Biofilm Development in Gram-Positive and Gram-Negative Bacteria

Abstract

Biofilms are the communities of microorganisms, especially bacteria attached to a biotic or abiotic surface. These biofilms live in a self-sustained matrix and produce different substances called extracellular polymeric substances (EPS) which are responsible for the pathogenicity of a number of bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Escherichia coli, etc. These EPS substance makes it difficult to eradicate the biofilm present on the surface. Biofilm formation is a five-step process. Biofilms can be monospecies or multispecies. In biofilms, cells communicate via Quorum Sensing (QS). QS is the regulation of gene expression in bacteria with respect to changes in cell population density. In QS, bacteria produce various signaling molecules called Auto-inducers (AI). AI concentration increases as the bacterial population increases. Bacteria respond to these AIs results in an alteration of gene expression, which results in the release of various virulence factors. QS involves a two-component signaling process which is different for both Gram-positive and Gram-negative bacteria. QS and EPS make the bacteria resistant to various antibiotics, which make the eradication difficult and hence requires more effective treatment. This article discusses the biofilm structure, phenomenon of biofilm formation, signaling, and pathogenicity to highlight the understanding of processes involved in biofilm formation.

1. Introduction

Microorganisms exist in nature primarily attached to biotic and abiotic surfaces. This is possible due to the development of biofilm. Biofilms are the group of microorganisms living within a self-produced matrix of polymeric substances which get attached to several surfaces [1]. Biofilms are different from the planktonic form of bacteria. Planktonic forms are the free-living forms of bacteria. Bacteria try to switch this planktonic form to biofilm due to a number of advantages which includes protection against environmental stresses such as extreme pH, oxygen, osmotic shock, heat, freezing, UV radiation, predators, etc [2]. Biofilm contains a group of microorganisms irreversibly attached to and grow on a surface. The substances produced by these microbes are known as extracellular polymeric substances (EPS) result in the alteration in the phenotype of the organism with respect to growth rate and gene transcription [3].

Biofilms are found to be present on liquid surfaces as floating mat and in a submerged state as well [4]. Biofilms appear either beneficial or detrimental. Biofilms are considered beneficial as these degrade hazardous substances which are present in the soil, but are detrimental to food and slaughterhouse equipment and are also found responsible for the pathogenesis of a number of diseases [5]. Biofilm has been used for the remediation of heavy metals for a long time. EPS as being poly-anionic in nature, forms complexes with positively charged metals (cations) result in metal immobilization within the exopolymeric network. Extracellular enzymatic activities in EPS assist the detoxification of heavy metals by transforming and subsequently participating in exopolymeric mass [6]. Microorganisms in biofilm help in the production and degradation of organic matter, remediation of environmental pollutants, nitrogen cycle, sulfur, and many metals. Some of the literature revealed that microbial biofilms are involved in sewage purification also [7].

Biofilms can grow on surfaces of many medical implants such as sutures, catheters, dental implants, etc [8]. Biofilm formation is an important virulence mechanism in the pathogenesis of many medically important organisms such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, etc [9, 10, 11] infections including biofilm formation such as vaginitis, colitis, gingivitis, otitis, urethritis, etc [12, 13, 14]

Biofilms are communities of bacteria embedded in the EPS matrix. EPS is composed mainly of a complex mixture of proteins, lipids, nucleic acids i.e. extracellular DNA (e-DNA) and polysaccharides [15]. EPS helps the biofilm to withstand mechanical stress. Biofilms are viscoelastic in nature and EPS provides physical support against mechanical and chemical stresses [16].

Depending on the interaction between surface and constituent cells, biofilms can be categorized as monolayer or multilayer [17]. Flagellum and pilus present on the surface of cells increase the attachment of bacteria to the surface which accelerates the formation of biofilm monolayer. In another type, the microbial adhesion is synthesized with the simultaneous transition to the permanent attachment [17]. When microorganisms are able to adhere to a surface and also to each other, they often develop multilayer biofilm. It has been noted in many cases that the bacterial surface characteristics lead to repulsion [17].

