Application of Biofilms in Applied Microbiology

Application of Biofilms in Applied Microbiology gives a complete overview on the structure, physiology and application of biofilms produced by microbes, along with their potential application in biotechnology. Sections cover new technologies for biofilm study, physiology of microorganisms in biofilms, bacterial biofilms, biofilm development, and fungal biofilms, summarizing various technologies available for biofilm study. Subsequent chapters describe biofilm developments with Bacillus subtillis, Escherichia coli, and Pseudomonas putida, along with several chapters on the study of microbial biofilm and their advantages and disadvantages in the area of environmental biotechnology.

The book closes with a chapter on the rapid development of new sequencing technologies and the use of metagenomics, thus revealing the great diversity of microbial life and enabling the emergence of a new perspective on population dynamics.

• Summarizes various technologies available for biofilm study
• Describes the physiological study of bacteria, fungi and algae present in biofilms
• Provides the potential parameters on biofilm development
• Gives insights on the ability to construct and maintain a structured multicellular bacterial community that critically depends on the production of extracellular matrix components
• Reveals the rapid development of new sequencing technologies and the use of metagenomics, the great diversity of microbial life, and the emergence of a new perspective on population dynamics

• Language: English
• Publisher: Academic Press
• Release date: Aug 9, 2022
• ISBN: 9780323905251

Book preview

Chapter 1

Bacterial extracellular polysaccharides in biofilm formation and function

Dibyajit Lahiri¹*, Moupriya Nag¹*, Bandita Dutta², Ankita Dey² and Rina Rani Ray²,    ¹Department of Biotechnology, University of Engineering & Management, Kolkata, West Bengal, India,    ²Department of Biotechnology, Maulana Abul Kalam Azad University of Technology, Kolkata, West Bengal, India

Abstract

Microbial cells produce extracellular polymeric substances (EPS) comprising polysaccharides, proteins, and extracellular DNA, which plays an important role in the mechanism of biofilm formation. The excess production of polysaccharide is responsible for the change in the morphology of the existing colonies. The polysaccharides that form an integral part of EPS help in the mechanism of adhesion, protection, and structural formation within the biofilm. These group of polysaccharides act as a molecular glue helping in the attachment of cells with the biotic and abiotic surfaces. This mechanism of attachment using EPS helps in protecting the sessile colonies from environmental stresses like that of predators, nutrient source, and immune effectors. This also helps in providing the development of the biofilm structure and stratification of the bacterial communities. This chapter isfocused on the recent status of research into the role played by EPS in the adhesion of the bacterial cells, genetics associated with the EPS development, utility of EPS, and targeting EPS by potent drug-like compounds.

Keywords

EPS; carbohydrate; protein; eDNA; biofilm; application

1.1 Introduction

The construction and stability of the microbial consortia depend on the production of extracellular polymeric matrix (EPS) (Sutherland, 2001). This acts as a keyfactor for the development of biofilm (Flemming et al., 2007). EPS are the biopolymers that are synthesized by the several stains of microorganisms, providing structural stability and nutrients during the adhesion of the sessile microcolonies and the development of the biofilm. EPS produced by the microbial communities vary greatly in their chemical composition, thus exhibiting varying chemical and physical properties. Some of these can be polyanionic, whereas others can be polycationic in nature (Sutherland, 2001). The structural units and their arrangement inside the EPS are complex, which have been studied from decades. The macromolecules that are predominantly available within the EPS comprise polysaccharides, proteins, peptidoglycans, lipids, enzymes, and extracellular DNA (eDNA). Nucleases that is present within the EPS acts as an important regulator for the formation of the biofilm (Kiedrowski et al., 2011). The polysaccharides are the widely studied component of EPS. The analysis of various polysaccharides obtained from varied microbial species showed that it varies largely in its composition. They are made up of one or more structural components and its arrangement varies from one type of EPS to other. The commonly found polysaccharides within the EPS are highly soluble within salt solution and in water. The polysaccharides forming the capsule help in adherence to the cell surface via covalent bonds with the other polymers that are present upon the surface. The extracellular polysaccharides are insoluble and cannot be separated from the cells thereby makes the determination of physical and chemical properties difficult. The polymers obtained from the biofilm of Pseudomonas aeruginosa were found to be highly heterogenous in nature. The exopolysaccharides being present within the matrix can be ordered or disordered. Low ionic concentrations and high temperatures result in the development of disordered form of polysaccharides. The polysaccharides are capable of interacting with various types of heterologous ions and molecules, thus resulting in the development of gels where various types of multivalent cations play an essential role. The proteins and polysaccharides play an important role in the development of matrix thus inducing structural and functional properties. The eDNA is not related to any genetic instructions or transmission of genetic information to the next generation, rather it is important in genetic exchange, signaling and attachment. Though EPS have multiple functions, the most important function is providing protection to the microbial cells present within the matrix from the antimicrobials, heavy metals and various types of environmental stresses like temperature and pH change, depletion of nutrients and water. Other functions like adhesion to the biotic abiotic surfaces, communication between the microbes through genetic exchange, and entrapment of the nutrients have been reported (Wang et al., 2015).

