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Mechanism Involved in Biofilm Formation of Enterococcus faecalis

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Ajay Kumar Oli, Palaksha K. Javaregowda, Apoorva Jain and Chandrakanth R. Kelmani

Submitted: January 21st, 2022 Reviewed: February 25th, 2022 Published: April 29th, 2022

DOI: 10.5772/intechopen.103949

IntechOpen
Bacterial Biofilms Edited by Theerthankar Das

From the Edited Volume

Bacterial Biofilms [Working Title]

Dr. Theerthankar Das

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Abstract

Enterococci are commensal bacteria in the gastrointestinal flora of animals and humans. These are an important global cause of nosocomial infections. A Biofilm formation constitutes an alternative lifestyle in which microorganisms adopt a multi-cellular behavior that facilitates and prolongs survival in diverse environmental niches. The species of enterococcus forms the biofilm on biotic and abiotic surfaces both in the environment and in the healthcare settings. The ability to form biofilms is among the prominent virulence properties of enterococcus. The present chapter highlights the mechanisms underlying in the biofilm formation by enterococcus species, which influences in causing development of the diseases.

Keywords

  • biofilm
  • Enterococcus faecalis
  • pathogenesis
  • microcolony
  • quorum sensing

1. Introduction

Gram Positive bacterium has been renowned as a pathogen of hospitals acquired infectious. One among these bacteria is Enterococcus species. Enterococcusspecies are ubiquitous, commensally inhabitants of the gastrointestinal tract of humans and animals. These can be frequently isolated from the environmental sources such as soil, surface water, raw plant and animal products. Even these can screen from female genital tract, oropharynx and skin. Enterococcus spsbelongs to the gram positive, facultative anaerobic cocci with an optimum growth temperature of 35°C [1]. There are around 36 species of enterococci have been reported; conversely 26 species are associated with human infection. The most predominant human pathogen is Enterococcus faecalis, even Enterococcus faeciumis one of the important pathogen which is prevalent increasing as hospital acquired infections. The other remaining enterococci species only accounts 5% of infections [2, 3, 4]. Some few examples of enterococcus species which are associated with human infections, E. avium, E. cecorum, E. cassseliflavus, E. durans, E. gallinarum, E. raffinosus[5, 6].

E. faecalishas now become the most common nosocomial pathogen and its virulence is increasing in clinical isolates. The presence and function of different suggested characteristics related virulence have been reported [7, 8]. The factor which influences the virulence is mediated through gelatinase production, enterococcus surface protein (ESP), aggregation substance (AS), and biofilm formation [9]. It cause the following infections such as pelvic and abdominal infections, infections in the mouth especially after root canal surgery, infections in open wounds, a lesser known form of meningitis called enterococcal meningitis, infections in the blood called bacteremia and urinary tract infections.

Biofilms are surface attached, organized microbial communities made up of sessile cells (bacteria and /or fungi) embedded in an extracellular matrix composed of polysaccharides, DNA and other components.

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2. Chronological background on biofilm

Generally bacterial cell grow in two modes; biofilm formation through aggregate and planktonic cell. It associated with microorganism in which cells stick to each other on a surface encased within matrix of extracellular polymeric substance produced by bacteria itself [10]. Antoni van Leeuwenhoek, the Dutch research, who discovered the simple microscope and observed ‘animalcule’ on surfaces of tooth and this event is known as discovery of biofilm. Characklis, in the year 1973 phrase that biofilms are not only tenacious but even resist to disinfectants (e.g. chlorine). In 1978, Costerton, defined the term biofilm and explained the importance of biofilm. Biofilms can be found in nature in all places like waste water, labs, and hospital settings. It forms as floating mat on the surface of liquid on both living and non-living surfaces [11].

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3. Components of biofilm

Biofilm are produced from different group of organisms, the microbes cells produces the extracellular polymeric substances (EPS) such as DNA <1%, Polysaccharides 1–2%, proteins(includes enzymes) with <1–2%, RNA <1% and water with 97% are the major part of biofilm which is responsible for the flow of nutrients inside biofilm matrix [12]. The main two components of the biofilm that is water channel for nutrients transport and a region of densely packed cells having no prominent pores in it [12]. Another way microbial cells in which biofilms are arranged with significant different physiology and physical properties. They will access of antibiotics and human immune system. The organism that produces biofilm has capability to bear and neutralize antimicrobial agents and result in prolonged treatment. The bacteria which produces the biofilm, switch on the genes that can activate the expression of stress genes which in turn switch to resistant phenotypes due to certain changes examples are as follows cell density, nutritional, temperature, pH and osmolarity. When the biofilm water channels are compared with system of circulations showed that biofilms are considered primitive multi-cellular organism [13, 14]. The compositions of biofilms like DNA, proteins, polysaccharides and water will signify the biofilm integrity and making it resistant against different environmental factors [15].

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4. Epidemiology of biofilm formation by Enterococcus faecalis

In the worldwide, the prevalence of production of biofilm varies to different part. The study reported in Rome, Italy, 80% of E. faecalisisolates have ability to form biofilms in the infected patients [16]. In India, a study has showed that 52% of E. faecalisisolated screened from clinical samples has showed the biofilm formation [17]. In China, Shenzhen Nanshan Hospital, the prevalence of E. faecalisbiofilm formation has showed 50.4% (57/113) in urinary tract infection isolates [18]. The biofilm formation in case of food isolates were less with 60% non-biofilm producers. The major ability in formation of biofilm was endodontic isolates with 73.7% was observed in the Department of Operative Dentistry and Periodontology, University of Freiburg Medical Center, Germany [19].

A study carried out Ahvaz teaching hospital, Iran demonstrated that high frequency 63% of biofilm formation in clinical isolates [20]. The E. faecalisbacterial isolated from patient with complicated UTI from department of Urology, Okayama University, Japan has showed the biofilm formation 64 (18.2%) and 156 (44.3%) exhibited strong and medium respectively [21]. A study reported at Malaysia, the E. faecalisisolates has showed the biofilm formation of 49% [22]. In the United Kingdom, 100% E. faecalisisolates produced biofilms, these isolates were from intravascular catheter-related bloodstream infections (CRBI) found to produce more biofilm than enterococcal isolates that cause non-CRBI [23]. A 93% of E. faecalisstrains isolated from clinical samples especially fecal isolates have showed more biofilm formation in the United States [24]. In Spain, 57% of E. faecalisclinical isolates represent the biofilm production [25]. Tertiary care hospital in India showed 26% isolates of E. faecalishaving capability in forming biofilm [26].

