Chapter 1: Biofilm: A Life for Microorganisms with Basic Biofilm Principles

In the 1970s, bacteria were mostly thought to be solitary, free-floating creatures, a concept that dates back to the ground-breaking research of Robert Koch, the father of modern microbiology. However, the advent of drug-resistant bacteria and the difficulty of eliminating some bacteria prompted a reassessment of the bacterial lifestyle. It is now recognised that bacterial cells within a self-produced matrix, known as bacterial aggregates, provide bacteria with methods to resist biocides. Later, in 1978, this bacterial aggregation was termed ‘biofilm’ by Costerton.¹  Biofilms are characterised as aggregates of microorganisms in which cells are frequently immersed in a self-produced matrix of extracellular polymeric substances (EPS) that adhere to one another and/or a surface. The term ‘aggregate’ was used as it accounts for the fact that most cells in multi-layered biofilms experience cell-to-cell contact. This can occur in flocs, which are mobile biofilms that grow in the absence of a substratum, or in surface-attached biofilms, in which only one layer is in direct contact with the substratum.²  Biofilm communities develop novel structures, activities, patterns, and features through self-organisation in complex systems. The morphologies of biofilms vary based on the bacteria that make them up and the environment in which they develop. The social and physical interactions between cells as well as the characteristics of the matrix set the biofilm lifestyle apart from that of free-living bacterial cells. Consequently, biofilm communities exhibit emergent traits, or novel characteristics that arise within the biofilm and are not anticipated based on examining free-living bacterial cultures.³  Therefore, they are often referred to as ‘biogenic habitat formers’.²  The biofilm EPS matrices, collectively termed the ‘matrixome’, also delineate internal biofilm activities and interactions with the outside world. They function as an interface, or preferably, an “interspace,” between the biofilm and its surroundings. Additionally, the biofilms receive a spatial organisation from the matrix, which provides them with high biodiversity, steep gradients, and intricate, dynamic, and cooperative interactions, such as improved horizontal gene transfer and cell-to-cell communication. This matrix comprises lipids, lipopolysaccharides, and glycopeptides, which act as a scaffold to hold the bacterial community in the biofilm together. The matrix provides structural and functional benefits to the biofilm, including hydration, resource capture, digestive capacity, and antimicrobial protection. It also facilitates intercellular interactions, which can improve metabolic capacity and antimicrobial resistance.^4–6  The development of biofilm is actually a multi-phased process with five primary processes: reversible attachment, irreversible attachment, EPS synthesis, biofilm maturation, and dispersal/detachment. At different stages of biofilms, different bacterial species exhibit a wide range of expression and control mechanisms. There is still much research to be done before scientists can fully comprehend how all bacterial biofilms are formed.^5,7  Biofilm-associated virulence has significantly affected therapeutic strategies to combat infection. According to the National Institutes of Health (NIH) statistics, microbial biofilms infect both tissues and medically implanted devices, accounting for around 65% and 80% of microbiological and chronic infections, respectively.⁸  According to data from the Centres for Disease Control published in 2007, there were over 1.7 million hospital-acquired infections, over 0.5 million related fatalities, and an approximate US$11 000 million financial burden associated with treating biofilm-associated diseases.⁹  Although biofilms are regarded as potentially dangerous in the clinical and various other industrial domains, many biofilms are useful, and there are countless reports of their use. Beneficial biofilms have a range of benefits in agricultural, medical, environmental, and food fields, including bio-fertilizers, antibacterial and antimicrobial agents, filtration, corrosion prevention, wastewater treatment, biofouling, microbial fuel cells, bioremediation, and food fermentation.^10–12  Therefore, understanding the fundamentals of the biofilm life cycle and its properties is crucial.

This chapter aims to understand the complex composition and mechanism involved in biofilm formation and its ecological success as a habitat former.

Biofilm is a diverse, structured community of microbial cells (2–5%) embedded in a self-secreted matrix made up of extracellular polymeric substances (75–90%). This biofilm matrix mostly consists of water (up to 97%), polysaccharides (1–2%), proteins (<1–2%), nucleic acids (<1–2%), lipids, and other biopolymers.¹³  The matrix organises biofilms, resulting in steep gradients, great biodiversity, and intricate interactions like cell-to-cell communication and horizontal gene transfer. The formation of EPS is influenced by the biofilm’s growth stage as well as environmental factors such as temperature, nutrition availability, and shear pressure.¹⁴  Furthermore, EPS formation was significantly affected by pH.¹⁵  It has been demonstrated that Streptococcus, which causes vaginal infections, preferentially forms biofilms at low pH levels. Additionally, it has been demonstrated that multivalent cations like Ca⁺⁺ improve the stability and stiffness of the majority of biofilms.¹⁶  Table 1.1 describes factors influencing biofilm development, including bacterial metabolism, EPS generation, and mechanical characteristics.

