Tsiry Rasamiravaka

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

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.

The flavanone naringenin reduces the production of quorum sensing-controlled virulence factors in Pseudomonas aeruginosa PAO1

Preliminary screening of the Malagasy plant Combretum albiflorum for compounds attenuating the production of quorum sensing (QS)-controlled virulence factors in bacteria led to the identification of active fractions containing flavonoids. In the present study, several flavonoids belonging to the flavone, flavanone, flavonol and chalcone structural groups were screened for their capacity to reduce the production of QS-controlled factors in the opportunistic pathogen Pseudomonas aeruginosa (strain PAO1). Flavanones (i.e. naringenin, eriodictyol and taxifolin) significantly reduced the production of pyocyanin and elastase in P. aeruginosa without affecting bacterial growth. Consistently, 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. Naringenin also dramatically reduced the production of the acylhomoserine lactones N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), which is driven by the lasI and rhlI gene products, respectively. In addition, using mutant strains deficient for autoinduction (ΔlasI and ΔrhlI) and LasR- and RhlR-based biosensors, it was shown that QS inhibition by naringenin not only is the consequence of a reduced production of autoinduction compounds but also results from a defect in the proper functioning of the RlhR-C4-HSL complex. Widely distributed in the plant kingdom, flavonoids are known for their numerous and determinant roles in plant physiology, plant development and in the success of plant-rhizobia interactions, but, as shown here, some of them also have a role as inhibitors of the virulence of pathogenic bacteria by interfering with QS mechanisms.

Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume.

Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms.

Endemic Malagasy Dalbergia species inhibit quorum sensing in Pseudomonas aeruginosa PAO1

Various species of the plant genus Dalbergia are traditionally used as medicine for sundry ailments and some of them have been shown recently to quench the virulence of Gram-positive and Gram-negative bacteria. Cell-to-cell communication mechanisms, quorum sensing (QS) in particular, are key regulators of virulence in many pathogenic bacteria. Screening n-hexane extracts of leaves, roots and bark of endemic Malagasy Dalbergia species for their capacity to antagonize QS mechanisms in Pseudomonas aeruginosa PAO1 showed that many reduced the expression of the QS-regulated genes lasB and rhlA. However, only the extract of Dalbergia trichocarpa bark (DTB) showed a significant reduction of QS gene expression without any effect on the aceA gene encoding a QS-independent isocitrate lyase. Further characterization of DTB impact on QS revealed that the QS systems las and rhl are inhibited and that swarming, twitching, biofilm formation and the production of pyocyanin, elastase and proteases are also hampered in the presence of the DTB extract. Importantly, compared with the known QS inhibitor naringenin, the DTB extract showed a stronger negative effect on twitching, biofilm formation and tobramycin resistance. Preliminary structural characterization of these potent biofilm disrupters suggests that they belong to the phytosterols. The strong inhibition of motility and biofilm formation suggests that the DTB extract contains agents disrupting biofilm architecture, which is an important observation in the context of the design of new drugs targeting biofilm-encapsulated pathogens.

Quorum-Sensing Mechanisms and Bacterial Response to Antibiotics in P. aeruginosa.

Emergence and worldwide spreading of resistant bacteria to antibiotic have raised the importance for finding therapeutic alternative to compensate antibiotic drawbacks. Quorum sensing (QS) is a cell-to-cell communication involved in the development of various common bacterial behaviors including virulence factors expression, and targeting QS seems to be relevant to the struggle against bacterial infection. In this report, relevant literature on intrication of QS system and antimicrobial sensitivity mechanisms in P. aeruginosa PAO1 are reviewed.

At a supra-physiological concentration, human sexual hormones act as quorum-sensing inhibitors.

N-Acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.

