*5.3.1 Derivatives of shikimic acid, phenols, and polyphenols*

Many phenolic compounds and derivatives with anti-QS and antibiofilm activities have been isolated from plants [79]. Cinnamaldehyde [the dominant compound of certain essential oils, in particular *Cinnamomum camphora* (L.) J. Presl] and its derivatives modulate a wide range of anti-QS and antibiofilm activities of *P. aeruginosa* [80–82]. *Curcuma longa* L. produces curcumin, which inhibits the expression of virulence genes of *P. aeruginosa* PA01 [83].

Ellagic acid derivatives from *Terminalia chebula* Retz. downregulate *lasIR* and *rhlIR* genes expression and decrease AHLs production, leading to an attenuation of virulence factor production and to an enhanced sensitivity of biofilm facing a tobramycin treatment [84].

Flavonoids have been investigated for their roles as QS modulating compounds. From these, naringenin and taxifolin reduced the expression of several QS-controlled genes (i.e., *lasI, lasR, rhlI, rhlR, lasA, lasB, phzA1*, and *rhlA*) in *P. aeruginosa* PAO1. Similarly, the flavan-3-ol catechin, extracted from the bark of *Combretum albiflorum* (Tul.) Jongkind, reduces the production of QS-dependent virulence factors, such as pyocyanin, elastase, and the formation of biofilm by *P. aeruginosa* PAO1 [85]. Interestingly, baicalin, an active natural compound extracted from the traditional Chinese medicinal *Scutellaria baicalensis*, has been demonstrated to inhibit the formation of *P. aeruginosa* biofilms and enhance the bactericidal effects of antibiotics such as amikacin. Moreover, at sub-minimal inhibitory concentration (256 μg/mL), this flavonoid has been shown to reduce LasA protease, LasB elastase, pyocyanin, rhamnolipids, and exotoxin A production and to downregulate the three QS-regulatory genes, including *lasI, lasR, rhlI, rhlR, pqsR*, and *pqsA* [86]. Consistently, *in vivo* experiments indicated that baicalin treatment reduces *P. aeruginosa* pathogenicity in *Caenorhabditis elegans* and enhances the clearance of *P. aeruginosa* from the peritoneal implants of infected mice.

Furocoumarins from grapefruit can inhibit the QS signaling (AHLs and AI-2) of *V. harveyi* BB886 and BB170 strains as well as biofilm formation in pathogens such as *E. coli* O157:H7, *Salmonella typhimurium* and *P. aeruginosa* [87]. These purified furocoumarins (dihydroxybergamottin and bergamottin), tested at the concentration of 1 μg/mL, cause 94% inhibition of autoinducers (AHLs) without affecting

bacterial viability. Biofilm inhibition was up to 58.3 and 72%, respectively, for *E. coli* O157:H7 but modest for *P. aeruginosa* (27.3 and 18.1%, respectively).

Malabaricone C, a diarylnonanoid isolated from the bark of *Myristica cinnamomea* King inhibited the QS-regulated pyocyanin production and biofilm formation in *P. aeruginosa* PAO1 [88].

A screening of various herbs revealed that a clove extract [*Syzygium aromaticum* (L.) Merr. Et Perry] inhibits QS-controlled gene expression (*las* and PQS systems) in *P. aeruginosa* with eugenol as major active constituent [89]. Recently, the effects of eugenol and its nanoemulsion on *P. aeruginosa* QS-mediated virulence factors and biofilm formation have been identified by Lou et al. [90] at a 0.2 mg/mL concentration. Similarly, the anthraquinone emodin from *Rheum palmatum* L., a traditional Chinese medicinal plant, was found to inhibit the *P. aeruginosa* biofilm formation at 20 μM, increasing the antibiotic activity of ampicillin [91]. Finally, the 6-gingerol, isolated from fresh ginger oil, reduces the production of several virulence factors, decreasing the mortality induced in mice by *P. aeruginosa*. A DNA microarray analysis revealed that the application of the 6-gingerol on biofilm-encapsulated cells downregulates several QS-related genes, notably those involved in the production of rhamnolipids, elastase, pyocyanin, all of which are involved in biofilm formation [92].

