Natural Medicine a Promising Candidate in Combating Microbial Biofilm

Studies on biofilm-related infections are gaining prominence owing to their involvement in most clinical infections and seriously threatening global public health. A biofilm is a natural form of bacterial growth ubiquitous in ecological niches, considered to be a generic survival mechanism adopted by both pathogenic and non-pathogenic microorganisms and entailing heterogeneous cell development within the matrix. In the ecological niche, quorum sensing is a communication channel that is crucial to developing biofilms. Biofilm formation leads to increased resistance to unfavourable ecological effects, comprising resistance to antibiotics and antimicrobial agents. Biofilms are frequently combated with modern conventional medicines such as antibiotics, but at present, they are considered inadequate for the treatment of multi-drug resistance; therefore, it is vital to discover some new antimicrobial agents that can prevent the production and growth of biofilm, in addition to minimizing the side effects of such therapies. In the search for some alternative and safe therapies, natural plant-derived phytomedicines are gaining popularity among the research community. Phytomedicines are natural agents derived from natural plants. These plant-derived agents may include flavonoids, terpenoids, lectins, alkaloids, polypeptides, polyacetylenes, phenolics, and essential oils. Since they are natural agents, they cause minimal side effects, so could be administered with dose flexibility. It is vital to discover some new antimicrobial agents that can control the production and growth of biofilms. This review summarizes and analyzes the efficacy characteristics and corresponding mechanisms of natural-product-based antibiofilm agents, i.e., phytochemicals, biosurfactants, antimicrobial peptides, and their sources, along with their mechanism, quorum sensing signalling pathways, disrupting extracellular matrix adhesion. The review also provides some other strategies to inhibit biofilm-related illness. The prepared list of newly discovered natural antibiofilm agents could help in devising novel strategies for biofilm-associated infections.


Introduction
Bacterial infections threaten public health worldwide, and the severity of the crisis has intensified due to the appearance and proliferation of drug-resistant bacterial species [1]. The majority of the microbes defend themselves by diverse survival mechanisms, including morphogenesis, proteolytic approaches, demographic heterogeneity, etc., to overcome environmental stress conditions. One of the prominent growth states for microorganisms that exist in 90% or more of bacteria strains is biofilm. Biofilms are considered heterogeneous assemblages of surface-associated microbes compressed in a matrix composed crocolonies that resemble mushrooms [12]. It has been established that the morphology of biofilms in microcolonies is regulated by fluid flow, nutritional makeup, and the messenger molecules called quoromones. N-Acyl homoserine lactones (AHLs) are utilized for bacterial communication. [13,14]. Biofilms can evolve on non-living and living surfaces and are frequently seen in the natural world and in healthcare facilities. In contrast to planktonic cells, single cells of the same organism that grow in biofilms show different physiological properties [15]. Research has shown that the microbial cells in the biofilms hold the ability to mutate, which is the leading cause of developing resistance to antibiotics used in eroding the biofilm [16,17]. Each and every component plays a critical role in biofilm structure; that is, Pel and psl polysaccharide helps provide a solid surface for biofilm, whereas the alginate provides the surface adhesion. In case of starvation, eDNA functions as a nutrient source [18,19].

Stages of Biofilm Formation
There are five steps found in forming biofilm, as shown in Figure 1. They start from the initial stage of attachment of planktonic microorganisms to a surface. The initial stage (reversible) is defined as a set of physiochemical factors that specify how the bacteria's cell surfaces interact with the prepared surface [20]. The secondary stage consists of the specific adhesion with the surface; the bacteria in this stage undergo both phenotypic and genotypic changes. The third stage is maturation, wherein the irreversibly attached bacteria communicate with the nearby organic and inorganic molecules [21]. Microcolonization of the bacteria can be defined as the fourth stage in which the association also becomes stable, and the bacteria start the step of intercommunication among different bacteria [22,23]. The final fifth stage is dispersion, where factors such as fluid dynamic and shear stress cause microbial dispersal from the older biofilm; thus, either shedding the daughter cells and causing the microorganisms to move to the newer locations or leading to the formation of other biofilms [24,25].

Mechanism of Biofilm Formation
The microbial cells in the biofilm are composed of extracellular polymeric substances (EPS) with a larger molecular weight, causing resistance to many acids as well as metals; as well as this, the other causes of this resistance are a genetic adaptation, cell growth, and

Mechanism of Biofilm Formation
The microbial cells in the biofilm are composed of extracellular polymeric substances (EPS) with a larger molecular weight, causing resistance to many acids as well as metals; as well as this, the other causes of this resistance are a genetic adaptation, cell growth, and metabolic changes [26]. The mechanisms of biofilm tolerance can be explained as the synthesis of EPS, prompting physical tolerance, leading to difficulty in antimicrobial diffusion inside the matrix. The second type of tolerance caused is passive tolerance, which is basically due to the enzymes of the biofilm matrix that cause the antimicrobial agents' neutralization [27]. The third one is physiological tolerance, in which the deeper layers of the biofilm have metabolically dormant cells that demonstrate adaptive stress responses Antibiotics 2023, 12, 299 4 of 26 that modulate biofilm resistance to different antimicrobials [28]. During biofilm maturation, the microbial cells inside the EPS begin to communicate and secrete specific proteins, which are responsible for efflux pump development. Finally, free planktonic cell dispersal from the produced biofilm encourages the creation of additional biofilms in the periphery [29]. Many naturally obtained biofilm-resistant drugs target various stages of biofilm production and inhibit growth. Specific strategies could be targeted to degrade the cells in the matrix to halt the biofilm mechanism that inhibits QS, preventing efflux pump expression, and inhibiting matrix formation and surface modification, as shown in Figure 2. metabolic changes [26]. The mechanisms of biofilm tolerance can be explained as the synthesis of EPS, prompting physical tolerance, leading to difficulty in antimicrobial diffusion inside the matrix. The second type of tolerance caused is passive tolerance, which is basically due to the enzymes of the biofilm matrix that cause the antimicrobial agents' neutralization [27]. The third one is physiological tolerance, in which the deeper layers of the biofilm have metabolically dormant cells that demonstrate adaptive stress responses that modulate biofilm resistance to different antimicrobials [28]. During biofilm maturation, the microbial cells inside the EPS begin to communicate and secrete specific proteins, which are responsible for efflux pump development. Finally, free planktonic cell dispersal from the produced biofilm encourages the creation of additional biofilms in the periphery [29]. Many naturally obtained biofilm-resistant drugs target various stages of biofilm production and inhibit growth. Specific strategies could be targeted to degrade the cells in the matrix to halt the biofilm mechanism that inhibits QS, preventing efflux pump expression, and inhibiting matrix formation and surface modification, as shown in Figure 2. Show the common antibiotic resistance mechanism that leads to biofilm development.

