www.nature.com/scientificreports 37C in a humidified 10%CO2 incubator. The bacteria count was adjusted to the 12. Coates, M. E. Gnotobiotic animals in research: Their uses and limitations. Lab oncentration of 10" bacteria per ml to be used for animal Anim.9,275-282(1975) Animals and Inoculation. Male SPF and GF C57BL/6 mice of 4-6 weeks old were storage. Proc Natl Acad Sci USA 101, 15718-15723 used for experiments. Male mice were used because they have been reported to show a 14. Tortorella, C et al. Ghrelin, an endogenous ligand for the growth hormone- ortex. Int J Mol M and GF)were maintained in sterile plastic isolators at the National Cancer Centre's germ-free facility(NCC)housed in the SingHealth Experimental Medicine Centre. 15. Kageyama, H et al. Visualization of ghrelin-producing ne in the nimals were maintained hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice. Regul Pept of 12(SPE, SPFH, GF and GFH). SPF and GF mice for inoculation with H- pylori were 16.Anastasia, K.A. et al. Ghrelin: are its levels related to obesity? Aristotle Uni Med. 36,9-17(2009) p). 17. Margetic, S, Gazzola, C, Pegg, G.G. Hill, R. A Leptin: a revie 8 weeks(N 5)and 16 weeks(n 5)post-colonization Mouse gastric wer actions and interactions. Int J Obes Relat Metab Disord. 26, 1407-1433(200 collected for H Pylori culturing, and plasma was used for hormone and cytokine 18 Bado, A ef al. The stomach is a source of leptin. Nature 394, 790-793(1998). 19. Vaillant, C.R.& Lund, P. K Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract. J Histochem Cytochem. 34, 1117-1121 Ethics Statements. All experimental procedures were apt 20. Parker, H. E et al. Nutrient-dependent secretion of g tee(IACUC; Application #2011/SHS/680)and Natio insulinotropic polypeptide from primary murine K cells. Diabetologia Cancer Centre Singapore Research Biosafety Committee/SingHealth Institutional 289-298(2009 Biosafety Committee(IBS; Application #o10/RBC/2011) 21. Schuit, F. C, Huypens, P, Heimberg, H. Pipeleers, D. G Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, Plasma Sample Collection. Blood was drawn into pre-chilled EDTA-coated tubes pancreas, and hypothalamus. Diabetes 50, 1-11(2001) ad mixed gently by inversion. Blood was transferred into a chilled Eppendorf tube 22. Stephen, L et al. Glucose Metabolism and Regulation: Beyond Insulin and containing 2 M 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, Glucagon. Diabetes Spectrum 17, 183-190(2004) Sigma-Aldrich, Buchs, Switzerland )to a final concentration of 0.2 M and mixe 23. Shradha, B, Subhash, P.& Natasha, S. Leptin hormone: it's association with tly. The tube was centrifuged at 3,000 x g for 3 min(@C). The resultant plasma sity: A review Intr J Drug Formulation Res. 1, 204-220(2010 as added to a cryotube containing IN HCl to give a final concentration of 0. IN and 24. Roth, J D. et al. Leptin responsive stored by amylin agonism in diet-induced om nonclinical and clinical studies. Proc Natl Acad Sci USa 105,7257-7262(2008) agar plates supplemented with se of 25. Taylor, I L. Distribution and rel peptide YY in dog measured by specifi rim(5 ug/ml), vancomycin(10 ug/ml) radioimmunoassay Gastroenterology 88, 731-737(1985) D)and amphotericin B(5 e added. All the antibiotics were from 26. Adam, P ef al. Central nervous system integration of satiety signals. Curr BioL. 23, under humidified conditions with 10% carbon dioxide. H. 27. Batterham, R L et al. Gut hormone PYY3-36 physiologically inhibits food intake dori-like colonies were confirmed by positivity with urease, oxidase and catalase Nature 418. 65 8. Yoshikatsu, N. et al. Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervos Multiplex Assay for Gut Hormones and Cytokines. Milliplex MAP Kit for Mouse metabolic Magnetic Bead Panel( Catalogue# MMHMAG-44K: Millipore Corp. MO, 29. Lender, N. et al. Review article: associations between Helicobacter pylori and esity-an ecological study. Aliment Pharmacol Ther. 40, 24-31(2014) ylated(active)ghrelin, leptin, active 30. Francois, F et al. The effect of H pylori eradication on meal-associated changes in plasma ghrelin and leptin. BMC Gastroenterol 11, 37(2011) polypeptide( GIP), total peptide YY (PrY) and pancreatic polypeptide(PP). Mouse 31. Spencer, S.I. et al. Ghrelin regulates the hypothalamic-pituitary-adr ne/Chemokine Magnetic Bead Panel( Catalogue# MPXMCYTO-70K nd cognitive function. Nature 477, 90-94(2011 with xPONENTTM software, version 3.1( Luminex, Austin, TX, USA) ylori infection and risk of Parkinsons disease in Denmark. Eur /Neurol. 