### *5.3.2 Alkaloids*

Recently, caffeine (a purine alkaloid) has been shown to inhibit AHLs production and swarming mobility in *P. aeruginosa* PAO1 without causing AHLs degradation [93].

### *5.3.3 Terpenoids and Triterpenoids*

The pentacyclic triterpenoid ursolic acid was identified as an inhibitor of biofilm formation from *Diospyros dendo* Welw, the tree used for ebony from Gabon, Africa [94]. Tested at a dose of 10 μg/mL, ursolic acid reduces biofilm formation by 79% in *E. coli* and 57–95% in *V. harveyi* and *P. aeruginosa* PAO1. Similarly, oleanolic acid inhibits the *in vitro* biofilm formation by *S. aureus* and *P. aeruginosa* [95]. However, these triterpenoids showed no inhibitory effect on QS mechanisms contrarily to triterpenoid coumarate esters isolated from *Dalbergia trichocarpa*, a tropical legume from Madagascar. Indeed, oleanolic aldehyde coumarate at 200 μM inhibits the formation/maintenance of *P. aeruginosa* PAO1 biofilm and the expression of the *las* and *rhl* QS systems as well as *gacA* gene [96]. Consequently, the production of QS-controlled virulence factors, including, rhamnolipids, pyocyanin, elastase, and extracellular polysaccharides, as well as twitching and swarming motilities is reduced. Other African plants harbor terpenoids and triterpenoids with antivirulence properties. Indeed, cassipourol and β-sitosterol (both at 100 μM), isolated from *Platostoma rotundifolium* (Briq.) A. J. Paton, a Burundian medicinal plant, inhibit quorum sensing-regulated and -regulatory gene expression in *las* and *rhl* systems. These triterpenoids can still disrupt the formation of biofilms at concentrations down to 12.5 and 50 μM [97].

### *5.3.4 Isothiocyanates and organosulfur compounds*

Isothiocyanates produced by many plants are also QS inhibitors in *P. aeruginosa* PAO1. For example, iberin, isolated from horseradish (*Armoracia rusticana* G. Gaertn et al.), specifically blocks the expression of QS-regulated genes in *P. aeruginosa* PAO1 at the concentration of 100 μM; its impact on biofilm formation has not been investigated [98]. Sulforaphane and erucin, two isothiocyanates isolated from

**43**

*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting…*

broccoli, inhibit the *P. aeruginosa* PAO1 *las* and *rhl* system as well as biofilm forma-

A further compound known to affect the QS-regulated genes in *P. aeruginosa*, including the rhamnolipids production, is ajoene, an allyl sulfide isolated from *Allium sativum* L. Ajoene, at the concentration of 100 μg/mL and combined with the antibiotic tobramycin, leads to killing of biofilm-encapsulated *P. aeruginosa*. In a mouse model of pulmonary infection, this synergy improves the clearance of *P. aeruginosa* from lungs [100]. The S-phenyl-L-cysteine sulfoxide and its derivatives, notably diphenyl disulfide, have shown a significant impact on the biofilm formation by *P. aeruginosa* [101]; the sulfoxide derivative seems to interfere with both *las*

A series of studies have indicated that marine organisms are a potential source of anti-QS [102–104]. The halogenated furanones produced by the red alga *Delisea pulchra* inhibit QS-induced activities in bacteria by competing with AHL signals related to their receptor site (LuxR) [104]. This protein-ligand binding is destabilized, causing rapid receptor recycling [102]. Inspired from natural compounds, the halogenated furanones C-30 and C-56 have been demonstrated to exhibit biofilm

Following a screening of 284 extracts from the marine sponge *Luffariella variabilis*, 36 extracts were revealed as inhibitors of *P. aeruginosa* QS, targeting the *las* system [103]; from these, the sesterterpenoids manoalide displays antibiofilm activities. Note that this molecule does not generate bactericidal effects on *P. aeruginosa* [103], but presents an antibiotic activity against Gram-positive

