The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative Antibiofilm Agents

Abstract

Biofilms are a community of microorganisms with accretions of their extracellular matrix that attach both to biological or non-biological surfaces, conferring a significant and incompletely understood mode of growth for bacteria. Biofilm formation represents a protected mode of growth of bacteria that allows cells to survive in hostile environments, facilitating the colonization of new areas. This biofilm formation appears to be produced by microorganisms to resist drug action, causing them to become resistant. Therefore, the search for alternative agents is necessary to counteract and reduce this production, creating suitable drugs against these biofilms. Natural products from medicinal plants possess an array of secondary metabolites and bioactive compounds that could have bioactive potentials that inhibit and eradicate biofilms.

1. Introduction

Biofilms are complex communities of microbes found attached to a surface or may form aggregates without adhering to a surface. Biofilms also display unique properties, such as multidrug tolerance and resistance to both opsonization and phagocytosis, enabling them to survive in hostile environmental conditions by resisting selective pressures [1]. Sometimes, the host immune system is immunocompromised, making it ineffective in clearing biofilms with evidence that immune cells are paralyzed with disrupted phagocytosis capacities or decreased burst responses, lowering the production of reactive oxygen species [2, 3]. Moreso, these communities of microorganisms are unique since they involve several species in a cooperative. The biofilm thus constitutes a microbial society, with its own set of social rules and patterns of behavior, including altruism and cooperation, both of which favor the success of the group with task-sharing behavior. All of these characteristic patterns are orchestrated by chemical or genetic communication. The biofilm thus constitutes a unique way to stabilize interactions between species, inducing marked changes in the symbiotic relationships [3, 4]. Moreover, biofilms protect invading bacteria against the host’s immune system via impaired activation of phagocytes and the complement system [5]. The use of antibiotics such as imipenem and colistin mostly reduces biofilms but does not eliminate the entire biofilm in most cases [6]. Due to their toxicity and side effects, it is not possible to reach the minimal concentration of antibiotics in vivo. This chapter describes the mechanisms of bacterial biofilm inhibition and eradication with the search for alternative antibiofilm agents.

2. Stages of biofilm formation

Bacteria form complex multicellular structures called biofilms. Biofilm formation is commonly considered to occur in four main stages [7]: (1) adhesion of planktonic cells, (2) microcolony formation, (3) biofilm maturation and (4) detachment (also termed dispersal) of bacteria, which may then colonize new areas (Figure 1). Sessile bacterial cells exist in the stationary or dormant growth phase, exhibiting phenotypes distinct from planktonic bacteria [8]. In biofilms, bacteria display exceptional resistance to environmental stresses, especially antibiotics. This makes biofilms a major public health problem, as they account for 60–80% of human microbial infections [9]. The different stages in biofilm formation involve different environments, as shown in Figure 1.

Figure 1.

Stages of biofilm development.

2.1 Attachment of planktonic cells

Biofilm formation starts with the attachment of microbial cells to abiotic or biotic surfaces. These biotic surfaces are living tissues such as endothelial lesions, mucosae, and nervous tissues, while abiotic surfaces are non-living cells including indwelling devices, prostheses, clinical environment surfaces, vascular and urinary catheters [10]. This initial attachment depends on the motility and adhesins expression (microbial factors). The extension is influenced by the planktonic strains migrating to specific sites to either adhere to existing lesion or surface or directly cause tissue infection [11]. The physiology of the cell’s changes affecting the surface membrane proteins making the removal of the attached cells laborious, necessitating the action of specific enzymes, sanitisers and detergent. The physicochemical properties of the surfaces (biotic and abiotic) controls microbial adherence making biofilms independent of surface extension [12].

2.2 The extracellular polymeric substance (EPS) matrix

The genes responsible for attachment and matrix assembly are activated when stimulated by factors such as population density and nutrient limitation [7]. The EPS matrix is composed of a mixture of biopolymers. The matrix produced is different and is surface- or medium-specific and differs between in vivo and in vitro conditions [11]. EPS is produced by planktonic cells, resulting in enhanced extension [13, 14].