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5. Pathogenesis of biofilm in causing disease

Generally infectious is connected with biofilm primarily confine to particular location and though time detachment may occur. Further, the detached biofilms may result in bloodstream or urinary tract infections or in the production of blockage of blood flow [26]. In another side cells in biofilms are mostly resistant to antimicrobial agents and the host immune system. E. faecalisisolates which produces biofilms is 1000 times more resistant to antibodies, antimicrobial agents and phagocytosis process than non-biofilm producers. Consequently, infections caused from E. faecalisassociated with biofilm aggravated in this case [27, 28].

In endocarditis infection a complex biofilm formed by E. faecalisand host components will be formed on cardiac valve. These biofilms causes disease is through three basic mechanisms. Firstly, the biofilms physically disrupts valve function and may cause leakage. Second, detachment of biofilm can be carried to a terminal point in the circulation and formation of emboli (blockage of the blood vessel). Finally, the biofilm provides continuous infection of the bloodstream even during antibiotic treatment. These can cause recurrent fever, chronic systemic inflammation and lead to other infection also [27, 29].

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6. Mechanism steps involved in E. faecalisbiofilm formation

It comprises of four stages; initial attachment, microcolony formation, biofilm maturation (which is in part governed by quorum sensing) and dispersal.

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7. Initial attachment

A surface adhesion is the first step in establishing a biofilm, and a number of surface adhesions, proteases, and lipids are involved. The endocarditis and biofilm-associated pilus (Ebp), which is composed of subunits A, B, and C, mediates the adherence of biofilms on surface in-vitroand in-vivo[30, 31, 32, 33, 34, 35]. The deletion of ebpABC attenuates binding to platelets, fibrinogen and collagen, reduces initial attachment, and thus impairs biofilm formation in-vitro[30, 32, 33].

In addition, Ebp contributed to early biofilm formation in in-vivomodels of urinary tract infection (UTI), catheter associated UTI (CAUTI), and infectious endocarditis, in which bacteria with deletions of pilus components were substantially attenuated [30, 32, 33, 36]. Additionally, the absence of surface adhesions, such as aggregation substance (Agg), enterococcol surface protein (ESP), and adhesion to collagen from E. faecalis(Ace), reduced adhesion to cultured human cells and prevented biofilm formation in-vivo[37, 38, 39, 40, 41]. Bacteria deficient for Esp showed reduced initial attachment and decreased bladder colonization in a UTI ascending model, which is not unexpected since Esp binds fibrinogen and collagen, and these ligands are present in the bladder because Esp binds fibrinogen and collagen, and these ligands are present in the bladder [41, 42].

Ace is also involved in interacting with collagen, laminin, and dentin and deletion of Ace resulted in reduced colonization in rat endocarditis and UTI models [43, 44, 45, 46, 47]. As a result, Ace deletion in the peritonitis model did not reduce bacterial burden suggesting Ace-mediated biofilm formation is not relevant to peritoneal infection. By disparity, deletion of Agg reduced adherence to renal epithelial cells [38, 39], binding to lipoteichoic acid (LTA) of other E. faecaliscells (and therefore inter-bacterial clumping) and bacterial titers recovered from endocarditis vegetation on aortic heart valves. Agg cannot colonize the urinary tract, suggesting that Agg-mediated biofilms aren’t necessary for ascending UTI’s [48, 49].

In-vitro, biofilm associated glycolipid synthesis A (BgsA) contributes to initial adhesion and biofilm development, but its role in-vivois unknown [50]. The extracellular secreted protein encoded by salB (Saga-Like Protein B) increased fibronectin and collagen binding but decreased biofilm formation paradoxically, which has hypothesized to be owing to the salB mutant cells decreased hydrophobicity. These investigations suggest that a variety of variables play a role in the initial attachment of bacteria, and that their contribution is likely to vary depending on the surface to which the bacteria adhere. As a result, focusing on a single component as anti-adherence or anti-biofilm strategy is unlikely to totally prevent enterococcal biofilm formation [37].

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8. Microcolony formation

Bacteria proliferate and produce modest amounts of biofilm matrix to form aggregates known as microcolonies after first adhesion [51]. However, the enterococcal mechanisms that drive the establishment of microcolonies are unknown, and no transcriptome data from early-stage biofilms or microcolonies is available. The importance of microcolonies for gut colonization has been demonstrated. E. faecaliscolonization of the stomach of germ free mice resulted in discrete microcolonies covered in a fibrous sweater-like matrix within a week, rather than the largely 2D biofilm sheets (2–3 cells high) that are normally observed in biofilm models in-vitro[52].

Despite the fact that microcolonies are commonly assumed to be a temporary stage of early biofilm production, these data imply that microcolonies may represent a mature biofilm stage in this niche that is particularly crucial for gut colonization. In addition, in-vitroenterococcal microcolonies emerge in response to antibiotic therapy [53, 54]. Biofilms treated with sub-inhibitory levels of daptomycin began to restructure extensively into microcolonies as early as 8 hours after drug exposure, in contrast to typical biofilm sheets. Even in the absence of antibiotics, deletion mutants of eapOX, which encodes a glycosyl-transferase involved in the formation of cell wall associated rhamnopolysaccharide (Epa), developed microcolonies in-vitro.In contrast to the monolayer biofilms, these epaOX microcolonies had lower structural integrity, as shown by their facile separation following washing.

