Prevention and Treatment of Staphylococcus aureus Biofilms Using Promising Agr-QS-Targeting Anti-Biofilm Agents

Abstract

Staphylococcus aureus (S. aureus), a leading cause of nosocomial infections, contributes significantly to increased morbidity and mortality, especially when it forms biofilms on medical devices. This pathogen, specifically methicillin-resistant S. aureus (MRSA), remains a challenge to treat due to its ability to form biofilms and rapidly develop resistance against antibiotics. Biofilm formation allows bacteria to adhere to biotic and abiotic surfaces, creating a protective matrix that shields them from immune responses and antibiotic therapies. The widespread prevalence of multidrug-resistant S. aureus biofilms poses a significant therapeutic challenge in clinical settings. Several novel therapeutic strategies have been developed to combat S. aureus biofilm-associated infections. Accumulating evidence suggests that natural plants and their derivatives possess antimicrobial and chemo preventive properties that can disrupt established biofilms. Several plant-derived compounds with anti-biofilm activities have been reported to target the regulatory proteins involved in the Agr quorum sensing (Agr-QS) system, underscoring their potential as therapeutic candidates for the prevention and treatment of biofilm-associated infections. However, despite these encouraging findings, clinical validation of these plant-based agents is essential to ensure their efficacy, safety, and optimal application in treating S. aureus biofilm infections. The continued exploration of natural biofilm inhibitors anticipates the urgent need for new treatments to combat biofilm-associated infections and multidrug-resistant pathogens like MRSA. This review provides a detailed overview of preventive and therapeutic interventions to eradicate biofilm-forming S. aureus infections.

1. Introduction

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, is a major human pathogen in both inpatient and outpatient settings and contributes significantly to morbidity and mortality [1]. About 30–50% of healthy individuals in the United States have S. aureus, and one in a hundred of these people is colonized with methicillin-resistant S. aureus (MRSA). Therefore, this antibiotic-resistant pathogen can easily be transmitted through direct contact, exposing a large population to infection [2]. In recent years, the cases of MRSA infections in hospitalized patients have reduced in several countries, but the COVID-19 pandemic had a significant impact on antimicrobial resistance, resulting in a 15% increase in nosocomial MRSA infections in the United States [3]. S. aureus colonizes indwelling medical devices, including catheters, implants, joints, and artificial heart valves, and produces biofilms [4,5]. The infections associated with S. aureus biofilms are difficult to treat, as biofilms provide a phenotypic resistance mechanism that further protects the pathogen from antibiotics and host defense [6].

The antibiotic resistance of S. aureus is primarily mediated through its ability to evade the host’s immune system. This is achieved through the bacterium’s ability to invade the epithelial cell linings and form recalcitrant biofilms [7,8]. Key cell wall components, such as teichoic acid polymers, and the production of virulence factors, including coagulase and toxins, play crucial roles in the adhesion and invasion processes [7,8].

Though antibiotics are the first choice to combat bacterial infections, resistance to last-resort antibiotics like vancomycin by vancomycin-resistant S. aureus (VRSA) has led to the emergence of multidrug-resistant strains, posing a major concern [9]. The infections caused by antibiotic-resistant S. aureus strains increase hospital stays and mortality, resulting in a substantial financial burden on the medical industry. Over the past decade, the total hospital costs to treat S. aureus infection have been estimated at $450 million [10,11]. Research on S. aureus biofilm formation has improved our understanding of the intricacy of S. aureus pathogenesis and advanced the development of therapies to prevent and treat biofilm infections. Recently, natural plants and their extracted phytochemicals have gained significant attention for their potential antimicrobial and anti-biofilm properties [12]. These green alternatives consist of complex mixtures of various compounds, making it difficult for bacteria to develop resistance to these multi-component treatments [13].

A thorough understanding of the cellular communication mechanisms within a biofilm matrix can lead to the development of new strategies and targets for identifying novel therapies against biofilm-associated S. aureus infections. This review outlines the mechanisms of S. aureus biofilm formation and highlights recent advances in the use of various natural compounds as alternative therapeutic approaches.

2. Prevalence of Staphylococcus aureus

S. aureus is a persistent resident of the human nasal passages and epidermal surfaces and substantially increases the risk of invasive infection. As an opportunistic pathogen, it can colonize various sites, including the skin, blood, soft tissues, lungs, bones, brain, and heart valves, causing diseases ranging from superficial skin infections to life-threatening bacteremia, endocarditis, pneumonia, and sepsis (

Table 1. Selected clinical and epidemiological contexts in which S. aureus infections and biofilms are implicated.

Clinical Epidemiology	Population	Metric Reported	Approximate Affected Population	Biofilm Resistance	References
Staphylococcal food poisoning (SFP)	Outbreak of S. aureus enterotoxin-mediated foodborne disease	Individuals exposed to these specific outbreaks	Up to ~85% of exposed persons	Consumption of enterotoxin-producing S. aureus-contaminated food and contact surfaces contributes to persistence biofilms	[15,16]
Sepsis (all causes)	Global population	Annual sepsis cases and deaths, proportion of all global deaths	~48.9 million cases and ~11 million deaths in 2017, ~20% of all deaths worldwide	MRSA leads to sepsis and bloodstream infection. It forms biofilms on intravascular devices and endovascular tissues	[17,18]
Healthcare-associated MRSA infections	Hospitalized patients	Proportion of healthcare-associated S. aureus infections due to MRSA	Commonly 20–50% depending on region	MRSA causes device-related and surgical site infections, frequently associated with biofilm and multidrug resistance	[19,20]
Community-associated MRSA skin and soft tissue infections	Patients with SSTIs in community settings	Proportion of purulent SSTIs caused by MRSA	Often >50% of purulent SSTIs (high variability reported)	MRSA SSTIs frequently involve biofilm on skin and soft tissue surfaces, complicating treatment and recurrence	[20,21]
Prosthetic joint and device-related infections	Patients with orthopedic implants and intravascular catheters	Proportion of device-related infections due to S. aureus	S. aureus commonly accounts for 20–40% of prosthetic joint infections and many catheter-related bloodstream infections	Biofilm formation on prosthetic materials and catheters is central to chronic, relapsing infection and antibiotic tolerance	[20]

Note: All abbreviations are listed at the end of the manuscript.

). The two most prevalent strains, MRSA and methicillin-susceptible S. aureus (MSSA), are known to cause mild skin infections that can progress to life-threatening sepsis and even death [14].

MSSA infections can be treated using beta-lactam antibiotics, whereas MRSA infections are commonly treated with sulfamethoxazole–trimethoprim, daptomycin, telavancin, clindamycin, linezolid, tigecycline, quinupristin–dalfopristin, and vancomycin [21,22]. However, the effectiveness of conventional antibiotics has substantially reduced due to increasing prevalence of antimicrobial resistance (Figure 1, Table 2), highlighting the urgent need to develop alternative therapeutics.

The clinical persistence of S. aureus is mostly attributed to the biofilm-mediated protection from antimicrobial agents that helps maintain chronic infection. Beyond humans, S. aureus is also a significant cause of mastitis in bovine, ovine, and caprine species, leading to a notable decrease in milk production and quality, resulting in substantial economic losses for the dairy industry [23]. Consequently, understanding the mechanisms regulating biofilm formation and virulence in S. aureus can pave a path in the development of effective strategies.

Table 2. Therapeutic limitations and emerging resistance associated with antibiotics used against S. aureus.