2.3 Accumulation of multi-layered clusters of microbial cells

The microbial assembly development process results in simultaneous bacterial aggregation and growth. This disposition is entrenched as a distinct model with the aid of a confocal laser microscopy. The distinct model indicates that active metabolism is exhibited by the cells in the outer biofilm layers while those deeper inside the biofilm downregulate their metabolism, making them inactive in a persistent state [12, 15, 16]. This accumulation mostly involves intercellular adhesion. Specific genes and polysaccharide intercellular adhesin (PIA) are responsible for their accumulation on a polymer surface. However, the purification and structural analysis of these clustered microbial cells indicate the presence of two forms of that PIA, major polysaccharide I (>80%) and a minor polysaccharide II [17].

2.4 Biofilm maturation

In the biofilm maturation phase, the canals are created in the biofilm structure, allowing gradient-based passage of nutrients and signaling molecules based on their metabolic state, favoring the organized agglomeration and differentiation of cells [7, 12, 18]. These gradient passages are necessary for nutrients to enter the cells inside the biofilm layers. Biofilm structuring is a disruptive process causing the detachment of cell clusters controlling the biofilm invasion during in vivo biofilm infection leading to systemic dissemination [19].

2.5 The disentanglement and scattering of planktonic bacteria

The biofilms grow more thicker and compact on the interior, while external layers begin separating. The disentanglement and scattering occurs as a results of nutritional imbalance with insufficient carbon accessibility, increasing the synthesis of extracellular polymeric substances [20]. The scattered cells or clusters travel as septic emboli colonizing new sites, causing infection with possibly novel biofilms [2]. The dispersed cells form biofilms as a result of growth and may return quickly to their normal planktonic phenotype.

3. Bacterial biofilm structure, characteristics and chemical composition

3.1 Bacterial biofilm structure and characteristics

The basic structural units of a biofilm are microcolonies and separate communities of bacterial cells embedded into the EPS matrix. These microcolonies are in most cases mushroom-shaped or rod-like and can consist of one or more types of bacteria. The microcolonies consist of 10–25% cells and 79–90% EPS matrix depending on the bacterial type. This EPS matrix protects biofilm cells from various environmental conditions, such as UV radiation, changes in pH values, draining and temperature. There are channels through which water flows between microcolonies. These water channels function in distributing nutrients to microcolonies and receiving harmful metabolites as a simple circulatory system. Biofilms under different hydrodynamic conditions, such as laminar and turbulent flow, show changes in biofilm structure depending on the flow type. In laminar flow, bacterial microcolonies become round, and in turbulent flow, they extend in the downstream direction [21].

3.2 Chemical composition

The matrix of extracellular polymeric substances (EPSs) are self-secreted substances that keep bacterial cells in a compact structure attaching them to surfaces which makes the physical aspect of a biofilm [16]. The major constituent of the biomass of the biofilm is the hydrated EPS ranging between 2–15% of the total biofilm mass [4]. The EPS contains mostly extracellular DNA (eDNA), polysaccharides, proteins and lipids (Table 1) [22]. The EPS matrix exhibit three important characteristic features which are enhancing antimicrobial resistance, nutrient capture and social cooperation [14]. The tissues of higher organisms are similar to biofilms structures which are architecturally different and extremely heterogeneous in gene expression, all participating to the resistance mechanisms of biofilms [5, 23].

1. Polysaccharides is one of the major constituents of the EPS matrix adhering to cell surfaces forming a compact network. The majority of these molecules are heteropolysaccharides constituted of a mixture of neutral, charged sugar residues, organic and inorganic substituents contributing to their charged (polyanionic or polycationic) nature [24, 25]. The exopolysaccharide composition differ between microorganisms of the same species [26, 27]. These exopolysaccharides are indispensable to biofilm formation and constitute the protective barrier of the EPS matrix despite the heterogeneity among biofilms [21]. Additionally, they are also responsible for water retention within the biofilm. The high amount of water in the biofilm provides a highly hydrated environment that protects cells from fluctuations in water potential. The presence of water confers the biofilm to a nonrigid structure with different viscosities that allow movement of the cells within the matrix [28].

