Biofilms: An Enigmatic Biochemistry of Microbial Life

Mini Review  Article

Biofilms: An Enigmatic Biochemistry of Microbial Life

Corresponding author: S. Rao.Biofilms: An Enigmatic Biochemistry of Microbial Life. J J Biochem. 2018; 3(2):004

Abstract

“Biofilms constitute a consortium of biotic elements like bacteria, cyanobacteria and algae attached to a substratum microbially produced extracellular polysaccharide matrix which entraps soluble and particulate matter, immobilizes extracellular enzymes and acts as a sink for nutrients and inorganic elements to further microbial life”.

The biofilm composition may vary both spatially and temporally and greatest differences are usually associated with shifts in the relative importance of autotrophic and heterotrophic microorganisms.  A prerequisite of biofilm formation is that bacteria should get close enough to a surface. As bacteria approach a surface several forces, both attractive and repulsive, come into play. At about 10–20 nm distance from the surface, the negative charges on the bacterial surface are repelled. This repulsion could, however, be overcome by attractive van der Waals forces between the bacterial cells and the surface as well as the use of fimbriae and flagella of a bacterial cell to provide mechanical attachment to the surface [1].

Generally, biofilms constitute a distinct growth phase of microorganisms that are distinctly different from the planktonic microbiota. During the adhesion process bacterial cells undergo phenotypic changes in response to the proximity of a surface. In the course of biofilm formation, sessile bacteria form micro-colonies which are in juxtaposition with cells of the same species. The microenvironment surrounding the micro-colonies, along with the copious exopolymeric matrix, result in biofilm formation on a surface. The microenvironments are specific for each microbe, with different growth patterns, and with time a structurally complex biofilm develops. The typical structure of biofilm formation can be described in three stages: attachment, maturation and dispersion. The attachment step is further categorized into a two-stage process: initial reversible attachment and irreversible attachment. The irreversible attached biofilm can tolerate stronger physical or chemical shear forces.  Flagella are critical for initial interactions between cells and surface. Microbial surface components recognizing adhesive matrix molecules are covalently linked to the peptidoglycan on the cell wall. For example, Staphyloccus aureus has >20 microbial surface components recognizing adhesive matrix molecule genes. Non-covalent adhesions, such as those mediated by autolysins, also contribute to initial attachment of biofilms [2,3,4].

Biofilm grows from a heterogeneous thin bio growth layer to a mushroom shape structure. In a thick biofilm (>100 µm), bacteria are arranged according to their metabolism and aero-tolerance. For example, anaerobic bacteria live in deeper layers to avoid exposure to oxygen. Bacteria within biofilm communities chemically communicate (chemotactic response) to each other and take specialized functions. As the biofilm matures, more biofilm scaffolds, such as proteins, DNA, polysaccharides, etc. are secreted into the biofilm by the entrapped bacteria. After biofilm maturation the dispersal step, which is also critical for the biofilm life cycle, follows. Biofilms disperse because of myriads of factors, such as lack of nutrients, intense competition, outgrown population, etc. Dispersal could occur in the whole biofilm or just a portion of it. Release of planktonic bacteria promotes the initiation of new biofilms at other sites (5, 6).

Biofilms are communities of microorganisms that are attached to a surface and play a significant role in persistent bacterial infections. Bacteria within a biofilm are several orders of magnitude more resistant to antibiotics, compared with planktonic bacteria. Thus far, no drugs are in clinical use that specifically target bacterial biofilms. This is probably because until recently the molecular details of biofilm formation were poorly understood. Bacteria integrate information from the environment, such as quorum-sensing auto-inducers and nutrients, into appropriate biofilm-related gene expression, and the identity of the key players, such as cyclic dinucleotide second messengers and regulatory RNAs are beginning to be uncovered.

Dating back to the seminal works of Robert Koch, till the 1970s, bacteria were largely considered as single free-floating microorganisms i.e., planktonic type. Using the planktonic pure bacterial culture model, scientists have been able to study many deadly bacteria and developed biocides/antibiotics to kill such bacteria. The emergence of drug-resistant bacteria and the difficulty in killing some bacteria led to a re-evaluation of the bacterial lifestyle and it is now acknowledged that the aggregation of bacteria within self-produced matrices, called biofilms, endows bacteria with mechanisms to resist biocides. Biofilms were observed a few centuries before their relevance to the persistence of disease was realized. In 1684, Antonie van Leeuwenhoek, saw dental plaque using his self-constructed microscope and he described it as scurf. During the early part of the 20th century many researchers reported that most bacteria were not free-floating but were attached to surfaces and bottom sediments. Scientists began to realize that some sessile bacteria were directly related to disease when in 1977 Pseudomonas aeruginosa aggregation was found in sputum from the lungs of infected cystic fibrosis patients. In 1978, The term ‘biofilm’ was formally introduced in 1978 by Costerton (7).

