Gut microbiota BMJ Changes in the gut microbiota gastroenterology composition and the plasma ghrelin level in patients with Helicobacter pylori-infected patients with eradication therapy Hidetaka Yanagi, Ayumi Tsuda, Masashi Matsushima, Shunsuke Takahashi Genki Ozawa, Yasuhiro Koga, Atsushi Takagi To cite: Yanagi H, Tsuda A, ABSTRACT Matsushima M. et al bjective To investigate the influence of antimicrobials on Summary box 3o9N9=N8 omposition and the plasma level using Helicobacter pylori-infected patients who What is already known about this subject? underwent eradication thera Gut microbiota plays a fundamental role in the elicobacter pylori-infected Design Twenty H pylori-infected patients(mean age 68.3 pathophysiology of obesity atients with eradication therapy. BM Open Gastro years old) who underwent eradication therapy participated Ghrelin is associated with obesity after the 2017: 4: e000182 do: 10.1136/ in the study. For the therapy, patients had 1 week of eradication of Helicobacter pylori megas-2017-000182 triple therapy consisting of amoxicillin, clarithromycin What are the new findings? and proton-pump inhibitors Stool and blood samples p Alteration in the gut microbiota may affect the 8gNz3 Received 9 october 2017 were obtained before(S1), immediately after(S2) and/ ccepted 8 November 2017 or 3 months after(S3)the therapies. The concentrations of ghrelin and leptin in the blood were assayed using How might it impact on clinical practice in the学 an ELISA. The V3-V4 region of the 16s rRNa gene was foreseeable future? amplified using bacterial DNA from the stool, and about Both the gut microbiota profiling and plasma 50000 high-quality amplicons per sample were grouped ghrelin level might be used as an earliest nto operational taxonomic units for bacteriological biomarker to foresee the insulin resistance or significantly greater at S3 than S1(P<0.01). This increase in the b F ratio between S3 and S1 was found in 15 out tested, route of administration, duration of of 20 patients. a significant decrease in the concentration follor follow-up and so on. However, in the eradi- of active ghrelin(P=0.003)in the plasma was observed cation therapy of Helicobacter pylori, the first- g etween $3 and s1. There was a statistically significant line therapy has been widely used, which correlation between the rate of patients whose B: F ratio consists of oral administration of clarithro- Check for updates increased and that of patients whose active ghrelin level epartment of Genera decreased between S3 and S1 according to Fisher's exact .ycin, amoxicillin and a proton-pump inhib- Q robability test(P=0.03) itor twice a day for 7 days. Therefore, it will 4 Medicine, Tokai University School of medicine. Isehara Conclusions Changes in the gut microbiota, such as the B: F ratio after treatment with antimicrobials, might cause to use H. pylorHinfected patients with erad- a change in the plasma ghrelin level, as the direct and ication therapy as subjects in the study of 8 Gastroenterology, Tokai earliest target of antimicrobials would be the microbiota the effect of antimicrobials on indigenous University School of Medicine, rather than the hormone-secreting system. bacteria in human hara, Japa It was reported that Technical Department, body mass index significantly increased l year Techno Suruga Laboratory Co Ltd, Shizuoka, Japan INTRODUCTION ter the eradication of H. pylori when exam- Laboratory for Infectious Although antimicrobials have definite effects d in 579 Japanese patients. Ghrelin Diseases, Tokai University on particular pathogenic bacteria, the effect a multifunctional hormone that facilitates School of medicine. isehata Japan community and metabolism has recently been ghrelin is predominantly synthesised and explored. Many of those studies performed secreted by the gastric mucosa, the obesity y Dr Yasuhiro Koga so far are characterised by significant hetero- associated with eradication of H. pylori yasuhiro@isicc. u-tokai ac ip geneity with respect to type of antimicrobials has been attributed to an increase in the Yanagi H, et al. BMJ Open Gastro 2017: 4: e000182 doi 10. 1136/bmjgast-2017-000187
Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 1 Changes in the gut microbiota composition and the plasma ghrelin level in patients with Helicobacter pylori-infected patients with eradication therapy Hidetaka Yanagi,1 Ayumi Tsuda,1 Masashi Matsushima,2 Shunsuke Takahashi,3 Genki Ozawa,3 Yasuhiro Koga,4 Atsushi Takagi1 To cite: Yanagi H, Tsuda A, Matsushima M, et al. Changes in the gut microbiota composition and the plasma ghrelin level in patients with Helicobacter pylori-infected patients with eradication therapy. BMJ Open Gastro 2017;4:e000182. doi:10.1136/ bmjgast-2017-000182 Received 9 October 2017 Accepted 8 November 2017 1 Department of General Medicine, Tokai University School of Medicine, Isehara, Japan 2 Department of Gastroenterology, Tokai University School of Medicine, Isehara, Japan 3 Technical Department, TechnoSuruga Laboratory Co. Ltd, Shizuoka, Japan 4 Laboratory for Infectious Diseases, Tokai University School of Medicine, Isehata, Japan Correspondence to Dr Yasuhiro Koga; yasuhiro@is.icc.u-tokai.ac.jp Gut microbiota Abstract Objective To investigate the influence of antimicrobials on both the gut microbiota structure and the plasma ghrelin level using Helicobacter pylori-infected patients who underwent eradication therapy. Design Twenty H. pylori-infected