Quorum Sensing and Its Association with Biofilm Development
Multi-drug resistance is mainly caused by the rigid development of biofilm, which is a complex structure developed in four to five different stages, starting with the irreversible attachment (initial) of planktonic bacteria, the second step is colony formation, the third one is bacterial growth, the fourth is extracellular matrix generation, and, finally, the biofilm's maturation leading to the final reversible detachment of cells from the older formed biofilm [30,31], see Figure 1. The exopolysaccharide or (EPS) is composed of so many essential nutrients among which Aap protein associated with the cell surface contains the G-5 domain, accountable for cell adhesion; during infection in Gram-negative time bacteria (Figure 3), Sortase A (SrtA), a transpeptidase, induces extracellular localization and biofilm generation and can bind cell surface proteins [32,33]. Thus, it can be concluded that inhibitors against this adhesion can potentiate the antibiofilm activity [34]. The communication among the cells is dependent on a chemical signal molecule termed as quorum sensing (QS) [35]. The QS mechanism varies between bacteria, as evidenced by Show the common antibiotic resistance mechanism that leads to biofilm development.

Quorum Sensing and Its Association with Biofilm Development
Multi-drug resistance is mainly caused by the rigid development of biofilm, which is a complex structure developed in four to five different stages, starting with the irreversible attachment (initial) of planktonic bacteria, the second step is colony formation, the third one is bacterial growth, the fourth is extracellular matrix generation, and, finally, the biofilm's maturation leading to the final reversible detachment of cells from the older formed biofilm [30,31], see Figure 1. The exopolysaccharide or (EPS) is composed of so many essential nutrients among which Aap protein associated with the cell surface contains the G-5 domain, accountable for cell adhesion; during infection in Gram-negative time bacteria (Figure 3), Sortase A (SrtA), a transpeptidase, induces extracellular localization and biofilm generation and can bind cell surface proteins [32,33]. Thus, it can be concluded that inhibitors against this adhesion can potentiate the antibiofilm activity [34]. The communication among the cells is dependent on a chemical signal molecule termed as quorum sensing (QS) [35]. The QS mechanism varies between bacteria, as evidenced by the production of signalling molecules in Gram-negative bacteria N-acyl-homoserine lactones (AHLs) as described in Figure 3, whereas in Gram-positive bacteria, the signalling molecule is peptides (autoinducer 2), which are described in Figure 4. Thus, similar to the adhesion inhibitors discussed above, some other enzyme shows the enzymatic degradation of QS Antibiotics 2023, 12, 299 5 of 26 signals caused by different enzymes such as lactonase, various acylase, and paraoxonase oxidoreductase, and the enzyme may provide a promising strategy for regulating biofilm development. As a result, the inhibition of QS and biofilm development has been reported in numerous studies [36][37][38]. It has been observed that many of the natural QSIs show a remarkable degradation of biofilm when combined with antibiotics [39], for example, usnic acid, which is a derived metabolite of lichen origin, and interfere with QS resulting in a changed morphology of S. aureus as well as of P. aeruginosa [36,40]. The biofilm has a specific relevance in terms of the QS system for a specific gene expression as well as communication among the cells; pyocyanin is a QS-mediated product helpful in the maturation of biofilm by interacting with eDNA released after the cell lysis and causing a viscous solution and thus aggregation of the cells and the production of an established mature biofilm [41,42]. In most of the microbes, the quorum signalling system, viz, rhl, iqs, pqs, and las. P. aeruginosa las and rhl are QS, is an expression for virulence genes. The las and rhl are further divisions in which LasR, acts as a transcriptional activator protein and is associated with AHL synthase LasI, which controls the production autoinducer, N-(3-oxododecanoyl)-l-homoserine lactone [43,44]. Likewise, the N-butyryl-l-homoserine lactone (AHL) of the Rhl system, which RhlI produces, is composed of the transcriptional activator RhlR [45].
the production of signalling molecules in Gram-negative bacteria N-acyl-homoserine lactones (AHLs) as described in Figure 3, whereas in Gram-positive bacteria, the signalling molecule is peptides (autoinducer 2), which are described in Figure 4. Thus, similar to the adhesion inhibitors discussed above, some other enzyme shows the enzymatic degradation of QS signals caused by different enzymes such as lactonase, various acylase, and paraoxonase oxidoreductase, and the enzyme may provide a promising strategy for regulating biofilm development. As a result, the inhibition of QS and biofilm development has been reported in numerous studies [36][37][38]. It has been observed that many of the natural QSIs show a remarkable degradation of biofilm when combined with antibiotics [39], for example, usnic acid, which is a derived metabolite of lichen origin, and interfere with QS resulting in a changed morphology of S. aureus as well as of P. aeruginosa [36,40]. The biofilm has a specific relevance in terms of the QS system for a specific gene expression as well as communication among the cells; pyocyanin is a QS-mediated product helpful in the maturation of biofilm by interacting with eDNA released after the cell lysis and causing a viscous solution and thus aggregation of the cells and the production of an established mature biofilm [41,42]. In most of the microbes, the quorum signalling system, viz, rhl, iqs, pqs, and las. P. aeruginosa las and rhl are QS, is an expression for virulence genes. The las and rhl are further divisions in which LasR, acts as a transcriptional activator protein and is associated with AHL synthase LasI, which controls the production autoinducer, N-(3-oxododecanoyl)-l-homoserine lactone [43,44]. Likewise, the N-butyryl-l-homoserine lactone (AHL) of the Rhl system, which RhlI produces, is composed of the transcriptional activator RhlR [45].  As a result of the ongoing drug resistance, it is essential to search for alternative antimicrobial medications. It has been observed that the las and rhl QS systems use either a direct or indirect method to regulate their expression of environmental changes. Quorum quenching is one of these, and it impairs microbial quorum sensing. It minimises the virulence factors' synthesis and biofilm formation. Only a few quorum quenching (QQ) methods use structurally similar QS receptor autoinducers (AI). They are natural or synthetic chemicals. Most of the QQ molecules that have been discovered are enzymes that can degrade signalling molecules, preventing the creation of biofilms [31,46]. Thus it can be concluded that quorum sensing inhibitors, whether from a natural or synthetic origin, when combined with antibiotics, can help erode biofilms [47][48][49]. As a result of the ongoing drug resistance, it is essential to search for alternative antimicrobial medications. It has been observed that the las and rhl QS systems use either a direct or indirect method to regulate their expression of environmental changes. Quorum quenching is one of these, and it impairs microbial quorum sensing. It minimises the virulence factors' synthesis and biofilm formation. Only a few quorum quenching (QQ) methods use structurally similar QS receptor autoinducers (AI). They are natural or synthetic chemicals. Most of the QQ molecules that have been discovered are enzymes that can degrade signalling molecules, preventing the creation of biofilms [31,46]. Thus it can be concluded that quorum sensing inhibitors, whether from a natural or synthetic origin, when combined with antibiotics, can help erode biofilms [47][48][49].