19. sis. Statistical analyses were perfor 64-86902012) One-way ANoVA(alpha= 0.05)and Tukey's honestly significant 34. Nagy, T.A. et al. Helicobacter pylori induction of eosinophil migration is mediated (HSD) Post-hoc test were performed. Parametric Pearson correlation by the cag pathoge island via microbial-epithelial interactions. Am J Pathol P-value of <0.05 was considered significant. 78,1448-1452(2011) 35. Khosravi, Y. et al. Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives Gut Pathog. 5. 25(2013). 36. Pritchard, D. M.& Przemeck, S M. Review article: How useful are the rodent animal models of gastric adenocarcinoma? Aliment Pharmacol Ther 19, 841-859 Evolution of mammals and their gut microbes. Science 320, 3. Fanaro, S, Chierici, R, Guerrini, P. vigi, v nal microflora in ear ancy: composition and development. Acta Paediatr Suppl. 91, 48-55(2003). 4. Musso, G, Gambino, R& Cassader, M Interactions between gut microbiota and Acknowled nts ost metabolism predisposing to obesity and diabetes. Annu Rev Med. 62, (1)U Ministry of Education(UM-MoE) 1-380(2012 High Impact Research(HIR) Grant UM C/625/1/HIR/MoE/CHAN/O2(Account No Chung. H et al. Gut immune maturation depends on colonization with a host- H-50001-A000013)to 1.V,(2)Singapore Polytechnic Grant(GL Account pecific microbiota. Cell. 149, 1578-1593(2012) 11-27801-45-3341)to K H.C. and (3)grants from Veten 6. Mitchell, H M. The epidemiology of Helicobacter pylori. Curr Top Microbiol TORNADO, Me Millennium Foundation. LKC School of nn.241,11-30(1999) medicine NTU and SCELSE, Microbiome Centre at NTU toS.P We thank Ms Lim Sor Sing 7. Sekirov, L, Russell, S L, Antunes, L C. Finlay, B. B Gut microbiota in healt eng from Merck Millipore Malaysia and Dr. Sylvia Peng, Fiel and disease. Physiol Rev. 90, 859-904(2010) 8. Heijtz, R D ef al. Normal gut microbiota modulates brain development and ILLIPLEX Kits, as well as Mr Ng Kim Tien(University of Malaya) for his help with the vior. Proc Natl Acad Sci USA. 108, 3047-3052(2011) uminex 200TM system. We also wish to thank Ms Nai Huey Shan for proof-reading and 9. Moodley, Y. ef aL Age of the association between Helicobacter pylori and valuable comments on the manuscript. PLos Pathog. 8, e1002693(2012) 10.Pounder,RE&Ng, D. The prevalence of Helicobacter pylori infecti Pharmaco! Ther. 9 Suppl 2, 33-40(1995). AUth。 r contributions 11.Wang.AY&Peura, D A. The prevalence and incidence of Helicobacter pylori- Y KW.Yw.Q-HP and IMD-S Carried out the lab investigation and data analysis associated peptic ulcer disease and upper gastrointestinal bleeding throughout the S.w.S. J.O.,A.A. A and R.M. B: Animal study S.P. M.F. LJ.V,KH C andT. LT- Planned world. Gastrointest Endosc Clin N Am. 21, 613-635(2011) the experiments. SCIENTIFIC REPORTS I 5: 8731 I DO1: 10.1038/ srep0873137uC in a humidified 10% CO2 incubator. The bacteria count was adjusted to the concentration of 109 bacteria per ml to be used for animal inoculation. Animals and Inoculation. Male SPF and GF C57BL/6 mice of 4–6 weeks old were used for experiments. Male mice were used because they have been reported to show a higher incidence of intestinal-type gastric carcinoma than females36. All mice (SPF and GF) were maintained in sterile plastic isolators at the National Cancer Centre’s germ-free facility (NCC) housed in the SingHealth Experimental Medicine Centre. Animals were maintained on autoclaved R36 Lactamin Chow (Lactamin, Sweden) and kept under 12-h light cycles condition. The animals were divided into four groups of 12 (SPF, SPFH, GF and GFH). SPF and GF mice for inoculation with H. pylori were fasted overnight and fed intragastrically with 0.1 ml of bacterial culture three times with a 1-day interval in between. Control animals were inoculated with sterile BHYC media in the same way. Subsequently, mice were sacrificed 2 weeks (N 5 3 per group), 8 weeks (N 5 5) and 16 weeks (N 5 5) post-colonization. Mouse gastrics were collected for H. pylori culturing, and plasma was used for hormone and cytokine analysis. Ethics Statements. All experimental procedures were approved