Antonie van Leeuwenhoek

, Volume 90, Issue 3, pp 201–210

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

  • Galina D. Stoyancheva
  • Svetla T. Danova
  • Ivo Y. Boudakov
Original Paper

Abstract

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.

Keywords

16S rDNA Identification Lactobacillus Ribotyping Vaginal microflora 

Abbreviations

ARDRA

amplified ribosomal

DNA

restriction analysis

BV

bacterial vaginosis

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References

  1. Andreu A (2004) Lactobacillus as a probiotic for preventing urogenital infections. Rev Med Microbiol 15:1–6Google Scholar
  2. Antonio MA, Hawes SE, Hillier SL (1999) The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 180:1950–1956PubMedCrossRefGoogle Scholar
  3. Bjorkroth J, Korkeala H (1996) rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime. Int J Food Microbiol 30:293–302PubMedCrossRefGoogle Scholar
  4. Brosius J, Ullrich A, Raker MA, Gray A, Dull TJ, Gutell RR, Noller HF (1981) Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli Plasmid 6:112–118PubMedCrossRefGoogle Scholar
  5. Bruce AW, Reid G (1988) Intravaginal instillation of lactobacilli for prevention of recurrent urinary tract infections. Can J Microbiol 34:339–43PubMedCrossRefGoogle Scholar
  6. Chagnaud P, Machinis K, Coutte LA, Marecat A, Mercenier A (2001) Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species. J Microbiol Methods 44:139–48PubMedCrossRefGoogle Scholar
  7. Chang CE, Pavlova SI, Tao L, Kim E-K, Kim SC, Yun HS, So J-S (2002) Molecular identification of vaginal Lactobacillus spp. isolated from Korean women. J Microbiol Biotechnol 12:312–317Google Scholar
  8. Charteris WP, Kelly PM, Morelli L, Collins JK (1997) Selective detection, enumeration and identification of potentially probiotic Lactobacillus and Bifidobacterium species in mixed bacterial populations. Int J Food Microbiol 35:1–27PubMedCrossRefGoogle Scholar
  9. Collins MD, Rodrigues UM, Ash C, Aguirre M, Farrow JAE, Martinez-Murcia A, Philips BA, Williams AM, Wallbanks S (1991) Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77:5–12CrossRefGoogle Scholar
  10. De Man JD, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135Google Scholar
  11. Delley M, Mollet B, Hottinger H (1990) DNA probe for Lactobacillus delbrueckii. Appl Environ Microbiol 56:1967–1970PubMedGoogle Scholar
  12. Dicks LM, Silvester M, Lawson PA, Collins MD (2000) Lactobacillus fornicalis sp. nov., isolated from the posterior fornix of the human vagina. Int J Syst Evol Microbiol 50:1253–1258PubMedGoogle Scholar
  13. Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, Critchlow CM, Holmes KK (1989) Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 27:251–256PubMedGoogle Scholar
  14. Giorgi A, Torriani S, Dellaglio F, Bo G, Stola E, Bernuzzi L␣(1987) Identification of vaginal lactobacilli from asymptomatic women. Microbiologica 10:377–384PubMedGoogle Scholar
  15. Hillier SL, Krohn MA, Klebanoff SJ, Eschenbach DA (1992) The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women. Obstet Gynecol 79:369–373PubMedCrossRefGoogle Scholar
  16. Holzapfel WH, Haberer P, Geisen R, Bjorkroth J, Schillinger U (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73:365S–373SPubMedGoogle Scholar
  17. Kilic AO, Pavlova SI, Alpay S, Kilic SS, Tao L (2001) Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: prevalence, morphology, host range, and DNA homology. Clin Diagn Lab Immunol 8:31–39PubMedCrossRefGoogle Scholar
  18. Massi M, Vitali B, Federici F, Matteuzzi D, Brigidi P (2004) Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina. J Appl Microbiol 96:777–786PubMedCrossRefGoogle Scholar
  19. McLean NW, Rosenstein IJ (2000) Characterisation and selection of a Lactobacillus species to re-colonise the vagina of women with recurrent bacterial vaginosis. J␣Med Microbiol 49:543–552PubMedGoogle Scholar
  20. Miteva V, Boudakov I, Ivanova-Stoyancheva G, Marinova B, Mitev V, Mengaud J (2001) Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA). J Appl Microbiol 90:909–918PubMedCrossRefGoogle Scholar
  21. Miteva V, Stefanova T, Budakov I, Ivanova I, Mitev V, Gancheva A, Ljubenov M (1998) Characterization of bacteriocins produced by strains from traditional Bulgarian dairy products. Syst Appl Microbiol 21:151–161PubMedGoogle Scholar
  22. Pavlova SI, Kilic AO, Mou SM, Tao L (1997) Phage infection in vaginal lactobacilli: An in vitro study. Infect Dis Obstet Gynecol 5:36–44PubMedCrossRefGoogle Scholar
  23. Schleifer KH, Stackebrandt E (1983) Molecular systematics of prokaryotes. Annu Rev Microbiol 37:143–187PubMedCrossRefGoogle Scholar
  24. Sieber R, Dietz UT (1998) Lactobacillus acidophilus and yogurt in the prevention and therapy of bacterial vaginosis. Int Dairy J 8:599–607CrossRefGoogle Scholar
  25. Silvester ME, Dicks LM (2003) Identification of lactic acid bacteria isolated from human vaginal secretions. Antonie Van Leeuwenhoek 83:117–123PubMedCrossRefGoogle Scholar
  26. Sobel JD (2000) Bacterial vaginosis. Annu Rev Med 51:349–356PubMedCrossRefGoogle Scholar
  27. Song YL, Kato N, Matsumiya Y, Liu CX, Kato H, Watanabe K (1999) Identification of Lactobacillus species of human origin by a commercial kit, API50CHL. RBJSKS 10:77–82Google Scholar
  28. Tilsala-Timisjarvi A, Alatossava T (1997) Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Int J Food Microbiol 35:49–56PubMedCrossRefGoogle Scholar
  29. Ventura M, Casas IA, Morelli L, Callegari ML (2000) Rapid amplified ribosomal DNA restriction analysis (ARDRA) identification of Lactobacillus spp. isolated from fecal and vaginal samples. Syst Appl Microbiol 23:504–509PubMedGoogle Scholar
  30. Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl Environ Microbiol 66:297–303PubMedCrossRefGoogle Scholar
  31. Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, Corfield A, Millar M (2004) Identification and H2O2 production of vaginal lactobacilli from pregnant women at high risk of preterm birth and relation with outcome. J Clin Microbiol 42:713–717PubMedCrossRefGoogle Scholar
  32. Zhong W, Millsap K, Bialkowska-Hobrzanska H, Reid G (1998) Differentiation of Lactobacillus species by molecular typing. Appl Environ Microbiol 64:2418–2423PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Galina D. Stoyancheva
    • 1
  • Svetla T. Danova
    • 1
  • Ivo Y. Boudakov
    • 2
  1. 1.Department of Microbial GeneticsInstitute of Microbiology, Bulgarian Academy of SciencesSofiaBulgaria
  2. 2.Toronto General HospitalTorontoCanada

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