1. Introduction

Biofilms are spatially organized microbial communities whose functionality relies on a complex web of symbiotic relationships [1, 2]. Organisms engage in complicated social interactions that can either be cooperative or competitive and take place within and between species [3]. Biofilms are formed depending on bacterial strain and environmental conditions such as pH, nutrients, fluid flow, surfactants, and temperature [4]. Biofilms may become more complicated due to increased cell density, which promotes chemical signals to communicate with responsive cells for social interactions [5]. Since biofilms commonly include dense cell populations, quorum sensing (QS) cell density–dependent control of gene expression becomes essential for the whole establishment of biofilms [6–8]. For example, the bacteria that form biofilms on the teeth produce peptide bacteriocins to compete with each other. Streptococcus bacteria such as Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguinis, and S. mitis produce bacteriocins and regulate the formation of biofilms controlled by the QS system [9, 10].

The QS system regs biofilm production on teeth for the process of bacterial adhesion, biofilm development, and maturation. In order to monitor species complexity and cell density in the population, QS is a communication system between microbial cells that is related to the detection of chemical signals [11–14]. QS controls the expression of genes related to virulence, competition, pathogenicity, and resistance by tracking the number of cells through chemical signals that allow bacteria to communicate each other [15]. QS systems commonly depend on the species and play a role in activities like horizontal gene transfer (HGT), biofilm development, and cell maintenance. Other activities involving the synchronization of the entire population, such as the generation of antibiotics, natural competence, sporulation, and the expression of secretion systems, are also affected by QS [16, 17]. QS activity can be inhibited through several mechanisms, namely, inhibition of autoinducer (AI) synthesis or transport, AI degradation and sequestration, and QS signal competition. QS inactivation agents are known as anti-QS with their signaling disruption called quorum quenching (QQ) [18]. Therefore, this review describes and explains about the role of QS in biofilm formation and potential natural sources for use as alternative anti-QS.

2. QS

Bacteria have a QS system that contributes to cell–cell communication by producing, detecting, and responding to cell signaling molecules referred to as AIs [19–21]. When the bacteria population increases, AIs accumulate in the environment, and bacteria will monitor changes in cell numbers and change gene expression collectively [22]. QS is a system that bacteria utilize to control gene expression that is dependent on cell density [23]. QS can control the production of antibiotics, sporulation, competition, bioluminescence, biofilm formation, and virulence secretion [24–26]. The three basic principles of the QS system are that community members produce AIs as signaling molecules. The AI will diffuse at low cell density so that it is at a concentration below the threshold required for detection. However, at high cell densities (HCDs), AIs are produced at high concentrations, enabling detection and response. Second, receptors on the cytoplasm or cell membrane detect the AI. Third, the detection of the AI may also activate the production of the AI [22].

QS has three types of signaling molecules: AI-1, AI-2, and AI-3 [27]. AI-1 and AI-2 are signaling molecules produced in Gram-negative bacteria. Both molecules are affected by an increase in cell density which will lead to gene expression. AI-1 is an N-acyl-L-homoserine lactone (AHL) [14, 24, 28]. In AI-1, there are proteins that produce bioluminescence signals, namely, LuxI and LuxR. Both proteins activate lux gene expression through the freely diffusing N-3-oxohexanoyl-homoserine lactone (3-O-C₆-HSL) as a proxy for cell density [29, 30]. AHLs are synthesized from Vibrio fischeri utilizing enzymatic reactions through LuxL signaling, and then, LuxR signaling activates gene expression resulting in AI-1. Some types of bacteria that produce AI-1 are Enterobacter cloacae, Rhodobacter capsulatus, and Escherichia coli which are composed of 14 to 18 carbon chains on AHLs and one or two double bonds [31, 32]. Then, commonly found in Vibrio harveyi and Pseudomonas aeruginosa are four carbon AHLs, and there are N-butyryl-L-homoserine lactone and N-3-hydroxy-butyryl-L-homoserine lactone [33–35].