Infection	Antibiotic	Clinical Implications	Major Therapeutic Limitations	Emerging Resistance	References
Endocarditis	Linezolid	To treat MRSA-like cases when IV access or oral step-down is needed	Myelosuppression, especially thrombocytopenia, dose- and duration-dependent toxicity	Linezolid resistance	[24,25,26,27]
Severe community- acquired pneumonia	Daptomycin	To treat MRSA, but generally not recommended for pneumonia	Inactivated by pulmonary surfactant	Increased daptomycin resistance	[26,28,29]
Bacteremia and vertebral osteomyelitis		To treat MRSA bacteremia and deep infections	Increased CPK levels, myopathy risk, need for higher dosing in severe infection	In vivo-acquired resistance and treatment failures in MRSA bacteremia	[30,31,32]
Sepsis and septic shock	Vancomycin	Standard therapy for severe MRSA infection	Nephrotoxicity; infusion reactions (red man syndrome)	Increasing vancomycin-intermediate S. aureus (VISA) prevalence and rare VRSA. Recent sepsis guidelines highlighted reduced efficacy with elevated MICs	[33,34,35]
Chronic skin ulcers		Used for susceptible isolates but often limited in biofilm-rich chronic disease	Toxicity limits prolonged courses, poor penetration, and tolerance in biofilms	Increasing prevalence of VISA/VRSA along with reduced vancomycin susceptibility	[35,36,37]
Skin and soft tissue infections	Clindamycin	Commonly used for susceptible MSSA/MRSA SSTIs	Gastrointestinal toxicity kills beneficial bacteria and allows harmful C. difficile to grow	Substantial inducible clindamycin resistance in MRSA and MSSA	[38,39,40]
Mixed (samples taken from hospital)	Quinupristin– dalfopristin	Used for highly resistant Gram-positive infections only	Infusion-related pain, arthralgia/myalgia, limited routine use	Plasmid-mediated resistance in S. aureus and other staphylococci	[26,41,42]
Mixed (samples taken from hospital)	Trimethoprim–sulfamethoxazole (TMP-SMX)	Useful for selected MRSA SSTIs and some invasive infections	Hypersensitivity; hematologic toxicity	TMP–SMX resistance in MRSA and MSSA	[42,43]
Mixed (samples taken from hospital)	Tigecycline	Alternative for complicated skin/soft tissue and intra-abdominal infections	Nausea/vomiting, low serum levels unsuitable for bacteremia	Emergence of tigecycline resistance in S. aureus and other Gram-positive pathogens	[26]

Note: All abbreviations are listed at the end of the manuscript.

Table 2. Therapeutic limitations and emerging resistance associated with antibiotics used against S. aureus.

Infection	Antibiotic	Clinical Implications	Major Therapeutic Limitations	Emerging Resistance	References
Endocarditis	Linezolid	To treat MRSA-like cases when IV access or oral step-down is needed	Myelosuppression, especially thrombocytopenia, dose- and duration-dependent toxicity	Linezolid resistance	[24,25,26,27]
Severe community- acquired pneumonia	Daptomycin	To treat MRSA, but generally not recommended for pneumonia	Inactivated by pulmonary surfactant	Increased daptomycin resistance	[26,28,29]
Bacteremia and vertebral osteomyelitis		To treat MRSA bacteremia and deep infections	Increased CPK levels, myopathy risk, need for higher dosing in severe infection	In vivo-acquired resistance and treatment failures in MRSA bacteremia	[30,31,32]
Sepsis and septic shock	Vancomycin	Standard therapy for severe MRSA infection	Nephrotoxicity; infusion reactions (red man syndrome)	Increasing vancomycin-intermediate S. aureus (VISA) prevalence and rare VRSA. Recent sepsis guidelines highlighted reduced efficacy with elevated MICs	[33,34,35]
Chronic skin ulcers		Used for susceptible isolates but often limited in biofilm-rich chronic disease	Toxicity limits prolonged courses, poor penetration, and tolerance in biofilms	Increasing prevalence of VISA/VRSA along with reduced vancomycin susceptibility	[35,36,37]
Skin and soft tissue infections	Clindamycin	Commonly used for susceptible MSSA/MRSA SSTIs	Gastrointestinal toxicity kills beneficial bacteria and allows harmful C. difficile to grow	Substantial inducible clindamycin resistance in MRSA and MSSA	[38,39,40]
Mixed (samples taken from hospital)	Quinupristin– dalfopristin	Used for highly resistant Gram-positive infections only	Infusion-related pain, arthralgia/myalgia, limited routine use	Plasmid-mediated resistance in S. aureus and other staphylococci	[26,41,42]
Mixed (samples taken from hospital)	Trimethoprim–sulfamethoxazole (TMP-SMX)	Useful for selected MRSA SSTIs and some invasive infections	Hypersensitivity; hematologic toxicity	TMP–SMX resistance in MRSA and MSSA	[42,43]
Mixed (samples taken from hospital)	Tigecycline	Alternative for complicated skin/soft tissue and intra-abdominal infections	Nausea/vomiting, low serum levels unsuitable for bacteremia	Emergence of tigecycline resistance in S. aureus and other Gram-positive pathogens	[26]

Note: All abbreviations are listed at the end of the manuscript.

3. Virulence Potential of Staphylococcus aureus

Among all staphylococcal bacteria, S. aureus is the most lethal pathogen, causing frequent outbreaks over the years. This is primarily due to its wide array of virulence factors, with biofilm formation being a significant contributor to its pathogenicity (

Table 3. Virulence factors responsible for recalcitrant S. aureus biofilm infections.

Virulence Factors	Associated Genes	Function	Clinical Symptoms	References
MSCRAMMs	clfA, clfB, fnbA, fnbB, cna, sdr, bbp	Adhesion with host tissues	Endocarditis, osteomyelitis, endoprosthesis	[47,48]
Biofilm	Locus ica, arg system	Persistence in the host	Chronic infections	[49,50]
Leukocidins (e.g., PVL and toxin γ)	luks-PV, lukF-PV	Deceive the host immune response	Invasive skin infections, pneumonia, abscesses	[51]
Capsular polysaccharides, protein A, extracellular matrix binding protein	hlg, cap5, cap8, spa, eap	Deceive the host immune response	Invasive skin infections, pneumonia, abscesses	[52,53]
RNAIII-dependent proteases, lipases, and nucleases	V8, hysA, hla, plc, sepA	Penetration into host tissue	Tissue lesions	[54,55]
Toxins	TSST-1, ETs, and enterotoxins	Biofilm dispersal and virulence	MTSS, NMTSS, SSSS, Bullous impetigo, SFD	[56]

Note: All abbreviations are listed at the end of the manuscript.

) [44]. In addition, the key feature of highly recalcitrant S. aureus infections is their strong adherence and invasion capabilities [45,46]. The rise in multidrug-resistant strains, along with the presence of antibiotic residues in food products such as dairy and meat, further intensifies the challenge.

4. Biofilm Formation by Staphylococcus aureus

Biofilm Development—Attachment, Proliferation, and Detachment

Biofilms are complex, structured communities formed by sessile microbes, allowing the bacteria to adhere and synthesize an organic matrix. The development of S. aureus biofilm can be divided into three phases: bacterial attachment to the host, proliferation leading to a mature biofilm formation, and detachment to initiate a new biofilm cycle (Figure 2). During initial attachment, MSCRAMM adhesions aid in host tissue colonization [47,48,57]. The LPXTG motifs in MSCRAMM adhesins form strong covalent bonds with various human matrix proteins, initiating biofilm formation at the infection site [58]. Other surface proteins, including Sdr proteins (Serin-aspartate repeat family), autolysins (Atl), and accumulation-associated proteins (Aap), are also involved during the initial stages of biofilm formation [57,59]. During maturation, the synthesis and excretion of polysaccharide intercellular adhesins (PIAs/PNAG), regulated by the icaADBC operon-coded enzyme, provide adhesion between the cells (Figure 2) [49,50,60]. PIA plays a vital role in determining the structure of the growing biofilm during the infection. Its expression is significantly upregulated by the sarA and sigB genes; luxS negatively regulates the expression of PIA and associated genes [60]. The expression of the ica operon is regulated by the activation of SarA and Pur proteins. N-acetyl-glucosaminyl transferase, a transmembrane protein produced by the expression of icaAD genes, is required to produce N-acetyl-glucosamine oligomers [52]. Additionally, the co-expressed IcaC gene is responsible for the elongation and translocation of the growing polysaccharide to the cell surface. Finally, the deacetylation of this poly-N-acetylglucosamine occurs through the action of a surface protein, IcaB, contributing to PIA development. However, PIA is not present in all S. aureus isolates. Other adhesion proteins, such as Aap (accumulation-associated protein), fibrinogen-binding proteins (FnbpA and FnbpB), protein A, extracellular matrix-binding protein (Embp), and surface protein G (SasG), are involved in biofilm formation and maturation [60,61].

