Obstetrics & gynecology science

Korean Society of Obstetrics and Gynecology (대한산부인과학회)
권호 표지

Microbiologic and Molecular Genetic Analysis of Lactobacillus spp. Isolated from Vagina of Korean Women and A Pilot Clinical Study on the Treatment of Vaginitis using the Best Lactobacillus Strain KLB 46

한국여성의 질에서 분리된 유산간균총의 미생물학적, 분자유전학적 분석 및 우량간균 KLB46을 이용한 질염 치료의 예비적 임상실험 결과

Park, Mi-Young;Park, Han-Moie;So, Jae-Seong;Kim, Seung-Cheol
박미영;박한뫼;소재성;김승철

  • Published : 20040600
⟨ Previous Next ⟩

Abstract

Objective: The aim of this study was to analize the biologic and molecular genetic characteristics of Lactobacillus spp. isolated from vagina of Korean healthy women and to perform a pilot clinical study on the treatment of vaginitis using the best Lactobacillus strain KLB 46. Methods: Vaginal specimens were obtained from 205 Korean women with healthy vaginal ecosystems. 155 lactobacillus strains were used for catalase test, cell surface hydrophobicity (CSH) test and antimicrobial activity test. Also we identified the species prevalence by PCR-RFLP analysis of 16S rDNA gene and performed the minimal inhibitory concentrationtest using14 antibiotics to determine their patterns of antibiotic susceptibility. A pilot clinical trial of 8 bacterial vaginosis, 1 trichomonas vaginitis and 6 atrophic vaginitis using Lactobacillus crispatus KLB 46 was done by vaginal instillation (10$^{10}$-10$^{11}$ cells/mL). Results: 155 lactobacilli strains were isolated and all isolates showed high CSH and 61% of the isolates had higher CSH. L. acidophilus and L. crispatus were the most prevalent species by PCR-RFLP analysis of their 16S rDNA gene. And PCR-RFLP analysis suggested that KLB 46 was L. crispatus. The clinical study showed that the vaginal epithelial adherence rate was high as 77% for bacterial vaginosis and 33.3% for atrophic vaginitis. Conclusion: The predominating lactobacilli were L. crispatus and L. acidophilus in Korean women’s vaginas. Although further study would be needed, L. crispatus KLB 46 is a good candidate of suitable probiotics for bacterial vaginosis.

목적: 한국 여성의 질에서 유산간균총을 분리하여 그 중 probiotics로서 사용될 수 있는 유력한 유산간균주를 선정하여 질염의 치료효과를 확인하고자 하였다. 연구 방법: 205명의 건강한 여자의 질 분비물로부터 분리된 유산균주들을 catalase test, 세포 표면 소수성 검사, 항균 활성도 측정, Restriction fragment length polymorphism (RFLP)을 이용한 분자 유전학적 분석 등을 시행하여 그 종류와 분포 및 항생제 감수성을 분석하였다. 또한 L. crispatus KLB 46 균주를 이용하여 세균성 질증 및 위축성 질염 환자를 대상으로 예비적 임상실험을 시행하였다. 결과: 분리된 유산간균주는 105종으로 대부분 항균활성을 보였다. 가장 많이 분포하는 우점균주는 L.crispatus와 L. acidophilus (51%)였고, 우량균주 KLB 46은 L. crispatus 였다. L. crispatus KLB 46을 질 내에 접종한 후 세균성 질증 환자와 위축성 질염 환자에서 우수한 치료효과를 보였다. 결론: 한국 여성의 질에서 분리된 유산간균총의 우점균주는 성장속도, 생존력, 항균활성에서 모두 좋은 결과를 보이며, L. crispatus KLB 46은 세균성 질증의 예비적 임상실험에서 우수한 치료결과를 보여 향후 probiotics로서 유망한 균주로 사료된다.

