# The Formation of Biofilms byPseudomonas aeruginosa: A Review of the Natural and Synthetic Compounds Interfering with Control Mechanisms

Authors: Tsiry Rasamiravaka; Quentin Labtani; Pierre Duez; Mondher El Jaziri
Journal: BioMed Research International
 (2015)

---

## Abstract

P. aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections. Beyond its natural resistance to many drugs, its ability to form biofilm, a complex biological system, renders ineffective the clearance by immune defense systems and antibiotherapy. The objective of this report is to provide an overview (i) on P. aeruginosa biofilm lifestyle cycle, (ii) on the main key actors relevant in the regulation of biofilm formation by P. aeruginosa including QS systems, GacS/GacA and RetS/LadS two-component systems and C-di-GMP-dependent polysaccharides biosynthesis, and (iii) finally on reported natural and synthetic products that interfere with control mechanisms of biofilm formation by P. aeruginosa without affecting directly bacterial viability. Concluding remarks focus on perspectives to consider biofilm lifestyle as a target for eradication of resistant infections caused by P. aeruginosa.


---

## Body

## 1. Introduction

The misuse and abuse of antibiotics are recognized to create selective pressure, resulting in the widespread development of resistant bacterial strains [1, 2]. Antibiotics are also known to kill “good/beneficial” indigenous bacteria, which may have protective role against pathogenic bacteria [3, 4]. Another important point to consider is that antibiotics have been found to be less effective in biofilm-growing bacteria [5].Facing these limitations of antibiotics, there is an increasing need for the discovery and the development of antimicrobial agents that present novel or unexplored properties to efficiently control and manage bacterial infectious diseases [6]. Inhibition of bacterial virulence and/or biofilm formation by targeting nonmicrobicidal mechanisms are examples of increasingly explored antipathogenic approaches [7–9]. Among opportunistic pathogenic bacteria,P. aeruginosa, which produces several virulence factors, is known to be an important human and plant pathogen, responsible for various infections, particularly in immunocompromised persons [10]. Besides this, the remarkable ability ofP. aeruginosa to form biofilms in many environments renders antibiotic treatments inefficient and therefore promotes chronic infectious diseases [5, 11].Three global nonmicrobicidal strategies have been proposed to struggle against pathogenic bacteria with biofilm formation ability by (i) avoiding microbial attachment to a surface; (ii) disrupting biofilm development and/or affecting biofilm architecture in order to enhance the penetration of antimicrobials; and (iii) affecting biofilm maturation and/or inducing its dispersion and degradation [8, 12, 13].The present review covers the scope of natural compounds from both prokaryote and eukaryote organisms that have been identified to disrupt biofilm lifestyle cycle inP. aeruginosa without affecting directly bacterial viability. As a prerequisite and for a better understanding of the proposed mechanisms of action of some of the identified compounds, relevant key molecular actors inP. aeruginosa biofilm formation and its regulation, such as the chemical signalization machinery involved in bacteria-environment interaction, including quorum sensing (QS) pathways, will be summarized.


## 2. Biofilm Lifestyle Cycle ofP. aeruginosa

Biofilm formation is an endless cycle, in which organized communities of bacteria are encased in a matrix of extracellular polymeric substances (EPS) that hold microbial cells together to a surface [14, 15]; these are thought to be determinant in 65–80% of all microbial infections [16–18]. In this microscopic world, biofilms are metaphorically called a “city of microbes” [19, 20] with EPS, which represents 85% of total biofilm biomass, as “house of the biofilm cells” [21]. EPS is composed mainly of biomolecules, exopolysaccharides, extracellular DNA (eDNA), and polypeptides that form a highly hydrated polar mixture that contributes to the overall structural scaffold and architecture of the biofilm [22–24].Depending onP. aeruginosa strains and/or nutritional conditions, different biofilm phenotypes can be developed [25]. For instance, in glucose minimal media, biofilm lifestyle cycle ofP. aeruginosa PAO1 can be subdivided into five major phenotypic steps (Figure 1). The process begins by the reversible adhesion of planktonic bacteria onto a surface suitable for growth (Figure 1(a), Stage I), followed by irreversible attachment of bacteria, which thereafter form microcolonies in EPS matrix (Figure 1(b), Stage II). Progressively, bacterial microcolonies expand and their confluences lead to a more structured phenotype with noncolonized space (Figure 1(c), Stage III). Then, noncolonized spaces are filled with bacteria, which finally cover the entire surface (Figure 1(d), Stage IV). Meanwhile, the growth of three-dimensional communities is observed (Figure 1, Stages III and IV). Finally, bacteria disperse from the sessile structure and reenter in planktonic state to spread and colonize other surfaces [15, 26] (Figure 1(e), Stage V).Figure 1
Biofilm lifestyle cycle ofP. aeruginosa PAO1 grown in glucose minimal media. In stage I, planktonic bacteria initiate attachment to an abiotic surface, which becomes irreversible in stage II. Stage III corresponds to microcolony formation. Stage IV corresponds to biofilm maturation and growth of the three-dimensional community. Dispersion occurs in stage V and planktonic bacteria that are released from the biofilm to colonize other sites. The biofilm formation byP. aeruginosa PAO1 was revealed with Syto9 and visualized in Leica DM IRE2 inverted fluorescence microscope with 400x magnification at 2 h (Stage I), 8 h (Stage II), 14 h (Stage III), 1 to 4 days (Stage IV), and 5 days (Stage V). Images represent a 250 × 250-μm field.P. aeruginosa produces at least three polysaccharides (alginate, Pel, and Psl) that are determinant for the stability of the biofilm structure [27, 28]. Mucoid and nonmucoidP. aeruginosa strains differ by the qualitative composition of their polysaccharides in the biofilm matrix, predominantly alginate or Psl/Pel, respectively [29–31]. Alginate, a linear unbranched polymer composed of D-mannuronic acid and L-guluronic acid [32], contributes to the structural stability and protection of biofilms as well as to the retention of water and nutrients [33]. The Pel polysaccharide is mainly a glucose-rich matrix material, with still unclarified composition [34, 35], while Psl comprises a repeating pentasaccharide consisting of D-mannose, L-rhamnose, and D-glucose [36]. Pel and Psl can serve as a primary structure scaffold for biofilm development and are involved at early stages of biofilm formation [30, 37, 38].eDNA constitutes an important functional component ofP. aeruginosa biofilm matrix; indeed (i)P. aeruginosa biofilm formation is prevented by exposition to DNase I [39]; (ii) biofilms that are deficient in eDNA have been shown to be more sensitive to the detergent sodium dodecyl sulfate [40]; (iii) eDNA facilitates the twitching motility-mediated biofilm expansion by maintaining coherent cell alignments [41]; (iv) eDNA has been proposed to play an important role in the initial and early development ofP. aeruginosa biofilms as a cell-to-cell interconnecting compound [24, 42, 43]; and (v) finally, eDNA constitutes a nutrient source for bacteria during starvation [44, 45].Beyond their role in bacterial motilities [46–48],P. aeruginosa extracellular appendages flagella, type IV pili and cup fimbriae, are also considered to be matrix components that play adhesive roles in the cell-to-surface interactions (irreversible attachment) as well as in microcolony formation in biofilms. Mutants defective in flagellar-mediated motility and mutants defective in biogenesis of the polar-localized type IV pili do not develop microcolonies compared to the wild type strains [49–51].


