**Abstract**

The biofilm lifestyle mode certainly represents one of the most successful behaviors to facilitate bacterial survival in diverse inhospitable environments. Conversely, the ability of bacteria to develop effective biofilms represents one of the major obstacles in the fight against bacterial infections. In *Pseudomonas aeruginosa*, the biofilm formation is intimately connected to the quorum sensing (QS) mechanisms, a mode of cell-to-cell communication that allows many bacteria to detect their population density in order to coordinate common actions. In this chapter, we propose an overview (i) on *P. aeruginosa* QS mechanisms and their implication in biofilm formation, and (ii) on natural products that are known to interfere with these QS mechanisms, subsequently disrupting biofilm formation. The concluding remarks focus on perspectives of these compounds as possible antibiotherapy adjuvants.

**Keywords:** biofilm, *las*, natural products, PQS, *pseudomonas*, quorum sensing, *rhl*

### **1. Introduction**

Bacterial infections are mainly related to the ability of bacteria to invade and disseminate through their hosts by using different types of motility, by releasing a myriad of virulence factors, by building structured biofilm which lead to host cell and tissue damage but also allow bacteria to evade the immune system and conventional antimicrobial agents [1]. For decades, antibiotics, although less effective in biofilm-growing bacteria [2], have represented our best weapon against bacterial diseases. However, the on-going emergence and worldwide spreading of resistant bacteria is considerably reducing the antibiotic pallet available for the treatment of bacterial infections [3]. This alarming situation forces researchers to consider other strategies to combat bacterial infections, notably the use of phages [4] or the use of alternative agents, such as essential oils [5], silver nanoparticles [6], bacteriocins [7], and antimicrobial peptides [8]. Some interesting strategies propose original compounds that disrupt biofilm formation without affecting the viability of invading bacteria; this strategy is expected (i) to reduce the bacterial aptitude to build protective barriers, but without exerting a selective pressure *per se* [4]; (ii) to allow

sufficient time for the immune defenses to effectively destroy invaders; and (iii) to minimize the use of effective antibiotics.

In most bacteria, the expressions of virulence factors are coordinated by quorum sensing (QS) mechanisms, a cell-to-cell communication which allows bacteria to detect their population density by producing and perceiving diffusible signal molecules to synchronize common actions [9]. This cell-to-cell communication has been largely investigated in *Pseudomonas aeruginosa,* an opportunistic pathogen which mainly affects people who are severely immunocompromised, in part due to its ability to evade from both innate and acquired immune defenses through adhesion, colonization, and biofilm forming and to produce various virulence factors that cause significant tissue damage [10, 11]. In this bacterium, QS regulates virulence factors production, motilities and, in particular, biofilm formation for which QS is one of the relevant key actors. Interestingly, within the two past decades, study papers reporting natural and synthetic compounds that interfere with QS and/or biofilm formation are regularly published; QS circuitry and biofilm formation control mechanisms indeed constitute promising targets to struggle against *P. aeruginosa* infection with potential huge clinical interests [12]. The present chapter covers the scope of natural compounds from both prokaryote and eukaryote organisms that have been identified to disrupt the biofilm lifestyle cycle in *P. aeruginosa* via modulation of QS mechanisms. An overview of the entanglement between QS circuitry and biofilm formation is reported as a prerequisite for a better understanding of the mechanisms of action proposed for some of the identified compounds. The concluding remarks focus on the perspectives of these compounds as possible antibiotherapy adjuvants for possible eradication of resistant infections caused by *P. aeruginosa*.

### **2.** *P. aeruginosa* **biofilm lifestyle**

Like most bacteria, *P. aeruginosa* can develop two distinct lifestyles, planktonic and sessile cells. The planktonic state is encountered when *P. aeruginosa* evolves freely in a liquid suspension, whereas on natural or synthetic surfaces, *P. aeruginosa* can form sticky clusters in permanent rearrangements characterized by the secretion of an adhesive and protective matrix [13]. Defined as "biofilm," this set of bacterial community adherent to a surface appears as an adaptive response to an environment more or less unsuited to growth in planktonic form [14].

