Investigation of microbial flora of pigment gallstones on the basis of 16S ribosomal RNA gene analysis
Abstract
Aim of investigation. Investigation of microbial flora of pigment gallstones by analysis of ribosomal 16S RNA gene.
Material and methods. Seven samples of pigment gallstones were obtained at cholecystectomy from children infected by H. pylori. DNA amplification method (PCR) was used for identification of microorganisms. Ligation, E. coli transformation and creation of clonal libraries were carried out. Nucleotide sequence was determined. Computer analysis of the obtained information was done; belonging of the read nucleotide sequences to certain microorganism by NCBI database was accomplished.
Results. In 4 samples of black pigment stones no bacterial DNA was revealed. In 3 samples of brown pigment stones libraries of 16S rRNA gene fragments were created, their sequences were determined and microorganisms to which they belong were identified.
Conclusion. At children infected by H. pylori, this microbe is not the cause of gallstones development.
About the Authors
A. K. LarinRussian Federation
P. L. Scherbakov
Russian Federation
L. A. Kharitonova
Russian Federation
O. A. Tsarkova
Russian Federation
K. T. Momynaliyev
Russian Federation
References
1. Abayli B., Colakoglu S., Serin M. et al. Helicobacter pylori in the etiology of cholesterol gallstones // J. Clin. Gastroenterol. – 2005. – Vol. 39, N 2. – P. 134–137.
2. Bik E.M., Eckburg P.B., Gill S.R. et al. Molecular analysis of the bacterial microbiota in the human stomach // Proc. Natl. Acad. Sci. U. S. A. – 2006. – Vol. 103, N 3. – P. 732–737.
3. Blaut M., Collins M.D., Welling G.W. et al. Molecular biological methods for studying the gut microbiota: the EU human gut flora project // Br. J. Nutr. – 2002. – Vol. 87 (suppl 2). – P. 203–211.
4. Burton J.P., Cadieux P.A., Reid G. Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation // Appl. Environ. Microbiol. – 2003. – Vol. 69, N 1. – P. 97–101.
5. Cetta F. Bile infection documented as initial event in the pathogenesis of brown pigment biliary stones // Hepatology. – 1986. – Vol. 6, N 3. – P. 482–489.
6. Cetta F. The role of bacteria in pigment gallstone disease // Ann. Surg. – 1991. – Vol. 213, N 4. – P. 315–326.
7. Cetta F., Dhamo A., Malagnino G. et al. Fewer infectious manifestations are induced by bacteria entrapped in cholesterol stones than by bacteria in brown pigment gallstone // J. Gastrointest. Surg. – 2008. – Vol. 12, N 5. – P. 988–990.
8. Edwards U., Rogall T., Blцcker H. et al. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA // Nucleic Acids Res. – 1989. – Vol. 17, N 19. – P. 7843–7853.
9. Farrelly V., Rainey F.A., Stackebrandt E. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species // Appl. Environ. Microbiol. – 1995. – Vol. 61, N 7. – P. 2798–2801.
10. Ferris M.J., Masztal A., Martin D.H. Use of species-directed 16S rRNA gene PCR primers for detection of Atopobium vaginae in patients with bacterial vaginosis // J. Clin. Microbiol. – 2004. – Vol. 42, N 12. – P. 5892–5894.
11. Graham D.Y., Osato M.S. H. pylori in the pathogenesis of duodenal ulcer: interaction between duodenal acid load, bile, and H. pylori // Am. J. Gastroenterol. – 2000. – Vol. 95, N 1. – P. 87–91.
12. Han S.W., Evans D.G., el-Zaatari F.A. et al. The interaction of pH, bile, and Helicobacter pylori may explain duodenal ulcer // Am. J. Gastroenterol. – 1996. – Vol. 91, N 6. – P. 1135–1137.
13. Hyman R.W., Fukushima M., Diamond L. et al. Microbes on the human vaginal epithelium // Proc. Natl. Acad. Sci. U. S. A. – 2005. – Vol. 102, N 22. – Р. 7952–7957.
14. Kawai M., Iwahashi M., Uchiyama K. et al. Gram-positive cocci are associated with the formation of completely pure cholesterol stones // Am. J. Gastroenterol. – 2002. – Vol. 97, N 1. – P. 83–88.
15. Klappenbach J.A., Dunbar J.M., Schmidt T.M. rRNA operon copy number reflects ecological strategies of bacteria // Appl. Environ. Microbiol. – 2000. – Vol. 66, N 4. – P. 1328–1333.
16. Larin A.K., Seleznyova O.V., Momynaliev K.T. A method for identification of vaginal epithelial microflora by the analysis of 16S ribosomal RNA gene // Bull. Exp. Biol. Med. – 2007. – Vol. 144, N 3. – P. 413–415.
17. Lee D.K., Tarr P.I., Haigh W.G., Lee S.P. Bacterial DNA in mixed cholesterol gallstones // Am. J. Gastroenterol. – 1999. – Vol. 94, N 12. – P. 3502–3506.