## 3. Overview of Global Regulating Systems Involved inP. aeruginosa Biofilm Formation

The complex regulation of biofilm formation involves multiple bacterial machineries, including the QS systems and the two-component regulatory systems that both interact mainly with EPS production [52]. Deficiency in the network regulation required for biofilm matrix formation effectively results in the alteration of the biofilm structure and architecture and, therefore, of its protective role. The main key actors relevant in the regulation of biofilm formation byP. aeruginosa are summarized in Figure 2.Figure 2
Relevant bacterial systems and factors implicated in the regulation ofP. aeruginosa biofilm formation. (1) Quorum sensing system; (2) Two-component regulatory systemGacS/GacA andRetS/LadS (RR: response regulator domain receiver; P: phosphorylation) pathway; (3) Exopolysaccharides production and c-di-GMP pool regulation. See text for explanation.


### 3.1. QS Mechanisms and Biofilm Formation

QS is a cell-to-cell communication used by many bacteria to detect their population density by producing and perceiving diffusible signal molecules that coordinate virulence factors production, motility, and biofilm formation [53, 54].P. aeruginosa possesses two main QS systems (las andrhl) which drive the production (throughout synthases LasI and RhlI) and the perception (by the transcription factors LasR and RhlR) of the autoinducer signaling moleculesN-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) andN-butanoyl-L-homoserine lactone (C4-HSL) (Figure 3(a)), respectively [54]. A third QS system, based on quinolone signals (PQS system), interacts with the acyl homoserine lactones (AHLs) systems in an intricate way [54].Figure 3
Structure of natural and synthetic AHL-based compounds which inhibit biofilm formation byP. aeruginosa. (a) NativeN-acyl-l-homoserine lactone, signal molecules ofP. aeruginosa (C4-HSL and 3-oxo-C12-HSL), (b) synthetic analogue of AHLs with side aromatics and synthetic analogues of AHLs with modified lactone rings, and (c) natural (manoalide, penicillic acid, and patulin), and synthetic (furanones) compounds with lactone ring analogues.
(a)
Native AHLs
(b)
AHLs analogues
(c)
Compounds with lactone ring analoguesDavies et al. [55] have evidenced the role of thelas system for biofilm formation and maturation; compared to wild type biofilm, the biofilm oflasI mutant appears flat, undifferentiated, and quickly dispersed from the surface upon exposure to sodium dodecyl sulfate. The precise implication oflas system in biofilm formation is not yet clear. However, Gilbert et al. [56] reported that the QS regulator LasR can bind to the promoter region of thepsl operon, suggesting that QS can regulatepsl expression. Therhl system has been reported to intervene inP. aeruginosa biofilm formation [57] by enhancing Pel polysaccharide biosynthesis; transcription of thepel operon is actually reduced inrhlI mutant. The PQS system, for its part, is linked to eDNA release during biofilm development; biofilm formed bypqsA mutant contains less eDNA than biofilm formed by the wild type [40, 42]. All together these data indicate that the three QS systems known inP. aeruginosa play roles in biofilm lifestyle cycle.Importantly, an indirect link between biofilm formation and QS has been reported, through the control of swarming and twitching motilities, as well as rhamnolipids and lectins production. The swarming motility, a form of organized surface translocation, depends on extensive flagellation and cell-to-cell contact [58, 59]; regulated by therhl system [60], swarming motility is implicated in early stages ofP. aeruginosa biofilm establishment. Strains grown under conditions that promote swarming motility (growth medium with glutamate or succinate as carbon source) form flat and uniform biofilm while strains with limited swarming motility result in biofilm containing nonconfluent cell aggregates [25]. Twitching motility, a flagella-independent form of bacterial translocation, occurs by successive extension and retraction of polar type IV pili [47]. Known to be regulated by therhl system on Fe-limited minimal medium [61], twitching motilities are necessary for the assembly of a monolayer ofP. aeruginosa cells into microcolonies [49].Beyond their biosurfactant and virulence factor roles [62], rhamnolipids, whose production is under therhl system control [63], present multiple roles in biofilm formation byP. aeruginosa. Indeed, they are believed to be involved in (i) forming microcolonies [64]; (ii) maintaining open channel structures that prevent bacterial colonization by disrupting both cell-to-cell and cell-to-surface interactions [26]; (iii) facilitating three-dimensional mushroom-shaped structures formation inP. aeruginosa biofilms [64]; and (iv) facilitating the cell dispersion from the biofilm asP. aeruginosa variants which produce more rhamnolipids than wild-typeP. aeruginosa exhibit hyper-detaching properties [65, 66]. Finally, the cytotoxic virulence factor, galactophilic lectins LecA and LecB, has been proposed to contribute to biofilm development inP. aeruginosa, since LecA and LecB mutants form thin biofilms as compared to the wild type bacteria [67, 68]. Both LecA and LecB expressions are regulated by therhl QS system [69].


### 3.2. Biofilm Regulation byGacS/GacA andRetS/LadS Two-Component Systems

Among the 60 two-components systems found in the genome ofP. aeruginosa [70], theGacS/GacA system acts as a super-regulator of the QS system and is involved in the production of multiple virulence factors as well as in biofilm formation [71]. The Gac system consists of a transmembrane sensor kinase (GacS) that, upon autophosphorylation, transfers a phosphate group to its cognate regulator (GacA) which in turn upregulates the expression of the small regulatory RNAs (RsmZ andRsmY).RsmZ andRsmY capture the small RNA-binding regulatory protein RsmA (encoded byrsmA gene), a repressor that posttranscriptionally regulates thepsl locus (pslA-L) [72–74]. TheGacS/GacA system also has a control on the AHL system as it inactivates free RsmA which negatively controls the synthesis of C4-HSL and 3-oxo-C12-HSL and therefore the extracellular virulence factors controlled by thelas andrhl systems [75–77].The hybrid sensor histidine kinase RetS is known to repress biofilm formation [78, 79] whereas the histidine kinase LadS antagonizes the effect of RetS [80]. Indeed, ΔretS mutant form more structured biofilms as compared to wild typeP. aeruginosa PAO1 [78]; the PA14 strain (naturally deficient inladS gene) displays attenuated biofilm formation compared to PA14LadS
+ strain [81]. It is reported that RetS and LadS interact with theGacS/GacA system by modulating the phosphorylation state of GacS, which consequently inhibits and promotes, respectively, the phosphorylation of GacA [82, 83].It is interesting to note thatGacS/GacA andRetS/LadS systems are proposed to be involved in mediating the transition of theP. aeruginosa phenotype from an acute to chronic phase infection [78].


### 3.3. C-di-GMP-Dependent Polysaccharides Biosynthesis and Biofilm Formation

Polysaccharides production is dependent on the intracellular pool of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [84, 85], a ubiquitous intracellular second messenger widely distributed in bacteria [86]. In bacterial cells, c-di-GMP is generated from two molecules of guanosine triphosphate by diguanylate cyclases and broken down into 2-GMP by specific phosphodiesterases [86].High levels of c-di-GMP promote the biosynthesis of polysaccharides (alginate and Pel). Indeed, a binding process of c-di-GMP to PelD and Alg44 proteins is required for Pel and alginate polymer formation, respectively [85, 87]. However, the exact molecular mechanism by which this interaction regulates the polymerization of sugar precursors is not known.Conversely, low levels of c-di-GMP promote bacterial motilities by enhancing flagellar formation and bacterial dispersion [85].