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9. Biofilm growth and maturation

Active growth and synthesis of extracellular matrix components such as extracellular DNA (eDNA), polysaccharides, LTA, and extracellular proteases are required for biofilm development. eDNA is the best studied matrix component of enterococcal biofilms:eDNA can be found at the bacterial septum, as part of intercellular filamentous structures, and as part of the larger biofilm matrix, and its release from cells is controlled by autolysin Atla [55, 56, 57].

eDNA-associated cells showed no significant cell lysis and had a membrane potential [55], implying that eDNA is liberated from metabolically active cells. As a result, DNase treatment decreased biofilm stability and increased detachment [58, 59], whereas atlA deletion decreased eDNA release and biofilm formation [56]. Despite the lack of evidence that eDNA influences the spatial organization of enterococcal biofilms (as has been postulated for other bacterial species), eDNA remains a potential therapeutic target.

Biofilm production is also aided by non-proteinaceous cell surface components such as glycoproteins, polysaccharides, and modified lipids. The dltABCD operons are involved in the production of D-alanine esters of LTA, which are an important component of Gram-positive bacteria’s cell wall, and deletion of this operons decreased biofilm formation in-vitro, decreased adherence to epithelial cells, and increased susceptibility to antimicrobial peptides [60]. Biofilm on plastic D (BopD), a potential sugar-binding transcriptional regulator, also promotes to biofilm development in-vitro[61].

The deletion of bopABC, which is located upstream of bopD, boosted biofilm growth in glucose but decreased biofilm growth and colonization levels in the murine gut, implying that the ability to utilize maltose is required for biofilm growth in the gut. MprF2, a paralogue of multiple peptide resistance factor (MprF), was likewise found to promote eDNA release and biofilm formation [61, 62, 63]. MprF2 reduces the net positive charge of the membrane via aminoacylating phosphatidylglyceroal to mediate electrostatic repulsion of cationic antimicrobial peptides.

While deletion of MprF2 had no effect on biofilm persistence in a mouse bacteremia model, deletion of both MprF1 and MprF2 reduced biofilm persistence in a wound infection model, suggesting that cell membrane charge may play a role in biofilm formation and pathogenicity in-vivo[63, 64]. These findings back up the theory that cell surface glycoproteins, membrane phosphatidylglycerol, and polysaccharides all play a role in biofilm development.

The quorum sensing response regulator FsrA regulates matrix remodeling by upregulating the expression of gelE, SprE, and altA [57, 58, 65, 66, 67]. The proteases gelE and sprE were found to diminish biofilm formation in-vitroand bacterial load in numerous in-vivomodels [68, 69, 70, 71]. However, in a rabbit endocarditis model, loss of gelE alone increased fibrinous matrix formation in aotic vegetation, leading to endocarditis as shown in the Table 1 [70].

Name of the GeneGene codeRole
D-alanine- d-alanine ligaseddlIt involved in metabolism process (d-ala) especially for bacterial peptidoglycan biosynthesis. Its role in cell wall integrity and biofilm formation.
CytolysincylIt a secreted toxin expressed in response to pheromones, contributes to the pathogenicity of E. faecalisby causing blood hemolysis.
GelatinasegelEIt hydrolyzes the gelatin and ability to damage host tissues plays a vital role in spreading of enterococci in their host. It promotes the aggregation of the cells in microcolonies which constitutes the initial step of biofilm formation.
Serine proteasesprEIt hydrolyzes the casein, quorum sensing and autolysis (release of eDNA)
Fecal streptococci regulator locus genesfsrA, fsrB, fsrCIt the major quorum sensing in E. faecalis, the fsrregulator locus, is encoded by fsrA, fsrBand fsrCgenes which regulate the expression of both gelatinase and serine protease. It controls biofilm development through regulating the production of gelatinase.
Biofilm associated piliebpIt is the protein organelles, anchored to the surface of the bacterium, that interact with the external environment. It role in biofilm formation, initial attachment and IE.
Adhesion to collagen of E. faecalisaceA surface protein that facilitates the bacterial adherence to collagen is the adhesion to collagen of E. faecalis. It play key role in adherence and colonization process.
Aggregation substanceaggA surface protein expressed in response to pheromone induction that mediates the adherence of E. faecalisto renal epithelial cells. It plays important role in adherence to and colonization of host tissues.
Enterococcal fibronectin-binding protein AefbAIt is an adhesin, localized on the outer surface of E. faecalisthat confers adhesion to immobilized fibronectin.
Enterococcal surface proteinespIt promotes primary attachment and biofilm formation.
LuxS/autoincuder −2 (AI-2) quorum sensing systemluxSIt plays role in interspecies communication and involved in bacterial virulence, persistence infections and biofilms

Table 1.

Different quorum sensing genes signaling molecules involved in Enterococcus quorum sensing system and virulence factors production.

In-vitro, sprE deletion increased autolysis and eDNA release and accelerated biofilm development, but gelE deletion inhibited eDNA releaseand elevated ace expression, which may increase surface attachment but make the biofilm detachable [71, 72].

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10. Quorum sensing

Population density-dependent signaling influences biofilm formation [73, 74]. Despite the fact that quorum sensing and peptide pheromone signaling are known to coordinate gene expression and direct enterococcus biofilm growth, there have been few research on these tiny signaling molecules and secondary messengers in enterococci. The cCF10 peptide pheromone, which facilitates the transfer of the conjugative plasmid pCF10, is an exception. This plasmid has the ability to transfer antibiotic resistance genes as well as virulence determinants like Agg across cells [75, 76, 77, 78, 79]. The buildup of cCF10, which stimulates conjugation proteins, is required for pCF10 transfer. The mechanism underpinning peptide pheromone-mediated gene regulation and plasmid transfer has been well documented, and it was recently demonstrated in mice to promote pCF10 transmission between E. faecaliscells in the gut [79, 80]. The immature peptide pheromones cAD1 and cCF10 are processed by the membrane protease Eep. Eep also facilities the proteolytic processing of RsiV, the anti-sigma factor for sigV, resulting in improved stress resistance. A sigV mutant showed similar symptoms, indicating that Eep is involved in the regulation of sigV production [81, 82, 83].

