Molecular Characterisation of the Faecal Microbiota in Patients with Type II Diabetes

Abstract

The investigation provides molecular analyses of the faecal microbiota in type 2 diabetic patients. In order to characterise the gut microbiota in diabetic patients and to assess whether there are changes in the diversity and similarity of gut microbiota in diabetic patients when compared with healthy individuals, bacterial DNAs from 16 type 2 diabetic patients and 12 healthy individuals were extracted from faecal samples and characterised by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specifically targeting V3 region of the 16S rRNA gene, as well as been sequenced for excised gel bands. The counts of Bacteroides vulgatus, Clostridium leptum subgroup and Bifidobacterium genus were assessed using quantitative PCR. By comparing species diversity profiles of two groups, we observed that there were no significant differences between diabetic and healthy group, although a few diabetic individuals (D6, D8) exhibited a remarkable decrease in species profiles. As for the similarity index, it was lower in inter-group than that in intra-group, which showed that the composition of gut microbiota in diabetic group might be changed due to diabetes status. Sequencing results also revealed that bacterial composition of diabetic group was different from that of the healthy group. B. vulgatus and Bifidobacterium genus were low represented in the microbiota of diabetic group, and the significant decrease was observed for Bifidobacterium by real-time PCR. Taken together, in this work we observed the characterisation of gut microbiota in diabetic patients, which suggestes that the gut microbiota of diabetes patients have some changes associated with occurrence and development of diabetes.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

References

  1. 1.

    Backhed F, Ding H, Wang T et al (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 101:15718–15723

    Article  PubMed  Google Scholar 

  2. 2.

    Backhed F, Ley RE, Sonnenburg JL et al (2005) Host-bacterial mutualism in the human intestine. Science 307:1915–1920

    Article  PubMed  Google Scholar 

  3. 3.

    Backhed F, Manchester JK, Semenkovich CF et al (2007) Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci USA 104:979–984

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Cani PD, Neyrinck AM, Fava F, Knauf C, Burcelin RG, Tuohy KM, Gibson GR, Delzenne NM (2007) Selective increases of bifidobacteria in gut microflora improves high-fat diet-induced diabetes in mice through a mechanism associated with endotoxemia. Diabetologia 50:2374–2383

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Cani PD, Amar J, Iglesias MA et al (2007) Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56:1761–1772

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Cani PD, Delzenne NM (2007) Gut microflora as a target for energy and metabolic homeostasis. Curr Opin Clin Nutr Metab Care 10:729–734

    Article  PubMed  Google Scholar 

  7. 7.

    Cani PD, Delzenne NM (2009) The role of the gut microbiota in energy metabolism and metabolic disease. Curr Pharm Des 15:1546–1558

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Creely SJ, McTernan PG, Kusminski CM et al (2007) Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes. Am J Physiol Endocrinol Metab 292:E740–E747

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Dorigo U, Volatier L, Humbert JF (2005) Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Water Res 39:2207–2218

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Fromin N, Hamelin J, Tarnawski S et al (2002) Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environ Microbiol 4:634–643

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Gafan GP, Lucas VS, Roberts GJ et al (2005) Statistical analyses of complex denaturing gradient gel electrophoresis profiles. J Clin Microbiol 43:3971–3978

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Gill SR, Pop M, DeBoy RT et al (2006) Metagenomic analysis of the human distal gut microbiome. Science 312:1355–1359

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Griffiths EA, Duffy LC, Schanbacher FL et al (2004) In vivo effects of bifidobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in Balb/c mice. Dig Dis Sci 49:579–589

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Guo X, Xia X, Tang R et al (2008) Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Lett Appl Microbiol 47:367–373

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444:860–867

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Kahn SE, Hull RL, Utzschneider KM (2006) Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444:840–846

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Kowalchuk GA, Stephen JR, De Boer W et al (1997) Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63:1489–1497

    CAS  PubMed  Google Scholar 

  18. 18.

    Ledder RG, Gilbert P, Huws SA et al (2007) Molecular analysis of the subgingival microbiota in health and disease. Appl Environ Microbiol 73:516–523

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Ley RE, Backhed F, Turnbaugh P et al (2005) Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 102:11070–11075

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Ley RE, Turnbaugh PJ, Klein S et al (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444:1022–1023

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Lyons SR, Griffen AL, Leys EJ (2000) Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria. J Clin Microbiol 38:2362–2365

    CAS  PubMed  Google Scholar 

  22. 22.

