Plant Derived Natural Products against Pseudomonas aeruginosa and Staphylococcus aureus: Antibiofilm Activity and Molecular Mechanisms

Bacteria are social organisms able to build complex structures, such as biofilms, that are highly organized surface-associated communities of microorganisms, encased within a self- produced extracellular matrix. Biofilm is commonly associated with many health problems since its formation increases resistance to antibiotics and antimicrobial agents, as in the case of Pseudomonas aeruginosa and Staphylococcus aureus, two human pathogens causing major concern. P. aeruginosa is responsible for severe nosocomial infections, the most frequent of which is ventilator-associated pneumonia, while S. aureus causes several problems, like skin infections, septic arthritis, and endocarditis, to name just a few. Literature data suggest that natural products from plants, bacteria, fungi, and marine organisms have proven to be effective as anti-biofilm agents, inhibiting the formation of the polymer matrix, suppressing cell adhesion and attachment, and decreasing the virulence factors’ production, thereby blocking the quorum sensing network. Here, we focus on plant derived chemicals, and provide an updated literature review on the anti-biofilm properties of terpenes, flavonoids, alkaloids, and phenolic compounds. Moreover, whenever information is available, we also report the mechanisms of action.


Introduction
The classical concept of microorganisms as solitary entities was revised when it appeared clear that bacteria, as well as fungi, are social organisms able to build complex communities, like biofilms. This condition facilitates survival in adverse conditions, allowing microorganisms to grow and colonize host tissues or inert surfaces, including implants and urinary catheters [1], with adverse effects on human health. Therefore, a great effort is needed to find new drugs able to counteract this phenomenon, with natural products in the hotspot as possible promising candidates.
(modification of surface properties of the biofilm carrier and mechanical stability of the biofilm, application of hydrolytic enzymes disrupting its structure and composition, and others) as well as acting on the regulatory system of its formation (QS and virulence factors) [17].
The aim of this review is to examine the most recent literature in the field of plant-derived natural products as potential novel anti-biofilm agents against P. aeruginosa and S. aureus. We briefly summarize the mechanisms of biofilm formation and QS for both bacteria objects of the present review. The anti-biofilm effects of natural products, mainly relying on the inhibition of formation of the polymer matrix, suppression of cell adhesion and attachment, and decrease of virulence factor production, thereby blocking QS network, are summarized. Furthermore, as mentioned above, biofilm formation is driven by sophisticated regulatory mechanisms, involving events both at singlecell level and at cell population level. Thus, in this paper we will discuss the molecular mechanisms associated with the anti-biofilm effects of terpene, flavonoids, alkaloids, and phenolic compounds.

Quorum Sensing Mechanism
Among the regulatory mechanisms that ensure timely adaptation of microorganisms to the environment, QS is the most studied since it plays a critical role in the formation of biofilm and its surrounding extracellular polymeric substance (EPS). The latter is important to keeping the basic architecture of a biofilm matrix, and forms the defense shield for bacteria inside the biofilm [18]. EPS quantification can directly correlate with the extent of biofilm formation. The EPS protects bacteria from the antimicrobial activity of antibiotics. It comprises 50-90% of the total organic mass of the biofilm and contains exopolysaccharides, extracellular DNA (eDNA), proteins, lipids, and humic substances [19]. Biofilm is not just a barrier to avoid the deleterious effect of antibiotics but also increases bacteria pathogenicity, through the activation of genes that control their virulence. QS is a cell-to-cell communication, depending on density population, and it is differently gene-controlled in gram-negative and gram-positive bacteria, as well as in fungi. It controls the expression of important bacterial genes that encode for virulence factors [20,21]. The QS system is mediated by autoinducers (AIs), identified as oligopeptides and acylated homoserine lactones (AHLs) in gram-positive and gram-negative bacteria, respectively.

