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There are primarily two different forms in which microbes can be found in nature; the planktonic and the sessile form, where bacteria fungi and yeasts can be found as free floating microorganisms in moist environments, or attached in large numbers to a surface and form slimy layers respectively. For a long time, bacteria were described as unicellular life forms and in order a certain microorganism to be studied it should be diluted to a single cell and studied in a liquid culture. Environmental microbiologists were the first to introduce the term ''bacterial communities'', after examining natural populations of different bacteria found in real ecosystems. Antoinie van Leuwenhoek in the late 1940's was one of the first scientists to describe communities of ''animalcules'' after examining tooth surfaces (Costerton, 1999) and not until recently, it has been established that a large number of microorganisms are able to adhere to surfaces, in moist environments, within a structured biofilm ecological community and not as free floating microorganisms, excreting a slimy, glue-like substance. (Costerton, 1995; Davey and O'Toole, 2000). Mitchell and Marshall examined the first stages of formation of these bacterial communities to surfaces in 1964, whilst in the early 1970's Charaklis showed that these communities where highly resistant to disinfectant agents and they could be formed in industrial systems when moisture was available (Charaklis, 1973). A few years later, in 1978, Costerton explained the mechanisms of microbial adhesion in different materials and surfaces, explaining the features and importance of biofilms and being the first to introduce the term ''biofilm'' (Prakash et al., 2003). Development of different techniques such as scanning electron microscopy (SEM) and culture techniques have contributed to a better investigation and understanding of biofilm formation and characterization.
A biofilm can therefore be defined as ''the sessile community of microbes characterized by cells that are irreversibly associated with a surface, embedded in a matrix of extracellular polymeric substances (EPS) and display an altered phenotype with respect to gene expression, protein production and growth'' (Cloete et. al., 2009). They are dynamic communities held together by extracellular polymeric substances which are sugary molecular strands and allow biofilms to develop complex, three-dimensional communities (Costerton, 1995). Biofilm formation is a phenomenon that occurs in a wide variety of different materials, both natural and man-made environments, such as metals, plastics, medical materials and most ecosystems where moisture and nutrients are available. This adhesion to different surfaces for the formation of microbial communities is a general strategy for survival, especially in cases where the level of nutrients is low; when, however, the conditions are unfavorable cells can detach and colonize to other surfaces. Biofilms can be composed by a single bacterial species; however, in nature almost always they consist of a community of multiple species of bacteria (as well as yeasts, algae, fungi and protozoa) (Costerton et al., 1994). In both situations multicellular development is essential for the biofilm growth. The role of biofilms in nature is quite important as they can produce and degrade organic matter and environmental pollutants and, rotate many metals. Development of biofilms can offer important advantages to cells; it allows close contact between different species and symbiotic relationship providing essential nutrients and potential exchange of genetic material, while the matrix of the biofilms allows them to tolerate higher levels of antibiotics, bacteriophages or predator organisms (Nuno Miguel, 2006).
Biofilm formation is characterized by features different than the ones microorganisms show in their suspended form; they consist of high levels of water (almost 90%), organic and inorganic substances such as debris or humic substances and as mentioned before, cells are embedded in a matrix of extracellular polymer substances which contributes to almost 90% of the organic matter and consists of proteins and polysaccharides. There are also differences between biofilm bacteria and their planktonic form in term of the genes they express, the rate at which the cells grow and their antibiotic or chemical biocide resistance (Cloete et. al., 2009; Jefferson, 2004). Finally they differ in yet another way in addition to their heterogeneity as a result of different diffusion rates of available oxygen or nutrients which allows different metabolic tasks within the biofilm (Jefferson, 2004).
2. General model for biofilm formation
Biofilm formation is a process which includes different stages; generally there are 3 main stages which describe the pattern of biofilm development as shown in figure 1 (a) and can be applied to different species. Those stages are:
a) The stage of the initial attachment of the microorganisms to a surface,
b) The formation of bacterial communities (microcolonies) on the surfaces to be colonized. In some cases, during the early formation of the bacterial biofilms, individual bacteria may form pillar-like or chain like structures as shown in figures 1(b) and 1(c).
c) The stage of differentiation of these communities into mature biofilms.
