1. Mario Vaneechoutte Mario.Vaneechoutte@UGent.be Laboratory Bacteriology Research (LBR) Faculty Medicine & Health Sciences University of Ghent Flanders, Belgium The similarities between the vaginal microflora and the gut microflora Mid-Atlantic Conference on Birth and Primal Health Research Honolulu 26-28 October 2012

2. Vagina (healthy condition):  Only one or two species of lactobacilli! vaginal epithelial cell lactobacilli Abundance and diversity of microbes on the human body but vaginal econiche is dominated by only few species 1. Why study vaginal microflora (VMF)?? 2. Why study possible correspondence of VMF and gut microflora?? Answer to Q1: Normal vaginal microflora is protective for mother and newborn Gut, skin, environment: Complex mixtures of bacteria

3. Why study the vaginal microflora?: Protective role of normal vaginal microflora Most lactic acid in vagina is of bacterial origin: Boskey et al. 2001, Human Repr. 16: 1809-1813. Boskey et al. 2001, Human Repr. 16: 1809-1813. Low vaginal pH: < 4.5

4. Co-evolution and symbiosis between humans and vaginal lactobacilli? Menarche: fertility period of women  oestrogen production starts at puberty  glycogen increases in vaginal epithelium  protection of fetus and newborn  inhibition of viral and bacterial pathogens  vaginal pH decrease: < 4.5  lactic acid production by lactobacilli selective stimulation of / advantage for metabolism of lactobacilli Why study the vaginal microflora?: Protective role of normal vaginal microflora Not in rats, mice, cattle, horse, monkey!!!: neutral pH, few lactobacilli

5. Why study the vaginal microflora?: Protective role of normal vaginal microflora But frequent disturbance: bacterial vaginosis (BV)= vaginal dysbiosis 10 9 10 2

6. Bacterial vaginosis (vaginal dysbiosis) Clue cell pH = 5-6 Atopobium vaginae - Gardnerella vaginalis Bacteroides Mobiluncus Mycoplasma Prevotella  Normal VMF pH < 4.5 Lactobacillus crispatus Why study the vaginal microflora?: Protective role of normal vaginal microflora But frequent disturbance: bacterial vaginosis

7. Prevalence 5-15% of women worldwide > 50% of women in equatorial Africa and of Afro-Americans Predominantly in sexually active women: sexually ‘enhanced’ disease Symptoms (only in half of the women with BV) Vaginal discharge – desquamation of vaginal epithelium Malodorious Prevalence and Symptoms of BV

8. Sequelae of BV: Infectious problems of the female urogenital tract, shown to be related to vaginal dysbiosis (BV) * BV: Gardnerella vaginalis, Atopobium vaginae, anaerobes, (Mycoplasma hominis, Ureaplasma urealyticum, Prevotella bivia) * Yeast vaginitis: Candida albicans * Group B streptococci: Streptococcus agalactiae neonatal meningitis * STD: pathogens from partner Trichomonas vaginalis Chlamydia trachomatis: da Silva. 2004. GOInvest. 58: 189 Mycoplasma genitalium Neisseria gonorrhoeae Treponema pallidum: syphilis Human Papilloma Virus  cervix carcinoma: da Silva. 2004. GOInvest. 58: 189 Herpes Simplex Virus 2: Cherpes et al. 2005. CID 40: 1422 HIV!!! * UTI: pathogens from intestine/skin?/environment? 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

9. BV 1. increases susceptibility for HIV-infection 2. increases HIV shedding: Cu-Uvin. 2004. CID 33: 894 Sewankambo. 1997. Lancet 350: 546  increases sexual HIV-transmission  increases perinatal mother-child HIV-transmission: Taha & Gray. 2000. Genital tract infections and perinatal transmission of HIV. Ann N Y Acad Sci. 918: 84-98. Sequelae of BV: increased HIV susceptibility and transmission