QS Molecular Signaling Network of Gram-Positive Bacteria
The Agr system has been identified as the most classical QS system in gram-positive bacteria. It therefore plays a major role in staphylococcal pathogenesis [24]. The Agr locus comprises two divergent transcriptional units, RNAII and RNAIII, containing genes responsible for the production of many virulence factors in Staphylococcus spp ( Figure 2).
Molecules 2020, 25, x 5 of 29 Figure 2. The Agr quorum sensing (QS) system in Staphylococcus spp. The Agr locus comprises two divergent transcriptional units, RNAII and RNAIII, containing genes responsible for the production of many virulence factors in S.aureus. AgrD encodes the precursor of AIP, which is then processed and transported through AgrB. The processed AIP interacts with a histidine sensor kinase receptor AgrC, which in turn leads to the phosphorylation (*P) of AgrA. This leads to the activation of the regulator AgrA, which binds to the chromosomal P2 and P3 promoter regions to upregulate transcription of RNAII and RNAIII. RNAIII can induce upregulation of virulence factor expression as proteases, toxins, and degradative enzymes.
RNAII encodes the core QS circuit protein AgrABCD, whereas RNAIII regulates the expression of multiple virulence genes. AgrB and D are involved in the production of the auto-inducing octapeptides (AIPs) [24]. AgrD encodes the precursor of AIP, which is then processed and transported to the extracellular environment by the integral membrane protease AgrB. When it is released in the environment at high concentration, AIP binds to the kinase receptors (AgrC) on the bacteria divergent transcriptional units, RNAII and RNAIII, containing genes responsible for the production of many virulence factors in S.aureus. AgrD encodes the precursor of AIP, which is then processed and transported through AgrB. The processed AIP interacts with a histidine sensor kinase receptor AgrC, which in turn leads to the phosphorylation (*P) of AgrA. This leads to the activation of the regulator AgrA, which binds to the chromosomal P2 and P3 promoter regions to upregulate transcription of RNAII and RNAIII. RNAIII can induce upregulation of virulence factor expression as proteases, toxins, and degradative enzymes.
RNAII encodes the core QS circuit protein AgrABCD, whereas RNAIII regulates the expression of multiple virulence genes. AgrB and D are involved in the production of the auto-inducing octa-peptides (AIPs) [24]. AgrD encodes the precursor of AIP, which is then processed and transported to the extracellular environment by the integral membrane protease AgrB. When it is released in the environment at high concentration, AIP binds to the kinase receptors (AgrC) on the bacteria membrane, which in turn leads to the phosphorylation of AgrA. This leads to the activation of the regulator AgrA, which binds to the chromosomal P2 and P3 promoter regions to upregulate transcription of RNAII and RNAIII [24]. RNAIII is thus the intracellular effector of the Agr system. Agr can downregulate the expression of cell surface-associated proteins (microbial surface components recognizing adhesive matrix molecules, MSCRAMMs) and upregulate the expression of virulence factors, including toxins (phenol-soluble modulins PSMs, alpha-toxin, delta-toxin (hld), etc.) and degradative exoenzymes (proteases SspA, SspB, Spl, etc.). In addition, Agr induces an increased expression of methicillin resistance genes [25]. Inhibition of AgrA and RNAIII transcription represent an effective strategy for suppressing the virulence of S. aureus.

Anti-Biofilm Activity of Natural Compounds Against Pseudomonas aeruginosa
P. aeruginosa is responsible for a wide range of opportunistic infections, but more complicated to manage are the biofilm-related nosocomial infections, including cystic fibrosis, urinary tract, and eye and burn wounds in immunocompromised patients. Increasing incidents of resistant biofilm infection have resulted in high mortality rates worldwide. Plant-derived anti-biofilm products identified against P. aeruginosa include alkaloids, organosulfur compounds, flavonoids, phenolic compounds, and terpenoids (Figures 3 and 4). A number of natural products have been tested for their anti-biofilm potential using mainly crystal violet, or safranin staining method, the evaluation of QS-related antivirulent activity, as well as the capacity to eradicate preformed-biofilm. The section below describes the molecular mechanisms associated with the anti-biofilm effects of the above-mentioned classes of natural products.

