Microalgal and bacterial strains
I. galbana lyophilized (Archimede Ricerche Srl, Camporosso, Imola, Italy) was solubilized in phosphate buffered saline (PBS) (36 mg/ml) and used for bacterial growth.
The adherent-invasive AIEC strain LF82 (kindly provided by Prof. Arlette Darfeuille-Michaud, Clermont Universite, Universite d’Auvergne, Clermont-Ferrand, France) was cultured in Tryptone Soy Agar (TSA; plates Oxoid, Basingstoke, UK) for 24 hours at 37°C and then sub-cultured in Tryptone Soy Broth (TSB; Oxoid, Basingstoke, UK) with overnight incubation at 150 rpm, 37°C.
Powder of L. reuteri DSM17398 (BioGaia, Stockholm, Sweden) was kept at -20oC, inoculated in commercial medium De Man, Rogosa and Sharpe (MRS; Sigma-Aldrich, St. Louis, USA) and incubated overnight, 37oC without agitation.
Human colorectal adenocarcinoma cell line, CACO2, was obtained from the American Type Culture Collection (ATCC, Rockville, MA, USA). Cells were grown at confluence at 37°C in Dulbecco’s minimum essential medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% inactivated fetal bovine serum (FBS; Euroclone, Milan, Italy) and 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (Biochrom, Berlin, Germany).
Anaerobic growth of L. reuteri
For anaerobic growth, 2x106 CFU/ml of L. reuteri were inoculated in MRS or in 5 ml of I. galbana solubilized in PBS (36 mg/ml). Each vial was capped and incubated anaerobically without agitation at 37oC for 120 hours (5 days).
The bacterial growth was evaluated at different times (24, 48, 72, 96, 120 hours) by plating serially diluted samples in PBS on MRS agar plates (1,2% agarose) and incubated at 37°C for 24 hours. Resulting colonies were counted and the viability (CFU/mL) value was calculated based on the plated dilution. After 5 days, the fermentation compound (FC) contained an average concentration of 3.5x107 CFU/ml.
Lipids were extracted from I. galbana (36 mg/ml) solubilized in PBS and FC of a single experiment and the analysis was performed in duplicate.
Samples were freeze-dried for 2 days at -40oC and 60 mBar pressure by freeze-dryer (Edwards). Each sample (5 mg) was resuspended with 1 ml of dichloromethane (DCM) and 0.5 ml of methanol/sulfuric acid (MeOH/ H2SO4) and sonicated for 1 hour at 50oC, 40 KHz frequency. Hexane (1 ml) was used as extracting solvent and, after agitation, calcium carbonate (16 mg) and H2O (1 ml) were added and samples were centrifugated for 5 min at 2000 rpm. The separation of polar from apolar phase was repeated twice and finally the latter was dried with nitrogen flow (4 ml for each sample).
Gas-chromatography mass-spectrometry (GC-MS)
GC-MS analysis was performed by a 7890A gas chromatograph (Agilent) with capillary columns SBP-2331 (Sigma-Aldrich) [60 m, 0.25 mm inner diameter (ID), 0.2 µm film thickness]. Helium was used as carrier gas at a linear velocity of 36.26 cm/sec and 1µl of each sample was injected splitless. The initial column temperature was 40o and held 4 min, ramped to 140o at the rate of 20o/min, ramped to 220o at the rate of 2o/min and held 1min and then finally increased to 260o at the rate of 10o/min and kept at this temperature for 5 min. The mass spectra were recorded using a 5975C mass spectrometer (Agilent) in full scan mode from 45-450 m/z and 240o. The fatty acids concentration of each sample was determined using the software Xcalibur (Thermo Scientific, Waltham, USA) and 37 Component FAME Mix (Supelco, USA) was used as external standard for calibration.
AIEC adhesion and invasion assay
CACO2 cells were grown on 24-well plates at confluence (3x105 cells) and infected with LF82 (3x106 CFU), or LF82+ L. reuteri (3.5x106 CFU), or LF82 + I. galbana (100µl), or LF82 + FC (100µl) at 37 °C for 3 hours. To quantify the adherence of LF82, we followed the protocol of A. Darfeuille-Michaud et al. . Briefly, infected cells were washed twice in PBS and lysed for 10 minutes with 0.5 ml of 0.1% Triton X-100 in PBS buffer. Adherent bacteria were recovered and plated on LB agar plates. The latter were incubated at 37°C overnight and then the colonies were counted for statistical analysis.
CACO2 cells were infected and incubated as above. For invasion assay, we followed the protocol of A. Darfeuille-Michaud et al . Briefly, after incubation, cells were washed twice in sterile PBS and then incubated in DMEM and McCoy’s medium, respectively with 0.1 mg/ml gentamicin for 1 hour to kill the extracellular bacteria. Cells were washed twice in sterile PBS. Lysis, incubation and counts were performed as in the adhesion assay. To ensure maximum reproducibility, accuracy and statistical significance, adhesion and invasion assays were carried out simultaneously in triplicates. To obtain an accurate count of adhesive bacteria, the number of invasive colonies was subtracted from the number of the adhesive ones.
Data are given as mean ± standard deviation. All experiments were repeated three times. Comparison between groups was performed by a two-tailed Student t-test (significance taken as P<0.05).