Natural Product as Antibiofilm Agent
Natural medicines have been used for centuries. Various plant-derived natural compounds exhibit in vitro antimicrobial and antibiofilm properties [50]. Numerous plantderived molecules or medicinal herbs, and natural compounds show remarkable prophylaxis effects for bacterial biofilm infections, and their mechanisms of action in antibiofilm activity have been identified [7,51]. The development of biofilm is a very complex process. The factors responsible for the antibiofilm activity of natural drugs prevent polymer matrix development, inhibit cell adhesion and attachment, prevent the formation of extracellular matrix (ECM), and limit the virulence factors' production, thus leading to the development of the QS system, and the biofilm is restrained. Targeting one or both of the initial two stages crucial for developing biofilms seems to be the best action to prevent their creation [52]. Antibiofilm activity of the natural product is divided into three subcategories based on their mode of action, as shown in Figure 5 and Table 1; the potential antibiofilm activity of a few natural antimicrobial drugs is shown.

Natural Product as Antibiofilm Agent
Natural medicines have been used for centuries. Various plant-derived natural compounds exhibit in vitro antimicrobial and antibiofilm properties [50]. Numerous plantderived molecules or medicinal herbs, and natural compounds show remarkable prophylaxis effects for bacterial biofilm infections, and their mechanisms of action in antibiofilm activity have been identified [7,51]. The development of biofilm is a very complex process. The factors responsible for the antibiofilm activity of natural drugs prevent polymer matrix development, inhibit cell adhesion and attachment, prevent the formation of extracellular matrix (ECM), and limit the virulence factors' production, thus leading to the development of the QS system, and the biofilm is restrained. Targeting one or both of the initial two stages crucial for developing biofilms seems to be the best action to prevent their creation [52]. Antibiofilm activity of the natural product is divided into three subcategories based on their mode of action, as shown in Figure 5 and Table 1; the potential antibiofilm activity of a few natural antimicrobial drugs is shown.

Phytochemicals
Plants produce substances that may not be essential for their primary metabolism but exhibit their potential to adapt to harsh abiotic and biotic environmental conditions. Extensive research was conducted on various plant extracts and their active ingredients to get rid of the Propionibacterium acne biofilm [53,54]. Studies showed that these extracts (Dolichos lablab, Polygonum cuspidatum, Malus pumila, Rhodiola crenulata, and Epimedium brevicornum) have significant antibiofilm activity [55]. The research also revealed that P. cuspidatum and E. brevicornum extracts with icartin and resveratrol as active components demonstrated considerable antibiofilm activity below their minimal inhibitory concentrations [56]. After being tested, Melia dubia bark extracts effectively regulate E. coli biofilm development [57][58][59] Extract from the caper bush (Capparis spinosa) effectively reduced the growth of biofilms and the production of EPS in S. marcescens, E. coli, P. aeruginosa, and P. mirabilis [60,61]. Southeast Asian native herb Lagerstroemia speciosa significantly reduced Antibiotics 2023, 12, 299 7 of 26 biofilm growth in P. aeruginosa PAO1 [62,63]. The aqueous extracts of Brazilian xeric show antibiofilm activity and serve as an in vitro screening of S. epidermidis [64]. According to the study, biofilm development was significantly decreased by extracts of Bauhinia acuruana, Chamaecrista desvauxii, and Pityrocarpa moniliformis. Moreover, Senna macranthera and Commiphora leptophloes fruit extracts were said to diminish biofilms by 67.3% and 66.7%, respectively [65]. Using qualitative and quantitative methods, a study on the growth of M. smegmatis biofilm revealed the role of various herbs, spices, and plants, including Vaccinium oxycoccus, Azadirachta indica, Juglans regia, and Hippophae rhamnoides. The extract of A. indica was shown to be the most effective on M. smegmatis biofilms [66,67].

