Gentamicin

To produce Tg frogs, we used the I-SceI meganuclease- [18] (link) and transposon-mediated gene trap [19] (link). Fertilized eggs were injected with I-SceI meganuclease (NEB) and the I-SceI-cleaved plasmid encoding Z-AR and V5, and the Tol II mRNA [19] (link) and the prrT2ARG plasmid encoding Z-AR and GFP, respectively, using a NANOJECT II injection apparatus (Drummond). Tg embryos were cultured in 0.1×MMR with 6% Ficoll PM400 (GE Healthcare) and 50 µg/ml gentamicin (Wako), developed to St. 20 at 18°C, and then transferred to water at room temperature. The tadpoles were continuously reared in water with or without T (50 ng/ml; 150 nM). To confirm the exogenous Z-AR integration into genomic DNA, we extracted DNA from the tail tip of all Tg frogs just after metamorphosis, using the AllPrep DNA/RNA Micro Kit (QIAGEN). The PCR primers used were: forward, 5′-GCGGTTTTTCCAACTTACCA-3′ and reverse, 5′-CGAGACCGAGGAGAGGGTTA-3′).

We used the following antibiotics: from Sigma (St. Louis, MO, USA), gentamicin and metronidazole; from Wako (Osaka, Japan), ampicillin and clindamycin. Freshly cultured P. gulae in the exponential phase of growth were employed so that antibiotics reactions could be observed for 24 h. Bacterial growth was measured on an SH-1000 Lab microplate reader (Corona Electric, Ibaraki, Japan) at 595 nm.

RAW264.7 cells were seeded on 24-well culture plates (Asahi Glass, Tokyo, Japan) at 1 × 10⁶ cell/well in DMEM supplemented with 10% (v/v) FBS. Cells were treated with 100 μg of the antibody and infected simultaneously with L. monocytogenes at multiplicity of infection (MOI) 10. After incubation for 30 min, the extracellular bacteria were eliminated with 50 μg/ml gentamicin (Wako). The intracellular bacteria were enumerated by serial dilution and spread on tryptic soy agar (TSA) plates. For adhesion assay, RAW264.7 cells were seeded and incubated with the antibody and L. monocytogenes at MOI 10 as described above. NMuLi cells were seeded at 5 × 10⁵ cell/well and incubated with the antibody and L. monocytogenes at MOI 100. After incubation for 15 min, the non-adhesive bacterial cells were washed with PBS for 5 times. The adhesive bacterial cells were enumerated on TSA plates. For combination of antibodies, 50 μg of each antibody were mixed to obtain a final amount of 100 μg.

The esophageal cancer cell line KYSE30 (94072011; European Collection of Authenticated Cell Cultures, Salisbury, UK) was cultured in a 1:1 mixture of Ham’s F12 medium (Wako Pure Chemical Industries, Tokyo, Japan) and RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biosera, Nuaillé, France) and 1% ampicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The human colon cancer cell line HCT116 (CCL-247; American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS and 1% ampicillin and streptomycin (Thermo Fisher Scientific). Murine colon adenocarcinoma cell line MC38 (ENH204-FP; Kerafast, Boston, MA, USA) was cultured in Dulbecco’s modified Eagle medium with high glucose (Wako Pure Chemical Industries) supplemented with 10% FBS, 1 mM sodium pyruvate (Thermo Fisher Scientific), 100 μM non-essential amino acids (Thermo Fisher Scientific), 50 μg/mL gentamicin (Wako Pure Chemical Industries), 10 μM 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (Thermo Fisher Scientific), and 1% penicillin-streptomycin-amphotericin B (Wako Pure Chemical Industries). Cells were cultured in an atmosphere of 5% carbon dioxide (CO₂) at 37°C. All experiments using cells in this study were performed for fewer than 20 passages after thawing.

HeLa cell (RCB #0007) was provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan, and cultured for three days in Dulbecco’s-modified Eagle’s medium, high glucose (DMEM; Sigma-Aldrich, #D6429) supplemented with 10% fetal bovine serum (FBS; Ausgene) and 50-µg/ml gentamicin (Wako, #078-06061) [DMEM (+)] at 37°C in 5% CO₂. Additionally, the human intestinal epithelial cell line, Caco-2 cells, was cultured in DMEM (+) for four days.
For the infection experiments, the medium was changed to DMEM without FBS and gentamicin [DMEM (−)]. The cells were infected at a multiplicity of infection of 100−200:1 in C. jejuni infection or 10−20:1 in S. Enteritidis infection, and infected cells were incubated at 37°C in 5% CO₂.