The structure of the extracellular polymeric substance (EPS) matrix of biofilms is composed of one or more extracellular polysaccharides, DNA and proteins. Channels in the biofilm allow water, air and nutrients to get to all parts of the microbial structure. Exopolysaccharides are both synthesized extracellularly or intra-cellular and secreted into immediate milieu. Exopolysaccharides serve as scaffolds for other carbohydrates, proteins, nucleic acids and lipids to adhere. The components, structures and properties of the exopolysaccharides differ from one bacterium to another. Most exopolysaccharides are not biofilm specific, but their production increases as a result of a stress response, such as colanic acid production in Escherichia coli and alginate synthesis in P. aeruginosa. Colanic acid (also known as M antigen) is an extracellular hetero-polysaccharide found in Enterobacteriaceae. Similarly, there are three exopolysaccharides associated with P. aeruginosa biofilms: Pel, Psl and alginate. Alginate is composed of D-mannuronic acid residues interspersed with L-guluronic acid residues. Alginate is not involved in biofilm initiation, but it is a critical factor in chronic infection. Alginate protects P. aeruginosa cells from antibiotic action such as ciprofloxacin, gentamicin, etc., and it also suppresses host immune response. Apart from alginate and LPS, Psl and Pelare also two major biofilm matrix exopolysaccharides. Psl polysaccharide is composed of a repeating pentamer consisting of D-mannose, L-rhamnose and D-glucose residues and it promotes the initial surface attachment process. Pel genes are responsible for glucose-rich exopolysaccharides formation and psl genes are involved in mannose-rich exopolysaccharides formation. In Staphylococcus, the biofilm-related exopolysaccharide is polysaccharide intercellular adhesin (PIA), which is also known as poly N-acetyl glucosamine, is a linear polymer composed of β-1,6-linked glucosamine residues. More than 80% of the PIA residues in Staphylococcus epidermidis are N-acetylated and the rest are positively charged. In Bacillus subtilis the biofilm-related exopolysaccharide can be EPS or poly-δ-glutamate, depending on the strain and the conditions (8).

Extracellular proteins are another major biofilm matrix component. Some proteins are attached to cell surfaces and polysaccharides to help with biofilm formation and stabilization. Amyloids are also insoluble fibrous proteins that play a supportive role in biofilm architecture. One example is the Fap amyloids in Pseudomonas spp. Overexpression of Fap amyloids leads to cell aggregation and increased biofilm formation. Amyloid protein TasA is one of the major components of B. subtilis biofilms. TasA forms strong fibers that can hold biofilm cells together and tolerate severe environment. Another example is the biofilm-associated protein (bap) family. The bap family includes Bap protein from S. aureus and Esp protein from E. faecalis. The bap proteins are involved in biofilm formation and in furthering the infection processes. Some enzymes are involved in degradation processes within biofilms. The enzymes’ substrates include polysaccharides, proteins, nucleic acids, cellulose, lipids, and other EPS matrix components. These enzymes can break down biopolymers and provide carbon and energy resources to biofilm cells, especially during starvation. The detachment and dispersal of biofilm also requires enzymatic functions. Degradation of EPS matrix internally by enzymes releases biofilm cells and initiate a new biofilm lifecycle [8, 4].

Extracellular DNAs (eDNAs) were previously considered as leftovers from lyzed cells until it was reported that DNase I could prevent P. aeruginosa biofilm formation. The fact that eDNA not only comes from lyzed cells but also is actively secreted indicates that eDNA has an important role in biofilm formation. It is also found to be a critical factor for biofilm attachment. Its negative charge works as a repulse force in the initial attachment, but when the distance between cell and surface becomes a few nanometres, eDNA interacts with chemical receptors on substratum surface to facilitate bacterial adhesion. Also, eDNA was found to coordinate cell movement in the twitching motility mode. Due to its negative charge, eDNA is able to chelate metal cations and some positively charged antibiotics. eDNA can chelate Mg2+ and activate the synthetic system leading to antimicrobial peptide resistance in P. aeruginosa. The current knowledge about the roles that eDNA plays in biofilm maturation and persistence is limited and more research is warranted to shed light on its role in biofilm development [5].

