Microbial Ecology

, Volume 65, Issue 3, pp 763–772 | Cite as

Microbiological Survey of the Human Gastric Ecosystem Using Culturing and Pyrosequencing Methods

  • Susana Delgado
  • Raúl Cabrera-Rubio
  • Alex Mira
  • Adolfo Suárez
  • Baltasar Mayo
Host Microbe Interaction

Abstract

Stomach mucosa biopsies and gastric juices samples of 12 healthy persons were analysed by culturing in selective- and non-selective-rich media. Microbial DNA from four mucosal samples was also amplified by nested PCR using universal bacterial primers, and the 16S rDNA amplicons pyrosequenced. The total number of cultivable microorganisms recovered from the samples ranged from 102 to 104 cfu/g or ml. The isolates were identified at the species level by PCR amplification and sequencing of the 16S rDNA. Isolates belonged mainly to four genera; Propionibacterium, Lactobacillus, Streptococcus and Staphylococcus. A total of 15,622 high-quality 16S rDNA sequence reads were obtained by pyrosequencing from the four mucosal samples. Sequence analysis grouped the reads into 59 families and 69 genera, revealing wide bacterial diversity. Considerable differences in the composition of the gastric microbiota were observed among the subjects, although in all samples the most abundant operational taxonomic units belonged to Streptococcus, Propionibacterium and Lactobacillus. Comparison of the stomach microbiota with that present in other parts of the human gastrointestinal tract revealed distinctive microbial communities. This is the first study in which a combination of culture and culture-independent techniques has been used to explore the bacterial diversity of the human stomach.

Supplementary material

248_2013_192_MOESM1_ESM.ppt (107 kb)
ESM 1(PPT 107 kb)