patients (mean age 68.3 years old) who underwent eradication therapy participated in the study. For the therapy, patients had 1week of triple therapy consisting of amoxicillin, clarithromycin and proton-pump inhibitors. Stool and blood samples were obtained before (S1), immediately after (S2) and/ or 3 months after (S3) the therapies. The concentrations of ghrelin and leptin in the blood were assayed using an ELISA. The V3-V4 region of the 16S rRNA gene was amplified using bacterial DNA from the stool, and about 50 000 high-quality amplicons per sample were grouped into operational taxonomic units for bacteriological analyses. Results The Bacteroidetes:Firmicutes (B:F) ratio was significantly greater at S3 than S1 (P<0.01). This increase in the B:F ratio between S3 and S1 was found in 15 out of 20 patients. A significant decrease in the concentration of active ghrelin (P=0.003) in the plasma was observed between S3 and S1. There was a statistically significant correlation between the rate of patients whose B:F ratio increased and that of patients whose active ghrelin level decreased between S3 and S1 according to Fisher’s exact probability test (P=0.03). Conclusions Changes in the gut microbiota, such as the B:F ratio after treatment with antimicrobials, might cause a change in the plasma ghrelin level, as the direct and earliest target of antimicrobials would be the microbiota rather than the hormone-secreting system. Introduction Although antimicrobials have definite effects on particular pathogenic bacteria, the effect of antimicrobials on the gut microbial community and metabolism has recently been explored. Many of those studies performed so far are characterised by significant heterogeneity with respect to type of antimicrobials tested, route of administration, duration of follow-up and so on. However, in the eradication therapy of Helicobacter pylori, the firstline therapy has been widely used, which consists of oral administration of clarithromycin, amoxicillin and a proton-pump inhibitor twice a day for 7 days.1 Therefore, it will be free from the problem of heterogeneity to use H. pylori-infected patients with eradication therapy as subjects in the study of the effect of antimicrobials on indigenous bacteria in human. It was reported that the body weight and body mass index significantly increased 1year after the eradication of H. pylori when examined in 579 Japanese patients.2 Ghrelin is a multifunctional hormone that facilitates fat storage and regulates body weight.3 As ghrelin is predominantly synthesised and secreted by the gastric mucosa, the obesity associated with eradication of H. pylori has been attributed to an increase in the Summary box What is already known about this subject? ► Gut microbiota plays a fundamental role in the pathophysiology of obesity. ► Ghrelin is associated with obesity after the eradication of Helicobacter pylori. What are the new findings? ► Alteration in the gut microbiota may affect the plasma ghrelin level. How might it impact on clinical practice in the foreseeable future? ► Both the gut microbiota profiling and plasma ghrelin level might be used as an earliest biomarker to foresee the insulin resistance or obesity. 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
Open Access 8 ghrelin level. However, some groups" have found that the test, H. pylori stool antigen test or culturing of biopsy plasma ghrelin concentration decreased in most patients specimens. The exclusion criteria were the patients with 12 weeks after successful H. pylori eradication therapy. gastric cancers and a history of gastrointestinal or hepato- A recent systemic review also reported that the relation- biliary surgery. The demographic data and clinical char- eradication therapy, patients had I week of triple therapy a ship between circulating ghrelin and eradication of H. acteristics of the subjects are summarised in table 1. For pylori is more complex. Recent studies have also suggested that the gut micro- consisting of lansoprazole(0mg twice a day), amox- biota play a fundamental role in the pathophysiology of icillin(750 mg twice daily)and clarithromycin(200mg obesity. Ley et al reported that the ratio of the phylum twice daily). The eradication of H pylori was confirmed by a Bacteroidetes to the phylum Firmicutes(B: F ratio)in the a urea breath test 3 months after the termination of erad- faeces increased when weight was reduced either by a ication therapy. Written informed consent was obtained fat-restricted or carbohydrate-restricted diet in humans, from all of the patients suggesting a critical role of the gut microbiota in the Hormone assay Therefore, in the present study, we investigated the The concentrations of des-acyl ghrelin and active ghrelin 8 influence of antimicrobials on both the gut microbiota in the plasma were assayed using a Desacyl-Ghrelin ELISA structure and the plasma ghrelin level, as we hypothe- Kit (SCETI,Tokyo, Japan) and Active Ghrelin ELISA sised that the change in the ghrelin level after H. pylori Kit(SCETI), respectively. The specific antibodies used eradication therapy was caused by elimination of H. pylori in the former and latter assay kits recognise the C-ter- in the stomach but also by an alteration in the gut micro- minal portion and the octanoyl-modified Ser 3 residue of biota due to antimicrobials ghrelin, respectively. The concentration of leptin in the serum was assayed using Human Leptin RIA