Type I porcine kidney acylase inactivates QS signals such as C6-HSL and 3-oxo-

Mammalian cells release enzymes called paraoxonases 1 (extracted from human and murine sera) that have lactonase activity; degrading *P. aeruginosa* AHLs. They prevent, in an indirect way, QS and biofilm formation [109]. Similarly, human epithelial cells and particularly human respiratory epithelia have the capacity to inactivate a *P. aeruginosa* QS signal by inactivating AHLs (3-oxo-C12HSL) produced by *P. aeruginosa* [108, 110]. However, the enzyme or enzyme-like compound involved in acyl-homoserine lactone inactivation have not been identified and characterized yet. Recently, Losa et al. [111] demonstrated that polarized airway epithelial monolayers, in contrast to nonpolarized cells, are also able to degrade 3-oxo-C12-HSL using membrane-associated paraoxonase 2 that catalyzes the

C12-HSL but not C4-HSL [50]. This type I acylase moderately reduces biofilm formation in *Aeromonas hydrophila*, *P. putida*, and probably *P. aeruginosa* [107]. This degradation is dependent on the length of the acyl chain, since only C6-HSL and

and *rhl* systems whereas the diphenyl sulfide only disturbs the *las* system.

reduction and target the *las* and *rhl* systems in *P. aeruginosa* [105].

*DOI: http://dx.doi.org/10.5772/intechopen.90833*

**5.4 From marine organisms**

*5.4.1 Furanones*

*5.4.2 Terpenoids*

bacteria [106].

*5.5.1 Enzymes*

**5.5 From animals and human**

3-oxo-C12-HSL are degraded [108].

opening of the lactone ring.

tion at concentrations of 50 and 100 μM, respectively [99].

*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting… DOI: http://dx.doi.org/10.5772/intechopen.90833*

broccoli, inhibit the *P. aeruginosa* PAO1 *las* and *rhl* system as well as biofilm formation at concentrations of 50 and 100 μM, respectively [99].

A further compound known to affect the QS-regulated genes in *P. aeruginosa*, including the rhamnolipids production, is ajoene, an allyl sulfide isolated from *Allium sativum* L. Ajoene, at the concentration of 100 μg/mL and combined with the antibiotic tobramycin, leads to killing of biofilm-encapsulated *P. aeruginosa*. In a mouse model of pulmonary infection, this synergy improves the clearance of *P. aeruginosa* from lungs [100]. The S-phenyl-L-cysteine sulfoxide and its derivatives, notably diphenyl disulfide, have shown a significant impact on the biofilm formation by *P. aeruginosa* [101]; the sulfoxide derivative seems to interfere with both *las* and *rhl* systems whereas the diphenyl sulfide only disturbs the *las* system.

### **5.4 From marine organisms**

### *5.4.1 Furanones*

*Bacterial Biofilms*

*5.3.2 Alkaloids*

degradation [93].

*5.3.3 Terpenoids and Triterpenoids*

trations down to 12.5 and 50 μM [97].

*5.3.4 Isothiocyanates and organosulfur compounds*

in *P. aeruginosa* PAO1 [88].

bacterial viability. Biofilm inhibition was up to 58.3 and 72%, respectively, for *E. coli*

Malabaricone C, a diarylnonanoid isolated from the bark of *Myristica cinnamomea* King inhibited the QS-regulated pyocyanin production and biofilm formation

A screening of various herbs revealed that a clove extract [*Syzygium aromaticum* (L.) Merr. Et Perry] inhibits QS-controlled gene expression (*las* and PQS systems) in *P. aeruginosa* with eugenol as major active constituent [89]. Recently, the effects of eugenol and its nanoemulsion on *P. aeruginosa* QS-mediated virulence factors and biofilm formation have been identified by Lou et al. [90] at a 0.2 mg/mL concentration. Similarly, the anthraquinone emodin from *Rheum palmatum* L., a traditional Chinese medicinal plant, was found to inhibit the *P. aeruginosa* biofilm formation at 20 μM, increasing the antibiotic activity of ampicillin [91]. Finally, the 6-gingerol, isolated from fresh ginger oil, reduces the production of several virulence factors, decreasing the mortality induced in mice by *P. aeruginosa*. A DNA microarray analysis revealed that the application of the 6-gingerol on biofilm-encapsulated cells downregulates several QS-related genes, notably those involved in the production of rhamnolipids, elastase, pyocyanin, all of which are involved in biofilm formation [92].

