Dg250

A. muciniphila strain (ATCC BAA-835) was cultured in modified Gifu Anaerobic Medium (GAM) broth (60 g/L). After incubation at 37°C under anaerobic conditions (10% H₂, 10% CO₂, 80% N₂; Don Whitley Scientific DG250, West Yorkshire, United Kingdom) for 48 h, A. muciniphila cells were harvested by centrifugation at 8,000 rpm for 10 min at 4°C, resuspended in sterile pre-reduced phosphate-buffered saline (PBS, containing 20% glycerol), and finally stored at −80°C until use. Bacterial suspensions were diluted with sterile pre-reduced PBS to 3.5 ×10⁸ CFU/mL (final glycerol concentration: 2%) and activated in a water bath at 37°C for 10 min before use. Pasteurization consisted of heat treatment at 70°C for 30 min, as previously described (Plovier et al., 2017 (link)).
A. muciniphila fermentation broth, cultured as mentioned above, was centrifuged at 8,000 rpm for 10 min and the supernatant was discarded. A solution containing 10% skimmed milk powder was then added to the precipitate, followed by mixing and vacuum freeze-drying for 24 h. The obtained lyophilized powder was stored at −20°C. The A. muciniphila concentration in the fermentation broth before freeze-drying was 1 × 10⁸ CFU/ml.

The bacterial strains and plasmids used in this study are listed in Table 1. All bacteria were grown in Luria-Bertani (LB) broth or on LB plates at 37 °C with shaking at 180 rpm. Complemented mutants harboring antibiotic-resistance genes were cultured in LB containing ampicillin (Amp, 100 μg/mL) or chloramphenicol (Cm, 34 μg/mL) when appropriate. Anaerobic growth was achieved via static culture at 37 °C in the anaerobic workstation (DG250, Don Whitley Scientific, Bingley, UK) with mixed gas (10% H₂, 10% CO₂, and 80% N₂). For RNA-Seq analysis, all strains were cultured under iron-limited, hypoxic, and nutrient-limited conditions. This stressful culture was achieved by culturing the S. Enteritidis wild-type (WT) strain and all the deletion mutants in a medium containing 0.05 mol/L KH₂PO₄ and 10 g/L trypsin with 0.2 mM 2, 2′-dipyridyl at 37 °C in an anaerobic workstation [12 (link)]. Chicken macrophage HD11 cells (accession number OTWO, HTX2259) were cultivated in Dulbecco’s Minimal Essential Medium (DMEM) (HyClone, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% chicken serum (VivaCell, Shanghai, China). Cells were maintained in an atmosphere of 5% CO₂ at 37 °C.

Clostridium difficile strains were cultured anaerobically on brain-heart infusion (BHI) agar or in BHI broth (Thermo Scientific, Waltham, MA, United States) supplemented with 0.05% L-cysteine. Anaerobic experiments were conducted inside a Don Whitley DG250 anaerobic workstation (Don Whitley Scientific Ltd, West Yorkshire, United Kingdom). E. coli strains were grown at 37°C in LB (Luria Broth, Thermo Scientific, Waltham, MA, United States).