In addition to PIA/PNAG, many clinical S. aureus isolates form biofilms whose matrices are predominantly proteinaceous or eDNA-rich rather than polysaccharide [62,63]. Protein-dependent biofilms are mediated by surface adhesins and secreted proteins, including SasG, protein A, fibronectin-binding proteins (FnBPA/FnBPB), clumping factor B, serine–aspartate repeat proteins, and biofilm-associated protein (Bap), which can drive robust biofilm formation even in ica-negative or PIA-independent strains [64,65,66]. Recent genetic and biochemical studies further showed that secondary messenger signaling and global regulators also contribute to these non-PIA biofilms. For example, the changes in the secondary messenger cyclic-di-AMP (mediated by the phosphodiesterase GdpP) and regulators like SarA and SigB determine how much eDNA is released and how the matrix is assembled, underscoring that PIA-independent, protein/eDNA-dominated biofilms are common and clinically relevant in S. aureus [50,67].

Biofilm-associated protein (Bap) promotes biofilm formation and proliferation, even in the absence of exopolysaccharides. Bap was first discovered in the S. aureus strain responsible for bovine mastitis [68]. The expression of both the ica operon and the bap gene significantly enhances the biofilm formation and invasion capabilities of S. aureus isolates. In clinical S. aureus strains, increased expression of the rbf gene activates the ica operon. However, the rbf gene has not been observed in any bovine S. aureus isolates [69].

Detachment, the final step in biofilm formation, involves the disruption of non-covalent interactions between biofilm cells and the supportive matrix [53].

5. Biofilm Development and Its Relation to Quorum Sensing

Within the biofilm matrix, bacteria use certain chemical signaling molecules to communicate with one another, a process known as quorum sensing (QS). The QS system plays an intriguing role in the development of biofilm resistance against different antimicrobial agents [50]. The QS system allows the bacteria to sense and respond to the accumulation of various signaling auto-inducing peptides (AIPs) secreted within the matrix [70,71]. During biofilm maturation and dispersion, the kinase receptors on the bacterial surface bind to high concentrations of these AIPs in the matrix and transmit signals to trigger the expression of sarA and an accessory gene regulator, Agr [53]. The S. aureus Agr system is essential for the production of several virulence factors, including toxins and degradative exoenzymes [56].

5.1. Role of Agr System in Biofilm Dispersal

In S. aureus isolates, the biofilm maturation and dispersal processes are tightly regulated by the Agr-QS system. Repression of agr operon genes contributes to biofilm formation, while activation of the agr system triggers the detachment of mature biofilms [53]. The Agr system is a highly integrated signaling network that regulates virulence by sensing the concentration of signaling molecules and bacterial cell density in the matrix [55]. The agr operon comprises two divergent promoters, P2 and P3, which drive the transcription of two distinct RNA molecules, RNAII and RNAIII (Figure 2) [54]. RNAII is a polycistronic mRNA that codes for four Agr proteins, including AgrC, AgrA, AgrB, and AgrD. A histidine kinase, AgrC, along with response regulator AgrA, forms a two-component signal transduction system [55]. AgrB is a multifunctional protein that functions both as an endopeptidase and a chaperone, aiding in the maturation and export of AIPs. AgrD serves as a precursor of the AIP pheromone, which is proteolytically processed by AgrB to form a thiolactone intermediate, as shown in Figure 2. This intermediate is subsequently exported across the membrane, where it is cleaved to generate a mature AIP pheromone. S. aureus strains can be divided into four different groups based on the polymorphic variant produced by the arg locus: agrI, agrII, agrIII, and agrIV (Figure 2) [72]. Species-specific variants have been widely reported; some studies have also reported the association of agr variants with different S. aureus features (Table 4). Notably, isolates with the agrI variant exhibit an increased tendency to invade MAC-T cells, while the isolates with agrII showed a greater reliance on biofilm formation [73].

When the level of mature AIP reaches its threshold, it activates AgrC. Upon activation, AgrC initiates a phospho-relay cascade that results in AgrA phosphorylation. Phosphorylated AgrA binds to the P2 promoter, and upregulates the transcription of RNAII (positive feedback mechanism) and the P3 operon. The P3 operon, which is an AgrA-dependent operon located adjacent to P2, encodes RNAIII (hld), a posttranscriptional regulator that controls the expression of multiple virulence factors, including proteases, nucleases, the biofilm dispersal gene (hla), toxins, and surfactants. This ultimately leads to biofilm dispersal as depicted in Figure 2 [74]. Activated AgrA also regulates the psm-mec gene in an RNAIII-independent manner, leading to the production of phenol-soluble modulins (PSMs). PSMs such as δ-hemolysin, PSMα1-4, PSMmec, and PSMβ1-2 contribute to the degradation of exopolysaccharides (EPSs), facilitating biofilm dispersal and promoting host cell perturbations. During the dispersal phase, mature biofilm ruptures, releasing bacterial aggregates that can seed the formation of new biofilms [48,61].

Table 4. Characteristic features of different Agr variants and their role in biofilm regulation.

Agr Variant	Characteristic Features	Biofilm Regulation	Reference
agrI	Predominantly reported in CA-MRSA and methicillin-resistant bovine isolates	Regulates a broad set of toxins, proteases, and adhesins; agrI dysfunction is linked to prolonged bacteremia and altered biofilm behavior.	[75,76]
	High prevalence of resistance towards beta-lactamase, glycopeptides, fluoroquinolones, aminoglycosides, tetracyclines, macrolides, lincomycins, and sulphonamides		[77]
agrII	Predominantly causes nosocomial MRSA and MRSA bloodstream infections	agrII activity influences biofilm maturation and dispersal. It is associated with distinct toxin/adhesin profiles.	[54,75]
	Toxic shock syndrome
	Prolific biofilm producers		[78]
agrIII	Predominantly causes CA-MRSA	Linked with toxin-mediated virulence, agrIII mutants show altered biofilm and persistence phenotypes.	[75,79]
	Potent biofilm producers		[80]
agrIV	Predominantly reported in swine farm isolates and generalized exfoliative syndromes	Regulates virulence and biofilm, but detailed functional data are limited.	[54,77]
	High prevalence of resistance to fluoroquinolones, aminoglycosides, tetracyclines, macrolides, lincomycins, and sulphonamides		[77]
	High prevalence of enterotoxin genes		[54]

Note: All abbreviations are listed at the end of the manuscript.

Table 4. Characteristic features of different Agr variants and their role in biofilm regulation.

Agr Variant	Characteristic Features	Biofilm Regulation	Reference
agrI	Predominantly reported in CA-MRSA and methicillin-resistant bovine isolates	Regulates a broad set of toxins, proteases, and adhesins; agrI dysfunction is linked to prolonged bacteremia and altered biofilm behavior.	[75,76]
	High prevalence of resistance towards beta-lactamase, glycopeptides, fluoroquinolones, aminoglycosides, tetracyclines, macrolides, lincomycins, and sulphonamides		[77]
agrII	Predominantly causes nosocomial MRSA and MRSA bloodstream infections	agrII activity influences biofilm maturation and dispersal. It is associated with distinct toxin/adhesin profiles.	[54,75]
	Toxic shock syndrome
	Prolific biofilm producers		[78]
agrIII	Predominantly causes CA-MRSA	Linked with toxin-mediated virulence, agrIII mutants show altered biofilm and persistence phenotypes.	[75,79]
	Potent biofilm producers		[80]
agrIV	Predominantly reported in swine farm isolates and generalized exfoliative syndromes	Regulates virulence and biofilm, but detailed functional data are limited.	[54,77]
	High prevalence of resistance to fluoroquinolones, aminoglycosides, tetracyclines, macrolides, lincomycins, and sulphonamides		[77]
	High prevalence of enterotoxin genes		[54]

Note: All abbreviations are listed at the end of the manuscript.

5.2. Alternative Anti-Biofilm Strategies

The global rise in MRSA and VRSA is significantly undermining the effectiveness of nearly all reported antibiotics [81]. Unfortunately, no vaccines have been approved to treat S. aureus infections. In addition, recalcitrant S. aureus biofilms require surgical debridement and prolonged antimicrobial therapy. The global market for antibiotics to treat S. aureus infections, especially MRSA, was valued at 984.6 million US dollars in the year 2020, with a projected Compound Annual Growth Rate (CAGR) of 4.4%, expected to reach approximately 1327.9 million US dollars by 2027 [82]. Consequently, various natural and synthetic anti-biofilm agents are being explored as an alternative to conventional antibiotics. These include the metabolites extracted from other prokaryotes to target the Agr-QS system or to degrade the biofilm matrix (

Table 5. Alternative anti-biofilm strategies and their respective limitations.