Recently, caffeine (a purine alkaloid) has been shown to inhibit AHLs production and swarming mobility in *P. aeruginosa* PAO1 without causing AHLs

The pentacyclic triterpenoid ursolic acid was identified as an inhibitor of biofilm formation from *Diospyros dendo* Welw, the tree used for ebony from Gabon, Africa [94]. Tested at a dose of 10 μg/mL, ursolic acid reduces biofilm formation by 79% in *E. coli* and 57–95% in *V. harveyi* and *P. aeruginosa* PAO1. Similarly, oleanolic acid inhibits the *in vitro* biofilm formation by *S. aureus* and *P. aeruginosa* [95]. However, these triterpenoids showed no inhibitory effect on QS mechanisms contrarily to triterpenoid coumarate esters isolated from *Dalbergia trichocarpa*, a tropical legume from Madagascar. Indeed, oleanolic aldehyde coumarate at 200 μM inhibits the formation/maintenance of *P. aeruginosa* PAO1 biofilm and the expression of the *las* and *rhl* QS systems as well as *gacA* gene [96]. Consequently, the production of QS-controlled virulence factors, including, rhamnolipids, pyocyanin, elastase, and extracellular polysaccharides, as well as twitching and swarming motilities is reduced. Other African plants harbor terpenoids and triterpenoids with antivirulence properties. Indeed, cassipourol and β-sitosterol (both at 100 μM), isolated from *Platostoma rotundifolium* (Briq.) A. J. Paton, a Burundian medicinal plant, inhibit quorum sensing-regulated and -regulatory gene expression in *las* and *rhl* systems. These triterpenoids can still disrupt the formation of biofilms at concen-

Isothiocyanates produced by many plants are also QS inhibitors in *P. aeruginosa* PAO1. For example, iberin, isolated from horseradish (*Armoracia rusticana* G. Gaertn et al.), specifically blocks the expression of QS-regulated genes in *P. aeruginosa* PAO1 at the concentration of 100 μM; its impact on biofilm formation has not been investigated [98]. Sulforaphane and erucin, two isothiocyanates isolated from

O157:H7 but modest for *P. aeruginosa* (27.3 and 18.1%, respectively).

**42**

A series of studies have indicated that marine organisms are a potential source of anti-QS [102–104]. The halogenated furanones produced by the red alga *Delisea pulchra* inhibit QS-induced activities in bacteria by competing with AHL signals related to their receptor site (LuxR) [104]. This protein-ligand binding is destabilized, causing rapid receptor recycling [102]. Inspired from natural compounds, the halogenated furanones C-30 and C-56 have been demonstrated to exhibit biofilm reduction and target the *las* and *rhl* systems in *P. aeruginosa* [105].

## *5.4.2 Terpenoids*

Following a screening of 284 extracts from the marine sponge *Luffariella variabilis*, 36 extracts were revealed as inhibitors of *P. aeruginosa* QS, targeting the *las* system [103]; from these, the sesterterpenoids manoalide displays antibiofilm activities. Note that this molecule does not generate bactericidal effects on *P. aeruginosa* [103], but presents an antibiotic activity against Gram-positive bacteria [106].

### **5.5 From animals and human**

### *5.5.1 Enzymes*

Type I porcine kidney acylase inactivates QS signals such as C6-HSL and 3-oxo-C12-HSL but not C4-HSL [50]. This type I acylase moderately reduces biofilm formation in *Aeromonas hydrophila*, *P. putida*, and probably *P. aeruginosa* [107]. This degradation is dependent on the length of the acyl chain, since only C6-HSL and 3-oxo-C12-HSL are degraded [108].

Mammalian cells release enzymes called paraoxonases 1 (extracted from human and murine sera) that have lactonase activity; degrading *P. aeruginosa* AHLs. They prevent, in an indirect way, QS and biofilm formation [109]. Similarly, human epithelial cells and particularly human respiratory epithelia have the capacity to inactivate a *P. aeruginosa* QS signal by inactivating AHLs (3-oxo-C12HSL) produced by *P. aeruginosa* [108, 110]. However, the enzyme or enzyme-like compound involved in acyl-homoserine lactone inactivation have not been identified and characterized yet. Recently, Losa et al. [111] demonstrated that polarized airway epithelial monolayers, in contrast to nonpolarized cells, are also able to degrade 3-oxo-C12-HSL using membrane-associated paraoxonase 2 that catalyzes the opening of the lactone ring.