EPS molecules shape the space between cells in biofilms, affecting their environment and providing mechanical stability. Mono-species and multi-species microbial communities differ in their EPS synthesis and spatial organisation. Numerous different types of biomolecules have been identified thus far. These are classified into two types: (i) those that are found on the cell surface and (ii) those that are secreted outside of the cell. Flagella, type IV pili, and functional amyloids are a few examples of cell-associated appendages that influence bacterial adhesion, mechanical stability, and autoimmune responses. Secreted bacterial exopolysaccharides, proteins, eDNA, and eRNA, which are released extracellularly, contribute to matrix scaffolding and function. It has been discovered that host proteins and glycoproteins, such as salivary proteins, support microbial adhesion and serve as a source of nutrients.²²  Minerals result from bio-mineralization processes closely regulated by the environment or microorganisms (see Figure 1.1). They too serve as a vital component of EPS.³²  Due to biofilm composition’s diversity and complexity, Karygianni et al.⁴  utilise the term ‘matrixome’ to describe the composition. The term ‘matrixome’ is derived from ‘matrisome’, which has long been employed in the field of eukaryotic cell biology to define the entire inventory of currently known biomolecules, as well as their molecular, structural, and functional diversity, associated with biofilm assembly and its virulence and physicochemical attributes.

The following is a concise description of the primary components of EPS.

Polysaccharides (PSs) act as a scaffold on which other lipids, proteins, nucleic acids, and carbohydrates can cling. The exopolysaccharides vary from one another in terms of their constituent parts, structures, and characteristics. The composition of PSs produced by microbial cells varies widely, which in turn affects their chemical and physical characteristics. PSs are found in ordered compositions with long, thin molecule chains with masses ranging from 0.5 to 2.0 × 10⁶ Da in the majority of natural and experimental situations.³³  These PSs are heteropolysaccharides since they are made up of both neutral and charged sugars. They may also have organic or inorganic substituents, which have a substantial impact on their physical and biological properties. For instance, alginate, which is made up of d-mannuronic and l-guluronic acids and formed in Pseudomonas aeruginosa biofilms, one of the most researched biofilm models, is one of the most prevalent exopolysaccharides.³⁴  The PS in the biofilm matrix governs the biofilm landscape. Exopolysaccharides fulfil various essential functions for the formation of biofilms, generally associated with their adhesion to surfaces and the maintenance of structural integrity. Multiple PSs are produced by bacteria to meet these needs in a multitude of ways. Here, PSs are categorised into three functional groups to emphasise their diversity and significance in the field of biofilm biology. Subjectively, these PSs are divided into three categories: architectural, protecting, and aggregative.³³  Several PSs have roles in each of these categories (see Table 1.2).

Aggregative PSs aid in adhesion, complex structure development, and biofilm breakdown. Bacteria produce various PSs based on environmental factors such as the surface substrate, nutrition, and flow rate, making them relevant to varied strains and situations. The necessity of bacteria continuing to be a part of the biofilm community is shown by the redundancy of aggregative PSs produced by numerous bacteria. Furthermore, the capacity to alter PS production offers compensatory strategies for adjusting to shifting surroundings. The principal PS involved in Staphylococcus aureus and Staphylococcus epidermidis biofilm formation is PS intercellular adhesion (PIA). Numerous studies have examined the significance of PIA during staphylococcus infections, and PIA has been linked to a number of functions, including enhancing virulence, protecting against host innate immune responses, and encouraging cellular aggregation and biofilm development. High NaCl, glucose, temperature, ethanol, anaerobiosis, and sub-inhibitory doses of certain drugs can all cause PIA, which increases virulence.³⁵  Staphylococci lack PIA hydrolytic enzymes for the detachment stage of the biofilm. Instead of scattering PIA, detergent-like peptides are overexpressed, disrupting non-covalent connections with bacterial cell surfaces.³⁶  Pel is another aggregative PS produced by P. aeruginosa. The term is derived from the thick pellicle found in strains that overexpress the pel operon. Pel’s structure and composition are unclear, although investigations are underway to find sugars and connections in this PS. Pel is believed to cause elevated levels of cyclic diguanylate (c-di-GMP), an intracellular second messenger, and regulate the flagellum.³⁷  Another significant PS of P. aeruginosa produced by the polysaccharide synthesis locus is Psl. Psl exists in two forms: a low molecular weight form that seems to be discharged from cells, and a high molecular weight form that is connected with cells.³⁸  d-Mannose, d-glucose, and l-rhamnose make up released Psl in the following ratio: 3 : 1 : 1.^38,39  Cell-associated Psl’s structure is unclear, although it is thought to be a polymer of mannose, glucose, rhamnose, and maybe galactose.³⁹  Colvin et al. found strain-specific requirements for PS synthesis in biofilms. In some strains, Psl is essential for biofilm formation, while in others Psl and Pel are redundant. All examined strains require one of two sticky PSs to generate a mature biofilm in vitro.⁴⁰  Irie et al.⁴¹  found that Psl can signal and promote biofilm development.

Originally, it was suggested that PSs in the matrix act as a diffusion barrier to prevent antibiotic penetration of biofilm-grown cells.⁴²  Nonetheless, research has indicated that a number of medicines can easily pierce the biofilm.⁴³  Diffusion might not be stopped, but it might be stopped long enough to cause the expression of genes involved in tolerance or stopped before it reaches the bacterial cell by matrix-resident enzymes.⁴⁴  Protective PSs are critical for maintaining protection against host and extrinsic stimuli, as well as maintaining a moist biofilm environment, which helps avoid desiccation and encourages biofilm fluidity. The first of the protecting Ps is alginate. It was found in Pseudomonas and Azotobacter vinelandii, and is more common in cystic fibrosis patients. By preventing both opsonic and non-opsonic phagocytosis, alginate also offers defence against the innate immune response. This effect is further mediated by the degree of alginate acetylation.^45–47  Alginate also provides protection by scavenging reactive oxygen intermediates and preventing cationic antimicrobial peptides from killing the microorganism. Moreover, alginate’s anionic character favours cation chelation (Ca⁺⁺).⁴⁸  Alginate biofilms chelate calcium, leading to type III secretion that may protect against host immunological responses.⁴⁹ 