## 3.1. QS Mechanisms and Biofilm Formation

QS is a cell-to-cell communication used by many bacteria to detect their population density by producing and perceiving diffusible signal molecules that coordinate virulence factors production, motility, and biofilm formation [53, 54].P. aeruginosa possesses two main QS systems (las andrhl) which drive the production (throughout synthases LasI and RhlI) and the perception (by the transcription factors LasR and RhlR) of the autoinducer signaling moleculesN-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) andN-butanoyl-L-homoserine lactone (C4-HSL) (Figure 3(a)), respectively [54]. A third QS system, based on quinolone signals (PQS system), interacts with the acyl homoserine lactones (AHLs) systems in an intricate way [54].Figure 3
Structure of natural and synthetic AHL-based compounds which inhibit biofilm formation byP. aeruginosa. (a) NativeN-acyl-l-homoserine lactone, signal molecules ofP. aeruginosa (C4-HSL and 3-oxo-C12-HSL), (b) synthetic analogue of AHLs with side aromatics and synthetic analogues of AHLs with modified lactone rings, and (c) natural (manoalide, penicillic acid, and patulin), and synthetic (furanones) compounds with lactone ring analogues.
(a)
Native AHLs
(b)
AHLs analogues
(c)
Compounds with lactone ring analoguesDavies et al. [55] have evidenced the role of thelas system for biofilm formation and maturation; compared to wild type biofilm, the biofilm oflasI mutant appears flat, undifferentiated, and quickly dispersed from the surface upon exposure to sodium dodecyl sulfate. The precise implication oflas system in biofilm formation is not yet clear. However, Gilbert et al. [56] reported that the QS regulator LasR can bind to the promoter region of thepsl operon, suggesting that QS can regulatepsl expression. Therhl system has been reported to intervene inP. aeruginosa biofilm formation [57] by enhancing Pel polysaccharide biosynthesis; transcription of thepel operon is actually reduced inrhlI mutant. The PQS system, for its part, is linked to eDNA release during biofilm development; biofilm formed bypqsA mutant contains less eDNA than biofilm formed by the wild type [40, 42]. All together these data indicate that the three QS systems known inP. aeruginosa play roles in biofilm lifestyle cycle.Importantly, an indirect link between biofilm formation and QS has been reported, through the control of swarming and twitching motilities, as well as rhamnolipids and lectins production. The swarming motility, a form of organized surface translocation, depends on extensive flagellation and cell-to-cell contact [58, 59]; regulated by therhl system [60], swarming motility is implicated in early stages ofP. aeruginosa biofilm establishment. Strains grown under conditions that promote swarming motility (growth medium with glutamate or succinate as carbon source) form flat and uniform biofilm while strains with limited swarming motility result in biofilm containing nonconfluent cell aggregates [25]. Twitching motility, a flagella-independent form of bacterial translocation, occurs by successive extension and retraction of polar type IV pili [47]. Known to be regulated by therhl system on Fe-limited minimal medium [61], twitching motilities are necessary for the assembly of a monolayer ofP. aeruginosa cells into microcolonies [49].Beyond their biosurfactant and virulence factor roles [62], rhamnolipids, whose production is under therhl system control [63], present multiple roles in biofilm formation byP. aeruginosa. Indeed, they are believed to be involved in (i) forming microcolonies [64]; (ii) maintaining open channel structures that prevent bacterial colonization by disrupting both cell-to-cell and cell-to-surface interactions [26]; (iii) facilitating three-dimensional mushroom-shaped structures formation inP. aeruginosa biofilms [64]; and (iv) facilitating the cell dispersion from the biofilm asP. aeruginosa variants which produce more rhamnolipids than wild-typeP. aeruginosa exhibit hyper-detaching properties [65, 66]. Finally, the cytotoxic virulence factor, galactophilic lectins LecA and LecB, has been proposed to contribute to biofilm development inP. aeruginosa, since LecA and LecB mutants form thin biofilms as compared to the wild type bacteria [67, 68]. Both LecA and LecB expressions are regulated by therhl QS system [69].


## 3.2. Biofilm Regulation byGacS/GacA andRetS/LadS Two-Component Systems

Among the 60 two-components systems found in the genome ofP. aeruginosa [70], theGacS/GacA system acts as a super-regulator of the QS system and is involved in the production of multiple virulence factors as well as in biofilm formation [71]. The Gac system consists of a transmembrane sensor kinase (GacS) that, upon autophosphorylation, transfers a phosphate group to its cognate regulator (GacA) which in turn upregulates the expression of the small regulatory RNAs (RsmZ andRsmY).RsmZ andRsmY capture the small RNA-binding regulatory protein RsmA (encoded byrsmA gene), a repressor that posttranscriptionally regulates thepsl locus (pslA-L) [72–74]. TheGacS/GacA system also has a control on the AHL system as it inactivates free RsmA which negatively controls the synthesis of C4-HSL and 3-oxo-C12-HSL and therefore the extracellular virulence factors controlled by thelas andrhl systems [75–77].The hybrid sensor histidine kinase RetS is known to repress biofilm formation [78, 79] whereas the histidine kinase LadS antagonizes the effect of RetS [80]. Indeed, ΔretS mutant form more structured biofilms as compared to wild typeP. aeruginosa PAO1 [78]; the PA14 strain (naturally deficient inladS gene) displays attenuated biofilm formation compared to PA14LadS
+ strain [81]. It is reported that RetS and LadS interact with theGacS/GacA system by modulating the phosphorylation state of GacS, which consequently inhibits and promotes, respectively, the phosphorylation of GacA [82, 83].It is interesting to note thatGacS/GacA andRetS/LadS systems are proposed to be involved in mediating the transition of theP. aeruginosa phenotype from an acute to chronic phase infection [78].


## 3.3. C-di-GMP-Dependent Polysaccharides Biosynthesis and Biofilm Formation

Polysaccharides production is dependent on the intracellular pool of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [84, 85], a ubiquitous intracellular second messenger widely distributed in bacteria [86]. In bacterial cells, c-di-GMP is generated from two molecules of guanosine triphosphate by diguanylate cyclases and broken down into 2-GMP by specific phosphodiesterases [86].High levels of c-di-GMP promote the biosynthesis of polysaccharides (alginate and Pel). Indeed, a binding process of c-di-GMP to PelD and Alg44 proteins is required for Pel and alginate polymer formation, respectively [85, 87]. However, the exact molecular mechanism by which this interaction regulates the polymerization of sugar precursors is not known.Conversely, low levels of c-di-GMP promote bacterial motilities by enhancing flagellar formation and bacterial dispersion [85].