Phytochemicals
Plants produce substances that may not be essential for their primary metabolism but exhibit their potential to adapt to harsh abiotic and biotic environmental conditions. Extensive research was conducted on various plant extracts and their active ingredients to get rid of the Propionibacterium acne biofilm [53,54]. Studies showed that these extracts (Dolichos lablab, Polygonum cuspidatum, Malus pumila,Rhodiola crenulata, and Epimedium brevicornum) have significant antibiofilm activity [55]. The research also revealed that P. cuspidatum and E. brevicornum extracts with icartin and resveratrol as active components demonstrated considerable antibiofilm activity below their minimal inhibitory concentrations [56]. After being tested, Melia dubia bark extracts effectively regulate E. coli biofilm development [57][58][59] Extract from the caper bush (Capparis spinosa) effectively reduced the growth of biofilms and the production of EPS in S. marcescens, E. coli, P. aeruginosa, and P. mirabilis [60,61]. Southeast Asian native herb Lagerstroemia speciosa significantly reduced biofilm growth in P. aeruginosa PAO1 [62,63]. The aqueous extracts of Brazilian xeric show antibiofilm activity and serve as an in vitro screening of S. epidermidis [64]. According to the study, biofilm development was significantly decreased by extracts of Bauhinia acuruana, Chamaecrista desvauxii, and Pityrocarpa moniliformis. Moreover, Senna macranthera and Commiphora leptophloes fruit extracts were said to diminish biofilms by 67.3% and 66.7%, respectively [65]. Using qualitative and quantitative methods, a study on the growth of M. smegmatis biofilm revealed the role of various herbs, spices, and plants, including Vaccinium oxycoccus, Azadirachta indica, Juglans regia, and Hippophae rhamnoides. The extract of A. indica was shown to be the most effective on M. smegmatis biofilms [66,67].
Natural substances with strong antibiofilm characteristics fall roughly into eight types, flavonoids, terpenoids, lectins, alkaloids, polypeptides, polyacetylenes and phenolics, and essential oils [68]. There are seven subclasses of phenols, and condensed tannins, which mainly show antibiofilm activity [64]. These compounds act on biofilms using different mechanisms, including substrate restriction, cell wall rupture, acting on adhesion groups and cellular membranes, protein binding, and DNA association, leading to blocking viral fusion [69,70]. According to a published research article, antibiofilm capabilities of Indian-originated herbs, Bischofia javanica, Syzygium Roxb, Rathakr. Mazz, and Cinnamomum glaucescens (Nees) showed positive results. Extract of Holigarna caustica (Dennst.), extract of gum Arabic tree Acacia nilotica (L.), Anacardiaceae family, extract of jack of orange jasmine Murraya paniculata (L.), and fruit extract of Buddha Coconut Pterygota alata, act on S. aureus against their biofilm development [71]. Salvia officinalis L., a medicinal herb from Algeria that contains 12-Methoxy-trans-carnosic acid and abietane Natural substances with strong antibiofilm characteristics fall roughly into eight types, flavonoids, terpenoids, lectins, alkaloids, polypeptides, polyacetylenes and phenolics, and essential oils [68]. There are seven subclasses of phenols, and condensed tannins, which mainly show antibiofilm activity [64]. These compounds act on biofilms using different mechanisms, including substrate restriction, cell wall rupture, acting on adhesion groups and cellular membranes, protein binding, and DNA association, leading to blocking viral fusion [69,70]. According to a published research article, antibiofilm capabilities of Indianoriginated herbs, Bischofia javanica, Syzygium Roxb, Rathakr. Mazz, and Cinnamomum glaucescens (Nees) showed positive results. Extract of Holigarna caustica (Dennst.), extract of gum Arabic tree Acacia nilotica (L.), Anacardiaceae family, extract of jack of orange jasmine Murraya paniculata (L.), and fruit extract of Buddha Coconut Pterygota alata, act on S. aureus against their biofilm development [71]. Salvia officinalis L., a medicinal herb from Algeria that contains 12-Methoxy-trans-carnosic acid and abietane diterpenoid, also known as carnosol, demonstrated antibiofilm efficacy against Candida [72]. Phytochemicals generally act on the inducers molecule such as AHL and may act on some other autoinducer and type 2 receptors, to stop the functioning of the QS signalling system [73]. Garlic extracts exhibited a significant inhibitory role in the QS signalling compound of P. aeruginosa biofilm and other species' biofilms such as Vibrio spp. [6,74]. Emodin is a potent inhibitor and facilitates the protein degradation of transcription factors involved in quorum sensing [75].
Numerous researchers have shown that quorum quenchers and antibiotics combination might be considered as the best non-traditional antibiofilm agents [46]. Furthermore, phytochemicals are essential for suppressing the genes that lead to biofilms forming and preventing bacterial adhesions [76]. Biofilms in their early phases can be disturbed by the fluid dynamic properties of the planktonic cell, an electrostatic force of attraction, and properties of sedimentation that encourage attachment to diverse surfaces [34]. Phytocompounds have the ability to stop the availability of essential nutrients required for bacterial attachment and growth. The Psidium guajava L. extracts in ethanol and acetone, as well as extracts from several Eugenia species, have been shown to have antiadhesive effects on C.
albicans [77]. Syzygium aromaticum (S. aromaticum) extract shows antibacterial activity in dental caries against various strains of streptococci [78] Norbgugaine significantly impacted P. aeruginosa biofilm by inhibiting cell motility caused by an inability to adhere to the surface [79]. Adiantum philippense L. crude extract has the potential to reduce the number of exopolysaccharides in biofilms [76]. They discovered that crude extract of Adiantum philippense L. blocks the generation of EPS, targets adhesin proteins, and deforms already-formed biofilms to prevent biofilm growth in the initial stages. In terms of different phytocompounds, numerous studies have discovered that polyphenols, particularly those such as 7-epiclusianone (tetraprenyl benzophenone derivative), casbane parent hydride of casbene, and tannic acid, a type of polyphenol, inhibit surface attachment [76,80,81]. Enterobacteriaceae's curli, an amyloid fibre expressed on the EPS surface, encourages the formation of biofilms, cell aggregation, and target attachment [82,83] They revealed that the flagellar operon flhDC was suppressed by the citrus sterol-sitosterol glucoside, which prevented the production of biofilms and cell motility in E. coli O157:H7. Curlicide derivative (Aβ-peptide) and pilicide phytocompounds are used as new therapeutic approaches to prevent Enterobacteriaceae biofilms [84]. Pyridones interfere with curli, leading to expression modification and biogenesis of the CsgA. (Curli, pilli) genes are regulated by phytochemicals from Malaysian plants, including phloretin, ginkgolic acid, and phytocompounds [84][85][86].
Garlic extract has been shown to inhibit QS in the research of Bjarnsholt. It was found that the administration of garlic extract for a lung infection (mouse model) reduces the drug resistance of tobramycin and the neutrophils (PMN) phagocytosis of P. aeruginosa [87]. Garlic extract diminishes QS signals, and virulent factors of P. aeruginosa are reported in the UTI model [88]. Six clinical bacterial isolates showed resistance to the biofilm-forming activities of garlic extracts [89,90]. Plant extracts from Coptis chinensis and C. trilobus may prevent germs from adhering to fibronectin-coated surfaces. They show the effects at the adhesion stage by inhibiting sortase (an enzyme of biofilm development) and enhanced the covalent attachment of surface proteins in Gram-positive bacteria [91,92]. The polyphenols found in cranberries prevent the growth of biofilms and the colonisation of pathogens [93,94]. Cranberry components exhibited promising effects for the deterrence and cure of oral infections, such as dental caries and periodontitis, by impacting binding proteins (glucan); the enzymes acted by splitting down the ECM, energy production, hydrophobicity of the biofilm, and degradation activities [95]. According to studies, Ginkgo biloba extract significantly reduced the development of fimbriae and biofilm in Escherichia coli O157:H7; this agreed with the inhibited curli and prophage genes [96,97]. E. coli's ability to swim was found to be impaired by cinnamaldehyde, which also allegedly affects biofilm formation and structure [98]. Citrus limonoids are distinct secondary triterpenoid metabolites.

