Components and functions of the bacterial biofilms’ matrix
One of the biggest concerns in terms of hygiene in the food industry is the formation of bacterial biofilms. Biofilms are groups of microorganisms that accumulate on a solid-liquid interface and are surrounded by a mucilaginous matrix[1]. The biofilm matrix is the extracellular material, mostly produced by the microorganisms themselves, in which the biofilm cells are embedded. It is integrated by a conglomerate of different types of biopolymers, known as Extracellular Polymer Substances (EPS) acting as a support for the biofilm’s three-dimensional structure. Biofilm formation gives cells a way of life completely different from the planktonic state, protecting them from adverse environments and facilitating their survival.
Biofilm formation on surfaces follows a sequential process represented in Figure 1, and it begins with the adhesion of cells to the surface. This results in the formation of microcolonies and it induces the grouped cells to undergo phenotypic changes used to adapt to the new environment. They start producing EPS to generate the biofilm matrix and make the biofilm grow. The mature biofilm has a structure that extends perpendicularly to the surface, often in a mushroom shape, with channels through which water can circulate.
Figure 1. Stages of a mature biofilm development on a surface.
The biofilm matrix retains the cells and keeps them close to each other, allowing a high degree of interaction that includes the intercellular communication and the formation of synergistic microconversions. In addition, the matrix protects organisms against drying, oxidants, biocides, some antibiotics and metal cations, ultraviolet radiation and immune defences. However, the biofilm cells are not completely immobilised, they can move inside the biofilm and detach from it.
There is a great variety of EPS that can form the biofilms matrix, depending on the microorganisms present, the shear forces experienced during their formation, the temperature and the availability of nutrients. In general, the main components of the biofilm matrix are water, polysaccharides, proteins, nucleic acids, lipids and other biopolymers. Figure 2 represents in a simplified way the different EPS that can be found in the biofilm matrix and how they are distributed among their cells.
Figure 2. Representation of the distribution of the biofilm matrix main components (polysaccharides, proteins and DNA) among the cells that inhabit the biofilm[2].
The biofilm structure is influenced by the EPS included in its matrix, as well as by many other factors, such as hydrodynamic conditions, nutrients concentration, bacterial mobility and intercellular communication and metal ions that may exist in the environment.
Some of the biofilm matrix main components are described below:
Exopolysaccharides
Polysaccharides are a predominant fraction of the extracellular matrix, and they appear ubiquitously in biofilms formed in different environments: salt water, fresh water, soils, infections in humans, laboratory cultures, etc. Most of them are long, linear or branched molecules, with molecular mass of approximately 106 D[3] and consist of a mixture of neutral and charged sugars, so they are heteropolysaccharides. They may also contain organic or inorganic substituents that significantly affect their physical and biological properties. For example, one of the most common exopolysaccharides is alginate, consisting of D-manuric and L-guluronic acids and produced in Pseudomonas aeruginosa biofilms, one of the most studied biofilm models [4].
Exopolysaccharides play several essential roles in the biofilm formation, generally associated with their adhesion to surfaces and maintenance of the structural integrity. Their chemical nature can vary depending on the microorganisms that produce them, although exopolysaccharides found in the biofilm matrix do not necessarily reflect the biofilm’s microbial distribution, due to the fact that cells unable to produce exopolysaccharides can be lodged in mixed-species biofilms[5].
Extracellular proteins
Proteins present in the extracellular matrix have functions that enable the biofilm to grow and the cells housed to survive by facilitating the access of nutrients or the integrity regulation and the biofilm stability.