Then, AI-2 is usually synthesized by E. coli bacteria through LuxS signals [36]. LuxS proteins can secrete and detect signal molecules in the presence of the expression of a specific set of genes to regulate the QS mechanism [37]. AI-2 molecules have properties that tend to be more hydrophilic and the process requires phosphorylation or bromination so that the molecules are not found in many microorganisms compared to AHLs [38, 39]. An example of AI-2 molecule is the furanosyl borate diester found in V. harveyi [40, 41]. In addition, E. coli can produce pyrazinone AI derivatives referred to as AI-3. This signaling molecule is commonly found in Salmonella sp. and Shigella sp [42, 43]. An example of the structure of AI-3 is phevalin.

In the synthesis of AI-1, S-adenosyl-L-methionine (SAM) interacts with acyl-ACP through the enzymatic process of AHLs synthase, and SAM binds to the acyl of acyl-ACP forming a peptide bond resulting in AHLs. This process produces a side product through esterification reaction in the form of 5′-methylthiadenosine (MTA). This product can be reused by bacteria to synthesize SAM through 5′-methylthiadenosine nucleosidase (MTAN) and 5-methylthioribose phosphate. Then, through methyltransferase, SAM is converted into S-adenosylhomocysteine (SAH) as the initial stage to synthesize AI-2. Furthermore, MTAN enzyme synthesizes S-ribosylhomoserine (SRH) as a precursor of homocysteine with LuxS as catalyst [44–46]. The biosynthesis pathway of AI-1 and AI-2 can be seen in Figure 1.

In QS systems, the AI-2 mechanism plays a role in bacterial interspecies communication. The LuxS enzyme that catalyzes the final enzymatic reaction for AI-2 formation produces 4,5-dihydroxy-2,3-pentanedione (DPD). Figure 1 shows that DPD is formed through a spontaneous reaction, and then, the complex of chemical compounds is produced and can be converted by other species through rearrangement into appropriate signals by those species [47]. Interspecies communication does not only occur between bacteria that produce AI-2, and bacteria that do not produce AI-2, such as Pseudomonas aeruginosa, can also recognize species producing AI-2 in the same environment [48]. Zhang et al. [36] reported that P. aeruginosa can respond to AI-2 through dCACHE domain generated from PctA and TlpQ chemoreceptors. The domain can activate AI-2 and produce chemotaxis, resulting in the formation of biofilms. The existence of dCACHE domain that becomes the AI-2 receptor is also produced in Bacillus subtilis through histidine kinase and in Rhodopseudomonas palustris through diguanylate cyclase. In addition, LuxP and LsrB enzymes are also receptors of AI-2. AI-2 is bound and delivered by the LsrB through the ATP-binding cassette (ABC) transporter for entry into the cell and regulation of gene expression, while LuxP enzyme is commonly found in Vibrio spp [49].

Furthermore, some environmental factors also affect the activation of AI-2 in bacteria. Several studies have reported that low pH affects enzymatic activity in bacteria. Gu et al. [50] reported that AI-2 production increased with decreasing pH. The data showed that at pH 4.5 and 5.5, AI-2 activity of L. fermentum was higher than at pH 6.5. Then, Wen and Burne [51] also reported acid tolerance testing on LuxS from S. mutans showed good adaptability at pH 5. In addition, temperature also affects enzymatic reactions in bacteria. AI-2 activity, metabolic rate, and cell density decreased at a lower temperature of 23°C, but luxS and pfs gene expression increased. AI-2 activity started to increase when testing at 44°C and doubled when the temperature was increased to 50°C [50, 52]. The appropriate amount of nutrients also affects the action of QS. Rutherford and Bassler [22] reported that the QS response by the CodY enzyme in Staphylococcus aureus is delayed when the environment is nutrient-poor.

3. QS in Gram-Negative Bacteria

QS systems of Gram-negative and Gram-positive bacteria are different. There are four characteristics shared by all existing Gram-negative QS systems. AHLs or other compounds derived from SAM are the molecules that function as AIs in these systems, and they are capable of passing through the bacterial membrane. Second, specialized receptors located in the cytoplasm or inside the inner membrane bind AIs. Third, QS often modifies a large number of genes that are involved in many different biological processes. Fourth, the mechanism known as autoinduction drives the increased production of AI through the activation of QS triggered by AI. As a result, a feed-forward loop is created, which is expected to promote synchrony gene production in the population [53, 54].