and carried out in accordance to the regulations and guidelines of the SingHealth Institutional Animal Care and Use Committee (IACUC; Application #2011/SHS/680) and National Cancer Centre Singapore Research Biosafety Committee/SingHealth Institutional Biosafety Committee (IBS; Application #010/RBC/2011). Plasma Sample Collection. Blood was drawn into pre-chilled EDTA-coated tubes and mixed gently by inversion. Blood was transferred into a chilled Eppendorf tube containing 2 M 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma-Aldrich, Buchs, Switzerland) to a final concentration of 0.2 M and mixed gently. The tube was centrifuged at 3,000 3 g for 3 min (4uC). The resultant plasma was added to a cryotube containing 1N HCl to give a final concentration of 0.1N and was stored at 280uC until analysis. Culturing H. pylori from mouse gastric stomach was homogenized and inoculated onto chocolate agar plates supplemented with 7% lysed horse blood and, in the case of selective media, trimethoprim (5 mg/ml), vancomycin (10 mg/ml), nalidixic acid (20 mg/ml) and amphotericin B (5 mg/ml), were added. All the antibiotics were from Sigma-Aldrich Corporation (St. Louis, MO, USA). The agar plates were simulta￾neously incubated at 37uC under humidified conditions with 10% carbon dioxide. H. pylori-like colonies were confirmed by positivity with urease, oxidase and catalase tests. Multiplex Assay for Gut Hormones and Cytokines. Milliplex MAP Kit for Mouse Metabolic Magnetic Bead Panel (Catalogue# MMHMAG-44K; Millipore Corp., MO, USA) was used to quantify eight gut hormones that are important regulators of food intake, energy expenditure and body mass: acylated (active) ghrelin, leptin, active amylin, insulin, active glucagon-like peptide-1 (GLP-1), total gastric inhibitory polypeptide (GIP), total peptide YY (PYY) and pancreatic polypeptide (PP). Mouse Cytokine/Chemokine Magnetic Bead Panel (Catalogue# MPXMCYTO-70K; Millipore Corp., MO, USA) was used to measure a panel of 32 different mouse cytokines/chemokines. Plasma was used and assays were carried out according to manufacturer’s instructions. Measurement was performed on Luminex 200TM system with xPONENTTM software, version 3.1 (Luminex, Austin, TX, USA). Statistical analysis. Statistical analyses were performed using the IBM SPSS version 22.0 software. One-way ANOVA (alpha 5 0.05) and Tukey’s honestly significant difference (HSD) post-hoc test were performed. Parametric Pearson correlation was carried out. P-value of , 0.05 was considered significant. 1. Qin, J. et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464, 59–65 (2010). 2. Ley, R. E. et al. Evolution of mammals and their gut microbes. Science 320, 1647–1651 (2008). 3. Fanaro, S., Chierici, R., Guerrini, P. & Vigi, V. Intestinal microflora in early infancy: composition and development. Acta Paediatr Suppl. 91, 48–55 (2003). 4. Musso, G., Gambino, R. & Cassader, M. Interactions between gut microbiota and host metabolism predisposing to obesity and diabetes. Annu Rev Med. 62, 361–380 (2012). 5. Chung, H. et al. Gut immune maturation depends on colonization with a host￾specific microbiota. Cell. 149, 1578–1593 (2012). 6. Mitchell, H. M. The epidemiology of Helicobacter pylori. Curr Top Microbiol Immunol. 241, 11–30 (1999). 7. Sekirov, I., Russell, S. L., Antunes, L. C. & Finlay, B. B. Gut microbiota in health and disease. Physiol Rev. 90, 859–904 (2010). 8. Heijtz, R. D. et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci U S A. 108, 3047–3052 (2011). 9. Moodley, Y. et al. Age of the association between Helicobacter pylori and man. PLoS Pathog. 8, e1002693 (2012). 10. Pounder, R. E. & Ng, D. The prevalence of Helicobacter pylori infection in different countries. Aliment Pharmacol Ther. 9 Suppl 2, 33–40 (1995). 11. Wang, A. Y. & Peura, D. A. The prevalence and incidence of Helicobacter pylori￾associated peptic ulcer disease and upper gastrointestinal bleeding throughout the world. Gastrointest Endosc Clin N Am. 21, 613–635 (2011). 12. Coates, M. E. Gnotobiotic animals in research: Their uses and limitations. Lab Anim. 9, 275–282 (1975). 13. Backhed, F. et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A. 101, 15718–15723 (2004). 14. Tortorella, C. et al. Ghrelin, an endogenous ligand for the growth hormone￾secretagogue receptor, is expressed in the human adrenal cortex. Int J Mol Med. 12, 213–217 (2003). 