## 4. Natural and Synthetic Products That AffectP. aeruginosa Biofilm Formation

Plants and animals are naturally exposed to bacterial infections and they respond to bacterial components and signal molecules in different manners, including the activation of defense mechanisms and/or the expression of stress management genes [88–93]. Therefore, it is obvious to expect that eukaryotes have developed chemical mechanisms to combat pathogens by killing them or silencing virulence mechanisms such as QS system and/or biofilm formation. Tables 1 and 2 summarize the reported natural and synthetic products that affectP. aeruginosa biofilm formation.Table 1
Natural inhibitory compounds forP. aeruginosa biofilm formation.
Natural products compounds
Origin
Class
Activities
References
QS inhibition
Biofilm inhibition(a)
Dispersion promotion
Synergy with antibiotic
Alginate lyase
P. aeruginosa
Enzyme
−
+
+
+(1)
[134]
Ursolic acid
Diospyros dendoWelw.
Triterpenoid
−
+ (24 h)motility
NC
NC
[141]
p-Coumaroyl-hydroxy-ursolic acid
Diospyros dendoWelw.
Coumarate ester of triterpene
NC
+ (24 h)
NC
NC
[142]
Zingerone
Zingiber officinaleRosc.
Phenolic compound
NC
+ (168 h)c-di-GMP
NC
+(2)
[138, 139]
Casbane diterpene
Croton nepetaefoliusBaill.
Diterpenoid
NC
+ (24 h)adherence
NC
NC
[140]
DNase I
Bovine pancreas
Enzyme
NC
+ (18–24 h)
+
+(3)
[137]
DNase-1L2
Humanstratum corneum
[136]
Paraoxonases 1
Human and murine sera
Enzyme (lactonase)
+
+ (24 h)
NC
NC
[116, 117]
Manoalide
Luffariella variabilis (Polejaeff, 1884)(marine organism)
Sesterterpenoid
+(las system)
+ (24 h)
NC
NC
[113, 114]
Solenopsin A
Solenopsis invicta (insect; ant)
Alkaloid
+ (rhl system)
+ (24 h)
NC
NC
[115]
Catechin
Combretum albiflorum (Tul.) Jongkind
Flavonoid
+ (las rhl systems)
+ (24 h)
NC
NC
[124]
Naringenin
Commercial
Flavonoid
+ (las rhl systems)
+ (48 h)
NC
NC
[125]
Coumarate ester
D. trichocarpaBaker.
Phenolic compound
+ (las rhl systems)
+ (48 h)
+
+(1)
[126]
Ajoene
Allium sativumL.
Organosulfur
+ (las rhl systems)
+ (96 h)
NC
+(1)
[130]
Ellagic acid derivatives
Terminalia chebula Retz.
Phenolic compound
+ (las rhl systems)
+ (72 h)
NC
+(1)
[120]
Rosmarinic acid
Ocimum basilicum L.
Phenolic compound
+ (las rhl systems)
+ (18 h)
−
NC
[89, 119]
Eugenol
Syzygium aromaticum (L.)Merr. Et Perry
Phenylpropanoid
+(las  pqs systems)
+ (24 h)
NC
NC
[128]
Curcumin
Curcuma longaL.
Phenolic compound
+ (AHLs)
+ (48 h)
NC
NC
[118]
Bergamottin and dihydroxybergamottin
Citrus paradisi Macfad.(Rio Red and Marsh White grapefruits)
Furocoumarins
+ (AI-1 and AI-2)
+ (24 h)
NC
NC
[121]
Penicillic acid
Penicillium species
Furanone
+ (LasR, RhlR)
NC
NC
NC
[111]
Patulin
Furopyranone
+ (LasR RhlR)
—‡
NC
+(1)
Emodin
Rheum palmatumL.
Anthraquinone
+ (docking traR)
+ (72 h)
NC
+(3)
[123]
Baicalein
Scutellaria baicalensisGeorgi.
Flavonoid
+ (docking traR)
+ (72 h)
NC
+(3)
[122]
+: yes; −: no; NC: not communicated.
‡Patulin does not affect the development of biofilm.
(a)Experiment duration.
(1)Aminoglycosides, (2)ciprofloxacin, (3)ampicillin.Table 2
Synthetic inhibitory compounds inP. aeruginosa biofilm formation.
Synthetic compounds(Natural compound origin)
Activities
References
QS inhibition
Biofilm inhibition(a)
Dispersion promotion
Synergistic antibiotic and/or immune defense effect
TAGE and CAGE (Bromoageliferin)
NC
+ (24 h)
+
NC
[132]
Dihydrosventrin (Sventrin)
NC
+ (24 h)
+
NC
[133]
N-(4-bromo-phenylacetanoyl)-l-HSL; N-(indole-3-butanoyl)-L-HSL (AHLs)
+ AHLs antagonist (las system)
+ (24 h)
NC
NC
[97]
3-oxo-C12-cyclohexanone (AHLs)
+ AHLs antagonist (las system)
+ (24 h)
NC
NC
[98]
C10-cyclopentylamide (AHLs)
+ (lasI and rhlA)
+ (24 h)
NC
NC
[99]
Furanone C-30 and C-56 (Furanone)
+ (las, rhl systems)
+ (24 h)
[55, 100, 101]
S-phenyl-L-cysteine sulfoxide (Cysteine sulfoxide alliin)
+ (las, rhl  systems)
+ (24 h)
NC
NC
[148]
Diphenyl disulfide (Disulfide derivatives of the alliinase mediated reactions)
+las system
+ (24 h)
NC
NC
Azythromycin‡ (Erythromycin)
+ (gacA, las and rhl systems)
+ (72 h)
+
+(1)
[103, 108, 109]
+: yes; −: no; NC: not communicated.
‡At subinhibitory concentration.
(a)Experiment duration.
(1)Aminoglycosides.