Alkaloids and Nitrogen-Containing Compounds
Alkaloids, a large group of basic (mostly) heterocyclic nitrogen containing natural products, are promising candidates for drug discovery. Hordenin is a dietary phytochemical from sprouting barley, traditionally known for its properties as an antimicrobial compound, inhibitor of monoamine oxidase B, stimulator of gastrin release, and as a vasoconstrictive [26]. Recently, Zhou et al. investigated the properties of hordenine as a QS-inhibitor anti-biofilm agent and as an aminoglycoside antibiotic-accelerant against P. aeruginosa PAO1 [27]. Hordenine reduced AHLs production and, subsequently, biofilm formation, motility, and virulence factors as protease, elastase, rhamnolipid, pyocyanin, and pyoverdine (Tables 1 and 2), all important indicators of QS operon in P. aeruginosa. The authors analyzed the effect of hordenine on the expression of four QS-related genes, that is lasI, lasR, rhlI, and rhlR, in P. aeruginosa PAO1. They observed a significant down-regulation of all genes after exposure to 1.0 mg/mL of hordenine. The interest of these results lies in the ability of hordenine to work as a competitive inhibitor of QS (Table 2), exerting a fine gene regulation of major virulence factors in P. aeruginosa, thus contrasting the infection. Rhamnolipids are a class of glycolipids regulated by the rhl system (Figure 1), and play a vital role in surface motility and biofilm initiation. They are important bacterial surfactants and a key virulence determinant in P. aeruginosa [28]. Rhamnolipids facilitate 3. Anti-Biofilm Activity of Natural Compounds against Pseudomonas aeruginosa P. aeruginosa is responsible for a wide range of opportunistic infections, but more complicated to manage are the biofilm-related nosocomial infections, including cystic fibrosis, urinary tract, and eye and burn wounds in immunocompromised patients. Increasing incidents of resistant biofilm infection have resulted in high mortality rates worldwide. Plant-derived anti-biofilm products identified against P. aeruginosa include alkaloids, organosulfur compounds, flavonoids, phenolic compounds, and terpenoids (Figures 3 and 4). A number of natural products have been tested for their anti-biofilm potential using mainly crystal violet, or safranin staining method, the evaluation of QS-related antivirulent activity, as well as the capacity to eradicate preformed-biofilm. The section below describes the molecular mechanisms associated with the anti-biofilm effects of the abovementioned classes of natural products.       Polyamines are small organic nitrogen-containing compounds, positively charged at the physiological pH required for normal cell growth in both eukaryotes and prokaryotes [32]. Among them, norspermidine displayed remarkable properties, since it can inhibit the formation of P. aeruginosa biofilm and eradicate established biofilms [33]. Norspermidine significantly inhibited the transcription level of lasR/I, rhlR/I, and mvfR, and modulated the QS-related virulence factors (pyocyanin, elastase activity, and protease) [33].