LuxI-type proteins act as AI synthases for the biosynthesis of AHL signaling molecules [53]. The concentration of AHLs increases as the cell population density increases. When the concentration of AHLs has reached a critical threshold, a LuxR protein binds to AHLs to form a LuxR–AI complex and activate transcription of QS-regulated genes. The genes include luxI gene encoding the LuxI protein that produces AHLs, enhancing the positive feedback [55, 56].

More than 100 species of Gram-negative bacteria have produced LuxI/LuxR homologs [30]. These homologs produce AHLs with different side chains such as acyl chains with C4 to C18 with some modifications, namely, the presence of carbonyl and hydroxy groups at C3 position [57]. These chemical structures identify intraspecies-specific bacterial intercellular communication. LuxR homologs work specifically with unique binding pockets to detect specific AHLs ligands and LuxI homologs have substrate binding site of different sizes and shapes so that they hold acyl carrier proteins for AI synthesis specifically [56].

4. QS in Gram-Positive Bacteria

Gram-positive bacteria use peptides as signaling molecules called oligopeptide permease (Opp) and autoinducing peptides (AIPs). Opp consists of five proteins guaranteed by ABC importer and first encountered in Salmonella typhimurium to translocate peptides in the periplasmic system. The five genes in Opp form the OppABCDF operon. The gene is also found in the bacteria Streptococcus thermophilus, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, Bacillus subtilis, and Bacillus cereus [58]. Moreover, one class of AIPs encoded as a precursor (pro-AIP) from QS operon requires specialized transporters to be processed, penetrate the cell membrane, and secrete the AIPs extracellularly [22, 59]. The obtained AIPs with five to 17 amino acids have linear or cyclic states of amino acids [22, 59–62]. A two-component sensor kinase bound to the membrane detects extracellular AIP. In the HCD state, the concentration of AIP is high and it binds to sensor histidine kinases that are AIP receptor bound in membrane [59]. The binding of AIP activates the receptor kinase, which autophosphorylates and cytoplasmic response regulator protein that regulates the transcription of QS-related genes [59, 63, 64].

Figure 2 shows one of the mechanisms of QS in Gram-positive bacteria. ComC enzyme forms the peptide precursor locus, and then, it translates into protein precursor, and a peptide-inducing signal is generated. The ABC transporter, which contains ComA and ComB enzymes, carries the peptide signal out of the cell [57]. The histidine protein kinase of the two-component signaling system which bind to ComD detects the peptide signal when its extracellular concentration is at a minimal level of stimulation. Subsequently, the conserved histidine when bound by AIP is autophosphorylated by sensor kinase. The phosphoryl group is passed to the aspartate of histidine and ComE expresses QS target genes which is controlled by a phosphorylation response regulator [55]. In this AIP-QS systems, the transporter, pro-AIP, response regulator, and cognate histidine receptor are encoded in an operon. QS controls the production of virulence factors in Gram-positive pathogens [55, 65, 66].

There are other systems in which the AIPs produced by extracellular processing of pro-AIPs with extracellular proteases are transported into the cell by an Opp to regulate gene expression [59, 67, 68]. Opp belongs to a large family of ABC transporters and appears to transport peptides relatively nonspecifically ranging in size from three to 20 amino acids [67].

5. The Role of QS in Biofilm Formation

During the formation of biofilms, microorganisms have the ability to communicate with each other by using QS. QS regulates the metabolic processes of planktonic cells and may promote pathogenicity and the formation of microbial biofilms. Biofilms are formed through a number of processes, including cell-to-cell adhesion, growth, maturation, and dispersion [69, 70]. Microcolonies are created by bacterial growth and are enclosed in a layer of hydrogel which serves as a wall between the microbial population and the outside world. Bacteria use QS systems to communicate with one another in their community. Chemical signals are used in these systems. Cellular functions, disease dependent on population density, food intake, the transfer of genetic material between cells, motility, and the creation of secondary metabolites are all impacted by communication. The growth of biofilms occurs concurrently with the production of extracellular polymeric substances (EPS). The final step involves removing bacterial strains from microcolonies, which may cause the growth of a new biofilm colony at a specific location [71–73].

