1. La microflore vaginale, la vaginose et sa recurrence Mario Vaneechoutte Laboratoire de Microbiologie Hôpital Universitaire de Gand Flandres, Belgique RICAI 2006 26ième Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse Palais des Congrés de Paris, Porte Maillot, Paris, France 7-8 décembre 2006

2. La Microflore Vaginale

3. Protective role of normal vaginal microflora Oestrogen Glycogen Lactic acid H2O2H2O2 Bacteriocines Lactobacillus Mucus Epitheli al cells Afweer- cellen

4. Genus Lactobacillus: currently some 80 species From 1980 onwards: update of the taxonomy of the L. acidophilus group La microflore vaginale: les lactobacilles vaginales L. acidophilus ss L. amylovorus L. amylolyticus L. crispatus L. gallinarum L. gasseri L. iners L. johnsonii L. kitasatonis Accurate and rapid identification of cultured lactobacilli: Baele M, Vaneechoutte M, Verhelst R, Vancanneyt M, Devriese LA, Haesebrouck F. 2002. 2002. Identification of Lactobacillus species using tDNA-PCR. J Microbiol Methods 50: 263-271. Until 1995 (biochemical identification): L. acidophilus

5. tDNA-PCR pattern of vaginal lactobacilli L. crispatus L. jensenii L. gasseri L. iners Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004. 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 4:16.

6. Fréquence (%) des lactobacilles vaginales dans le vagin en condition saine et en condition perturbé à base de cultivation + identification par tDNA-PCR Verhelst, R., H. Verstraelen, G. Claeys, G. Verschraegen, L. Van Simaey, C. De Ganck, E. De Backer, M. Temmerman, and M. Vaneechoutte. 2005. Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora. BMC Microbiol. 2005, 5: 61.

7. Normal vaginal microflora Grade I Ison et al. 2002 Lactobacillus crispatus L. jensenii - L. gasseri Grade Ib Grade Ia Verhelst et al. BMC 2005 The normal vaginal microflora

8. Identification génotypique: Bifidobacterium spp. Normal vaginal microflora? Microscopie : lactobacilles atypiques? Grade I-like

9. La Vaginose Bactérienne (VB)

10. Infectious problems of the female urogenital tract, shown to be related to disturbance of normal vaginal microflora * VB: "lactobacilli deficiency syndrome", nonspecific vaginitis, G. vaginalis vaginitis Gardnerella vaginalis, Atopobium vaginae, anaerobes, (Mycoplasma hominis, Ureaplasma urealyticum) * UTI: Escherichia coli (Gupta et al. 1998, Atassi et al. 2006), Gram negatives (Chan et al. 1984, Fraga et al. 2005, Osset et al. 2000) Staphylococcus aureus * STD: Chlamydia trachomatis Mycoplasma genitalium Neisseria gonorrhoeae Treponema pallidum: syphilis HPV: cervix carcinoma (BV is co-factor?) HIV: BV causes increased HIV shedding (Sewankambo et al. 1997) Herpes Simplex Virus 2 Trichomonas vaginalis * Yeast vaginitis: Candida albicans (VB protective? (Rodrigues AG ea 1999) * Group B streptococci: Streptococcus agalactiae neonatal meningitis

11. UTI in women: les femmes sont plus vulnérables Cerveau 300 million - 1 milliard de cas par an (Reid 2001. Am J Clin Nutr 73: S437-S443) one of the most common reasons for women to visit the family physician. Each episode: on average 6 days of symptoms, often very painful Uropathogens: E. coli (approx. 70%), Enterobacteriacae Enterococcus faecalis, Staphylococcus spp. Increasing drug resistance among uropathogens Sequelae: kidney infection (pyelonephritis) --> preterm birth preterm birth