Terpenoids
Terpenes are a wide group of natural compounds characterized by enormous structural diversity, and originating from the coupling of isoprene units. Monoterpenes present in essential oils as well as di-and triperpenoids have long been used as natural medicaments, because of their antimicrobial and anti-biofilm properties. Terpinen-4-ol, the main bioactive constituent of tea tree oil, exhibited QS inhibition at sub-MIC (sub minimum inhibitory concentration) values [36], and reduced the expression of QS genes (lasI, lasR, rhlI, rhlR, rhlAB, lasB, aprA, toxA, and plcH). Further analyses showed the decrease of virulence factors in treated P. aeruginosa PAO1, thus confirming the results of QS gene expression analyses. Moreover, Terpinen-4-ol acted synergistically when used in combination with ciprofloxacin, enhancing the effectiveness of the antibiotic against P. aeruginosa. This feature makes this natural product very remarkable, because the combined use of old drugs in association with a new antimicrobial, able to potentiate or restore their efficacy, appears as a good strategy to safeguard the future effectiveness of critically important antibiotics. Parthenolide ( Figure 3) is a sesquiterpene lactone obtained from Tanacetum parthenium, a plant with well-known medicinal properties, attributable to the active components, sesquiterpenes and sesquiterpene lactones [37]. A study by Kalia et al. [38] demonstrated the ability of parthenolide to contrast P. aeruginosa PAO1 biofilm formation, reducing the production of 3-oxo-C12 HSL. Significant decrease in virulence factors and biofilm formation was observed when P. aeruginosa was treated with a sub-MIC concentration (Table 4) of parthenolide. At this concentration bacterial growth was not affected. Real time PCR demonstrated the down-regulation of autoinducer synthases (lasI, rhlI), as well as their receptors (LasR and RhlR), correlated with the down-regulation of various virulence factors like pyocyanin, protease, and swarming (Tables 3 and 4). All the analyzed virulence factors were reduced to a level equivalent to that of the double negative mutant ∆lasI∆rhlI. The addition of autoinducers restored the virulence phenotypes, thus suggesting that parthenolide might interfere with either the synthesis or the reception of AHL. Finally, molecular docking studies evidenced the binding of parthenolide to the active site of the LasR, which may be responsible for the repression of its expression.
Ghosh et al. [40] investigated the anti-biofilm properties against P. aeruginosa of tormentic acid and 23-hydroxycorosolic acid, two ursane triterpenes isolated from Sarcochlamys pulcherrima (Roxb.) Gaud, an ethnomedicinal plant traditionally used for its anti-microbial and anti-inflammatory properties [48]. Ghosh et al. [40] observed that tormentic and 23-hydroxycorosolic acids (Figure 3) inhibited the growth of planktonic P. aeruginosa MTCC 2488 bacteria at MIC of 55 and 40 µg/mL, respectively, in comparison to untreated control. At sub-inhibitory doses, they did not inhibit bacterial growth, while being effective at reducing biofilm formation. Both compounds significantly increased the membrane potential of P. aeruginosa at the MIC values, enhancing cell membrane damage and, consequently, cell death. Notably, tormentic and 23-hydroxycorosolic acids reduced the swarming motility and the secretion of proteases and pyoverdine (Tables 3 and 4), and in vitro and in vivo toxicity studies suggested that they were non-toxic. It was also observed that the treatment with these two triterpenes significantly reduced the bacterial load on a catheter, as well as in liver and spleen. The authors demonstrated that both triterpenoids reduced lasR, lasI, lasB, rhlI, and rhlR gene expression with respect to the untreated control. These genes are all interconnected and represent a valid tool to verify the QS modulation by natural compounds. The lasB gene encodes the metalloproteinase elastase, an important virulence factor in P. aeruginosa, since a lasB mutation decreases the virulence of the bacterium [49]. It is under the transcriptional control of lasI, which encodes a synthase that leads to formation of 3O-C12-HSL. The latter diffuses toward the surrounding cells initiating QS, interacts lasR with the transcription factor, and activates multiple virulence genes, including lasB. In silico docking studies with proteins, like the las family (lasA, lasI, and lasR), luxR, and pil family (pilB, pilT, and pilY1), showed that tormentic and 23-hydroxycorosolic acids [40], as well as a third ursane triperpene, 23-hydroxytormentic acid from Mussaenda roxburghii [41], have good binding affinity with all the selected proteins.

Organosulfur Compounds
Jakobsen et al. [35] reported the anti-biofilm properties of ajone, an organosulfide which represents a natural remedy for some human diseases. To determine the QSI (QS Inhibitor) activity of ajoene the authors performed fine experiments by using three reporter systems, which contain fusions of the QS-controlled lasB promoter and rhlA promoter to gfp (ASV), encoding an unstable GFP variant in a P. aeruginosa background. The third was a QS reporter system harbored in an E. coli background, where the luxR gene and the promoter region of the luxI were fused to gfp (ASV). Microarray analysis showed that ajoene induced a concentration-dependent down-regulation of a few, but central, QS-controlled virulence genes of P. aeruginosa (lasA, chiC, lecA, rhlA, rhlB, prpL, cbpD), with the best activity at 80 µg/mL. Attempts to repress more genes were successful only with higher concentrations, also affecting cell growth. DNA microarray studies represent an important tool in the investigation of a plethora of QS-regulating genes. Microarray data were confirmed by RT-PCR analysis of two QS-regulated genes lasB and rhlA. Due to rhlA gene down-regulation, the rhamnolipid content was drastically reduced when the cells were treated with 80 µg/mL ajoene (Table 2). Ajoene demonstrated a clear synergistic effect, with tobramycin killing bacteria embedded in biofilm, and inhibited the lytic necrosis of polymorphonuclear leukocytes. Furthermore, during in vivo studies on a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group.
Isothiocyanates, another class of compounds containing sulfur, known for their antimicrobial activity, also showed significant activity in the treatment of biofilm-related infections caused by P. aeruginosa. In particular allylisothiocyanate (AITC), benzylisothiocyanate (BITC), and phenylethylisothiocyanate (PEITC), found in plants such as nasturtium (Tropaeolum majus) and horseradish (Armoracia rusticana), were analyzed on mature and developing biofilms of clinical P. aeruginosa (blood culture isolates, multidrug-resistant (MDR) and extensively drug-resistant (XD) Pa strains from invasive and non-invasive clinical samples) isolated either from clinical patients with signs and symptoms of infection, or from the hospital environment [34]. PEITC was the most effective on the development of P. aeruginosa biofilms (500 µg/mL) while AITC preparations showed effectiveness on established P. aeruginosa biofilms, reducing their metabolic activity (between 200 and 800 µg/mL) to a level comparable to the mixture of all three compounds (ITCM, 500-1000 µg/mL). The combination of isothiocyanates with the antibiotic meropenem showed a synergistic effect, with better results when compared to either preparation alone [34].