Biofilm formation in bacteria involves several stages, namely, the initial stage by adhesion through the formation of EPS. Generally, sucrose-dependent EPS formation is catalyzed by glucosyltransferase. Then, in the second stage, cell division occurs causing reversible bacterial attachment to become irreversible. Bacterial attachment that begins to flatten on the host cell forms a microcolony into a young biofilm and the activity continues until the mature biofilm. In the final stage, bacterial cells are dispersed and cell autolysis occurs. During maturation and toward cell autolysis, QS controls the secretion of EPS by bacteria. This is because EPS plays an important role in biofilm evolution and harms or helps other cells in the same environment. The control of EPS secretion by QS is different for each microbe. Research by Sakuragi & Kolter [74] reported that at HCD, activation of EPS by QS increased in P. aeruginosa. This is different from Vibrio cholerae, after the bacterium attaches to the host cell surface and flagellar activity decreases, EPS secretion begins to be activated by QS. Then, EPS activity is stopped when cell density is high [21, 75, 76]. Gram-negative and Gram-positive microorganisms can also produce biofilms on medical equipment, and the most common forms include Staphylococcus aureus, Proteus mirabilis, Enterococcus faecalis, E. coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, Streptococcus viridans, and Klebsiella pneumoniae and are the most prevalent types [77, 78]. There are several QS systems of some bacteria which contribute to biofilm formation. These bacteria synthesize specific virulence factors that cause dental infection through the QS mechanisms. These virulence factors are described in Table 1.

5.1. QS System of Streptococcus mutans

One health issue of particular concern is oral infections due to the formation of biofilm on the teeth. Streptococcus mutans is one of Gram-positive bacteria that play a role in biofilm formation. This occurs due to the interaction between S. mutans bacterial cells through QS. The QS mechanism in S. mutans is regulated by two ComDE enzymes to express virulence genes. This system is based on HCD where competence-stimulating peptide (CSP) will activate Com-dependent QS enzymes in S. mutans [92]. In this mechanism, QS is regulated by the ComABCDE enzyme for genetic modification, bacteriocin development, and acid tolerance. ComDE plays a role in responding to CSP [93, 94]. The biosynthesis of QS in S. mutans can be seen in Figure 3. ComC enzyme synthesizes propeptide as 21-amino acid, and then, 21-CSP maturation occurs by ABC transporters (ComA and ComB). The peptide chain is synthesized by SepM enzyme into active signal 18-CSP and secreted outside the cell [95]. The ComD enzyme bound to the histidine kinase sensor detects the active signal, resulting in phosphorylation with ComE enzyme which results in the expression of virulence genes as AI signals on QS [96, 97]. Further studies showed ComDE signaling also plays a role in regulating the expression of glucosyltransferase B, C, and D (GtfBCD) proteins, glucan binding protein B (gbpB), and fructosyl transferase (FTF) [98].

In addition to expressing signaling molecules, ComE can also drive the ComRS pathway with ComS as a transcriptional regulator of ComR [99]. ComS will be processed into XIP peptide upon transfer to extracellular environment, and then, XIP will enter the cell via Opp. Next, ComR forms a complex with XIP to activate the ComX and ComS enzymes [100]. ComX will express SigX as an important molecule for genetic competence. As the biofilm matures, the XIP peptide is required to increase ComX expression [101].

6. Advantage and Disadvantage of QS

In pathogenic bacteria, QS can control coordinated virulence. In P. aeruginosa and V. cholerae, it is reported that when the number of cells is sufficient, the bacteria will produce harmful toxins so that the bacteria can infect against host cells [123]. In the other condition, las and rhl in P. aeruginosa positively control Type VI secretion (H2-T6SS), but rhl negatively controls Type III secretion (T3SS) [124, 125]. Furthermore, QS facilitates biofilm formation in bacteria. The release of extracellular DNA is facilitated by QS, and it is very essential at each stage of biofilms for maintaining the stability and protecting antibiotic-induced lysis. In addition, gene expression regulated by QS when cell density is high is advantageous to bacteria because it can control the necessary resources. QS will not activate gene expression when metabolic processes are sufficient for virulence factors [126]. For example, Gram-positive bacteria have oligopeptide transport systems or two-component systems that detect signaling molecules. A transcriptional regulator or phosphate (cognate regulator) will interact with the imported peptide or signaling molecule. The regulator will also control virulence, conjugation, competence, and presence factors. This system has been reported in groups of bacilli, streptococci, and enterococci [127].