2. Extracellular proteins: structural proteins and enzymes. These are also critical components of the matrix and are present in higher amounts than polysaccharides. The structural proteins are mainly involved in the stabilization of the biofilm architecture by connecting cells to the EPS [29]. The enzymes are essentially involved in the degradation of other matrix components, such as polysaccharides (dispersin B), matrix proteins (proteases), and eDNA (DNases). Thus, the enzymatic activity within the biofilm provides nutrients to bacterial cells and promotes biofilm reorganization and dispersal [29]. In addition to polysaccharides and proteins, eDNA also contributes to the structural integrity of the matrix. The contribution of this component to the three-dimensional structure of the biofilm differs greatly among species [29]. The EPS matrix has an important role in biofilm formation, progression and durability as a result to its multiplex constitution and organization. It is also a protective barrier against external factors, a source of nutrients, enzymes and an intercellular connector. These unique features of the matrix participate in the high antimicrobial forbearance and/or recalcitrance of biofilms [15, 29].

Components	Percentage of matrix	Functions in biofilm	References
Microbial cells	2–5%	Cohesion of the structure	[5]
DNA and RNA	<1–2%	Cohesion of the structure; Nutrient source; Exchange of genetic information	[22]
Polysaccharides	1–2%	Cohesion of the structure; Nutrient source; Water retention; Protective barrier; Sorption of organic compounds and inorganic ions	[22]
Structural Proteins	<1–2%	Cohesion of the structure; Nutrient source; Protective barrier; Sorption of organic compounds and inorganic ions; Electron donor and acceptor	[5]
Enzymes	<1–2%	Enzymatic activity,; Nutrient Source	[22]
Lipids and biosurfactants	<1–2%	Nutrient source	[22]
Water	Up to 97%	Lubricates the environment, simple circulatory system distributing; nutrients to microcolonies	[22]

Table 1.

Chemical composition of biofilms.

4. Factors influencing bacterial biofilm formation and development

The formation of biofilms is a dynamic and complex process that includes the initial attachment of bacterial cells to the substratum, physiological changes within the microbe, multiplication of adhered cells to form microcolonies and finally biofilm maturation [30]. Biofilm-associated bacteria demonstrate distinct features from their free-living planktonic counterparts, such as different physiologies and high resistance to immune systems and antibiotics that render biofilms a source of chronic and persistent infections [2, 31]. It is known that the change in phenotype from planktonic to the sessile form occurs in response to changes in environmental conditions [3].

Environmental factors, such as nutrient level, temperature, pH, and ionic strength, can influence biofilm formation, as shown in Figure 2 [30]. These factors influence bacterial adhesion; cell surface properties, such as hydrophobicity, flagellation, and motility; surface properties, such as hydrophobicity and roughness; and environmental factors, such as temperature, pH, availability of nutrients and hydrodynamic conditions [21, 30, 32]. The cell surface properties, specifically the presence of extracellular appendages, such as fimbriae and flagella, the interactions involved in cell-to-cell communication and EPS production, such as surface-associated polysaccharides or proteins, possibly provide a competitive advantage for one organism in a mixed microbial community [3, 12]. Bacteria with hydrophobic properties are more likely to attach to surfaces than hydrophilic bacteria; however, the attachment of biofilms will occur readily on surfaces that are rough, hydrophobic, and coated by surface conditioning films.

Figure 2.

Factors affecting biofilm formation.

The physicochemical properties of the substratum, such as texture (rough or smooth), hydrophobicity and charge, can also be modified by environmental conditions, such as pH, temperature, and nutrient levels [4, 10, 30]. In aquatic environments, the rate of microbial attachment can be increased by increasing the velocity of the flow, water temperature or nutrient concentration, providing that these factors do not exceed critical levels [6, 15].

Quorum Sensing: This is a bacterial cell–cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers (AIs) [33]. In Gram-positive bacteria, oligopeptides are used as signaling molecules to form biofilms, and quorum sensing is used for intraspecies communication. Quorum sensing controls processes such as bioluminescence, sporulation, competence, antibiotic production, biofilm formation, and virulence factor secretion [34]. Three main types of quorum sensing systems exist:

• Acyl-homoserine lactone quorum sensing system (AHL) in Gram-negative bacteria,

• Autoinducing peptide (AIP) quorum sensing system in Gram-positive bacteria

• Autoinducer-2 (AI-2) system in both gram-negative and positive bacteria [34].