Flavonoids
Flavonoids are natural products ubiquitously present in the plant kingdom. They are classified based on the chemical functionalization of the C ring in: flavones (α-β unsaturated ketone), flavanones (ketone at C-4), flavonols (the 3-hydroxy derivative of flavones), and flavan-3-ol (hydroxyl at C-3). These compounds are often also present in glycoside form. Several flavonoids have been evaluated for their anti-biofilm activities, mainly QS-activities. Baicalein (Figure 4) is the most abundant flavone monomer extracted from the roots of Scutellaria baicalensis, and used as a medicine in the Chinese Pharmacopoeia for the treatment of fever, sore throat, and upper respiratory tract infection [50,51]. Baicalein is commercially produced as oral tablets for the treatment of bacteria-induced diarrhea. In addition to its antimicrobial properties baicalein has demonstrated important anti-inflammatory properties [52]. The latter is an important result since a hallmark of P. aeruginosa pulmonary infection is the secretion of various proinflammatory cytokines and a massive recruitment of neutrophils to the infection site. Such excessive inflammatory responses are harmful to the host, contributing to severe tissue damage and organ dysfunction. Therefore, the contemporary administration of an anti-inflammatory drugs is necessary, to slow the progression of chronic infectious diseases by interrupting the infection and inflammation.
Along with anti-QS activity (attenuation of P. aeruginosa virulence factors, including swarming and twitching, and down-regulation of QS-regulated genes transcription) baicalein (128 µg/mL) significantly attenuated IL-1β, IL-6, IL-8, and TNFα secretion at sub-MIC level compared with the PAO1-infected group in the absence of baicalein treatment. At the same concentration, baicalein significantly prevented P. aeruginosa-induced IκBα phosphorylation and the subsequent nuclear translocation and DNA-binding activity of NFκB (p65), compared to the untreated P. aeruginosa PAO1. In summary, the results showed that baicalein represents a promising candidate for combating P. aeruginosa infection, since it can attenuate bacterial pathogenesis by interfering with the QS system, and for its notable anti-inflammatory effect. The flavanones naringenin and taxifolin [53], as well as the flavan-3-ol catechin [54], also showed promising anti-biofilm properties, due to the ability to reduce the production of QS-controlled virulence factors in P. aeruginosa PAO1 (e.g., pyocyanin and elastase) and to modulate the expression of several QS-controlled genes (Tables 5 and 6). Naringin, a glycoside of naringenin, was screened for its capacity to inhibit the QS-controlled factors, and its antibiofilm efficacy by Vandeputte [53], and recently by Dey et al. [55]. Although naringin showed antibiofilm activities, in addition to its combinatorial performances with antibiotics ciprofloxacin and tetracycline [55], RT-PCR showed that this compound did not reduce the expression of any of the selected QS genes (lasI, lasR, lasB, rhlI, rhlR, rhlA, and aceA) [53].  1 Down arrow (↓) indicates decrease of tested activity with respect to the control (ctr). Tested concentrations are reported in parenthesis. White box shows a not performed assay. 3-oxo-C12-HSL AIs N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL).
Thanks to the low risk and contextual multitargeted actions, the combination of nanoparticles (NPs) and natural compounds has gained a lot of attention in biomedical applications [58]. Zinc and copper play vital roles in several biological processes. Due to their biomedical applications and selective binding to phytochemicals, zinc oxide nanoparticles or zinc and copper thin film techniques are becoming attractive in biomedical applications. Recently, flavonoid-loaded nanoparticles were assessed for their anti-biofilm properties in order to evaluate the potential antibacterial effects, in comparison to the parent flavonoid. In particular, the use of dual drug-like molecules (rutin-benzamide) loaded in a poly vinyl alcohol (PVA) surface modified single nanocarrier (PEG−PLGA) represents a potential anti-biofilm therapy, based on interesting results in term of EPS reduction as well as the extent (%) of biofilm inhibition compared to the control [59]. In addition to pure flavonoids, crude extracts containing flavonoid derivatives as principal constituents also attenuated QS-mediated virulence and biofilm formation. In particular, the binding affinity of mosloflavone for RhlR, detected in the methanolic extract of Plectranthus tenuiflorus [60], was observed to be comparatively higher than its natural ligand, while kaempferol constituted the major constituent of Centella asiatica, a herb with proven anti-QS properties [61].