2. Biofilm structure

The structure of biofilm consists of matrix of EPS which comprises e-DNA, polysaccharides, and proteins [18]. Channels in this biofilm allow water, air, and nutrients transport to all parts of the biofilm [19].

Exopolysaccharides: These are the high molecular-weight sugar polymers that are secreted outside the matrix act as a scaffold for proteins, nucleic acids, carbohydrates, and lipids to adhere to the surface [20]. Mannose, galactose, and glucose are the most abundant carbohydrates in EPS. Most of the exopolysaccharides are not biofilm specific but their production increases as an environmental stress response.

Extracellular Proteins: This is another major class of EPS. These are found attached to the surface and polysaccharides to help with biofilm formation and stabilization. E.g. Amyloids play a supportive role in biofilm formation. Fap amyloids in P. aeruginosa lead to cell aggregation and increased biofilm formation [21]. The dispersal and detachment of biofilm also require some enzymes which release biofilm cells and initiate a new biofilm lifecycle. For E.g. Dsp B protein is responsible for the detachment of Actinobacillus pleuropneumoniae biofilms [22].

e-DNA: It comes from both lyzed cells and also actively secreted [23]. It plays an important role in biofilm formation critical for attachment. It interacts with receptors present on the substratum surface to facilitate adhesion [24]. It also coordinates with the cell movement in twitching motility mediated P. aeruginosa biofilm expansion [25]. It also inhibits the transportation of antibiotics within biofilm thus protects the bacteria within the biofilm. E.g. In Staphylococcus epidermis, e-DNA inhibits the transportation of vancomycin and thus protect the biofilm [26]. Vancomycin is a glycopeptide antibiotic that penetrates the biofilm and kills the growing biofilm including gram-positive bacteria. Figure 1 shows components of the EPS matrix.

Figure 1.

Components of EPS matrix.

3. Steps of biofilm formation

Biofilms are three-dimensional communities of microorganisms that adhere to a surface and form a matrix of EPS. Both gram-positive and gram-negative bacteria develop biofilm but the most common species are E. faecalis, S. aureus, S. epidermidis, S. viridans, E. coli, K. pneumoniae, P. mirabilis, and P. aeruginosa [27]. Biofilm formation takes place over five main stages including: 1. Initial reversible attachment; 2. Irreversible attachment; 3. Maturation Stage I; 4. Maturation Stage II and 5. Dispersion [28, 29].

1. Initial reversible attachment: Bacteria generally adhere to a surface that is rich in organic molecules (e.g. nutrients, salivary proteins, large macromolecules). These molecules promote the adherence of bacteria to the surface. Initial attachment is mediated through weak van der Waals force which later turns to stronger dipole-dipole interaction, hydrogen, ionic or hydrophobic interactions. There is a stronger adhesin-receptor mediated attachment. It is an attachment between adhesins, adhesive structures present on the surface of microorganisms and receptors, complementary adhesive structures present on the surface of host cells [6]. These interactions are mediated through the surface structures present on the bacterial cell such as fimbriae, flagella, lipopolysaccharides (LPS), outer membrane proteins (OMPs), and exopolysaccharides [30].

2. Irreversible attachment: Initial reversible attachment further changes to the irreversible attachment. In this stage, the forces of attraction are greater than the forces of repulsion. Initially immobilized bacterial cells attach to the surface irreversibly [31]. The structures present on the surface overcome the physical repulsive forces of the electrical double layer of the cell and consolidate the interaction between bacteria and the surface [32]. The hydrophobic interactions between the surface and bacteria also reduce the repulsive forces between them [4].

In the first and second stages, bacteria reversibly adhere to the surface which is further replaced by irreversible interaction.

1. Maturation Stage I: The bacterial cells start communicating in this stage by the production of AI signals which results in the expression of biofilm-specific genes [33]. The bacteria start producing EPS which stabilizes the biofilm. In this stage, the thickness of biofilm increases up to 10 μm.

2. Maturation Stage II: In this stage, the thickness of biofilm further increases to 100μm. Multispecies microconsortia develops on the surface which results in increase in substrate exchange between bacteria, distribution of metabolic products, and removal of toxic end-products produced by the bacteria [34]. Syntrophic association develops between distinct bacteria in which these utilize certain substrates as energy sources [34]. In this stage, biofilm adapts with the external conditions by manipulating its structure, physiology, and metabolism.