Fig.1: (a) different stages of biofilm formation, (b) pillar-like structures, (c) individual bacteria forming chain-like structures (Armitage, 2005)
It is commonly believed that nonspecific interactions (such as hydrophobic interactions) intervene during the initial attachment of bacteria to abiotic surfaces, whereas in the case of bacterial attachment to living tissue specific molecular docking mechanisms take part (Carpentier and Cerf, 1993)
Different environmental factors such as temperature, pH, iron availability and oxygen tension, usually act as the initiatory power that trigger the transition from planktonic growth to biofilm formation (O'Toole and Kolter, 1998a,b). Adhesion of microorganisms to different surfaces is usually affected and controlled by different variables including the environmental factors mentioned above, as well as, the surface composition on which bacteria will adhere, the species of bacteria and essential gene products. It has been proposed that the initial attachment of bacteria into surfaces and a potential biofilm growth is a two-step process; the primary, reversible stage (or docking stage) when bacteria make weak attachments, and the secondary, irreversible stage (or locking stage) in which bacteria must stay in contact with the substratum and grow a biofilm, a stage which mainly depends on the properties of the bacterial cells and the colonized surfaces. When the conditions for microbial attachment are favourable the reversible attachment is followed by the irreversible. It has been also suggested an additional stage, called surface conditioning, which occurs when films of organic molecules attach to clean, fluid surfaces and describes the interaction between the substratum and the surrounding environment (An et. al., 2000; Gristina, 1987).
The development of biofilms involves a cell-to-cell communication between bacteria, through excreted chemical signals, known as Quorum Sensing (QS). These signals are referred as auto-inducer molecules (AI's); when the microcolonies reach a quorum level (threshold concentration) the auto-inducer molecules bind to specific transcription regulators and this form of communication regulates gene expression, as well as, enhances access to nutrients and protects against environmental stresses (Cloete et. al., 2009). Quorum sensing signals are essential for the formation of biofilms and closely regulated to the auto-inducer molecules and also play a significant role in the cell detachment and triggers cell dispersion, when the microcolonies reach a threshold concentration (Hentzer et al., 2002).
2.1. Primary stage of attachment
The primary stage of attachment is characterized by instability and, is in fact the first step of contact between planktonic microorganisms and conditioned surfaces and this interaction between the bacterial cell surface and conditioned surfaces is characterized by a number of physiochemical interactions; phenomena of chemotaxis and bacterial motility facilitates the microorganisms to be brought into close contact with the surface to be colonized, in a directed or random fashion. Electrostatic, van der Waals's forces and hydrophilic interactions, temperature and steric hindrance between the two surfaces determine the level of adhesion between the two surfaces after the organism reaches critical proximity to a surface (~1nm) (Cloete et. al., 2009; Liu et al., 2004; An et. al, 2000;).
Motile and non-motile bacteria species show a different pattern in biofilm formation. When conditions for subsequent biofilm formation are favourable, non-motile species display an increased adhesin expression and become more sticky, which promotes both cell to cell and cell to surface interaction (figure 2a). This behaviour is met in some staphylococcal species, in which a group of surface proteins involved in the formation of biofilms (Bap proteins) promote cell to cell adherence and contribute to extracellular matrix(Gotz 2002, Lasa and Penades 2006). On the other hand, when conditions are propitious, motile species (figure 2b) show quite a different pattern than the non-motile; once they adhere to a surface they completely change they characteristics. They lose their motility and produce an extracellular matrix which helps the cells to be held together (Lemon et. al., 2007).
It is also characteristic that during the primary stage of attachment a contact and subsequent stick between planktonic microorganisms and other species or surface bound organisms is likely, resulting in the formation of aggregates on the substratum. In fact, in some cases presence of one species on a surface can facilitate the adhesion of another (Merritt and An. 2000). Multiple adhesins are produced by the bacteria which allow the change from sessile to planktonic form under different environmental conditions. Such is the case in some staphylococcal species, including S. epidermidis which produces a polysaccharide intercellular adhesin (PIA) and promotes a cell to cell adhesion with other bacteria to an already existing biofilm, resulting in a multilayer biofilm (Dunne, 2002)
2.2. Secondary stage of attachment
This, irreversible stage of bacterial attachment, is characterized by molecularly binding between the surface and specific bacterial adhesins. The successful attachment of loosely bound organisms is ''secured'' by the production of exopolysaccharides and the development of a complex between receptor ligands on pili and surface materials (Dunne, 2002). Firm attachment is seconded by proteins on the surface of bacteria, motility structures such as flagella and pili or exopolymers secreted by bacteria and when conditions for attachment are propitious, bacterial cells, under dipolic, ionic or hydrophobic interactions, switch to a more stable, irreversible attachment. The role of these structures is very distinct during biofilm formation; motility structures for example, such as flagella, are important in the primary stages of biofilm formation and essential for some bacteria to overcome repulsive forces during attachment to abiotic materials (Cloete et. al., 2009).
After this stable attachment a number of genotypic and phenotypic changes start to take place to create a more protective environment and stabilize the development and maturation of the biofilm, resulting in a more frequent gene exchange, increased synthesis of EPS and higher levels of tolerance against antibiotics and UV light (O' Toole et. al., 2000).