The biofilm formation can be delimited in five main stages (**Figure 1**, image A). A first reversible phase corresponds to the initial adhesion of bacteria to surfaces; this adhesion becomes irreversible in the second stage (image B). Then, thanks to a proliferation period corresponding to the third stage, microcolonies are built concomitantly with the production of extracellular matrix (image C), leading to the fourth stage of biofilm structuration and organization in which the growth of three dimensional communities is observed with amplified extracellular matrix production (image D). This biofilm cycle is completed by a dispersion step (image E) [12].

The secreted extracellular matrix mainly consists of proteins, nucleic acids, lipids, and exopolysaccharides (EPS). These account for 50–90% of total organic matter [16]. *P. aeruginosa* produces at least three types of EPS that are required for biofilm formation and architecture [17]. (i) Alginate a linear polysaccharide composed of L-guluronic and D-mannuronic acids linked by β-1,4 bonds [18], (ii) Pel polysaccharide, a glucose-rich matrix material, with unclarified composition, and (iii) Psl polysaccharide, a repeating pentasaccharide consisting of D-mannose, L-rhamnose, and D-glucose. In mucoid strains, EPS are predominantly characterized by the presence of alginate. The alginate participates in the structuring of the

**37**

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

biofilm [19], but its real importance is still controversial since some authors claim that it is not essential; indeed architecture and antibiotic resistance profiles of wild-type and alginate-deficient biofilms are identical [20, 21]. Nevertheless, the overexpression of alginate was shown to protect *P. aeruginosa* from phagocytosis and host responses [22]. In "nonmucoid" *P. aeruginosa* strains, such as the PAO1 strain isolated from an infected wound [23], alginate is even considered poorly produced at the expense of exopolysaccharides rich in glucose and mannose [24], Pel and Psl, which have been described as being more important in the formation

*Sketch of the different steps of a biofilm development (A) [15]. Several representative scanning electron microscopy (SEM-JEOL JSM-7200F) images of the* P. aeruginosa *biofilm at different steps of development and with different magnifications (B = reversible and irreversible stages at 8 h growth, C = microcolonies stage at 30 h growth, D = mature biofilm stage at 120 h growth, and E = dispersion stage at 144 h growth).*  P. aeruginosa *PAO1 colonies were grown at 37°C with Centers for Disease Control and Prevention (CDC)* 

*biofilm reactor (biosurface technologies, MT) on tryptone soy broth (TSB).*

Extracellular DNA (eDNA) is an important component of *P. aeruginosa* biofilm

matrix, which particularly intervenes in the establishment, maintenance, and perpetuation of structured biofilms [26]. Its importance has been demonstrated since *P. aeruginosa* biofilm formation is prevented by exposition to DNase I [27] and biofilms that are deficient in eDNA have been shown to be more sensitive to the detergent sodium dodecyl sulfate [28]. It has been established that eDNA plays roles in bacterial adhesion and in the structural stability of biofilms by maintaining coherent cell alignments [29]; interestingly, its contribution to antimicrobial resistance has also been proposed as eDNA, a highly anionic polymer, is believed to bind cationic antibiotics, such as aminoglycosides and antimicrobial peptides [30].

**3. QS mechanisms and their implication in biofilm formation**

The complex regulation of biofilm formation involves multiple bacterial machineries including the QS systems. In *P. aeruginosa*, this mechanism is involved in the development of various common bacterial behaviors, including virulence factors expression and biofilm formation, which are mostly implicated in infection success. Three QS systems have been clearly characterized: (i) the *las* system and the *rhl* system, two LuxI/R type systems using the signal molecules of the family of acyl-homoserine lactones (AHLs); and (ii) the PQS (pseudomonas quinolone signal) system based on molecules of the 2-alkyl-4-quinolone class [10, 31]. The mechanisms of QS in *P. aeruginosa* are summarized in **Figure 2** while the main

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

and maintenance of the biofilm [25].