In-vitro, Eep, together with AhrC and the ArgR family transcriptional regulators, leads to biofilm formation, and deletion of the genes encoding either protein lowered bacterial burden in UTI and endocarditis models [84, 85, 86]. Furthermore, eep deletion mutants develop tiny aggregates unlike wild-type biofilms. FsrABC is another quorum-sensing system. FsrC is a membrane sensor kinase that detects density-dependent accumulation of the FsrB peptide and triggers a signal to the FsrA response regulator [87]. Because this system controls multiple biofilm-related genes and operons (such as bopABCD, ebpABC, GelE, and SprE), knocking down fsrABC entirely eliminates biofilm formation [88]. FsrD, a precursor for the cyclic peptide gelatinase biosynthesis activating pheromone (GBAP), is also controlled by the Fsr quorum sensing system as shown in the Table 1 [89]. Finally, autoinducer 2 (Al-2) is involved in E. faecalisbiofilm formation and is produced by S-ribosylhomocysteinelyase (LuxS). In-vitrobiofilm development of E. faecalisis increased by Al-2 supplementation, while luxS deletion causes aberrant biofilm production with aggregation a dense structure, in contrast to the confluent monolayers of wild type in-vitrobiofilms [90, 91].

11. Factors influencing for the formation of biofilms in E. faecalis

11.1 Dlt gene

A Lipoteichoic Acid, component of E. faecalis, the most common organism in root canals, develops colonies on the dentin surface (LTA). LTA is a biofilm-forming component of E. faecalisthat functions as a receptor molecule on receptor cells during the aggregation process. E. faecalisantigen recognizes immune cells via pattern recognition receptors (PRRs) and induces the release of proinflammatory cytokines like TNF alpha (TNFα), interleukin 1 beta (IL-1β), IL-6, and IL-8 [92]. LTA causes cells to produce cytokines, which is followed by the activation of Nuclear Factors kβ (NF-kβ), which promotes cytokines release as shown in the Table 2 [93].

FactorsFunction
dltgeneIt as acts biofilm forming component during aggregation process. It causes cells to produce cytokines. It controls cationic homeostasis and autolytic activity
Cytolysin lytic enzymesIt is the virulence factors, play role in lysing erythrocytes and collagen fragmentation. The cylLLand cylLSgenes on cytolysin promoted for longer survive of E. faecalis.
HyaluronidaseIt acts as toxin protein for the progression of host tissue increase damage and inflammation. It beneficial protein for the development of E. faecalis.
Dentine MatrixIt increases the enhancement of biofilm formation through dentin. It also resists the antimicrobial treatment by delay penetration of the drug through the biofilm matrix by altering/changing the physiological shaper of biofilm growth in dentin.
NutrientsGlucose is the major determinate in the formation of E. faecalis.It utilizes as the carbon source and hydrolyzes the substrate for its survival.
EnvironmentalPhysicochemical properties of the surface may exert a strong influence on the rate and extent of attachment. Temperature, cations, and presence of antimicrobial agents influence the attachment. The optimum temperature 37°C, pH -8.5 increase the production biofilm formation.

Table 2.

Factors influencing for the formation of biofilms in E. faecalis.

The release of these cytokines causes the dlt gene in LTA to fabricate D-alanine instantly, causing other bacteria to assist in the formation of biofilms [94, 95]. The D-Ala-LTA gene is triggered by the surface protein of Gram-Positive bacteria. Cationic homeostasis and autolytic activity are controlled by this gene. Additionally, it is involved in the assimilation of metal cations as well as the electromechanical repair of bacterial cell walls [94]. These capabilities will enhance bacterial cell system transfer while even increasing autolytic activity. The host’s defense system will be weakened by the modified tick.

11.2 Cytolisin lytic enzymes

A lytic enzyme operated on by cytolysin is the one of E. faecalisbacteria’s virulence factors. Apart from lysing erythrocytes, collagen fragmentation caused by this enzyme can cause tissue injury at the site of inflammation. The cylLL and cylLs genes on cytolysin promote this role, allowing E. faecalisto survive longer. E. faecalis is the most common microbe found in root canals [92, 96]. Other bacteria will be inhibited by E. faecaliscytolysin. The cylLL and cylLS genes in E. faecaliscytolysin encode structural cytolysin subunits. They create cytolysin in anaerobic circumstances and respond to oxygen depletion in root canals by producing cytolysin as shown in the Table 2.

11.3 Hyaluronidase

Hyaluronidase is a protein to be found in E. faecalisthat helps the bacteria and toxins progress to the host tissue. Other bacteria will continue to migrate from the root canal to the periapical lesions as a result of hyaluronidase. Furthermore, hyaluronidase stimulates the production of toxins by other bacteria, which increases damage and inflammation. This stipulation is very beneficial for the development of E. faecalis[97, 98].

11.4 Dentine matrix structurization

E. faecaliswill increase resistance to antimicrobial treatments by increasing the biofilm structural characteristics at the primary site of E. faecalisinvasion, notably dentin. As a result, E. faecalisis known to delay antimicrobial agent penetration through the biofilm matrix by altering the growth rate of other microbes in biofilm development and encouraging changes in the physiological shape of biofilm growth in dentin.

When E. faecalisis cultivated in nutrient-poor media, it forms thicker biofilms than when cultured in nutrient-rich media [99]. Under stress inducing mechanism in other bacteria that can cause a more resilient E. faecalisbiofilm. Besides E. faecalisbiofilms profitably renew themselves. Furthermore, E. faecaliswill receive vital carbon by hydrolyzing the substrate required for survival [23].

E. faecaliswill continue to grow and develop in environments with or without oxygen with extreme alkaline pH by penetrating cell membrane ions and increasing the cytoplasmic’s buffer capacity [100]. The pH balance of the biofilm is always maintained by bacteria by assimilation of protons into the cell, resulting in a lower internal cell pH. As a result, the dentin buffer capacity is unable to keep the pH in the dentinal tubule constant, and E. faecalissurvives [101].

Other investigations found in E. faecalisthat the ability to promote apatite re-deposition in the forming biofilm is responsible for its persistence after root canal therapy. Besides this, the dentin matrix is composed of chlorapatite Ca5 (PO4)3 [102]. Different varieties of apatite have different dissolving tolerances. Till date, chlorapatite has been considered as a weaker apatite than hydroxyapatite and fluorapatite in terms of nanostructure [102, 103]. Although it is known that calcium hydroxide can stimulate the formation of hard tissue by raising the Ca2+ ion to increase defense through dentin mineralization, the type of apatite that makes up the host dentin will influence the results [104, 105].