3. Dispersion: In this stage, dispersion of bacteria takes place and bacteria return to motile form [35]. In this stage, the microbial community produces different saccharolytic enzymes which break the biofilm stabilizing polysaccharides that releases the bacteria present on the top of the biofilm and colonize to the new surface. The microorganism upregulates the expression of flagella proteins and bacteria return to motile form to translocate to the new site. Figure 2 shows the process of biofilm formation.

Figure 2.

Stages of biofilm formation.

4. Quorum sensing

QS in bacteria is the regulation of gene expression with respect to the fluctuations in the cell-population density. In QS, bacteria produce chemical signal molecules called AI which increase in concentration as a function of cell density [36]. Bacterial populations coordinate their gene expression by producing and responding to a variety of intra and inter-cellular signals called AIs [37]. Microorganisms communicate by producing and responding to small diffusible molecules AIs that acts as signals. When a single bacterium releases AIs into the environment, the concentration is too low to be detected but when mass bacteria releases AIs, the concentration reaches a threshold level which allows the bacteria to sense a critical cell mass, and in response to this it activates or represses target genes. Many classes of AIs have been described to date and N-acyl homoserine lactones (AHLs) are most studied AIs of gram-negative bacteria. A class of AIs termed AI-2 with unknown structure in most cases and the peptides of gram-positive bacteria are most studied [38].

5. Quorum sensing in gram-negative bacteria

In gram-negative bacteria, the QS circuit involves at least two regulatory proteins called LuxR and LuxI. These proteins bind with the protein receptor bound to the bacterial cell membrane/wall. The signaling molecules bind with the receptor proteins then enter the cell. The LuxI protein is responsible for the biosynthesis of AHL, which is utilized as signaling molecules. The AHL concentration increases with the increase in cell population density. The LuxR protein is responsible for binding to cognate AHL AIs that have achieved a threshold concentration; these complexes also activate target gene transcription. The following Figure 3 shows protein involved in QS and signaling pathway in gram-negative bacteria.

Figure 3.

Proteins & two-component signaling pathway in gram-negative bacteria.

6. Quorum sensing in Pseudomonas aeruginosa

P. aeruginosa can be best understood in terms of the virulence factors regulated and the role of QS plays in pathogenicity. P. aeruginosa is found to be an opportunistic pathogen as it primarily infects individuals who are immune-compromised, such as patients with cancer or AIDS or those having breaches in normal barriers caused by burns, indwelling medical devices, or prolonged use of broad-spectrum antibiotics [39]. P. aeruginosa is an impressive armament of both cell-associated and extracellular virulence factors. P. aeruginosa involves two intertwined QS systems in virulence, biofilm development, and many other processes. Iglewski and colleagues discovered the first system (Las) consists of LasI encoded acyl-HSL synthase and the LasR encoded transcriptional activator. LasI is homologous to LuxI. A number of investigators found the second system (Rhl) consists of an rhlI-encoded acyl-HSL synthase and an rhlR-encoded transcriptional activator. In the respective QS systems, each produces and responds to a specific acyl-HSL; LasI directs the synthesis of 3-oxo-dodecamoyl-HSL (3-oxo-C12-HSL) and RhlI directs the synthesis of butyryl-HSL (C4-HSL) [40].

Using P. aeruginosa, lasI, and rhlI double mutant recently, Whiteley et al identified nearly 40 QSc genes that showed a fivefold or greater response to exogenously added acyl-HSL signals. On the basis of the pattern of the responses to cells grown in presence of Las signal, 3-oxo-C12-HSL and/or the Rhl signal, CH-HSL, the QSc genes were classified. A number of early QSc genes were found that responded immediately to exogenously added signals suggesting that these genes behave like the Lux genes of V. fischeri and the carbapenem biosynthesis genes of Ervinia. By seminal observations, a number of proteins have been found that support this hypothesis including the stationary phase sigma factors RpoS, RsmA, a third LuxR homolog (QScR), and stringent response proteins RelA, all of them are involved in modulating the expression of genes. QScR gene was found to be the negative regulator of both rhlI and lasI genes. In P. aeruginosa, early activation of QSc genes and premature synthesis of signals like C4-HSL and 3-oxo-C12-HSL were found in QScR mutant varieties. Overexpression of rsmA gene product resulted in decreased production of QSc virulence factors and acyl-HSLs whereas rsmA deletion led to early activation of LasI and thus the early synthesis of 3-oxo-C12-HSL [41].