### *5.5.2 Alkaloids*

The *P. aeruginosa* pyocyanin production is inhibited by a molecule found and isolated from the ant *Solenopsis invicta*, the piperidine alkaloid Solenopsin A alkaloid. The biofilm formation is also reduced in a dose-dependent manner. This molecule probably disrupts the signals from the *rhl* system [112].

## **6. Concluding remarks**

This review presents natural compounds reported to exhibit anti-QS and antibiofilm properties against *P. aeruginosa* (summarized in **Table 1**); these highlight the great potentiality of living organisms as reservoir of compounds susceptible to modulate virulence mechanisms without affecting bacterial viability. Overall, it appears that prokaryotes as well as animals and humans are sources for enzymes that degrade or antagonize AHLs, whereas plants harbor larger panels of anti-QS and antibiofilm compounds with very diverse chemical structures, including alkaloids, organosulfurs, phenolics, and terpenoids. Contrarily to animals and humans, plants are not able to deploy elaborate defense through humoral and cell-mediated immunity (antibodies and phagocytes) to struggle against bacterial invasions [113]. Plants immune defenses rely on the secretion of antibacterial compounds (bactericide and/or bacteriostatic agents [114]), including resistance modulating compounds [115] (e.g., inhibitors of efflux pumps [116]), and mostly on their ability to recognize molecules released from pathogens through plant cell surface receptors. This recognition triggers specific signaling cascades, activating series of defense responses, including the synthesis of antimicrobial lytic proteins, enzymes, phytoalexins, and other secondary metabolites. Some of these exert nonmicrobicidal antivirulence properties [117, 118]. Finally, marine organisms and fungi produce also bioactive secondary metabolites (halogenated furanones and antibiotics, respectively) and other original and promising compounds, such as terrein which was identified as the first dual inhibitor of QS and c-di-GMP signaling at 30 μM.

The increasing presence of antibiotic-resistant bacteria certainly pushes scientists to reorient the strategy of fight against bacterial infections to defer entry into a post-antibiotic era where major antibiotics would not be effective even for banal infections. Antivirulence approaches and antivirulence drugs are being increasingly considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. For example, oleanolic aldehyde coumarate and cassipourol, anti-QS compounds, exert interesting antibiofilm properties, restoring the effectiveness of the antibiotic tobramycin in the clearance of biofilm-encapsulated *P. aeruginosa* (**Figure 4**); also the association between biofilm formation and antimicrobial resistance has been highlighted in carbapenem-resistant *P. aeruginosa* [119]. Such nonmicrobicidal drugs inhibit virulence factors essential for establishing infection and pathogenesis through targeting nonessential metabolic pathways which should not lead to activation of bacterial evasion mechanisms. This approach should reduce the selective pressure and consequently could slow down the development of resistance. Compounds that target QS may be particularly interesting as they impact planktonic and biofilm lifestyles, by reducing at the same time the production of virulence factors and the generation of biofilms. This should lead to less severe infections at levels that can be cleared by the host's immune defense and with increased activity of antibiotics.

Despite these important prospects, however, the big breakthrough in antibacterial strategies is still out of reach. This is probably due to a very complex

**45**

*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting…*

Fungi *Penicillium* species [66] Penicillic acid (Furanone) LasR and

**Origin Compounds (class) Target (QS) Synergy** 

Acetic acid, lactic acid, phenyl lactic acid

(alkylcyclopentanone)

derivative

halogenated furanones and

Cassipourol (terpenoid), β-sitosterol (terpenoid)

Oleanolic aldehyde Coumarate (Phenolic compound)

Ellagic acid derivatives (Phenolic compound)

*Allium sativum* L. [100] Ajoene (Organosulfur) *las* and *rhl*

compound)

*Rheum palmatum* L. [91] Emodin (Anthraquinone) docking

6-gingerol (Phenolic compound)

Bergamottin and dihydroxybergamottin (Furocoumarins)

