World Journal of Microbiology and Biotechnology

, Volume 22, Issue 10, pp 1055–1060

Assessment of Lactobacillus species colonizing the vagina of apparently healthy Nigerian women, using PCR-DGGE and 16S rRNA gene sequencing

  • Kingsley C. Anukam
  • Emmanuel O. Osazuwa
  • Ijeoma Ahonkhai
  • Gregor Reid


The identity of lactobacilli colonizing Africa-American women, have previously been based on culture-dependent methods. This led to some misleading speculations that black women lack lactobacilli in their vagina and are therefore highly susceptible to Bacterial vaginosis and STDs including HIV. In this study we used culture-independent procedures.About 241 vaginal swabs were obtained from ‘apparently’ healthy premenopausal women, between 18 and 48 years. Samples were Gram stained for the Nugent score evaluation. DNA was extracted from the bacteria on the vaginal swabs and amplified using polymerase chain reaction (PCR), with Lactobacillus primers. Samples with PCR products were separated with denaturing gradient gel electrophoresis (DGGE) and cut bands were re-amplified with Lactobacillus primers without the GC clamp. The re-amplified product was purified and sequenced with ABI PRISM Big-Dye Terminator. The sequence was identified with BLAST algorithm having the highest Gene Bank Accession number.

Out of the 241 vaginal swab samples, 207 (85.8%) had PCR products, indicating the presence of lactobacilli, while 34 (14.2%) showed absence of lactobacilli and the Nugent scores were synonymous to either intermediate bacterial vaginosis or bacterial vaginosis (BV). Out of the 207 samples that had PCR product for Lactobacillus, 149 (72%) had sequence results as revealed by the BLAST algorithm. Most of the women (64%) were colonized by Lactobacillus iners as the predominant strain. Lactobacillus gasseri had 7.3%, followed by L. plantarum and L suntoryeus (6.0%) each. Others were colonized by Lactobacillus crispatus (3.0%), Lactobacillus rhamnosus (2.7%), Lactobacillus vaginalis (2.7%), Lactobacillus rennanqilfy (2.7%), followed by Lactobacillus fermentum (1.3%), Lactobacillus helveticus (1.3%), Lactobacillus johnsonii (1.3%) and Lactobacillus salivarus (1.3%). To our knowledge, this is the first report of Lactobacillus iners being the predominant species colonizing the vagina of the Nigerian women examined. This finding is in line with those from a recent study conducted among Canadian and Swedish women.


