Review on the Role of Biofilm Formation in Bacterial Pathogenesis

    

    

    Figure 1: The development of a biofilm, depicted as a five-stage process.
Source: (Lasa, 2006) [30]
Stage1: Reversible or Initial Attachment: 2. Irreversible Attachment: 3. Microcolony Formation. 4. Biofilm Maturation. 5. Biofilm Dispersal.
    

Reversible or initial attachment: The transition from planktonic to biofilm mode is marked by bacterial surface adhesion [54]. Planktonic microorganisms interact with a conditioned surface to form reversible adhesion. However, the interaction, which comprises van der Waals forces, electrostatic forces, and hydrophobic contacts, is relatively weak. Surfaces that are rough, hydrophobic, and coated with various organic compounds will work best for the attachment [12] The link between bacteria and the surface of attachment is strengthened by bacterial features such as fimbriae, pili, and flagella. In general, cell appendages engaged in reversible attachment and bacteria commit tothe biofilm lifestyle or depart the surface and return to a planktonic lifestyle at this stage [54].

Irreversible attachment: Loosely bound organisms consolidate the attachment process at this stage by producing extracellular polymeric substances that complex with surface materials and/or receptor-specific ligands located on pili, fimbriae, and fibrillae, or both. After microorganisms are attached to preconditioned and permissive surfaces, the cell begins an irreversible adhesion and accumulates as a multilayered cell. The creation of a biofilm begins with a layer of polymeric substances (EPS) on which microbial cells swarm, followed by the biofilm's expansion. A number of physiological and anatomical changes have happened during this step, including nonmotility of the attached cells [49].

Microcolony formation: Micro colonies are formed when microbial cells anchored within the extracellular matrix proliferateina coordinated community. Micro colony formation, according to Dunne, is caused by the simultaneous aggregation and growth of microorganisms, as well as the creation of EPS [13].

Micro colonies, which are the fundamental units of biofilm, are divided into channels with varied microenvironments. After cells are securely connected to conductive surfaces, a slew of bacteria emerges and exudes polymeric compounds that can act as a "glue" to keep microorganisms attached to various surfaces. Micro colonies are formed as a result of these consecutive occurrences [59].

Biofilm maturation: A biofilm may develop into spatially well-arranged, three-dimensional mature biofilm structures such as mushroom or tower-like structures interspersed with fluid-filled channels in which nutrients, oxygen, and essential substances can be diffused and circulated if conditions are suitable for sufficient growth and differentiation [41].

The establishment of biofilm is governed by density-dependent chemical signals released by bacterial populations embedded in a self-produced extra cellular matrix in each micro environment. Quorum sensing is a signaling mechanism that is utilized to communicate and orchestrate group behaviors like as virulence factor secretion and biofilm formation. Quorum sensing triggers a coordinated maturation and breakdown of the biofilm. Cell-to-cell signaling is important for cell attachment and dissociation from biofilms in general [46].

Biofilm dispersal: Biofilm development is a cyclical process in which bacterial cells are detached from the mature biofilm and revert to their prior state of life, planktonicity. Disconnected bacterial cells will look for new surfaces to cling to and begin a fresh round of biofilm formation. Microbial cells will select whether to live together or “fall apart” at this step, based on environmental cues [29]. This stage is critical for disseminating bacteria into food products from the standpoint of food contamination. Biofilm cells can be separated from actively growing cells or the impoverished environment, and aggregates can be removed. Nutrient scarcity has been shown to push microbes to seek out new habitats [59].

Biofilm Formation Factors

Biofilm creation is a dynamic and complicated process that comprises initial bacterial cell attachment to the substrate, physiological changes within the microbe, multiplication of adherent cells to form micro colonies, and finally biofilm maturation. Biofilm-associated bacteria have different physiology and greater tolerance to the immune system and drugs than their free-living planktonic counterparts, making biofilm a source of chronic and persistent infections [42].

Biofilm Formation Causes

Many bacteria have the ability to transition between planktonic and biofilm forms. Planktonic bacteria have a fast rate of cell development and reproduction. The biofilm state, on the other hand, appears to be a natural and prevalent condition of bacteria. A bacterial biofilm condition may be required for a variety of reasons [24]. For starters, biofilm can improve bacteria's endurance to hostile environmental circumstances. Bacteria cancelling to a surface or tissue to avoid being was headway by water move mentor blood flow. Oral biofilms may withstand repeated high shear stresses. Second, the EPS matrix protects bacterium cells in deeper levels from antimicrobial agents, most likely by reducing antimicrobial agent diffusion. Biofilms limit bacterial mobility and enhance cell density, creating an ideal environment for eDNA (plasmid) exchange (through conjugation), which encodes for antibiotic resistance in some cases [44].