18. Leung J.W., Liu Y.L., Leung P.S. et al. Expression of bacterial beta-glucuronidase in human bile: an in vitro study // Gastrointest. Endosc. – 2001. – Vol. 54, N 3. – P. 346–350.
19. Maurer K.J., Rogers A.B., Ge Z. et al. Helicobacter pylori and cholesterol gallstone formation in C57L/J mice: a prospective study // Am. J. Physiol. Gastrointest. Liver Physiol. – 2006. – Vol. 290, N 1. – P. 175–182.
20. Misra V., Misra S.P., Dwivedi M. et al. Helicobacter pylori in areas of gastric metaplasia in the gallbladder and isolation of H. pylori DNA from gallstones // Pathology. – 2007. – Vol. 39, N 4. – P. 419–424.
21. Monstein H.J., Jonsson Y., Zdolsek J., Svanvik J. Identification of Helicobacter pylori DNA in human cholesterol gallstones // Scand. J. Gastroenterol. – 2002. – Vol. 37, N 1. – P. 112–119.
22. Myung S.J., Kim M.H., Shim K.N. et al. Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis // Dig. Dis. Sci. – 2000. – Vol. 45, N 7. – P. 1405–1412.
23. Neri V., Margiotta M., de Francesco V. et al. DNA sequences and proteic antigens of H. pylori in cholecystic bile and tissue of patients with gallstones // Aliment. Pharmacol. Ther. – 2005. –Vol. 22, N 8. – P. 715–720.
24. Nugent R.P., Krohn M.A., Hillier S.L. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation // J. Clin. Microbiol. – 1991. – Vol. 29, N 2. – P. 297–301.
25. Pei Z., Bini E.J., Yang L. et al. Bacterial biota in the human distal esophagus // Proc. Natl. Acad. Sci. U. S. A. – 2004. – Vol. 101, N 12. – P. 4250–4255.
26. Pei Z., Yang L., Peek R.M. et al. Bacterial biota in reflux esophagitis and Barrett’s esophagus // World J. Gastroenterol. – 2005. – Vol. 11, N 46. – P. 7277–7283.
27. Petti C.A., Polage C.R., Schreckenberger P. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods // J. Clin. Microbiol. – 2006. – Vol. 44, N 9. – P. 3469–3470.
28. Soloway R.D., Crowther R.S. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis // Gastroenterology. – 1995. – Vol. 108, N 3. – P. 934–936.
29. Soloway R.D., Malet P.F. Pigment stones // Bockus Gastroenterology / Ed. J.E. Berk. – 4th ed. Philadelphia: WB Saunders. – 1985. – P. 3510–3517.
30. Swidsinski A., Khilkin M., Pahlig H. et al. Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstones // Hepatology. – 1998. – Vol. 27, N 3. – P. 662–665.
31. Swidsinski A., Lee S.P. The role of bacteria in gallstone pathogenesis // Front. Biosci. – 2001. – Vol. 6. – P. 93–103.
32. Trotman B.W., Soloway R.D. Pigment gallstone disease: summary of the National Institutes of Health International Workshop // Hepatology. – 1982. – Vol. 2, N 6. – P. 879–884.
33. Vaishnavi C., Kochhar R., Kaur S., Singh G. Estimation of endotoxin in infected bile from patients with biliary diseases // Trop. Gastroenterol. – 2006. – Vol. 27, N 1. – P. 22–25.
34. Verhelst R., Verstraelen H., Claeys G. et al. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis // BMC Microbiol. – 2004. – Vol. 4. – P. 16.
35. Vitetta L., Best S.P., Sali A. Single and multiple cholesterol gallstones and the influence of bacteria // Med. Hypotheses. – 2000. – Vol. 55, N 6. – P. 502–506.
36. Wilmotte A., van der Auwera G., de Wachter R. Structure of the 16S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF (‘Mastigocladus laminosus HTF’) strain PCC7518, and phylogenetic analysis // FEBS Lett. – 1993. – Vol. 317. – P. 96–100.
37. Wilson K.H., Blitchington R.B. Human colonic biota studied by ribosomal DNA sequence analysis // Appl. Environ. Microbiol. – 1996. – Vol. 62, N 7. – P. 2273–2278.
38. Wilson K.H., Blitchington R.B., Greene R.C. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction // J. Clin. Microbiol. – 1990. – Vol. 28, N 9. – P. 1942–1946.
39. Woo P.C., Lau S.K., Teng J.L. et al. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories // Clin. Microbiol. Infect. – 2008. – Vol. 14, N 10. – P. 908–934.
40. Wu X.T., Xiao L.J., Li X.Q., Li J.S. Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction // World J. Gastroenterol. – 1998. – Vol. 4, N 3. – P. 234–237.
41. Zhou X., Bent S.J., Schneider M.G. et al. Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods // SGM Microbiol. – 2004. – Vol. 150, N 8. – P. 2565–2573.
Review
For citations:
Larin A.K., Scherbakov P.L., Kharitonova L.A., Tsarkova O.A., Momynaliyev K.T. Investigation of microbial flora of pigment gallstones on the basis of 16S ribosomal RNA gene analysis. Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2009;19(5):49-54. (In Russ.)
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