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

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 planktonic to biofilm lifestyle [49].

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

### **5.1 From prokaryotes**

### *5.1.1 Enzymes*

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*  *(Acinetobacter, Agrobacterium tumefaciens, Alteromonas, Comomonas, Halomonas, Hyphomonas, Klebsiella pneumoniae, P. aeruginosa, Ralstonia, Stappia*, and *Variovorax paradoxus*) [50–56].

Four types of enzymes are known to degrade AHLs [57, 58], a phenomenon sometimes described as "quorum quenching" (QQ ) [59]; these include AHLlactonases and decarboxylases that attack the lactone ring (*Bacillus indicus, B. pumilus*, and *B.* sp. SS4 cause significant inhibition of QS-dependent activities in Gram-negative bacteria such as *P. aeruginosa* PAO1, *Serratia marcescens*, and *Vibrio*), AHL-acylases that cleave the acyl side chain (*B. pumilus* S8-07 degrades 3-oxo-C12- HSL into the corresponding lauric acid [60]), and deaminases that separate the lactone ring from the acyl side chain. Recently, lactonases and acylases were identified in *Erythrobacter, Labrenzia***,** and *Bacterioplanes* found in Red Sea sediments; these both degrade AHLs of different acyl chain lengths, particularly the 3-oxo-C12-HSL, and inhibit the formation *P. aeruginosa* PAO1 biofilm [59].

*Mycobacteroides abscessus* subspecies, emerging pathogens, are capable of degrading both PQS and HHQ. *M. abscessus* subsp. *abscessus*, in coculture with *P. aeruginosa* PAO1, reduced PQS levels through a PQS dioxygenase (encoded by the *aqdC* gene), *M. abscessus* subsp. *massiliense*, a recombinant strain overexpressing the *aqdC* gene, reduces the level of the virulence factors pyocyanin, pyoverdine, and rhamnolipids, suggesting that AqdC is a QQ enzyme [61]. Its impact on biofilm formation would have been interesting to investigate as another dioxygenase, the 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase (HodC), was described to cleave PQS, attenuate the production of virulence factors but conversely increase the viable biomass, in both newly formed and established biofilms, by increasing iron availability [62].

### *5.1.2 Organic acids*

The acetic and phenyl lactic acids, found in the supernatant of probiotic strains *Lactobacillus paracasei* subsp. *paracasei* CMGB isolated from newborn feces, were shown to inhibit, at nonbacteriostatic/bactericide levels, the expression of QS genes in *P. aeruginosa*, preventing adherence of bacteria to an inert substratum [63, 64]. Similarly, the lactic acid produced by a potential probiotic *Pediococcus acidilactici* M7 strain, also isolated from newborn feces, inhibits the production of *P. aeruginosa* short-chain AHLs, elastase, protease, pyocyanin, and biofilm as well as the swarming-swimming-twitching motilities [65].

### **5.2 From fungi**

### *5.2.1 Antibiotics and mycotoxins*

Penicillin produced by *Penicillium* spp. has been shown to be effective in controlling a bacterial infection. Recently, about 33 *Penicillium* spp. have been recognized as producers of QS inhibitors such as the small lactone mycotoxins patulin and penicillic acid. The use of patulin can significantly reduce lung infection caused by *P. aeruginosa* on a mouse model. Interestingly, a synergy has been shown *in vitro* between patulin and tobramycin toward *P. aeruginosa* PAO1 biofilms, whereas patulin alone does not affect the development of biofilm [66]. Although the antiinfective property of patulin has been demonstrated, its genotoxicity and potential carcinogenic properties [67] probably preclude clinical applications.

Erythromycin, a macrolide antibiotic isolated from *Saccharopolyspora erythraea*, has been recently demonstrated to reduce virulence factors in *P. aeruginosa* PAO1, including various motilities, biofilm formation, and production of rhamnolipids, total protease, elastase, and pyocyanin at nonmicrobicidal level (1.6 μg/mL) [68]. Comparably,

**41**

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

the erythromycin derivate, azithromycin, shows a strong *P. aeruginosa* QS and biofilm inhibitory effect [69–71] with inhibition of alginate synthesis [69], a reduction of each type of bacteria movement [72] and a diminution of *gacA* gene expression [73]. At weak antibiotic concentration (2 μg/mL), a biofilm inhibition is observed, probably explained by a lower production of both AHL signal molecules, C4-HSL and 3-oxo-

Recently, Kim et al. [77] indicated that the alkylcyclopentanone terrein, isolated from *Aspergillus terreus*, reduced virulence factors (elastase, pyocyanin, and rhamnolipids) and biofilm formation via antagonizing QS receptors without affecting *P. aeruginosa* cell growth. Beyond a negative impact on the production of QS signaling molecules and expression of QS-related genes, terrein also reduced c-di-GMP levels, an important secondary messenger for the switch from planktonic to biofilm lifestyle mode, by decreasing the activity of a diguanylate cyclase

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

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

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

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

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

C12-HSL, and of virulence factors [74–76].

required for c-di-GMP biosynthesis [78].

virulence genes of *P. aeruginosa* PA01 [83].

tobramycin treatment [84].

implants of infected mice.

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

*5.2.2 Alkylcyclopentanone*

**5.3 From Plants**

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

the erythromycin derivate, azithromycin, shows a strong *P. aeruginosa* QS and biofilm inhibitory effect [69–71] with inhibition of alginate synthesis [69], a reduction of each type of bacteria movement [72] and a diminution of *gacA* gene expression [73]. At weak antibiotic concentration (2 μg/mL), a biofilm inhibition is observed, probably explained by a lower production of both AHL signal molecules, C4-HSL and 3-oxo-C12-HSL, and of virulence factors [74–76].