Category	Source	Targeted Action	Limitations	Level of Evidence	Reference
Synthetic Compounds	RNAIII-inhibiting protein and its derivatives	Inhibit the expression of agr and biofilm-producing genes	High production cost, limited sustainability	In vitro and animal models	[85,86]
	Savirin	Inhibits auto-induction and quorum sensing		In vitro and animal models	[87,88,89]
Probiotics	Lactobacillus casei, Lactococcus lactis V7, Lactobacillus rhamnosus ATCC 7469	Inhibit adhesion, invasion, and biofilm formation	Side effects on beneficial bacteria of the host and emergence of antimicrobial resistance. Variable colonization efficiency; delivery challenges	In vitro and animal models	[90,91,92]
Bacterial	Streptomyces sp. N174	Antimicrobial and anti-biofilm properties	Survival of live cells during the gastrointestinal transit and their effective delivery to target tissues. Cross-species variability	In vitro	[93]
	Staphylococcus schleiferi	Inhibits Agr expression		In vitro	[94]
	Marine bacteria	Competitive inhibitor of AgrC		In vitro	[95]
	3-oxo-C12-HSL, (HQNO) from Pseudomonas aeruginosa	Inhibits auto-induction of AIPs		In vitro
Bacteriophages	Isolated from farmyard slurry, host: S. aureus DPC5246	Inhibits biofilm proliferation	Bacterial resistance, immunogenicity, co-evolutionary dynamics, narrow host range, and difficulty in phage delivery to the target site. Host specificity	In vitro	[96]
	ᶲ SA012			In vitro	[97]
	Polyvalent phage K	Inhibits biofilm formation		In vitro and animal models	[98,99]
	Bacteriophage K and DRA88	Reduced biofilm		In vitro	[100]
Snake venom lectins	Bothrops jararacussu	Biofilm disruption	Small-size peptide hinders its large-scale production and toxicity evaluation	In vitro	[101]
Nanoparticles	Silver and gold	Anti-biofilm properties	Expensive production, high doses exert cytotoxic and genotoxic effects	In vitro	[83,84]
	Silver and Zinc oxide with nitric oxide	Inhibit biofilm formation		In vitro	[102]
	Silver and Zinc oxide nanoparticles in combination with Antibiotics	Dispersion of biofilm		In vitro	[103]
	Phosphatidylcholine-decorated gentamicin-loaded gold nanoparticles	Anti-biofilm		In vitro and animal models	[104,105]
	Magnesium fluoride and yattrium fluoride nanomaterials	Reduce colonization		In vitro	[106,107]
Hormones	17β-Estradiol	Invasion	High cost, less sustainability	In vitro	[108]
AIP and AIP derivatives	Truncated AIP-I, II, III,	Inhibits auto-induction of AIPs	High cost	In vitro and animal models	[59,109]
Fungal	12 compounds from marine-derived fungi	Inhibit biofilm formation	High cost, less sustainability	In vitro	[110]
Enzymes	Lysostaphin	Disrupts biofilms	Poor retention, enzymatic stability, and activation of immune responses	In vitro, animal models, and early clinical evaluation	[111,112,113,114]
	Cysteine histidine-dependent amidohydrolase/peptidase	Disrupts biofilms		In vitro	[115]
	Endolysins	Disrupt biofilms		In vitro and animal models	[116,117,118]
	V8 protease	Inhibits biofilm formation and promotes biofilm detachment		In vitro	[119]
	Staphopains	Affects biofilm integrity		In vitro	[119,120]
	Aureolysin	Inhibits biofilm formation and disperses preformed biofilms		In vitro	[121]
	Cysteine proteases	Anti-biofilm activity		In vitro	[119]
	DNases	Disrupting mature biofilms		In vitro and animal models	[122,123]
	Dispersin B	Inhibits adherence and attachment		In vitro and animal models	[116,117,124]
	Neutrase from Bacillus Amyloliquefaciens	Anti-biofilm activity		In vitro	[125]
Bio-surfactants	Mannosylerythritol lipids	Biofilm disruption	Expensive production	In vitro	[126]
Prokaryotes	Cochinmicin from Actinomycetes	Competitive inhibitor of AgrC	Survival of cells during the gastrointestinal transit and their effective delivery to target tissues upon ingestion	In vitro	[95]
	Avellanin from sponges			In vitro
Chelators	EDTA, EGTA, and TSC	Inhibit biofilm formation	Health hazards, cytotoxic, weakly genotoxic, intracellular metal accumulation, unsuitable for systemic applications	In vitro and limited clinical adjunct use	[127]
Sulfhydryl compounds	DTT, betamercaptoethanol, and cysteine	Inhibit biofilm formation	Skin irritation, organ toxicity, unsuitable for systemic applications	In vitro	[52,128]

Note: ᶲ a virus that specifically targets S. aureus. All abbreviations are listed at the end of the manuscript.

), as well as “green synthesized” nanoparticles and bacteriophages to combat S. aureus biofilms [83,84].

5.3. Green Alternatives as Potential Medicinal Therapeutic Agents

To combat this growing threat, it is crucial to explore unconventional therapies with antimicrobial and anti-biofilm properties. While approaches like bacteriophages, synthetic compounds, and nanotechnologies hold potential, they often face limitations such as cytotoxicity and high cost (Table 5). This dire situation urges us to reconsider “traditional” green alternatives, though for most biofilm applications the current reports are largely limited to in vitro and early preclinical studies. Plants, for instance, serve as a huge reservoir for secondary metabolites (phytochemicals) with known medicinal properties. Over 80% of commercialized medicines are derived directly or indirectly from natural sources, with more than 50% containing active compounds isolated from plants, herbs, and minerals [13]. These compounds play an important role in modern healthcare. However, some remedies still require experimental validation, and clinical trials are needed to commercialize their use.

Plants produce two types of active metabolites: primary and secondary metabolites. Primary metabolites are the building blocks of an organism undergoing various metabolic processes to produce secondary metabolites. Secondary metabolites assist them in surviving and reproducing [129]. These metabolites play a crucial role in chemical defense, protecting plants from abiotic stress while also contributing to floral scents and pigments. The most indispensable benefit, however, is their role as a source of medicines and industrial additives. Based on their biosynthetic pathways, secondary metabolites can be classified into several categories, including alkaloids, glycosides, steroids, phenols, terpenoids, and tannins. These compounds exhibit biodynamic medicinal properties, applicable to both human and animal health [129,130].

Phenols and polyphenols are the most potent compounds, extensively reported for their antimicrobial activities. They have a unique ability to bind to a wide range of proteins and glycoproteins and are often used to enhance the antimicrobial properties of antibiotics [131,132]. Tannins, which are polymeric phenolic compounds, have demonstrated antibacterial and anti-biofilm-forming properties due to their ability to inhibit enzymes involved in adhesion and transport [133,134,135]. Flavonoids, the most diverse group of metabolites, are well-known for their antibacterial effects. Based on their chemical structure, they are classified as flavones, isoflavones, flavonols, flavanonols, flavanones, chalcones, and anthocyanides. Flavonoids are known to induce oxidative stress and block the transport of electrons during respiration [136]. Quinones, particularly anthraquinones, are another class of highly reactive compounds that can form irreversible complexes with bacterial adhesins, leading to biofilm dysfunction [132]. Plants also produce terpenes and terpenoids, which interact with other species. Most essential oils, rich in terpenoids, exhibit both antibacterial and anti-biofilm activities [137,138].

Alkaloids are well-known nitrogen-containing natural bioactive compounds that are a rich source for drug discovery. These compounds inhibit bacterial growth by interfering with DNA intercalation and suppress bacterial proliferation and accumulation during biofilm formation [131,132,136]. Numerous alkaloids extracted from medicinal plants and herbs contribute to several biological and pharmacological uses. Compared to commonly used antibiotics, alkaloids provide enhanced resistance, which has led to cutting-edge research aimed at exploring novel therapeutic approaches [132,136]. Phytochemicals represent a complex reservoir of active compounds with a broad though often vague spectrum of activity. Many plant-based anti-biofilm targets mainly interfere with adhesion, attachment, formation of a polymer matrix, or blocking the QS system, which ultimately leads to biofilm inhibition (Figure 3).