Role of Biofilm on Bacterial Pathogenesis

Biofilms originate on inert surfaces or dead tissue, and are usually found on medical equipment and fragments of dead tissue like sequestra of dead bone; nevertheless, they can also form on living tissues, as in the case of endocarditis [56]. Antigens are released by sessile bacterial cells, which trigger antibody formation. However, antibodies are in effective at killing bacteria within biofilms, and they may cause immunological complex harm to near by tissues. Biofilm infections are rarely healed by the host defensive mechanisms, even In people without standing cellular and humoral immune responses [7].

Animal Health Importance of Biofilm Forming Bacteria

Quorum sensing, swarming, and biofilm formation are examples of collaborative group behaviors used by bacteria to adapt to a variety of settings, including human hosts [50]. In general, quorum sensing during host tissue colonization is linked to virulence gene expression and acute-phase infections, whereas biofilm formation aids the establishment of chronic infections, immune evasion, and treatment tolerance [16]. The role of biofilm in human and animal disease processes is now generally acknowledged. The risk of infection in animal species is likely to be higher than in humans. Because of the differences in animal housing and living conditions [57]. The natural bacterial microflora in the mouths of dogs and cats is made up of a variety of aerobic, facultative, and totally anaerobic bacteria. Teeth provide continual dampness and adhesive surfaces in the oral cavity, allowing vast bacterial deposits to form [40].

Biofilm Forming Microorganisms

Almost all microorganisms (99.9%) have the ability to produce biofilm on a variety of surfaces, including biological and inert surfaces. Micro organisms create Extracellular Polymeric Substance (EPS) and form biofilm when they bind to a surface. Due to its antibiotic resistance and sickness associated within dwelling medical devices, biofilm poses

Significant threat to public health [48]. H. influenza has been discovered to have the ability to develop biofilm in the human body and to evade the immune system.

P. aeruginosa, S. epidermidis, E. coli spp, S. aureus, E. cloacae, and K. pneumoniae are among the bacteria that can produce biofilms [17]. Several gram-positive (e.g.Bacillus spp., Listeria monocytogenes, Staphylococcus spp., and Lactobacillus plantarum and Lactococcuslactis) and gram-negative (e.g. Escherichia coli, Pseudomonas aeruginosa). In aquatic conditions, cyano bacteria also create biofilms [53].

a. Escherichiacoli

E. coli is a Gram-negative rod-shaped bacteria that causes a wide range of nosocomial and community diseases, including UTIs and prostatitis. It can secrete toxins and polysaccharide, as well as produce biofilm. In vitro, it can also create biofilm [38]. E. colicapsules are large molecules with a high molecular weight that bind to the cell surface. E. colicapsules play an indirect function in biofilm formation by preventing bacterial adherence tothe surface. The ability of E. coli to generate biofilm is affected by a variety of environmental factors. Because of the existence of exopolymers, E. coli biofilms can be hundreds of microns thick, making antibiotic therapy challenging [33].

b. Staphylococcusaureus

Staph aureus is a multi drug-resistant bacteria that causes a variety of nosocomial infections. As a biofilm, it grows on catheters and chronic wounds [39] S. aureus recycles proteins in the cytoplasm to produce the extracellular matrix. The ability of cy toplasmic proteins to act as matrix proteins provides for more flexibility and adaptation to S. aureus in the creation of biofilms in infectious settings, and it may favor the production of mixed-species biofilms in chronic wounds (Lucy et al. 2014).

Diseases Related Biofilm

Microorganisms cling to surfaces and form biofilms, which have been linked to a number of diseases of major public health relevance (Mahami and Adu-Gyamfi 2011). Biofilms have been connected to a number of diseases. Many bacterial infections, such as chronic lung, wound, and ear infections, are caused by biofilms. Bioflms can colonize medical equipment like catheters and implants as well. Biofilms are responsible for more than 80% of all microbial illnesses in bacterial infections; the existence of biofilms can increase the pathogenicity of the bacteria while also protecting it from being killed by external treatment.