12. Bacterial vaginosis (BV): Symptoms and diagnosis BV - nonspecific vaginitis - G. vaginalis vaginitis - Prevalence –5-35% of Caucasian women –> 50% of black African women Microscopy: clue cells + overgrowth of bacteria pH raised from 4-4.5 to 6 Only 50-60% symptomatically: –Itch –Vaginal discharge: due to desquamation of vaginal epithelial cells (clue cells: covered with bacteria) –Malodor: due to production by anaerobe bacteria of polyamines (triethylamine, putrescine, cadaverine)

13. Sequelae of BV = Conséquences de microflore vaginale perturbé BV: increases HSV2 infection (Cherpes TL 2005. CID 40: 1422). increases susceptibility for HIV-infection increases HIV shedding (Cu-Uvin S. 2004. CID 33: 894). (Sewankambo. 1997. Lancet 350: 546). --> increases perinatal mother-child HIV-transmission --> increases sexual HIV-transmission increases infection with CT and HPV (da Silva CS. 2004. GOInvest. 58: 189).

14. BV (more general: disturbed vaginal microflora) is associated with recurrent UTI Hooton TM. 2001. IJAA 17: 259-268 Gupta et al. 1998. Inverse association of H 2 O 2 -producing lactobacilli and vaginal Escherichia coli colonization in women with recurrent urinary tract infections. J Infect Dis 178: 446-450. responsible for 30% of PTB --> 70% of all neonatal mortality & morbidity (PTB & PTL: 4 billion US$/year) associated with PID, postpartum endometritis,... Jacobsson et al. 2002. Bacterial vaginosis in early pregnancy may predispose for preterm birth and postpartum endometritis. Acta Obstet Gynecol Scand 81:1006-1010. cause of asthma? Benn et al. 2002. Maternal vaginal microflora during pregnancy and the risk of asthma hospitalization and use of antiasthma medication in early childhood. J Allergy Clin Immunol 110: 72- 77. Sequelae of BV = Implications de microflore vaginale perturbé

15. Results of the culture independent characterization of the vaginal microflora by cloning Cloning of 16S rRNA genes amplified directly from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae and bacterial vaginosis. What is the role of L. iners? Nombre de clones Age Species (N = 38) Verhelst et al. BMC Microbiology 2004

16. Vaginose: Atopobium vaginae Association with BV and G. vaginalis independently described Ferris MJ, Masztal A, Martin DH. 2004. Ferris MJ, Masztal A, Martin DH. 2004. Use of species-directed 16S rRNA gene PCR primers for detection of Atopobium vaginae in patients with bacterial vaginosis. J Clin Microbiol 42:5892-4. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004. 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 4:16. Other simultaneous publications on A. vaginae Burton JP, Devillard E, Cadieux PA, Hammond J-A, Reid G. 2004 Burton JP, Devillard E, Cadieux PA, Hammond J-A, Reid G. 2004. Detection of Atopobium vaginae in postmenopausal women by cultivation-independent methods warrants further investigation J Clin Microbiol 42: 1829-1831.

17. Results of species specific PCR for A. vaginae and G. vaginalis GradeN A+G+A+G-A-G+ A-G- I11210 2060 II2615203530 III1080010 % Verhelst et al. BMC Microbiology 2004

18. Microscopy of vaginal lactobacilli L. crispatus L. iners A. vaginae L. gasseri L. crispatus L. jensenii

19. Bacterial vaginosis Number of germs Normal Symptoms Lactobacillus Gardnerella vaginalis Anaerobes pH 4.0 - 4.5pH 5.0 - 6.0 Etiology of Bacterial Vaginosis (BV)

20. Etiology for BV Oestrogen is protective: BV prevalence lower in women using combined oral contraception (Yen et al. 2003). BV lower during pregnancy (lower during third trimester) (Hay et al. 1994) Clinical trials with oestradiol show cure of BV, restoration pH,... (Kanne & Jenny 1991, Raz & Stamm 1993, Parent et al. 1996, Ozkinay et al. 2005) Progesterone treatment induces BV-like microflora in mice (Furr & Taylor-Robinson 1991). Hay P. 2005. Hay P. 2005. Life in the littoral zone: lactobacilli losing the plot. Sex Transm Infect. 81:100-102. "The vagina is not a steady state ecosystem. As the menstrual cycle becomes established, there are marked cyclical changes in the vaginal environment, which can be compared to life in the littoral zone of the seashore: Levels of oestrogen and progesterone alter, changing the endogenous environment for lactobacilli by influencing levels of glycogen and glucose as substrate, and levels of vaginal pH."

