Quantificazione mediante LC-MS/MS delle Molecole di segnalazione del quorum sensing di Pseudomonas aeruginosa in diverse condizioni di crescita

Pseudomonas aeruginosa is a great threat for patients with cystic fibrosis, cancer, or AIDS since its infection may lead to morbidity and mortality in immunocompromised individuals. P. aeruginosa infections are difficult to treat due to bacterial resistance to most of available antibiotics. To treat persistent infections and to reduce mortality due to P. aeruginosa infections, non-antibiotic therapeutic approaches, like inhibition of quorum sensing (QS), are necessary. QS refers to the interbacterial communication process to control their gene expression in cell density-dependent manner by controlling production of quorum sensing signalling molecules (QSSMs). P. aeruginosa produces four major QSSMs which are butanoyl-homoserine lactone (C4HSL), 2-heptyl-3-hydroxy-4-quinolone (PQS), 4-hydroxy-2-heptylquinoline (HHQ), and 3-oxo-dodecanoyl-homoserine lactone (3OC12HSL). The development of novel QS-inhibiting antimicrobials requires accurate and sensitive methods to detect and measure concentration of QSSMs. The objective of this study was the development of a novel analytical method based on liquid chromatography-mass spectrometry for the detection and quantification of QSSMs. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with electrospray ion source and triple quadrupole mass spectrometer in multiple-reaction monitoring mode was applied to quantify the concentration of QSSMs produced by P. aeruginosa in different growth conditions. Each of the four QSSMs utilised in this study was detected and quantified by LC-MS/MS displaying distinctive peak. The calibration curves were all linear with the lowest R2 value 0.9545. This sensitive method can detect the four QSSMs with limit of detecting ranging between 1 to 5 ng/mL. The addition of benzanilide as internal standard decreased the inconsistency in the results which increased the precision of the analysis. Sample preparation by liquid-liquid extraction (LLE) was useful to separate the QSSM from the growth media and to concentrate the QSSM. Double LLE gave higher recovery than single LLE, especially for C4HSL and PQS. Quantification of QSSMs in supernatant culture of P. aeruginosa showed that P. aeruginosa grown with agitation generally produced higher concentrations of QSSMs rather than without agitation, especially for C4HSL and 3OC12HSL. This method could be a great starting point for further optimisation of the LC-MS/MS method in quantification of QSSMs.