A biofilm is a multi-layered community of the bacterial cells, which form syntropic consortia that remain embedded within the hydrated solid biotic as well as abiotic surfaces. Though both the sessile bacterial cells are responsible for the biofilm formation, cell growth of the planktonic cells rarely exists in nature. The biofilm was identified long ago around 17th Century by Anthonie van Leeuwenhoek, after investigating the deposited microbial aggregated upon the tooth surface using his home-made microscope. That was the first microscopic visualization of bacteria and the discovery of the biofilm (Costerton, 2007). An observation of the Bottle effect of the marine microorganism by Heukelekian and Heller, explained that the bacterial growth and activity can be enhanced substantially due to the surface attachment (Heukelekian, 1940). Depending upon the several observations it became evident that bacterial survival is related to the bacterial functions and growth within the population and also related to the typical life style of the microorganism.

The term biofilm, which was coined and described in 1978, is related to the microbial community, distributed through the matrix or glycocalyx on the hard non-shedding surface material. Microbes are present at the bottom layers of the biofilm and they are bound together through a polysaccharide matrix containing both the organic and inorganic materials. The upper layer of the biofilm is amorphous in nature to extend towards the surroundings. Biofilm provides the protection towards the microbial community from the environmental stresses. EPS is one of the important components of the biofilm, which comprises glycocalyx and chemically made up of water (96%–97%), microbial cells (2%–5%), polysaccharides (1%–2%), proteins (<1%–2% including extracellular and lysed), nucleic acids (<1%–2%), and ions (both bounded and nonbounded). EPS helps the bacterial cells to adhere into the embedded slimy layer and provides the nutrients to the developing cells within the biofilm. Thus, it helps in pathogenesis of biofilm associated with infection and resistance due to the formation of impermeable layers to the penetration of antibiotics and drugs. Biofilm can be formed by the microbes depending on various factors comprising of specific and non-specific site of attachment, nutritional sources and exposure to various sub-inhibitory concentration of antibiotics. The present chapter describes the structural features of component exopolysaccharides in a mature biofilm matrix as evident from their extraction, their functional attributes and their industrial applicability.

1.2 Exopolysaccharides associated with the matrix of biofilm

EPSs are the building material of bacterial attachment either upon the cell’s outer surface or are secreted into its growth medium. They vary in their composition, localization, and charges (Table 1.1). The exopolysaccharides being present within the biofilm play a vital role in its formation, persistence of pathogens and have various potent application in medical and food industries, thus considered as a potent component of the biofilm (Sutherland, 2001). Some studies also predicted that the exopolysaccharides are not important for the process of the formation of the biofilm. The polysaccharides that are being present within the biofilm may be comprised of homopolymers like that of dextran or curdlan, cellulose as well as heteropolymers like that of gellans or xanthan, emulsan and alginate. The exopolysaccharides that are present can be branched or linear in nature. The EPS comprise of monosaccharides along with various noncarbohydrate sources like succinate, phosphate, acetate and pyruvate.