    Mariat D, Firmesse O, Levenez F et al (2009) The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age. BMC Microbiol 9:123

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Matsuki T, Watanabe K, Fujimoto J et al (2004) Quantitative PCR with 16S rRNA-Gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol 70:167–173

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Matsuki T, Watanabe K, Fujimoto J et al (2002) Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol 68:5445–5451

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Matsuki T, Watanabe K, Fujimoto J et al (2004) Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces. Appl Environ Microbiol 70:7220–7228

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    McBain AJ, Bartolo RG, Catrenich CE et al (2003) Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms. Appl Environ Microbiol 69:177–185

    Article  CAS  PubMed  Google Scholar 

  27. 27.

    Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of Complex Microbial-Populations by Denaturing Gradient Gel-Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes-Coding for 16 s Ribosomal-Rna. Appl Environ Microbiol 59:695–700

    CAS  PubMed  Google Scholar 

  28. 28.

    Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73:127–141

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Phillips ML (2009) Gut reaction environmental effects on the human microbiota. Environ Health Perspect 117:A198–A205

    PubMed  Google Scholar 

  30. 30.

    Satokari RM, Vaughan EE, Akkermans AD et al (2001) Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis. Appl Environ Microbiol 67:504–513

    Article  CAS  PubMed  Google Scholar 

  31. 31.

    Scanlan PD, Shanahan F, O’Mahony C et al (2006) Culture-independent analyses of temporal variation of the dominant fecal microbiota and targeted bacterial subgroups in Crohn’s disease. J Clin Microbiol 44:3980–3988

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Seksik P, Lepage P, de la Cochetiere MF et al (2005) Search for localized dysbiosis in Crohn’s disease ulcerations by temporal temperature gradient gel electrophoresis of 16S rRNA. J Clin Microbiol 43:4654–4658

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    Silva EP, Russo CAM (2000) Techniques and statistical data analysis in molecular population genetics. Hydrobiologia 420:119–135

    Article  CAS  Google Scholar 

  34. 34.

    Tresse O, Lorrain MJ, Rho D (2002) Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis. Appl Microbiol Biotechnol 59:585–590

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Tuohy KM, Rouzaud GCM, Bruck WM et al (2005) Modulation of the human gut microflora towards improved health using prebiotics—assessment of efficacy. Curr Pharm Des 11:75–90

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Turnbaugh PJ, Ley RE, Mahowald MA et al (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1031

    Article  PubMed  Google Scholar 

  37. 37.

    van der Gast CJ, Whiteley AS, Lilley AK et al (2003) Bacterial community structure and function in a metal-working fluid. Environ Microbiol 5:453–461

    Article  PubMed  Google Scholar 

  38. 38.

    Vanhoutte T, Huys G, De Brandt E et al (2004) Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers. Fems Microbiol Ecol 48:437–446

    Article  CAS  PubMed  Google Scholar 

  39. 39.

    Walter J, Tannock GW, Tilsala-Timisjarvi A et al (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl Environ Microbiol 66:297–303

    Article  CAS  PubMed  Google Scholar 

  40. 40.

    Wang RF, Cao WW, Cerniglia CE (1996) PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl Environ Microbiol 62:1242–1247

    CAS  PubMed  Google Scholar 

  41. 41.

    Wellen KE, Hotamisligil GS (2005) Inflammation, stress, and diabetes. J Clin Invest 115:1111–1119

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Ministry of Science and Technology, The People’s Republic of China with a 973 project (2004CB418503). We gratefully acknowledge Xuan Xie and Yang Jiao from Department of Endocrinology, the Second Affiliated Hospital, School of Medicine of Xi’an Jiaotong University for sample collection and the Center for Disease Control and Prevention of Xi’an, Shaanxi Province, for equipments and technical support. We gratefully acknowledge Ivan Yuan from St. Catherine’s College, University of Cambridge, UK for careful revision of the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Jiru Xu.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wu, X., Ma, C., Han, L. et al. Molecular Characterisation of the Faecal Microbiota in Patients with Type II Diabetes. Curr Microbiol 61, 69–78 (2010). https://doi.org/10.1007/s00284-010-9582-9

Download citation

Keywords

  • Diabetic Group
  • Bacteroidetes
  • Intestinal Microbiota
  • Healthy Group
  • Faecal Microbiota