### 4.1. Antibiofilm Compounds with Anti-QS Activity

Several classes of molecules have been reported to present both antibiofilm formation and anti-QS properties inP. aeruginosa [94–96].Some AHL analogues (Figure3(b)) have been shown to exhibit this double inhibitory activity. Geske et al. [97] have reported that synthetic analogues of AHLs with additional aromatic moieties [N-(indole-3-butanoyl)-L-HSL andN-(4-bromo-phenylacetanoyl)-L-HSL] display inhibitory activity on LasR-based QS system as well as biofilm formation inP. aeruginosa PAO1. Synthetic AHLs analogues, where the homoserine lactone ring is replaced by a cyclohexanone ring, downregulate expression of theLasI AHL synthase, resulting in a reduced expression of the virulence factors pyocyanin and elastase and in an alteration of biofilm morphology/phenotype [98]. Nonhydrolysable cyclopentyl analogues of AHLs (N-acyle cyclopentylamides) inhibit thelasI andrhlA expression, the production of virulence factors, including elastase, pyocyanin, and rhamnolipids, and the biofilm formation, without affecting bacterial growth [99].Halogenated furanones (particularly furanones C-30 and C-56), inspired from natural compounds produced by the marine macroalgaDelisea pulchra, exhibit biofilm reduction and target thelas andrhl systems inP. aeruginosa [55, 100, 101]. Besides, in mouse lungs infected withP. aeruginosa, they were found to inhibit bacterial colonization to improve the clearance of bacteria from the host and to reduce the tissue damage [102].Among the macrolide antibiotics, azithromycin, derived fromSaccharopolyspora erythraea, has been the most investigated anti-QS antibiotic that presents a strong QS and biofilm inhibitory effect inP. aeruginosa [103–105]. Indeed, at subinhibitory azithromycin concentration (2 μg/mL),P. aeruginosa produces lower AHL signal molecules and virulence factors [106, 107] suggesting that the observed biofilm inhibition is at least partially due to the reduction of both C4-HSL and 3-oxo-C12-HSL production [108]. Interestingly, azithromycin has been reported to diminish the expression ofGacA but alsoRsmA at translational level [109], to inhibit the synthesis of alginate [103] and to reduce the three types of motility (swimming, swarming, and twitching) [110].Penicillic acid and patulin, two secondary fungal metabolites fromPenicillium species, were shown to effect QS-controlled gene expression inP. aeruginosa, most likely by affecting the RhlR and LasR regulatory proteins at posttranscriptional level.In vitro studies showed thatP. aeruginosa PAO1 biofilms treated with patulin and tobramycin were considerably more susceptible to the antibiotic as compared to control biofilms exposed to either tobramycin or patulin alone [111]. However, treatment with patulin alone did not affect development of the biofilm and no hypothesis of mechanisms of action was proposed by authors. The genotoxicity of patulin certainly limits its potential usefulness [112].Manoalide, a sesterterpenoid from the marine organismLuffariella variabilis, exhibits antibiofilm and anti-QS activities (las system) inP. aeruginosa without bactericidal effects [113], although presenting antibiotic activity against gram-positive bacteria [114].Solenopsin A alkaloid, isolated from the antSolenopsis invicta, inhibitsP. aeruginosa pyocyanin production, probably through disruption of therhl signaling system and reduces biofilm production in a dose-dependent manner [115].Mammalian cells release enzymes called paraoxonases 1 (extracted from human and murine sera) that have lactonase activity; degradingP. aeruginosa AHLs, they prevent, in an indirect way, QS and biofilm formation [116, 117].The phenolic compound curcumin, a major constituent of turmeric roots (Curcuma longa L.), downregulates virulence factors (pyocyanin, elastase, and protease) inP. aeruginosa PAO1 and inhibits adherence of the bacteria to polypropylene surfaces. This was correlated with a decrease in 3-oxo-C12-HSL production [118]. Rosmarinic acid, a natural phenolic compound produced by the root ofOcimum basilicum L. uponP. aeruginosa infection, prevents biofilm formation but fails to penetrate mature biofilm underin vivo andin vitro conditions [89]. Structure-based virtual screenings against LasR and RhlR receptor proteins effectively indicate that rosmarinic acid is a potential QS inhibitor [119]. Ellagic acid derivatives, fromTerminalia chebula Retz., have been shown to downregulatelasIR andrhlIR genes expression with a concomitant AHLs decrease, resulting in the attenuation of virulence factor production and in an enhanced sensitivity of biofilm towards tobramycin [120]. Girennavar et al. [121] demonstrated that the furocoumarins from grapefruit juice, bergamottin and dihydroxybergamottin, inhibit the activities of the autoinducers AI-1 (N-3 hydroxybutanoyl-homoserine lactone) and AI-2 (furanosyl borate diester) in aV. harveyi bioassay. Besides, these authors showed that AI-1 and AI-2 inhibit biofilm formation inE. coli O157:H7,Salmonella typhimurium, andP. aeruginosa without affecting bacterial growth. However, the mechanisms of action remain unclear.Docking screening for QS inhibitors predicted that the flavone baicalein, obtained from the roots ofScutellaria baicalensis Georgi, could interact withA. tumefaciens QS transcription activator protein TraR. Effectively, at 20 μM, baicalein promotes the proteolysis of the signal receptor TraR protein inEscherichia coli biosensor, significantly inhibiting the biofilm formation byP. aeruginosa [122]. Similarly, the screening of traditional Chinese medicinal plants identified the anthraquinone emodin, extracted from rhubarb (Rheum palmatum L.); emodin actually inhibits theP. aeruginosa biofilm formation at 20 μM, increasing the activity of ampicillin [123].The flavan-3-ol catechin, isolated from the bark ofCombretum albiflorum (Tul.) Jongkind, as well as the flavanone naringenin, both at 4 mM final concentration, do interfere with QS mechanism inP. aeruginosa PAO1 by affecting autoinducers perception and biofilm formation [124–126]. A coumarate ester isolated from the bark extract of Malagasy endemicDalbergia trichocarpa Baker interferes withP. aeruginosa QS systems (las andrhl), inhibits the biofilm formation and increases the effectiveness of the antibiotic tobramycin in killing biofilm-encapsulatedP. aeruginosa [126] (Figures 4 and 5).Figure 4
Phenolic compounds and derivatives with antibiofilm and anti-QS proprieties.Figure 5
P. aeruginosa biofilm phenotypes and effectiveness of tobramycin treatment in presence of DMSO 1% or coumarate ester (CE) at 300 µg/mL. (a) After 1 day of incubation,P. aeruginosa fails to form structured confluent aggregate in presence of CE as compared to DMSO treatment. (b) CE considerably increases the susceptibility ofP. aeruginosa to tobramycin (100 µg/mL), as shown by the increased proportion of dead cells compared with DMSO. The bacterial viability was assessed by staining the cells with SYTO-9 (green areas—live bacteria) and propidium iodide (red areas—dead bacteria) furnished in the LIVE/DEADBacLight kit. Cells were visualized using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and colored images were assembled using Adobe Photoshop.Recently,Meliaceae,Melastomataceae,Lepidobotryaceae, andSapindaceae, collected from neotropical rainforests in Costa Rica, presented significant anti-QS activities in aChromobacterium violaceum bioassay and/or inhibition of biofilm formation byP. aeruginosa PA14 [127]. Although the exact natures of the active constituents are not yet elucidated, the authors suggest that they could belong to polar polyphenols similar to tannic acid.A recent screening of various herbal extracts revealed that clove extract (Syzygium aromaticum (L.) Merr. Et Perry) inhibits QS-controlled gene expression (las andpqs systems) inP. aeruginosa with eugenol as major active constituent [128]. Eugenol, at subinhibitory concentrations (400 μM) inhibited virulence factors production including elastase, pyocyanin and biofilm formation. In agreement with this finding, subinhibitory concentrations of the clove essential oil significantly reduceslas- andrhl-regulated virulence factors, exopolysaccharide production, and biofilm formation byP. aeruginosa PAO1 [129].Ajoene, an allyl sulfide isolated from garlic (Allium sativum L.), has been reported to affect QS-regulated genes inP. aeruginosa, including the production of rhamnolipids. Additionally, ajoene synergizes with the antibiotic tobramycin in killing biofilm-encapsulatedP. aeruginosa, improving the clearance ofP. aeruginosa from lungs in a mouse model of pulmonary infection [130]. A naturally-inspired organosulfur compound (S-phenyl-L-cysteine sulfoxide) and its derivative (diphenyl disulfide) have been reported to significantly reduce the amount of biofilm formation byP. aeruginosa [131]. TheS-phenyl-L-cysteine sulfoxide antagonizes both thelas andrhl QS systems whereas the diphenyl disulfide only interferes with thelas system.