Other Phenolic Compounds
Curcumin, present in the rhizome of turmeric (Curcuma longa L.), has many properties and a long-term use in traditional Indian medicine as an antimicrobial agent [62]. Anti-biofilm properties (see Table 7) at sub-MIC concentration are ascribed to curcumin, that down-regulate the P. aeruginosa PAO1 QS system and related virulence factor (pyocyanin, protease and elastase, Table 8) [63]. To overcome its poor water solubility, and enhance its antimicrobial properties, curcumin has been loaded onto zinc oxide nanoparticles (ZnO-NCs), excellent drug carriers due to their low toxicity and biodegradable nature [64]. This considerably improved the anti-QS effect of curcumin against P. aeruginosa PAO1. ZnC-NCs suppressed the LasR-RhlR transcriptional activators and was capable of triggering ROS generation. The ZnC-NC-induced O 2− generation was responsible for its anti-biofilm effect against P. aeruginosa PAO1. Molecular docking analysis confirmed the molecular mechanism, showing how curcumin better fits inside the binding site of LasR protein (−5.9730) and RhlR protein (−6.5435).
Another natural product showing good antibacterial and anti-biofilm properties against P. aeruginosa (MTCC 424, MTCC 2488) is the naphthoquinone plumbagin [65]. This compound has been used as a traditional medicine in India for its antiparasitic, antioxidant, anticancer, and antimicrobial properties, and can be isolated from the roots of Plumbaginaceae plants [66]. It was demonstrated that plumbagin alone, and in combination with gentamicin, significantly reduced the secretion of virulent enzymes and virulence factors against both strains of P. aeruginosa. The expression of lasB, lasI, and lasR genes was also significantly reduced following plumbagin treatment of P. aeruginosa MTCC 424 and MTCC 2488, at 250 and 150 µg/mL, respectively. In addition, plumbagin showed a synergistic interaction with gentamicin. This combinatorial approach, which represents a novel strategy for the reduction of biofilm formation by P. aeruginosa, also encourages the use of existing antibiotics at lower doses. Plumbagin's mechanism of action was assessed by protein-ligand docking analysis. The compound showed good affinity for the ligand binding site of Las family and Pil family proteins: the former is related to QS, while the latter to pilus assembly. This result led the authors to hypothesize that plumbagin may affect pilus assembly, inhibiting the QS and swarming motility.

Anti-Biofilm Properties of Natural Compounds against Staphylococcus aureus
Staphylococcus aureus is a gram-positive pathogen, frequently the cause of biofilm-associated infections on indwelling medical devices [77]. Like for P. aeruginosa, staphylococcal biofilms show enhanced resistance toward antibiotics and the immune response, thus representing an important therapeutic challenge in clinics worldwide. A recent study has already provided an accurate overview of natural products isolated from plants and microorganisms with activity against the major virulence factors of S. aureus [78]. In this section, we report the latest updates and, when the information is available, the molecular mechanisms associated with the anti-biofilm effects of terpenes, flavonoids, and phenolic compounds (Figures 5 and 6, Tables 9-11).
information is available, the molecular mechanisms associated with the anti-biofilm effects of terpenes, flavonoids, and phenolic compounds (Figures 5 and 6, Tables 9-11).

