Open Access after eradication therapy(S3). On the day of sample the genera representing >0. 1% of the total microbiota, collection, the patients were instructed to take a fresh which was approximately >85% in this study. Hierarchical stool in a test tube. Those samples were immediately clustering based on the unweighted pair method sealed in a plastic bag containing a disposable oxygen-ab- with arithmetic mean was performed using JMP V.8.0 sorbing and carbon dioxide-generating agent, and then (SAS Institute, Cary, North Carolina, USA)to analyse the 3 transported to the laboratory within several hours on ice. overall bacterial community structure and the similarity 8 To count the number of anaerobic bacteria using the in the frequency of identification of each taxon among =t culturing method, fresh stool was spread over glucose the samples. The similarity between the bacterial commu- blood liver(BL)agar plates containing 5% horse serum, nities was determined using Euclidean distance and then incubated at 37.C for 72 hours in 10%H 10%CO, and 80%N, The other samples were stored Statistical analyses at-80C until the 16S rDNA assay For the extraction of The paired t-test or Wilcoxon signed-rank test was used to DNA, faecal solids in the suspension were broken down compare the bacterial abundance and clinical laboratory using a Fast Prep 24 Instrument(MP Biomedicals, Santa data between different time points. The difference in Ana, California, USA)with zirconia beads at 5m/s for the rate between groups was examined by Fisher's exact 0) 2 min Bacterial DNA extraction from the suspension was probability test. An analysis of similarity was used to test performed using a Magtration System 12GC(Precision for statistical differences in bacterial community similari- s System Science, Tokyo, Japan) ties using the vegan package (V2.3-4)implemented inr (V.3.2.4). All P values were two sided and considered to B PCR amplification of the 16S rRNA gene and next be significant at P<0. 05 generation sequencing The hypervariable v3-v4 region of the bacterial 16s rDNA was amplified by PCR with 341F and R8062 RESULTS R primers. PCR was performed in accordance with the Patients with eradication therapy 9 method reported by Takahashi et al.Sequencing was A total of 20 patients who had been diagnosed with H. o conducted using a paired end and modified to 2x300 bp pylori infection and underwent eradication therapy 5 cycle run on an Illumina Miseq sequencing system(Illu- participated in this study from April 2016 to March 2017. mina,San Diego, California, USA) with MiSeq Reagent The mean age (SD)of them was 63.8(18.2)years and 8 Kit version 3 chemistry. male/female ratio was 10/10. Three patients whose ID numbers were 8.16 and 18 were revealed to be in failure o A 16S rDNA-based operational taxonomic unit (OTU) for eradication(table 1) analysis The paired-end reads for each sample were joined using Change in the gut microbiota community structure after The number of quality filter passed reads were 984 296, The bacterial diversity within individual stool samples 1 187681 and 1 022 232 in the samples at Sl, S2, and S3, (alpha diversity)was evaluated using both the chao respectively. Those high-quality reads were then sorted 1 index and the observed OTU number. Rarefaction and grouped into OTUs using the Quantitative Insights curves showed no significant difference in the degree of E he reads were next clustered into OTUs at 97% pair- eradication therapy (data not shown) wise identity. To analyse the bacterial composition, Tech- 2-OSuruga Lab Microbial Identification Database DB-BA Alteration in the gut microbiota composition after 0(Techno Suruga Laboratory, Shizuoka-City, Japan) eradication therapy were used in accordance with the method reported by The number of anaerobes by the culturing method s Hisada et al In this composition analysis, we focused on was marginally lower at $2 than at SI but increased to g Table 2 Effect of eradication therapy on the number/abundance of bacteria dication therapy P value Before(S1) Immediately after(S2)3 months after (S3) A(S3-S1) 2 Anaerobes by culturing method 10.11(9.60-10.40)965(941-10.28) 10.10(9.61-10.35) 0.76 (xLog,, CFU/g) Bacteroidetes Firmicutesratio(x100) 0.93(0.. 