Ampicillin

Colony forming units (CFU) of S. Typhimurium in the intestine of worms were performed as described previously [55 (link)]. For measurement of S. Typhimurium CFU, mild-L4 worms were exposed to NGM plates with S. Typhimurium expressing GFP (the vector for GFP expression is a gift from Dr. XP Qi, Kunming Institute of Zoology, CAS) for 48 hours at 25°C. Then worms were collected and soaked in M9 buffer containing 25 mM levamisole hydrochloride (Sangon Biotech Co.) and 100 μg/ml ampicillin (Sangon Biotech Co.) for 30 minutes at room temperature. Then worms were washed three times with M9 buffer. Some of worms were immediately mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss, Jena, Germany) with a digital camera. At least 15 worms were examined per assay in four independent experiments. Meanwhile, about 50 of nematodes per group were transferred into 50 μl PBS plus 0.1% Triton and ground. The lysates were diluted by 10-fold serial dilutions in sterilized water and spread over Chromogenic/Fluorogenic Culture Media agar (Sangon Biotech Co.) plates with 100 μg/ml ampicillin. After incubation overnight at 37°C, the colonies of GFP-S. Typhimurium were counted. For each group, 6–10 independent experiments were carried out.

Antibiotic treatment was carried out together with calorie restriction. According to a previously published protocol^{61 (link)}, 9-week old mice in AB and AB + CR groups received a combination of four nonabsorbable antibiotics: ampicillin, neomycin, metronidazole and vancomycin (Sangon Biotech, Shanghai, China) via oral gavage (0.2 mL) for 5 consecutive days (10 mg of each antibiotic per mouse per day) followed by administration in drinking water (ampicillin, neomycin and metronidazole: 1 g/L; vancomycin: 500 mg/L) which was renewed every week for the duration of the experiment. Mice in CTRL and CR groups received the same amount of sterile water by oral gavage in the first 5 days of treatment.

At the beginning of the 5th week of CD or HSD supplementation, the mice were provided with drinking water containing an antibiotic cocktail (ampicillin, 1 g/L; neomycin sulfate, 1 g/L; metronidazole, 1 g/L; and vancomycin, 500 mg/L; Sangon Biotech (Shanghai) Co., Ltd.) for 4 weeks ad libitum in the meantime (27 (link)). The antibiotics were renewed every other day. After 4 weeks of antibiotic treatment, the mice were treated with zymosan A, and the peritoneal fluid was collected for further experiments.

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-glyceol (DMPG), 1,2-Dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) were purchased from Larodon (Solna, Sweden). The expression vectors pET21a and pET28a were purchased from Stratagene (La Jolla, CA, USA). Escherichia coli Shuffle T7 Express Competent cells were purchased from New England BioLabs (Beijing, China). IPTG (isopropyl β-d-1-thiogalactopyranoside) and ampicillin were obtained from Sangon Biotech, Shanghai Co., Ltd. (Shanghai, China). Ni²⁺-NTA Sepharose fast-flow and anion exchange chromatography (Q-Sepharose XL) columns were purchased from GE Healthcare (Boston, MA, USA). Bicinchoninic acid (BCA) Protein Assay Kit was from Sangon Biotech, Shanghai Co., Ltd. (Shanghai, China). All other regents were of analytical grade.

Following the results of the resistance gene analysis, the minimum inhibitory concentrations (MICs) of GE2270A (Adipogen, San Diego, CA, USA) were determined against L. rhamnosus X253 and L. rhamnosus GG. In addition, the MICs of other antibiotics (Sangon Biotech, Shanghai, China)—including ampicillin, chloramphenicol, erythromycin, gentamicin, streptomycin, tetracycline, and vancomycin—were determined for these two strains in accordance with the ISO 10932:2010 standard (https://www.iso.org/standard/46434.html, accessed on 30 May 2022). In 96-well plates, suspensions of the two strains were combined with antibiotics at varying doses and incubated anaerobically at 37 °C for 48 h. The optical density at 625 nm was measured using a microplate reader (Thermo Labsystems, Franklin, MA, USA). The threshold values for resistance to each antibiotic were derived from the European Food Safety Authority (EFSA) [32 ].

E.coli infection was performed as previously described (Nguyen-Chi et al., 2014 (link)). In brief, single colony of E.coli which expressing GFP (Olson et al., 2014 (link)) were incubated in LB Broth Miller (MKCL4658; Sigma-Aldrich) containing the antibiotic ampicillin (A100339; Sangon Biotech) at 37°C on orbital shaker for at least 24 h before experimentation. After washing cells in 1×PBS and centrifuging at 500 g for 5 min, the E.coli were resuspended and diluted to the desired concentration in 1×PBS. E.coli were injected into the otic vesicle of zebrafish larvae and observed at the desired developmental stage.