Kit(Milli- S pore, Belmopan, Belize) R METHODS Patients Stool sample collection and DNA extraction Patients were defined as positive for H. pyloni infect Stool sample collection from patients was done three if positive results were obtained on aC-urea bi times before(S1), immediately after(S2)and 3months Table 1 Demographic and clinical data of the patients After eradication therapy ID of the patient Ag Gender Endoscopy ofthe stomach Eradication B: F ratio Ghrelin level AG Success Down 2 F Success Down 9Nz<3gN=oosoa3 Success Down AG. RE Down Down AG Failure Success Down 10 AG Success Down AG, HG Success M AG. GU scar Success F Success 15 ccess Down Down AG. DU scar Failure Down AG 18 51 AG Failure Down 19 72 M GU active Success Dowt Success AG, atrophic gastritis; B F, Bacteroidetes: Firmicutes; DU, duodenal ulcer, F, female; GU, gastric ulcer, HG, haemorrhagic gastritis; M, male Yanagi H, et al. BM Open Gastro 2017: 4: 0000182. doi: 10. 1136/bmigast-2017-000182
2 Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 Open Access ghrelin level.4 However, some groups5 6 have found that the plasma ghrelin concentration decreased in most patients 12 weeks after successful H. pylori eradication therapy. A recent systemic review also reported that the relationship between circulating ghrelin and eradication of H. pylori is more complex.7 Recent studies have also suggested that the gut microbiota play a fundamental role in the pathophysiology of obesity.8 Ley et al9 reported that the ratio of the phylum Bacteroidetes to the phylum Firmicutes (B:F ratio) in the faeces increased when weight was reduced either by a fat-restricted or carbohydrate-restricted diet in humans, suggesting a critical role of the gut microbiota in the obesity. Therefore, in the present study, we investigated the influence of antimicrobials on both the gut microbiota structure and the plasma ghrelin level, as we hypothesised that the change in the ghrelin level after H. pylori eradication therapy was caused by elimination of H. pylori in the stomach but also by an alteration in the gut microbiota due to antimicrobials. Methods Patients Patients were defined as positive for H. pylori infection if positive results were obtained on a 13C-urea breath test, H. pylori stool antigen test or culturing of biopsy specimens. The exclusion criteria were the patients with gastric cancers and a history of gastrointestinal or hepatobiliary surgery. The demographic data and clinical characteristics of the subjects are summarised in table 1. For eradication therapy, patients had 1week of triple therapy consisting of lansoprazole (30mg twice a day), amoxicillin (750mg twice daily) and clarithromycin (200mg twice daily). The eradication of H. pylori was confirmed by a urea breath test 3months after the termination of eradication therapy. Written informed consent was obtained from all of the patients. Hormone assay The concentrations of des-acyl ghrelin and active ghrelin in the plasma were assayed using a Desacyl-Ghrelin ELISA Kit (SCETI, Tokyo, Japan) and Active Ghrelin ELISA Kit (SCETI), respectively. The specific antibodies used in the former and latter assay kits recognise the C-terminal portion and the octanoyl-modified Ser 3 residue of ghrelin, respectively.10 The concentration of leptin in the serum was assayed using Human Leptin RIA Kit (Millipore, Belmopan, Belize). Stool sample collection and DNA extraction Stool sample collection from patients was done three times before (S1), immediately after (S2) and 3months Table 1 Demographic and clinical data of the patients ID of the patient Age Gender Endoscopy ofthe stomach After eradication therapy Eradication B:F ratio Ghrelin level 1 47 F AG Success Down Up 2 66 F AG Success Down Up 3 42 M AG Success Up Down 4 62 F AG Success Up Down 5 73 F AG Success Down Up 6 42 M AG, RE Success Down Down 7 80 M AG Success Up Down 8 42 M AG Failure Up Down 9 76 M AG Success Up Down 10 63 F AG Success Up Down 11 58 F AG, HG Success Up Up 12 78 M AG, GU scar Success Up Down 13 71 F AG Success Up Down 14 65 F AG Success Up Down 15 57 M AG Success Down Down 16 78 F AG, DU scar Failure Up Down 17 74 M AG Success Up Down 18 51 F AG Failure Up Down 19 72 M GU active Success Up Down 20 79 M AG Success Up Down AG, atrophic gastritis; B:F, Bacteroidetes:Firmicutes; DU, duodenal ulcer; F, female; GU, gastric ulcer; HG, haemorrhagic gastritis; M, male; RE, reflux esophagitis. 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
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-2017-000182
Yanagi 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-absorbing 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 nextgeneration 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 (Illumina, 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% pairwise identity. To analyse the bacterial composition, TechnoSuruga 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 communities 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 similarities 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