15. Kageyama, H. et al. Visualization of ghrelin-producing neurons in the hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice. Regul Pept. 145, 116–121 (2008). 16. Anastasia, K. A. et al. Ghrelin: are its levels related to obesity? Aristotle Uni Med J. 36, 9–17 (2009). 17. Margetic, S., Gazzola, C., Pegg, G. G. & Hill, R. A. Leptin: a review of its peripheral actions and interactions. Int J Obes Relat Metab Disord. 26, 1407–1433 (2002). 18. Bado, A. et al. The stomach is a source of leptin. Nature 394, 790–793 (1998). 19. Vaillant, C. R. & Lund, P. K. Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract. J Histochem Cytochem. 34, 1117–1121 (1986). 20. Parker, H. E. et al. Nutrient-dependent secretion of glucose-dependent insulinotropic polypeptide from primary murine K cells. Diabetologia 52, 289–298 (2009). 21. Schuit, F. C., Huypens, P., Heimberg, H. & Pipeleers, D. G. Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 50, 1–11 (2001). 22. Stephen, L. et al. Glucose Metabolism and Regulation: Beyond Insulin and Glucagon. Diabetes Spectrum 17, 183–190 (2004). 23. Shradha, B., Subhash, P. & Natasha, S. Leptin hormone: it’s association with obesity: A review. Intr J Drug Formulation Res. 1, 204–220 (2010). 24. Roth, J. D.et al. Leptin responsiveness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies. Proc Natl Acad Sci U S A. 105, 7257–7262 (2008). 25. Taylor, I. L. Distribution and release of peptide YY in dog measured by specific radioimmunoassay. Gastroenterology 88, 731–737 (1985). 26. Adam, P. et al. Central nervous system integration of satiety signals. Curr Biol. 23, R379–R388 (2013). 27. Batterham, R. L.et al. Gut hormone PYY3-36 physiologically inhibits food intake. Nature 418, 650–654 (2002). 28. Yoshikatsu, N. et al. Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervosa. Eur J Endocrinol 149, R1–R3 (2003). 29. Lender, N. et al. Review article: associations between Helicobacter pylori and obesity - an ecological study. Aliment Pharmacol Ther. 40, 24–31 (2014). 30. Francois, F.et al. The effect of H. pylori eradication on meal-associated changes in plasma ghrelin and leptin. BMC Gasteroenterol 11, 37 (2011) 31. Spencer, S. J. et al. Ghrelin regulates the hypothalamic-pituitary-adrenal axis and restricts anxiety after acute stress. Biol Psychiatry 72, 457–465 (2012). 32. Villeda, S. A. et al. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature 477, 90–94 (2011). 33. Nielsen, H. H., Qiu, J., Friis, S., Wermuth, L. & Ritz, B. Treatment for Helicobacter pylori infection and risk of Parkinson’s disease in Denmark. Eur J Neurol. 19, 864–869 (2012). 34. Nagy, T. A.et al. Helicobacter pylori induction of eosinophil migration is mediated by the cag pathogenicity island via microbial-epithelial interactions. Am J Pathol. 178, 1448–1452 (2011). 35. Khosravi, Y. et al. Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives. Gut Pathog. 5, 25 (2013). 36. Pritchard, D. M. & Przemeck, S. M. Review article: How useful are the rodent animal models of gastric adenocarcinoma? Aliment Pharmacol Ther. 19, 841–859 (2004). Acknowledgments This study was supported by (1) University of Malaya-Ministry of Education (UM-MoE) High Impact Research (HIR) Grant UM.C/625/1/HIR/MoE/CHAN/02 (Account No. H-50001-A000013) to J.V., (2) Singapore Polytechnic Grant (GL Account: 11-27801-45-3341) to K.H.C., and (3) grants from Vetenskapsrådet, EU-project TORNADO, Me´rieux Institute, Singapore Millennium Foundation, LKC School of medicine NTU and SCELSE, Microbiome Centre at NTU to S.P. We thank Ms. Lim Sor Sing and Mr. Goh Chee Meng from Merck Millipore Malaysia and Dr. Sylvia Peng, Field Application Scientist from Merck Millipore Singapore for their technical support for MILLIPLEX Kits, as well as Mr. Ng Kim Tien (University of Malaya) for his help with the Luminex 200TM system. We also wish to thank Ms. Nai Huey Shan for proof-reading and valuable comments on the manuscript. Author contributions Y.K., W.Y.W., Q.H.P. and I.M.D.S.: Carried out the lab investigation and data analysis. S.W.S., J.O., A.A.A. and R.M.B.: Animal study. S.P., M.F.L., J.V., K.H.C. and T.L.T.: Planned the experiments. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8731 | DOI: 10.1038/srep08731 6