Expression of a number of virulence factors is regulated by QS in P. aeruginosa and QS plays an important role in the pathogenicity of this organism. This presumption has been confirmed by using a number of different animal models. A lasR deficient strain of P. aeruginosa was found to have decreased virulence compared to that of the parent in a neonatal mouse model of pneumonia. Analysis of the P. aeruginosa mutant varieties such as lasI mutant, rhlI mutant, and a lasI, rhlI double mutant in the same model revealed markedly decreased virulence and the most remarkable reduction was found in the double I mutant variety [42]. Figure 4 shows the QS in P. aeruginosa.

Figure 4.

Quorum sensing in P. aeruginosa.

7. Quorum sensing in gram-positive bacteria

QS systems are found to be involved in the pathogenicity and biofilm formation of a number of gram-positive bacteria and these systems use different signal molecules from those of gram-negative bacteria which produce AHLs as AIs. In gram-positive bacteria, no AHL production has been observed in biofilm. Small post-translationally processed peptide signal molecules are used by the gram-positive bacteria QS system. These peptide signals interact with the sensor element of a histidine kinase two-component signal transduction system. Development of bacterial competence in B. subtilis and S. pneumoniae, conjugation in E. faecalis, and virulence in S. aureus is regulated by using QS system. A wide variety of disease states caused by S. aureus ranges from mild skin infections to life-threatening endocarditis. The virulence of this organism is dependent on the temporal expression of a diverse array of virulence factors which include cell-associated products, such as collagen and fibronectin-binding protein A, and secreted products including lipases, proteases, alpha-toxin, toxin-1, beta-hemolysin, and enterotoxin [43]. Figure 5 shows the signaling pathway in gram-positive bacteria.

Figure 5.

Signaling pathway in gram-positive bacteria.

8. Quorum sensing in Staphylococcus aureus

Surface proteins involved in attachment during the early stages of S. aureus infection (collagen and fibronectin-binding protein) and defense protein (protein A) predominate. Expression of S. aureus surface proteins is decreased and secreted proteins are preferentially expressed when once a high cell density is achieved at the infection site. Two pleiotropic regulatory gene loci called agr (accessory gene regulator) and sar (staphylococcus accessory gene regulator) determine the genetic basis for this temporal gene expression [44].

The agr locus of S. aureus consists of two promoters P2 and P3 with two divergent operons, RNAII and RNAIII. The RNAII operon contains the agr BDCA genes which encode the response regulator (AgrA) and signal transducer (AgrC), and AgrB and AgrD which are involved in generating the QS signal molecule. δ-hemolysin is encoded by the RNAIII and is itself a regulatory RNA that plays a key role in agr response. In response to the octapeptide signal molecule, the AgrC signal transducer is autophosphorylated during S. aureus QS, which in turn leads to the phosphorylation of the AgrA response regulator. The transcription of RNAIII is stimulated by phosphorylated AgrA and in turn RNAIII upregulates the expression of numerous S. aureus exoproteins as well as the agr BDCA locus. The latter leads to a rapid increase in the synthesis and the export of the octapeptide signal molecules. The AgrA gene product (AgrA) functions as a regulatory DNA-binding protein to induce the expression of both RNAII and RNAIII operons of the agr locus at the second regulatory locus [45, 46]. Figure 6: Showing the QS in S. aureus.

Figure 6.

Quorum sensing in S. aureus.

9. Role of biofilm in pathogenesis

Biofilms play a major role in the pathogenesis of many diseases [47]. A large number of nosocomial infections result due to the colonization of bacteria on the surface. Almost 95% of urinary tract infections are associated with urinary catheters which include S. aureus infections. S. aureus and P. aeruginosa are responsible for frequent biofilm infections.

