Anaeropouch

Fresh feces were obtained from 14 healthy infants (aged between 5 and 12 months) with informed consent from their primary care givers. Approval #16/NTA/154, dated 7 October 2016, was obtained from the Health and Disability Ethics Committees, Ministry of Health, New Zealand. Relevant details of the infant donors who donated feces as a source of gut microbiota are provided as a supplementary table (Table S1).
The nappy liners containing fresh feces were transferred to gastight bags containing one Anaeropouch™ (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan) and transported in an insulated lunch bag containing an icepack to the laboratory. The feces were processed within 1 h of defecation into a 25% v/v fecal slurry by homogenizing with chilled sterile pre-reduced glycerol in phosphate-buffered saline with 0.05% w/v cysteine, and aliquots were stored at −80 °C. All processing of feces was carried out anaerobically under an atmosphere of CO₂:H₂:N₂ at 5:5:90 in a Coy anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI, USA). Two hours before the fermentation, one aliquot of each of 14 fecal slurries was removed from −80 °C, thawed in the anaerobic chamber, and pooled in equal proportions for immediate use as the inoculum.

A laboratory strain of cariogenic bacteria, S. mutans MT8148 strain (Biosafety level 1), was used for this S. mutans-infected model. After 16 hrs of preculture, fresh S. mutans were cultured with 5 ml of brain heart-infusion broth (BHI; Becton, Dickinson and Company, Sparks, MD, USA) at 37°C under anaerobic conditions using O₂-absorbing and CO₂-generating AnaeroPouch (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) for 16 hrs, then centrifuged at 3,500 rpm (1330g), 4°C for 10 minutes as described elsewhere (Gyo et al., 2008 (link)). The pellet was dissolved with sterile phosphate buffer saline (PBS). The optical density of the bacterial suspension was adjusted by adding up PBS until an optical density of 490 nm (OD₄₉₀) = 1.0 [approximately 3.3 × 10⁷ colony-forming unit (CFU)/ml]. The optical density was recorded using a spectrophotometer (Model 680 Microplate Reader: Bio-Rad, Hercules, CA, USA).

Minimum inhibitory concentrations (MICs) were determined as the lowest concentration of antibiotics that prevented visible growth, as described previously (Kusada et al., 2017 (link)). In brief, an overnight full-growth culture of L. paragasseri JCM 5343^T was inoculated on MRS agar plates containing a selected β-lactam antibiotic. All plates were incubated at 37°C for 3 days under anaerobic conditions (AnaeroPouch, Mitsubishi Gas Chemical Co, Inc., Tokyo, Japan). The tested β-lactam antibiotics were penicillin G, penicillin V, ampicillin, amoxicillin, carbenicillin, cephalosporin C, cephalexin, and cefadroxil at final concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 20.0, 30.0, and 40.0 μg/ml. All experiments were performed in triplicates.

Feces from four healthy adult Japanese females who did not have any gastrointestinal disease (21–54 years of age) (HF1, HF2, HF3, HF4) were individually collected on paper sheets and immediately transferred into sterilized containers (Sarstedt K.K., Tokyo, Japan) that were placed in an AnaeroPouch with a CO₂ generator (Mitsubishi Gas Chemical Company), Tokyo, Japan and these feces were stored in the freezer at −80°C. This study was performed under the guidelines of the Helsinki Declaration (1964). The Human Investigations Review Board of the Food Research Institute approved the study protocol and informed consent was obtained from the subjects.

Using the BIONIX-1 hypoxic culture kit (Sugiyama-Gen, Tokyo, Japan), HeLa-Fucci cells were made hypoxic (pO₂ < 0.1 %) as described previously [13 (link)] and irradiated as described above. Briefly, 24 h after cells (5 × 10⁴ cells) were seeded on a 35-mm glass-bottom dish, the medium was replaced by 300 μl of fresh growth medium to quickly equilibrate the gas and liquid phases. The dish was placed in an AnaeroPouch (Mitsubishi Gas Chemical, Tokyo, Japan) along with an AnaeroPack-Anaero 5 % oxygen absorber (Mitsubishi Gas Chemical) and an OXY-1 oxygen monitor (JIKCO, Tokyo, Japan), and the pouch was sealed. Irradiation was performed 2 h after verifying an oxygen monitor reading of 0 %, which confirms pO₂ < 0.1 % in the gas phase, to ensure equilibration between the gas and liquid phases. The oxygen enhancement ratio (OER) obtained from dose–cell survival curves (at a surviving fraction of 0.1) was ~2.1. Time-lapse imaging was performed under normoxic conditions.

Fresh fecal samples were transferred by the subjects into tubes and immediately enclosed in plastic bags containing AnaeroPouch (Mitsubishi Gas Chemical, Tokyo, Japan) to create an anaerobic environment.

Fresh faecal samples were obtained from five healthy infants (between the ages of 6 and 10 months), with written consent, approval #16/NTA/151 from Health and Disability Ethics Committees, Ministry of Health, New Zealand. The infants had never taken any antibiotics or medications.
Immediately after voiding, nappy liners containing the faecal samples were transferred to anaerobic conditions in gas-tight bags with one Anaeropouch (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan), and chilled with ice packs while transported to the laboratory. The samples were processed within 1 h into 20% v/v faecal slurries in chilled, sterile, anaerobic, glycerol-buffered saline with 0.05% w/v cysteine, aliquots dispensed and stored at − 80 °C. All processing of faecal samples and subsequent inoculation for the fermentation was carried out in a controlled atmosphere of 5% CO₂, 5% H₂ and 90% N₂ within the anaerobic chamber (Coy Laboratory Products Inc., Michigan, USA).