Terpenes
Among monoterpenes, 1,8-cineole ( Figure 5) and carvacrol [79,80] were shown to act against biofilm formation, while eugenyl acetate was active against alfa-hemolysin [81]. The sesquiterpene (+)-nootkatone is present in essential oils from Alaska yellow cedar trees, some herbs, and grapefruit. It has been approved by the Food and Drug Administration (FDA) as a flavoring agent in citrus-flavored foods and beverages. Farha et al. [82] demonstrated that (+)nootkatone at 200 µg/mL significantly disrupted S. aureus preformed biofilm, and reduced the viability of cells within matured biofilm, suggesting that the compound penetrates through the biofilm. Additionally, the molecular analysis showed that (+)-nootkatone suppressed the expression levels of sarA, icaA, agrA, RNAIII, and spa; major genes involved in biofilm formation. The compound was also able to inhibit the sliding motility of S.aureus, thus contrasting the initial phase of bacterial   Figure 6. Chemical structures of flavonoids active against S. aureus.
The sesquiterpene (+)-nootkatone is present in essential oils from Alaska yellow cedar trees, some herbs, and grapefruit. It has been approved by the Food and Drug Administration (FDA) as a flavoring agent in citrus-flavored foods and beverages. Farha et al. [82] demonstrated that (+)-nootkatone at 200 µg/mL significantly disrupted S. aureus preformed biofilm, and reduced the viability of cells within matured biofilm, suggesting that the compound penetrates through the biofilm. Additionally, the molecular analysis showed that (+)-nootkatone suppressed the expression levels of sarA, icaA, agrA, RNAIII, and spa; major genes involved in biofilm formation. The compound was also able to inhibit the sliding motility of S.aureus, thus contrasting the initial phase of bacterial surface colonization and biofilm formation. Moreover, up to 50 µg/mL, sub-MIC concentration, at which the inhibition of biofilm formation was observed, (+)-nootkatone was non-toxic to normal fibroblast cells.
The diterpenes, salvipisone and aethiopinone, isolated from hairy roots of Salvia sclarea, showed activity against methicillin-resistant S. aureus. They reduced the resistance to the antibiotic oxacillin, and caused a reduction of the biofilm biomass, as well as the disruption of the biofilm structure [83]. The triterpene celestrol (Table 9) was shown to inhibit biofilm formation, and to possess antimicrobial activity against S. aureus ATCC 29,213 (a reference strain of methicillin-sensitive S. aureus (MSSA)) and a clinical methicillin-resistant S. aureus (MRSA) isolate [84]. The compound was not only active on planktonic cells (with a MIC of 2 µM and a MBC of 32 µM), but it was also effective in dispersing preformed biofilms of the clinical MRSA isolates, as evaluated by confocal laser scanning microscopy. Furthermore, it inhibited the secretion of EPS, which are crucial for the formation of the matrix that facilitates the adherence of these microorganisms on the target surfaces. Therefore, the compound has a great potential, since it is not only inhibiting to the formation of biofilms, but it can further act by eradicating preformed biofilms, while also being active on the planktonic cells. However, this compound also showed a certain cytotoxicity against hFOB 1.19 cells (osteoblast).
Ursolic acid (Table 9) was active against the formation of biofilm by S. aureus subsp. aureus COL, a MRSA strain, resistant to several antibiotics, including penicillin and tetracycline. The RNA-Seq-based transcriptome analysis showed that ursolic acid reduces the metabolism of some amino acids and the expression of adhesins [85].
A mixture of triterpenoid saponins, known as Bacoside A ( Figure 5), was reported for its antimicrobial and anti-biofilm activity against S. aureus MTCC 96. It is very likely that these saponins alter the structure and permeability of the bacterial cell membrane. Furthermore, Bacoside A, also dispersed preformed biofilm. The treated biofilm showed altered cell structure and a loss of EPS that caused biofilm dispersion [43].

