mario vaneechoutte [email protected] laboratory bacteriology research (lbr)

Mario [email protected]

Laboratory Bacteriology Research (LBR)Faculty Medicine & Health Sciences

University of GhentFlanders, Belgium

The similarities between the vaginal microflora and the gut microflora

Mid-Atlantic Conference on Birth and Primal Health ResearchHonolulu 26-28 October 2012

Vagina (healthy condition): Only one or two species of lactobacilli!

vaginal epithelial cell lactobacilli

Abundance and diversity of microbes on the human bodybut 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

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.

Low vaginal

pH: < 4.5

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

Why study the vaginal microflora?:Protective role of normal vaginal microfloraBut frequent disturbance: bacterial vaginosis (BV)= vaginal dysbiosis

109

102

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 microfloraBut frequent disturbance: bacterial vaginosis

Prevalence5-15% of women worldwide> 50% of women in equatorial Africa and of Afro-AmericansPredominantly in sexually active women: sexually ‘enhanced’ disease

Symptoms (only in half of the women with BV)Vaginal discharge – desquamation of vaginal epitheliumMalodorious

Prevalence and Symptoms of BV

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

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

Gupta et al. 1998. J Infect Dis 178: 446-450.Inverse association of H2O2-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)

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?

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)

PTB

SI

DrugsSmoking

Vaginal douching UTI

STD/ChlamydiaGonorrhoe

BV

Previous PTB

SED

AdvancedMaternal 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

BV: Vaginal dysbiosis: Summary

Prevalence:5-15% of women worldwidePredominantly sexually active women

Symptoms:Half of the women: disturbing symptoms80% of women with BV: recurrent - chronic

Sequelae:Increases Pelvic Inflammatory Disease (PID) infertility, ectopic pregnancyIncreases susceptibility to UTI and STDs (HIV)Increases (mother-child) transmission of HIVDirect cause of 40% of PTBs + indirect cause (via UTI, STD)?

Normal VMF: what is the source of the protective lactobacilli?

STD: pathogens come from partnerUTI: 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 vaginalisBacteroides Mobiluncus Mycoplasma Prevotella

Answer to Q2:

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?

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

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

Lactobacilli losing the plot? Follow up study of vaginal microflora during 2 menstrual cycles

17 volunteers, age 20-35, self sampled the vagina dailyduring two menstrual cycles

= 50-60 samples per volunteer

Microscopy of the vaginal smearsCulture and identification of the bacteriaQuantitative PCR of the vaginal swabs

Subject #5, with predominant normal VMF: dysbiosis during mensesLog cells / ml

Lactobacilli loosing the plot? Follow up study of vaginal microflora during 2 menstrual cycles

9

8

7

6

5

4

3

Week: 1 2 3 4 5 6 7 8 9

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

Effect van antimicrobiële behandelingLactobacilli 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

Answering Q2: Comparison of the vaginal and rectal (intestinal) microflora

in pregnant women

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 gestationCulture for the presence of GBS

Our studies:We asked to also swab separately vagina and rectum:

Vaginorectal swabVaginal swabRectal swab

Comparison of vaginal and intestinal microflora:Methods: study set up

Culture

Vaginal swab

Comparison of vaginal and intestinal microflora:Methods: study set up132 pregnant women

Rectal swab

Isolate 4 colonies frommost abundant colony types

V1, V2, V3, V4

I. Identify species

Isolate 4 colonies frommost abundant colony types

R1, R2, R3, R4

I. Identify species

Culture

III. qPCR 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

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-PCRtDNA-PCR fingerprinting of cultured bacteria: species specific patterns are obtained

145, 158, 180 bp

158, 180, 228 bp

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 found2. Of these 63: 28 species (44%) were found both in vagina and rectum

Most other species only in rectum3. 47 women (36%) carried same species in both rectum and vagina4. 3 women even carried two species in common in both in rectum and vagina

For the 50 cases where the same species was found simultaneouslyboth 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