Open Access Eradication therapy tore (Immediately after) (3 months after) Firmicutes 76.3 718% Actinobacteria 14.6 Archaea Figure 1 Proportion of the dominant five phyla in the faecal samples. The per cent proportions of the five dominant phyla of a N=ggNz3gN→o Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes and Archaea in all faecal samples at S1, S2 and S3 are depicted in the bar charts. The S1, S2 and S3 samples include 984 296, 1 187 681 and 1 022 232 high-quality reads, respectively the pretherapy level at S3(table 2). The OTUs gener- Bifidobacterium and Ruminococcaceae including Faecali- 5 ted from the 16s reads were then classified into phyla bacterium in the analysis of similarity in the frequency of according to a similarity search(figure 1). At S2, there identification among samples in the clusterin was a marked increase in the abundance of both Bacte- rial families roidetes((0.9% to 4.5%)%)and Archaea but a decrease in that of both Actinobacteria(20.5% to 15.6%)%) Change in the biomarkers regarding obesity/type 2 diabetes and Proteobacteria. The increase in Bacteroidetes and mellitus(T2DM)and their association with gut microbiota decrease in Actinobacteria persisted even at $3(0.9% to during the therapy 4.0%and 20.5% to 14.6%, respectively). The abundance Among the clinical and laboratory data conce of Firmicutes, the predominant phylum, showed little obesity/T2DM examined in the present study(table 3) change between SI and $3(69.9% to 71.8%)%). Thus, statistically significant decreases and increases were the median B: F ratio was significantly greater at S than observed in the values of active ghrelin(P=0.00%)and respectively, between S3 and Sl. We a quite obvious, with 15 of the 20 patients(75%)showing a therefore examined whether the change in the levels of 3 higher ratio at S3 than at SI (table I and figure 2) 6 We then examined the abundance of particular those adipogenesis-regulating hormones was associated acteria considered to be associated with dysbiosis in with the alteration of the ratio of B: F in each patient A obesity(table 2). The abundance of the genus Bifidobac- (figure 2). The rate of decrease in active ghrelin levels s terium,which is included in phylum Actinobacteria, was among patients with an increased B: F ratio was 14 out 8 the abundance of Faecalibacterium prausnitz was greater levels among patients with a decreased B: F ratio in o significantly lower at S3 than at SI(P<0.01). In contrast, of 15, whereas the rate of decrease in active ghrelin o at S3 than at SI(P-0.02). A double hierarchical clus- only 2 out of 5(table 1). The difference in the rates 2 tering analysis demonstrated an opposite but closely between these two groups was statistically significant relate ed change in the abundance after therapy betwe (P=0.03) by Fisher's test No significant association was T Bifidobacterium and Faecalibacterium(figure 3), with a found between the change in the leptin level and the distinct cluster formed by Bifidobacteriaceae including B: F ratio. Yanagi H, et al. BM Open Gastro 2017: 4: 0000182. doi: 10. 1136/bmigast-2017-000182
4 Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 Open Access the pretherapy level at S3 (table 2). The OTUs generated from the 16S reads were then classified into phyla according to a similarity search (figure 1). At S2, there was a marked increase in the abundance of both Bacteroidetes ((0.9% to 4.5%)%) and Archaea but a decrease in that of both Actinobacteria ((20.5% to 15.6%)%) and Proteobacteria. The increase in Bacteroidetes and decrease in Actinobacteria persisted even at S3 (0.9% to 4.0%and 20.5% to 14.6%, respectively). The abundance of Firmicutes, the predominant phylum, showed little change between S1 and S3 ((69.9% to 71.8%)%). Thus, the median B:F ratio was significantly greater at S3 than at S1 (P<0.01, table 2). This increase in the B:F ratio was quite obvious, with 15 of the 20 patients (75%) showing a higher ratio at S3 than at S1 (table 1 and figure 2). We then examined the abundance of particular bacteria considered to be associated with dysbiosis in obesity (table 2). The abundance of the genus Bifidobacterium, which is included in phylum Actinobacteria, was significantly lower at S3 than at S1 (P<0.01). In contrast, the abundance of Faecalibacterium prausnitzii was greater at S3 than at S1 (P=0.02). A double hierarchical clustering analysis demonstrated an opposite but closely related change in the abundance after therapy between Bifidobacterium and Faecalibacterium (figure 3), with a distinct cluster formed by Bifidobacteriaceae including Bifidobacterium and Ruminococcaceae including Faecalibacterium in the analysis of similarity in the frequency of identification among samples in the clustering of bacterial families. Change in the biomarkers regarding obesity/type 2 diabetes mellitus (T2DM) and their association with gut microbiota during the therapy Among the clinical and laboratory data concerning obesity/T2DM examined in the present study (table 3), statistically significant decreases and increases were observed in the values of active ghrelin (P=0.003) and leptin (P=0.03), respectively, between S3 and S1. We therefore examined whether the change in the levels of those adipogenesis-regulating hormones was associated with the alteration of the ratio of B:F in each patient (figure 2). The rate of decrease in active ghrelin levels among patients with an increased B:F ratio was 14 out of 15, whereas the rate of decrease in active ghrelin levels among patients with a decreased B:F ratio was only 2 out of 5 (table 1). The difference in the rates between these two groups was statistically significant (P=0.03) by Fisher’s test. No significant association was found between the change in the leptin level and the B:F ratio. Figure 1 Proportion of the dominant five phyla in the faecal samples. The per cent proportions of the five dominant phyla of Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes and Archaea in all faecal samples at S1, S2 and S3 are depicted in the bar charts. The S1, S2 and S3 samples include 984 296, 1 187 681 and 1 022 232 high-quality reads, respectively. 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