Numerous medicinal plants and plant-derived phytochemicals have been reported to inhibit S. aureus biofilm formation. This protection is achieved by inhibiting initial bacterial adhesion, membrane disruption, and modulating the regulatory genes (agr, sarA, and ica) [52,53].

Table 6. List of the most reported selected medicinal plants and phytochemicals reported to inhibit S. aureus biofilm formation.

Plant	Main Bioactive Compound(s)	Molecular Target	Anti-Biofilm Mechanism	Effect on Agr-QS	Experimental Model Used	Effective Concentration	Stage of Development	Reference
Camellia sinensis	Catechins, especially EGCG and its derivatives	Bacterial membrane, efflux pumps, amyloid-like biofilm matrix	Membrane disruption, anti-adhesion, anti-biofilm	Interferes with the AgrA response regulator and downregulates RNAIII	S. aureus and MRSA clinical isolates	Reported active at concentrations lower than MIC, 10 μg/mL to 60 μg/mL	Preclinical phase	[139,140,141,142]
Moringa oleifera	Phytochemicals, fatty acids, including palmitoleic, linolenic, and oleic acids	Quorum sensing and virulence	Inhibits biofilm formation, reduces CFU in biofilms, lowers MIC	Interferes with agr locus targeting AgrA or AgrC	S. aureus and MRSA from PVC-surface biofilm model	0.5 to 2.0 mg/mL, resulting in up to 99% inhibition	In vitro and early animal stage (feed only)	[140,143,144,145]
Rosmarinus officinalis	Diterpene carnosic acid, camphor, micromeric acid, oleanolic acid, ursolic acid	Early attachment and biofilm formation	Inhibits initial attachment, formation, and promotes dispersal of preformed biofilms	Reduces AgrA and RNAIII expression	S. aureus and MRSA isolates	0.05 mg/mL (0.1%) extract reported to inhibit biofilm development by 94%	Early preclinical translational stage	[146,147,148]
Psidium guajava	Benzyl isocyanate, phenolics, L-5-propylthiomethylhydantoin	Biofilm formation and Quorum sensing	Inhibits biofilm formation	Downregulates agr, icaAD, and sarA	MRSA, other S. aureus isolates, and BGM cell-line	100 μg/mL to 1000 μg/mL (sub-minimal inhibitory concentration)	Preclinical phase	[149,150,151,152]
Eucalyptus globulus	Essential oil, 1,8-cineole	Early adhesion/attachment	Inhibits initial attachment and adhesion, decreases virulence and biofilm formation	Targets AgrA-AIP (auto-inducing peptide)	MRSA and S. aureus isolates	2.5 mg/mL or less	Preclinical phase	[153,154,155]
Eucalyptus sideroxylon	Flower extract enriched in phloroglucinols	Biofilm formation and Quorum sensing	Inhibits biofilm formation	Targets Agr system	MRSA and S. aureus isolates	0.05 mg/mL achieves up to 95.9% inhibition at sublethal doses	In vitro	[156,157]
Azadirachta indica	Crude and methanolic leaf extracts	Disrupting quorum sensing and downregulating virulence	Reduces biofilm formation	Repressing the Agr system	MRSA/MSSA and S. aureus isolates	62.5 µg/mL to 125 µg/mL sub-minimum inhibitory concentrations	Preclinical phase	[158,159]
Curcuma longa	Curcumin; curcuminoids; diacetyl curcumin	Disrupting quorum sensing and inhibiting swimming and swarming ability	Suppresses biofilm formation, antibiotic synergy, anti-adhesive effects, and photodynamic killing with blue LED	Targets agrA and agrC operons and RNAIII	MRSA and S. aureus isolates	100% inhibition at 20 μM Curcumin plus blue light, 62.5 μg/mL to 125 μg/mL sub-inhibitory concentrations	Preclinical phase	[160,161,162]
Sanguisorba officinalis	Triterpenoid saponins; tannins; polyphenolic root extract	Targets the ica locus and agr system genes	Reduces biofilm formation and density	Interacts with ica and agr system genes	MRSA and S. aureus isolates	256 μg/mL half-maximal inhibitory concentration (IC50)	Preclinical phase	[163,164]

Note: preclinical means in vitro and in vivo studies before human testing.

summarizes major findings of the most reported plants, including major bioactive constituents, proposed targets, anti-biofilm mechanisms, experimental models, and developmental stage.

5.4. Camellia sinensis

Camellia sinensis (C. sinensis) is a flowering plant of the Theaceae family with evergreen shrubs whose leaves and leaf buds are used to make green tea. This tea plant is a hub of beneficial theophylline, flavonoids, saponins, and several polyphenol derivatives including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) [165,166]. Tea leaf extract is reported to inhibit the growth of various bacterial species, including S. aureus, and possesses antimicrobial, antiviral, and anti-biofilm properties [166,167]. Green tea extract contains a putative anti-adhesive component that inhibits bacterial attachment and development of biofilms, and disperses pre-existing biofilms at concentrations even lower than MIC, suggesting the potential for pharmaceutical applications [140,141,168,169].

5.5. Moringa oleifera

Moringa oleifera (M. oleifera), commonly known as drumstick or Miracle tree, is a fast-growing Moringaceae family tree particularly valued for its rich nutritional content, including fatty acids, carotenoids, minerals, and vitamins [170]. M. oleifera has been shown to possess significant antimicrobial and anti-biofilm properties, especially against S. aureus. Leaves and seeds of M. oleifera are reported to inhibit biofilm formation, with an inhibition rate of up to 99% [171,172]. Further research, particularly in animal models, is needed to better understand its full therapeutic potential.

5.6. Rosmarinus officinalis

Rosmarinus officinalis (rosemary) is a well-known medicinal plant. Experimental studies have demonstrated that methanol extracts of rosemary effectively inhibit S. aureus biofilm formation, with significant reductions in the minimum inhibitory concentration (MIC) values, indicating its potential as an antimicrobial agent [173,174,175,176]. Recent studies using NMR and MS fragmentation identified bioactive metabolites from rosemary extracts, such as micromeric acid and oleanolic acid. These compounds demonstrated complete inhibition of MRSA biofilm formation, supporting the plant’s potential as a treatment for biofilm-related infections [177].

5.7. Psidium guajava

Psidium guajava is a fruiting plant known for its broad range of therapeutic activities, including antimicrobial effects [178,179,180,181]. Several studies have reported that the methanol extract of P. guajava leaves inhibits biofilm formation by downregulating the expression of critical biofilm formation genes such as icaAD, sarA, and agr [151,182]. Ultra-performance liquid chromatography (UPLC) and GC-MS analyses identified compounds like L-5-Propylthiomethylhydantoin and several phenolics that inhibit biofilm formation without causing cytotoxicity. Therefore, their pharmaceutical use may be a justification for clinical trials [182,183].

5.8. Eucalyptus

Eucalyptus is particularly known for its medicinal, commercial, and ornamental applications [184]. Eucalyptus globulus (E. globulus) is a prominent evergreen tree whose leaves are used to produce essential oils with antimicrobial, antioxidant, and anti-inflammatory properties [185,186,187]. Studies have shown that the essential oil, aqueous and methanolic leaf extracts of E. globulus significantly inhibit the initial attachment and adhesion of S. aureus and lead to decreased virulence and biofilm inhibition [130,187,188]. The Eucalyptus sideroxylon (E. sideroxylon) species is well-known for antibacterial and anti-fungal properties [156,189]. It has been reported that the flower extract of E. sideroxylon possesses potent anti-biofilm activity against S. aureus isolates at sublethal doses (95.9% inhibition) in a dose-dependent manner [156,190].

5.9. Azadirachta indica

Azadirachta indica (A. indica) is a well-known medicinal plant that has been extensively studied for its antimicrobial and anti-biofilm properties against S. aureus [178,179,191]. Crude extracts of A. indica showed a 60–70% reduction in MRSA and MSSA biofilm formation and up to 80% when used in combination with Ocimum sanctum [192,193]. These studies underline the need for further research and clinical trials to explore the full therapeutic potential of A. indica.