The biofilm matrix contains several extracellular enzymes, generally associated with exopolysaccharides, many of which are involved in the biopolymer degradation. The presence of enzymes that degrade extracellular matrix components converts this matrix into an external digestive system that degrades biopolymers to low molecular mass products that can be assimilated and used as sources of energy and carbon. For example, the exopolysaccharides degradation is mainly due to hydrolases and lyases. These enzymes can act on EPS produced by the same bacteria that produces the enzyme or on EPS from other species. Likewise, the structural EPS degradation plays an important role in the biofilm’s development, since it enables the dispersion of sessile cells of biofilms, allowing the formation of new biofilms. This dispersion takes place in response to environmental changes such as nutrients shortage or sudden availability of nutrients.
Moreover, other enzymes can even degrade the surfaces that lodge the biofilm, as is the case of redox enzymes that contribute to microbial corrosion [6].
The matrix non-enzymatic proteins, such as those associated with cell walls or lectins (carbohydrate-binding proteins), are involved in the formation and stabilisation of the matrix polysaccharide network and are a bond between the bacterial surface and extracellular EPS. These proteins foster biofilm formation in several bacterial species and play roles such as adhesion to inanimate surfaces and host cells [7].
Finally, protein appendages, such as pili, fimbriae and flagella, can also act as structural elements through their interaction with other EPSs of the biofilm matrix.
Extracellular DNA
Extracellular DNA (eDNA) is an integral part of the biofilm matrix and its way of life[8]. For example, it has been shown that eDNA is a major component in the P. aeruginosa biofilms matrix, where it acts as an intercellular connector, and in fact the presence of DNAase inhibits the formation of these biofilms[9]. In other species’ biofilms, eDNA acts as an adhesive or even as an antimicrobial agent, causing cell lysis by chelating cations that stabilise lipopolysaccharides and the bacterial outer membrane.
Surfactants and lipids
Unlike polysaccharides, proteins and DNA, which are hydrophilic molecules, other EPSs with hydrophobic properties are present in the biofilm matrix. This hydrophobic character has been associated with methyl and acetyl substituents in polysaccharides or lipids, which are essential for bacteria adhesion to hydrophobic surfaces.
In addition, other EPS with surfactant capacity, such as surfactin, viscosin and emulsan, can disperse hydrophobic substances and facilitate their availability. Biosurfactants have been identified as factors that promote the initial formation of microcolonies, favouring the migration of bacteria associated to the surface and the formation of mushroom-shaped structures, preventing the colonisation of channels and contributing to the biofilm dispersion.
Water
The main component of the biofilm matrix is water. The exopolymer matrix provides a highly hydrated environment that loses water more slowly than its surrounding environment, thus protecting biofilm cells against fluctuations in water potential. Bacteria actively respond to drying by producing EPS[10]. In addition, the exopolymer matrix can act as a molecular filter, retaining cations, anions, non-polar components and particles in the aqueous stage. EPS contain non-polar regions, groups with potential to form hydrogen bonds, anionic groups (in uronic acids and proteins) and cationic groups (in amino sugars).
EPS and biofilm mechanical properties
In general, biofilms show viscoelastic properties. They can experience both reversible elastic responses and irreversible deformations, depending on the forces acting on the exopolymer matrix. This fact suggests that there are fluctuating binding points between EPS components that remain bonded by weak physical chemical interactions, such as hydrogen bonds, van der Waals forces and electrostatic interactions (see Figure 2). The biopolymers cross-linking contributes to the matrix stability[11]. Likewise, the interaction of multivalent inorganic ions with EPS can significantly influence the biofilms mechanical properties. For example, the presence of Ca2+ increases the mechanical stability of the Pseudomonas aeruginosa biofilms, due to the crosslinking with alginate polyanionic molecules[9].
In conclusion, the bacterial biofilm matrix is an extremely complex environment that enables the lodged cells a way of life completely different from that of the planktonic state. Exopolymer substances (EPS) are essential for biofilm formation and their variety provides various functions that enable the cells expansion and survival in the biofilm against external aggressions. The biofilm matrix complexity and the protection level that this life form provides to microorganisms mean that its formation poses a significant risk to food safety and that biofilms control requires applying specific hygiene procedures and using specialised tools.
Bibliography
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