Aloa plates

Seven- to 11-week-old male mice (C57BL/6 mEcad E16P KI^{77 (link)}) were infected intravenously in the tail vein as previously described^{77 (link)}. Fraternities were kept together in a cage during the whole experiment, except when separated to exclude that carriage was due to coprophagy. To quantify carriage at 30-days post-inoculation, faeces were collected from each individual mouse and weighted before being homogenised in 2 ml of PBS. CFU count was performed by serial dilution of homogenised faeces on ALOA plates (Biomérieux) as described in ref. ⁸ . Separate faecal pellets were collected pre-inoculation and/or 30-days post-inoculation and stored at −20 °C for DNA extraction for 16S rRNA gene sequencing. DNA was extracted with DNeasy PowerSoil Pro Kit (Qiagen).
For experiments with antibiotic treatment, 5-week-old female mice (C57BL/6 mEcad E16P KI^{77 (link)}) received orally every 12 h during 4 days PBS (as control) or the following antibiotics (similarly to^{57 (link)}): ampicillin (100 mg/kg, Sigma-Aldrich A9618), vancomycin (50 mg/kg, Sigma-Aldrich 75423), metronidazole (100 mg/kg, Abcam ab141218), neomycin (100 mg/kg, Gibco 11811-031), amphotericin B (1 mg/kg, ApexBio Technology B1885). The mice were inoculated, as described above, 4 weeks after the antibiotic treatment.

As indicated in Section 2.1.2, B. safensis inoculum was prepared according to the previous monospecies biofilm formation study. One week later, L. monocytogenes was inoculated on the surfaces where biofilms of B. safensis were previously formed (see Section 2.2.3). For this, a new culture of L. monocytogenes was made from the stock strains, which had been kept in inclined tubes, by streaking them on TSA plates. These plates were incubated at 37 °C for 18 to 24 h. The initial microbial concentration was estimated with the densitometer DENSIMAT (bioMérieux, Marcy l’Etoile, France). The concentration of the pathogen was estimated by means of a previous standard curve established between McFarland units and CFU/mL of L. monocytogenes [37 ]. The inoculum was prepared by transferring multiple isolated colonies in TSB until reaching a turbidity of 0.1 McFarland units, equivalent to approximately 10⁶ CFU/mL. The bacterial suspension was diluted decimally in 9 mL peptone water tubes. The initial concentration of the inoculum from the different strains of L. monocytogenes was determined using ALOA plates (bioMérieux, Marcy l’Etoile, France).