Identification lactobacilli nugent score polymerase chain reaction sequencing vagina 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215: 403–410CrossRefGoogle Scholar
  2. Antonio M.A., Hawes S.E. and Hillier S.L. (1999). The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. Journal of Infectious Diseases 180: 1950–1956CrossRefGoogle Scholar
  3. Anukam K.C., Osazuwa E.O., Reid G. and Katsivo M.N. (2004a). Receptivity for probiotic products among pre-menopausal female students in an African university. Sexually Transmitted Diseases 31: 460–464CrossRefGoogle Scholar
  4. Anukam K.C., Osazuwa E.O., Reid G. and Ozolua R.I. (2004b). Feeding probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 does not significantly alter hematological parameters of Sprague-Dawley rats. HAEMA 7: 497–501Google Scholar
  5. Anukam K.C., Osazuwa, E.O. & Reid, G. 2005. Improved appetite of pregnant rats and increased birth weight of newborns following feeding with probiotic Lactobacillus rhamnosus GR-1 and L. fermentum RC-14. Journal of Applied Research. In PressGoogle Scholar
  6. Burton J.P. and Reid G. (2002). Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (Nugent Score) and molecular (Polymerase Chain Reaction and Genaturing Gradient Gel Electrophoresis) techniques. Journal of Infectious Diseases 186: 1770–1780CrossRefGoogle Scholar
  7. Burton J.P., Cadieux P. and Reid G. (2003). Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Applied and Environmental Microbiology 69: 97–101CrossRefGoogle Scholar
  8. Chang C.H., Simpson D.A., Xu Q., Martin P.K., Lagenaur L.A., Schoolnik G.K., Ho D.D., Hillier S.L., Holodniy M., Lewicki J.A. and Lee P.P. (2003). Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two domain CD4. Proceedings of the National Academy of Sciences, USA 100: 11672–11677CrossRefGoogle Scholar
  9. Cohn S.E. and Clark R.A. (2003). Sexually transmitted diseases, HIV and AIDS in women. Medical Clinics of North America 87: 971–95CrossRefGoogle Scholar
  10. Falsen E., Pascual C., Sjoden B., Ohlen M. and Collins M.D (1999). Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. International Journal of Systematic Bacteriology 49: 217–221CrossRefGoogle Scholar
  11. Hillier S.L., Nugent R.P., Eschenbach D.A., Krohn M.A., Gibbs Martin R.S. D.H., Cotch M.F., Edelman R., Pastorek J.G., Rao A.V., Mcnellis D., Regan J.A., Carey J.C. and Klebanoff M.A (1995). Association between bacterial vaginosis and preterm delivery of a low-birth weight infant. New England Journal of Medicine 333: 1732–1736CrossRefGoogle Scholar
  12. Klebanoff M.A., Schwebke J.R., Zhang Nansel J. T.R., Yu K.F. and Andrews W.W (2004). Vulvovaginal symptoms in women with bacterial vaginosis. Obstetrics and Gynecology 104: 267–72Google Scholar
  13. Lachlak N., Ageron Zampatti E. O., Michel G. and Grimont A.D (1996). Composition of the Lactobacillus acidophilus complex isolated from vaginal flora. Microbiologica 19: 123–132Google Scholar
  14. Martino J.L. and Vermund S.H. (2002). Vaginal douching: evidence for risks or benefits to women’s health. Epidemiological Review 24: 109–124CrossRefGoogle Scholar
  15. Nugent Krohn R.P. M.A. and Hillier S.L (1991). Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. Journal of Clinical Microbiology 29: 297–301Google Scholar
  16. Redondo-Lopez Cook V. R.L. and Sobel J.D (1990). Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Review of Infectious Diseases 12: 856–872Google Scholar
  17. Reid G., Bruce A.W., Fraser N., Heinemann C., Owen J. and Henning B (2001). Oral probiotics can resolve urogenital infections. FEMS Immunology and Medical Microbiology 30: 49–52CrossRefGoogle Scholar
  18. Reid G., Burton J., Hammond J.A. and Bruce A.W (2004). Nucleic acid-based diagnosis of bacterial vaginosis and improved management using probiotic lactobacilli. Journal of Medicinal Food 7: 223–228CrossRefGoogle Scholar
  19. Rosenstein I.J., Morgan D.J., Sheehan M., Lamont R.F. and Taylor-Robinson D (1996). Bacterial vaginosis in pregnancy: distribution of bacterial species in different Gram-stain categories of the vaginal flora. Journal of Medical Microbiology 45: 120–126CrossRefGoogle Scholar
  20. Sewankambo N., Gray R.H., Wawer M.J., Paxton L., McNaim D., Wabwire-Mangen F., Serwadda D., Li C., Kiwanuka N., Hillier S.L., Rabe L., Gaydos C.A., Quinn T.C. and Konde-Lule J. (1997). HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet 350: 530–531CrossRefGoogle Scholar
  21. Song Y.-L., Kato N., Matsumiya Y., Liu Kato C-X. H. and Watanabe K (1999). Identification of and hydrogen peroxide production by fecal and vaginal lactobacilli isolated from Japanese women and newborn infants. Journal Clinical Microbiology 37: 3062–3064Google Scholar
  22. Vasquez A., Jakobsson T., Ahrne S., Forsum U. and Molin G (2002). Vaginal Lactobacillus flora of healthy Swedish women. Journal Clinical Microbiology 40: 2746–2749CrossRefGoogle Scholar
  23. Zhong W., Millsap K., Bialkowska-Hobrzanska H. and Reid G (1998). Differentiation of lactobacillus species by molecular typing. Applied, Environmental Microbiology. 64: 2418–2423Google Scholar
  24. Zhou X., Bent S.J., Schneider M.G., Davis C.C., Islam M.R. and Forney L.J (2004). Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods. Microbiology 150: 2565–2573CrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Kingsley C. Anukam
    • 1
    • 2
  • Emmanuel O. Osazuwa
    • 2
  • Ijeoma Ahonkhai
    • 2
  • Gregor Reid
    • 1
    • 3
  1. 1.Canadian Research & Development Centre for ProbioticsLawson Health Research InstituteLondonCanada
  2. 2.Department of Pharmaceutical Microbiology, Faculty of PharmacyUniversity of BeninBenin CityNigeria
  3. 3.Department of Microbiology and Immunology and SurgeryUniversity of Western OntarioLondonCanada

Personalised recommendations