**Figure 1.**

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

### **Figure 1.**

*Bacterial Biofilms*

minimize the use of effective antibiotics.

resistant infections caused by *P. aeruginosa*.

**2.** *P. aeruginosa* **biofilm lifestyle**

sufficient time for the immune defenses to effectively destroy invaders; and (iii) to

Like most bacteria, *P. aeruginosa* can develop two distinct lifestyles, planktonic and sessile cells. The planktonic state is encountered when *P. aeruginosa* evolves freely in a liquid suspension, whereas on natural or synthetic surfaces, *P. aeruginosa* can form sticky clusters in permanent rearrangements characterized by the secretion of an adhesive and protective matrix [13]. Defined as "biofilm," this set of bacterial community adherent to a surface appears as an adaptive response to an

The biofilm formation can be delimited in five main stages (**Figure 1**, image A). A first reversible phase corresponds to the initial adhesion of bacteria to surfaces; this adhesion becomes irreversible in the second stage (image B). Then, thanks to a proliferation period corresponding to the third stage, microcolonies are built concomitantly with the production of extracellular matrix (image C), leading to the fourth stage of biofilm structuration and organization in which the growth of three dimensional communities is observed with amplified extracellular matrix production (image D). This biofilm cycle is completed by a dispersion step (image E) [12]. The secreted extracellular matrix mainly consists of proteins, nucleic acids, lipids, and exopolysaccharides (EPS). These account for 50–90% of total organic matter [16]. *P. aeruginosa* produces at least three types of EPS that are required for biofilm formation and architecture [17]. (i) Alginate a linear polysaccharide composed of L-guluronic and D-mannuronic acids linked by β-1,4 bonds [18], (ii) Pel polysaccharide, a glucose-rich matrix material, with unclarified composition, and (iii) Psl polysaccharide, a repeating pentasaccharide consisting of D-mannose, L-rhamnose, and D-glucose. In mucoid strains, EPS are predominantly characterized by the presence of alginate. The alginate participates in the structuring of the

environment more or less unsuited to growth in planktonic form [14].

In most bacteria, the expressions of virulence factors are coordinated by quorum sensing (QS) mechanisms, a cell-to-cell communication which allows bacteria to detect their population density by producing and perceiving diffusible signal molecules to synchronize common actions [9]. This cell-to-cell communication has been largely investigated in *Pseudomonas aeruginosa,* an opportunistic pathogen which mainly affects people who are severely immunocompromised, in part due to its ability to evade from both innate and acquired immune defenses through adhesion, colonization, and biofilm forming and to produce various virulence factors that cause significant tissue damage [10, 11]. In this bacterium, QS regulates virulence factors production, motilities and, in particular, biofilm formation for which QS is one of the relevant key actors. Interestingly, within the two past decades, study papers reporting natural and synthetic compounds that interfere with QS and/or biofilm formation are regularly published; QS circuitry and biofilm formation control mechanisms indeed constitute promising targets to struggle against *P. aeruginosa* infection with potential huge clinical interests [12]. The present chapter covers the scope of natural compounds from both prokaryote and eukaryote organisms that have been identified to disrupt the biofilm lifestyle cycle in *P. aeruginosa* via modulation of QS mechanisms. An overview of the entanglement between QS circuitry and biofilm formation is reported as a prerequisite for a better understanding of the mechanisms of action proposed for some of the identified compounds. The concluding remarks focus on the perspectives of these compounds as possible antibiotherapy adjuvants for possible eradication of