Extracts of Cordia gilletii de wild (Boraginaceae) quench the quorum sensing of Pseudomonas aeruginosa PAO1

Aim: The fight against infectious diseases and antimicrobial resistances needs the exploration of new active compounds with new proprieties like disrupting quorum sensing (QS) mechanisms, which is a cell-to-cell communication that regulates bacterial virulence factors. In this work, leaves and root barks extracts of a Congolese medicinal plant, Cordia gilletii, were investigated for their effect on the production of Pseudomonas aeruginosa major virulence factors regulated by QS. Materials and Methods: The effect of C. gilletii extracts on virulence factors of P. aeruginosa PAO1 was studied by the evaluation of the production of pyocyanine, elastase and biofilm; and by the measurement of the expression of QS-related genes. Results: The dichloromethane extract from root barks was found to quench the production of pyocyanin, a QS-dependent virulence factor in P. aeruginosa PAO1. Moreover, this extract specifically inhibits the expression of several QS-regulated genes (i.e. lasB, rhlA, lasI, lasR, rhlI, and rhlR) and reduces biofilm formation by PAO1. Conclusion: This study contributes to explain the efficacy of C. gilletii in the traditional treatment of infectious diseases caused by P. aeruginosa.

Terpenoids from Platostoma rotundifolium (Briq.) A. J. Paton Alter the Expression of Quorum Sensing-Related Virulence Factors and the Formation of Biofilm in Pseudomonas aeruginosa PAO1

Platostoma rotundifolium (Briq.) A. J. Paton aerial parts are widely used in Burundi traditional medicine to treat infectious diseases. In order to investigate their probable antibacterial activities, crude extracts from P. rotundifolium were assessed for their bactericidal and anti-virulence properties against an opportunistic bacterial model, Pseudomonas aeruginosa PAO1. Whereas none of the tested extracts exert bacteriostatic and/or bactericidal proprieties, the ethyl acetate and dichloromethane extracts exhibit anti-virulence properties against Pseudomonas aeruginosa PAO1 characterized by an alteration in quorum sensing gene expression and biofilm formation without affecting bacterial viability. Bioguided fractionation of the ethyl acetate extract led to the isolation of major anti-virulence compounds that were identified from nuclear magnetic resonance and high-resolution molecular spectroscopy spectra as cassipourol, β-sitosterol and α-amyrin. Globally, cassipourol and β-sitosterol inhibit quorum sensing-regulated and -regulatory genes expression in las and rhl systems without affecting the global regulators gacA and vfr, whereas α-amyrin had no effect on the expression of these genes. These terpenoids disrupt the formation of biofilms at concentrations down to 12.5, 50 and 50 µM for cassipourol, β-sitosterol and α-amyrin, respectively. Moreover, these terpenoids reduce the production of total exopolysaccharides and promote flagella-dependent motilities (swimming and swarming). The isolated terpenoids exert a wide range of inhibition processes, suggesting a complex mechanism of action targeting P. aeruginosa virulence mechanisms which support the wide anti-infectious use of this plant species in traditional Burundian medicine.

An Active Fraction from DalbergiaTrichocarpa Baker Disrupts the Formation and Maintenance of Biofilms in Pseudomonas Aeruginosa PAO1

The bark of Dalbergiatrichocapra Baker is traditionally used in Madagascar as an anti-infective remedy. Beyond the recently known anti-quorum sensing (QS) properties of the D. trichocarpabark n-hexane extract, QSindependent anti-infective activities have been also detected. Indeed, chromatographic fractionation allowed the elution of fraction F1 that affects neither bacterial growth nor the expression of QS-related genes (lasB and rhlA) but significantly reduces the formation of biofilm (55.8 ± 2.3%, as compared to control conditions). Moreover,F1 is able to disrupt the structure of one-day old preformed biofilms, which consequently increases the effectiveness of an antibiotic, levofloxacin, on biofilm-encapsulated bacteria (dead bacteria in presence of levofloxacin-F1 were two-fold higher compared to levofloxacin alone). This F1-triggered disruption of biofilm formation is presumably due to an induced reduction in flagellar-dependent motilities (swimming and swarming) as well as in exopolysaccharides production. The inhibitory effect on biofilm appears reversible as the biofilm formation resumes when F1 is discarded from the culture medium. This interesting non-bactericidal mechanism of action may justify the traditional uses of D. trichocarpa in Malagasy medicine. Further work aims at identifying the compound(s) responsible for this biofilm disruption.