Table 1.1

1.2.1 Various types of architectural polysaccharides associated with the biofilm

The previously described polysaccharides all are related to the biofilm structure formation. The architectural polysaccharides regulate the biofilm formation and their structure mainly the observable phenotypes due to the mutations or overproduction of the structural components. For example, The rugose phenotype of the Vibrio cholerae as well as the gene regulation and second messenger signaling and the assembly of the biofilm matrix is regulated by the Vibrio polysaccharide (Berk et al., 2012).

1.2.1.1 Bacterial alginates

Alginates are the common type of polysaccharides that are found predominantly with brown algae and various types of bacterial species like P. aeruginosa and Azotobacter vinelandii (Gorin et al., 1966). It is of high molecular weight and comprise uronic acid residues, β-D-mannuronate, and α-L-guluronate. It comprise of a block of copolymer possessing homopolymeric regions comprising of poly-β-D mannuronate (M component) and poly-α-L-guluronate (G component) along with heteropolymeric regions. The presence of poly-α-L-guluronate makes the alginate being produced by P. aeruginosa from the alginates that are being produced by other groups of algae (Remminghorst et al., 2006). The functional property of the alginate is dependent on the ration of M to G along with the presence of the uronic acid sequence. In has been observed that 24 genes that are present within the chromosome of P. aeruginosa is responsible for the production of alginate. Studies has also further shown that eight genes are responsible for the purpose of exportation of the alginic acid and the remaining four genes are responsible for the mechanism of synthesizing the alginates (Fig. 1.1). Alginates can result in the development of gel in the presence of divalent cations. The mechanical property exhibited by alginates depends on the presence of guluronic acid associated with the polymer (Shah et al., 2020) Alginate: Alginate is one of the main components of the P. aeruginosa PS as it is required for the transformation of the nonmucoid to mucoid phenotype of the P. aeruginosa during the course of infection. The 90% of the clinical isolates of P. aeruginosa have the overproduced alginate causing the cystic fibrosis (CF) pulmonary infections (Elston et al., 1967). In case of the CF patients initially the nonmucoid (Psl and/or Pel producing) P. aeruginosa colonizes and then mucoid phenotypes occur after the conversion to increase the morbidity and mortality of the CF patients, which is the significant clinical challenges in eradicating the P. aeruginosa infections (Gaspar et al., 2013). Alginate is a random linear polymer consists of acetylated 1,4-linked β-D-mannuronic acid and C5 epimer α-L-guluronic acid, provides the highly hygroscopic PS promoting biofilm fluidity and resistance to desiccation (Sutherland et al., 2001).

Figure 1.1 Synthesis of bacterial alginate.

Alginate synthesis is regulated by the alginate biosynthetic operon (algD-A), which is dependent of the alternative sigma factor AlgT (alternatively known as AlgUAσE/o22/RpoE). The anti-sigma factor MucA has the antagonist effect to the activity of the AlgT, by preventing the interaction with AlgT-dependent promoters (Ramsey et al., 2005). Due to the mutation of the mucA gene alginate is constitutively overproduced in CF mucoid isolates and mutant mucA is unable to inactivate the AlgT. Along with the MucA there are other regulators including AlgR, IHF, AlgB, and AmrZ (algD transcriptional regulators) and MucB to E and P, AlgW, KinB, and ClpX proteases (regulation of MucA stability) are regulating the alginate production (Damron and Goldberg, 2012).

1.2.1.2 Cellulose

Cellulose is the most abundant polymeric substance that is available on planet. This chemical substance is found in plants, fungi, animals, and bacteria like that of E. coli, Acetobacter, Salmonella, Rhizobium, and Agrobacterium. The extracellular matrix of E. coli and Salmonella comprise cellulose as the vital component. The glucose units that are present within cellulose comprise of β-1–4 linkage. The glucose chains that persist within cellulose fibers are held by hydrogen bonds. The sheets formed by cellulose are highly stable and varies depending on the environment. Cellulose forms a crystalline structure possessing various glycans in parallel orientation. It consists of reducing component at one end and non-reducing component at the other end. Cellulose has the ability to form gel at adequate temperature. Synthesis of cellulose in S. typhimurium and E. coli is associated with genes termed as bacterial cellulose synthesis (bcs). The four types of the bcs genes like bcsA, bcsB, bcsZ and bcsC are together associated within the operon (Fig. 1.2). Further the operon is regulated by AgfD that help in the enhancement of cellulose production. The solution of cellulose is liquid at room temperature and forms gel at higher temperature than 50°C and lower than 10°C (Cai et al., 2006).

