4.2. P.a Clinical Isolates

P.a isolates from sputum of CF patients followed at Grenoble-Alpes University Hospital were obtained from the Grenoble-Alpes University Hospital Microbiology Laboratory. Strains have been isolated and purified according to the national guidelines [57]. Isolates were stored at −80 °C in cryotubes with beads. P.a isolates were identified by standard biochemical testing and proteomic profiling by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Wissenbourg, France). Three to 5 isolates per patient (see cohort selection section) were arbitrarily selected and cultivated in SCFM2 medium for further analysis.

If not specified, all pre cultures and cultures were done at 37 °C, 230 rpm, in SCFM2 medium in aerobic conditions. Pre-cultures were done in 2.5mL SCFM2, cultures in 2 ∗ 2.5 mL SCFM2, pooled together prior to experimental procedure. SCFM2 medium was prepared as indicated by Turner et al. [32]. Mucin was discarded from the medium composition in order to allow a precise follow-up of bacterial growth by Optical Density measurements at 595 nm (OD₅₉₅).

PFGE analysis of SpeI restricted genomic DNA was performed as described previously [58]. PFGE was performed using a CHEF-DR III apparatus (Bio-Rad), set at 5.0 V/cm, with a linear ramping from 5 to 25 s for 11 h and 5 to 60 s for 13 h. PFGE gels were pictured using ImageLab 5.1 software (Bio-Rad). Images were then aligned and analyzed using BioNumerics software (version 7.1, Applied Maths, Sint-Martens-Latem, Belgium) DNA patterns (or pulsotypes) were converted into a 0/1 discrete matrix of presence/absence of bands at each molecular weight, as described previously by Lavenir et al. [59]. Hamming’s distances have been calculated using R software version 3.3.2 [60] (Vienna, Austria), and the number of different bands between isolates have been interpreted following the criteria defined in Römling et al. (1995) [31]. Two or more pulsotypes sharing less than 7 different bands have been defined as a PFGE Clonal Complex (CC), attesting for a recent common ancestor.