Rectal

Vaginal

Vaginal

Genotyping (RAPD) results for subject RVS 90:2 rectal and 2 vaginal isolates of Lactobacillus gasseri

Comparison of vaginal and intestinal microflora:II. Strain genotyping of cultured bacterial colonies

of the same species in the same subject

29

B

B

B

B

B

B

Genotyping (RAPD) results for subject RVS 86:4 rectal and 2 vaginal isolates of Enterococcus faecalis

Rectal

Rectal

Rectal

Rectal

Vaginal

Vaginal



30

F

E

D

Rectal

Rectal

Rectal

Vaginal

Vaginal F

F

Genotyping (RAPD) results for subject RVS 31:3 rectal and 2 vaginal isolates of group B Streptococcus



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

132 women from which each 4 vaginal and 4 rectal isolates = 1056 isolates844 identified

For the 50 cases where the same species was foundStrain 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

Culture

Vaginal swab

Comparison of vaginal and intestinal microflora:III. Quantification by qPCR

Pregnant women

Rectal swab

Culture

qPCR qPCRI. 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

Real-time PCR or quantitative PCR: qPCR

qPCR assays were developed to quantify the DNA present in V and R samplesfor 7 target bacterial species:

Normal VMFLactobacillus crispatusLactobacillus jensenii

Normal VMF and known to be intestinal:Lactobacillus gasseri

BV associatedAtopobium vaginaeGardnerella vaginalis

BV associated?Lactobacillus iners

Neonatal meningitisStreptococcus agalactiae (group B streptococci)


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 230 = 109 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



Threshold

Number of bacteria initially present in the clinical sample: 109 108 107 106 105 104 103 102 cells/sample

Quantification of Lactobacillus crispatus from rectum and vaginaEach dot = quantity in R and V for one female

Number of cellsin vagina (log10)


Number of cellsin rectum (log10)

L. crispatus L. jensenii L. gasseri

L. iners G. vaginalis A. vaginae



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

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

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)

108

107

106

105

104

103

107

106

105

104

103

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

Conclusion

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

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?

Evidence for the role of maternal (vaginal) microflorain initial colonisation of the newborn

M oral

M vagina

B Caesarean

M skin

B Vaginal

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: 1013

Number of bacterial cells inhabiting per human body: 1014

Our gene number (= genes of one eukaryotic species): 20 000Bacterial species: 1000-2000 in the gutBacterial 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 individualand 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/AdiposityCancer

2. immune homeostasis: Inflammatory Bowel Diseases (IBD) Hygiene hypothesis Allergies – Asthma

Psoriasis?3. mental health?: Autism?

Schizophrenia?

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

De Theije et al. 2011. Eur J Pharmacol 668 Suppl 1:S70-80.

2. Roles of gut microbes in behaviour/mental health?

3. Roles of gut microbes in development of immune homeostasis

Studies in mice

Olszak et al. 2012. Microbial exposure during early life has persistent effects on naturalKiller 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 NKCsno 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

3. Roles of gut microbes in development of immune homeostasisInfluence of birth mode on asthma?

Final conclusionsStrong correspondence between maternal vaginal and intestinal microflora

Strong evidence that delivery mode influences GMF of newborn

Indications for the importance of maternal VMFfor 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

With many thanks to my team

Mario [email protected]

Laboratory Bacteriology Research (LBR)Faculty Medicine & Health Sciences

University of GhentFlanders, Belgium

The similarities between the vaginal microflora and the gut microflora

Mid-Atlantic Conference on Birth and Primal Health ResearchHonolulu 26-28 October 2012

Thanks for your attention

But: Besides changing mode of deliverymany 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?

mario vaneechoutte [email protected] laboratory bacteriology research (lbr)

Documents

vaginal lactobacilli

study vaginal microflora

vaginal dysbiosis bv

low vaginal ph

human bodybut vaginal

bacterial vaginosis

bacterial vaginosisprevalence5

bacterial origin