ORIGINAL ARTICLE Phospholipid Content of Pseudomonas aeruginosa PAO1 Is Modulated by the Growth Phase Rather Than the Immobilization State Caroline Le Sénéchal 1 · Marc Crouzet 1 · Patricia Costaglioli 2 · Christophe Barthe 1 · Corinne Buré 3 · Sébastien Vilain 2 Received: 1 April 2019 / Revised: 15 July 2019 / Accepted: 16 July 2019 © 2019 AOCS Abstract Biofilms have significance in medical, industrial, and environmental settings, and can cause important damage. As biofilms are tolerant to various stresses, including antibi- otics, it is necessary to better understand their formation. For this reason, we characterized the phospholipidome of Pseudo- monas aeruginosa, an opportunistic pathogen involved in numerous infections, during the first steps of the biofilm devel- opment. By a liquid chromatography-tandem mass spectrome- try time-course analysis over a 24-h period, we compared the phospholipid (PL) composition of immobilized (attached) and planktonic (unattached) P. aeruginosa PAO1 cells. Our results showed that the PL content of P. aeruginosa PAO1 was mainly modulated by the incubation time, thus related to bacte- rial growth but also, more modestly, by the immobilization state. We observed that relative amounts of PL varied over time with two main profiles and that these profiles are corre- lated to its fatty acid composition, including the degree of unsaturation. A statistical analysis revealed that the PL con- tents of both attached and unattached PAO1 cells were signifi- cantly different mainly after 3 and 6 h of incubation and that the amounts of two PL presented a statistical difference between attached and unattached cells all along the 24-h period: PtdEtn 16:0_18:1 and PtdEtn 18:1_18:1. Keywords Attachment  Biofilm  Lipidomics  Mass spectrometry  Phospholipids  Pseudomonas aeruginosa Lipids (2019). Abbreviations AC attached cell C carbon ESI electrospray ionization GW glass wool LB lysogeny broth MRM multiple reaction monitoring PCA principal component analysis PL phospholipid(s) PtdCho phosphatidylcholine PtdEtn phosphatidylethanolamine PtdGro phosphatidylglycerol PtdIns phosphatidylinositol PtdOH phosphatidic acid PtdSer phosphatidylserine RSD relative standard deviation RPLC- MS/MS reverse-phase liquid chromatography-mass spectrometry SFA saturated fatty acid U unsaturation UC unattached cell UFA Unsaturated Fatty Acid Introduction Biofilms are defined as matrix-enclosed bacterial populations that are adherent to surfaces or interfaces (Costerton et al., 1995). These biological structures are Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article. * Sébastien Vilain sebastien.vilain@bordeaux-inp.fr. 1 CBMN, University of Bordeaux, UMR 5248, F-33600, Pessac, France 2 CBMN, Bordeaux INP, UMR 5248, F-33600, Pessac, France 3 CBMN, CNRS, UMR 5248, F-33600, Pessac, France Lipids (2019) DOI 10.1002/lipd.12184 Lipids (2019)