Figure 1.2 Synthesis of cellulose being present within the matrix of biofilm.

1.2.1.3 Poly-N-acetyl glucose amine

The bacterial adhesion and formation of biofilm are mediated by poly-N-acetyl-glucoseamine (PNAG) and polysaccharide intercellular adhesion (PIA). These polysaccharides were first observed within Staphylococcus aureus and thereafter in E. coli. PNAG are the group of positively charged linera homoglycans comprising of β-1,6-N-acetylglucosamine residues out of which 20% is found to be acetylated. The genes that are associated in the production of PIA is ica which aids in the process of intercellular adhesion. The operon is regulated by icaR gene and four biosynthetic gene icaADBC (Gerke et al., 1998). The bacterial cells are involved in cell-to-cell communication with the help of the protective matrix formed by PNAG. It has been further observed that the interaction between eDNA result in the development of the matrix of the biofilm.

Capsular polysaccharide: Capsular polysaccharides (CPSs) are covalently attached to the phospholipids or lipid-A molecules of the bacterial cell-surface to make the biofilm hydrated. CPSs are diverse in nature which composed of not only the monosaccharide joined through the glycosidic linkage but also some other noncarbohydrate constituents joined through glycosidic linkage, branching and substitution (Whitfield et al., 1999).

A variety of the CPSs are present in both the gram-positive and gram-negative bacteria to regulate the polymer synthesis, and 80 CPSs (K antigens) are present in E. coli and 93 CPSs (serotypes) in Streptococcus pneumoniae (van der Woude, 2011). The nucleotide diphosphosugar precursors present in the cytoplasm is required for the capsule synthesis which further assemble to the polymer present in the periplasmic face of the plasma membrane (Whitfield et al., 1999).

1.2.1.4 Capsular polysaccharides

It plays an important role to protect the cells from the environmental conditions by protecting the cells from desiccation, complement-mediated, and cationic antimicrobial peptide-mediated killing and opsonophagocytosis. With the change of the environmental conditions the capsule production may varies from low CPS-producing phase to high CPS-producing phase (Yother, 2011).

1.2.1.5 Levan

This polymer provides protection to the bacteria from any desiccation and bacteriophages and also helps to develop virulence and store the nutrients. Bacterial mucoid phenotype produces the levan, which is a high molecular weight neutral homopolymers of β-D-fructans. Levan is produced from extracellular sucrose, catalyzed by excreted levansucrase, which is activated in presence of sucrose and regulated by two-component sensor kinases (LadS in P. syringae); SacX/SacY and DegS/DegU in B. subtilis; RscC/RscB, GacS/GacR (GrrS/GrrA), and EnvZ/OmpR in E. amylovora; and CovS/CovR (VicK/VicR) in S. mutans (Li and Kim, 2013). In B. subtilis levansucrase which is secreted through SecA pathway is regulated and modified by the sacB and sacC gene (Shida et al., 2002).

1.2.1.6 Colonic acid

Colanic acid (CA, also known as M antigen) is a polysaccharide comprising glucose, galactose, and glucuronic acid branched together to form a complex structure. In various species of Enterobacteriaceae the 19-gene wca cluster (formerly known as cps cluster, which was changed after several discovery of CA) is responsible for the production of CA. There are few enzymes, which are included in the wca cluster are responsible for the synthesis of the CA, whereas other enzymes which are producing the CA precursors like UDP-D-glucose and UDP-D-galactose, are located in other loci (Stevenson et al., 1996; Anderson et al., 1963; Grant et al., 1969). Rcs phosphorelay system regulates the wca gene cluster, through the transfer of the phosphate from RcsC to RcsD to RcsB. RcsB is a helix-turn-helix response regulator of LuxR family, which activates the transcription by binding to the promoter of wca operon. RcsA which is a highly unstable accessory protein acts as enhancer in this reaction. The upregulation of wca cluster and production of CA is regulated by the signaling cascade which allows the bacterium to endure in the environmental stress (Majdalani et al., 2005).