10. Gupta et al. 1998. J Infect Dis 178: 446-450. Inverse association of H 2 O 2 -producing lactobacilli and vaginal E. coli colonization in women with recurrent urinary tract infections. BV increases risk for UTI (Hooton. 2001. IJAA 17: 259-268) UTI: *300 million - 1 billion cases annually (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 (increasingly resistant to antibiotics): intestinal origin: E. coli (approx. 70%), Enterobacteriacae, Enterococcus faecalis, Staphylococcus spp. * Sequelae of UTI: preterm birth kidney infection (pyelonephritis)  preterm birth * Women are 5 times more susceptible than men Brain Sequelae of BV: increased susceptibility for Urinary Tract Infection (UTI)

11. Amnion Vagina Cervix Sequelae of BV: APO: adverse pregnancy outcome PTB: Preterm birth Goldenberg et al. 2000. Intrauterine infection and preterm delivery. New Engl J Med 342: 1500-1507. http://www.cdc.gov/std/bv Most importantly: 40-50% of PTBs are caused by BV! Anaerobes degrade mucus plug with mucinases?

12. PTB (= birth < 37 weeks) causes 75% of neonatal mortality 90% of very preterms have lifelong sequels: motoric handicaps, retardation, deafness Cost: 0.5-1 million USD/ child born between 26 and 37 weeks Belgium: 7-8% of births are preterm PTB is increasing worldwide Sequelae of BV: APO: adverse pregnancy outcome Preterm birth (PTB)

13. PTB SI Drugs Smoking Vaginal douching UTI STD/ Chlamydia Gonorrhoe BV Previous PTB SED Advanced Maternal age Multiple pregnancy Direct and indirect links between vaginal dysbiosis (BV) and STD in APO & PTB Importance of BV for PTB may be underestimated Genetic predisposition? Wrong lactobacilli? 40% Pyelo- nephritis

14. BV: Vaginal dysbiosis: Summary Prevalence: 5-15% of women worldwide Predominantly sexually active women Symptoms: Half of the women: disturbing symptoms 80% of women with BV: recurrent - chronic Sequelae: Increases Pelvic Inflammatory Disease (PID)  infertility, ectopic pregnancy Increases susceptibility to UTI and STDs (HIV) Increases (mother-child) transmission of HIV Direct cause of 40% of PTBs + indirect cause (via UTI, STD)?

15. Normal VMF: what is the source of the protective lactobacilli? STD: pathogens come from partner UTI: pathogens come from intestine/skin?/environment? Lactobacilli? Endogenous to vagina? Intestine? Anaerobes in BV? Partner, intestine, vagina, environment? 1. Why study vaginal microflora (VMF)?? 2. Why study possible correspondence of VMF and gut microflora?? Vaginal dysbiosis (BV): what is the source of? Atopobium vaginae Gardnerella vaginalis Bacteroides Mobiluncus Mycoplasma Prevotella Answer to Q2:

16. What causes dysbiosis of the VMF? HYPOTHESIS: Lactobacilli cause acidification of the vaginal econiche: pH 4.5  inhibition of growth of anaerobes  monopolisation, sole colonisation by lactobacilli But: Regular disturbances – alkalinisations occur: menses: nutrient rich fluid with neutral pH sexual intercourse  sexually enhanced disease (SED): vaginal douching: increasing pH, disturbing the epithelium, the lactobacilli antibiotics: killing the lactobacilli In case lactobacilli do not succeed in acidifying the vaginal econiche rapidly after each disturbance  lactobacilli loose the plot  overgrowth by anaerobic bacteria: dysbiosis  BV. Question 2: What is the origin of these BV bacteria? vagina, partner, intestine, other?