### 4.2. Antibiofilm Compounds without or with Unspecified Anti-QS Activity

Various organisms, including prokaryotes and eukaryotes (marine organisms, animals, and plants) have been reported to produce secondary metabolites which exert antibiofilm activity. Some of those natural compounds have been used as models to build synthetic antibiofilm compounds againstP. aeruginosa.Bromoageliferin, pyrrole-imidazole alkaloids from marine sponges (Agelas conifer,Agelaceae), has been the scaffolding for the development of two derivatives,trans-bromoageliferin analogue 1 (TAGE) andcis-bromoageliferin analogue 2 (CAGE). Both synthetic derivatives inhibit biofilm formation and furthermore are able to disperse preexistingP. aeruginosa PAO1 biofilms without demonstrating a bactericidal or growth-inhibiting effect [132]. Analogues based upon the oroidin template, parent molecules of bromoageliferin, have been synthesized and screened inP. aeruginosa for their antibiofilm ability [133]. The authors found that the most potent analogue turned out to be dihydrosventrin, a variant of the pyrrole-imidazole alkaloids sventrin (fromAgelas sventres) which exhibits biofilm inhibition and biofilm dispersion for different strains ofP. aeruginosa without any microbicidal activity.Alginate lyase, produced byP. aeruginosa itself, promotes biofilm dispersion and acts synergically with antibiotics for successful elimination of mucoid strains ofP. aeruginosa established in the respiratory tracts of cystic fibrosis patients [134]. However, a recent study demonstrated that this effect cannot be attributed to the catalytic activity of the enzyme. Indeed, bovine serum albumin or simple amino acids lead to the same results. The authors postulate that alginate lyase acts simply as a nutrient source, modulating cellular metabolism and thus inducing cellular detachment and enhancing tobramycin efficacy [135].Bovine pancreatic Dnase I and Dnase-1L2, extracted from humanstratum corneum, exhibited strong antibiofilm activity inP. aeruginosa [136]. Indeed, the degradation of extracellular DNA leads to an altered biofilm that permits increased antibiotics penetration [137].Extracts of Ginger (Zingiber officinale Rosc.), long used by Indians, Asians, and Arabs to treat numerous ailments [137], inhibitP. aeruginosa PA14 biofilm formation through the reduction of c-di-GMP production and consequent reduction of total polysaccharides production [138]. The ginger extract revealed no AHL-based QS inhibition in theChromobacterium violaceum CV026 andAgrobacterium tumefaciens NT1 reporter biosensor systems. The major component of dry ginger root, zingerone (vanillyl acetone), has been shown to inhibit biofilm formation, to increase the susceptibility ofP. aeruginosa PAO1 to ciprofloxacin [139] and to inhibit swimming, swarming, and twitching motilities. However, authors did not propose any mechanism of action.The casbane diterpene, isolated from the ethanolic extract ofCroton nepetaefolius Baill., a plant native from northeastern Brazil, inhibits biofilm formation in several clinical relevant species, includingP. aeruginosa (at 250 μg/mL) without affecting the planktonic growth. Authors suggest that this inhibition of biofilm formation may be related to an interaction between casbane diterpene and lipopolysaccharides present on the cell surface, which might affect their adherence properties [140].Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) fromDiospyros dendo Welw. is identified to inhibit biofilm formation without interfering with QS systems inE. coli,P. aeruginosa, andV. harveyi; ursolic acid, at 10 μg/mL, has been found to reduce 72% ofE. coli JM109 biofilm. Transcriptomic analyses led to the conclusion that ursolic acid inhibits biofilm formation by inducing motility [141]. The3β-O-cis-p-coumaroyl-20β-hydroxy-12-ursen-28-oic acid, isolated from the same plant, strongly inhibits biofilm formation byP. aeruginosa PAO1 [142]. However, the mechanism of activity was not investigated.