However, no further research into the drug resistance of this inorganic dentin material’s nanostructures has been done. Furthermore, dentin deterioration is not solely dependent on inorganic elements. Collagen makes up 20% of the organic dentin, which accounts for 85% of the total [103]. Gelatinase, an E. faecalisvirulence component, is required for hydrolyzing host collagen, High gelatinase levels have been linked to dentin organic matrix degradation [106, 107].

11.5 Tolerance for antimicrobial therapy

Antimicrobial therapy is known to be limited to eliminating free microbes but not to remove cells bound to the biofilm so that re-infection can occur [100]. As a root canal medication, calcium hydroxide is currently the most popular option among dentists. E. faecalisis known to be resistant to calcium hydroxide. This is a serious clinical problem. Every root canal treatment failure, which is documented widely, has linked to E. faecalis[101]. Calcium hydroxide is known to prevent the acid reaction that happens as a result of the inflammatory response. This lactic acid generated by osteoclasts to absorb hard tissue will be neutralized by the alkaline pH [102, 103].

12. Conclusion

Enterococcus faecalisis one of the most predominant organism in nosocomial infection and also developed the drug resistance. The intrinsic virulence factors E. faecalisare associated in biofilm formation and other environmental factor and signals are alarming the biofilm formation. A genome wide study is required to know the role of genetic and environmental factors in development of biofilm and mounting the superior strategies for biofilm control in E. faecalisisolates.