31) 2.04 (1.40-10.62 2.40(1.40-7.84) Abundance(%) Genus Bifidobacterium 12.6(7.6-232) 0.1(0.006-5275) 4.8(0.3-17.1) <0.01 prausnitz 0.95012-2.18)0.590454)120.10-283) 0.02 Akkermansia muciniphila 0.002(00.288) 0(0-0 0(00.69) 0.66 anagi H, et al. BMJ Open Gastro 2017: 4: 0000182. doi: 10.1136/bmjgast-2017-000182Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 3 Open Access after eradication therapy (S3). On the day of sample collection, the patients were instructed to take a fresh stool in a test tube. Those samples were immediately sealed in a plastic bag containing a disposable oxygen-ab￾sorbing and carbon dioxide-generating agent, and then transported to the laboratory within several hours on ice. To count the number of anaerobic bacteria using the culturing method, fresh stool was spread over glucose blood liver (BL) agar plates containing 5% horse serum, and then incubated at 37ºC for 72hours in 10%H2 , 10% CO2 and 80%N2 . The other samples were stored at –80ºC until the 16S rDNA assay. For the extraction of DNA, faecal solids in the suspension were broken down using a FastPrep 24 Instrument (MP Biomedicals, Santa Ana, California, USA) with zirconia beads at 5m/s for 2min. Bacterial DNA extraction from the suspension was performed using a Magtration System 12GC (Precision System Science, Tokyo, Japan). PCR amplification of the 16S rRNA gene and next￾generation sequencing The hypervariable V3-V4 region of the bacterial 16S rDNA was amplified by PCR with 341F11 and R80612 primers. PCR was performed in accordance with the method reported by Takahashi et al. 13 Sequencing was conducted using a paired end and modified to 2×300bp cycle run on an Illumina Miseq sequencing system (Illu￾mina, San Diego, California, USA) with MiSeq Reagent Kit version 3 chemistry. A 16S rDNA-based operational taxonomic unit (OTU) analysis The paired-end reads for each sample were joined using the Fastiq-Join and then processed with quality filtering.14 The number of quality filter passed reads were 984 296, 1 187 681 and 1 022 232 in the samples at S1, S2, and S3, respectively. Those high-quality reads were then sorted and grouped into OTUs using the Quantitative Insights Into Microbial Ecology pipeline15 with default settings. The reads were next clustered into OTUs at 97% pair￾wise identity. To analyse the bacterial composition, Tech￾noSuruga Lab Microbial Identification Database DB-BA 10.0 (TechnoSuruga Laboratory, Shizuoka-City, Japan) were used in accordance with the method reported by Hisada et al. 16 In this composition analysis, we focused on the genera representing >0.1% of the total microbiota, which was approximately >85% in this study. Hierarchical clustering based on the unweighted pair group method with arithmetic mean was performed using JMP V.8.0 (SAS Institute, Cary, North Carolina, USA) to analyse the overall bacterial community structure and the similarity in the frequency of identification of each taxon among the samples. The similarity between the bacterial commu￾nities was determined using Euclidean distance. Statistical analyses The paired t-test or Wilcoxon signed-rank test was used to compare the bacterial abundance and clinical laboratory data between different time points. The difference in the rate between groups was examined by Fisher’s exact probability test. An analysis of similarity was used to test for statistical differences in bacterial community similari￾ties using the vegan package (V.2.3–4) implemented in R (V.3.2.4). All P values were two sided and considered to be significant at P<0.05. Results Patients with eradication therapy A total of 20 patients who had been diagnosed with H. pylori infection and underwent eradication therapy participated in this study from April 2016 to March 2017. The mean age (SD) of them was 63.8 (13.2) years and male/female ratio was 10/10. Three patients whose ID numbers were 8, 16 and 18 were revealed to be in failure for eradication (table 1). Change in the gut microbiota community structure after eradication therapy The bacterial diversity within individual stool samples (alpha diversity) was evaluated using both the chao 1 index and the observed OTU number. Rarefaction curves showed no significant difference in the degree of diversity between before (S1) and 3months after (S3) the eradication therapy (data not shown). Alteration in the gut microbiota composition after eradication therapy The number of anaerobes by the culturing method was marginally lower at S2 than at S1 but increased to Table 2 Effect of eradication therapy on the number/abundance of bacteria Before (S1) Eradication therapy P value Immediately after (S2) 3months after (S3) Δ (S3-S1) Anaerobes by culturing method (×Log10 CFU/g) 10.11 (9.60–10.40) 9.65 (9.41–10.28) 10.10 (9.61–10.35) 0.76 Bacteroidetes:Firmicutesratio (×100) 0.93 (0.30–3.31) 2.04 (1.40–10.62) 2.40 (1.40–7.84) <0.01 Abundance (%) Genus Bifidobacterium 12.6 (7.6–23.2) 0.1 (0.006–5.275) 4.8 (0.3–17.1) <0.01 Faecalibacterium prausnitzii 0.95 (0.12–2.18) 0.59 (0.006–4.54) 1.20 (0.10–2.83) 0.02 Akkermansia muciniphila 0.002 (0–0.288) 0 (0–0) 0 (0–0.69) 0.66 copyright. on 4 July 2018 by guest. Protected by http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2017-000182 on 26 November 2017. Downloaded from