CA is well studied in the E. coli, where it is the first PSs formed in the biofilm. In mutant E. coli, the two phenotypes, one wild type and the mutant both the strain have the similar attachment capabilities, and second the mutant strain produces the three dimensional structure of the biofilm, which is densely packed or collapsed against the substrate.

CA plays different role in different bacterial strain. CA produced in the pathogen like Salmonella enterica serovar Typbimurium helps them to bind to the abiotic surfaces. The CA mutant in S. enterica is unable to build a three dimensional biofilm on Hep-2 cells and chicken intestinal epithelial.

1.2.1.7 Vibrio polysaccharide

Vibrio polysaccharide (VPS) is the polysaccharide produced by the V. cholerae, a Gram-negative aquatic bacterium, which brings about the diarrheal disease cholera after entering the human gastrointestinal track. VPS is essential for the biofilm formation and give rise to thr resistance to the chlorine. Glucose (52.6%) and galactose (37.0%), with small amounts of N-acetylglucosamine, mannose, and xylose (5.1%, 3.8%, and 1.5%, respectively) are the main components of the VPS, where 4-linked glucose and 4-linked galactose are present to form the linear backbone. The vps operon is allowing the PS production which is regulated by the NtrC family regulator VpsR. The two operons vpsA through vpsK and vpsL through vpsQ, which are positively regulated by the VpsR and VpsT, encode VPS. VpsR is epistatic to the VpsT, which regulates the positive feedback loop in VPS production. the negative regulator HapR represses the VpsR and VpsT, to regulate the vps expression. Deletion of the hapR from smooth strain results transition to rugose colony whereas deletion of vpsR or vpsT from the rugose strain generate smooth strain. The intracellular level of the second messenger c-di-GMP regulates the expression of the vps gene cluster. VpsT binds to the c-di-GMP to regulate oligomerization and upregulation of the vps operon. When the pool of c-di-GMP is altered by the diguanylate cyclases and phosphodiesterases, the VPs production get altered. vps expression is reduced by the phosphodiesterase VieA through the degradation of c-di-GMP, in contrast of which diguanylate cyclases CdgA, H, K, L, and M activate VpsT to elevate the level of c-di-GMP. When the VPS is absent the biofilm is formed in monolayer stage, bacterial cells form spherical foci on the cell surface in presence of VPS.

1.3 Variation in structural components of bacterial EPS

A biofilm is defined as a community of microorganisms networked within an exopolysaccharide (EPS) matrix with a distinct architecture. This EPS architecture serves both structural and protective functions. It forms channels that facilitate the transport of nutrients, enzymes, metabolites, and disposal of waste products within and outside of the biofilm matrix. In addition to the EPS, there are proteins, nucleic acids, peptidoglycan, lipids, phospholipids, and other cell components present in the matrix of biofilm communities. These components of the EPS and subsequent layers of cells can retard the penetration of antimicrobials, in addition to facilitating the transfer of nutrients in and waste products out of the biofilm structure. As a result of these characteristics, the EPS matrix confers properties in biofilm-associated communities distinct from their planktonic counterparts.

The nature of the matrix exopolysaccharide greatly varies counting on growth conditions, medium, and substrates. The exopolysaccharides secreted by gram negative and gram positive bacteria are different and may vary with strains. Gram-negative P. aeruginosa produces three exopolysaccharides: alginate, Pel, and Psl.

Gram-positive bacterium B. subtilis produces two exopolysaccharides: the polysaccharide EPS and poly-δ-glutamate (PGA). Both of these play a crucial role in the process of biofilm formation (Stanley, 2005).