**36**

*Sketch of the different steps of a biofilm development (A) [15]. Several representative scanning electron microscopy (SEM-JEOL JSM-7200F) images of the* P. aeruginosa *biofilm at different steps of development and with different magnifications (B = reversible and irreversible stages at 8 h growth, C = microcolonies stage at 30 h growth, D = mature biofilm stage at 120 h growth, and E = dispersion stage at 144 h growth).*  P. aeruginosa *PAO1 colonies were grown at 37°C with Centers for Disease Control and Prevention (CDC) biofilm reactor (biosurface technologies, MT) on tryptone soy broth (TSB).*

biofilm [19], but its real importance is still controversial since some authors claim that it is not essential; indeed architecture and antibiotic resistance profiles of wild-type and alginate-deficient biofilms are identical [20, 21]. Nevertheless, the overexpression of alginate was shown to protect *P. aeruginosa* from phagocytosis and host responses [22]. In "nonmucoid" *P. aeruginosa* strains, such as the PAO1 strain isolated from an infected wound [23], alginate is even considered poorly produced at the expense of exopolysaccharides rich in glucose and mannose [24], Pel and Psl, which have been described as being more important in the formation and maintenance of the biofilm [25].

Extracellular DNA (eDNA) is an important component of *P. aeruginosa* biofilm matrix, which particularly intervenes in the establishment, maintenance, and perpetuation of structured biofilms [26]. Its importance has been demonstrated since *P. aeruginosa* biofilm formation is prevented by exposition to DNase I [27] and biofilms that are deficient in eDNA have been shown to be more sensitive to the detergent sodium dodecyl sulfate [28]. It has been established that eDNA plays roles in bacterial adhesion and in the structural stability of biofilms by maintaining coherent cell alignments [29]; interestingly, its contribution to antimicrobial resistance has also been proposed as eDNA, a highly anionic polymer, is believed to bind cationic antibiotics, such as aminoglycosides and antimicrobial peptides [30].

### **3. QS mechanisms and their implication in biofilm formation**

The complex regulation of biofilm formation involves multiple bacterial machineries including the QS systems. In *P. aeruginosa*, this mechanism is involved in the development of various common bacterial behaviors, including virulence factors expression and biofilm formation, which are mostly implicated in infection success. Three QS systems have been clearly characterized: (i) the *las* system and the *rhl* system, two LuxI/R type systems using the signal molecules of the family of acyl-homoserine lactones (AHLs); and (ii) the PQS (pseudomonas quinolone signal) system based on molecules of the 2-alkyl-4-quinolone class [10, 31]. The mechanisms of QS in *P. aeruginosa* are summarized in **Figure 2** while the main

functions regulated by QS systems and involved in the pathogenesis of *P. aeruginosa* are presented in **Figure 3**.

Evidence that the *las* system is implicated in biofilm formation has been firstly established when Davies et al. [32] demonstrated that the biofilm formed by *lasI* mutant appears flat, undifferentiated, and quickly dispersed from the surface upon exposure to sodium dodecyl sulfate, compared to wild type biofilms.

Furthermore, Gilbert et al. [33] observed the binding of the QS regulator LasR to the promoter region of the *psl* operon, suggesting that the *psl* expression may be regulated by the QS. Considering that the *psl* operon is implied in biofilm modulation, the QS then plays a role in the biofilm formation and architecture. The transcription of the *pel* operon seems to be reduced in *rhlI* mutant, suggesting that the *rhl* system plays a biofilm formation role in *P. aeruginosa* by modulating the biosynthesis of the Pel polysaccharide [34]. The *pqsA* mutant produces a biofilm with less eDNA than the wild type biofilm, suggesting that the PQS system also plays a role in biofilm formation, more particularly in the eDNA releasing [34].

Notably, the production of rhamnolipids and lectins is under QS control, indicating a further indirect link between biofilm formation/degradation and QS.

