Membrane phospholipid composition of Pseudomonas aeruginosa grown in a cystic fibrosis mucus-mimicking medium

https://doi.org/10.1016/j.bbamem.2020.183482Get rights and content
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Highlights

  • Growth media altered the membrane phospholipid quantities and fatty acid composition.

  • Growth media altered the membrane phospholipid isomer ratio of Pseudomonas aeruginosa.

  • A low phosphatidylcholine content was observed in synthetic cystic fibrosis medium.

  • Phosphatidylcholine levels linked to susceptibility of P. aeruginosa to carbenicillin.

Abstract

Background

Pseudomonas aeruginosa is a bacterium able to induce serious pulmonary infections in cystic fibrosis (CF) patients. This bacterium is very often antibiotic resistant, partly because of its membrane impermeability, which is linked to the membrane lipid composition. This work aims to study the membrane phospholipids of P. aeruginosa grown in CF sputum-like media.

Methods

Three media were used: Mueller Hilton broth (MHB), synthetic cystic fibrosis medium (SCFM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) complemented SCFM (SCFM-PC). Lipids were extracted and LC-MS/MS analyses were performed. Growth curves, atomic force microscopy images and minimal inhibitory concentration determination were performed in order to compare the growth and potentially link lipid modifications to antibiotic resistance.

Results

Semi-quantification showed phospholipid quantity variation depending on the growth medium. Phosphatidylcholines were detected in traces in SCFM. MS/MS experiments showed an increase of phospholipids derived from DOPC in SCFM-PC. We observed no influence of the medium on the bacterial growth and a minor influence on the bacterial shape. MIC values were generally higher in SCFM and SCFM-PC than in MHB.

Conclusions

We defined a CF sputum-like media which can be used for the membrane lipid extraction of P. aeruginosa. We also showed that the growth medium does have an influence on its membrane lipid composition and antibiotic resistance, especially for SCFM-PC in which P. aeruginosa uses DOPC, in order to make its own membrane.

General significance

Our results show that considerable caution must be taken when choosing a medium for lipid identification and antibiotic testing —especially for phospholipids-enriched media.

Abbreviations

CF
cystic fibrosis
MHB
Mueller Hilton broth
SCFM
synthetic cystic fibrosis medium
DOPC
1,2-dioleoyl-sn-glycero-3-phosphocholine
SCFM-PC
DOPC complemented SCFM
LB
lysogeny broth
ASM
artificial sputum medium
PE
phosphatidylethanolamines
PC
phosphatidylcholines
PG
phosphatidylglycerols
CL
cardiolipins
LC-MS
liquid chromatography - mass spectrometry
MS/MS
tandem mass spectrometry
MIC
minimal inhibitory concentration
ESI-MS
electrospray ionisation-mass spectrometry
PAK
P. aeruginosa strain PAK
CFU
colony forming units
OD600
optical density at 600 nm
AFM
atomic force microscopy
(n)
number of replicates

Keywords

Pseudomonas aeruginosa
CF sputum
Membrane lipids
Antibiotic resistance
Culture medium
Mass spectrometry

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