Open Access Active Ghrelin Leptin Bacteroidetes/ Firmicutes oo 四生gE 88°o…oo°9 5 20032 39N9=89Nzo3N=o Figure 2 Difference in the bacterial ratios and hormone levels between S1 and S3. Each symbol represents the logarithmic differential ratio of Bacteroidetes: Firmicutes(B: F)(middle), the concentration of plasma ghrelin(left), and the concentration of serum leptin(right)in a patient at S1 and S3. Symbols representing the same patients are connected with lines. P values at the bottom indicate the statistical significance according to Fisher's exact probability test. DISCUSSION (500 mg twice daily), whose dose was pretty greater Q In recent studies of gut microbiota using high-throughput than that used in the present study. Those authors also 16S rRNA gene sequencing, loss of diversity and marked observed not an increase but a decrease in the B: F ratio short-term changes in several bacterial taxa have been osteradication consistent finding after treatment with antimicrobials In the present study using H pylori-infected patients who treatment, the ecosystem of gut microbiota exhibits no S After external perturbation, such as antimicrobial were treated with first-line therapy using both amoxi- change, resilience, adaptation or failing resilience. The cillin and clarithromycin for l week, the alpha diversity robustness of the microbiota ecosystem is considered to o in the stool samples showed no significant changes at be a major factor determining which of those responses 3 months after the therapy. Given that only a slight tran- the microbiota exhibit. The difference in the age and race sient decrease in the number of bacteria was observed by between our subjects (apanese, 42-79 years of age)and a culturing method just after the therapy(see table 1), the subjects of Yap et al(Malaysian, 18-30 years of age) g the antimicrobials used in the present study might have might induced in a difference in the robustness of the 5 exerted only a moderate perturbing effect on the micro- gut microbiota ecosystem, thereby resulting in different 3 biota, thereby resulting in no marked decrease in the outcomes in the B: F ratio after antimicrobial treatment. diversity of the gut microbiota after the In addition, differences in the dose of antimicrobials s Of note, however, in the bacterial composition analysis used for eradication and the duration of follow-up might at the phylum level, the B: F ratio significantly increased also have affected the outcomes of these two studies. just after the therapy and remained increased even at Furthermore, as the subjects without H pylori infection t 8months after the therapy. Yap et al also reported that were not included in our study, it is possible that some 5 the alpha diversity was similar between pre-eradication bias due to long-lasting H. pylori infection on the B: F ratio and 6months posteradication in stool samples obtained at SI might be involved from H. pylor-infected young adults treated with When analysing the existence of any similarities in the amoxicillin(1000 mg twice daily) and clarithromycin frequency of identification among samples(see figure 3) Yanagi H, et al. BMJ Open Gastro 2017: 4: e000182 doi 10. 1136/bmjgast-2017-000187
Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 5 Open Access Discussion In recent studies of gut microbiota using high-throughput 16S rRNA gene sequencing, loss of diversity and marked short-term changes in several bacterial taxa have been a consistent finding after treatment with antimicrobials.17 In the present study using H. pylori-infected patients who were treated with first-line therapy using both amoxicillin and clarithromycin for 1week, the alpha diversity in the stool samples showed no significant changes at 3months after the therapy. Given that only a slight transient decrease in the number of bacteria was observed by a culturing method just after the therapy (see table 1), the antimicrobials used in the present study might have exerted only a moderate perturbing effect on the microbiota, thereby resulting in no marked decrease in the diversity of the gut microbiota after the therapy. Of note, however, in the bacterial composition analysis at the phylum level, the B:F ratio significantly increased just after the therapy and remained increased even at 3months after the therapy. Yap et al18 also reported that the alpha diversity was similar between pre-eradication and 6months posteradication in stool samples obtained from H. pylori-infected young adults treated with amoxicillin (1000mg twice daily) and clarithromycin (500mg twice daily), whose dose was pretty greater than that used in the present study. Those authors also observed not an increase but a decrease in the B:F ratio posteradication. After external perturbation, such as antimicrobial treatment, the ecosystem of gut microbiota exhibits no change, resilience, adaptation or failing resilience.19 The robustness of the microbiota ecosystem is considered to be a major factor determining which of those responses the microbiota exhibit. The difference in the age and race between our subjects (Japanese, 42–79 years of age) and the subjects of Yap et al (Malaysian, 18–30 years of age) might induced in a difference in the robustness of the gut microbiota ecosystem, thereby resulting in different outcomes in the B:F ratio after antimicrobial treatment. In addition, differences in the dose of antimicrobials used for eradication and the duration of follow-up might also have affected the outcomes of these two studies. Furthermore, as the subjects without H. pylori infection were not included in our study, it is possible that some bias due to long-lasting H. pylori infection on the B:F ratio at S1 might be involved. When analysing the existence of any similarities in the frequency of identification among samples (see figure 3), Figure 2 Difference in the bacterial ratios and hormone levels between S1 and S3. Each symbol represents the logarithmic differential ratio of Bacteroidetes:Firmicutes (B:F) (middle), the concentration of plasma ghrelin (left), and the concentration of serum leptin (right) in a patient at S1 and S3. Symbols representing the same patients are connected with lines. P values at the bottom indicate the statistical significance according to Fisher’s exact probability test. 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
Open Access N→ogNzs3gN→ooo3 Figure 3 Double-hierarchical clustering analysis. The faecal samples from 20 patients at three time points for 36 dominant the samples at the family level. Sample ID numbers 1-20, 21-40 and 41-60 on the left side represent those obtained at Sz o families are represented on a double-hierarchical clustering heat map. The clusters on the right side indicate similarity amor S2 and S3, respectively. IDs 1, 21 and 41, and likewise 2, 22 and 42 and so on correspond to the same patient. The clusters at the bottom indicate the families showing similarity in the frequency of identification among samples. the Z-score column represents the relative ratio of abt an opposite pattern of colonisation was found between In the present study, the gut microbiota after the eradi Bifidobacteriaceae and Ruminococcaceae whose major genera cation therapy showed a phenotype not of obesity but of 9 That is, a sample with a high density (columns with ratio and an abundance of E prausnitzii. Furthermore, the 9 red colour) of Bifidobacteriaceae contained a low density plasma level of the active form of ghrelin was significantly Columns with blue colour) of Ruminococcaceae, and vice lower after eradication therapy than before the therapy. A common niche in the gut ecosystem. Thus, the adminis- between such changes in the B: F ratio and the ghrelin oo tration of both genera together as probiotics may result in level. The majority of circulating ghrelin is produced in the x competition for nutrition and colonisation sites, despite gastric mucosa and stimulates the appetite, thus increasing their being considered beneficial bacteria for improving the fat mass deposition and body weight. 0 Therefore, in A energy balance 20 21 Indeed, Faecalibacterium is known energy homeostasis, the increase in the B F ratio in the gut butyrate in the human gut microbiota &g-s that produce microbiota was comparable with the decrease in the plasma to be one of the major bacterial group ghrelin level. It has been reported that the plasma ghrelin a Yanagi H, et al. BM Open Gastro 2017: 4: 0000182. doi: 10. 1136/bmigast-2017-000182
6 Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 Open Access an opposite pattern of colonisation was found between Bifidobacteriaceae and Ruminococcaceae whose major genera were Bifidobacterium and Faecalibacterium, respectively. That is, a sample with a high density (=columns with red colour) of Bifidobacteriaceae contained a low density (=columns with blue colour) of Ruminococcaceae, and vice versa. This suggests that these two genera may share a common niche in the gut ecosystem. Thus, the administration of both genera together as probiotics may result in competition for nutrition and colonisation sites, despite their being considered beneficial bacteria for improving energy balance.20 21 Indeed, Faecalibacterium is known to be one of the major bacterial groups that produce butyrate in the human gut microbiota.22 In the present study, the gut microbiota after the eradication therapy showed a phenotype not of ‘obesity’ but of ‘lean’, which was represented by an increase in both the B:F ratio9 and an abundance of F. prausnitzii. 22 Furthermore, the plasma level of the active form of ghrelin was significantly lower after eradication therapy than before the therapy. A statistical analysis demonstrated a significant correlation between such changes in the B:F ratio and the ghrelin level. The majority of circulating ghrelin is produced in the gastric mucosa and stimulates the appetite, thus increasing the fat mass deposition and body weight.3 10 Therefore, in energy homeostasis, the increase in the B:F ratio in the gut microbiota was comparable with the decrease in the plasma ghrelin level. It has been reported that the plasma ghrelin Figure 3 Double-hierarchical clustering analysis. The faecal samples from 20 patients at three time points for 36 dominant families are represented on a double-hierarchical clustering heat map. The clusters on the right side indicate similarity among the samples at the family level. Sample ID numbers 1–20, 21–40 and 41–60 on the left side represent those obtained at S1, S2 and S3, respectively. IDs 1, 21 and 41, and likewise 2, 22 and 42 and so on correspond to the same patient. The clusters at the bottom indicate the families showing similarity in the frequency of identification among samples. The Z-score column represents the relative ratio of abundance. 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