Essential Oils
Essential oils (EOs) are aromatic compounds that plants produce; they are nonbiocidal and show promise as a treatment for bacterial biofilm illness and increased drug resistance in vitro [120,121]. In the case of S. epidermidis strains, EOs primarily function as biofilm inhibitors. Results have demonstrated that they can destabilise biofilms at deficient concentrations without compromising bacterial viability due to their effectiveness against P. aeruginosa. The effectiveness of cumin oil in contradiction of the biofilm production by pneumonia strains was investigated [122]. This study showed that this EO decreased biofilm growth and improved the antibiotic ciprofloxacin's effectiveness. The research reveals that cinnamon oil is effective against the biofilm of S. mutans and Lactobacillus plantarum [123,124]. Cinnamon oil effectiveness was reported in S. epidermidis biofilm [125]. The Enteropathogenic E. coli attachment stage in biofilm is suppressed by Cinnamomum oil [126]. The essential oil derived from oregano showed disruptive biofilm properties of staphylococci and E. coli species [127]. Oregano EO exerts antibacterial potency on different species of Staphylococcus and E. coli planktonics, and prevents biofilm formation [127,128]. Recently, studies have been conducted on the antibiofilm properties of vegetable oil (Brazil nut oil) on dentifrice biofilm [129]. It was claimed that using commercially available dentifrice with vegetable oil could help prevent and treat dental caries and periodontal disease because of its antibacterial properties. With regards to tea tree oil, the tea tree EO (TTO) and ciprofloxacin (CIP) combination and their antibacterial activity were evaluated on P. aeruginosa. The result revealed that the synergistic effect of the combination reduces the biofilm biomass by 70%. According to reports, EOs (thymol, cinnamon oils and oregano) were found to be effective against the biofilm-forming strains of Acinetobacter, Sphingomonas, and Stenotrophomonas at sublethal concentrations [130,131].