QS can also facilitate HGT for antibiotic-resistant bacteria transferring genes to antibiotic-sensitive bacteria, thereby increasing their ability to survive. Gene transfer can be done through conjugation by transferring conjugating plasmids, then transduction through the use of bacteriophages to transfer DNA, and through transformation by importing DNA in the outer area of fragmented cells. The original DNA of the recipient bacteria will be replaced by the DNA that was successfully transferred by the donor bacteria through recombination [128]. In the study of Wang et al. [129], it was reported that through the artificial biofilm test, Treponema denticola, which had resistance in its shuttle plasmid to erythromycin, could be translated with S. gordonii. Other studies report HGT can occur through membrane vesicles. This process is most common in Gram negatives, but also occurs in some Gram positives. E. coli has successfully exported an antibiotic resistance gene, virulence gene, and green fluorescent protein encoding or gft in membrane vesicles to Salmonella sp [130]. Then, eDNA was also successfully exported through membrane vesicles by S. mutans to the biofilm mechanism, thus becoming an important genetic source for its formation cycle [131].

However, the presence of QS can also be a disadvantage in pathogenic bacteria. Cell density–dependent QS activity can hinder pathogenicity when the number of colonies on the host cell is not reached due to host cell attack or antibiotic activity. For example, oxidant activity produced by the host cell causes QS inference in S. aureus to produce virulence factors. This results in the disruption of S. aureus pathogenicity [132]. Then, research by Kapadia et al. [133] showed that ethyl acetate extract of P. aeruginosa containing cyclo(L-prolyl-L-valine), cyclo(Pro-Leu), and cyclo(D-phenylalanine-L-prolyl) compounds successfully inhibited the growth of Lelliottia amnigena RCE biofilm at HCDs. Furthermore, QS-dependent biofilm activity becomes a therapeutic target as a new antimicrobial agent by inhibiting or damaging the pathway of QS. This activity is referred to as QQ which only inhibits the expression of pathogenicity but does not cause cell death [134]. For example, inhibition of AHL biosynthesis is an efficient strategy to inhibit QS. Enzymes encoded from the aiiA gene of an AI invasion of Bacillus spp with two homologous domains of metallo-B lactamase, glyoxalase II, and arylsulfatase are able to inhibit AI production [135]. In addition, a monoclonal antibody (mAb) has also been developed as an anti-AHLs that has been able to inhibit QS in P. aeruginosa [123].

Moreover, the presence of QS also benefits nonpathogenic bacteria through HGT. QS allows them to adapt to their environment for nutritional needs and adjust their metabolic activities to thrive in certain ecosystems [136]. In addition, HGT can also facilitate the spread of infection in nonpathogenic bacteria through intra- and interspecies interactions [137]. S. aureus can transfer genes to nonpathogenic bacteria resulting in methicillin resistance to antibiotics [138]. However, QS can also cause disadvantage to nonpathogenic bacteria due to resource competition. In environments with limited resources or nutritional factors, nonpathogenic bacteria will lose out to pathogenic bacteria that use QS more dominantly to survive [132].

7. Anti-QS

In the discovery of traditional antibiotics, the inhibition of QS system could be a potential target. Some of the factors that can be seen from the QS inhibitory system are the process of producing a signal, signal detection, the signal itself, and the source used as an inhibitor. Sources of inhibitory that can be used are natural, bacterial, and eukaryotic products. The researchers focused on synthesizing mimic compounds of AHLs or similar to AHLs. It is expected to inhibit the signal receptor, synthase, or both. In addition, AHLs-like compounds have the ability to diffuse through Gram-negative bacterial cell membranes and work specifically on QS [139]. One of the compounds to be the first QS inhibitor was furanone isolated from the macroalgae Delisea pulchra which has the structural similarities with AHLs [140, 141]. Natural products have been of particular interest due to their therapeutic effects in the field of traditional medicine [142]. The biological values contained in them have the potential to provide activity as anti-QS on pathogenic bacteria [143]. Determination of anti-QS by therapeutic agents is generally tested by biofilm formation assays using crystal violet and microscopy, then QS inhibition assays to see the effectiveness of antimicrobial agents against inhibition of the QS mechanism. In addition, some researchers also use quantitative RT-PCR to determine its activity at the molecular level [144–146]. In addition, a biomolecular approach to the inhibitory interactions of antimicrobial agents against specific enzymes in QS can be predicted by a computational approach, namely, molecular docking. The more advanced prediction can be done using molecular dynamic [146]. Tables 2 and 3 show some plants and compounds which have the activity as anti-QS. Interventional studies involving animals or humans, and other studies that require ethical approval, must list the authority that provided approval and the corresponding ethical approval code.

