Targeted and untargeted quantification of quorum sensing signalling molecules in bacterial cultures and biological sampl
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RESEARCH PAPER
Targeted and untargeted quantification of quorum sensing signalling molecules in bacterial cultures and biological samples via HPLC-TQ MS techniques Federica Dal Bello 1 & Michael Zorzi 1 & Riccardo Aigotti 1 & Davide Medica 2 & Vito Fanelli 2 & Vincenzo Cantaluppi 3 & Eleonora Amante 4 & Viviana Teresa Orlandi 5 & Claudio Medana 1 Received: 9 September 2020 / Revised: 9 October 2020 / Accepted: 30 October 2020 # The Author(s) 2020
Abstract Quorum sensing (QS) is the ability of some bacteria to detect and to respond to population density through signalling molecules. QS molecules are involved in motility and cell aggregation mechanisms in diseases such as sepsis. Few biomarkers are currently available to diagnose sepsis, especially in high-risk conditions. The aim of this study was the development of new analytical methods based on liquid chromatography-mass spectrometry for the detection and quantification of QS signalling molecules, including N-acyl homoserine lactones (AHL) and hydroxyquinolones (HQ), in biofluids. Biological samples used in the study were Pseudomonas aeruginosa bacterial cultures and plasma from patients with sepsis. We developed two MS analytical methods, based on neutral loss (NL) and product ion (PI) experiments, to identify and characterize unknown AHL and HQ molecules. We then established a multiple-reaction-monitoring (MRM) method to quantify specific QS compounds. We validated the HPLC-MS-based approaches (MRM-NL-PI), and data were in accord with the validation guidelines. With the NL and PI MS-based methods, we identified and characterized 3 and 13 unknown AHL and HQ compounds, respectively, in biological samples. One of the newly found AHL molecules was C12-AHL, first quantified in Pseudomonas aeruginosa bacterial cultures. The MRM quantitation of analytes in plasma from patients with sepsis confirmed the analytical ability of MRM for the quantification of virulence factors during sepsis.
Keywords Quorum sensing molecules . Homoserine lactones . Hydroxyquinolones . Pseudomonas aeruginosa . Mass spectrometry . Triple quadrupole
Federica Dal Bello and Michael Zorzi contributed equally to this work. * Federica Dal Bello [email protected] 1
Department of Molecular Biotechnology and Health Sciences, University of Turin, via Pietro Giuria 5, 10, 125 Torino, Italy
2
Department of Surgical Science Anesthesia and Critical Care, AOU Città della Salute e della Scienza, University of Turin, Corso Bramante 88, 10, 126 Torino, Italy
3
Department of Translational Medicine, University of Eastern Piedmont, Via Solaroli, 17, 28, 100 Novara, Italy
4
Department of Chemistry, University of Turin, via Pietro Giuria 5, 10, 125 Torino, Italy
5
Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21, 100 Varese, Italy
Abbreviations 3-oxoN-(3-Oxododecanoyl)-L-homoserine lactone C12-AHL AHLs N-Acyl homoserine lactones AI Autoinducer C4-AHL N-Butanoyl-L-homoserine lactone C7 HQ 2-Heptyl-3-hydroxy-4(1H)-quinolone (or PQS) HPLC High-performa
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