17. Answering Q1: Characterization of the VMF with culture-based as well as molecular techniques

18. Sexual intercourse:  alkalinisation of vaginal econiche by semen  wet  overflow of rectal microflora Frequent SI  lactobacilli fail to acidify the vaginal econiche BV prevalent in women becoming sexually active having several partners having a new partner

19. Lactobacilli losing the plot? Follow up study of vaginal microflora during 2 menstrual cycles 17 volunteers, age 20-35, self sampled the vagina daily during two menstrual cycles = 50-60 samples per volunteer Microscopy of the vaginal smears Culture and identification of the bacteria Quantitative PCR of the vaginal swabs

20. Subject #5, with predominant normal VMF: dysbiosis during menses Log cells / ml Lactobacilli loosing the plot? Follow up study of vaginal microflora during 2 menstrual cycles 98765439876543 Week: 1 2 3 4 5 6 7 8 9

21. Subject #9, with predominantly BV microflora Week: 1 2 3 4 5 6 7 8 9 11 10 9 8 7 6 5 4 3 Log cells / ml Lactobacilli loosing the plot? Follow up study of vaginal microflora during 2 menstrual cycles

22. Effect van antimicrobiële behandeling Lactobacilli loosing the plot? Follow up study of vaginal microflora during 2 menstrual cycles 11 10 9 8 7 6 5 4 3 Log cells / ml Week: 1 2 3 4 5 6 7 8 9 Subject #2, taking antimicrobials at the start of the study period

23. 23 Answering Q2: Comparison of the vaginal and rectal (intestinal) microflora in pregnant women

24. Belgium: routine screening of pregnant women for the presence of GBS Aim: prevention of neonatal GBS meningitis  GBS positive women are treated with penicillin Method: Vaginorectal swabbing at 35-37 weeks of gestation Culture for the presence of GBS Our studies: We asked to also swab separately vagina and rectum: Vaginorectal swab Vaginal swab Rectal swab Comparison of vaginal and intestinal microflora: Methods: study set up

25. Culture Vaginal swab Comparison of vaginal and intestinal microflora: Methods: study set up 132 pregnant women Rectal swab Isolate 4 colonies from most abundant colony types V1, V2, V3, V4 I. Identify species Isolate 4 colonies from most abundant colony types R1, R2, R3, R4 I. Identify species Culture III. qPCR Same species present in both R and V? II. Genotype strains of same species: same strain (lineage of descent) in both R and V? YES

26. L. crispatus L. jensenii L. gasseri L. iners Baele, M., M. Vaneechoutte, R. Verhelst, M. Vancanneyt, L. A. Devriese, and F. Haesebrouck. 2002. Identification of Lactobacillus species using tDNA-PCR. J. Microbiol. Methods 50: 263-271. Comparison of vaginal and intestinal microflora: I. Species identification of cultured bacterial colonies Method: Molecular identification by tDNA-PCR tDNA-PCR fingerprinting of cultured bacteria: species specific patterns are obtained 145, 158, 180 bp 158, 180, 228 bp

27. 132 women with each 4 vaginal and 4 rectal isolates = 1056 isolates 844 isolates could be identified with tDNA-PCR-fingerprinting 1.A total of 63 different bacterial species were found 2.Of these 63: 28 species (44%) were found both in vagina and rectum Most other species only in rectum 1.47 women (36%) carried same species in both rectum and vagina 2.3 women even carried two species in common in both in rectum and vagina For the 50 cases where the same species was found simultaneously both in rectum and vagina in the same woman: is it also the same strain?  Strain genotyping (RAPD-fingerprinting) Comparison of vaginal and intestinal microflora: I. Species identification of cultured bacterial colonies

28. Rectal A A A A Vaginal Genotyping (RAPD) results for subject RVS 90: 2 rectal and 2 vaginal isolates of Lactobacillus gasseri 28 Comparison of vaginal and intestinal microflora: II. Strain genotyping of cultured bacterial colonies of the same species in the same subject

29. 29 B B B B B B Genotyping (RAPD) results for subject RVS 86: 4 rectal and 2 vaginal isolates of Enterococcus faecalis Rectal Vaginal Comparison of vaginal and intestinal microflora: II. Strain genotyping of cultured bacterial colonies of the same species in the same subject

30. 30 F E D Rectal Vaginal F F Genotyping (RAPD) results for subject RVS 31: 3 rectal and 2 vaginal isolates of group B Streptococcus Comparison of vaginal and intestinal microflora: II. Strain genotyping of cultured bacterial colonies of the same species in the same subject

