Fecal specimen collection.
Fecal samples were obtained from 12 healthy Japanese volunteers, who had not been treated with antibiotics for more than six months prior to the experiment. All participants were recruited according to the following inclusion criteria: age 20 to 60 years, Japanese, non-smoking, and with good health and physical condition. All subjects provided written informed consent prior to specimen collection. Immediately after collection, each fecal sample was stored in an anaerobic culture swab (212550 BD BBL Culture Swab; Becton, Dickinson and Company, New Jersey, USA) and used within 24 h. The study was performed in accordance with the guidelines of Kobe University Hospital, and was approved by the Institutional Ethics Review Board of Kobe University. All methods used in this study were in accordance with the principles of the Declaration of Helsinki.
Operation of the model culture system with and without IgA.
We used a small-scale multi-channel fermenter (Bio Jr. 8; ABLE, Tokyo, Japan) comprising eight parallel and independent anaerobic culturing vessels, as described by Sasaki et al. . Each vessel contained autoclaved Gifu anaerobic medium (GAM [Code 05422]; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), with the initial pH adjusted to 6.5. The anaerobic conditions in the vessel were achieved by purging with a mixture of N2 and CO2 (80:20, 15 mL/min), which was filter-sterilized through a 0.2-μm polytetrafluoroethylene membrane filter (Pall Corporation, Port Washigton, Ny, USA) at 37°C for 1 h prior to cultivation. To prepare the fecal suspension, the fecal sample in the swab was suspended in phosphate buffer (0.1 M, 2.0 mL, pH 6.5, comprising of mixture of NaH2PO4 and Na2HPO4 at 61.65:28.35) supplemented with L-ascorbic acid (1.0% w/v; Wako Pure Chemical Industries, Osaka, Japan) in aerobic conditions.
W27 IgA was prepared as described previously (Okai et al. 2016). Mouse IgA-LE/AF was purchased from Southern Biotechnologies (0106-14). The fecal suspension was preincubated with IgA or without IgA at 37°C under aerobic condition for 3 h. Cultivation in the fermantation jar was initiated by inoculating one fecal suspension or mixture with IgA (approximately 250 μL) into each vessel. During fermentation at 37°C, the culture broth was stirred at 300 rpm with a magnetic stirrer and continuously purged with a filter-sterilized gas mixture. Aliquots of the culture broth were collected from the vessel 48 h after initiating the culture. The fecal suspensions and culture broth samples were then stored at −20°C until use.
Profiling of bacterial 16S rRNA
Microbial genomic DNA was extracted from the fecal suspension and culture broth obtained from KUHIMM, as described previously . Purified DNA was eluted into TE buffer (10 mM Tris–HCl, 1.0 mM EDTA) and stored at –20°C until use. Bacterial 16S rRNA genes (V3‒V4 region) were amplified using genomic DNA as the template with the primers S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′) . PCR and amplicon pool preparation were performed according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). PCR amplicons were purified using AMPure XP DNA purification beads (Beckman Coulter, Brea, CA, USA) and were eluted in 25 μL of 10 mM Tris (pH 8.5). Purified amplicons were quantified using the Agilent Bioanalyzer 2100 with DNA 1000 chips (Agilent Technology, Santa Clara, CA, USA) and the Qubit 2.0 instrument (Thermo Fisher Inc., Waltham, MA, USA), and were pooled at equimolar concentrations (5 nM). The 16S rRNA genes and an internal control (PhiX control v3; Illumina) were subjected to paired-end sequencing using the MiSeq instrument (Illumina) and the MiSeq Reagent Kit v3 (600 cycles; Illumina). The PhiX sequences were removed, and paired-end reads with Q scores ≥ 20 were joined using the Automated CASAVA 1.8 paired-end demultiplexed fastq, which was performed with the FASTQ Generation program on the Illumina Basespace Sequence Hub (https://bases pace.illum ina.com/). Sequence quality control and feature table construction of the sequence data were performed and corrected using QIIME 2 version 2020.8 (https://qiime 2.org) (Bolyen et al. 2019) and the DADA2 pipeline (Callahan et al. 2016). The taxonomic compositions of the OTUs were classified using the naive Bayes classifier trained on the Greengenes 13_8 99% OTU full-length sequence database (https://data.qiime2.org/2020.8/common/gg-13-8-99-nb-classifier.qza). The OTU data were then used for α-diversity estimation of Faith’s phylogenetic diversity (Faith 1992) and Shannon’s indices (Shannon 1948, Shannon 1948).
Real-time PCR analysis
Real-time PCR was performed to quantify total bacteria, using the LightCycler 96 system (Roche, Basal, Switzerland) with a universal primer set (5′-ACTCCTACGGGAGGCAGCAGT-3′ and 5′-GTATTACCGCGGCTGCTGGCAC-3′) targeting eubacteria (Nordeste et al. 2017). PCR amplification was performed as described previously (Takagi et al. 2016).
Isolation of E. coli from human feces
Fresh fecal samples derived from one human volunteer were prepared and cultured in Gifu anaerobic medium as described above. The human fecal fermentation culture was plated on the surface of autoclaved nutrient broth agar with 0.5% NaCl (composition per liter was 15 g agar, 5 g Bacto peptone, 3 g beef extract, and 5 g NaCl). The agar plate was incubated at 37°C under aerobic conditions for 1 d. A single colony was picked, subcultured in nutrient broth medium with 0.5% NaCl, and then stored as the stock culture at –80°C after adding glycerol (20% [vol/vol]). Genomic DNA was extracted from the 24-h culture in each culture medium as described previously.
Growth assayof isolated E. coli
Isolated E. coli were pre-cultured overnight in nutrient broth medium with 0.5% NaCl at 37°C under aerobic conditions. The culture was then diluted to 800 cells/mL in phosphate-buffered saline and preincubated with or without 200 μg/mL of W27 IgA at 37°C under aerobic conditions for 1 h. Then, E. coli with or without W27 IgA (final concentration: 5 μg/mL) was cultured in Gifu anaerobic medium at 37°C under anaerobic conditions (N2: 80%, H2: 10%, CO2: 10%) for 24 h. Finally, the OD600 was measured using a spectrophotometer (UVmini-1240; Shimadzu, Japan).
Data were compared between groups using the Wilcoxon signed-rank test in JMP version 12. Statistical significance was set at p < 0.05.
Data Availability Statement
All sequences from the original fecal samples and corresponding KUHIMMs were deposited in MG-RAST as “Model Culture System of Human Colonic Microbiota IgA” under accession number mgm4922092.3-mgm4922148.3.