10. Pseudomonas aeruginosa pathogenicity

P. aeruginosa is a gram-negative bacterium that is found to be responsible for a number of infections. It is an opportunistic human pathogen capable of causing both acute and chronic infections [48]. The lungs are one of the common niches for its colonization. It is found to be associated with respiratory infections like cystic fibrosis, lung infections [49]. Its greater adaptability and opportunistic sense enable its association with other infections also like wounds, burns, etc. [50]. Multidrug-resistant P. aeruginosa is emerging nowadays which makes the treatment more difficult. P. aeruginosa shows resistance to a number of antibiotics like β-lactams, aminoglycosides, quinolones, etc due to mechanisms such as low outer membrane permeability, efflux system, inactivating enzymes like β-lactamases [51]. It can also acquire resistance genes from other micro-organisms by horizontal gene transfer such as in the case of biofilm [52].

P. aeruginosa shows adaptation which is related to complex mechanisms. A number of factors are found to be responsible for the pathogenic potential of bacteria which play a key role in biofilm formation and dispersion. These include flagella, pili, enzymes like proteases, siderophores like pyoverdine, surfactants like rhamnolipids and toxins like exotoxin A and pyocyanin, etc. [53].

11. Staphylococcus aureus pathogenicity

Both gram-positive and gram-negative bacteria are found to be pathogenic in nature. S. aureus is a gram-positive bacteria frequently found on the mucosal surface of the nose and respiratory tract and skin [51]. It is easily transmitted by direct contact. It is also found to be methicillin-resistant which makes it difficult to treat. Methicillin is a narrow-spectrum β-lactam antibiotic of the penicillin family. S. aureus is very often found to be associated with nosocomial infections. Multidrug-resistant S. aureus (MRSA) has the ability to evolve and adapt easily which is being considered as a threat according to W.H.O [54]. In addition to this, MRSA is also developing resistance to other antibiotics via mutations and horizontal gene transfer [55]. It has been reported that the presence of S. aureus in heterogeneous biofilms increases the rate of plasmid horizontal transfer which increases the resistance of antibiotics in biofilm [56]. S. aureus shows the ability to survive host-defense mechanisms through different factors such as cell wall-anchored proteins like clumping factors, fibronectin-binding protein A, collagen adhesion which enables tissue attachment, evasion, and biofilm formation [57]. Extracellular toxins (including hemolysin, leukotoxin, entero-toxin) and enzymes (including coagulase, proteases, staphylokinase) help in tissue penetration and host invasion [58]. Surface-associated factors are down-regulated and surfactants are also expressed in the later stages which lead to biofilm dispersion and the spread of infection [59].

12. Conclusion

Biofilms are made up of bacteria that consist of monospecies or multispecies. Bacterial biofilms are found to be present on a number of surfaces and for this purpose, bacteria secrete and produce EPS matrix which makes adherence easier. Biofilm formation has become a ubiquitous phenomenon found on both living and non-living surfaces. In this biofilm, bacteria interact by producing various toxins, virulence factors that are pathogenic in nature. Both gram-positive and gram-negative bacteria show different QS systems. QS leads the bacteria to evade the immune response and increase cell density. QS is found to be responsible for the virulence shown by the bacteria. Many bacteria show virulence characteristics such as S. aureus, P. aeruginosa, E. faecalis, V. cholerae, S. pneumoniae, etc. S. aureus produces alpha-hemolysin, toxins, various proteases whereas P. aeruginosa is found to produce exoenzymes, cell-cell spacing and sis also resistant to chloramphenicol. S. aureus and P. aeruginosa are two of the most common bacteria which show biofilm formation. These bacterial biofilms are difficult to eradicate from the surface due to strong adhesive forces and resistance against a number of antibiotics. Current therapeutic approaches are not effective to prevent biofilm formation and thus there is a requirement for new strategies and drugs for the treatment of biofilm infection.

EPS	Extracellular polymeric substances
AI	Auto-inducers
AHL	N-acyl homoserine lactones
MRSA	Multidrug resistant S.aureus
QS	Quorum sensing