5.10. Curcuma longa

Curcuma longa (C. longa) is widely recognized for its broad medicinal applications [194,195,196]. Recent research has highlighted significant antibacterial and anti-biofilm properties of C. longa rhizome extracts (containing curcumin) against biofilm-producing S. aureus isolates [168]. MRSA isolates were completely eradicated at 20 μM curcumin combined with light, with lower doses also significantly reducing survival rates, highlighting curcumin’s potential in photodynamic therapy [197,198]. These findings suggest the fact that C. longa derivatives can be used as potential anti-biofilm agents against S. aureus infections.

5.11. Sanguisorba officinalis

Sanguisorba officinalis (S. officinalis) is commonly known in traditional Chinese medicine for treating wounds and stopping bleeding [199]. The root extract of S. officinalis is rich in triterpenoid saponins and tannins, compounds known for significantly inhibiting the growth of Gram-positive bacteria as compared to Gram-negative bacteria [200,201,202]. Further studies identified that the reduction in biofilm-forming ability is due to the interaction of triterpenoid saponins with the ica locus and agr system genes [163,203,204].

5.12. Others

Natural products have been one of the most important sources of novel antimicrobial agents for the past decade because of their structural diversity [205]. Recent advances in isolation and separation technologies have led to the discovery of various novel metabolites that can serve as potential candidates to cure persistent bacterial infections all over the world. The screening of natural anti-biofilm agents has consistently widened. In addition to the above-mentioned anti-biofilm agents, several other plants are also reported for their role in the inhibition of biofilms, including Allium sativum [206], Annona senegalensis [207], Jatropha curcas L. [208], Orostachys japonicus [209], Moringa stenopetala [205,210], Spondias purpurea [211], Juglans regia L. [212], Citrus sinensis [213] and several others enlisted in

Table 7. List of plants with their respective phytochemicals targeting the Agr-QS system with potential anti-biofilm properties.