Flavonoids
Among the flavonoids, baicalein (which is also active against P. aeruginosa) was active against the QS system, by inhibiting the transcription of AgrA and RNAIII, and inhibits biofilm formation [88]. The biofilm formation was also inhibited by myricetin [89]. Myricetin, quercetin, farrerol, isorhamnetin, dracorhodin, lysionotin, diosmetin, silibinin, apigenin, epicallocatechin gallate, oroxylin A, and baicalin ( Figure 6) were active against alfa-haemolysin [89][90][91][92][93][94][95][96][97][98][99][100]. The flavonoid rutin showed a concentration dependent reduction of biofilm formation (Table 10). However, it did not significantly decrease the biomass, while it reduced the secretion of EPS. Therefore, it probably acts by interfering with the adhesion, and with all the other functions, of EPS [101]. Pro-antocyanidin A2 inhibited de-novo biofilm formation, without showing bactericidal activity, nor inhibiting activity on planktonic growth. Furthermore, it also appeared to have no activity on mature biofilm [86]. Two flavonoids isolated from Teucrium polium, namely 3 ,4 ,5-trihydroxy-6,7-dimethoxyflavone and 5,6,7,3 ,4 -pentahydroxyflavone, inhibited biofilm growth of Staphylococcus aureus (Table 10) AH133 strain [102] A recent study explored the capacity of kaempferol to inhibit S. aureus biofilm formation, and the associated potential molecular mechanisms [103]. Kaempferol inhibited the attachment phase of biofilm formation, by reducing S. aureus adhesion, since its action was evident only if added immediately after the inoculation of bacteria to plates. This was mediated by blocking the activity of Sortase A (SrtA), an enzyme essential in the anchoring of surface proteins to the cell wall of gram-positive bacteria. This has important consequences for the onset of acute infection by S. aureus, since the bacteria cannot display functional surface adhesins in the cell wall envelope. In addition, the authors analyzed the expression of adhesion-related genes. They demonstrated that the compound reduced the expression of clfA and clfB, which encode clumping factor A (ClfA), and ClfB, fnbA, and fnbB which encode fibronectin-binding proteins (FnbpA and FnbpB), and sarA, a global regulator gene that is closely related to biofilm formation, and positively regulates fnbA and fnbB. The results reported suggest that kaempferol represents a potential compound with a novel mechanism of biofilm inhibition.
Besides these, gallic and ferulic acid were also tested for their activity against S. aureus, although they were active only at relatively high concentrations. Only ferulic acid completely inhibited colony spreading. Furthermore, it was hypothesized that changes in motility could affect the ability of the bacteria to form a biofilm [71] The tannin, hamamelitannin, was shown to inhibit the quorum sensing regulator RNAIII [110,111], while punicalagin was active against α-hemolysin [112], and exerted a remarkable inhibitory effect on biofilm formation [113]. The activity of punicalagin against S. aureus was further investigated, with the aim of understanding the possible mode of action. Punicalagin exhibited a MIC of 0.25 mg/mL and induced morphological damage to the cell membrane, also inducing an efflux of potassium.
Tannic acid showed antibacterial and anti-biofilm formation activity, although further studies are needed to understand the mechanism of action [114].
1,2,3,4,6-Penta-O-galloyl-d-glucopyranose (PGG) prevented biofilm formation at 6.25 µM of several strains of S. aureus, while showing no bactericidal activity at this concentration [115]. Arylbenzylfuran was active against clinical strains of methicillin-resistant S. aureus (MRSA), and was able to induce a significant reduction in S. aureus ATCC 12600S biofilm viability [116]. Table 11. Phenolic compounds inhibiting S. aureus biofilm formation and production of virulence Figure 1. Several aromatic polyketides isolated from plants have been reported, in particular aloe-emodin [117], acting against the Agr quorum-sensing system. The same compound and the structurally related rhein [117] were able to inhibit biofilm formation.

Compounds
Finally, noteworthy is the activity of capsaicin, which acts against α-hemolysin by suppressing the expression of Hla and AgrA [95].

Conclusions
Biofilms represent one of the most successful strategies used by bacteria to increase their survival in terms of resistance to antibiotics and antimicrobial agents. If biofilm forming microorganisms are a big challenge, even more complex is the fight against polymicrobial biofilms, like the ones formed by S. aureus and P. aeruginosa. Therefore, finding new anti-biofilm chemicals is crucial. In this context, plants are an extraordinarily rich source of compounds endowed with several different biological activities, including antimicrobial and antibiofilm properties. These compounds often act via modes of action that are different than the ones of currently used antibiotics, thus also offering a tool for combating antibiotic resistance.
Many studies have been published on the topic in recent years, and the latest advances in the discovery of plant-derived natural products with anti-biofilm properties against P. aeruginosa and S. aureus have been herewith reviewed. Knowing the molecular mechanisms underlying the biological activity is very important, especially if these compounds are to be further studied for possible applications. Therefore, when known, the molecular mechanisms were also herewith reported and discussed, with the aim of providing a clear overview of the state of the art.
Funding: This research received no external funding.