Microalgal and bacterial strains
Dried powder of I. galbana, (freeze dried biomass for aquaculture, batch number ISO15SPRI2, Archimede Ricerche srl, Camporosso, Italy) was sterilized by UV under hood, weighed in sterile conditions, solubilized with phosphate buffered saline (PBS) and left on the rocker for 30 min.
The adherent-invasive AIEC strain LF82 (kindly provided by Prof. Arlette Darfeuille-Michaud, Université Clermont-Auvergne, Clermont-Ferrand, France) was cultured in Tryptone Soy Agar (TSA; plates Oxoid, Basingstoke, UK) for 24 h 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 − 20 °C, inoculated in commercial medium De Man, Rogosa and Sharpe (MRS; Sigma-Aldrich, St. Louis, USA) and incubated overnight, 37 °C without agitation.
Cell culture
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
In order to ensure no air/oxygen contact during fermentation, 2 × 106 CFU/ml of L. reuteri were inoculated in 10 ml of MRS or I. galbana solubilized in PBS (36 mg/ml). The solutions were aliquoted in 5 vials (2 mL each), fill up to the edge, then closed and sealed with parafilm. Vials were incubated anaerobically without agitation at 37 °C for 120 h (5 days) and opened only the day of the experiment.
The bacterial growth was evaluated at different times (24, 48, 72, 96, 120 h) by plating serially diluted samples in PBS on MRS agar plates (1.2% agarose) and incubated at 37 °C for 24 h. Resulting colonies were counted and the viability (CFU/ml) value was calculated based on the plated dilution.
Lipid extraction
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 − 40 °C 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 h at 50 °C, 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/s and 1 µl of each sample was injected splitless. The initial column temperature was 40° and held 4 min, ramped to 140° at the rate of 20°/min, ramped to 220° at the rate of 2°/min and held 1 min and then finally increased to 260° at the rate of 10°/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 to 450 m/z and 240°. 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.
Co-culture of AIEC LF82 and L. reuteri in I. galbana
To test the ability of AIEC LF82 and L. reuteri to grow in I. galbana, 1.3 × 106 CFU/ml of L. reuteri and 1.4 × 107 CFU/ml of LF82 were inoculated in 10 ml of I. galbana solubilized in PBS (36 mg/ml), aliquoted in 5 vials, capped and incubated anaerobically without agitation at 37 °C for 120 h (5 days).
The L. reuteri and LF82 growth was evaluated at different times (24, 48, 72, 96, 120 h) by plating serially diluted samples in PBS respectively on MRS agar plates (1.2% agarose) and TSA and incubated at 37 °C for 24 h. Resulting colonies were counted and the viability (CFU/mL) value was calculated based on the plated dilution.
AIEC adhesion and invasion assay
Adhesion assay
CACO2 cells were grown on 24-well plates at confluence (3 × 105 cells) and infected with LF82 (3 × 106 CFU), or LF82 + L. reuteri (3.5 × 106 CFU), or LF82 + I. galbana (100 µl), or LF82 + FC (100 µl) at 37 °C for 3 h. The final volume was 1 ml/well and 100 µl of I. galbana and FC were taken before and after fermentation without any further concentration step. To quantify the adherence of LF82, we followed the protocol of Darfeuille-Michaud et al.27. Briefly, infected cells were washed twice in PBS and lysed for 10 min with 0.5 ml of 0.1% Triton X-100 in PBS buffer. Adherent bacteria were recovered and plated on TSA plates. The latter were incubated at 37 °C overnight and then the colonies were counted for statistical analysis.
Invasion assay
CACO2 cells were infected and incubated as above. For invasion assay, we followed the protocol of A. Darfeuille-Michaud et al.32. 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 h 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.
Statistics
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).