References

  1. 1. Murray BE. The life and times of the Enterococcus. Clinical Microbiology Reviews. 1990;3:46-45
  2. 2. Gordon S, Swenson JM, Hill BC, Pigott NE, Facklam RR, Cooksey RC, et al. Antimicrobial susceptibility patterns of common and unusual species of enterococci causing infections in the United States. Journal of Clinical Microbiology. 1992;30:2373-2378
  3. 3. Ruoff KL, de La Maza L, Mj M, Spargo JD, Ferraro MJ. Species identities of enterococci isolated from clinical specimens. Journal of Clinical Microbiology. 1990;28:435-437
  4. 4. Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. Journal of Clinical Microbology. 1989;27:731-734
  5. 5. Kohler W. The present state of species within the genera Streptococcus and Enterococcus. International Journal of Medical Microbiology. 2007;297(3):133-150
  6. 6. Transupawat S, Sukontasing S, Lee JS.Enterococcus thailandicusSpnov, isolated from fermented sausage (“mum”) in Thailand. International Journal of Systematic and Evolutionary Microbiology. 2008;58(7):1630-1364
  7. 7. Klare I, Konstable C, Muller BS, Werner G, Strommenger B, Kettlitz C, et al. Spread of ampicillin/vancomycin-resistantEnterococcus faeciumof the epidemic virulent clonal complex-17 carrying the gene esp and hy1 in German hospitals. European Journal of Clinical Microbiology & Infectious Diseases. 2005;24(12):815-825
  8. 8. Mundy LM, Sahm DF, Gilmore M. Relationships between enterococcal virulence and antimicrobial resistance. Clinical Microbiology Reviews. 2000;13:513-522
  9. 9. Carolina BC, Cr MC, Juliana C, Pedro AA. Presence of virulence factors inEnterococcus faecalisandEnterococcus faeciumsusceptible and resistant to vancomycin. Memórias do Instituto Oswaldo Cruz. 2013;108(5):590-595
  10. 10. Hall-Stoodley L et al. Bacterial biofilms: From the natural environment to infectious diseases. Nature Reviews. Microbiology. 2004;2:95-108
  11. 11. Costern J et al. Bacterial biofilms: A common cause of persistent infections. Science. 1999;284:1318-1322
  12. 12. Available from:http://www.horizonpress.com/biofilms
  13. 13. Fux C et al. Survival strategies of infectious biofilms. Trends in Microbiology. 2005;13:34-40
  14. 14. Gilbert P et al. Biofilm susceptibility to antimicrobials. Advances in Dental Research. 1997;11:160-167
  15. 15. Vinodkumar CS et al. Utility of lytic bacteriophage in the treatment of multidrug-resistantPseudomonas aeruginosasepticaemia in mice. Indian Journal of Pathology & Microbiology. 2008;51:360-366
  16. 16. Baldassarri L, Betuccini L, Ammedolia MG, Cocconcelli P, Arciola CR, Montanaro L, et al. Receptor-mediated endocytosis of biofilm- formingEnterococcus faecalisby rat peritoneal macrophage. The Indian Journal of Medical Research. 2004;199(Suppl):131-135
  17. 17. Oli AK, Raju S, Rajeshwari S, Nagaveni S, Kelmani Chandrakanth R. Biofilm formation by multidrug resistantEnterococcus faecalis(MDEF) originated from clinical samples. Journal of Microbiology and Biotechnology Research. 2012;2(2):284-288
  18. 18. Jin XZ, Bing B, Zhi-wei L, Zhang-ya P, Wei-ming Y, Zhong C, et al. Characterization of biofilm formation byEnterococcus faecalisisolates derived from urinary tract infections in China. Journal of Medical Microbiology. 2018;67(1):60-67
  19. 19. Annette CA, Jonas D, Ingrid H, Lamprini K, Johan W, Elmar H, et al.Enterococcus faecalisfrom food, clinical specimens, and Oral sites: Prevalence of virulence factors in association with biofilm formation. Frontiers in Microbiology. 2015;6(1534)
  20. 20. Fatemeh S, Hajar H, Saeed K, Golshan M, Aram AD, Ahamd FS. Virulence determinants and biofilm formation in clinical isolates of Enterococcus: A cross-sectional study. Journal of Acute Disease. 2020;9(1):27-32
  21. 21. Yuko S, Reiko K, Ritsuko M, Koichi M, Hiromi K. Clinical implications of biofilm formation byEnterococcus faecalisin the urinary tract. Acta Medica Okayama. 2005;59(3):79-87
  22. 22. Poh LW, Ramliza R, Rukman AH. Antibiotic susceptibility patterns, biofilm formation and esp gene among clinical enterococci: Is there any association. International Journal of Environmental Research and Public Health. 2019;16:3439
  23. 23. Sandoe JA, Witherden IR, Cove JH, Hertiage J, Wilcox MH. Correlation between enterococcal biofilm formation in vitro and medical-device related infection potential in vivo. Journal of Medical Microbiology. 2003;52:547-550
  24. 24. Mohamed JA, Huang W, Nallapareddy SR, Teng F, Murray BE. Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation byEnterococcus faecalis. Infection and Immunity. 2004;72:3658-3663
  25. 25. Toledo AA, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, et al. The enterococcal surface protein, Esp, is involved inEnterococcus faecalisbiofilm formation. Applied and Environmental Microbiology. 2001;67:4538-4545
  26. 26. Prakash VP. Clinical prevalence, identification and molecular characterization of enterococci, PhD thesis, Pondicherry University India. 2005;1-150
  27. 27. Parsek M, Singh P. Bacterial biofilms: An emerging link to disease pathogenesis. Annual Review of Microbiology. 2003;57:677-701
  28. 28. Oliveria M, Santos V, Fernandes A, Bernardo F, Vilela C. Antimicrobial resistance and in vitro biofilm-forming ability of enterococci from intensive and extensive farming broilers. Poultry Science. 2010;89:1065-1069
  29. 29. Donlan R. Biofilms: Microbial life on surfaces. Emerging infectious Diseases. 2002;9(8):881-890
  30. 30. Mohamed J, Huang W, Nallapareddy S, Teng F, Murray B. Influence of origin of isolates especially endocarditis isolates, and various genes on biofilm formation byE. faecalis. Infection and immunity. 2004;72(6):3653-3663
  31. 31. Nallapareddy SR et al. Endocarditis and biofilm associated pili ofEnterococcus faecalis. The Journal of Clinical Investigation. 2006;116:2799-2807
  32. 32. Bourgogne A, Thomson LC, Murray BE. Bicarbonate enhances expression of the endocarditisand biofilm associated pilus locus, ebpR- ebpABC, inEnterococcus faecalis. BMC Microbiology. 2010;10:17
  33. 33. Nallapareddy SR et al. Conservation of Ebp- type pilus genes among enterococci and demonstration oftheir role in adherence ofEnterococcus faecalisto human platelets. Infection and Immunity. 2011;79:2911-2920
  34. 34. Nallapareddy SR, Singh KV, Sillanpaa J, Zhao M, Murray BE. Relative contributions of Ebp pili and the collagen adhesin ace to hostextracellular matrix protein adherence andexperimental urinary tract infection byEnterococcus faecalisOG1RF. Infection and Immunity. 2011;79:2901-2910
  35. 35. Singh KV, Nallapareddy SR, Murray BE. Importance of the ebp (endocarditis and biofilm associated pilus) locus in the pathogenesis ofEnterococcus faecalisascending urinary tract infection. The Journal of Infectious Diseases. 2007;195:1671-1677
  36. 36. Nielsen HV et al. The metal ion dependent adhesionsite motif of theEnterococcus faecalisEbpA pilin mediates pilus function in catheter- associated urinary tract infection. MBio. 2012;3:e00177-e00112
