Patients and samples
The survey was conducted from 2014 to 2016 in Indonesia to evaluate the correlation between GERD and H. pylori infection. Endoscopy and biopsy samples were taken, including 1,074 samples included in our previous study . Biopsy samples were taken in the lesser curvature of the antrum, ~ 3 cm from the pyloric ring. Samples were then stored in a transport medium containing 10% glycerol. We collected demographic data by questionnaire, consisting of age, gender, smoking and alcohol-drinking habit as risk factors for dyspepsia and GERD, as mentioned in our previous study . All subjects aged more than 18 years old, had dyspeptic symptoms (postprandial fullness, early satiety, epigastric pain, and heartburn), and never received H. pylori eradication therapy or other antibiotic treatments within 2 weeks prior to examination. We excluded subjects with incomplete specimens and subjects with gastric cancer, duodenal ulcer, gastric ulcer, and severe intestinal metaplasia (gastritis score more than 2). Disease characterization was based on symptoms and upper-endoscopy evaluation. In the beginning, all patients with H. pylori positive were classified as gastritis group. The patients presented with reflux symptoms and mucosal lesion (esophageal erosions), which fulfilled the Los Angeles classification were diagnosed with ERD. If the patients presented with reflux symptoms without any mucosal lession (esophageal erosions), NERD was diagnosed. We included subjects without reflux symptoms and no intestinal metaplasia or atrophy score of more than 1 (mild) for histology evaluation in the gastritis group. Randomization was performed to obtain 204 samples for ERD, NERD and gastritis for sequencing. We included only the samples with complete data, especially the questionnaire data. Next, after filtering with a 500 reads cutoff, we finally included 197 subjects for DNA extraction and sequencing (see Additional file 1: Fig. S1).
For microbiome investigation, gastric mucosal specimens from the lesser curvature of the antrum were preserved in a transport medium. For H. pylori culture, specimens were placed into 500 µL phosphate-buffered saline, while for DNA extraction we used specimens stored at − 80 °C. Histologic examination was performed with two biopsy specimens from the corpus and antrum to identify the presence of inflammation, atrophy and intestinal metaplasia. All specimens were obtained using Radial Jaw 4 forceps.
All participants signed an informed consent form. The Ethics Committee of Dr. Soetomo Teaching Hospital (Surabaya, Indonesia) and Oita University, Faculty of Medicine (Yufu, Japan) approved the study concept and protocol.
Determination of H. pylori infection
H. pylori obtained from antrum were cultured and incubated at a selective agar plate in microaerophilic conditions for 10 days at 37 °C. Colonies were then sub-cultured in antibiotic-free agar medium; Mueller–Hinton II (Becton Dickinson, Heidelberg, Germany) supplemented with 10% Horse Blood (Nissui Pharmaceutical, Tokyo, Japan), incubated in microaerophilic conditions. H. pylori stock was stored at − 80 °C in Brucella Broth (Becton Dickinson, Heidelberg, Germany). All biopsy specimens were fixed in 10% buffered formalin and embedded in paraffin for histologic examination. Hematoxylin eosin, and May-Grunwald Giemsa stain (Beckman-Coulter, California, USA) were used to stain serial sections, and H. pylori was detected in samples with bacterial burdens grade 1 or above. Furthermore, we also determined H. pylori positive based on the 16S rRNA analysis that reflect colonization with the H. pylori abundance cut-off more than 2%, as previously described [15, 32,33,34].
DNA extraction and PCR amplification of 16S rRNA sequences
DNA extraction and PCR amplification methods were done as previously described . QIAGEN’s DNeasy Blood & Tissue Kit (QIAGEN, Santa Clarita, California) was used to extract DNA, and DNA Clean & Concentrator was used for concentration (Zymo Research, Irvine, California, USA). Extracted DNA was stored at − 20 °C. Preparation for the 16S rRNA gene library was under the manufacturer’s protocol (Illumina Inc, San Diego, CA). The amplification of V3-V4 of bacterial 16S rRNA gene used universal primers 341F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and 805R (5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3-3′).
PCR amplification was performed using KAPA HiFi HotStart Ready Mix (KAPA Biosystem Inc). Eight cycles of PCR were performed using Nextera XT Index kit (Illumina Inc, San Diego, CA). Agencourt AMPure XP magnetic beads (Beckman Coulter, Tokyo, Japan) were used for amplicons purification. Validation of DNA library was achieved with bioanalyzer MCE-202 MultiNA system (Shimadzu, Kyoto, Japan) and QuantiFluor dsDNA system (Promega, Madison, USA). Pooled 5 pM DNA library was denatured with 0.2 N NaOH and combined with PhiX Control v3 (Illumina Inc, San Diego, CA) to 15% of the final concentration, according to Illumina's protocol. MiSeq platform (Illumina Inc, San Diego, CA) and MiSeq Reagent Kit version 3 2 300 bp Paired-End Reads (Illumina Inc, San Diego, CA) were used to perform paired-end sequencing.
Sequence data analysis
Reads obtained from the Illumina Miseq platform were analyzed in the Qiime2 (Version 2021.2) pipeline . Demultiplexed reads were uploaded and trimmed using cutadapt and filtered to remove low-quality filtering. Denoising, Chimera removal, and clustering were performed by Deblur pipeline that was integrated into the Qiime2. Output was assigned into taxonomy to SILVA 138 reference database with 99% identity parameters . These sequences were aligned using MAFFT, and a phylogenetic tree was constructed using FastTree [37, 38]. We imported the OTU table, representative sequences, and phylogenetic tree as artefacts to Qiime2 analysis platform (https://qiime2.org). The OTU tables, metadata, taxonomy, and phylogenetic tree were exported to phyloseq object using biom-convert and ‘phyloseq’ package in R environment (ver 4.02) for diversity and abundance analysis. The α-diversity analysis, including Observed richness, ACE, Chao1, Fisher, Simpson’s and Shannon's diversity index, were analyzed using ‘vegan’ package in R and produced the figure by ‘ggplot2’ . The β-diversity analysis measured the weighted Unifrac. A Principal component analysis was drawn in R. The Adonis function from the vegan package was used to calculate the permutational analysis of variance (PERMANOVA) with Bonferroni correction. Using the ‘microbiomeMarker’ package in R, we performed linear discriminant analysis effect size (LEfSe) to determine OTUs that are likely to explain differences between GERD, NERD, and gastritis (version 4.02). We also used analysis of variance (ANOVA) to see if there were any differences in the relative abundance of specific OTUs within ethnic groups and diseases.