Many different species have bacterial capsular polysaccharides (CPSs) on their cell surfaces. The phospholipid or lipid-A molecules that the CPS covalently attaches to are what hold the bacterial cell surface securely together. Comprising repeated monosaccharides connected by glycosidic bonds, CPSs are highly hydrated molecules.⁵⁰  CPSs have a nearly infinite variety of structures due to their great diversity in terms of monosaccharide composition, glycosidic connections, branching, and substitutions with non-carbohydrate residues. CPSs are frequently downregulated in response to interaction with epithelial cells and during biofilm formation.^51,52  Reduced CPSs are thought to strengthen the quiescent state that is frequently linked to biofilm formation, strengthening immune evasion, reducing virulence, and increasing persistence. Research has also indicated that CPSs could play a crucial role in established biofilms, supporting the preservation of biofilm dispersion and size. Levans are neutral homopolymers with a high molecular mass that are made up of β-d-fructans that exhibit widespread and irregular branching.⁵³  Levan production has been found in a number of plant diseases and gives bacteria that express it a mucoid phenotype. The majority of the literature on levan formation in biofilms has been published about Streptococcus mutans dental biofilms.⁵⁴  Levan builds up on tooth plaque after being exposed to sucrose and is converted to acid when the ambient sugar supply runs low. Long-term exposure encourages dental caries. Levan is a low viscosity, water-soluble PS produced by Bacillus subtilis. Levan stays fluid at ordinary concentrations, suggesting that it might not form an adhesive structure, in contrast to other PSs that show gel behaviour.⁵⁵ 

Some of the earliest biofilm-related PSs discovered were investigated due to their significance in biofilm building processes. These PSs have provided substantial insight into the regulation of biofilm development and structure, particularly due to the easily observed abnormalities associated with mutants or overproducers of these products.

Colanic acid (CA) or M antigen is a branching PS made up of glucose, galactose, and glucuronic acid. Early in vitro studies revealed that an Escherichia coli mutant unable to produce CA had two intriguing phenotypes. Initially, the CA mutant showed attachment properties that were comparable to those of the wild-type strain. Secondly, even though the CA mutant could adhere at wild-type levels, the biofilms it produced had a lower three-dimensional structure than those made by the strain that is wild-type.⁵⁶  The term “collapsed” was used to describe these biofilms, which had tightly packed cells against the substrate. Since it was one of the first findings to link PSs to the structural components of the biofilm matrix, this work was crucial to the biofilm research. Vibrio cholerae, a Gram-negative aquatic bacterium, causes the diarrheal illness cholera. The observation of V. cholerae rugose variations has led to much of the knowledge that exists today about the function of Vibrio polysaccharide (VPS) in the biology of biofilms. The creation of the VPS plays a crucial role in the ongoing infectious cycle. The overproduction of VPS has resulted in a highly structured and wrinkled morphology in these colonies. When a rugose strain’s VPS genes are removed, the colony returns to having a smooth shape. Rugged strains have rough colony shapes in addition to improved adhesion and biofilm production traits. Super-resolution confocal microscopy revealed that VPS encourages the biofilm to hold onto daughter cells and the build-up of RbmA, Bap1, and RbmC, the biofilm matrix proteins.⁵⁷ 

One of the most prevalent PSs in nature is cellulose. Over the course of evolution, the structure of cellulose fibrils has remained constant, and bacterial cellulose is identical to cellulose made by fungi, plants, and higher-order algae. This polymer is made up of repeated chains of d-glucose joined by β-1,4 linkages, forming fibrils that have a cable-like appearance. The earliest accounts of cellulose’s role in Gluconacetobacter xylinus pellicles suggested that cellulose caused cell aggregation in the pellicle, which enabled the biofilm to float to the culture’s surface where the bacteria could easily access oxygen.⁵⁸  Apart from this function, cellulose also offers defence against the mutagenic consequences of UV radiation.^59,60  Numerous Enterobacteriaceae, such as Klebsiella pneumoniae, Salmonella enteritidis, E. coli, and Salmonella typhimurium, are reported to produce cellulose.⁶⁰  Cellulose and curli fimbriae in these species frequently interact to form the biofilm matrix that gives rise to the rdar (rough, dry, and red) colony phenotype. In the probiotic strain of E. coli Nissle 1917,⁶¹  cellulose synthesis improves epithelial cell attachment and decreases immunological reaction to the bacteria. These are essential functions for the establishment of a commensal relationship with the host epithelium.

Another important element of the EPS matrix is extracellular proteins. The combination of secreted extracellular proteins, protein subunits of cell appendages including pili and flagella, cell surface adhesions, and outer membrane vesicle proteins makes up the extracellular proteins in the biofilm matrix. Their interactions with exopolysaccharides and nucleic acid constituents aid in the stabilisation of the biofilm matrix, surface colonisation, and preservation of the biofilm’s integrity and architecture.⁵⁷  Some proteins, such as proteases, aid in the breakdown and dissemination of biofilms.⁶²  Enzymes can act on both EPS produced by the same bacterium and those from other species. Furthermore, structural EPS breakdown is critical in biofilm growth because it permits sessile cells to disperse from biofilms, allowing new biofilms to form. Environmental changes, such as nutrient scarcity or availability, can cause dispersion. Enzymes that perform this function include dispersin B, which breaks down extracellular polysaccharides containing N-acetylglucosamine,⁶³  and surface protein-releasing enzyme (SPRE), which liberates adhesin P1 from bacterial surfaces to enable Streptococcus mutants to separate from surfaces.⁶⁴  In addition, some DNases disrupt extracellular nucleic acids.⁶⁵  Some enzymes can even break down the surfaces that support the biofilm; redox enzymes, for example, are implicated in microbial corrosion.⁶⁶  Non-enzymatic proteins primarily function structurally, serving as a bridge between the outer membrane of the bacteria and the exopolysaccharide matrix. One class of extracellular carbohydrate-binding proteins called lectins, which is thought to be involved in cell-to-cell contacts within biofilms, facilitates the creation of microcolonies and the maturation of biofilms.^67,68 