Open Access Table 3 Clinical and laboratory data regarding obesity/ significantly decreased in the patients after eradication T2DM therapy. While we still need to clarify the relationship Eradication therapy between changes in the B: F ratio and the ghrelin level after the treatment with antimicrobials, changes in the S1(n=20)$3(n=20) P value ut microbiota, such as B: F ratio and abundance of both Body weight(kg) 52.9 53.9 dobacterium and Faecalibacterium, might cause a reduc- (51.6-70.2)”(49.7-70.8) tion in the plasma level of ghrelin, as the direct and earliest= Percent body fat(%)26.7±8.3t26.4±8.10.43 Body mass index 23.0=3. 4 22.9=3.6 0.26 hormone-secreting system. Moreover, Lin et aP reported S Serum triglycerides 80.5 109.0 0.35 that butyrate, one of the major short-chain fatty acids (mg/dl (698-1393)(5.8-1553) formed by gut microbiota such as Faecalibacterium, whose Fasting blood 0.99 abundance actually enlarged in the present study, reduced an glucose(mg/dL)(91-102) (87-103) the plasma ghrelin level in the murine system. It has been a Insulin(ulU/mL) 4.48 3.17 (262-681)(1.86-9.56) the host energy balance by affecting the energy yield from 9 HOMA-R 1.02 2.05 and modulating diet-derIved compounds that alter 3 (0.62-1.99)(0.42-3.14) the host metabolic pathway. Taken together, we suggestec HbA1C(NGSP % 5.6 5.6 0.10 that ghrelin, an appetite-regulating hormone of stomach (53-6.0)(54-5.9) origin, might be a novel factor by which the gut microbiota a Adiponectin(ug/mL)9.2 10.4 0.41 affect the energy balance of the host. Furthermore, bot (7.1-128)(6.5-14.1) the gut microbiota composition and the plasma ghrelin 8 CRP (ng/mL) 0.18 R (187-1083)(202-1180) visceral obesity or insulin resistance 9 yl- ghrelin89.8±40.877.6±36.70.14 Why such a 'lean phenotype was found in both the o gut microbiota composition and plasma ghrelin level at Z 3months after the eradication therapy in the present study Active ghrelin 0.003 (6-10.5) (0-6.25) Leptin(ng/mL) 0.03 Months after the eradication therapy was also reported in s bears further investigation. A lower plasma ghrelin level at 8.1 8.5 Japanese patients in other studies. b In contrast, however, 9 58-13.0)(5.9-152) a decrease in the B: F ratio, a phenotype of obesity, was observed at 6months posteradication, which is 3months later than the time-point for our assay. This suggests that reactive protein; HbAlc, glycated haemoglobin type 2 diabetes mellitus HOMA-R, homeostasis model an assay at 3months after the termination of therapy might assessment-insulin resistance be too early to accurately assess the resilience of gut micro- a biota to reversely fluctuate towards a 'fat' phenotype in level is regulated by multiple factors, including food intake, vulnerable patients, which may have increased the ghrelin body weight and gastric ghrelin production. Because the level and then the body weight later. As such, the short dura eradication of H. pylori is known to increase the produc- tion of follow-up after the eradication therapy may be one s ion of ghrelin in the gastric mucosa, it appeared unlikely limitation associated with the present study that the elimination of H pylori caused the reduction in the plasma ghrelin level. Indeed, such reduction in the plasma odin inn ih Hl, H si d no om red binet a, pyominnticne pt nds cn dergoing ghrelin was observed even in the three patients in the performed bacteriological, genetical and statistical analyses YK was involved in the 5 present study who failed H. prylonieradication therapy. There- study design and drafting the manuscript. AtT was involved in the study design and fore, some event other than the eradication of H. pylori itself data interpretation. may have caused the decrease in the plasma ghrelin level Funding This study was funded by Meji Co, Ltd (okyo, Japan) after eradication therapy in the present study, while it still Competing interests None declared needs further studies using sufficient number of H. pyloni-in- Ethics approval The ethics committee of Tokai University Hospital approved the 2 fected subjects to exclude the H. pylori infection as a cause of study(15R-160) the plasma ghrelin reduction Provenance and peer review Not commissioned; extermally peer reviewed. Queipo-Ortuno et af highlighted the association Open Access This is an Open Access article distributed in accordance with the between the gut microbiota and appetite-regulating Creative Commons Attribution Non Commercial(CC BY-NC4.0)license, which hormones using rat models with different nutritional permits others to distribute, remix, adapt, build upon this work non-commercially, onditions and levels of physical activity. In their study, and license their derivative works on different terms, provided the original work i plasmaghrelinlevelwassignificantlycorrelatedwiththelicenses/dy-ncOeuseisnon-commercial.SEe:http:/lcreativecommons.org/ properly cited and quantity of several kinds of bacteria, including Bifidobac- aRticle authors) for their employer(s) unless otherwise stated in the text of the terium and Bacteroides. Also in the present study, both the article) 2017. All rights reserved. No commercial use is permitted unless otherwise plasma ghrelin level and the abundance of Bifidobacte expressly granted. Yanagi H, et al. BMJ Open Gastro 2017: 4: e000182 doi 10. 1136/bmjgast-2017-000187