Andrographis paniculata (Andrographolide)
Andrographis paniculata (A. paniculata), commonly found in China, belong to the Acanthaceae family [132]. It has proven successful in treating bacterial infections and has a sizable impact on P. aeruginosa, S. aureus, and E. coli biofilm generation [133,134] and decreases the synthesis of extracellular pathogenic factors in P. aeruginosa regulated by the QS system, such as anthocyanins and elastase. Andrographolide (AG) is the active metabolite of A. paniculata; AG inhibits biofilm formation by inhibiting the QS system by regulating SarA factor, which is responsible for biofilm development in S. aureus [135]. E. coli adhesion can be reduced by AG as the amount of PIA/PNAG declines because this also affects the ability to produce biofilms [136].

Polygonum cuspidatum (Emodin)
Polygonum cuspidatum's constituents are anthraquinone, emodin, etc. [137]. One goal of caries prevention is to control dental plaque. Since biofilm formation in the dental cavity starts with the salivary membrane and planktonic bacteria adhering to the surface of teeth, reduced planktonic bacterial loads may help regulate biofilm development [138].

Curcuma longa (Curcumin)
Curcuma longa of the Zingiberaceae family produces sesquiterpenes, turpentine, and fatty acids that disrupt antibiofilm activity, impairing the bacterial cell membrane's normal barrier function, and is crucial for the growth and metabolism of bacteria [139]. It modulates various QS-dependent pathogenic factors, such as Vibrio spp. swarming, alginate synthesis, and mobility [140,141]. It works by blocking the growth of EPS and decreasing the flagellum effect, which restricts bacteria from swimming and the development of biofilms [140,142]. Reduced synthesis of QS signals and decreased pathogenicity. [61] Carvacrol (terpenoid) Origanum vulgare L.

Pseudomonas aeruginosa
Inhibition of lasI, which affects AHL production.
It acts by post-translational inhibition enzyme and interrupts the QS signaling mechanism [143] Polyphenols (cranberry) Vaccinium oxycoccos

Pseudomonas aeruginosa
Inhibits EPS production, regulating the pathogenic factors.
Inhibits EPS production, reduces bacterial adherence at the primary stages of biofilm generation, and acts on the QS system by altering the expression of virulence factors. [147] Hordenine Hordeum vulgare L.
Limits the generation of AHL resulting in bacterial biofilm generation [101] Vitexin (flavonoid) Vitex species P. aeruginosa Inhibit EPS, by acting on LasA, Las B, and Lux R Reduces the production of proteolysis enzyme, and surface protein as well as EPS and components linked with QS. Attenuates LasA, Las B, and Lux R  [156] Guava alcoholic extract Psidium guajava L C. albicans Inhibits cell motility enzyme.
Inhibits primary-stage adhesion due to a lack of motility, impacted on biofilm development Ajoene exhibits QS inhibitory activity in Allium sativum, where organic sulphide is the main antibacterial component and decreases the rhlA gene in P. aeruginosa, lowering the concentration of rhamnolipids encrypted by rhlA. Garlic extract inhibits the biofilm formation in L. monocytogenes and P. aeruginosa [116], and garlic checks the virulence factors and QS signal generation in P. aeruginosa [88].

Biosurfactants
Biosurfactants (BS) impede the capacity of cells to attach to surfaces by affecting the hydrophilic nature of the cell membrane, perforating membranes, and limiting the electron transport chain, thus decreasing the energy need of cells [157]. Medical implants such as urinal catheters and bone implants can be coated with biosurfactants to prevent biofilms from harmful organisms without synthetic medications [158]. Staphylococcus aureus was tested for how biosurfactants from Lactobacillus Plantarum and Pediococcus acidilactici affected the expression of biofilm-related genes and quorum sensing signalling molecules [146,159]. As claimed from the study, biosurfactants control gene expression related to biofilms, such as dltB, icaA, cidA, etc., which control the development of S. aureus biofilm [158]. At 50 mg/mL, Pediococcus acidilactici biosurfactant inhibits gene production such as (AI-2) signalling factor and staphylococcal accessory regulatory-sar A [158]. According to earlier research, BS-loaded liposomes made from Lactobacillus were more efficient than free BS, preventing the development and removal of S. aureus (MRSA) biofilms [160,161]. According to earlier research, BS-loaded liposomes produced from Lactobacillus were more efficacious than plain BS at preventing the development and removal of S. aureus (MRSA) biofilms. Table 2 summarizes some biosurfactants with their effect on biofilm development. The lipopeptide component of the anionic bacteria Acinetobacter junii is found to selfassemble and create vesicles filled with biosurfactant sheets. A lipidic biosurfactant of the fungus Beauveria bassiana is essential for M. canis to function as a biofilm inhibitory agent in ex vivo settings [162]. It impacted membrane permeability and started to break down the integrity of the cell membrane. Cyclical metal complexes were discovered to be highly efficient against infections brought on by C. albicans biofilms [163]. Surfactin does not have the same antiadhesive and antibiofilm properties as Pseudomonas aeruginosa MN1 rhamnolipids [164]. The glycolipid produced by Burkholderia sp. WYAT7 showed ant biofilm activity against Staphylococcus aureus [165] as an endophyte of Artemisia nilagirica (Clarke) Pamp [166]. Burkholderia cepacia is resistant to biofilm formation Burkholderia pnomenusa MS5 [167]. According to reports, rhamnolipids and sophorolipids may be effective inhibitors of the biofilms developed by all classes of microorganisms [168]. Several studies have demonstrated that when exposed to cell-associated biosurfactants from Lactobacillus acidophilus, S. aureus, and Proteus vulgaris cannot develop biofilms on (PDMS)-based implants [169]. Biosurfactants from L. rhamnosus promote cell lysis by upsetting the structure of the membrane, making them an effective antibiofilm agent for diverse microbial biofilms on silicone implants, such as voice prostheses in laryngectomy situations [170]. Caprylic acid, which prevents the growth of biofilms from P. aeruginosa, E. coli, and B. subtilis, can significantly increase the antibiofilm efficacy of biosurfactants [171]. Fluconazole and (AmB) work together to prevent the growth of a biofilm and the existing biofilm of Candida albicans [172]. Surfactants such as sodium dodecyl sulfate caused P. aeruginosa PAO1 biofilm's inhibition. [173].