Extracellular DNAs (eDNAs) were originally thought to be the remains of lyzed cells, but Mattick and colleagues discovered that P. aeruginosa biofilm formation could be inhibited by DNase I.⁶⁹  The fact that eDNA is actively released and derived from lyzed cells suggests that eDNA plays a significant part in the production of biofilms. It turns out to be essential for the adhesion of biofilms.⁷⁰  eDNA interacts with receptors on the substratum surface to promote adhesion when the distance between the cell and surface drops to a few nanometers, although initially its negative charge acts as a repulsive force during attachment.⁷¹  Additionally, it was discovered that eDNA coordinated cell movement in the P. aeruginosa biofilm expansion mediated by twitching motility.⁷²  eDNA can chelate some positively charged antibiotics as well as metal cations because of its negative charge. P. aeruginosa,^73,74  Salmonella enterica serovar Typhimurium,⁷⁵  and other Gram-negative bacteria can develop antimicrobial peptide resistance as a result of eDNA’s ability to chelate Mg⁺⁺ and activate the PhoPQ/PmrAB two-component systems. Additionally, it was discovered that eDNA in S. epidermidis inhibits the movement of vancomycin within biofilms, protecting the bacteria trapped within the biofilm.^76,77 

The major element in the EPS matrix of biofilm appears to be water. Even when there are fluctuations in the water content of the surrounding environment, it prevents desiccation and keeps the biofilm hydrated.⁶  Additionally, the quantity of water present affects the flow and upkeep of vital nutrients within a biofilm.⁷⁸  In a nutshell, the exopolymer matrix can function as a molecular filter, holding onto particles, cations, and anions in the aqueous phase. Apolar areas, groups that can establish hydrogen bonds, anionic groups (found in proteins and uronic acids), and cationic groups (found in amino sugars) are all found in EPS.

EPS might also contain lipids. Although their exact function in the growth and production of biofilms is unknown, they are thought to enhance adherence on hydrophobic surfaces, especially when paired with amphiphilic polysaccharides.⁷⁹  Furthermore, lipidic extracellular polymers (EPS) including emulsan, surfactin, and viscosin may help retain hydrophobic materials in the biofilm to promote cell development.⁶  Biosurfactants promote the formation of microcolonies, allowing bacteria to migrate to the surface and form mushroom-shaped structures. They also prevent channel colonisation and contribute to biofilm dispersion. The function of biosurfactants in the binding of heavy metals and the synthesis of virulence factors was reported by Ron and Rosenberg.⁸⁰  Rhamnolipids are a significant family of surfactants that have been examined in P. aeruginosa. They aid in the production of microcolonies, shape biofilms, and promote the dispersal of biofilms.⁸¹ 

Biofilm is defined as a habitat because it is a spatial organisation that allows for steep gradients, high biodiversity, and intricate, dynamic, and synergistic interactions, such as improved horizontal gene transfer and cell-to-cell communication. The EPS promotes cell adhesion to solid substrates and cohesion among bacterial cells, eventually leading to the development of structured cell clusters, often termed microcolonies.⁸²  In addition to the well-known adhesin–receptor interactions, the initial bacterial attachment frequently involves fascinating surface-scanning and sensing mechanisms. Additionally, the EPS encourages interspecies recognition and cell-to-cell cohesion, which facilitates microbial aggregation and the creation of biofilms. Certain adhesin (protein)–receptor (saccharide) pairings or mechanosensors are required for other interspecies interactions. Biofilms exhibit a diverse ecosystem capable of resource capture through sorption, enhanced digestive functionality via enzyme retention, and remarkable tolerance or resistance to antibiotics facilitated by social interactions, such as quorum sensing and gene transfer. These features make biofilms adaptable and resilient (see Figure 1.2).² 

Biofilms capture nutrients more efficiently than free-living bacterial cells (see Figure 1.3). They utilise the passive sorption capabilities of the sponge-like EPS matrix to facilitate the movement of nutrients, gases, and chemicals between the environment and biofilms on a global scale.⁸³  Biofilms retain ‘sorbed’ substances from the water phase, including absorption in the matrix and adsorption to biopolymers. Biofilm cells and surface materials can use biodegradable materials as nutrients.⁶  Nutrients from surface materials are concentrated at the biofilm’s base layer, resulting in a reverse nutritional gradient relative to the water phase. Biofilms are complex sorbent systems with various sorption mechanisms and binding sites in the cytoplasm, cell walls, and EPS of the matrix. Binding sites contain anionic and cationic exchangers, allowing for the accumulation of various chemicals for ingestion by biofilm cells. Because the biofilm’s sorption is not compound-specific, hazardous materials as well as nutrients can build up in biofilms.⁸⁴  Despite its hydrophilic nature and lack of lipophilic binding sites, the EPS matrix can accumulate non-polar molecules such as benzene. Sorbed chemicals may be discharged into the water phase if there is a concentration gradient, or persist in the biofilm until its decomposition. Therefore, biofilms may serve as both a drain and a source of contamination.⁸⁵  Cells that die and lyse leave debris in the matrix, which surviving cells can use as nutrition. Biofilms trap both organic and inorganic particles, such as clay and silicate, which contribute to global lithification.³¹  Inorganic particles in biofilms including electrically conductive particles can facilitate interspecies electron transfer (IET) which further supports cell-to-cell interactions in the matrix.⁸⁶ 