AHL-acylase (Enzyme) AHL

Patulin (Furopyranone) LasR and

Erythromycin (Macrolide) *rhl system*

AHL-lactonase (Enzyme) NC

**with antibiotics**

NC

NC

NC

+1

NC

NC

+1

+1

NC

+1

+1

NC

+1

NC

NC

NC

+2

+1

NC

degradation

AHL antagonist

RhlR

RhlR<sup>ǂ</sup>

and *GacA*

LasR and RhlR antagonist; c-di-GMP

AHL antagonist

*las* and *rhl* systems

systems

systems

systems

systems

AHLs inhibition

AHLs inhibition

traR\*

PQS systems

docking lasR

*las* and *rhl* systems

*las* and *rhl* systems

Manoalide (Sesterterpenoid) *las* system NC

Catechin (Flavonoid) *las* and *rhl*

Iberin (Isothiocyanate) *las* and *rhl*

Eugenol (Phenylpropanoid) *las* and PQS

Baicalin (Flavonoid) *las*, *rhl* and

*DOI: http://dx.doi.org/10.5772/intechopen.90833*

*pumilus, B*. sp. [60]; *Erythrobacter, Labrenzia, Bacterioplanes* [59]

*Lactobacillus paracasei subsp. Paracasei* [64]*; Pediococcus acidilactici*

*Saccharopolyspora erythraea* [68]

*Delisea pulchra* [102, 104]

Plants *Platostoma rotundifolium*

*Luffariella variabilis* (Polejaeff, 1884) [103]

(Briq,) A, J, Paton [97]

*Combretum albiflorum* (Tul.) Jongkind [85]

*Dalbergia trichocarpa* Baker. [96]

*Armoracia rusticana* G. Gaertn et al. [98]

[84]

[89, 90]

*Terminalia chebula* Retz.

*Curcuma longa* L. [83] Curcumin (Phenolic

*Syzygium aromaticum* (L.) Merr. Et Perry

*Citrus paradisi* Macfad. (Rio Red and Marsh White grapefruits) [87]

*Scutellaria baicalensis* Georgi. [86]

*Zingiber officinale* Rosc.

[92]

*Aspergillus terreus* [77] Terrein

Prokaryotes *Bacillus indicus, B.* 

M7 [65]

marine organisms


*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting… DOI: http://dx.doi.org/10.5772/intechopen.90833*

*Bacterial Biofilms*

*5.5.2 Alkaloids*

ing at 30 μM.

**6. Concluding remarks**

The *P. aeruginosa* pyocyanin production is inhibited by a molecule found and isolated from the ant *Solenopsis invicta*, the piperidine alkaloid Solenopsin A alkaloid. The biofilm formation is also reduced in a dose-dependent manner. This molecule

This review presents natural compounds reported to exhibit anti-QS and antibiofilm properties against *P. aeruginosa* (summarized in **Table 1**); these highlight the great potentiality of living organisms as reservoir of compounds susceptible to modulate virulence mechanisms without affecting bacterial viability. Overall, it appears that prokaryotes as well as animals and humans are sources for enzymes that degrade or antagonize AHLs, whereas plants harbor larger panels of anti-QS and antibiofilm compounds with very diverse chemical structures, including alkaloids, organosulfurs, phenolics, and terpenoids. Contrarily to animals and humans, plants are not able to deploy elaborate defense through humoral and cell-mediated immunity (antibodies and phagocytes) to struggle against bacterial invasions [113]. Plants immune defenses rely on the secretion of antibacterial compounds (bactericide and/or bacteriostatic agents [114]), including resistance modulating compounds [115] (e.g., inhibitors of efflux pumps [116]), and mostly on their ability to recognize molecules released from pathogens through plant cell surface receptors. This recognition triggers specific signaling cascades, activating series of defense responses, including the synthesis of antimicrobial lytic proteins, enzymes, phytoalexins, and other secondary metabolites. Some of these exert nonmicrobicidal antivirulence properties [117, 118]. Finally, marine organisms and fungi produce also bioactive secondary metabolites (halogenated furanones and antibiotics, respectively) and other original and promising compounds, such as terrein which was identified as the first dual inhibitor of QS and c-di-GMP signal-