21. Etiology of BV Disturbance of the normal vaginal econiche: variation in oestrogen levels --> differences in glycogen concentration menses --> nutrient (iron) rich vaginal douching --> disturbance Frequency and kind of intercourse: new male sexual partner more male sexual partners: promiscuity = STD? female sexual partner cfr. Vallor et al. 2001: Sexual intercourse once a week was the only risk factor associated with loss of H 2 O 2 producing lactobacilli. Again: frequent disturbance of pH by alkaline sperm --> raise of pH (Boskey et al. 1993: acidification by lactobacilli takes several hours)

22. Etiologie de VB: hypothèse/conclusions Le risque à développement de VB dépend largement de la fréquence et de l'intensité de perturbance de la microflore vaginale lactobacillienne qui dépend à son tour: de la microflore même originelle du vagin: –des forts vers des faibles protecteurs de l'intensité de la menstruation de l'age de la femme: –femmes en menarche sont mieux protégés par la production des oestrogènes de l'intensité de l'acitivité sexuelle d'autres practiques perturbants (vaginal douching)

23. La vaginose recurrente

24. Recurrence rates of up to 80% within 3 months after treatment have been reported: Hay P. 2000. Recurrent bacterial vaginosis. Curr Infect Dis Rep 2:506-512. Larsson PG & U. Forsum. 2005 Bacterial vaginosis, a disturbed bacterial flora and treatment enigma. APMIS 113:305-316. This recurrence might be due to the survival of metronidazole or clindamycin resistant bacteria in the vagina, although Beigi et al. [2004] showed that less than one percent of vaginal anaerobes is metronidazole resistant: Beigi RH et al. 2004. Antimicrobial resistance associated with the treatment of bacterial vaginosis. Am J Obstet Gynecol 191:1124-1129. Vaginose recurrente Résistance antibiotique ou Biofilm?

25. Vaginose recurrente: Atopobium vaginae? Metronidazole resistant? Geissdörfer et al. 2003. J Clin Microbiol 41:2788-2790. Ferris et al. 2004. Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis. BMC Infect Dis 4:5: ensemble: 4 souches testés: MIC metronidazole: > 32 µg/ml Possible reason for BV recurrence problem? Gram-positive elliptical cocci Strictly anaerobic Very fastidious Recently described (Falsen, 1999) One case report (PID, 2003) Metronidazole resistant (?) (2003, 2004) De Backer, E., R. Verhelst, H. Verstraelen, G. Claeys, G. Verschraegen, M. Temmerman, and M. Vaneechoutte. 2006.. Antibiotic susceptibility of Atopobium vaginae. BMC Infectious Diseases 2006, 6:51.

26. Atopobium vaginaeGardnerella vaginalis (n=9)(n=4) Antimicrobial agent Range (mg/L) Ampicillin< 0.016 - 0.94< 0.016 - 0.047 Azithromycin< 0.016 - 0.32< 0.016 - 0.047 Bacitracin1 - 40.75 -2 Cefuroxim0.016 - 0.25< 0.016 - 0.125 Ciprofloxacin0.023 - 0.250.75 - 2 Clindamycin< 0.016< 0.016 - 0.047 Colistin> 1024 Doxycycline0.19 - 0.750.25 - 32 Kanamycin8 - 1616 - 32 Linezolid0.016 - 0.1250.125 - 0.19 Metronidazole2 - 2560.75 - 16 Nalidixic acid> 256 Penicillin0.008 - 0.25 Rifampicin< 0.002> 256 Vancomycin1 - 40.004 - 0.047 Susceptibilité de A. vaginae et de G. vaginalis 500 mg metronidazole intravaginal = maximal vaginal concentration of 2-10 mg/L Alper et al. 1985. Obstetr Gynecol 65: 781-784. Mattila et al. 1983. AAC 23: 721-725.