Compared to free-living bacterial cells, cells in the biofilm utilise their extracellular degradative enzymes far more efficiently. Extracellular enzymes secreted by planktonic cells are diluted in the aqueous environment, while those secreted by biofilm cells are retained in the biofilm which then interact with EPS components like polysaccharides. As a result, an active matrix is produced, which functions as an external digestive system.⁶  Extracellular enzymes interact with matrix constituents to generate stable complexes that are remarkably resistant to proteolysis, dehydration, and heat denaturation. Enzymatic activity persists, protecting the biofilm community from fluctuations in dissolved organic matter content in the bulk water phase. For instance, during P. aeruginosa infection, the extracellular enzymes lipase (LipA) and elastase link to the EPS matrix by generating electrostatic contacts with alginate. This results in an improvement in heat tolerance and protection against enzymatic destruction. Consequently, the matrix can unexpectedly change the characteristics of released enzymes.⁸⁷ 

The matrix-based structure of bacterial biofilms facilitates close interactions among various organisms. This allows for the interchange of metabolites, signalling molecules, genetic material, and defence substances, which influence relationships between species. Opportunities for cooperation arise from heterogeneity, such as cells with different physiological gradients or metabolic capacities.

Biofilms exhibit diverse physiological activity, leading to significant differences in electron acceptors, donors, pH, and redox states.⁸⁸  These distinct biofilm characteristics are not only seen in dense, multilayer biofilms but also appear even when a comparatively small number of cells have adhered to a surface.⁸⁹  Physiological stratification and heterogeneity in biofilms allow for spatial organisation of both mixed and mono-species biofilms. This is most likely due to phenotypic variation resulting from varying gene expressions of individual cells over time and from distinct cells expressing different genes. The creation of gradients and other spatial heterogeneities is facilitated by the localized physiological activity of these spatially separated, immobilised cells. This leads to an increase in multilayered biofilms, such as microbial mats or flocs.⁶  Oxygen and other electron acceptors are among the most significant external factors that initiate the formation of gradients. In an aquatic environment of biofilm, the upper layer of the biofilm is aerobic. Aerobic microcolonies use oxygen quicker than it diffuses through the biofilm, resulting in anaerobic zones formed in its deep layers. Gradients in oxygen availability can occur over a short distance, separating the aerobic and anaerobic portions of the biofilm by only a few micrometres.^90,91 

The term ‘sociomicrobiology’²  refers to the complex network and coordinated division of labour created by the near proximity of cells in the biofilm matrix. Sociomicrobiology relies on intercellular signalling, which is heavily impacted by biofilm matrix properties. However, metabolic activity also plays a crucial role in social interactions inside biofilms. The high cell density and diversity of biofilms make them ideal for exchanging metabolic byproducts between species.⁹²  Planktonic cell suspensions cannot undergo these processes. Amino acid auxotrophy is a common strategy used by microbial communities to reduce the metabolic burden of biosynthesis and stabilise cooperation.⁹³  This is likely also true for sugars and nucleotides. As a result, it is possible to regard the exchange of sugars and amino acids as typical mutualistic interactions among sub-communities that coexist side by side.⁹⁴  High cell densities in the biofilm are crucial for social interactions, as evidenced by the formation of synergistic multispecies consortia that are most noticeable when metabolic substrates and intermediates have short diffusion distances to minimise loss.⁹⁵  An intriguing example of metabolic interactions between diverse species in biofilms is the process of nitrification, in which ammonia-oxidizing bacteria turn ammonium into nitrite, which is then oxidized by nitrite-oxidizing bacteria. Nitrospira moscoviensis uses urease to create ammonia, which is then oxidized by bacteria without the enzyme.⁹⁶  Thus, N. moscoviensis uses ammonia to produce nitrite, indicating a reciprocal metabolic relationship. Nitrite-oxidizing bacteria and several metagenomic datasets have been found to harbour genes predicted to encode ureases. This suggests that many of these bacteria form reciprocal metabolic interactions in which ammonia is exchanged for nitrite, rather than merely being recipients of the nitrite produced by ammonia-oxidizing bacteria. A further intriguing consortium of synergistic biofilms consists of cyanobacteria and fungi found in biofilms on desert rocks or on building surfaces. In this community, the cyanobacteria nourish the fungus, which then helps the cyanobacteria by releasing vital metals from the rock.⁹⁷  In a study of a biofilm including P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae, stress tolerance was found to be equal across all three community members. A three-species biofilm community degraded the phenylurea herbicide linuron synergistically, which none of the associated monospecies was able to degrade individually.⁹⁸ 