In addition, essential oil (EO) consists of several compounds in the group of monoterpenoids, sesquiterpenoids, phenylpropanoids, and oxygen derivatives that have low molecular lipophilic properties and are volatile. EO is potentially antibacterial by damaging the function and integration of cytoplasmic membranes. Therefore, some studies also utilized the EO component as an anti-QS agent. The anti-QS activity on EO extracted from plant fibers can be seen in Table 4.

Specific compounds isolated from plants also provide anti-QS effects, some of which belong to the terpenoid, flavonoid, or phenolic acid group [177]. The terpenoid compounds have anti-QS effects against Chromobacterium violaceum and P. aeruginosa, namely, carvacrol, sesquiterpene lactone, and eugenol by inhibiting the formation of biofilms, reducing AHLs production, and inhibiting QS mediators [178–181]. Later, linalool inhibits the formation of biofilms in Acinetobacter baumannii, D-limonene inhibits biofilm formation in E. coli, and (−)-α-pinene reduces QS communication [182–184].

Epigallocatechin, kaempferol, and morin are flavonoid compounds which have effect to inhibit biofilm formation and QS mediated by AI-2 of S. aureus and Eikenella corrodens [185–187]. Then, naringin can inhibit S. mutans by suppressed biofilm maturation [188, 189]. Quercetin, quercetin 4′O-β-D-glucopyranoside, taxifolin, and naringenin can inhibit P. aeruginosa by decreased biofilm formation, the LasR expression, and the QS-regulated gene expressions [190–192].

The phenolic acid compounds such as rosmarinic acid, chlorogenic acid, salicylic acid, cinnamic acid, and phenylacetic acid can inhibit P. aeruginosa by decreased the QS regulator, biofilm formation, QS gene expression, QS-dependent virulence factor, and AHL signaling [193–195]. Then, 4-dimethyl-aminocinnamic acid and 4-methoxycinnamic acid can inhibit Chromobacterium violaceum by downregulated QS-related metabolites and QS gene expressions [196]. p-Coumaric acid can inhibit Chromobacterium violaceum by reduced the QS responses [197]. Caffeic acid can inhibit Staphylococcus aureus by reduced bacterial adhesion [198]. Ellagic acid can inhibit Burkholderia cepacia by reduced biofilm formations [185]. Some compounds have also been tested for their anti-QS activity in vitro and in silico as in Tables 5 and 6. The chemical structure of these compounds is shown in Figures 4 and 5.

The test of antibiofilm inhibitory activity on compounds from natural plants is not only limited to in silico. Research from Dzoyem et al. [217] reported emodin (62) and kaempferol (63) isolated from Cassia alata L. leaves were active as antibiofilms with MBIC values of 70.81 μg·mL⁻¹ and 65.65 μg·mL⁻¹ and then MBEC values of 63.65 μg·mL⁻¹ and 82.66 μg·mL⁻¹ against Candida albicans, respectively. In addition, both compounds were also active in inhibiting violacein production in the QS system with IC₅₀ values of 28.08 μg·mL⁻¹ and 26.44 μg·mL⁻¹. Research from Tamfu et al. [218] also reported that three compounds from seeds of Annona senegalensis have activity as antibiofilm. Asimicin (64) provided antibiofilm activity of 6.3%–37.9% against C. albicans and 18.8%–43.2% against E. coli and inhibited violacein production in C. violaceum by 26.7%–43.8%. Then, N-cerotoyltryptamine (65) inhibited S. aureus biofilm by 26.7%–43.8%. N-cerotoyltryptamine inhibited violacein formation in C. violaceum by 9.66%–100%. 3,5,7-Trihydroxyflavone (20) also inhibited the biofilm formation about 50%–85% [208]. Terpinen-4-ol (66) inhibited biofilm formation of MRSA by 73.70% and violacein production of 69.3%. Then, it inhibited swarming motility of P. aeruginosa by 25% [219]. In addition, 4-hydroxycinnamic acid (54) inhibited biofilm formation of S. mutans by 34.3%–49.3% [220]. The violacein production of C. violaceum was inhibited by quercetin (17) and quercetin-3-O-arabinoside (67) of 50 and 100 μg·mL⁻¹, respectively [221]. The structure of these compounds is shown in Figures 4, 5, 6.