## 4.1. Antibiofilm Compounds with Anti-QS Activity

Several classes of molecules have been reported to present both antibiofilm formation and anti-QS properties inP. aeruginosa [94–96].Some AHL analogues (Figure3(b)) have been shown to exhibit this double inhibitory activity. Geske et al. [97] have reported that synthetic analogues of AHLs with additional aromatic moieties [N-(indole-3-butanoyl)-L-HSL andN-(4-bromo-phenylacetanoyl)-L-HSL] display inhibitory activity on LasR-based QS system as well as biofilm formation inP. aeruginosa PAO1. Synthetic AHLs analogues, where the homoserine lactone ring is replaced by a cyclohexanone ring, downregulate expression of theLasI AHL synthase, resulting in a reduced expression of the virulence factors pyocyanin and elastase and in an alteration of biofilm morphology/phenotype [98]. Nonhydrolysable cyclopentyl analogues of AHLs (N-acyle cyclopentylamides) inhibit thelasI andrhlA expression, the production of virulence factors, including elastase, pyocyanin, and rhamnolipids, and the biofilm formation, without affecting bacterial growth [99].Halogenated furanones (particularly furanones C-30 and C-56), inspired from natural compounds produced by the marine macroalgaDelisea pulchra, exhibit biofilm reduction and target thelas andrhl systems inP. aeruginosa [55, 100, 101]. Besides, in mouse lungs infected withP. aeruginosa, they were found to inhibit bacterial colonization to improve the clearance of bacteria from the host and to reduce the tissue damage [102].Among the macrolide antibiotics, azithromycin, derived fromSaccharopolyspora erythraea, has been the most investigated anti-QS antibiotic that presents a strong QS and biofilm inhibitory effect inP. aeruginosa [103–105]. Indeed, at subinhibitory azithromycin concentration (2 μg/mL),P. aeruginosa produces lower AHL signal molecules and virulence factors [106, 107] suggesting that the observed biofilm inhibition is at least partially due to the reduction of both C4-HSL and 3-oxo-C12-HSL production [108]. Interestingly, azithromycin has been reported to diminish the expression ofGacA but alsoRsmA at translational level [109], to inhibit the synthesis of alginate [103] and to reduce the three types of motility (swimming, swarming, and twitching) [110].Penicillic acid and patulin, two secondary fungal metabolites fromPenicillium species, were shown to effect QS-controlled gene expression inP. aeruginosa, most likely by affecting the RhlR and LasR regulatory proteins at posttranscriptional level.In vitro studies showed thatP. aeruginosa PAO1 biofilms treated with patulin and tobramycin were considerably more susceptible to the antibiotic as compared to control biofilms exposed to either tobramycin or patulin alone [111]. However, treatment with patulin alone did not affect development of the biofilm and no hypothesis of mechanisms of action was proposed by authors. The genotoxicity of patulin certainly limits its potential usefulness [112].Manoalide, a sesterterpenoid from the marine organismLuffariella variabilis, exhibits antibiofilm and anti-QS activities (las system) inP. aeruginosa without bactericidal effects [113], although presenting antibiotic activity against gram-positive bacteria [114].Solenopsin A alkaloid, isolated from the antSolenopsis invicta, inhibitsP. aeruginosa pyocyanin production, probably through disruption of therhl signaling system and reduces biofilm production in a dose-dependent manner [115].Mammalian cells release enzymes called paraoxonases 1 (extracted from human and murine sera) that have lactonase activity; degradingP. aeruginosa AHLs, they prevent, in an indirect way, QS and biofilm formation [116, 117].The phenolic compound curcumin, a major constituent of turmeric roots (Curcuma longa L.), downregulates virulence factors (pyocyanin, elastase, and protease) inP. aeruginosa PAO1 and inhibits adherence of the bacteria to polypropylene surfaces. This was correlated with a decrease in 3-oxo-C12-HSL production [118]. Rosmarinic acid, a natural phenolic compound produced by the root ofOcimum basilicum L. uponP. aeruginosa infection, prevents biofilm formation but fails to penetrate mature biofilm underin vivo andin vitro conditions [89]. Structure-based virtual screenings against LasR and RhlR receptor proteins effectively indicate that rosmarinic acid is a potential QS inhibitor [119]. Ellagic acid derivatives, fromTerminalia chebula Retz., have been shown to downregulatelasIR andrhlIR genes expression with a concomitant AHLs decrease, resulting in the attenuation of virulence factor production and in an enhanced sensitivity of biofilm towards tobramycin [120]. Girennavar et al. [121] demonstrated that the furocoumarins from grapefruit juice, bergamottin and dihydroxybergamottin, inhibit the activities of the autoinducers AI-1 (N-3 hydroxybutanoyl-homoserine lactone) and AI-2 (furanosyl borate diester) in aV. harveyi bioassay. Besides, these authors showed that AI-1 and AI-2 inhibit biofilm formation inE. coli O157:H7,Salmonella typhimurium, andP. aeruginosa without affecting bacterial growth. However, the mechanisms of action remain unclear.Docking screening for QS inhibitors predicted that the flavone baicalein, obtained from the roots ofScutellaria baicalensis Georgi, could interact withA. tumefaciens QS transcription activator protein TraR. Effectively, at 20 μM, baicalein promotes the proteolysis of the signal receptor TraR protein inEscherichia coli biosensor, significantly inhibiting the biofilm formation byP. aeruginosa [122]. Similarly, the screening of traditional Chinese medicinal plants identified the anthraquinone emodin, extracted from rhubarb (Rheum palmatum L.); emodin actually inhibits theP. aeruginosa biofilm formation at 20 μM, increasing the activity of ampicillin [123].The flavan-3-ol catechin, isolated from the bark ofCombretum albiflorum (Tul.) Jongkind, as well as the flavanone naringenin, both at 4 mM final concentration, do interfere with QS mechanism inP. aeruginosa PAO1 by affecting autoinducers perception and biofilm formation [124–126]. A coumarate ester isolated from the bark extract of Malagasy endemicDalbergia trichocarpa Baker interferes withP. aeruginosa QS systems (las andrhl), inhibits the biofilm formation and increases the effectiveness of the antibiotic tobramycin in killing biofilm-encapsulatedP. aeruginosa [126] (Figures 4 and 5).Figure 4
Phenolic compounds and derivatives with antibiofilm and anti-QS proprieties.Figure 5
P. aeruginosa biofilm phenotypes and effectiveness of tobramycin treatment in presence of DMSO 1% or coumarate ester (CE) at 300 µg/mL. (a) After 1 day of incubation,P. aeruginosa fails to form structured confluent aggregate in presence of CE as compared to DMSO treatment. (b) CE considerably increases the susceptibility ofP. aeruginosa to tobramycin (100 µg/mL), as shown by the increased proportion of dead cells compared with DMSO. The bacterial viability was assessed by staining the cells with SYTO-9 (green areas—live bacteria) and propidium iodide (red areas—dead bacteria) furnished in the LIVE/DEADBacLight kit. Cells were visualized using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and colored images were assembled using Adobe Photoshop.Recently,Meliaceae,Melastomataceae,Lepidobotryaceae, andSapindaceae, collected from neotropical rainforests in Costa Rica, presented significant anti-QS activities in aChromobacterium violaceum bioassay and/or inhibition of biofilm formation byP. aeruginosa PA14 [127]. Although the exact natures of the active constituents are not yet elucidated, the authors suggest that they could belong to polar polyphenols similar to tannic acid.A recent screening of various herbal extracts revealed that clove extract (Syzygium aromaticum (L.) Merr. Et Perry) inhibits QS-controlled gene expression (las andpqs systems) inP. aeruginosa with eugenol as major active constituent [128]. Eugenol, at subinhibitory concentrations (400 μM) inhibited virulence factors production including elastase, pyocyanin and biofilm formation. In agreement with this finding, subinhibitory concentrations of the clove essential oil significantly reduceslas- andrhl-regulated virulence factors, exopolysaccharide production, and biofilm formation byP. aeruginosa PAO1 [129].Ajoene, an allyl sulfide isolated from garlic (Allium sativum L.), has been reported to affect QS-regulated genes inP. aeruginosa, including the production of rhamnolipids. Additionally, ajoene synergizes with the antibiotic tobramycin in killing biofilm-encapsulatedP. aeruginosa, improving the clearance ofP. aeruginosa from lungs in a mouse model of pulmonary infection [130]. A naturally-inspired organosulfur compound (S-phenyl-L-cysteine sulfoxide) and its derivative (diphenyl disulfide) have been reported to significantly reduce the amount of biofilm formation byP. aeruginosa [131]. TheS-phenyl-L-cysteine sulfoxide antagonizes both thelas andrhl QS systems whereas the diphenyl disulfide only interferes with thelas system.


## 4.2. Antibiofilm Compounds without or with Unspecified Anti-QS Activity

Various organisms, including prokaryotes and eukaryotes (marine organisms, animals, and plants) have been reported to produce secondary metabolites which exert antibiofilm activity. Some of those natural compounds have been used as models to build synthetic antibiofilm compounds againstP. aeruginosa.Bromoageliferin, pyrrole-imidazole alkaloids from marine sponges (Agelas conifer,Agelaceae), has been the scaffolding for the development of two derivatives,trans-bromoageliferin analogue 1 (TAGE) andcis-bromoageliferin analogue 2 (CAGE). Both synthetic derivatives inhibit biofilm formation and furthermore are able to disperse preexistingP. aeruginosa PAO1 biofilms without demonstrating a bactericidal or growth-inhibiting effect [132]. Analogues based upon the oroidin template, parent molecules of bromoageliferin, have been synthesized and screened inP. aeruginosa for their antibiofilm ability [133]. The authors found that the most potent analogue turned out to be dihydrosventrin, a variant of the pyrrole-imidazole alkaloids sventrin (fromAgelas sventres) which exhibits biofilm inhibition and biofilm dispersion for different strains ofP. aeruginosa without any microbicidal activity.Alginate lyase, produced byP. aeruginosa itself, promotes biofilm dispersion and acts synergically with antibiotics for successful elimination of mucoid strains ofP. aeruginosa established in the respiratory tracts of cystic fibrosis patients [134]. However, a recent study demonstrated that this effect cannot be attributed to the catalytic activity of the enzyme. Indeed, bovine serum albumin or simple amino acids lead to the same results. The authors postulate that alginate lyase acts simply as a nutrient source, modulating cellular metabolism and thus inducing cellular detachment and enhancing tobramycin efficacy [135].Bovine pancreatic Dnase I and Dnase-1L2, extracted from humanstratum corneum, exhibited strong antibiofilm activity inP. aeruginosa [136]. Indeed, the degradation of extracellular DNA leads to an altered biofilm that permits increased antibiotics penetration [137].Extracts of Ginger (Zingiber officinale Rosc.), long used by Indians, Asians, and Arabs to treat numerous ailments [137], inhibitP. aeruginosa PA14 biofilm formation through the reduction of c-di-GMP production and consequent reduction of total polysaccharides production [138]. The ginger extract revealed no AHL-based QS inhibition in theChromobacterium violaceum CV026 andAgrobacterium tumefaciens NT1 reporter biosensor systems. The major component of dry ginger root, zingerone (vanillyl acetone), has been shown to inhibit biofilm formation, to increase the susceptibility ofP. aeruginosa PAO1 to ciprofloxacin [139] and to inhibit swimming, swarming, and twitching motilities. However, authors did not propose any mechanism of action.The casbane diterpene, isolated from the ethanolic extract ofCroton nepetaefolius Baill., a plant native from northeastern Brazil, inhibits biofilm formation in several clinical relevant species, includingP. aeruginosa (at 250 μg/mL) without affecting the planktonic growth. Authors suggest that this inhibition of biofilm formation may be related to an interaction between casbane diterpene and lipopolysaccharides present on the cell surface, which might affect their adherence properties [140].Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) fromDiospyros dendo Welw. is identified to inhibit biofilm formation without interfering with QS systems inE. coli,P. aeruginosa, andV. harveyi; ursolic acid, at 10 μg/mL, has been found to reduce 72% ofE. coli JM109 biofilm. Transcriptomic analyses led to the conclusion that ursolic acid inhibits biofilm formation by inducing motility [141]. The3β-O-cis-p-coumaroyl-20β-hydroxy-12-ursen-28-oic acid, isolated from the same plant, strongly inhibits biofilm formation byP. aeruginosa PAO1 [142]. However, the mechanism of activity was not investigated.