  37. 37. Nielsen HV et al. Pilin and sortase residues critical for endocarditis and biofilm- associated pilusbiogenesis inEnterococcus faecalis. Journal of Bacteriology. 2013;195:4484-4495
  38. 38. Mohamed JA, Teng F, Nallapareddy SR, Murray BE. Pleiotrophic effects of 2Enterococcus faecalissagA- like genes, salA and salB, which encode proteins that are antigenic during human infection,on biofilm formation and binding to collagen type I and fibronectin. The Journal of Infectious Diseases. 2006;193:231-240
  39. 39. Rozdzinski E, Marre R, Susa M, Wirth R, Muscholl SA. Aggregation substance mediated adherence ofEnterococcus faecalisto immobilized extracellular matrix proteins. Microbial Pathogenesis. 2001;30:211-220
  40. 40. Sussmuth SD et al. Aggregation substancepromotes adherence, phagocytosis, and intracellularsurvival ofEnterococcus faecaliswithin human macrophages and suppresses respiratory burst. Infection and Immunity. 2000;68:4900-4906
  41. 41. Sillanpaa J et al. Characterization of the ebp (fm) pilus encoding operon ofEnterococcus faeciumand its role in biofilm formation and virulence in a murine model of urinary tract infection. Virulence. 2010;1:236-246
  42. 42. Toledo AA et al. The enterococcal surface protein, Esp, is involved inEnterococcus faecalisbiofilm formation. Applied and Environmental Microbiology. 2001;67:4538-4545
  43. 43. Shankar N et al. Role ofEnterococcus faecalissurface protein Esp in the pathogenesis of ascending urinary tract infection. Infection and Immunity. 2001;69:4366-4372
  44. 44. Nallapareddy SR, Qin X, Weinstock GM, Hook M, Murray BE.Enterococcus faecalisadhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infection and Immunity. 2000;68:5218-5224
  45. 45. Nallapareddy SR, Singh KV, Duh RW, Weinstock GM, Murray BE. Diversity of ace,a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains ofEnterococcus faecalisand evidence for production of ace during human infections. Infection and Immunity. 2000;68:5210-5217
  46. 46. Kowalski WJ et al.Enterococcus faecalisadhesin,Ace, mediates attachment to particulate dentin. Journal of Endodontia. 2006;32:634-637
  47. 47. Singh KV, Nallapareddy SR, Sillanpaa J, Murray BE. Importance of the collagen adhesin acein pathogenesis and protection againstEnterococcus faecalisexperimental endocarditis. PLoS Pathogens. 2010;6:e1000716
  48. 48. Lebreton F et al. Ace, which encodes an adhesin inEnterococcus faecalis, is regulated by Ers and is involved in virulence. Infection and Immunity. 2009;77:2832-2839
  49. 49. Waters CM et al. An amino- terminal domain ofEnterococcus faecalisaggregation substance isrequired for aggregation, bacterial internalizationby epithelial cells and binding to lipoteichoic acid. Molecular Microbiology. 2004;52:1159-1171
  50. 50. JohnsonJR CC, Hirt H, Waters C, Dunny G. Enterococcal aggregation substance andbinding substance are not major contributors tourinary tract colonization byEnterococcus faecalisin a mouse model of ascending unobstructed urinary tract infection. Infection and Immunity. 2004;72:2445-2448
  51. 51. Theilacker C et al. Glycolipids are involved inbiofilm accumulation and prolonged bacteraemia inEnterococcus faecalis. Molecular Microbiology. 2009;71:1055-1069
  52. 52. Monds RD, O’Toole GA. The developmental model of microbial biofilms: Ten years of aparadigm up for review. Trends in Microbiology. 2009;17:73-87
  53. 53. Barnes AMT et al.Enterococcus faecalisreadily colonizes the entire gastrointestinal tract and formsbiofilms in a germ- free mouse model. Virulence. 2017;8:282-296
  54. 54. Dale JL, Nilson JL, Barnes AMT, Dunny GM. Restructuring ofEnterococcus faecalisbiofilmarchitecture in response to antibiotic induced stress. NPJ Biofilms Microbiomes. 2017;3:15
  55. 55. Dale JL, Cagnazzo J, Phan CQ , Barnes AM, Dunny GM. Multiple roles forEnterococcus faecalisglycosyltransferases in biofilm associated antibiotic resistance, cell envelope integrity, and conjugative transfer. Antimicrobial Agents and Chemotherapy. 2015;59:4094-4105
  56. 56. Barnes AM, Ballering KS, Leibman RS, Wells CL, Dunny GM.Enterococcus faecalisproduces abundant extracellular structures containing DNA in the absence of cell lysis during early biofilm formation. MBio. 2012;3:e00193-e00112
  57. 57. Guiton PS et al. Contribution of autolysin and sortase a duringEnterococcus faecalisDNAdependentbiofilm development. Infection and Immunity. 2009;77:3626-3638
  58. 58. Thomas VC et al. A fratricidal mechanism isresponsible for eDNA release and contributes to biofilm development ofEnterococcus faecalis. Molecular Microbiology. 2009;72:1022-1036
  59. 59. Dunny GM, Hancock LE, Shankar N. In: Gilmore M, editor.Enterococci: From Commensals to Leading Causesof Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary; 2014
  60. 60. Vorkapic D, Pressler K, Schild S. Multifacetedroles of extracellular DNA in bacterial physiology. Current Genetics. 2016;62:71-79
  61. 61. Fabretti F et al. Alanine esters of enterococcallipoteichoic acid play a role in biofilm formation andresistance to antimicrobial peptides. Infection and Immunity. 2006;74:4164-4171
  62. 62. Hufnagel M, Koch S, Creti R, Baldassarri L, Huebner J. A putative sugar binding transcriptional regulator in a novel gene locus inEnterococcus faecaliscontributes to production of biofilm and prolonged bacteremia in mice. The Journal of Infectious Diseases. 2004;189:420-430
  63. 63. Creti R, Koch S, Fabretti F, Baldassarri L, Huebner J. Enterococcal colonization of the gastro-intestinal tract: Role of biofilm and environmental oligosaccharides. BMC Microbiology. 2006;6:60
  64. 64. Bao Y et al. Role of mprF1 and mprF2 in the pathogenicity ofEnterococcus faecalis. PLoS One. 2012;7:e38458
  65. 65. Chong KKL et al.Enterococcus faecalismodulates immune activation and slows healing during wound infection. The Journal of Infectious Diseases. 2017;216:1644-1654
  66. 66. Waters CM, Antiporta MH, Murray BE, Dunny GM. Role of theEnterococcus faecalisGelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation offibrin and misfolded surface proteins. Journal of Bacteriology. 2003;185:3613-3623
  67. 67. Qin X, Singh KV, Xu Y, Weinstock GM, Murray BE. Effect of disruption of a gene encoding an autolysin ofEnterococcus faecalisOG1RF. Antimicrobial Agents and Chemotherapy. 1998;42:2883-2888
  68. 68. Kristich CJ et al. Development and use of anefficient system for random mariner transposon mutagenesis to identify novel genetic determinants of biofilm formation in the coreEnterococcus faecalisgenome. Applied and Environmental Microbiology. 2008;74:3377-3386
  69. 69. Hancock LE, Perego M. Systematic inactivation and phenotypic characterization of two component signal transduction systems ofEnterococcus faecalisV583. Journal of Bacteriology. 2004;186:7951-7958
  70. 70. Kristich CJ, Li YH, Cvitkovitch DG, Dunny GM. Esp independent biofilm formation byEnterococcus faecalis. Journal of Bacteriology. 2004;186:154-163
