade wild-types ‘cooperators’ of pathogenic bacteria until cooperator populations go to extinction or overall population ‘cooperators and cheaters ’ go to extinction.

Dental plaque

Dental plaque is an oral biofilm that adheres to the teeth and consists of many species of both bacteria and fungi (such as Streptococcus mutans and Candida albicans), embedded in salivary polymers and microbial extracellular products. The accumulation of microorganisms subjects the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease.

The biofilm on the surface of teeth is frequently subject to oxidative stress and acid stress. Dietary carbohydrates can cause a dramatic decrease in pH in oral biofilms to values of 4 and below (acid stress). A pH of 4 at body temperature of 37 °C causes depurination of DNA, leaving apurinic (AP) sites in DNA, especially loss of guanine.

The dental plaque biofilm can result in the disease dental caries if it is allowed to develop over time. An ecologic shift away from balanced populations within the dental biofilm is driven by certain (cariogenic) microbiological populations beginning to dominate when the environment favours them. The shift to an acidogenic, aciduric, and cariogenic microbiological population develops and is maintained by frequent consumption of fermentable dietary carbohydrate. The resulting activity shift in the biofilm (and resulting acid production within the biofilm, at the tooth surface) is associated with an imbalance between demineralization and remineralisation leading to net mineral loss within dental hard tissues (enamel and then dentin), the sign and symptom being a carious lesion. By preventing the dental plaque biofilm from maturing or by returning it back to a non-cariogenic state, dental caries can be prevented and arrested. This can be achieved though the behavioural step of reducing the supply of fermentable carbohydrates (i.e. sugar intake) and frequent removal of the biofilm (i.e. toothbrushing).

A peptide pheromone quorum sensing signaling system in S. mutans includes the Competence Stimulating Peptide (CSP) that controls genetic competence. Genetic competence is the ability of a cell to take up DNA released by another cell. Competence can lead to genetic transformation, a form of sexual interaction, favored under conditions of high cell density and/or stress where there is maximal opportunity for interaction between the competent cell and the DNA released from nearby donor cells. This system is optimally expressed when S. mutans cells reside in an actively growing biofilm. Biofilm grown S. mutans cells are genetically transformed at a rate 10- to 600-fold higher than S. mutans growing as free-floating planktonic cells suspended in liquid.

When the biofilm, containing S. mutans and related oral streptococci, is subjected to acid stress, the competence regulon is induced, leading to resistance to being killed by acid. As pointed out by Michod et al., transformation in bacterial pathogens likely provides for effective and efficient recombinational repair of DNA damages. It appears that S. mutans can survive the frequent acid stress in oral biofilms, in part, through the recombinational repair provided by competence and transformation.

Streptococcus pneumoniae

S. pneumoniae is the main cause of community-acquired pneumonia and meningitis in children and the elderly, and of septicemia in HIV-infected persons. When S. pneumonia grows in biofilms, genes are specifically expressed that respond to oxidative stress and induce competence. Formation of a biofilm depends on competence stimulating peptide (CSP). CSP also functions as a quorum-sensing peptide. It not only induces biofilm formation, but also increases virulence in pneumonia and meningitis.

It has been proposed that competence development and biofilm formation is an adaptation of S. pneumoniae to survive the defenses of the host. In particular, the host’s polymorphonuclear leukocytes produce an oxidative burst to defend against the invading bacteria, and this response can kill bacteria by damaging their DNA. Competent S. pneumoniae in a biofilm have the survival advantage that they can more easily take up transforming DNA from nearby cells in the biofilm to use for recombinational repair of oxidative damages in their DNA. Competent S. pneumoniae can also secrete an enzyme (murein hydrolase) that destroys non-competent cells (fratricide) causing DNA to be released into the surrounding medium for potential use by the competent cells.

Biofilms in medicine

The rapidly expanding worldwide industry for biomedical devices and tissue engineering related products is already at $180 billion per year, yet this industry continues to suffer from microbial colonization. No matter the sophistication, microbial infections can develop on all medical devices and tissue engineering constructs.

