Effect of quorum quenchers on virulence factors production and quorum sensing signalling pathway of non-mucoid, mucoid, and heavily mucoid Pseudomonas aeruginosa.

Quorum quenching (QQ), a mechanism which inhibits, interferes or inactivates quorum sensing, has been investigated for control of biofilms instigated by quorum sensing process. Application of quorum quenchers (QQs) provides the possibility to investigate how different phenotypes of Pseudomonas aeruginosa (non-mucoid, mucoid, and heavily mucoid strains) modulate their gene expression to form biofilms, their quorum sensing (QS) mediated biofilm to be formed, and their virulence expressed. The mRNA expression of the AHL-mediated QS circuit and AHL-mediated virulence factors in P. aeruginosa was investigated in presence of QQs. qPCR analysis showed that farnesol and tyrosol actively reduce the expression of the synthase protein, LasI and RhlI, and prevent production of 3OC12-HSL and C4-HSL, respectively. Also, the use of farnesol and tyrosol significantly moderated gene expression for exo-proteins toxA, aprA, LasB, as well as rhlAB, which are responsible for rhamnolipid production. Our findings were promising, identifying several suppressive regulatory effects of furanone and Candida albicans QS signal molecules, tyrosol, and farnesol on the AHL-mediated P. aeruginosa QS network and related virulence factors.

Antifungal effect of triclosan on Aspergillus fumigatus: quorum quenching role as a single agent and synergy with liposomal amphotericin-B.

The purpose of this research was to determine Aspergillus fumigatus conidial viability and its biofilm formation upon treatment with triclosan and amphotericin-B loaded liposomes. A. fumigatus was treated with the antimicrobials, triclosan and liposomal amphotericin-B (L-AMB), in single and combined supplementation. To quantify the cells' viability upon treatments, resazurin-based viability assay was performed. Confocal laser scanning microscopy was done by applying FUN-1 stain to screen the role of the agents on extracellular polymeric substances. Total A. fumigatus biomass upon treatments was estimated by using crystal violet-based assay. To study the agents' effect on the conidial viability, flow cytometry analysis was performed. Expression levels of A. fumigatus genes encoding cell wall proteins, α-(1,3)-glucans and galactosaminogalactan were analysed by real-time polymerase chain reaction assay. A synergistic interaction occurred between triclosan and L-AMB when they were added sequentially (triclosan + L-AMB) at their sub-minimum inhibitory concentrations, the triclosan and L-AMB MICs were dropped to 0.6 and 0.2 mg/L, respectively, from 2 to 1 mg/L. Besides, L-AMB and triclosan contributed to the down-regulation of α-(1,3)-glucan and galactosaminogalactan in A. fumigatus conidia and resulted in less conidia aggregation and mycelia adhesion to the biotic/abiotic surfaces; A. fumigatus conidia-became hydrophilic upon treatment, as a result of rodlet layer being masked by a hydrophilic layer or modified by the ionic strength of the rodlet layer. In A. fumigatus, the potential mechanisms of action for L-AMB might be through killing the cells and for triclosan through interrupting the cells' development as a consequence of quorum quenching.

Quorum quenchers affect the virulence regulation of non-mucoid, mucoid and heavily mucoid biofilms co-cultured on cell lines.

Biofilm formation conferring pathogenicity is a survival strategy for Pseudomonas aeruginosa. P. aeruginosa's virulence may differ due to differences in host-microbe interactions and the growth environment. The epithelial cell line within the respiratory system and the keratinocytes on the skin form the first physical barrier of defence. P. aeruginosa spp. biofilm formation and virulence factor secretion with and without quorum quenching (QQ) treatment was studied in co-culture using A549 and HaCaT cell lines; pyocyanin and rhamnolipid productions and elastolytic activity as virulence factors were quantified by independent assays. Biofilm formation was evaluated under dynamic conditions by quantifying total carbohydrates, alginate, proteins and eDNA. A sandwich ELISA was performed to study IL-8 secretion by the epithelial cells. The difference in gene expression of the quorum sensing (QS) and virulence factors between strains during individual and combination treatments was analysed by qPCR. Combination treatment by farnesol and tyrosol was more effective against P. aeruginosa biofilms when grown in co-cultures. The strain RBHi was found to be 3 to 4 times more virulent compared to PAO1 and NCTC 10,662, respectively, and combination treatment was more effective against RBHi strain when grown in co-culture with A549 cell line. The addition of quorum quenchers (QQs) individually and in combination reduced IL-8 secretion by A549 cells. Relative mRNA expression showed upregulation of the QS genes and virulence factors. Co-culture of P. aeruginosa and HaCaT cell line showed a general decrease in gene expression, especially in the case of P. aeruginosa RBHi when treated with farnesol and tyrosol combination.Key points• Differentiating the interactions of biofilm formed by different phenotypes of P. aeruginosa, NCTC 10,662 (non-mucoid), PAO1 (semi mucoid) and RBHi (heavily mucoid).• Biofilm formed by these P. aeruginosa strains on two commonly afflicted tissues represented by A549 (lung) and HaCaT (skin) cell lines.• Anti-biofilm/anti-virulence roles of quorum quenchers, tyrosol and farnesol in co-cultures.