At the end of the secondary stage of adhesion bacteria are firmly attached to the surface, the boning becomes permanent, physical or chemical interventions no longer take place and attachment becomes irreversible (Stoodley et. al., 2002) the following figure illustrates the stages of reversible and irreversible biofilm development, maturation and cell detachment. (Figure 3). Finally if the conditions are no longer favorable, cells can detach and return to their planktonic form.
2.3. Microcolony formation
After the secondary stage of attachment the conditions become stable for the formation of microcolonies and bacteria cells start to emit chemical signal that act as ''communication signals'' promoting multiplication. Exopolysaccharide production is then activated, bacterial multiplication and a subsequent microcolony formation starts to take place within the embedded extracellular polysaccharide matrix and microorganisms eventually become immobilised in relatively close proximity to one another (Prakash et. al., 2000). The development of microcolonies is a complex process governed by a several mechanisms and once again motility plays a significant role. For example, flagella and type IV pili in Ps. aeruginosa and flagella, type I pili and fimbriae in E. coli, are surface motility structures that assist the redistribution of cell attachment in motile bacteria while in the case of the non-motile S. epidermidis protein adhesins and polysaccharides were found to be involved in attachment (Cloete et. al., 2009).
Larger amount of cells within the extracellular polysaccharide matrix lead to the development of macro-colonies with increased metabolic and physiological heterogeneity resulting in an increased development of physical and chemical microgradients respectively, such as nutrients, pH and oxygen. Clonal growth of cells, direct attachment of planktonic cells in the already existing microcolonies and translocation of surface cells in the formed microcolonies result in the macro-colony development mentioned above. Synergistic interactions and homeostatic mechanisms can also develop during the growth of microcolonies, protecting the community from external dangers and additional location of bacteria within the biofilm establishes interactive groups of microorganisms achieving physiological cooperation.
Is not easy to define biofilm thickness since it is mainly depended on the different environmental factors; however it can be proposed that when the thickness is around 20-25nm biofilms enter the stage of maturity characterized by stability and a switch from aerobic to anaerobic conditions.
2.4. Biofilm maturation
The irreversible attachment of the microorganisms follows the stage of maturation in which a number of physicochemical changes take place; bacteria start to actively replicate, they are redistributed away from the substratum, the complexity and the density of the existing biofilm is increased and the glycocalyx is created after the interaction between the bacteria and both organic and inorganic molecules. According to Mittelman (1985), "Attachment is mediated by extracellular polymers that extend outward from the bacterial cell wall (much like the structure of a spider's web). This polymeric material, or glycocalyx, consists of charged and neutral polysaccharides groups that not only facilitate attachment but also act as an ion-exchange system for trapping and concentrating trace nutrients from the overlying water. The glycocalyx also acts as a protective coating for the attached cells which mitigates the effects of biocides and other toxic substances." (Mittelman, 1985). Furthermore, three dimensional architecture is developed and the structure of the biofilm is mushroom-like with a number of pores and water channels which deliver nutrients to most parts of the community (figure 4) (Reisner et. al., 2003). The availability and perfusion of nutrients, as well as, the the removal of waste are factors that limit the biofilm growth. Other factors that may affect the biofilm maturation are carbon sources, the level of the internal pH, osmolarity, oxygen perfusion and the type of the surface. O' Toole and Kolter have described two types of surfaces; high surface energy materials which are negatively charged and low surface energy materials which are either low positively or low negatively charged (O' Toole and Kolter, 1998a,b). When the biofilm reaches a critical mass the external layers generate planktonic organisms which can detach from the biofilm and colonize other surfaces (Dunne, 2002)
2.5. Detachment and dispersion of the cells
Detachment and dispersion of cells is considered as a step which determines the maximum accumulation and the thickness of a biofilm, influenced by a number of factors including external forces, fluid dynamics and shear effects of the bulk fluid (Brugoni et. al., 2007). Characklis and Tulear showed that when biofilms are grown under low shear conditions detachment is more frequent whilst more compact biofilms with decreased levels of detachment is witnessed under high shear conditions (Characklis and Tulear, 1982).
The level of nutrients has been also suggested as a potential factor for detachment. It has been observed that low level of nutrients promotes the formation of biofilms, as bacteria prefer forming communities rather than staying at their planktonic form. On the other hand, bacteria are unable or form loose instable floc-like communities under high level of nutrients and prefer to remain in their bulk liquid environment. This behaviour was observed by Peyton and Characklis who showed that under low nutrients loads Ps. aeruginosa releases a low number of cells whilst large number are released under high level of nutrients (Danhorn et al. 2004; Jackson et al. 2002, Peyton and Characklis 1993). It is not easy though to determine the role of nutrient sufficiency on biofilm detachment since numerous reports suggest the alternative hypothesis, that low levels of nutrients promote the detachment and cell dispersion. For instance, a study conducted by Hunt and his colleagues in 2004 revealed that nutrient starvation in A. hydrophila and Ps. aeruginosa led to increased detachment (Hunt et. al., 2004).