Environmental factors influence bacteria to switch between planktonic to biofilm form. The planktonic bacteria have relatively high cell growth and reproduction rate. However, the biofilm state appears to be natural and predominant state of bacteria. Several reasons could account for the need for a bacterial biofilm state. First, biofilm can enhance the tolerance of bacteria to harsh environmental conditions. Bacteria can avoid being washed away by water flow or blood stream by simply attaching to a surface or tissue. Cells in biofilms are about 1000 times more resistant than their planktonic cells. Second, the EPS matrix protects bacteria cells, in deeper layers, against antimicrobial agents probably by limiting the diffusion of these agents. Biofilm restricts bacterial mobility and increases cell density, which also provides an optimal environment for eDNA (plasmid) exchange (via conjugation), some of which encode for antibiotic resistance. The horizontal gene transfer rate is significantly higher in biofilms than in the planktonic cells. Understanding how biofilms form, respond to environmental cues and contribute to disease is a complex, multi-faceted challenge. The roles of signalling by cyclic-di-GMP; the relationship between quorum sensing and biofilm formation; and the role of efflux pumps and persister cells aid in biofilm propagation and promote antibiotic tolerance and resistance [9].

The biochemistry of the extracellular material of bacterial biofilms, enables pathogenic bacteria to avoid host immunity, Three Gram-negative bacteria (Pseudomonas aeruginosa, Haemophilus influenzae, and Salmonella enterica) are extensively studied. The extracellular material in the biofilms of these organisms has four major components; exopolysaccharides, extracellular DNA, several types of proteins, and outer membrane vesicles, all of which contribute to promote resistance to host immunity. The physical structure of the biofilm impedes penetration and engulfment by host cells, the DNA and polysaccharides bind and sequester antimicrobial molecules produced by host cells. The transition between motile and sessile life styles during biofilm formation involves metabolic, physiological and phenotypic changes that are coordinated by intracellular signalling pathways that respond to environmental cues. The second messenger cyclic-di-GMP acts as the transducing signal for many of these pathways. Cellular levels of c- di-GMP depend on the relative activities of two families of enzymes, diguanylate-cyclases and phosphor-diesterases. While each family has a characteristic structural motif, individual members have additional domains that may segregate their activity temporally or spatially, thus enabling different members of each family to respond to different signals and regulate different cellular processes. Emerging opportunities and challenges include new imaging methods for detecting heterogeneous c-di-GMP levels in individual cells, investigating cross-talk between c-AMP and c-di-GMP regulated pathways, and understanding the role of c-di-GMP in regulating antimicrobial resistance in biofilms. The quorum sensing system is turned on at high cell densities and controls the production of exo-toxins and exo-enzymes required for infection and biofilm production and dispersal. The signalling pathway consists of an inducing peptide (AIP), a histidine kinase that is activated by AIP, and the response regulator, a protein that, when phosphorylated by the kinase, binds to four promoters on the chromosome.  One of these promoters (P2) is the promoter for the operon for the signalling pathway, which is therefore auto-inducible. The second (P3), controls the production of RNAs, which up regulates both exo-toxin and exo-enzyme production via protein-RNA interactions. Despite these advances in understanding quorum sensing at the molecular level still there is no concrete evidence with molecular proof in biofilm development. Similarly, efflux pumps work to maintain low intracellular concentrations of the antibiotic by removing it from the cell. At the cellular level, a subpopulation of cells called persister cells has a phenotype that enables them to survive exposure to high levels of antibiotics, and to begin growing again when the antibiotic is removed.  The mechanisms that give rise to persister cells are not well understood and an area of vital research importance. A key question, still under debate, is whether persister cells are simply dormant cells with inactive antibiotic targets, or cells with a different metabolism that is actively maintained. Since several factors appear to be involved in the persister phenotype cells such as the oxidative stress, stringent as well as SOS responses, toxin-antitoxin modules, and levels of metabolic activity [2,5].

During the past decade, substantial progress has been made in recognizing the importance of biofilms in chronic infections, and understanding the biochemical and cellular processes that lead to biofilm formation. Still much less is known about how biofilm forming pathogens interact with their hosts in vivo, and about the mechanisms that enable biofilms to resist the action of antibiotics and both innate and acquired immune factors both in vitro and in vivo. Advances on both fronts are essential for preventing and treating infections. Progress will require technologies that will enable sophisticated analyses of the biochemistry and cell biology of biofilm-forming micro-organisms in vivo, as well on-going development of animal models that mimic chronic infections. In addition, there is a need for innovative methods for disrupting biofilms that do not require antibiotics. Nosocomial infections, frequently involving biofilms, are a leading cause of death and thus a major problem not only for individuals but also for health care facilities. The on-going international and interdisciplinary efforts to understand both the basic biology and pathogenesis of biofilms gives confidence that there will be substantial progress in developing new avenues for therapeutic interventions in the coming years.

 

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