Species in these ecosystems consume comparable resources, which may lead to competition. Biofilm competition mechanisms, such as antibiotics, bacteriocins, extracellular membrane vesicles, and type VI secretion systems, have been extensively studied.⁹⁹  Competitive tactics involve inhibiting early adherence to biofilms and surface blanketing. For instance, P. aeruginosa cells can spread on surfaces by twitching their motility, preventing Agrobacterium tumefaciens cells from adhering, or producing antimicrobial biosurfactants.⁹⁹  Invaders can disrupt biofilm development and dissemination by reducing adhesin synthesis, preventing cell-to-cell communication, and degrading matrix polysaccharides, nucleic acids, and proteins.^100,101 

The terms ‘resistance’ and ‘tolerance’ refer to an organism’s greater ability to tolerate exposure to substances that would kill susceptible organisms. The term ‘resistance’ refers to a heritable genetic trait that can be acquired through mutation or gene exchange and persists even after cell dispersal in the biofilm. The word ‘tolerance’ refers to a property of biofilms that is lost during dispersal to free-living bacterial cells.² 

The EPS matrix may logically appear to represent a diffusion barrier on an intuitive level. Nevertheless, it has been demonstrated that antimicrobials that do not interact with EPS molecules permeate through biofilms just as readily as they do through water.¹⁰²  Although EPS does not appear to be a physical barrier to antimicrobial diffusion, it can significantly inhibit their activity through diffusion-reaction inhibition. This can occur through chelation, enzymatic degradation, or even sacrificial reactivity with disinfectants.¹⁰²  Antimicrobial resistance in biofilm cells that can tolerate antimicrobial stress may be selected via diffusion-reaction inhibition, which lowers effective antimicrobial concentrations to sublethal levels.

Bacteria in biofilms exposed to antimicrobials can survive by slowing their development and going dormant.¹⁰³  Antimicrobials that rely on bacterial cell metabolism are less susceptible to cells in the stationary phase.¹⁰⁴  At least 1% of bacterial cells in stationary biofilms develop antibiotic tolerance. As the biofilm grows, more cells become dormant.¹⁰⁵ 

Horizontal gene transfer can promote antimicrobial resistance in biofilm cells. Biofilms are favourable for horizontal gene transfer due to their high cell density, improved genetic competence, and accumulation of mobile genetic elements, including resistance genes.¹⁰⁶  Biofilm contains antibiotic resistance genes (ARGs, the resistome) that provide pathogens with antibiotic resistance via HGT, conjugation, transformation, and transduction. HGT helps bacteria adapt to changing environments, promoting biofilm development and antibiotic resistance.¹³  Fan et al.¹⁰⁷  found that HGT supports the structural stability, integrity, and robustness of microbial communities coexisting. HGT leads to pathogenicity and antibiotic resistance in E. faecalis, which causes persistent endodontic infection.

The matrix creates a stable environment for gene transfer and serves as a source of eDNA.¹⁰⁸  Cell lysis and active secretions produce eDNA, which has been shown to enhance microbial adherence, limit antimicrobial diffusion, and chelate cations,⁵⁶  as well as reduce innate immune response.¹⁰⁹  eDNA has been linked to cation gradients, genomic DNA release, and induced antibiotic resistance. Chiang et al.¹¹⁰  showed that positively charged aminoglycosides and antimicrobial peptides can be attached and chelated by eDNA in P. aeruginosa biofilms. According to Jakubovics and Burgess,¹¹¹  eDNA plays a crucial role in bacterial adhesion, structural integrity, genetic recombination and HGT, antibiotic resistance, and phosphorus supply.

Efflux pumps cause antibiotic resistance in bacteria by relocating intracellular toxins, including antibiotics, to extracellular space. Although, efflux pumps are active in planktonic bacteria, they are increased in biofilms, resulting in multidrug resistance (MDR). In P. aeruginosa, overexpressed RND efflux pumps such as BCAL1672-1676 (RND-3) give biofilm resistance to tobramycin and ciprofloxacin, while BCAM0925-0927 (RND-8) and BCAM1945-1947 (RND-9) protect biofilms from tobramycin in Burkholderia cepacia.¹¹² 

In reaction to stressful circumstances such as low nutrition, extreme pH and temperature, high salt concentrations, pressure, UV radiation, desiccation, and antimicrobial chemicals, microorganisms form biofilms.^113–115  They change from free-swimming planktonic to sessile form. External factors like as pH, temperature, Brownian movements, surface properties, quorum sensing, gravitational forces, hydrodynamic forces, secondary messengers, and signalling molecules all influence the formation process. The process of biofilm development begins with the reversible attachment of bacteria to a surface, which is then followed by the irreversible attachment, which is often facilitated by the bacterium’s adhesive structures and spatial interactions. The generation of EPS advances their reversible attachment. Subsequently, they grow into a structured system trapped in an EPS matrix. Finally, bacterial cells have the ability to break out from the developed biofilm and propagate across the surroundings to occupy fresh niches.^7,33,116 

The five key phases of the transition from free-living planktonic life to a sedentary “biofilm” existence are outlined below and illustrated in Figure 1.4.