31. V1 V2 V3 V4 R1 R2 R3 R4 Comparison of vaginal and intestinal microflora: Results: species identification and strain genotyping of cultured bacterial colonies Genotyping results for 50 cases in which the same species could be isolated from the same subject vaginally and rectally Comparison of vaginal and intestinal microflora: Overview Results

32. 132 women from which each 4 vaginal and 4 rectal isolates = 1056 isolates 844 identified For the 50 cases where the same species was found  Strain genotyping: For most species: different strains per woman Despite this diversity: for 34 of the 50 species pairs (68%), at least one vaginal isolate and one rectal isolate are identical Comparison of vaginal and intestinal microflora: Results: species identification and strain genotyping of cultured bacterial colonies

33. Culture Vaginal swab Comparison of vaginal and intestinal microflora: III. Quantification by qPCR Pregnant women Rectal swab Culture qPCR I. Is the same species present simultaneously in rectum and vagina? II. If same species is present: Is it also the same strain? DNA extraction III. If same species is present in both vagina and rectum: Are the quantities in vagina and rectum comparable? DNA extraction

34. Real-time PCR or quantitative PCR: qPCR qPCR assays were developed to quantify the DNA present in V and R samples for 7 target bacterial species: Normal VMF Lactobacillus crispatus Lactobacillus jensenii Normal VMF and known to be intestinal: Lactobacillus gasseri BV associated Atopobium vaginae Gardnerella vaginalis BV associated? Lactobacillus iners Neonatal meningitis Streptococcus agalactiae (group B streptococci) Comparison of vaginal and intestinal microflora: III. Quantification by qPCR

35. Principle of quantification of DNA by means of qPCR: PCR: Exponential amplification of DNA of target bacterial species: doubling of initially present DNA after every cycle of 2 min. 1 hour = 30 cycles: 1 copy  2 30 = 10 9 copies With each doubling of DNA  doubling of fluorescent dye bound to dsDNA Fluorescence is monitored after each cycle The instrument can detect fluorescence above a certain threshold The more bacterial cells initially present = the more DNA initially present  the faster the threshold is crossed. Comparison with threshold crossing of dilution series with known concentration of bacterial cells: quantification Comparison of vaginal and intestinal microflora: III. Quantification by qPCR

36. Comparison of vaginal and intestinal microflora: III. Quantification by qPCR Threshold Number of bacteria initially present in the clinical sample: 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 cells/sample

37. Quantification of Lactobacillus crispatus from rectum and vagina Each dot = quantity in R and V for one female Number of cells in vagina (log10) Comparison of vaginal and intestinal microflora: III. Quantification by qPCR Number of cells in rectum (log10)

38. L. crispatusL. jenseniiL. gasseri L. inersG. vaginalisA. vaginae Comparison of vaginal and intestinal microflora: III. Quantification by qPCR

39. Comparison of vaginal and intestinal microflora: III. Quantification by qPCR 1. For at least 36% of the women, the same species was present simultaneously in vagina and rectum 2. For those women where the same species was present, also the same strain was present simultaneously in vagina and rectum in at least 68% of the cases 3. The number of bacterial cells corresponds significantly between vagina and rectum for 6/7 species tested

40. A small plastic ruler was used to measure the anovaginal distance = anatomic distance from the posterior fourchette to the anus with the participant in the lithotomy position. The mean anovaginal distance was 3.22 cm (range 1.8–5.2) for controls 3.37 cm (range: 1.8–5.7) for cases of BV. There was no correlation … Comparison of vaginal and intestinal microflora: Other studies

41. 2012. J. Infect. Dis. 205: 1580-1588. Rectal colonisation with G. vaginalis and L. crispatus in women with BV and women with normal VMF Gardnerella vaginalis Lactobacillus crispatus BV Normal BV Normal Number of Cells in rectum (log10) 10 8 10 7 10 6 10 5 10 4 10 3 10 7 10 6 10 5 10 4 10 3 Conclusions: Women with normal VMF have more L. crispatus in the rectum Women with BV have more G. vaginalis in the rectum Comparison of vaginal and intestinal microflora: Other studies