The acyl homoserine lactone-dependent QS system is a prominent cellular signaling molecules of homoserine lactones involved in quorum sensing regulation used primarily by Gram-negative bacteria. The AHL molecules have the homoserine lactone ring in common varying in length and substituents, synthesized by a specific AHL synthetase. The concentration of AHL contributes to bacterial growth. Autoinducing peptide (AIPs) are signal molecules secreted by membrane transporters and synthesized by Gram-positive bacteria. The AIPs bind to the histidine kinase sensor phosphorylating, consequently altering gene expression as the environmental concentration of AIPs augments [32, 35, 36]. These genes control the formation of innumerable toxins and decomposable exoenzymes [21, 36, 37]. The microorganisms can sense and translate the signals from distinct strains in AI-2 or autoinducer-2 interspecific signals, catalyzed by LuxS synthase as part of their cooperation and communication strategies [6, 25, 38]. Moreover, LuxS is involved in the activation of the methylation cycle and has been demonstrated to control the expression of hundreds of genes associated with the microbial processes of surface adhesion, detachment, and toxin production [24, 39, 40]. The QS system is a paramount target for the treatment of biofilm-associated infections [12].

5. Biofilm-producing bacteria and infections

Biofilm formation is present in approximately 65% of all bacterial infections and approximately 80% of all chronic infections according to the statistics of the National Institute of Health (NIH) (Table 2) [12]. Indwelling devices by bacteria settlement was associated with infections in 4% of the cases when pacemakers and inhaler were utilized and 2% in breast implant cases [35]. The device-related infections were estimated to be about 40% in ventricular-assisted devices, 2% in joint prostheses, 4% in mechanical heart valves and 6% in ventricular shunts [12, 25]. The heart infection (infective valve endocarditis) occurs as a result of the adherence of bacteria cells to the endothelium. The most frequent microbes being staphylococci and streptococci, members of the HACEK group, gram-negative bacteria and fungal strains [42]. The implanting of the endothelium generally occurs from colonization or the infection of different tracts (the genitourinary and gastrointestinal tract) or through the direct crossing of the skin barrier, either due to wounds or through injecting drugs [41]. Some biofilm-driven infections are chronic wounds, diabetic foot infections, and pulmonary infections in patients with cystic fibrosis and specific bacterial species (Table 2) [21, 37, 43].

Bacterial strain	Gram stain	Types of infections	References
Staphylococcus aureus	Gram-positive	Chronic biofilm infections: chronic wound infection, right valve endocarditis, lung infections in patients with cystic fibrosis	[20]
Staphylococcus epidermidis	Gram-positive	Endocarditis: catheter-related infection, joint prosthesis infection	[20]
Streptococcus pneumoniae	Gram-positive	Lung infections, bacterial meningitis, acute or chronic otitis	[36]
Listeria monocytogenes	Gram-positive	Coculture interactions with Pseudomonas, Vibrio strains, listeriosis, contamination of food products	[36]
Burkholderia cepacia	Gram-negative	Opportunistic infections in patients with blood cancer	[41]
Escherichia coli	Gram-negative	Hemolytic uremic syndrome, acute diarrheic syndrome, urinary tract infections	[36]
Klebsiella pneumoniae	Gram-negative	Bacteremia, liver abscess, urinary tract infections	[20]
Pseudomonas putida	Gram-negative	Urinary tract infection	[36]
Pseudomonas aeruginosa	Gram-negative	Osteomyelitis, ventilator-associated pneumonia, lung infections in patients with cystic fibrosis, opportunistic infections in neutropenic patients, nosocomial infections.	[41]
Pseudomonas fluorescens	Gram-negative	Bioremediation, biocontrol- Pythium, Fusarium, antimicrobial properties –	[20]
Rhizobium leguminosarum	Gram-negative	Biocontrol properties – Pythium	[36]
Lactobacillus plantarum	Gram-positive	Salmonella infection	[41]
Lactococcus lactis	Gram-positive	Gastrointestinal tract infections	[20]

Table 2.

Examples of bacterial species involved in biofilm formation with their biological effects.