The increasing presence of antibiotic-resistant bacteria certainly pushes scientists to reorient the strategy of fight against bacterial infections to defer entry into a post-antibiotic era where major antibiotics would not be effective even for banal infections. Antivirulence approaches and antivirulence drugs are being increasingly considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. For example, oleanolic aldehyde coumarate and cassipourol, anti-QS compounds, exert interesting antibiofilm properties, restoring the effectiveness of the antibiotic tobramycin in the clearance of biofilm-encapsulated *P. aeruginosa* (**Figure 4**); also the association between biofilm formation and antimicrobial resistance has been highlighted in carbapenem-resistant *P. aeruginosa* [119]. Such nonmicrobicidal drugs inhibit virulence factors essential for establishing infection and pathogenesis through targeting nonessential metabolic pathways which should not lead to activation of bacterial evasion mechanisms. This approach should reduce the selective pressure and consequently could slow down the development of resistance. Compounds that target QS may be particularly interesting as they impact planktonic and biofilm lifestyles, by reducing at the same time the production of virulence factors and the generation of biofilms. This should lead to less severe infections at levels that can be cleared by the host's immune defense and with

Despite these important prospects, however, the big breakthrough in antibacterial strategies is still out of reach. This is probably due to a very complex

probably disrupts the signals from the *rhl* system [112].

**44**

increased activity of antibiotics.


*+, yes; NC, not communicated.*

*ǂ Patulin alone does not affect the development of biofilm.*

*\* LuxR-type transcription factor of Agrobacterium tumefaciens.*

*1 Aminoglycosides.*

*2 Ampicillin.*

### **Table 1.**

*Natural compounds inhibiting* P. aeruginosa *QS and biofilm formation.*

### **Figure 4.**

P. aeruginosa *biofilm phenotypes and effectiveness of tobramycin treatment in presence of dimethyl sulfoxide (DMSO 1%) or, cassipourol (CAS: 100 μM) or oleanolic aldehyde coumarate (OALC: 200 μM). (a) After 1 day of incubation,* P. aeruginosa *fails to form structured confluent aggregate in presence of CAS or OALC as compared to DMSO treatment. (b) CAS and OALC considerably increase the susceptibility of* P. aeruginosa *to tobramycin (100 μg/mL), as shown by the increased proportion of dead cells compared with DMSO. Similar results are observed when tobramycin is added simultaneously with CAS or OALC to one-day old untreated biofilms. The bacterial viability was assessed by staining the cells with SYTO-9 (green areas zones—live living bacteria) and propidium iodide (red areas zones—dead bacteria) furnished in the LIVE/DEAD BacLight kit. Cells were visualized using a LeicaDMIRE2 inverted fluorescence microscope using equipped with a 40× objective lens and colored images were assembled using Adobe Photoshop.*

entanglement between different QS systems, to the ability of *Pseudomonas* to compensate deficient systems and to the intervention of key actors involved in biofilm formation, outside of QS circuitry [12]. Millenia of coevolution between plants and bacteria have led to complex defense strategies, with plants producing cocktails of bioactive compounds with multiple targets [114] and/ or compounds such as terrein that impact dual inhibitory targets. In the current state of research, much remains to be done in understanding these mechanisms and the real impact of such combinations before arriving at a commercial use. Nevertheless, following a combined approach for "adjuvant antibiotherapy" and "combined antibiotherapy" will undeniably lead to a renewed concept of "complex drugs for complex diseases," a well-known presupposed in traditional medicines [120].

**47**

provided the original work is properly cited.

, Amandine Nachtergael1

\*

2 Université Libre de Bruxelles, Brussels, Belgium

\*Address all correspondence to: travaka@yahoo.fr

3 University of Antananarivo, Antananarivo, Madagascar

*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting…*

The authors declare that there is no conflict of interests regarding the publica-

The authors would like to thank ARES (Académie de Recherche et d'Enseignement Supérieur, Belgium) for financial support throughout PRD

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Pierre Duez1

, Mondher El Jaziri<sup>2</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.90833*

**Acknowledgements**

**Conflict of interest**

tion of this paper.

**Author details**

and Tsiry Rasamiravaka<sup>3</sup>

1 University of Mons, Mons, Belgium

Julie Carette1

projects.

*Natural Compounds Inhibiting* Pseudomonas aeruginosa *Biofilm Formation by Targeting… DOI: http://dx.doi.org/10.5772/intechopen.90833*