60-70% of nosocomial or hospital acquired infections are associated with the implantation of a biomedical device. This leads to 2 million cases annually in the U.S., costing the healthcare system over $5 billion in additional healthcare expenses.

If an infection develops a biofilm, it becomes even harder to treat. As the bacteria change, they becomes more resistant to antibiotics and the body's own host defenses.

Biofilms in the food industry

Biofilms have become problematic in several food industries due to the ability to form on plants and during industrial processes. Bacteria can survive long periods of time in water, animal manure, and soil, causing biofilm formation on plants or in the processing equipment. The buildup of biofilms can affect the heat flow across a surface and increase surface corrosion and frictional resistance of fluids. These can lead to a loss of energy in a system and overall loss of products. Along with economic problems biofilm formation on food poses a health risk to consumers due to the ability to make the food more resistant to disinfectants As a result, from 1996 to 2010 the Center for Disease Control and Prevention estimated 48 million foodborne illnesses per year. Biofilms have been connected to about 80% of bacterial infections in the United States.

In produce, microorganisms attach to the surfaces and biofilms develop internally. During the washing process, biofilms resist sanitization and allow bacteria to spread across the produce. This problem is also found in ready to eat foods because the foods go through limited cleaning procedures before consumption Due to the perishability of dairy products and limitations in cleaning procedures, resulting in the buildup of bacteria, dairy is susceptible to biofilm formation and contamination. The bacteria can spoil the products more readily and contaminated products pose a health risk to consumers. One bacteria that can be found in various industries and is a major cause of foodborne disease is Salmonella. Large amounts of salmonella contamination can be found in the poultry processing industry as about 50% of salmonella strains can produce biofilms on poultry farms. Salmonella increases the risk of foodborne illnesses when the poultry products are not cleaned and cooked correctly. Salmonella is also found in the seafood industry where biofilms form from seafood borne pathogens on the seafood itself as well as in water. Shrimp products are commonly affected by salmonella because of unhygienic processing and handling techniques The preparation practices of shrimp and other seafood products can allow for bacteria buildup on the products.

New forms of cleaning procedures are being tested in order to reduce biofilm formation in these processes which will lead to safer and more productive food processing industries. These new forms of cleaning procedures also have a profound effect on the environment, often releasing toxic gases into the groundwater reservoirs.

Eukaryotic biofilms

See also phototrophic biofilms.

Along with bacteria, biofilms are often initiated and produced by Eukaryotes. The biofilms produced by eukaryotes is usually occupied by bacteria and other Eukaryotes alike, however the surface is cultivated and EPS is secreted initially by the Eukaryote. Both fungi and microalgae are known to form biofilms in such a way. Biofilms of fungal origin are important aspects of human infection and fungal pathagenicity, as the fungal infection is more resistant to antifungals.

In the environment, fungal biofilms are an area of ongoing research. One key area of research are fungal biofilms on plants. For example, in the soil, plant associated fungi including mycorrhiza have been shown to decompose organic matter, protect plants from bacterial pathogens.

Biofilms in aquatic environments are often founded by diatoms. The exact purpose of these biofilms is unknown, however there is evidence that the EPS produced by diatoms facilitates both cold and salinity stress. These Eukaryotes interact with a diverse range of other organisms within a region known as the phycosphere, but importantly are the bacteria associated with diatoms, as it has been shown that although diatoms excrete EPS, they only do so when interacting with certain bacteria species.

Biofilms in aquaculture

In shellfish and algae farms, biofouling species tend to block nets and cages and ultimately outcompete the farmed species for space and food. Bacterial biofilms start the colonization process by creating microenvironments that more favorable for biofouling species. In the marine environment, biofilms could reduce the hydrodynamic efficiency of ships and propellers, lead to pipeline blockage and sensor malfunction, and increase the weight of appliances deployed in seawater. Numerous studies have shown that biofilm can be a reservoir for potentially pathogenic bacteria in freshwater aquaculture. As mentioned previously, biofilms can be difficult to eliminate even when antibiotics or chemicals are used in high doses. The role that biofilm plays as reservoirs of bacterial fish pathogens regarding has not been explored in detail but it certainly deserves to be studied.