Antimicrobial Peptides
Broad-acting antimicrobial medications (AMPs) are commonly used against bacterial and fungal biofilms. Infections caused by S. aureus, K. pneumoniae, P. aeruginosa, Acinetobacter, and Enterobacter spp. (ESKAPE) as well as non-ESKAPE microorganisms are interrupted by these peptides acting on medical specimens such as valves, stents, and dentures [174,175]. Antimicrobial AMPs can block various biofilm formation molecular pathways [176]. The several AMPs in amphibian skin are efficient against different microbes that cause biofilms. AMP Japonicin-2LF, which inhibits MRSA biofilms by permeabilizing membranes, is a derived secretion from the skin of Limnonectes fujianensis, a frog from Fujian. Both sessile and planktonic pathogens are eliminated from biofilms by the detergent-like behaviour of Japonicin-2LF. Esculentin-1a, also known as Esc, is an AMP obtained from frog skin; its Esc contains D-amino acids . By disrupting membranes, -1c inhibited the biofilms of P. aeruginosa [172]. Three processes work together to prevent P. aeruginosa from forming biofilms. The fleN gene, which determines the flagella number in P. aeruginosa, is first downregulated, which prevents flagella-mediated swimming. Second, it inhibits P. aeruginosa twitching motility, which is crucial for microcolony formation and colonization during biofilm growth. It reduces type IV pili synthesis genes' mRNA levels at a low concentration. Third, it prohibits the lasB gene, which codes for the virulence factor elastase LasB, and the lasI gene, which codes for the QS protein (AHL) synthase. It also carries out its function by electrostatically binding to the negatively charged bacterial cytoplasmic membrane, causing the creation of pores as a result of the solubilization of the membrane, which results in its destruction [177]. Due to their unique qualities of limitless sequence space and ability to produce antimicrobial action, these AMPs become the perfect candidate for overcoming microbial resistance. Additionally, some low molecular weight AMPs that penetrate the cellular structure act by becoming bound to the microbial genetic material, triggering the ROS-dependent pathway for their antimicrobial effect. The AMPs are mainly classified as defensins and bacteriocins. The defensins are the class of AMPs and peptides obtained from natural sources. This class of AMPs is also known as HDPs (host defence peptides), which act as broad-spectrum antibiotics and perform their action by electrostatic interaction at the microbial membrane, causing membrane destruction and causing a bactericidal effect [178,179]. On the other hand, bacteriocins are peptides obtained from bacteria, which are synthesized in the ribosomes. The first class of bacteriocins were discovered in 1925, after which a number of different forms of bacteriocins were discovered [180]. Although functionally they also perform antimicrobial and antibiofilm activities very similar to that of defensin, they are different from defensin in that this cationic amphiphilic molecule, in addition to becoming attached to the bacterial cell, causes the destruction of the phospholipid membrane, and hence obtains access to the DNA component and organelles of the cell, causing direct interference with the protein synthesis process. Thus, both forms of AMPs have proven to be valuable weapons in dealing with resistant microbes, which usually avoid killing via biofilm formation.

Other Ways to Inhibit Biofilm Resistance
The increasing case of 'the non-effectiveness' of antibiotics while dealing with common microbial infections has opened the door for in-depth study and research on the cause of microbial resistance and dealing with the strategies to combat such resistance. The search for new alternatives to the current conventional therapies should be able to act as broad-spectrum antimicrobial agents while keeping minimal side effects [181]. Some novel methods for combating resistance are the application of chitosan and its derivatives, antimicrobial peptide use of metallic nanoparticles and (QACs) [182], stimuli-responsive materials, and the application of Phage therapy. The details of these new methods are discussed below [183] and summarized in Figure 6.

Other Ways to Inhibit Biofilm Resistance
The increasing case of 'the non-effectiveness' of antibiotics while dealing with common microbial infections has opened the door for in-depth study and research on the cause of microbial resistance and dealing with the strategies to combat such resistance. The search for new alternatives to the current conventional therapies should be able to act as broad-spectrum antimicrobial agents while keeping minimal side effects [181]. Some novel methods for combating resistance are the application of chitosan and its derivatives, antimicrobial peptide use of metallic nanoparticles and (QACs) [182], stimuli-responsive materials, and the application of Phage therapy. The details of these new methods are discussed below [183] and summarized in Figure 6.

Use of Chitosan and Its Derivatives
Chitosan is obtained from its natural precursor, chitin, which is commonly found in organisms such as algae, crustaceans, insects, and fungus, etc. When Chitosan undergoes the process of deacetylation either enzymatically or under the effect of an alkaline solution, chitosan formation takes place. Since, chitosan is obtained naturally, it is known for its biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and ease of availability. Owing to its various beneficial properties, several studies from the literature have reported its use as an antioxidant, antimicrobial, and anticancer agent [184]. Chitosan, being a positively charged agent, has been used in various drug delivery systems either as the main component or applied as a coating material to impart a positive charge to the particles. The positive charge on the chitosan has been utilized for electrostatic interaction between surfaces that are either negatively charged and/or have been altered to induce a negative charge on the surface. The prime reason behind using chitosan as an antibiofilm agent is the cellular component of biofilms such as (lipids, exopolysaccharides,