During the first phase of biofilm development, planktonic cells are loosely and reversibly attached to substrate surfaces, resulting in the existence of polarly attached microorganisms. This process is influenced by factors such as Brownian motion, sedimentation, and convection. Specifically, reversible bacterial attachment occurs when bacteria adhere to a substrate through two-dimensional Brownian motion, but they can be easily removed by bacterial mobility or fluid shear forces.¹¹⁷  Briefly, bacterial cells move along a concentration gradient in mobile fluids towards a source of nutrients or chemo-attractants (such as sugars and amino acids). This process is also known as chemotaxis.¹¹⁸  It is present in almost all microbes and, by promoting contact between cells and surfaces, can encourage the development of bacteria on surfaces. When cells approach a surface, their interaction with the conditioned surface is determined by the net repulsive or attraction forces between them. Bacteria adhere to surfaces when attractive forces outweigh repulsive forces, and vice versa. Non-specific physical forces including electrostatic, hydrophobic, and Lifshitz–van der Waals interactions contribute to the first attachment.¹¹⁹  Figure 1.5 summarizes the variables important in cell attachment and biofilm formation.

At this stage, several factors can affect the attachment for example:

• Substratum effects: the physicochemical aspects of a substrate surface can influence bacterial adhesion. According to studies, imperfections on abiotic surfaces encourage bacterial adhesion and biofilm growth because they have lower shear pressures and a greater surface area. Surface characteristics can significantly affect the attachment rate and extent. Most researchers have discovered that bacteria cling more quickly to hydrophobic, nonpolar surfaces such as Teflon and other plastics than to hydrophilic materials like glass or metals.^120,121  In contrast, few studies found that surface roughness had no effect on bacterial adhesion.^122,123  The contradictory results might be attributed to differences in extracellular structures and physicochemical qualities amongst bacteria, as well as the variable physicochemical features of a substratum surface with varying hydrophobicity, surface charge, and conditioning films. Studies have found that bacteria appear to easily settle on hydrophobic surfaces than hydrophilic ones.^124,125  This is most likely the result of hydrophobicity decreasing the repulsive forces that exist between the colonisation substratum and the bacterial surface. One major exception is that Listeria monocytogenes is more likely to cling to hydrophilic substrates like stainless steel than hydrophobic ones like polytetrafluoroethylene.¹²⁶ 

• Conditioning films: exposure to an aqueous solution causes a material surface to become coated with polymers, affecting the pace and degree of microbial adhesion. The host-produced conditioning films such as blood, tears, urine, saliva, intervascular fluid, and respiratory secretions affect bacterial adhesion to biomaterials, for instance, “acquired pellicle,” a protein-based conditioning layer that forms on tooth enamel surfaces in the oral cavity. Pellicles include albumin, lysozyme, glycoproteins, phosphoproteins, lipids, and gingival crevicular fluid. Bacteria from the oral cavity may colonise pellicle-conditioned surfaces within hours of contact.¹²⁷ 

• Hydrodynamics: cells behave like particles in a liquid, and the pace at which they settle and associate with a submerged surface is primarily determined by the liquid’s velocity characteristics. Under extremely low linear velocities, the cells must transverse a sizable hydrodynamic boundary layer, and their interaction with the surface is heavily influenced by cell size and motility. As the velocity increases, the boundary layer thins and cells experience more turbulence and mixing. Therefore, it would be predicted that higher linear velocities would correspond to faster surface attachment, at least until velocities reach a point where they impose significant shear stresses on the attached cells, causing these cells to separate.^128,129 

• Characteristics of the aqueous medium: the aqueous medium’s properties, including temperature, ionic strength, pH, and nutrition levels, may affect how quickly microorganisms adhere to a substratum. Increases in the concentrations of sodium, calcium, lanthanum, and ferric iron were found to have an impact on the adhesion of Pseudomonas to glass surfaces. This effect was likely caused by a decrease in the repulsive forces between the negatively charged bacterial cells and the glass surfaces.¹³⁰  Another study found that higher nutrient concentrations correlated with an increase in connected bacterial cells. As nutrient levels increase, the proportions of species both in the liquid and on surfaces changed, with a greater increase in total bacterial numbers in the liquid.¹³¹ 

Short-range interactions involving bacterial structural adhesions, such as dipole–dipole, hydrogen, ionic, and covalent bonding interactions, and hydrophobic interactions, are responsible for the irreversible attachment. The surface of bacteria is gifted with different adhesins such as fimbriae, other proteins, LPS, EPS, and flagella, which not only play a vital role in the attachment process but also influence the rate and extent of attachment of microbial cells. The hydrophobicity of the cell surface is important for adhesion because hydrophobic interactions tend to increase with increasing non-polarity of one or both of the surfaces involved (i.e., the microbial cell surface and the substratum surface). Fimbriae, non-flagellar appendages that do not transfer viral or bacterial nucleic acids (known as pili), contribute to the hydrophobicity of the cell surface. The majority of fimbriae analysed have a significant proportion of hydrophobic amino acid residues.¹³²  Fimbriae enhance cell surface hydrophobicity and attachment by bypassing the electrostatic repulsion barrier between the cell and substratum.¹³³  Fimbriae are found in many aquatic bacteria and play a role in their adhesion to mammalian cells. Studies have demonstrated that treating adsorbed cells with proteolytic enzymes results in the release of attached bacteria,^134,135  indicating the importance of proteins in attachment. The O antigen of lipopolysaccharide (LPS) can make Gram-negative bacteria more hydrophilic. Korber et al.¹³⁶  studied motile and non-motile strains of Pseudomonas fluorescens and discovered that motile cells adhere more frequently and against the flow (backgrowth) than non-motile cells. Additionally, bacterial pathogens produce unique adhesins that help them internalise themselves and cling to receptors on the surface of eukaryotic cells. For example, the protein invasin produced by Yersinia pseudotuberculosis and Yersinia enterocolitica binds to b1 integrins on the surface of M-cells, facilitating Yersinia entry into M-cells.^137,138  Additionally, individual cells are coordinated by a cell-to-cell signalling system known as quorum sensing (QS) to start the creation of bacterial biofilms.¹³⁹  To facilitate cell-to-cell communication within a bacterial population, bacteria use QS to synthesise and release first messengers, such as chemical signals (autoinducers, or AIs).^140,141  To control the production of biofilms, both Gram-positive and Gram-negative bacteria use cell-to-cell signalling systems. Muhammad et al.¹⁴²  found that Gram-positive bacilli utilise oligopeptides, while Gram-negative bacilli utilise acyl homoserine lactones.