When external forces are high, larger numbers of cells and polymers are transferred to the bulk liquid leading to increased biomass detachment and cell transfer. As the biofilm grows the thickness of the extracellular polymeric substances increases resulting in the development of anaerobic conditions within the biofilm and as a consequence the film sloughs off from the surface of the substrate. Different polysaccharides produced during the growth of the biofilm by the microorganisms degrade EPS and contribute to detachment as well (Spiers et.al., 2003, Prakash et. al., 2003).
There have been suggested four different mechanisms for biofilm detachment in accordance to the chemical and physical environment acting upon a biofilm (Figure 5) those being abrasion, which is used to describe the removal of biofilm biomass and the release of cells due to collision between biofilm carriers, grazing, which also describes the loss of biofilm as a result of feeding activity of eukaryotic organisms and can reduce the overall biomass in significantly low levels if feeding activity is high. The removal of small particles of biofilm as a result of a fluid shear in a flowing system is referred as erosion, and finally, sloughing is the rapid removal of large particles of biofilm containing bacteria within the extracellular matrix (Davies, 2010)
In a different mechanism of detachment, the dispersion, active, live bacteria are released as a result of internal or external stimuli. The main difference between detachment and dispersion is that in the second case bacteria change their behaviour. It is characterized as the terminal stage of the biofilm's development where bacteria return to their planktonic form and it occurs as a continuing process. The process of dispersion is not affected by fluid shear to overcome binding forces and is mainly a response mechanism by bacteria when conditions are not favourable. During dispersion bacterial cells are released from the inner part of the biofilm leaving void spaces. Studies have revealed a predispersion behaviour before the actual dispersion when bacteria are found floating within the biofilm. The biofilm then starts to increase its volume, a breach is made and bacteria through this breach can migrate and enter the bulk liquid as planktonic bacteria (Figures 6 and 7). A study conducted by Tolker and his colleagues showed that the determinative factor that starts dispersion is the size dependant rather than the age of the microcolonies; microcolonies must reach a certain size in order the dispersion to take place (Tolker et. al., 2000)
3. Extracellular polysaccharides (EPS) and their role in biofilms
The biofilm extracellular polysaccharides is a polymeric conglomeration, involved in the production of microbial aggregates, highly hydrated as it can incorporate high levels of water into its structure, and despite the fact that the term implies that it is composed by polysaccharides it may contain other molecules such as proteins, DNA, lipids and nucleic acids which in some cases may prevail those polysaccharides (Allison et al., 2000). Tsuneda and his colleagues estimated that almost 90% of the EPS is comprised by proteins and polysaccharides. Most of the biofilms are characterized by different amount of EPS synthesis which are age dependant and greatly influenced by the balance between carbon substrates and other limiting nutrients such as phosphate potassium or nitrogen; the older the biofilms, the higher the amount of EPS. Limitations of those nutrients and high amounts of carbon substrates will also promote the synthesis of EPS. It is characterized as a heterogeneous polymeric conglomeration mainly consisted of high molecular weight polymers produced by microorganisms, with a different composition amongst different microbial communities, controlled by different processes including cell lysis, active secretion and absorption from the environment (Wingender et. a.l, 1999). The schematic view of the EPS components are illustrated in the following figure.
Figure 8: Components of the EPS (Kristensen et. al., 2008)
The production of EPS is facilitated by different parameters such as the composition of the substratum, the hydrodynamic shear forces and the conditions (e.g temperature) of the culture as well as, the nutrient status of the medium. The main functions include providing stability to the biofilm structure and protection against antibiotic, biocides or other harmful effects, promotes microbial aggregation, surface adhesion and accumulation of enzymatic activities. In terms of cell aggregation it has been suggested that EPS allows communication and cooperation between the cells (Laspidou and. Rittmann, 2001).
The attachment of cells to surfaces is also related to the production of EPS; cells may attach easier in a surface after a change in the physicochemical properties of the attachment surface facilitated by the production of EPS. It has been found that production of high amounts of EPS increased cell attachment due to interactions between the polymeric substances. On the other hand electrostatic interactions developed under the production of low amounts of EPS inhibit the bacterial attachment (Tsuneda et. al., 2003). Another function of this substance is its ability to act as a protective layer against penetration of antimicrobial agents into the cells by preventing access or effectively reducing the concentration of those compounds. Finally EPS have the ability to bind high amount of water, protecting the inner biofilm cells against dehydration by forming a hard protective outer layer under water limited conditions (Sutherland, 2001). Interestingly, a number of studies concluded that cells that are unable to synthesize EPS, may form microcolonies, they are however unable to form mature biofilms (Watnick and Kolter, 1999).