Study subjects

IBS-D patients were recruited from the Department of Gastroenterology and Hepatology, The First Affiliated Hospital of Sun Yat-sen University, from June 2019 to December 2020. All patients met Rome IV criteria and aged between 18 and 65. They were required to perform a colonoscopy to exclude organic diseases. The protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, and all patients had signed informed consent.

Study design and procedure

All patients took 2000 mg Golden bifid (INNER MONGOLIA Shuangqi Pharmaceutical Co., Ltd., Inner Mongolia, China. 1 × 107 CFU/g Bifidobacterium Longum, 1 × 106 CFU/g Lactobacillus bulgaricus and 1 × 106 CFU/g Streptococcus thermophilus) triple daily for 4 weeks. One fasting fecal sample was collected from all patients for 16S rRNA gene-targeted pyrosequencing at baseline (T0) and the end of treatment (T28) to evaluate the characteristics of gut microbiota. Meantime, all patients completed GI symptom severity scale, Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS) questionnaires and lactulose hydrogen breath test (LHBT).

Small intestinal bacterial overgrowth

The LHBT was completed for all IBS-D patients according to a standard procedure. And according to our previous research [13], when the baseline value of H2 ≥ 20 p.p.m and/or a rise of > 20 p.p.m. from baseline in H2 within 90 min of lactulose administration, the LHBT should be considered a presence of SIBO.

GI symptoms and psychological symptoms

Three questionnaires including GI symptom severity scale [13], SAS [14] and SDS [14] were performed in all IBS-D patients at T0 and T28. The questionnaire of GI symptom severity scale includes abdominal discomfort, abdominal distension, abdominal pain, diarrhea, defecatory urgency and incomplete evacuation, six GI symptoms with seven-point Likert responses, and the final score is negatively correlated with GI symptoms. The SAS and SDS contain 20 items with four-point Likert responses about anxiety and depression, respectively, and the final score is negatively correlated with anxiety or depression symptoms too.

16S rRNA gene-targeted pyrosequencing

Fecal sample collection and DNA extraction

Fecal samples were collected by germ-free disposable sampling spoon and frozen immediately at − 80 °C. Fecal microbial DNA was extracted from 180–220 mg feces using E.Z.N.A.® soil Kit (Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s protocols. The DNA concentration and purification were determined by NanoDrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis. The qualified fecal bacterial genomic DNA samples were stored in Tris–HCl buffer, pH 8.0, at − 20 °C.

PCR amplification

The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor-ST (Promega, USA) according to the manufacturer’s protocol.

Illumina MiSeq sequencing

Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Major Bio-Pharm Technology, Shanghai, China) according to the standard protocols by Majorbio Bio-Pharm Technology Co. , Ltd. (Shanghai, China).

Processing of sequencing data

Raw fastq files were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap is longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE, and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm against the Silva (SSU123) 16S rRNA database using confidence threshold of 70%.

Microbiological analysis

Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE version 7.1, and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm against the Silva (138/16s_bacteria) 16S rRNA database using confidence threshold of 70%. Then, the rank abundance curves, rarefaction curves, Alpha-diversity analysis, Beta-diversity analysis and community bar chart were used to analyze and compare the characteristics of fecal microbiota in patients with IBS-D before and after Golden bifid treatment. The rank abundance curves were plotted to determine the species abundance and community evenness of each sample, and the range of the curve on the horizontal axis and the gentle degree of the curve on the vertical axis reflect the species abundance and community evenness of the sample, respectively. Rarefaction curves were drawn to determine whether the sample size is sufficient and sequencing data of each sample. Alpha-diversity analyses including community richness parameters (Sobs, Ace, Chao1), community diversity parameters (Shannon, Simpson) and community coverage parameters (Good’s coverage) were calculated using the Mothur software. Principal coordinates analysis (PCoA), which is a Beta-diversity analyses, was used to compare the community composition of different samples. Community composition analysis (community bar chart) was used to analyze the species composition drawn with R package software (version 3.3.1). In addition, bacterial taxonomic distributions of sample communities were visualized using the R package software. Microbiome features differences between two groups (before and after Golden bifid treatment) were analyzed with Wilcoxon signed-rank test. PICRUSt2 analysis (KEGG level) was used to predict the functional profiling of microbial communities [12, 15].

Statistical analysis

SPSS 23.0 (SPSS, Inc., Chicago, IL, USA) and Graph Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) were used for statistical analysis and chart making. The measurement data were expressed in mean differences ± standard deviation ((overline{x} pm s)). Paired T test or Wilcoxon test was used for comparison. For all results of statistical analysis, P < 0.05 was considered statistically significant and if P < 0.01, the difference was statistically significant.

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