6. Mechanisms of biofilm inhibition and eradication

1. Antibiofilm molecules and their mechanism of action:

The material matrix of implanted medical devices and biomaterials provides an ideal site for bacterial adhesion promoting mature biofilm formation [3]. Methods that prevent bacterial attachment to these materials represent a preventative strategy. The most common method for preventing bacterial extension is a surface modification (Table 3). The exterior surface of the implanted medical device or biomaterial is altered, either directly or with the aid of a cover-producing barrier that is hostile to bacteria [45, 46]. This strategy has shown significant promise for preventing biofilm-related infections resulting from orthopedic implants. Thus, the area of surface modification to prevent biofilm formation is a large field [46, 47, 48]. The use of small molecule biofilm inhibitors is another approach used to prevent biofilm formation (Figure 3). The antibiofilm properties of a biofilm inhibitor are often employed to passivate the surface of an implanted medical device or biomaterial [41, 49, 50]. The use of biofilm inhibitors is one of the largest areas in biofilm remediation research, with a plethora of unique biofilm inhibitors currently described (phenols, imidazoles, furanone, indole, bromopyrrole) [51].

Resistance mechanism	Characteristics	References
Glycocalyx	The capsule is an important part of the biofilm in both Gram positive and negative bacteria. Its contribution to the maturation step relies on the electrostatic and hydrogen bonds established on the matrix and the abiotic surface. The composition in glycoprotein and polysaccharides varies with biofilm progression, permitting pathogens to live in difficult environment. The antimicrobial resistance is supported by the glycocalyx with the external layer acquiring antimicrobial compounds, serving as adherent for exoenzymes and protecting against antibacterial activity.	[6, 44]
Enzyme mediated resistance	The presence of heavy metals, such as cadmium, nickel, silver, zinc, copper, cobalt, and induces diversity of resistant phenotypes. This causes the enzymatic reduction of ionic particles mediating the transformation of toxic molecules to nontoxic or inactive.	[30]
Metabolism and growth rate heterogeneity	The bacterial metabolic activity and growth rate are influenced by the nutrients and oxygen concentrations within biofilms. This limits the metabolic activity inside the biofilm resulting in the reduction of the growing rate of strains. The enzymatic process inside biofilms is controlled by the changes in cell growth cycle regulating the metabolic and growth rate variations. These microbial communities increase the level of antimicrobial resistance inducing the expression of certain genes in different conditions.	[31, 32]
Cellular persistence	The infections’ chronicity become tolerant to antibacterial agents with the persistent strains being responsible eliciting multidrug forbearance. The glycocalyx improves protection of the immune system inducing the growth of bacterial biofilm competing for antibiotic targets with multi-medicament resistance (MDR) protein synthesis.	[33, 34, 35]
Metabolic state	The inaccessibility of nutrients due the exposition to bactericidal agent’s inhibitory concentration affects the constitution of the prokaryotic envelope modifying it and conditioning the resistant cell population to exhibit phenotypic adjustment.; The genetic profile. The mar operons are involved in the control of various genes’ expression in E. coli assisting the MDR phenotype. The stress response cells display increase resistance to impaired factors within hours of exposure. The exposition of bacterial strains to molecular oxidants causes the diversified regulatory genes (oxyR and soxR) to exhibit persistence of the intracellular redox potential and the activation of stress response.	[38, 39]
Quorum sensing (QS)	QS regulates the heterogeneous organization with nutrient supply during the cell migration procedure. QS deficiency is linked with thinner microbial biofilm growth consequently lowering the EPS production.	[40, 42]
Stress response	The stress response acts as a preventive factor for cell damage more than repair. The causes of stress induction include starvation, decrease or increase temperature, high osmolality and low pH. The altered gene expression due to the stress response in immobilized strains result in increased resistance to antibiotics.	[41, 43, 45]
External membrane structure	The lipopolysaccharide layer prevents hydrophilic antimicrobials from entering through the outer membrane while the external membrane proteins reject hydrophobic molecules. Most antibacterial agents must penetrate the bacterial cells to target a specific site, modifying the cellular membrane that control antibiotic resistance.	[46, 47]
Efflux systems	The efflux pumps facilitate bacterial endurance under utmost environmental conditions exerting inherent and gained resistance to diverse antimicrobials of similar or divergent classes. The combination of similar recalcitrance processes leads to the overproduction of efflux pumps regulating the multi-medicament non-compliances. The efflux pumps are major player in the MDR of Gram-negative bacteria due to their clear mechanisms provided in drug discovery platforms of targeted bacterial pathogens.	[48, 49, 50, 51, 52]

Table 3.

