Miniaturized dispersive liquid-liquid microextraction and MALDI MS using ionic liquid matrices for the detection of bacterial communication molecules and virulence factors
The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10 μL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250 fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication.
KeywordsBiofilm Cystic fibrosis Pseudomonas aeruginosa Pyocyanin Quorum sensing Resistance
This work was funded by the SFB1182 “Function and Origin of Metaorganisms,” project A1, and by the Cluster of Excellence “Inflammation at Interfaces.” We thank Nadja Schmitz for the assistance with the experiments and Heidrun Ließegang as well as Corinna Wriedt for the technical help in the preparation of the bacterial cultures.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human or animal subjects. Based on the approval of the Ethics committee (Ethik-Kommission) of the Medical Faculty of the Christian Albrecht University of Kiel (D479/15 amendment of Aug 18, 2016) and according to the ethical standards, sputum samples were obtained from patients with cystic fibrosis after informed consent and Pseudomonas aeruginosa isolates were cultivated.
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