Keywords

References

  1. Berg AO, Heidrich FE, Fihn SD, Bergman JJ, Wood RW, Stamm WE, et al. Establishing the cause of genitourinary symptoms in women in a family practice. Comparison of clinical examination and comprehensive microbiology. JAMA 1984 3; 251(5): 620-5.
  2. Hillier S, Holmes KK. Bacterial vaginosis. In: Holmes KK, Mrdh P-A, Sparling PF, Wiesner PJ, eds. Sexually transmitted diseases. 2nd ed. New York: McGraw-Hill 1990: 547-59.
  3. Holmes KK. Bacterial vaginosis and women and babies. Int J Gynaecol Obstet 1999; 67 Suppl 1: S9-11.
  4. Hillier SL, Lipinski C, Briselden AM, Eschenbach DA. Efficacy of intravaginal 0.75% metronidazole gel for the treatment of bacterial vaginosis. Obstet Gynecol 1993; 81(6): 963-7.
  5. Barbes C, Boris S. Potential role of lactobacilli as prophylactic agents against genital pathogens. AIDS Patient Care STDS 1999; 13(12): 747-51. https://doi.org/10.1089/apc.1999.13.747
  6. Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, Critchlow CM, et al. Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 1989; 27(2): 251-6.
  7. FAO/WHO. Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. Food and Agriculture Organization of the United Nations and World Health Organization Report. 2001. http://www.fao.org/es/ESN/Probio/probio.htm.
  8. McLean NW, Rosenstein IJ. Characterisation and selection of a Lactobacillus species to recolonise the vagina of women with recurrent bacterial vaginosis. J Med Microbiol 2000; 49(6): 543-52.
  9. Mastromarino P, Brigidi P, Macchia S, Maggi L, Pirovano F, Trinchieri V, et al. Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets. J Appl Microbiol 2002; 93(5): 884-93. https://doi.org/10.1046/j.1365-2672.2002.01759.x
  10. Reid G, Burton J. Use of Lactobacillus to prevent infection by pathogenic bacteria.Microbes Infect 2002; 4(3): 319-24. https://doi.org/10.1016/S1286-4579(02)01544-7
  11. Pavlova SI, Kilic AO, Mou SM, Tao L. Phage infection in vaginal lactobacilli: An in vitro study. Infect Dis Obstet Gynecol 1997; 5: 36-44. https://doi.org/10.1002/(SICI)1098-0997(1997)5:1<36::AID-IDOG9>3.0.CO;2-V
  12. Kilic AO, Pavlova SI, Ma WG, Tao L. Analysis of Lactobacillus phage and bacteriocins in American dairy products and characterization of a phage isolated fromyogurt. Appl Envion Microbiol 1996; 62: 2111-6.
  13. Pae KH, So JS. Increased cell surfacehydrophobicity of a lipopolysaccharide-defective mutant of Bradyrhzobium Japonicum. J Microbiol Biotechnol 1995; 5: 241-3.
  14. Ahn C, Stiles ME. Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat. Appl Envion Microbiol 1990; 56: 2503-10.
  15. McAuliffe O, Ryan MP, Ross RP, Hill C, Breeuwer P, Abee T. Lacticin 3127, a broad-spectrum bacteriocin which selectively dissipates the membrane potential. Appl Envion Microbiol 1998; 64: 439-45.
  16. Jo YB, Heo K, Kim SK, Baik HS, Jun HK. Properties of the Fusants of Lactobacillus acidophilus 88and Lactobacillus casei subsp. casei KCTC 1121. J Microbiol Biotechnol 1997; 7: 25-31.
  17. Choi YO, Ahn C. Plasmid-associated bacteriocin production by Leuconostoc sp. LAB145-3A Isolatedfrom Kimchi. J Microbiol Biotechnol 1997; 7: 409-16.
  18. Rodriguez AV, Nadra MC. Effect of pH and hydrogen peroxide produced by Lactobacillus hilgardii onPediococcus pentosaceus growth. FEMS Microbiol Lett 1995; 128: 59-62. https://doi.org/10.1111/j.1574-6968.1995.tb07500.x
  19. Chang CE, Pavlova SI, Tao L, Kim EK, Kim SC, Yun HS, et al. Molecular identification of vaginal lactobacillus spp. isolated from Korean women. J Microbiol Biotechnol 2002; 12: 312-7.
  20. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991; 173(2): 697-703.
  21. Verellen TLJ, Bruggeman G, Reenen CAV, Dicks LMT, Vandamme EJ. Fermentation optimization of Plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423. J Ferment Bioeng 1998; 86: 174-9. https://doi.org/10.1016/S0922-338X(98)80057-3
  22. Redondo-Lopez V, Cook RL, Sobel JD. Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis 1990; 12(5): 856-72. https://doi.org/10.1093/clinids/12.5.856