Scientific Name	Common Name	Part of a Plant	Bioactive Component	Phytochemical	Function	Mechanism	Reference
Bacopa monnieri (Plantaginaceae)	Brahmi (herb)	-	Bacoside A and saponins	Terpenoids	Inhibits microbial adhesion, biofilm formation, and ability to disrupt biofilms	Binding with IcaA	[214,215]
Cinnamomum spp. (Lauraceae)	Cinnamon	Essential oil and bark	-	-	Cellular shrinkages, cell wall damages, and decreased biofilm densities	-	[216,217]
Lavandula angustifolia (Lamiaceae)	Lavender	Essential oil	Camphor, caryophyllene, eucalyptol, lavendulyl acetate, limonene, linalool, linalyl acetate, cis-ocimene, α-pinene, transocimene, terpinen-4-ol	Terpenoids	Inhibits proliferation and biofilm formation	-	[218,219,220]
Origanum onites and Origanum vulgare (Lamiaceae)	Oregano	Seeds and essential oil	Carvacrol, γ-terpinene, p-cymene, and thymol	Terpenoids	Inhibits biofilm formation and disrupts pre-formed biofilms	-	[219,221]
Leopoldia comosa (Asparagaceae)	Tassel hyacinth	Bulb	-	-	Inhibits biofilm formation	-	[222]
Mentha × piperita (Lamiaceae)	Pepper mint	Leaves	HCAs, rosmarinic, 1,8-cineole, and menthol	Polyphenol and terpenoids	Inhibits biofilm formation	-	[219,220]
Ballota nigra (Lamiaceae)	Black horehound	Aerial parts	Phenylpropanoid glycosides and phenylpropanoid derivatives	Flavonoids, glycosides, and terpenes	Inhibits biofilm growth and adherence	Inhibits δ-hemolysin, a small peptide encoded by RNAIII transcript	[1,222,223]
Sanguisorba officinalis L. (Rosaceae)	Great burnet	Dried roots	Saponins	Terpenoids and tannins	Anti-biofilm activity	ica-dependent manner	[163,202,203]
Juglans regia L. (Juglandaceae)	Walnut	Immature fruits and leaves	Naphtoquinones	Quinones, polyphenols and flavonoids	Anti-biofilm activity	-	[222]
Rhodomyrtus tomentosa (Myrtaceae)	Kemunting, rose, myrtle	Leaf	Rhodomyrtone	Flavonoid	Inhibits adherence and biofilm formations	Possible cure for mastitis, even better than vancomycin	[224]
Rosa damascene (Rosaceae)	Damask rose	Flower	-	-	Eradicates biofilms	-	[225]
Rosa canina (Rosaceae)	Rosehip	Fruit	-	Flavonoids and polyphenols	Inhibits biofilm formation	Inhibition of exopolysaccharides	[222,226]
Sambucus nigra and Sambucus ebulus (Adoxaceae)	Elder and dwarf elder	Leaves and stems	-	Flavonoids	Inhibits biofilm formation	Inhibits δ-hemolysin, a small peptide encoded by RNAIII transcript	[1,227]
Cyclamen hederifolium (Myrsinaceae)	Ivy-leaved cyclamen	Tubers	Saponins	Terpenoids	Inhibits biofilm formation	Inhibits δ-hemolysin, a small peptide encoded by RNAIII transcript	[222,227]
Ocimum sanctum (Lamiaceae)	Basil	Leaves	-	Eugenol and tannins	Inhibits biofilm formation	-	[193,228,229]
Lonicera alpigena (Caprifoliaceae)	Alpine honeysuckle	Woody parts and leaves	-	-	Inhibits biofilm formation	Inhibits δ-hemolysin, a small peptide encoded by RNAIII transcript	[222,227]
Nigella sativa (Ranunculaceae)	Black cumin	Seed oil	Thymoquinone	Quinones	Inhibits biofilm formation	-	[230,231]
Castanea sativa (Fagaceae)	European chestnut	Leaves	Ursene and oleanene	Flavonoids and terpenoids	Inhibits biofilm formation	Inhibits the agr system	[222,227]
Malva sylvestris (Malvaceae)	Common mallow	Stems and flowers	Menthol and sorbitol	Terpene	Anti-biofilm activity	Used in commercial and mouth rinses	[222,227]
Thymus vulgaris (Lamiaceae)	Red thyme	Essential oils	Thymol	Terpenoids	Anti-biofilm activity	-	[232,233,234]
Alcea rosea L. (Malvaceae)	Hollyhock	Leaves, stems, flowers, and roots	Menthol	Terpene	Anti-biofilm activity	Inhibits δ-hemolysin, a small peptide encoded by the RNAIII transcript	[1,227]
Hydrastis canadensis (Ranunculaceae)	Goldenseal	Leaves	Berberine and Mycopyranone: A 8,8′-binaphthopyranone	Alkaloids	Anti-biofilm activity	Inhibits the agr QS system by blocking signal transduction of the AgrCA two-component system	[235,236]
Solanum nigrum (Solanacae)	Black nightshade	Leaves	-	-	Anti-biofilm activity	-	[228,230]
Rhanterium suaveolens (Asteraceae)	Arfej (shrub)	Essential oil	Carvacrol, linalool, and citrals	Polyphenols	Anti-biofilm activity	-	[237]
Rosmarinus officinalis (Lamiaceae)	Rosemary	Leaves	Pinene, camphor, micromeric acid, oleanolic acid, and ursolic acid	Terpenoids	Inhibits biofilm formation and disrupts pre-formed biofilms	-	[174,175,176,238,239,240]
Cananga odorata (Annonaceae)	Fragrant cananga	Essential oils	p-cresyl methyl ether, linalool, geranyl acetate, geraniol, eucalyptol	Polyphenols and terpenoids	Anti-biofilm activity	-	[218,232]
Rubus ulmifolius (Rosaceae)	Elm-leaf blackberry	Leaves, stem, and roots	Ellagic acid derivatives	Polyphenols and glycosides	Anti-biofilm activity	Inhibits δ-hemolysin, a small peptide encoded by RNAIII transcript	[222,227,241,242,243]
Melissa officinalis (Lamiaceae)	Lemon balm	Essential oil	Citrals (geranial + neral, citronellal, limonene, geraniol, β-caryophyllene, β-caryophyllene oxide, and germacrene D)	Terpenoids	Anti-biofilm activity	-	[218,219]
Cocculus trilobus (Menispermaceae)	Queen coralbead	Rhizome	Isoquinoline	Alkaloids and quinones	Inhibits microbial adhesion and biofilm formation	-	[244]
Coriandrum sativum L. (Apiaceae)	Coriander	Seeds and essential oil	p-cymene, g-terpinene, linalool, geranyl acetate	Terpenoids	Anti-biofilm activity	-	[219,220,245,246]
Zanthoxylum armatum (Rutaceae)	Winged prickly ash	Fruit	-	Alkaloids and others	Anti-biofilm activity	-	[247]
Ficus sansibarica (Moraceae)	Knobbly fig	Fruits, leaves, and stem bark	5,7,4′-trihydroxyflavan-3-ol and isovitexin	Flavonoids and triterpenes	Inhibits microbial adhesion and biofilm formation	-	[248,249]
Marrubium vulgare (Lamiaceae)	White horehound	Roots, leaves, stem, and flowers	-	Terpenes, sterols, and flavonoids	Inhibits microbial adhesion and biofilm formation	Inhibits δ-hemolysin, a small peptide encoded by the RNAIII transcript	[222,227]
Jatropha curcas (Euphorbiaceae)	Purging nut	Pressed cake of whole plant and seed oil	Saponins, linoleic acid, and oleic acid	Alkaloids and polyphenols	Biofilm inhibition and degradation	-	[208,250]
Pimpinella anisum L. (Apiaceae)	Anise	Seeds and essential oil	(E)-anethole and estragol	Phenyl propanoids	Anti-biofilm activity	-	[219,220]
Dischidia rafflesiana (Apocynaceae)	Ant plant	Polyherbal formulations	-	-	Anti-biofilm activity	-	[246,251]
Krameria argentea (Krameriaceae)	Brazilian Rhatany	Roots	Chelerythrine, sanguinarine, dihydroxybenzofuran, and proanthocyanidin	Alkaloids and quinones	Anti-biofilm activity	Interferes with the arg-QS system	[243]
Lawsonia inermis (Lythraceae)	Henna tree	Leaves	Lawsone	Quinones	Anti-biofilm activity	-	[246,252]
Olea europaea L. (Oleaceae)	Olives	Leaves	Oleuropein	Polyphenols and flavonoids	Anti-biofilm activity	-	[253]
Glycyrrhiza glabra (Fabaceae)	Liquorice	Root	Glycyrrhizin, triterpinoid saponin, and glabridin	Terpenoids	Preventing biofilm formation and adherence	Interferes with the arg-QS system and inhibits exotoxin production	[246,254]
Leopoldia comosa (Hyacinthaceae)	Tassel grape hyacinth	Bulb	-	-	Preventing biofilm formation and adherence	-	[222]
Annona senegalensis (Annonaceae)	Wild custard apple	Seeds	N-cerotoyltryptamine, asimicin, and ent-19-carbomethoxykauran-17-oic acid	Polyphenols	Anti-biofilm activity	Interferes with the arg-QS system	[207]
Quercus cerris L., (Fagaceae)	Oak	Leaves, stem, and fruit	-	-	Anti-biofilm activity	Inhibits δ-hemolysin, a small peptide encoded by the RNAIII transcript	[222,227,255]
Orostachys japonicus (Crassulaceae)	Rock pine	Whole plant	-	Quinones	Inhibits cell-surface attachment	Downregulation of the psm-mec gene	[209]
Phyllanthus emblica (Phyllanthaceae)	Indian Gooseberry	Fruits	Gallic acid	Polyphenols	Inhibits cell-surface attachment	-	[246,256]
Melaleuca alternifolia (Myrtaceae)	Tea tree	Essential oil	4-Terpineol and terpinolene	Terpenoids	Inhibits biofilm adhesion	Alters threonine, purine, pyrimidine, and amino acid biosynthesis pathways	[257,258]
Terminalia bellirica (Combretaceae)	Beleric nut tree (Baheda)	Fruit	Termilignan, thannilignan, and anolignan	Tannins	Inhibits biofilm formation	-	[246,259]
Vanilla planifolia (Orchidaceae)	Vanilla	Pods and essential oil	Ethylvanillin, 4-hydroxybenzaldehyde, methyl anisate, 4-hydroxybenzyl methyl ether, piperonal, vanillic acid, vanillin, carvacrol, and thymol	Terpenoids, flavonoids, and polyphenols	Inhibits biofilm formation	Interferes with the agr-QS system	[202,218]
Dendrobium chrysotoxum (Orchidaceae)	Fried-egg orchid	Whole plant	Erianin isovitexin and parthenolide	Flavonoids	Inhibits biofilm adhesion	Interferes with the agr-QS system	[260]
Allium sativum (Amaryllidaceae)	Garlic	Essential oil	Allicin	Alkaloids	Inhibits biofilm formation	Downregulates the expression of icaA and interferes with agr expression	[261,262]
Vaccinium macrocarpon (Ericaceae)	American cranberry	Leaves and fruit	Urell R and proanthocyanins	Quinones and polyphenols	Inhibits microbial adhesion and biofilm formation	Non-toxic, cyto- compatible metabolites	[243,263]
Myristica fragrans (Myristicaceae)	Nutmeg	Seed essential oil	Eugenol, isoelemicin, isoeugenol, methoxy eugenol, myristic acid, myristicin, kayeassamin A, surangin C, theraphin B,	Polyphenols and alkaloids	Inhibits biofilm formation	-	[246]
Arundo donax (Poaceae)	Giant reed	Reed nodes	Bufotenidine and gramine	Alkaloids	Inhibits biofilm formation and disrupts already established biofilms	-	[222]
Citrus × paradisi (Rutaceae)	Grapefruit	Seeds and essential oil	Naringenin	Flavonoid	Inhibits microbial adhesion and biofilm formation	Reduces agrA and hla	[264,265]
Spondias purpurea (Anacardiaceae)	Spanish Plum	Leaves and fruit juice pulp	-	Terpenoids and flavonoids, polyphenols	Inhibits biofilm formation	Interferes with the agr-QS system	[211,266]
Aesculus hippocastanum (Sapindaceae)	Horse chestnut	Whole plant	Chelerythrine, sanguinarine, umbelliferone-3, aesculetin, dihydroxybenzofuran, and proanthocyanidin	Quinones and polyphenols	Inhibits microbial adhesion and biofilm formation	-	[241,243]
Moringa stenopetala (Moringaceae)	Cabbage tree/ African horse radish tree	Leaves and seeds	-	-	Inhibits biofilm formation	-	[205,210]
Vetiveria zizanioides (Poaceae)	Vetiver (Khus)	Roots	β-vetivenene, vetiselinenol, isovalencenol, vetivenic acid, α-vetivone, and β-vetivone	Terpenoids and polyphenols	Inhibits microbial adhesion and biofilm formation	Inhibits EPS and α-hemolysin toxin production via the agr-QS system	[213,267,268]
Citrus sinensis (Rutaceae)	Sweet orange	Fruits and essential oils from the peel	Limonene, myrcene, α-farnesene, γ-terpinene, α-pinene, and sabinene	Terpenes	Inhibits biofilm formation	-	[213,269]
Pogostemon cablin (Lamiaceae)	Patchouli	Whole plant	α-guaiene, β-caryophyllene, δ-cadinene, pogostol, patchoulol, seychellene, α- and β-patchoulene	Terpenoids, flavonoids, glycosides	Biofilm eradication	Upregulates biofilm-related bacterial genes luxR (inhibitor for the arg-QS system)	[246,270]
Hymenocallis littoralis (Amaryllidaceae)	Spider lily	Leaves	4-methylesculetin, methylisoeugenol, Quercetin 5,7,3′,4′-tetramethyl ether 3-rutinoside	Polyphenols and flavonoids	Inhibits microbial adhesion and biofilm formation	Blocks the active site residues of adhesion proteins	[271,272]
Cymbopogon flexuosus (Poaceae)	Lemon grass	Essential oil	Citral and β-Geranial	Aliphatic aldehydes	Inhibits microbial adhesion and proliferation to disrupt biofilm matrix	Inhibits PIA and arg-QS system	[273,274]
Dracaena cochinchinensis (Asparagaceae)	Chinese dragon’s blood (red resin)	Resin powder	Homo isoflavans and homo isoflavanones	Flavonoids	Disrupts biofilm	Downregulates biofilm regulatory genes saeR, saeS, and hla	[275,276]
Duabanga grandiflora (Lythraceae)	Duabanga	Leaves	F-10 fraction	Alkaloids, tannins, saponins, steroids, glycosides, and flavonoids	Inhibits cell-surface attachment and biofilm formation	Interfere with the agr-QS system and competitive inhibitor of PBP2a	[277,278]
Cymbopogon nardus (Poaceae)	Citronella grass	Essential oil	Geraniol and citronellal	Terpenes	Inhibits microbial adhesion and proliferation	Non-toxic, cytocompatible metabolites as an alternative for future mouthwashes formulations	[279,280]
Chelidonium majus (Papaveraceae)	Great celandine	Whole plant	Chelerythrine, sanguinarine, dihydroxybenzofuran, and proanthocyanidin	Alkaloids	Inhibits cell-surface attachment and biofilm formation	-	[236,243,281]
Ocimum gratissimum (Lamiaceae)	Clove basil	Essential oil and leaves	Eugenol, 1,8-cineole, α-terpineol, γ-terpinene	Alkaloids and terpenes	Inhibits cell-surface attachment and biofilm formation		[282,283]

