From Vasodilator to Virulence Disarmer: Glyceryl Trinitrate Modulates Quorum Sensing in Pseudomonas Aeruginosa Biofilms

Abstract

Antimicrobial resistance, biofilm formation and quorum sensing (QS) are all involved in the persistence and therapeutic challenge of Pseudomonas aeruginosa, an opportunistic pathogen. This study aimed to determine the effect of glyceryl trinitrate (GTN) on pre-established biofilms and QS regulation in clinical isolates of P. aeruginosa. Conventional and automated methods were used to identify clinical isolates and their antimicrobial susceptibility was assessed as well as their ability to form biofilms. The biomass of the biofilms was measured by OD₆₃₀ of crystal violet staining and the antibacterial activity of GTN was measured by resazurin based broth micro dilution assay. The influence of GTN on existing biofilms was phenotypically tested and then analysed in detail by field-emission scanning electron microscopy (FE-SEM). lasR, rhlR and mvfR expression was studied by quantitative real-time PCR. The 83 clinical isolates differed substantially in their ability to form a biofilm (F(82,166) = 15.01, p < 0.0001) and there was only a weak positive non-significant correlation between biofilm biomass and the number of resisted antibiotics (Spearman’s ρ = 0.222, p = 0.0538). GTN could not completely inhibit growth even at the highest concentration of 2 mg/mL. However, GTN decreased the amount of established biofilm biomass in 20/28 isolates (71.4%), of which 6 isolates showed significant decrease in biomass, while the remaining eight isolates showed an increase in the biomass. The corresponding isolate-dependent changes in the architecture of the biofilm were similarly found using FE-SEM. QS transcription was also a mixture with downregulation of mvfR and rhlR in 5 out of 6 isolates, and lasR in varying isolates. Interestingly, all three QS regulators were found to be up-regulated at the same time in PA1 with biofilm biomass being increased. These results show that GTN can affect the existing P. aeruginosa biofilms and the QS-related regulatory responses without the ability to detectably inhibit growth, although its impact is highly isolate dependent. GTN should be further investigated as an anti-virulence therapeutic agent, not as a conventional antibiotic.