  71. 71. Thurlow LR et al. Gelatinase contributes to the pathogenesis of endocarditis caused byEnterococcus faecalis. Infection and Immunity. 2010;78:4936-4943
  72. 72. Thomas VC, Thurlow LR, Boyle D, Hancock LE. Regulation of autolysis dependent extracellular DNA release byEnterococcus faecalisextracellular proteases influences biofilm development. Journal of Bacteriology. 2008;190:5690-5698
  73. 73. Pinkston KL et al. The Fsr quorum- sensing system ofEnterococcus faecalismodulates surface display of the collagen- binding MSCRAMM ace through regulation of gelE. Journal of Bacteriology. 2011;193:4317-4325
  74. 74. Krasteva PV, Giglio KM, Sondermann H. Sensing the messenger: The diverse ways that bacteria signal through c- di-GMP. Protein Science. 2012;21:929-948
  75. 75. Camilli A, Bassler BL. Bacterial small moleculesignaling pathways. Science. 2006;311:1113-1116
  76. 76. Cook LC, Federle MJ. Peptide pheromone signaling inStreptococcusandEnterococcus. FEMS Microbiology Reviews. 2014;38:473-492
  77. 77. Li YH, Tian X. Quorum sensing and bacterial socialinteractions in biofilms. Sensors. 2012;12:2519-2538
  78. 78. Cook L et al. Biofilm growth alters regulation of conjugation by a bacterial pheromone. Molecular Microbiology. 2011;81:1499-1510
  79. 79. Antiporta MH, Dunny GM. ccfA, the genetic determinant for the cCF10 peptide pheromone inEnterococcus faecalisOG1RF. Journal of Bacteriology. 2002;184:1155-1162
  80. 80. Dunny GM. The peptide pheromone inducible conjugation system ofEnterococcus faecalisplasmid pCF10: Cell- cell signaling, gene transfer, complexityand evolution. Philosophical Transactions of the Royal Society B. 2007;362:1185-1193
  81. 81. Hirt H et al.Enterococcus faecalissex pheromonec CF10 enhances conjugative plasmid transfer in vivo. MBio. 2018;9:e00037-e00018
  82. 82. An FY, Sulavik MC, Clewell DB. Identificationand characterization of a determinant (eep) on theEnterococcus faecalischromosome that is involvedin production of the peptide sex pheromone cAD1. Journal of Bacteriology. 1999;181:5915-5921
  83. 83. Chandler JR, Dunny GM. Characterization of the sequence specificity determinants required forprocessing and control of sex pheromone by the intramembrane protease Eep and the plasmid encoded protein PrgY. Journal of Bacteriology. 2008;190:1172-1183
  84. 84. Varahan S, Iyer VS, Moore WT, Hancock LE. Eep confers lysozyme resistance toEnterococcusfaecalisvia the activation of the extracytoplasmic function sigma factor SigV. Journal of Bacteriology. 2013;195:3125-3134
  85. 85. Frank KL et al. AhrC and Eep are biofilm infectionassociatedvirulence factors inEnterococcus faecalis. Infection and Immunity. 2013;81:1696-1708
  86. 86. Frank KL et al. Evaluation of theEnterococcus faecalisbiofilm associated virulence factors AhrC and Eep in rat foreign body osteomyelitis and in vitro biofilm associated antimicrobial resistance. PLoS One. 2015;10:e0130187
  87. 87. Frank KL et al. Use of recombinase based in vivoexpression technology to characterizeEnterococcus faecalisgene expression during infection identifiesin vivo- expressed antisense RNAs and implicates the protease Eep in pathogenesis. Infection and Immunity. 2012;80:539-549
  88. 88. Ali L et al. Molecular mechanism of quorum- sensinginEnterococcus faecalis: Its role in virulence and therapeutic approaches. International Journal of Molecular Sciences. 2017;18:960
  89. 89. Hancock LE, Perego M. TheEnterococcus faecalisfsr two- component system controls biofilm developmentthrough production of gelatinase. Journal of Bacteriology. 2004;186:5629-5639
  90. 90. Nakayama J et al. Revised model forEnterococcus faecalisfsr quorum sensing system: The small open reading frame fsrD encodes the gelatinase biosynthesis activating pheromone pro-peptide corresponding to staphylococcal agrd. Journal of Bacteriology. 2006;188:8321-8326
  91. 91. Shao C et al. LuxS dependent AI-2 regulates versatile functions inEnterococcus faecalisV583. Journal of Proteome Research. 2012;11:4465-4475
  92. 92. He Z et al. Effect of the quorum- sensing luxS gene on biofilm formation byEnterococcus faecalis. European Journal of Oral Sciences. 2016;124:234-240
  93. 93. Kayaoglu G, Orstavik D. Virulence factors ofEnterococcus faecalis: Relationship of endodontic disease. Critical Reviews in Oral Biology and Medicine. 2004;15(5):308-320
  94. 94. Albiger B, Dahlberg S, Henriques-Normark B, Normark S. Role of the innate immune system in host defence against bacterial infections: Focus on the toll-like receptors. Journal of Internal Medicine. 2007;261:511-528
  95. 95. Neuhaus FC, Baddiley J. A continuum of anionic charge: Structures and functions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiology and Molecular Biology Reviews. 2003;67:686-723
  96. 96. Fabretti F, Theilacker C, Baldassarri L, Kaczynski Z, Kropec A, Holst O, et al. Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infection and Immunity. 2006;74:4164-4171
  97. 97. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. Journal of Endodontia. 2002;28:689-693
  98. 98. Abou-Rass M, Bogen G. Microorganisms in closed periapical lesions. International Endodontic Journal. 1998;31:39-47
  99. 99. Sunde PT, Olsen I, Debelian GJ, Tronstad L. Microbiota of periapical lesions refractory to endodontic therapy. Journal of Endodontia. 2002;28:304-310
  100. 100. Shin SJ, Lee JI, Baek SH, Lim SS. Tissue levels of matrix metalloproteinases in pulps and periapical lesions. Journal of Endodontia. 2002;28:313-315
  101. 101. Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Australian Dental Journal. 2007;52(1 Suppl):S64-S82
  102. 102. Evan M, Davies JK, Sundqvist G, Fidgor D. Mechanisms involved in the resistance of theEnteococcus faecalisto calcium hydroxide. International Endodontic Journal. 2002;35:221-228
  103. 103. Nasution AI, Soraya C, Nati S, Alibasyah ZM. Effect of ethylene diamine tetra acetic acid and rcprep to microstrain of human root dentin. Journal of International Oral Health. 2016;8(1):32-33
  104. 104. Avery JK, Chiego DJ. Essential of Oral histology and embryology. In: A Clinical Approach. 3rd ed. Missouri: Mosby Elsevier; 2006. pp. 108-113
  105. 105. Cwikla S, Bellanger M, Giguere S, Fox A, Verticci F. Dentinal tubulus disinfection using three calcium hydroxide formulation. Journal of Endodontia. 2000;31:50-52
  106. 106. Estrella C, Pimenta FC, Ito IY, Bamman LL. Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules. Journal of Endodontia. 1999;25:416-418
  107. 107. Tjaderhane L, Palosaari H, Wahlgren J, Larmas M, Sorsa T, Salo T. Human odontoblast culture method: The expression of collagen and matrix metalloproteinases (MMPs). Advances in Dental Research. 2001;15:55-58

Written By

Ajay Kumar Oli, Palaksha K. Javaregowda, Apoorva Jain and Chandrakanth R. Kelmani

Submitted: January 21st, 2022 Reviewed: February 25th, 2022 Published: April 29th, 2022