Yanagi H, et al. BMJ Open Gastro 2017;4:e000182. doi:10.1136/bmjgast-2017-000182 7 Open Access level is regulated by multiple factors, including food intake, body weight and gastric ghrelin production.23 Because the eradication of H. pylori is known to increase the production of ghrelin in the gastric mucosa,5 it appeared unlikely that the elimination of H. pylori caused the reduction in the plasma ghrelin level. Indeed, such reduction in the plasma ghrelin was observed even in the three patients in the present study who failed H. pylori eradication therapy. Therefore, some event other than the eradication of H. pylori itself may have caused the decrease in the plasma ghrelin level after eradication therapy in the present study, while it still needs further studies using sufficient number of H. pylori-infected subjects to exclude the H. pylori infection as a cause of the plasma ghrelin reduction. Queipo-Ortuño et al24 highlighted the association between the gut microbiota and appetite-regulating hormones using rat models with different nutritional conditions and levels of physical activity. In their study, plasma ghrelin level was significantly correlated with the quantity of several kinds of bacteria, including Bifidobacterium and Bacteroides. Also in the present study, both the plasma ghrelin level and the abundance of Bifidobacterium significantly decreased in the patients after eradication therapy. While we still need to clarify the relationship between changes in the B:F ratio and the ghrelin level after the treatment with antimicrobials, changes in the gut microbiota, such as B:F ratio and abundance of both Bifidobacterium and Faecalibacterium, might cause a reduction in the plasma level of ghrelin, as the direct and earliest target of antimicrobials would be the microbiota, not the hormone-secreting system. Moreover, Lin et al25 reported that butyrate, one of the major short-chain fatty acids formed by gut microbiota such as Faecalibacterium, whose abundance actually enlarged in the present study, reduced the plasma ghrelin level in the murine system. It has been reported that the gut microbiota significantly influence the host energy balance by affecting the energy yield from foods and modulating diet-derived compounds that alter the host metabolic pathway.8 Taken together, we suggested that ghrelin, an appetite-regulating hormone of stomach origin, might be a novel factor by which the gut microbiota affect the energy balance of the host. Furthermore, both the gut microbiota composition and the plasma ghrelin level might be used as an earliest biomarker to foresee the visceral obesity or insulin resistance. Why such a ‘lean’ phenotype was found in both the gut microbiota composition and plasma ghrelin level at 3months after the eradication therapy in the present study bears further investigation. A lower plasma ghrelin level at 3months after the eradication therapy was also reported in Japanese patients in other studies.5 6 In contrast, however, a decrease in the B:F ratio, a phenotype of obesity, was observed at 6months posteradication, which is 3months later than the time-point for our assay.18 This suggests that an assay at 3months after the termination of therapy might be too early to accurately assess the resilience of gut microbiota to reversely fluctuate towards a ‘fat’ phenotype in vulnerable patients, which may have increased the ghrelin level and then the body weight later. As such, the short duration of follow-up after the eradication therapy may be one limitation associated with the present study. Contributors HY, AyT and MM recruited the H. pylori-infected patients undergoing eradication therapy and performed biochemical examinations. ST and GO performed bacteriological, genetical and statistical analyses. YK was involved in the study design and drafting the manuscript. AtT was involved in the study design and data interpretation. Funding This study was funded by Meiji Co, Ltd (Tokyo, Japan). Competing interests None declared. Ethics approval The ethics committee of Tokai University Hospital approved the study (15R-160). Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/ © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted. Table 3 Clinical and laboratory data regarding obesity/ T2DM Eradication therapy S1 (n=20) S3 (n=20) P value Body weight (kg) 52.9 (51.6–70.2)* 53.9 (49.7–70.8) 0.35 Percent body fat (%) 26.7±8.3† 26.4±8.1 0.43 Body mass index 23.0±3.4 22.9±3.6 0.26 Serum triglycerides (mg/dL) 80.5 (69.8–139.3) 109.0 (65.8–155.3) 0.35 Fasting blood glucose (mg/dL) 105 (91–102) 108 (87–103) 0.99 Insulin (μIU/mL) 4.48 (2.62–6.81) 3.17 (1.86–9.56) 0.93 HOMA-R 1.02 (0.62–1.99) 2.05 (0.42–3.14) 0.78 HbA1c (NGSP %) 5.6 (5.3–6.0) 5.6 (5.4–5.9) 0.10 Adiponectin (μg/mL) 9.2 (7.1–12.8) 10.4 (6.5–14.1) 0.41 CRP (ng/mL) 511 (187–1083) 408 (202–1180) 0.18 Desacyl – ghrelin (fmol/L) 89.8±40.8 77.6±36.7 0.14 Active ghrelin (fmol/L) 8 (6–10.5) 5 (0–6.25) 0.003 Leptin (ng/mL) 8.1 (5.8–13.0) 8.5 (5.9–15.2) 0.03 *Median (IQR). †Mean±SD. CRP, C reactive protein; HbA1c, glycated haemoglobin; T2DM, type 2 diabetes mellitus. HOMA-R, homeostasis model assessment-insulin resistance. copyright. on 4 July 2018 by guest. 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