Indeed, the *rhl* system controls the production of rhamnolipids [35], that play multiple roles in *P. aeruginosa* biofilm formation: (i) as biosurfactant and virulence factor [36]; (ii) in the formation of microcolonies [37]; (iii) in the maintenance of open channel structures necessary for nutrient circulation [38]; (iv) in the development of biofilm mushroom-shaped structures [37]; and (v) in cell dispersion from the biofilm [39]. Indeed, a hyper-detaching property has been observed in the *P. aeruginosa* mutants that produce more rhamnolipids compared to wild type strains [40]. Moreover, the *rhl* system also controls the expression of the cytotoxic virulence factors LecA and LecB. Data obtained on mutant strains indicate that these galactophilic lectins probably contribute to the biofilm development [41, 42]. Similarly, two types of *P. aeruginosa* motilities implicated in biofilm formation are also QS-regulated. The first movement, swarming motility, accomplishes an organized surface translocation, dependent on cell-to-cell

#### **Figure 2.**

*Systems involved in* P. aeruginosa *QS circuitry. The main QS systems in* P. aeruginosa *are the* las, rhl*, and PQS systems. The* las *system consists of a* lasR *regulatory gene coding for the LasR protein, a* lasI *gene coding for a LasI synthase involved in the synthesis of a signal molecule of the acyl-homoserine lactone (AHL) family, the 3-oxo-C12-HSL. The LasR/3-oxo-C12-HSL complex is a transcriptional activator of virulence genes (protease, elastase, and exotoxin) and* lasI *gene. According to the same model, the* rhl *system consists of* rhlR, rhlI *genes, and another AHL, the C4-HSL. This system activates genes in common with the* las *system and also specific genes, such as those coding for the synthesis of rhamnolipids, pyocyanin, and swarming/twitching motilities. The* las *system controls the* rhl *system. The third PQS system is interposed between the two main systems. The PqsABCDE operon produces the precursor 2-heptyl-4-quinolone (HHQ ), and PqsH catalyzes conversion of HHQ to 2-heptyl-3-hydroxy-4-quinolone (PQS), detected by the receptor PqsR [10, 31].*

**39**

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

contacts and extensive flagellation [43]; this has been observed during the first stage of *P. aeruginosa* biofilm development and seems to be regulated by the *rhl* system [44]. Flat and uniform biofilms are formed when the strains grow under conditions promoting swarming motility, for example, a growth medium with glutamate or succinate as carbon sources; by contrast, a biofilm without confluent cell aggregates is formed by strains with limited swarming motility [45]. The second movement, a flagella-independent form of translocation, is described as a successive extension and retraction of polar type IV pili [46]. This kind of movement, regulated by the *rhl* system on a Fe-limited minimal medium [47], is necessary to assemble bacteria in monolayers that form microcolonies [38].

The QS systems are not the sole key actors intervening in biofilm formation by *P. aeruginosa*. Indeed, the complex regulation of biofilm formation involves multiple bacterial machineries that also include the membrane-bound sensor kinase GacS, the transcriptional response regulator GacA (GacS/GacA two-component regulatory system), and the intracellular second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Briefly, the GacS/GacA system acts as a super-regulator of the *las* and *rhl* systems [48], whereas c-di-GMP is important for the biosynthesis of alginate and Pel polysaccharides and for the switch from

**5. Natural products that affect QS and biofilm formation by** *Pseudomonas* 

Microorganisms known to have the ability to produce anti-QS enzymes are still limited to a few bacteria from the families of (i) *Actinobacteria (Rhodococcus* and *Streptomyces)*; (ii) *Firmicutes-Arthrobacter (Bacillus* and *Oceanobacillus)*; (iii) *Cyanobacteria (Anabaena)*; (iv) *Bacteroidetes (Tenacibaculum);* (v) *Proteobacteria* 

**4. Other mechanisms implied in biofilm formation**

planktonic to biofilm lifestyle [49].