27. StrainAMAZBAXMCICMCODC KMKM LZMZNAPGRIVA CCUG 420990.094< 0.01630.250.064< 0.016> 10240.75120.0942> 2560.25< 0.0022.0 CCUG 441160.032< 0.01630.1250.25< 0.016> 10240.19120.0324> 2560.064< 0.0021.5 CCUG 442580.023< 0.01640.190.064< 0.016> 10240.38160.023> 256 0.094< 0.0021.5 PB2003/009-T1-4< 0.016 10.0160.023< 0.016> 10240.38160.12512> 2560.008< 0.0021.0 PB2003/017-T1-2< 0.016 1.50.1250.032< 0.016> 10240.25160.125> 256 0.008< 0.0021.5 CCUG 38953 T 0.094< 0.01630.0230.032< 0.016> 10240.2580,125> 256 0.125< 0.0021.5 CCUG 441250.047< 0.01630.250.064< 0.016> 10240.19120.0478> 2560.19< 0.0021.0 CCUG 440610.023< 0.01630.190.047< 0.016> 10240.38120.02316> 2560.19< 0.0021.5 PB2003/189-T1-40.0160.3230.1250.19< 0.016> 10240.38160.0166> 2560.008< 0.0021.0 Susceptibilité de Atopobium vaginae De Backer E, Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Temmerman M, and Vaneechoutte M. 2006: Antibiotic susceptibility of Atopobium vaginae. BMC Infect Dis. 6:51.

28. L'importance du biofilm dans l'infection: Public announcement of US National Institute of Health: "Biofilms are medically important, accounting for over 80% of microbial infections in the body" (Davies 2003. Nature Reviews 2: 114-122) infection of URT in CF-patients: Pseudomonas aeruginosa chronic otitis media: Haemophilus influenzae, Alloiococcus otitidis? burn wounds: Pseudomonas aeruginosa, Staphylococcus aureus foreign object infections: catheters, valves,...: Staphylococcus spp. acne: Propionibacterium acnes recurrent UTI: uropathogenic Escherichia coli bacterial vaginosis: Gardnerella vaginalis, Atopobium vaginae Vaginose recurrente Résistance antibiotique ou Biofilm?

29. Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale LP, Lochs H. 2005. Adherent biofilms in bacterial vaginosis. Obstetrics & Gynecol. 106: 1013-1023. 86% of the G. vaginalis biofilms were associated with Atopobium, which could reach up to 40% of the biofilm mass (i.e. 4 x 10 10 bacteria per mm 2 ) G. vaginalis – A. vaginae biofilm démontré à base de FISH avec probes G. vaginalis et A. vaginae fluorescentes Lumen Epithèle vaginale Biofilm

30. Grade III: Bacterial vaginosis. Clue cells: Gardnerella vaginalis + Atopobium vaginae + anaerobes

31. Hypothèses: 1. La recurrence peut être causée par des souches résistentes pour la métronidazole. Bienque 99% des anaerobes soient susceptibles (Beigi et al. 2004), plusieurs de souches de G. vaginalis et de A. vaginae sont très résistant. 2. La recurrence est probablement surtout la conséquence de la formation du biofilm par G. vaginalis en association avec A. vaginae. Traitement de VB avec des antibiotiques fait disparaître temporellement les symptomes cliniques, mais le biofilm ne peut pas être éradicé et résuscite après quelques jours/semaines/mois. En accordance avec l'apparition des 'clue cells' La vaginose recurrente: Conclusions/Hypothèses

32. La microflore vaginale, la vaginose et sa recurrence Mario.Vaneechoutte@UGent.be Laboratoire de Microbiologie Hôpital Universitaire de Gand Flandres, Belgique Presentation downloadable at http//users.ugent.be/~mvaneech/LBR.htm Merci pour votre attention