In adverse conditions, bacteria have long used biofilm production to survive. UV radiation, pH stress, chemical exposure, phagocytosis, dehydration, and antibiotics are among threats that biofilms protect cells from (Gupta et al., 2015). Biofilm-associated illnesses, unfortunately, are resistant to both conventional biocides and the host immune system. As a result, diagnosing and treating the condition is difficult [19].

a. Cysticfibrosis

The lung disease Cystic Fibrosis (CF) involves the creation of thick, sticky mucus, which restricts airways and makes it difficult for patients to breathe. P.aeruginosa infects 80% of CF patients on a long-term basis. P. aeruginosa can also be found on medical apparatus, devices, and tools [26], but unfortunately, there are few effective antibiotics for treating persistent P.aeruginosa infections.

b. Dental plaque

Biofilms play a significant function in dentistry, and many studies on dental biofilms have been conducted [35]. In dental plaque, about 700 species of bacteria and archaea have been identified [58]. Many oral cavity disorders, such as dental caries, periodontitis, gingivitis, and others, are caused by dental plaque. For initial colonization and subsequent biofilm formation on the enamel surfaces, bacteria must communicate with one another. If bacteria do not stick to the tooth surface, they are rinsed away with saliva [52].

c. Wounds

Biofilms are commonly found in chronic wounds. Compared with an acute wound, which is usually not associated with biofilm, a biofilm-related chronic wound persists and only slowly heals. Biofilms usually form on the outer layer of wound but some biofilm are also embedded in the deep layers of wounds [23].

d. Urinaryinfection

Biomaterials in the urinary tract, such as catheters, enhance the likelihood of bacterial biofilm formation, which can result in urine infection. Bacteria can cling to the synthetic foreign body's surfaces. Bacterial biofilms infect almost all urinary catheters (both the inner and exterior surfaces). P. mirabilis biofilms, for example, which can be crystalline in nature, can block catheters, requiring patients to replace the clogged catheters [22].

e. Prosthetic joint infection

Gram-positive bacteria, such as staphylococci, are commonly seen in prosthetic joint infections [36]. Bacteria (from the blood or lymph) bind to the surface of prosthetic joints to develop biofilms almost immediately after surgery. Unlike conventional bacterium infections, which cause symptoms such as fever, biofilm infections on these implants may take time to manifest symptoms such as pain [55].

f. Cardiac Valve Infection

Bacterial biofilm on mechanical cardiac valve causes a disease called prosthetic valve endocarditis. The species involved in endocarditis are S.epidermidis, S.aureus, Streptococcus spp., Coryne bacterium spp., Enterococcus spp. and Candida spp [9] Accumulated biofilm can disrupt or block the artificial cardiac valve, resulting in diminished flow, turbulence or even leaking. Detached biofilm cells can migrate along with the blood stream and cause infection in other organs. Biofilm fragments in the blood circulation can block blood at the terminal points, such as the brain and kidney.

Signaling in Biofilm Formation

The connection between environmental stimuli and the microorganisms' reciprocation of the appropriate signaling events is required for the formation of a biofilm. Many sensor systems are capable of incorporating environmental cues into signaling pathways. Two-component systems and Quorum sensing can be induced by these Signaling systems (Jonas et al.2009).

Quorum Sensing

At high cell density, a process of cell-to-cell communication regulates gene expression. The concentration of an auto inducer, which is produced by bacteria into the environment, exceeds a particular threshold at high cell density, resulting in gene expression and regulation of many physiological activities. Bacteria must synchronize their gene expression in order to build a biofilm, and some use quorum-sensing systems to doit. QS appears to be a process through which bacteria manage collective activities and monitor cell density [32].

Two-Component System

Histidine kinase and response regulator proteins make up the two-component signaling system. HK is a sensor protein with a ligand-binding domain on the N-terminus and a kinase domain on the C-terminus. The transfer of phosphoryl groups from Adenosine Triphosphate (ATP) to a particular conserved histidineresi due in HK causes signal transduction. Following that, HK moves the phosphoryl group from histidine to RR's as part at eresi due [51]. This phosphate activate RR, which regulates transcription, In general, GacS (HK)/GacA (RR) two-component systems are implicated in the production of Pseudomonas aeruginosa biofilms [43]. The rsm genes, which code for RsmY and RsmZ, which control the transition between planktonic and sedentary forms, are induced by this system [3]. A progressively coordinated network of gene expression is essential for the creation of biofilm. As a result, these signaling events play a critical role in the creation of microbial biofilms by producing adaptive responses to external and internal stimuli [4].