*aeruginosa*

**Figure 3.**

*5.1.1 Enzymes*

**5.1 From prokaryotes**

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

*Functions positively regulated by QS in* P. aeruginosa *[10, 31].*

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

### **Figure 3.**

*Bacterial Biofilms*

are presented in **Figure 3**.

functions regulated by QS systems and involved in the pathogenesis of *P. aeruginosa*

Evidence that the *las* system is implicated in biofilm formation has been firstly established when Davies et al. [32] demonstrated that the biofilm formed by *lasI* mutant appears flat, undifferentiated, and quickly dispersed from the surface upon

Furthermore, Gilbert et al. [33] observed the binding of the QS regulator LasR to the promoter region of the *psl* operon, suggesting that the *psl* expression may be regulated by the QS. Considering that the *psl* operon is implied in biofilm modulation, the QS then plays a role in the biofilm formation and architecture. The transcription of the *pel* operon seems to be reduced in *rhlI* mutant, suggesting that the *rhl* system plays a biofilm formation role in *P. aeruginosa* by modulating the biosynthesis of the Pel polysaccharide [34]. The *pqsA* mutant produces a biofilm with less eDNA than the wild type biofilm, suggesting that the PQS system also plays a role in

Notably, the production of rhamnolipids and lectins is under QS control, indicating a further indirect link between biofilm formation/degradation and QS.

Indeed, the *rhl* system controls the production of rhamnolipids [35], that play multiple roles in *P. aeruginosa* biofilm formation: (i) as biosurfactant and virulence factor [36]; (ii) in the formation of microcolonies [37]; (iii) in the maintenance of open channel structures necessary for nutrient circulation [38]; (iv) in the development of biofilm mushroom-shaped structures [37]; and (v) in cell dispersion from the biofilm [39]. Indeed, a hyper-detaching property has been observed in the *P. aeruginosa* mutants that produce more rhamnolipids compared to wild type strains [40]. Moreover, the *rhl* system also controls the expression of the cytotoxic virulence factors LecA and LecB. Data obtained on mutant strains indicate that these galactophilic lectins probably contribute to the biofilm development [41, 42]. Similarly, two types of *P. aeruginosa* motilities implicated in biofilm formation are also QS-regulated. The first movement, swarming motility, accomplishes an organized surface translocation, dependent on cell-to-cell

*Systems involved in* P. aeruginosa *QS circuitry. The main QS systems in* P. aeruginosa *are the* las, rhl*, and PQS systems. The* las *system consists of a* lasR *regulatory gene coding for the LasR protein, a* lasI *gene coding for a LasI synthase involved in the synthesis of a signal molecule of the acyl-homoserine lactone (AHL) family, the 3-oxo-C12-HSL. The LasR/3-oxo-C12-HSL complex is a transcriptional activator of virulence genes (protease, elastase, and exotoxin) and* lasI *gene. According to the same model, the* rhl *system consists of* rhlR, rhlI *genes, and another AHL, the C4-HSL. This system activates genes in common with the* las *system and also specific genes, such as those coding for the synthesis of rhamnolipids, pyocyanin, and swarming/twitching motilities. The* las *system controls the* rhl *system. The third PQS system is interposed between the two main systems. The PqsABCDE operon produces the precursor 2-heptyl-4-quinolone (HHQ ), and PqsH catalyzes conversion of* 

*HHQ to 2-heptyl-3-hydroxy-4-quinolone (PQS), detected by the receptor PqsR [10, 31].*

exposure to sodium dodecyl sulfate, compared to wild type biofilms.

biofilm formation, more particularly in the eDNA releasing [34].

**38**

**Figure 2.**

*Functions positively regulated by QS in* P. aeruginosa *[10, 31].*

contacts and extensive flagellation [43]; this has been observed during the first stage of *P. aeruginosa* biofilm development and seems to be regulated by the *rhl* system [44]. Flat and uniform biofilms are formed when the strains grow under conditions promoting swarming motility, for example, a growth medium with glutamate or succinate as carbon sources; by contrast, a biofilm without confluent cell aggregates is formed by strains with limited swarming motility [45]. The second movement, a flagella-independent form of translocation, is described as a successive extension and retraction of polar type IV pili [46]. This kind of movement, regulated by the *rhl* system on a Fe-limited minimal medium [47], is necessary to assemble bacteria in monolayers that form microcolonies [38].