However, further research development reported that the use of natural products as natural antibiofilms or anti-QS can also have the potential for resistance. This is because natural products may have the possibility of disrupting normal flora when suppressing a pathogenic microcolony, possible accidentally affecting beneficial nonpathogenic bacteria [222]. In addition, some natural compounds can be toxic to the host cell, which can lead to other inflammatory responses [223]. Furthermore, the presence of complex QS mechanisms allows pathogenic bacteria to increase biofilm production so that they can adapt to interference from antimicrobials [224]. Therefore, several studies continue to develop formulations of natural antibacterial agents. The previous research reported in increasing the effectiveness of antibiofilms with a long half-life, greater solubility, and stronger resistance to enzymatic hydrolysis, and nanocurcumin formulations were developed. This is because nanocurcumin provides a larger surface area. Nanocurcumin can reduce biofilm formation to 58% [225]. In addition, some studies increased the biocompatibility and biodegradability by modified the compounds using biopolymer such as chitosan, cellulose, collagen, hyaluronic acid, alginates [226], and polyethylene glycol (PEG) [227]. A nanohybrid thymol drug delivery system with zinc-coated hydroxide salt or ZnLHS was developed to improve the aqueous solubility of thymol and better distribution to the infection target. This study showed suppression of P. aeruginosa reaching 90%. This is because the hydroxide salt can oxidize due to its anionic nature, facilitating the inhibition of polysaccharides, thereby enhancing the ability of thymol to inhibit pathogenic bacterial biofilms [228]. Furthermore, eugenol has also been modified by nanoemulsion and can enhance the ability to inhibit the virulence factors of QS such as 3-O-C₁₂-HSL and C₄-HSL in P. aeruginosa [229]. So far for dental infection, clinical trials that have been reported are using thymol combined with chlorhexidine. This was tested on 90 patients (3–17 years old) who had caries disease. Anesthesia with thymol and chlorhexidine formulation was performed on salivary of Streptococcus mutans and lactobacilli. Results showed a decrease in the number of bacteria after treatment for 12 months [230].

8. Conclusions

QS is a communication system between bacterial cells through the response of cell signals called AIs. AIs are divided into three classes. AI-1 is AHL, AI-2 is furanosyl borate diester, and AI-3 is phevalin. Gram-negative bacteria produce AIs such as acyl-homoserine that produced by LuxR and LuxI proteins. Gram-positive bacteria produce autoinducing signal peptides that are followed in the process by ABCs, sensor kinases, and response regulators. In the process, Gram-positive bacteria involve several enzymes such as ComABCDE for genetic modification, bacteriocin development, and acid tolerance. QS is involved in biofilm formation at the maturation stage. Some of the bacteria involved in biofilm formation are Staphylococcus aureus, Proteus mirabilis, Enterococcus faecalis, E. coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, Streptococcus viridans, and Klebsiella pneumonia. In addition, studies on the discovery of natural antibacterial agents as anti-QS are widely developed to date. Studies show that there are 64 plant extracts, 39 EOs, and 36 natural compounds that have potential as anti-QS in vitro and in silico. Therefore, the review of the importance of QS in biofilm can serve as a foundation for the discovery of targets that are essential in inhibiting biofilm formation. Furthermore, these in vitro and in silico studies can serve as preliminary findings for the development of natural antibiofilm drugs against dental infections. Plant extracts that have been tested in vitro can be further developed to isolate single compounds that are highly active against pathogenic bacteria so that in the future, single compounds that have been found to be active in inhibiting pathogenic bacteria can be further tested for in vivo to clinical trials.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This research was supported by the scholarship of the Masters to Doctoral Program for Superior Scholars, Indonesia (014/E5/PG.02.00/PL.PMDSU/2024: May 03, 2024, and 3164/UN6.3.1/PT.00/2024: May 06, 2024), and the Academic Leadership Grant (ALG) Prof. Dikdik Kurnia, M.Sc., Ph.D, Indonesia (1439/UN6.3.1/PT.00/2024: March 18, 2024); Universitas Padjadjaran.