  23. Pavlova SI, Kilic AO, Kilic SS, So JS, Nader-Macias ME, Simoes JA, et al. Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences. J Appl Microbiol 2002; 92(3): 451-9. https://doi.org/10.1046/j.1365-2672.2002.01547.x
  24. Antonio MA, Hawes SE, Hillier SL. The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 1999; 180(6): 1950-6. https://doi.org/10.1086/315109
  25. Giorgi A, Torriani S, Dellaglio F, Bo G, Stola E, Bernuzzi L. Identification of vaginal lactobacilli from asymptomatic women. Microbiologica 1987; 10(4): 377-84.
  26. Johnson JL, Phelps CF, Cummins CS, London J, Gasser F. Taxonomy of the Lactobacillus acidophilus group. Int J Systemic Bacteriology 1980; 30: 53-68. https://doi.org/10.1099/00207713-30-1-53
  27. Du Plessis EM, Dicks LM. Evaluation of random amplified polymorphic DNA(RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Curr Microbiol 1995; 31(2): 114-8. https://doi.org/10.1007/BF00294286
  28. Song YL, Kato N, Matsumiya Y, Liu CX, Kato H, Watanabe K. Identification of and hydrogen peroxide production by fecal and vaginal lactobacilli isolated from Japanese women and newborn infants. J Clin Microbiol 1999; 37(9): 3062-4.
  29. Center for disease control and prevention guideline. 2002 sexually transmitted diseases treatment guidelines. MMWR 2002; 51(RR-6): 42-7.
  30. Gravett MG, Hummel D, Eschenbach DA, Holmes KK. Preterm labor associated with subclinical amniotic fluid infection and with bacterial vaginosis. Obstet Gynecol 1986; 67: 229-37. https://doi.org/10.1097/00006250-198602000-00013
  31. Hillier SL, Nugent RP, Eschenbach DA. Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. N Engl J Med 1995; 333: 1737-42. https://doi.org/10.1056/NEJM199512283332604
  32. Kurki T, Sivonen A, Renkonen OV, Savia E, Ylikorkala O. Bacterial vaginosis in early pregnancy and pregnancy outcome. Obstet Gynecol 1992; 80: 173-7.
  33. Soper DE. Gynecologic sequelae of bacterial vaginosis. Int J Gynaecol Obstet 1999; 67 Suppl 1: S25-8.
  34. Carey JC, Klebanoff MA, Hauth JC, Hillier SL, Thom EA, Ernest JM, et al. Metronidazole to prevent preterm delivery in pregnant women with asymptomatic bacterial vaginosis. N Engl J Med 2000; 342(8): 534-40. https://doi.org/10.1056/NEJM200002243420802
  35. McDonald HM, O'Loughlin JA, Vigneswaran R, Jolley PT, Harvey JA, Bof A, et al. Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora(Gardnerella vaginalis): a randomised, placebo controlled trial. Br J Obstet Gynaecol 1997; 104(12): 1391-7. https://doi.org/10.1111/j.1471-0528.1997.tb11009.x
  36. Hay PE, Morgan DJ, Ison CA, Bhide SA, Romney M, McKenzie P, et al. A longitudinal study of bacterial vaginosis during pregnancy. Br J Obstet Gynaecol 1994 Dec; 101(12): 1048-53. https://doi.org/10.1111/j.1471-0528.1994.tb13580.x
  37. McDonald HM, O'Loughlin JA, Vigneswaran R, Jolley PT, Harvey JA, Bof A, et al. Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora(Gardnerella vaginalis): a randomised, placebo controlled trial. Br J Obstet Gynaecol 1997; 104(12): 1391-7. https://doi.org/10.1111/j.1471-0528.1997.tb11009.x
  38. Osset J, Bartolome RM, Garcia E, Andreu A. Assessment of the capacity of Lactobacillus to inhibit the growth of uropathogens and block their adhesion to vaginal epithelial cells. J Infect Dis 2001; 183(3): 485-91. https://doi.org/10.1086/318070
  39. Dominique P, Michel B, Dominique B. Christine K, Federico G, Fay EW, et al. Therapy of bacterial vaginosis using exogenously-applied Lactobacillus acidophilus and a low dose of estriol. Drug Res 1996; 46: 68-73.
  40. Sobel JD. Vaginitis. N Engl J Med 1997; 337(26): 1896-903. 40. Simoes JA, Aroutcheva AA, Shott S, Faro S. Effect of metronidazole on the growth of vaginal lactobacilli in vitro. Infect Dis Obstet Gynecol 2001; 9(1): 41-5.
  41. Edwards DI. Nitroimidazole drugs--action and resistance mechanisms. I. Mechanisms of action. J Antimicrob Chemother 1993; 31(1): 9-20.
  42. McLean NW, McGroarty JA. Growth inhibition of metronidazolesusceptible and metronidazole-resistant strains of Gardnerella vaginalis by Lactobacilli in vitro. Appl Environ Microbiol 1996; 62(3): 1089-92.