References

  1. 1.
    Marshall BJ, Warren JR (1984) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1:1311–1315PubMedCrossRefGoogle Scholar
  2. 2.
    Merrell DS, Goodrich ML, Otto G, Tompkins LS, Falkow S (2003) pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun 71:3529–3539PubMedCrossRefGoogle Scholar
  3. 3.
    Adamson I, Nord CE, Lundquist P, Sjöstedt S, Edlund C (1999) Comparative effects of omeprazole, amoxycillin plus metronidazole versus omeprazole, clarithromycin plus metronidazole on the oral, gastric and intestinal microflora in Helicobacter pylori-infected patients. J Antimicrob Chemother 44:629–640CrossRefGoogle Scholar
  4. 4.
    Monstein HJ, Tiveljung A, Kraft CH, Borch K, Jonasson J (2000) Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis. J Med Microbiol 49:817–822PubMedGoogle Scholar
  5. 5.
    Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F, Perez-Perez G, Blaser MJ, Relman DA (2006) Molecular analysis of the bacterial microbiota in the human stomach. Proc Natl Acad Sci USA 103:732–737PubMedCrossRefGoogle Scholar
  6. 6.
    Andersson A, Lindberg M, Jakobsson H, Bäckhed F, Nyrén P, Engstrand L (2008) Comparative analysis of human gut microbiota by barcoded pyrosequencing. PlosOne 3:e2836Google Scholar
  7. 7.
    Li XX, Wong GL, To KF, Wong VW, Lai LH, Chow DK, Lau JY, Sung JJ, Ding C (2009) Bacterial microbiota profiling in gastritis without Helicobacter pylori infection or non-steroidal anti-inflammatory drug use. PlosOne 4:e7985Google Scholar
  8. 8.
    Tiveljung A, Borch K, Jonasson J, Mårdh S, Petersson F, Monstein HJ (1998) Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pillory-associated gastritis? J Med Microbiol 47:695–704PubMedCrossRefGoogle Scholar
  9. 9.
    Lawson RD, Coyle WJ (2010) The noncolonic microbiome: does it really matter? Curr Gastroenterol Rep 12:259–262PubMedCrossRefGoogle Scholar
  10. 10.
    Zoetendal EG, Heilig HG, Klaassens ES, Booijink CC, Kleerebezem M, Smidt H, de Vos WM (2006) Isolation of DNA from bacterial samples of the human gastrointestinal tract. Nat Protoc 1:870–873PubMedCrossRefGoogle Scholar
  11. 11.
    Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2329Google Scholar
  12. 12.
    Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, O’Toole PW (2010) Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38:e200PubMedCrossRefGoogle Scholar
  13. 13.
    Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659PubMedCrossRefGoogle Scholar
  14. 14.
    Holland SM (2003) Analytic Rarefaction 1.3. UGA Stratigraphy LabGoogle Scholar
  15. 15.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267PubMedCrossRefGoogle Scholar
  16. 16.
    Domínguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA 107:11971–11975PubMedCrossRefGoogle Scholar
  17. 17.
    Charlson ES, Chen J, Custers-Allen R, Bittinger K, Li H, Sinha R, Hwang J, Bushman FD, Collman RG (2010) Disordered microbial communities in the upper respiratory tract of cigarette smokers. PLoS One 5:e15216PubMedCrossRefGoogle Scholar
  18. 18.
    Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI (2009) The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci Transl Med 1:6ra14PubMedCrossRefGoogle Scholar
  19. 19.
    Nawrocki EP, Kolbe DL, Eddy SR (2009) Infernal 1.0: inference of RNA alignments. Bioinformatics 25:1335–1337PubMedCrossRefGoogle Scholar
  20. 20.
    Price MN, Dehal PS, Arkin AP (2009) FastTree: computing large minimum-evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26:1641–1650PubMedCrossRefGoogle Scholar
  21. 21.
    Hamady M, Lozupone C, Knight R (2010) Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME J 4:17–27PubMedCrossRefGoogle Scholar
  22. 22.
    Peek RM, Blaser MJ (2002) Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2:28–37PubMedCrossRefGoogle Scholar
  23. 23.
    Dicksved J, Lindberg M, Rosenquist M, Enroth H, Jansson JK, Engstrand L (2009) Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls. J Med Microbiol 58:509–516PubMedCrossRefGoogle Scholar
  24. 24.
    Cui Y, Wang CL, Liu XW, Wang XH, Chen LL, Zhao X, Fu N, Lu FG (2010) Two stomach-originated Lactobacillus strains improve Helicobacter pylori infected murine gastritis. World J Gastroenterol 16:445–452PubMedCrossRefGoogle Scholar
  25. 25.
    Ryan KA, Jarayaman T, Daly P, Canchaya C, Curran S, Fang F, Quigley EM, O’Toole PW (2008) Isolation of lactobacilli with probiotic properties from the human stomach. Lett Appl Microbiol 47:269–274PubMedCrossRefGoogle Scholar
  26. 26.
    Gagliardi D, Makihara S, Corsi PR, Viana Ade T, Wiczer MV, Nakakubo S, Mimica LM (1988) Microbial flora of the normal esophagus. Dis Esophagus 11:248–250Google Scholar
  27. 27.
    Savage DC (1977) Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 31:107–133PubMedCrossRefGoogle Scholar
  28. 28.
    Miura Y, Ishige I, Soejima N, Suzuki Y, Uchida K, Kawana S, Eishi Y (2010) Quantitative PCR of Propionibacterium acnes DNA in samples aspirated from sebaceous follicles on the normal skin of subjects with or without acne. J Med Dent Sci 57:65–74PubMedGoogle Scholar
  29. 29.
    Saulnier DM, Kolida S, Gibson GR (2009) Microbiology of the human intestinal tract and approaches for its dietary modulation. Curr Pharm Des 15:1403–1414PubMedCrossRefGoogle Scholar
  30. 30.
    Delgado S, Suárez A, Mayo B (2011) Identification, typing and characterisation of Propionibacterium strains from healthy mucosa of the human stomach. Int J Food Microbiol 149:65–72PubMedCrossRefGoogle Scholar
  31. 31.
    Zoetendal EG, von Wright A, Vilpponen-Salmela T, Ben-Amor K, Akkermans AD, de Vos WM (2002) Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl Environ Microbiol 68:3401–3407PubMedCrossRefGoogle Scholar
  32. 32.
    Delgado S, Suárez A, Mayo B (2006) Identification of dominant bacteria in feces and colonic mucosa from healthy Spanish adults by culturing and by 16S rDNA sequence analysis. Dig Dis Sci 51:744–751PubMedCrossRefGoogle Scholar
  33. 33.
    Qin J, Li R, Raes J et al (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464:59–65PubMedCrossRefGoogle Scholar
  34. 34.
    Dal Bello F, Hertel C (2006) Oral cavity as natural reservoir for intestinal lactobacilli. Syst Appl Microbiol 29:69–76PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Susana Delgado
    • 1
    • 4
  • Raúl Cabrera-Rubio
    • 2
  • Alex Mira
    • 2
  • Adolfo Suárez
    • 3
  • Baltasar Mayo
    • 1
  1. 1.Departamento de Microbiología y Bioquímica de Productos LácteosInstituto de Productos Lácteos de Asturias (IPLA-CSIC)VillaviciosaSpain
  2. 2.Departamento de Genómica y SaludCentro Superior de Investigación en Salud Pública (CSISP)ValenciaSpain
  3. 3.Servicio de DigestivoHospital de CabueñesGijónSpain
  4. 4.Instituto de Productos Lácteos de Asturias (IPLA-CSIC)VillaviciosaSpain

Personalised recommendations