Mechanism of biofilm-mediated antimicrobial resistance.

Figure 3.

The different steps in biofilm formation.

Anti-biofilm molecules are diverse compounds that inhibit biofilm formation. The identified anti-biofilm compounds are mainly isolated from natural sources, and some synthetic compounds, chelating agents, and antibiotics possess antibiofilm activity. The different antibiofilm molecules along with their target microorganisms are listed in Table 2. These antibiofilm molecules follow different mechanisms to inhibit biofilm formation in different bacteria, as listed in Table 3.

1. Using Natural Products:

The formation and development of biofilms is a complicated procedure involving different stages that can be the target of natural antibiofilm agents for the prevention of biofilm development. Natural anti-biofilm agents either act solely or synergistically by diverse mechanisms.

There are five broad classes of natural compounds that have high antibiofilm properties, including phenolics, essential oils, terpenoids, lectins, alkaloids, polypeptides, and polyacetylenes [52]. Phenolics are a group of compounds. It has seven subclasses, which include phenolic acids, quinones, flavonoids, flavones, flavonols, tannins, and coumarins, out of which tannins, specifically condensed tannins, have anti-biofilm activity. These compounds act on biofilms by six main mechanisms, such as substrate deprivation, membrane disruption, binding to the adhesin complex and cell wall, binding to proteins, interacting with eukaryotic DNA, and blocking viral fusion [52, 53]. Many bioactive compounds from medicinal plants for the discovery of novel natural antibiofilm compounds are ongoing. The antibiofilm properties of Indian medicinal plants were studied with Cinnamomum glaucescens (Nees) Hand.-Mazz, Syzygium praecox Roxb. Rathakr. & N. C. Nair, Bischofia javanica Blume, Elaeocarpus serratus L., Smilax zeylanica L., Acacia pennata (L.) Willd., Trema orientalis (L.) Blume, Acacia pennata (L.) Willd., Holigarna caustica (Dennst.) Oken, Murraya paniculata (L.) Jack, and Pterygota alata (Roxb.) R. Br. extracts have promising antibiofilm activity against S. aureus [36, 53, 54]. Phytochemicals inhibit the quorum sensing mechanism mainly by blocking quorum sensing inducers such as AHL, autoinducers, and autoinducer type 2. Garlic extracts play a vital role in the inhibition of quorum sensing signaling molecules of Pseudomonas and Vibrio spp. Biofilms [5, 36, 52, 55]. Phytochemicals also play a significant role in inhibiting bacterial adhesion and suppressing genes related to biofilm formation. Biofilm development at the initial stages can be outlined by interfering with the forces (van der Waals force of attraction, electrostatic attraction, sedimentation and Brownian movements) that are responsible for the support of bacterial attachment to various surfaces [56]. Some phytocompounds have the potential to interfere with the extension along with the capability to stop the accessibility to nutrients essential for adhesion and bacterial growth. An alkaloid (norbgugaine) had a significant effect on P. aeruginosa biofilms by preventing adhesion due to loss of cell motility [9, 24, 55, 57]. A very recent study on Adiantum philippense L. crude extract showed a promising role in decreasing the content of biofilm exopolysaccharides [44, 58, 59]. It was reported that A. philippense L. crude extract restrained biofilms at the initial stages by targeting adhesin proteins, destroying the preformed biofilms inhibiting EPS assembly. Diverse group of phytocompounds especially polyphenols such as 7-epiclusianone, tannic acid, and casbane, have been identified and proved to protect cell surface. Members of Enterobacteriaceae express curli, an amyloid fiber on the cell surface that helps in attachment to characteristics and cell aggregation and enhances biofilm formation as well as a cellular invasion [41, 49, 60]. The phytocompounds of curlicide and pilicide nature can be exploited in therapeutic strategies of Enterobacteriaceae biofilm prevention [57, 61, 62]. These phytocompounds with fewer side effects are better therapeutic agents for biofilm-related infections, but recent reports suggest a combined approach that is always better than the individualistic approach [24, 44, 50, 51]. A few plant-based antimicrobials with the potential of anti-biofilm activity are summarized in Table 4 [53].