## 5. Concluding Remarks and Perspectives

There is increasing evidence that biofilm-mediated infection facilitates the development of chronic infectious diseases and recurrent infections [143–145]. Relevance in using antibiofilm compounds is based on the restoration of antibiotic effectiveness by facilitating their penetration through compromised biofilm structure. Moreover, a degradation of the biofilm matrix could render infectious bacteria reachable to immune defenses (e.g., polymorphonuclear leukocytes, innate, and specific antibodies) [146, 147]. Thus, antibiofilm compounds could be interesting antibiotic adjuvants to prevent or treat chronic infections. Similarly, relevance in using anti-QS compounds is based on the concomitant drastic reduction of virulence factors expression, which gives the necessary time for immune defense systems to elaborate appropriate responses by the recruitment of immune cells and production of specific antibodies. Unlike antibiofilm compounds, anti-QS compounds are interesting to prevent or jugulate acute infection. However, it should also be noted that (i) anti-QS and antibiofilm compounds may lose their appeal in immune compromised patients who often harbor bacteria that are still alive but present in a disorganized and less virulent stage; (ii) QS systems do not control the totality of virulence factors expression; and (iii) the development of anti-QS bacterial resistance cannot be excluded [148]. These facts partly explain why the discovery of QS modulators has not yet led to major therapeutic breakthroughs. In our opinion, such bioactive compounds will probably not substitute antibiotics but rather optimize the effectiveness of infectious diseases treatment, notably through biofilm disruption and antibiotic dose reduction; their use is also appealing to optimize the use of microbicidal products by reducing biofilm encroachment on biomaterials and medical devices.In the perspective of therapeutic application, very few studies have been progressed to clinical trial. To the best of our knowledge, garlic is the only extract with anti-QS and antibiofilm to have been tested in a clinical trial with nonsignificant results, contrary to its drasticin vitro bioactivity effect [149]. One reason of this fact is that behavior of clinical isolates may be different when grown in laboratory condition and in human body which could lead to unexpected biofilm development. Thus, before progressing in clinical trial of relevant bioactive compounds, effort on the improvement of experimentalin vitro andin vivo conditions should be addressed and clinical trial protocols should be discussed.Potent antibiofilm agents are considered interesting if they exert a sustainable bioactivity; this can be indicated by an activity that resists accumulating bacterial toxins, enzymes, and metabolites for more than 48 h in culture media. As less than half of bioactive products have been tested up to 48 hours, further investigations are warranted to select those compounds with sustained activities, which would have more chances to be active in clinical conditions. Halogenated furanones have been widely studied for their powerful anti-QS and antibiofilm activities (<10μM) [100]. However, their toxic and carcinogenic properties relegate them so far to the role of positive QS inhibitory controls in laboratory experiments [150, 151]. In this regard, herbal phenolic compounds and their derivatives, frequent in food components, and more particularly those already present in popular and approved herbal drugs (i.e., rosmarinic acid inMelissa officinalis L.), are promising candidates to develop antibiofilm agents; however, structure-activity studies are still required to better assign essential structural features responsible for antibiofilm activity. In the same perspective, searching for compounds active at nanomolar levels should be privileged as these could presumably present lower toxicity risks. The QS system is an obvious target for biofilm-associated infections as QS interacts, directly and/or indirectly, in different steps of biofilm formation. Intriguingly, even if QS inhibition is the most extensively studied approach againstP. aeruginosa, several anti-QS natural compounds have not been yet investigated for their antibiofilm activity (e.g., human sexual hormones and some antibiotics at subinhibitory concentration, notably ceftazidime and ciprofloxacin) [103, 152]. Attractive therapeutic agents are those which modulate QS system(s) with an extending or particular impact on biofilm lifestyle; they could then be helpful as a preventive or curative approach and at every step of infectious diseases (acute and chronic). However, finding universal antibiofilm compounds represents a challenge as biofilm lifestyle, composition, and phenotype strongly depend on several parameters, such as nutritional conditions. In this regard, we support the hypothesis that compounds which targetGacS/GacA pathway are worthy of interest with respect to the pathway hierarchically upstream position that controls positively both QS system and exopolysaccharides biosynthesis (Psl) (Figure 2). Such compounds could possibly impair almost all the biofilm lifestyle cycle ofP. aeruginosa, from irreversible attachment to dispersion stages (Table 3) and could be powerful allies for conventional antibiotics in the struggle against bacterial biofilm-mediated infections [8, 12, 95].Table 3
Factors affecting biofilm lifestyle regulation.
Biofilm lifestyle cycle
Stage IReversible attachment
Stage IIIrreversible attachment
Stage IIIMaturation-1(microcolony development)
Stage IVMaturation-2(maintenance)
Stage VDispersion
Implicated factors
c-di-GMP
(i) Flagella and type IV pili(ii) Adhesins(iii) Psl, Pel
(i) Cell-to-cell communicationlasI (ii) Type IV fimbriae(iii) Matrix components: Psl, Pel, eDNA(iv) Lectin A, B(v) rhlA
(i) Cell-to-cell communication(ii) Matrix components: Psl, Pel, eDNA(iii) rhlA
Rhamnolipids
General target
(i) Promoting planktonic lifestyle(ii) Blocking switch from planktonic to biofilm lifestyle
Reducing initial adhesion and interaction
Interfering with QS communication
(i) Reactivating metabolic activity for antibiotic efficiency.(ii) Interfering with QS communication
Promoting dispersion and degradation
Examples of extracts or compounds
(i) Ginger extract(ii) Ursolic acid
(i) Casbane diterpene(ii) Coumarate ester
Coumarate ester
(i) Solenopsin A(ii) Naringenin(iii) Furanone C-30 and -56(iv) Flavan-3-ol Catechin(v) Ajoene(vi) Coumarate ester
DNAse I, 1L2



---

*Source: 759348-2015-03-19.xml*