During this phase, bacteria begin to establish micro-colonies. A biofilm contains a number of micro-communities. At this stage, EPS are synthesised and secreted, which constitute a crucial part of the biofilm extracellular matrix. EPS can mediate bacterial cohesion and biofilm adherence to surfaces through hydrophobic and ion-bridging interactions. These micro-colonies generated inside the matrix work together to enable metabolic product dispersion, substrate exchange, and excretion. The components of a bacterial matrix depend on the surrounding environment, different bacterial species, and strain kinds.¹⁴³  EPS is involved in several processes, including adhesion to surfaces, cell–cell recognition, biofilm development, biofilm structure, water retention, signalling, cell protection, plant symbiosis, nutrient trapping, and genetic exchange. Simultaneously, the extracellular polymeric material that the cells released includes proteins (i.e., enzymes), protein structures (i.e., fimbriae), lipids, nucleic acids (eDNA), and polysaccharides.¹⁴⁴  This complex mixture serves as a protection against immune reactions and antimicrobial agents in addition to its role in collecting nutrients and providing structural support.¹⁴⁵  Furthermore, the second messenger, c-di-GMP, promotes bacterial adherence from reversible to irreversible, owing to its ability to generate the EPS matrix and construct bacterial cell surface structures.¹⁴⁶ 

At this point, bacteria continue to proliferate and create signalling molecules known as auto-inducers (AIs).¹⁴⁷  These auto-inducers are essential for quorum sensing, which allows bacterial cells to sense cell density via cell-to-cell communication with cells from the same or other species. When a minimum threshold is attained, these signalling molecules detect each other’s presence and regulate gene expression. Increased EPS synthesis serves as the biological “glue” that holds embedded bacterial cells together.¹⁴⁸  At this stage, microcolonies frequently exhibit a three-dimensional multicellular structure in the shape of a pyramid or mushroom. Maturation restricts movement within microcolonies by inhibiting the formation of bacterial surface structures, and the gene expression pattern of sessile cells differs dramatically from that of planktonic cells. The three-dimensional matrix creates interstitial gaps that serve as conduits for water. This tube serves as a circulatory system, distributing nutrients and removing waste from microcolonies.¹⁴⁹ 

The ultimate step of the biofilm life cycle is detachment or dispersal of cells generated within the biofilm matrix. It is thought to be a tactic used by bacterial cells to break free from biofilms and start a new biofilm life cycle. The process of detachment of bacterial biofilms can be either active or passive.^150,151  Bacterial dispersal involves three stages: dissociation from microcolonies, migration to a new substrate, and attachment.

• Active behaviour: when the bacterial biofilm is exposed to matrix-degrading enzymes, antibiotics, and nutrition deprivation, the bacteria in the biofilm undergo their detachment in order to respond to changes in the environment. This process is known as seeding dispersion.

• Passive behaviour: passive behaviours like sloughing and erosion are influenced by external pressures like shear. Seeding dispersal occurs when microcolonies or planktonic cells are rapidly released from the centre of a biofilm, resulting in an empty hollow space. Sloughing is the rapid removal of a major piece of a biofilm, while erosion is the discharge of a small amount of bacteria from the biofilm.

Bacterial biofilm development can be inhibited and biofilm separation can be promoted by low expression levels of c-di-GMP.¹⁵¹  Therefore, bacterial biofilms can be successfully dispersed by blocking the c-di-GMP signalling pathway. The dispersion process of bacterial biofilms can also be impacted by changes in environmental parameters such as temperature, pH, oxygen level, and nutrition content. Low oxygen settings have the potential to promote the dispersion of bacterial biofilms by quickening the breakdown of c-di-GMP. Elevated glucose levels can decrease the organism’s c-di-GMP levels and encourage the manufacture of flagella, thereby delaying the process of separation.¹⁵² 

This chapter covers the formation, clinical aspects, and ecology of biofilms. As awareness grows regarding both the beneficial and harmful roles of biofilms, future research will focus on their negative impact on healthcare, agriculture, the food industry, drinking water, and oceans. Consequently, understanding the fundamental biology of biofilms is essential for developing innovative treatment strategies or conjugates. Therefore, understanding the fundamental biology of biofilms is crucial to developing novel treatment tools or conjugates. Thus, fundamental knowledge of biofilms may benefit:

• Designing new drugs or adjuvants to combat resistance based on the mechanism of resistance and components involved in the process, viz. QS, gene transfer, and efflux pumps.

• In agricultural and aquatic settings, information on certain essential bacteria and their biofilm cycle can help increase yield.

• To study environmental ecology and evolution.

• Since biofilms have an essential role in breaking down sewage and decontaminating polluted sites, their mechanism and secretory functional knowledge can change waste disposal techniques.

• It can be intriguing to understand the anatomy and physiology of biofilms from the perspectives of host–cell, cell–cell, and cell–surface dynamics to construct new equipment and fittings.