Compound	Source	Pathogenic species	Experimental details	Molecular mechanism	References
Allicin	Allium sativum L.	Pseudomonas aeruginosa	Invitro (1pqsABCD knockout strain)	It decreases the bacterial attachment in the initial stages of biofilm formation as it reduces EPS formation; It controls the expression of virulence factors hence interfere with the QS system	[52]
Ajoene		P. aeruginosa; P. aeruginosa; Staphylococcus aureus	In vitro (PMNs killing assays) and in vivo; (Pulmonary infection mice model)	It downregulates rhamnolipid production; It inhibits small regulatory RNA molecules (rsmY, rsmZ, and rnaIII) that operate in the later phase of QS signaling	[51]
Carvacrol (monoterpenoid)	Origanum vulgare L.	P. aeruginosa	In vitro (qPCR for relative; expression of lasI/lasR; genes) and ocking modeling of proteins LasI and LasR	It mainly acts on QS; Machinery.; The posttranslational inhibition against lasI, which effects AHL production.	[20]
Emodin (anthraquinone)	Polygonum cuspidatum; Siebold & Zucc.; Rheum palmatum L.	S. aureus	In vitro crystal violet biofilm assay and SEM analysis	It decreases the release of eDNA and downregulates the expression of biofilm-forming related genes like cidA, icaA, dltB, agrA, sortaseA, and sarA	[22]
Emodin (anthroquinone)	R. palmatum L.	Candida albicans; Candida krusei; Candida; parapsilosis; Candida tropicalis	In vitro (microdilution assay, kinase assay) and molecular docking for emodin in CK2 (Autodock Vina)	The biofilm formation is inhibited by targeting cellular kinase signaling; It acts on planktonic cells by reducing hyphal formation.; It acts as a competitive inhibitor of CK2.	[41]
Aloe-emodin	Rheum officinale aill.	S. aureus	In vitro (CLSM assays and Congo red assay)	It reduces the production of extracellular proteins and polysaccharide intercellular adhesin; It inhibits biofilm formation on; polyvinyl chloride surfaces	[52]
Hordenine	Hordeum vulgare L.(sprouting)	P. aeruginosa	In vitro (SEM and CLSM assays, qPCR for QS-related genes)	It decreases AHL production; It reduces the exhibition of virulence factors (proteases, elastase, pyocyanin,; rhamnolipid, alginate, and pyroviridine).; It impedes the swimming and swarming activity; It negatively regulates the expression of lasI, lasR, rhlI and rhlR genes	[52]
Pulverulentone A	Callistemon citrinus (Curtis) skeels leaves	S. aureus	In vitro broth microdilution assay, CLSM, TEM analysis Methicillin-resistant	It reduces styphyloxanthin production, thus inhibiting biofilm formation; It disrupts the cell membrane.	[52]

Table 4.

Anti-biofilm activity of phytocompounds and their mechanism of action [53].

7. Conclusion

Biofilm infections are highly resistant to antibiotics and physical treatments. Many strategies support biofilm antibiotic resistance and tolerance, such as persistent cells, adaptive responses, and limited antibiotic penetration. Thus, the underlying mechanisms of antibiotic forbearance and recalcitrance in biofilms are controlled by genes. In human infections, most organized bacterial cells gradually induce immune responses to form biofilms causing chronic infections leading to tissue destruction with permanent pathology. Therefore, biofilms arrangement is a vital perturbation in medical care environment. The exploration of alternative treatment procedures for biofilm-associated infections is of utmost importance. There are little novel and efficient antibiotic strategies which are scattering of biofilms, merging of antimicrobials with quorum sensing inhibitors, and a mixture of these procedures. Although the mentioned anti-biofilm strategies are key research areas, they are still in their infancy and has to be improved to upgrade and implement the strategies. The administration of a single antibiotic is often not enough to eradicate bacterial invasions, and a high concentration of the antibiotic can be extremely toxic. Also, some natural compounds as well as quorum sensing inhibitors, may be toxic and less effective. A possible solution might be the coadministration of antibiotics with antibiofilm peptides that allow the use of low antibiotic concentrations. New anti-biofilm molecules from natural substances with low or no harmful effects and synergistic effects with commonly used antibiotics are necessary. Moreso, natural products from medicinal plants and quorum sensing inhibiting compounds with little or no toxic effects will be of great importance in the fight against biofilms.