Pseudomonas aeruginosa has already been stipulated as a “critical” pathogen, emphasizing the urgent need for researching and developing novel antibacterial agents due to multidrug resistance. Bacterial biofilm formation facilitates cystic fibrosis development and restricts the antibacterial potential of many current antibiotics. The capacity of P. aeruginosa to form biofilms and resist antibiotics is closely correlated with quorum sensing (QS). Bacterial QS is being contemplated as a promising target for developing novel antibacterial agents. QS inhibitors are a promising strategy for treating chronic infections. This study reported that the active compound PT22 (1H-pyrrole-2,5-dicarboxylic acid) isolated from Perenniporia tephropora FF2, one endophytic fungus from Areca catechu L., presents QS inhibitory activity against P. aeruginosa. Combined with gentamycin or piperacillin, PT22 functions as a novel antibiotic accelerant against P. aeruginosa. PT22 (0.50 mg/mL, 0.75 mg/mL, and 1.00 mg/mL) reduces the production of QS-related virulence factors, such as pyocyanin and rhamnolipid, and inhibits biofilm formation of P. aeruginosa PAO1 instead of affecting its growth. The architectural disruption of the biofilms was confirmed by visualization through scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Real-time quantitative PCR (RT-qPCR) indicated that PT22 significantly attenuated the expression of QS-related genes followed by docking analysis of molecules against QS activator proteins. PT22 dramatically increased the survival rate of Galleria mellonella. PT22 combined with gentamycin or piperacillin presents significant inhibition of biofilm formation and eradication of mature biofilm compared to monotherapy, which was also confirmed by visualization through SEM and CLSM. After being treated with PT22 combined with gentamycin or piperacillin, the survival rates of G. mellonella were significantly increased compared to those of monotherapy. PT22 significantly enhanced the susceptibility of gentamycin and piperacillin against P. aeruginosa PAO1. Our results suggest that PT22 from P. tephropora FF2 as a potent QS inhibitor is a candidate antibiotic accelerant to combat the antibiotic resistance of P. aeruginosa.

Quorum sensing (QS) inhibition is recognized as a novel antimicrobial target for infections caused by drug-resistant pathogens and is an attractive strategy for antipathogenic agent development. We designed and synthesized three parts of 3-(2-isocyanobenzyl)-1H-indole derivatives and tested their activity as novel quorum sensing inhibitors (QSIs). 3-(2-Isocyanobenzyl)-1H-indole derivatives demonstrated promising QS, biofilms, and prodigiosin inhibitory activities against Serratia marcescens at subminimum inhibitory concentrations (sub-MICs). In particular, 3-(2-isocyano-6-methylbenzyl)-1H-indole (IMBI, 32) was identified as the best candidate based on several screening assays, including biofilm and prodigiosin inhibition. Further studies demonstrated that exposure to IMBI at 1.56 mu g/mL to S. marcescens NJ01 significantly inhibited the formation of biofilms by 42%. The IMBI treatment on S. marcescens NJ01 notably enhanced the susceptibility of the formed biofilms, destroying the architecture of the biofilms by up to 40%, as evidenced by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). For interference of virulence factors in S. marcescens NJ01, IMBI at 3.12 mu g/mL inhibited the activity of protease and extracellular polysaccharides (EPS) by 17% and 51%, respectively, which were higher than that of the positive control vanillic acid (VAN). Furthermore, IMBI downregulated the expression of QS- and biofilm-related genes fimA, bsmA, pigP, flhC, rssB, fimC, and rsmA by 1.02- to 2.74-fold. To confirm these findings, molecular docking was performed, which indicated that the binding of IMBI to SmaR, RhlI, RhlR, LasR, and CviR could antagonize the expression of QS-linked traits. In addition, molecular dynamic simulations (MD) and energy calculations indicated that the binding of receptors with IMBI was extremely stable. The biofilms of S. marcescens NJ01 were markedly reduced by 50% when IMBI (0.39 mu g/mL) was combined with kanamycin (0.15 mu g/mL). In conclusion, this study highlights the potency of IMBI in inhibiting the virulence factors of S. marcescens. IMBI has all the potential to be developed as an effective and efficient QS inhibitor and antibiofilm agent in order to restore or improve antimicrobial drug sensitivity.