Use of Chitosan and Its Derivatives
Chitosan is obtained from its natural precursor, chitin, which is commonly found in organisms such as algae, crustaceans, insects, and fungus, etc. When Chitosan undergoes the process of deacetylation either enzymatically or under the effect of an alkaline solution, chitosan formation takes place. Since, chitosan is obtained naturally, it is known for its biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and ease of availability. Owing to its various beneficial properties, several studies from the literature have reported its use as an antioxidant, antimicrobial, and anticancer agent [184]. Chitosan, being a positively charged agent, has been used in various drug delivery systems either as the main component or applied as a coating material to impart a positive charge to the particles. The positive charge on the chitosan has been utilized for electrostatic interaction between surfaces that are either negatively charged and/or have been altered to induce a negative charge on the surface. The prime reason behind using chitosan as an antibiofilm agent is the cellular component of biofilms such as (lipids, exopolysaccharides, and e-DNA), and external components that constitute bacterial membranes such as (lipopolysaccharides or teichoic acids) are all anionic in nature. Thus, positively charged polymeric agents such as chitosan are electrostatically bound to the component at the membrane and disrupt the cellular structure of microbes, leading to the destabilization and leakage of the bacterial membrane. This causes leakage of the internal cellular components, such as proteins and electrolytes, through pore formation, thus completing the bacteriostatic and bactericidal effect [185]. Usually, the science of nanotechnology has been applied in the formulation of chitosan nanoparticles, which have been used for antimicrobial and antibiofilm therapy. With the chitosan nanoparticles being small in size, they can penetrate deep inside the biofilm membrane, thus causing the lysis, disruption, and disaggregation of the biofilm [186,187]. A different group of researchers worked with Chitosan either as a single agent or in combination with other herbal or synthetic molecules with promising antimicrobial effects.
In one such study, Hongbin et al. developed a system of minocycline-loaded chitosan and alginate, which reportedly destroyed adherent and planktonic bacterial colonies [188]. Subhaswaraj et al. formulated cinnamaldehyde-loaded chitosan NPs, which showed effective inhibition of P. aeruginosa-led biofilm formation effecting QS function and altering mortality [189]. In another study, Jinhong et al. designed a system in which chitosan and heparin were deposited in alternate manners on poly (ethylene terephthalate) film to carry out an antibacterial effect. A critical outcome of this study was that the system was found to be antiadhesive; hence could be used in implants [190]. Another form of chitosan, quaternized chitosan (QCS), known as HACC (hydroxypropyltrimethyl ammonium chloride chitosan), is antibacterial in nature. It has been widely used in coating implants, tissue engineering, and wound healing. Peng et al. conducted their research and suggested that 18% of substituted HACC showed firm antibacterial activity, keeping toxicity to the minimum [49].

Phage Therapy
Phage is the virus that specifically invades and multiplies in bacterial cells. This inherent quality of the virus has been exploited to solve the issue of drug resistance in bacteria due to the biofilm formation. These phages have been explored as they only attack bacterial cells leaving behind healthy host cells and micro flora untouched [191,192]. Since phage therapy involves the steps of virus attachment to the host and transfer of the genetic material into the bacteria, this property was utilized to destroy the extracellular biofilm matrix to cause the bacterial elimination [191]. As the research progressed further, the cocktail of bacteriophage and antibiotics was explored with many studies showing a positive synergistic effect on disrupting biofilms [193]. The group, Gu et al., worked on the step-by-step technique for the combined delivery of phages to eradicate Klebsiella pneumonia by administering three phages (GH-K1, GH-K2, and GH-K3). The findings showed that when combined phages were delivered, they produced a stronger response against Klebsiella pneumonia than individual phages in the murine K7 strain model. They used data to prove their hypothesis that the lowest effective dose of the combined phage level was significantly lower than that of a single phage. Although this idea looks very effective for combating biofilm resistance, much effort still needs to be applied to formulate bacteriophage into some dosage forms. Needs for standardization, optimization, and guarantees of safety and efficacy are some of the questions that need to be addressed before such therapies can be used as a generalized treatment method for the larger population.

Conclusions and Perspective
Bacterial biofilms are widespread and play a crucial role in antibiotic resistance. Therapy of biofilm-associated infections is nowadays a problematic task for healthcare profes- sionals. There is the urge to develop novel antimicrobial techniques to overcome problems associated with bacterial resistance. As revealed in this review, a vast library of natural resources is available to examine antibiofilm compounds. A number of researches have shown the inhibitory effects of natural drugs on the formation and growth of bacterial biofilms, revealing their potential as substitute treatments for bacterial illnesses. Their possible regulation mechanism was primarily caused by the inhibition of the QS system at different phases of biofilm development. Many plant extracts have also shown potential antibiofilm activity; there molecular structures are not identified, indicating more clinical trials need to be performed. This review also focuses on the quorum quenching (QQ) molecules and natural extracellular polymeric substance inhibitory enzymes and their mechanism of action on biofilm. Most of the natural drugs' mechanisms are still unknown. More research in this area can overcome drug resistance. Clinical investigations that are now underway mostly concentrate on the external use of oral biofilms formed in dental plaque, periodontitis, and gingivitis. A promising therapy approach for biofilm infections is the combination of antibiotic drugs and herbal antibiofilm drugs for effective remedies. It is vital to conduct in vivo research to assess the dependability of the combined medications because most investigations on these drugs are now conducted in vitro. Based on fingerprint efficacy, QC modelling, and PK-PD (pharmacokinetic-pharmacodynamic) correlation in in vivo investigations, the formation of combined pharmaceutical therapies will be aided by the ability to show the mechanisms of the combined medications.