Microbiological Survey of the Human Gastric Ecosystem Using Culturing and Pyrosequencing Methods
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.
KeywordsLactobacillus Proteobacteria Actinobacteria Firmicutes Mucosal Sample
This research was funded by projects from the Spanish Ministry of Economy and Competitiveness (Ref. AGL2011-24300 and SAF2009-13032-C02-02) and FICYT (Ref. IB08-005). S. Delgado was supported by a contract under Juan de la Cierva program (Ref. JCI-2008-02391).
- 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.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
- 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.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.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
- 11.Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2329Google Scholar
- 14.Holland SM (2003) Analytic Rarefaction 1.3. UGA Stratigraphy LabGoogle Scholar
- 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
- 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