Certain bacteria also produce adhesive proteins which help them to bind with the surface and remain in close contact to the other cells. S. aureus is one such bacterium which possesses biofilm associated protein (Bap). These are adhesive proteins that are required for biofilm formation. These proteins are present in the cell wall of the bacteria and they help to hold the cells together by interacting with other surface proteins of the neighboring cells. In some bacteria the presence of Bap eliminates the requirement of the exopolysaccharides for the formation of biofilm. On the other hand, B. subtilis, a Gram-positive bacterium expresses a sole chief protein which is related with extracellular matrix, known as Tas A. This protein is crucial for biofilm formation as bacteria who lack this protein fail to produce biofilms despite producing exopolysaccharide.

The matrix also consists of DNA apart from proteins and exopolysaccharide. DNA (eDNA) provides structural integrity to the biofilm. Thus the addition of DNase to the culture can inhibit the formation of biofilms. Biofilms of S. aureus and P. aeruginosa have a considerable amount of eDNA present. This might be due to the lysis of the cells and release of the genomic DNA.

1.4 EPS variation in gram-positive and gram-negative bacteria

1.4.1 Gram-positive bacteria

Quorum sensing plays an important role in regulating the PIA expression. Though the luxS gene negatively regulates the PIA expression, mutant luxS gene increases biofilm formation and enhances the virulence of the biofilm mediated infections in S. epidermidis (Xu et al., 2005). Another two genes σB and RsbU positively regulate PIA, by repressing transcription of icaR to activates the ica operon (Knobloch et al., 2001) Ica operon is positively and negatively regulated by the different proteins which are also regulating the biofilm formation along with that. In S. aureus Spx and SarA protein regulating the ica operon and biofilm formation, in negative and positive way respectively. SarA is the global regulator which is also regulating the PIA production (Pamp et al., 2006).

In case of Bacillus subtilis, a Gram positive bacteria the exopolysaccharide is of levan type I, which consists b-2,6-linked D-fructose units and type II which is a fructose polymer, where a glucose residue is linked to the terminal fructose by a-glycoside bond. Levan is widely found in various microorganisms including different strains of B. subtilis 327UH, ISS3119, QB112 and Pseudomonas sp. (Shida et al., 2002).

Apart from the levan, different exopolysaccharides have been found in different Bacillus sp. It was reported that in exopolysaccharide, glucose, galactose, fucose, glucuronic acid and O-acetyl groups are in 2:2:1:1:1.5 ratio approximately. Some genes are regulating the exopolysaccharides by encoding the protein. The gene yhxB of B. subtilis synthesizes the uncharacterized exopolysaccharide components and also encodes an a-phosphoglucomutase and phosphomannomutase putatively. It was studied that the deletion of yhxB gene from the B. subtilis 3610 (mutant B. subtilis) is able to produce the fragile surface pellicle in liquid culture and flat undifferentiated colonies in solid media, while on contrary the wild type B. subtilis forms a robust pellicle in liquid culture and bundled structures in solid media. There are total 16 genes involved in eps operon (yveKyv) which are regulating the polysaccharide biosynthesis, modification and export. The two major genes namely epsG (yveQ) and epsH (yveR) belonging to the eps operon involved in the synthesis of the exopolysaccharides. The protein responsible for the EPS polymerization is encoded by epsG whereas glycosyl-transferase is encoded by epsH.

1.4.2 Gram-negative bacteria

The EPS production of the gram-negative bacteria is different from the gram-positive bacteria. Pseudomonas aeruginosa, a gram-negative bacterium is well known for its quorum sensing activity which is manly regulated by the pel and psl operon. P. aeruginosa produce an aggregative polysaccharide which is known as pel due the presence of thick pellicle because of the overexpressed pel operon. This operon encodes seven enzymes which are homologous to the PS synthesis proteins. There are other genes which are essential for the pel production ae absent in this operon (Mann et al., 2012). Studies are still going on to identify the structure of pel and the composition of the sugar as well as the linkage between them. As the pslABCD genes are missing in the Pseudomonas aeruginosa PA14 strain, the aggregative property of this strain is regulated by the pel production. Cyclic diguanylate (c-di-GMP), an intracellular second messenger is able to increase the pel production post-transcriptionally and also modulate the PelD. The flagellum regulator FleQ, which acts as a promoter to regulate the pel operon, can be changed to the pel operon activator when the cellular pool of the c-di-GMP sense the FleQ (Baraquet et al.,