Materials and Methods

Biofilm Laboratory Diagnosis is Test

Tissue Culture Plate: The TCP assay is most widely used and is considered a standard test for detection of biofilm formation. The microorganisms are grown in polystyrene tissue culture plates for 24 hours then after washing fixed with sodium acetate (2%) and stained with crystal violet (0.1% w/v). Biofilm formation is detected by measuring optical density with ELISA reader [6].

Congo Red Agar: Biofilm generation by Staphylococcus isolates is tested using a specially prepared solid medium called brain Heart Infusion Broth (BHI), which is supplemented with 5% sucrose andCongo red. BHI (37 gms/L), sucrose (50 gms/L), agar no. 1 (10 gms/L), and congo red stain (0.8 gms/L) made up the medium. Congo red was made as a concentrated aqueous solution that was autoclaved at 121°C for 15 minutes, separate from the other medium elements, and then added after the agar had cooled to 55°C. Plates were inoculation and aerobically incubated at 37°C for 24 to 48 hours. Black colonies with a dry crystalline quality indicated a positive result. Weak slime producers usually remained pink, though occasional darkening at the centers of colonies [15].

Bioluminescent Assay: To analyze the bio-activity of the bacterial biofilm, the ATP content of the biofilm was assayed using the ATP bioluminescence assay. A linear relationship between the ATP content and the number of the planktonic Pseudomonas aeruginosa cells. Attenuated Total Reflecting Spectros copy used to monitor the conditioning films that are an early harbinger of biofilm formation [15].

Antimicrobial Resistance

The emergence and spread of antibiotic resistance among bacteria is one of the world's most serious health issues [5]. One of the outcomes of bacterial biofilm communities that lead to chronic illnesses is antibiotic resistance. Biofilm-forming Klebsiella Pneumoniae is a Multidrug-Resistant (MDR) bacteria that affects people and is a leading cause of hospital infections with high morbidity and mortality due to restricted treatment choices [21]. Biofilm development is away for bacteria to with stand harmful environmental impacts like antibiotics and anti microbial drugs. Biofilm, according to Verderosa et al., is resistant to antibiotic therapy and is a primary source of persistent and recurring infections by clinically important pathogens over the world. This is due to the fact that biofilm formation and subsequent encasement of bacterial cells in a complex matrix can increase resistance to antimicrobials and sterilizing treatments, making these organisms difficult to eliminate and manage [28]. The host immune system responds to bacterial infections by activating a number of signaling pathways, cytokines, and genes involved in stress management [20]. Many bacterial diseases that are initially thought to be strictly extracellular can remain inside the host by forming biofilms as a result of a process of adaptation that allows the bacteria to evade the host's innate defense. The escape of biofilms from the host's innate response is damaging to the host since the inflammatory influx released by the body in response to the bacterial infection may cause tissue damage [1]. Antibiotics are protected by the Extracellular Polymeric Substances (EPS) matrix, which prevents drug entry at bactericidal concentrations. When compared to planktonic bacteria, bacteria within a biofilm are several orders of magnitude more resistant to antibiotics. Biofilms, for example, can withstand antimicrobial doses 10–1000 times higher than those required to inactivate genetically similar planktonic bacteria [37].

Conclusions

Biofilms are microbial communities that live in close proximity to one another and can cling to both biotic and a biotic surfaces. They biofim build structures that function as a barrier to sanitizer. Sanitizer, cleaner, and disinfectant disturb microbial cells and their attachment on surfaces and environments enables bacterial pathogens to colonize in harsh situations. It is preferable to develop appropriate materials with technology that will decrease bacterial attachment and make cleaning easier. It is also crucial to understand their genes, which are involved in encoding bacterial cell surfaces that are important for adhesion. The release of signaling molecules that might alert others for survival in hazardous situations is another important aspect of bacterial biofilm formation. To disrupt their communication networks, effective methods should be created. In the medical field, biofilm-forming microbes are a major issue. Biofilm-forming bacteria are wrapped in a matrix that protects them from antibiotics and the immune system of the host.

Biofilm formation by bacteria and their subsequent resistance to antibiotic and bactericidal is a slow but serious threat to health. Biofilm formation has become a ubiquitous phenomenon not only for human and animal infections, but also on non biological aspects. Biofilms are formed on food items and water which are considered as the basic necessities of daily life. Current therapeutic approaches for prevention of biofilms is limited to use of antimicrobial agents and post infection remedy lies in surgical removal of the biofilm followed by continued antibiotic administration. But nonetheless novel strategies are also being used to combat the problem. Option of vaccination against specific biofilm-associated bacteria is also being explored and one can hope that prevention and inhibition of biofilms by bacteria can be achieved in near future.

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