33. Traitement: probiotiques vs antibiotiques

34. Rationale pour la dévéloppement des probiotiques vaginales Disturbed vaginal microflora is important: medically and commercially: Urogenital tract infections are a major problem, directly (patient) and with consequences (PTB, HIV, STD,...) There is a clear hypothesis about role of lactobacilli (<> intestine?): The protective role of vaginal lactobacilli is clearly established. Several mechanisms of protective activity have been proposed and can serve as selection criteria for probiotic strains. Lactobacilli are predominant in the vagina (<> intestine) Application can be topical (<> intestine) --> reaching high inocula Clinical trials are easy to perform (<> intestine): Sampling is easy during clinical trials. Re-isolation of probiotic lactobacilli after application is easy. Several products are already available.

35. Vaginal probiotic strain(s) Lactic acid Hydrogen peroxide Bacteriocins Arginine deaminase [Arginine] Nitric oxide (NO) Inflammation Polyamines trimethylamine BV organisms Exfoliation - Discharge - Malodor Adherence competition Persistent effect Well-adhering Resistant to bacteriophages bacteriocins Biosurfactant production L. jensenii only Characteristics of a vaginal probiotic preparation Coaggregation molecules

36. Les probiotiques vis à vis les antibiotiques: quelques remarques Probiotics vs antibiotics? Antibiotics (clindamycine more than metronidazole) damage commensal microflora Antibiotics can increase the occurrence of resistant bacteria Antibiotics can have adverse side effects (especially in pregnant women) Probiotics can be used in adjunction to antibiotics to restore the commensal microflora Antibiotics may be needed as initial therapy to increase the chance that probiotics can colonize the vagina Boskey et al. 2001: all vaginal products should be tested for absence of toxicity vs lactobacilli before being admitted.

37. History of characterization of vaginal microflora and vaginal probiotics 1892: Döderleins' bacilli: vaginal microflora consists of one kind of Gram-positive bacilli 1892: Lactic acid is primary molecule responsible for low vaginal pH (Döderlein 1892). 1897: Glycogen is direct precursor of lactic acid in the vagina 1960: First clinical trial with vaginal probiotic bacteria? (Eschbach, W., Kludas, M. 1960). 1969: Wylie et al.: L. acidophilus 1980. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser. Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30:53–68. Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30:53–68. 1983. Cato, E. P., W. E. C. Moore, and J. L. Johnson. Synonymy of strains of “Lactobacillus acidophilus” group A2 (Johnson et al. 1980) with the type strain of “Lactobacillus acidophilus” group A2 (Johnson et al. 1980) with the type strain of Lactobacillus crispatus (Brygoo and Aladame 1953) Moore and Holdeman 1970. Lactobacillus crispatus (Brygoo and Aladame 1953) Moore and Holdeman 1970. Int. J. Syst. Bacteriol. 33: 426-428. Int. J. Syst. Bacteriol. 33: 426-428. 1987: Giorgi et al.: L. crispatus, L. gasseri, L. jensenii (L. delbrueckii group) 1995: Andreu et al.: 10 7 -10 8 cfu of lactobacilli/ml vaginal fluid 1999: Antonio et al.: Lactobacillus 1086V = L. iners (no growth on Man Rogosa Sharpe)

38. Clinical trials with vaginal probiotics Eschbach, W., Kludas, M. 1957. Über die Fortdauer einer Besiedlung der menschlichen Scheide mit lebenden, lyophilisierten Döderleinbakterien auf Wattetampons. Ärtzliche Wochenschrift 12: 739-742. Eschbach, W., Kludas, M. 1960. On the maintenance and restoration of ideal vaginal states in sexually mature women by Doederlein bacteria. Med. Klin. 55: 1114-1116. Mohler, R., and C. Brown. 1933. Döderlein's bacillus in the treatment of vaginitis. Am J Obstet Gynecol 25:718-723.