42. Conclusion The close proximity of the rectum to the vagina The substantial correspondence between vaginal and rectal bacterial species and strains The correspondence in bacterial loads of species present in vagina and rectum  the intestine may play a role as a reservoir for vaginal microorganisms

43. What is the source of the intestinal bacteria of the neonate? Environment? Other people? Mother? Gut? Vagina? Skin? Correspondence between vaginal and intestinal microflora in (pregnant) women: intestinal microflora (GMF) is source of vaginal microflora (VMF)  is maternal GMF and VMF also source of neonatal intestinal microflora during birth? If so  Birth mode for initial seeding of the GMF may be important: vaginal delivery vs C-section delivery Does the vaginal microflora of the mother play a role in initial colonisation of the intestine of the newborn?

44. Evidence for the role of maternal (vaginal) microflora in initial colonisation of the newborn M oral M vagina B Caesarean M skin B Vaginal

45. The importance of gut microflora for our general health is being increasingly recognized: Science 2012: Special Section Gut Microbiota. Science 336: 1245-1272. Science 26 november 2010, p. 1168, 1 april 2011: p. 32 Number of human cells per body: 10 13 Number of bacterial cells inhabiting per human body: 10 14 Our gene number (= genes of one eukaryotic species): 20 000 Bacterial species: 1000-2000 in the gut Bacterial genes: 2000 per genome  1000 species x 2000 genes = 2 000 000 bacterial genes/alleles in the gut Gut microflora is now considered as a separate, flexible organ, offering additional genetic possibilities to the animal individual and with which the animal must co-habit (Putative) Roles of gut microbes for human health The importance of our gut microflora for our health 1. metabolism: Diabetes Obesitas/Adiposity Cancer 2. immune homeostasis: Inflammatory Bowel Diseases (IBD) Hygiene hypothesis  Allergies – Asthma Psoriasis? 3. mental health?:Autism? Schizophrenia?

46. 1. Roles of gut microbes in metabolism (& adiposity)

48. De Theije et al. 2011. Eur J Pharmacol 668 Suppl 1:S70-80. 2. Roles of gut microbes in behaviour/mental health?

49. 3. Roles of gut microbes in development of immune homeostasis Studies in mice

50. Olszak et al. 2012. Microbial exposure during early life has persistent effects on natural Killer T cell function. Science 336: 489-493. Studies in germ free mice (born with C-section, raised in sterile environment) Absence of bacteria at birth causes lifelong increased inflammation increase of Natural Killer Cells (NKCs) Germ free mice, exposed to bacteria as adults: inflammation stays Germ free mice, exposed to bacteria on day 1: no increase in NKCs no increased risk for Ulcerative Colitis  Only early (= first weeks) exposure to bacteria restores immune homeostasis 3. Roles of gut microbes in development of immune homeostasis

51. Influence of birth mode on asthma?

52. Final conclusions Strong correspondence between maternal vaginal and intestinal microflora Strong evidence that delivery mode influences GMF of newborn Indications for the importance of maternal VMF for normal development of newborn GMF  for immune homeostasis and balanced metabolism FIRST WEEKS ARE MOST IMPORTANT for normal immunity and metabolism! Time window …. Primal health determines further life quality

53. With many thanks to my team

54. Mario Vaneechoutte Mario.Vaneechoutte@UGent.be Laboratory Bacteriology Research (LBR) Faculty Medicine & Health Sciences University of Ghent Flanders, Belgium The similarities between the vaginal microflora and the gut microflora Mid-Atlantic Conference on Birth and Primal Health Research Honolulu 26-28 October 2012 Thanks for your attention

55. But: Besides changing mode of delivery many other factors may play a role in the increase of allergy and inflammatory diseases: Breast feeding vs formula feeding Allergens UP  Allergy UP Hygiene UP  Th1/Th2 balance disturbed  Allergy UP Infection altered: helminths DOWN  Th1/TH2 balance disturbed  allergy UP Number of siblings DOWN  Allergy UP Palaeolithic diet changed: Consumption of grains and milk UP  Influence on GMF, on allergy?