Note: All abbreviations are listed at the end of the manuscript.

.

Most of the above-conducted experiments are pilot studies to evaluate the effectiveness of these anti-biofilm agents against infectious S. aureus isolates. Furthermore, most of these anti-biofilm agents usually alter the metabolic pathways involved in biofilm adhesion and proliferation. Some studies have also identified potential agents that can disperse the pre-established biofilms on biotic and abiotic surfaces by inhibiting the agr-QS system and related proteins. However, the molecular mechanisms underlying the inhibition of biofilm formation and proliferation need further experimental validation and clinical trials.

5.13. Translational Challenges in the Clinical Development of Natural Anti-Biofilm Agents

Despite the potential in vitro activity, plant-derived anti-biofilm agents have several translational challenges that limit their progression toward clinical use [284]. This translational gap reflects a series of interconnected challenges other than effectiveness of antibiotics. In addition to being a potent biofilm inhibiting agent, these phytochemicals require several standardization and quality control checks to be marked. The lack of phytochemical standardization is the major limitation in clinical development. The chemical composition of these plant extracts can vary depending on the cultivar, species, harvesting season, extraction method, storage conditions, and geographic origin, thereby reducing reproducibility across studies [285]. This variability resulted in inconsistent batch-to-batch activity and variability in dosage and therapeutic efficiency across different studies [286]. Furthermore, most studies rely on crude plant extracts rather than chemically characterized active constituents, which further limits reproducibility and regulatory approval.

Limited understanding of the pharmacokinetic properties of many phytochemicals is another major constraint [285]. Several plant-derived compounds have suboptimal pharmacokinetic properties, including limited aqueous solubility, poor intestinal absorption, rapid metabolism, low systemic bioavailability, and inadequate tissue penetration, thereby limiting their in vivo efficacy [284]. The reported studies do not reflect these physiological characteristics accurately leading to the reduced translation of in vitro findings.

The toxicity and safety evaluation of phytochemicals also remains understudied specifically regarding long-term exposure, high dose administration, and use of combination therapies for systemic treatment [284,287]. The stability of plant-derived phytochemicals is another hurdle, as several bioactive compounds are prone to degradation when exposed to light, temperature, pH, or oxidation, thereby reducing their bioactivity and stability during administration and storage [286]. To overcome these challenges, advanced delivery approaches such as nano formulations, encapsulation, and surface-targeted delivery systems may improve the stability and bioavailability of these phytochemicals. Although these approaches seem promising, they require rigorous safety, toxicological, and pharmacological validations before clinical use [288].

The lack of well-designed clinical trials remains one of the most significant challenges, because most available evidence for plant-derived anti-biofilm agents is still limited to in vitro studies or early preclinical models [289]. Consequently, future development should prioritize standardized extract characterization, identification of active principles, pharmacokinetic and toxicological profiling, optimized formulation, and controlled clinical evaluation for realistic therapeutic options.

6. Conclusions and Future Prospects

S. aureus is included in the World Health Organization’s list of antibiotic-resistant priority pathogens that pose a hazard to human health. Biofilm production is one of the most effective survival strategies used by S. aureus and is extremely difficult to treat using conventional antibiotics. S. aureus biofilm infections are usually managed by either removing the lesions or by administering high doses of systemic/topical antibiotics, necessitating repetitive hospitalization and multiple surgical operations, which ultimately increases the cost and risks of treatment. These challenges highlight the critical importance of finding alternative therapeutics to treat and prevent staphylococcal biofilms. The identification of effective bioactive drugs provides a feasible and cost-effective approach to address the overwhelming threat of antibiotic resistance. Several innovative therapeutic strategies have been proposed to combat these infections, but only a handful have been tested in clinical trials. Most anti-biofilm agents reported in vitro require additional in vivo validation because many of these compounds have significant disadvantages and safety concerns.

Quorum sensing, specifically through the agr system in S. aureus, operates as a genetic “see-saw”. This agr activation triggers virulence toxins (e.g., hemolysins, proteases) for tissue invasion and these proteases also facilitate biofilm dispersal. The downstream effector RNAIII acts as a master regulator of the Agr system by shifting the bacteria from a biofilm-forming state to a tissue-invasive state. Inhibiting the agr system blocks the production of these matrix-degrading enzymes, allowing the biofilm to become thicker, denser, and more resistant to mechanical or chemical clearance. This trade-off often creates a dilemma in clinical settings; while anti-virulence therapies reduce acute toxicity, they can inadvertently promote long-term bacterial persistence. Most of the phytochemicals discussed in this study, including quercetin, ferulic acid, carvacrol, curcumin, tea polyphenols, and salicylic acid derivatives, act directly on the Agr-QS system. These phytochemicals have been shown to downregulate agr and RNAIII expression, resulting in reduced virulence and biofilm formation in preclinical studies. However, disruption of the Agr-locus has been reported to enhance adherence, increase biofilm formation, bacterial persistence, and adaptation to chronic infection. Clinical and experimental studies show that isolates with agr-dysfunction form thicker and robust biofilms, indicating that indiscriminate inhibition of Agr system could be detrimental during chronic stage infections. The therapeutic outcome is highly dependent on the host, infection site, and stage of infection. Recent in vivo studies reported that Agr primarily contributes to virulence rather than biofilm formation, highlighting the complexity of targeting this regulatory system. Nevertheless, phytochemicals that modulate Agr-QS signaling not only attenuate virulence but also disrupt biofilms, providing a more balanced therapeutic strategy to combat S. aureus infections and requiring further validation. Several in vitro studies reported inhibition of both MRSA and MSSA infections in response to treatment with these bioactive derivatives. Additionally, they can also synergistically work with several antibiotics or photodynamic therapies to enhance biofilm disruption in experimental models. Although inhibiting agr alone can promote biofilm stabilization, phytochemicals in combination with traditional antibiotics have been reported with increasing frequency to be effective against treating S. aureus biofilms. By pairing a QS inhibitor with an antibiotic, clinicians aim to achieve virulence attenuation alongside the clearance of persistent, non-dispersing biofilm cells.

Despite substantial progress, several limitations impede the clinical translation and therapeutic applications of plant-derived anti-biofilm compounds. The clinical application of many phytochemicals is restricted due to unfavorable pharmacokinetic properties such as low oral bioavailability, poor aqueous solubility, rapid metabolism, and limited stability under physiological conditions. Moreover, the effective in vitro concentrations required to inhibit biofilm formation may not be achievable in vivo due to potential cytotoxicity. In addition, poor standardization, batch-to-batch variability, and the lack of pharmacokinetic and toxicological studies of plant extracts pose significant challenges to clinical development. Consequently, optimization of active compounds, improved drug delivery systems, and rigorous preclinical validations are needed before these agents can be used as viable therapeutic candidates to prevent and treat S. aureus biofilm-associated infections.