Microbiome and Vaginal Microbiome
Human as a "supraorganism," composed of both human and non-human cells We have about 10 times as many microbial cells as human cells you are having about 200g of non-human cells
The microbiome entire habitat including microorganisms (composed of bacteria, bacteriophage, fungi, protozoa and viruses) their genomes and environment
Human microbiome projects and initiatives • Trying to generate new knowledge and evidence base – NIH, Human microbiome project (HMP), US DHHS (2008-2012) – International human microbiome consortium (IHMC) – International Human Microbiome Congress – Human Metagenomics international conference – Vaginal human microbiome project (VaHMC) funded by NIH – Vaginal microbiome consortium (VMC)
Site specific terms nasal passage microbiome oral cavity microbiome skin microbiome gastrointestinal tract microbiome urogenital tract microbiome vaginal microbiome
Early stages or establishment of Microbiome • “The womb is a sterile environment,” explains Maskell, but as soon as you’re out, your microbiome starts taking shape. • You acquire your first microbes coming through the vaginal canal, and breast milk. • So, if you’re a C-section baby or formula fed baby, you’re slightly behind from the get-go. • Later, antibiotics play a huge role: While they’re good at destroying the bad guys, they kill indiscriminately, taking out innocent bystanders, the microbes who just want to help, along the way.
Probiotics & Prebiotics Probiotics are live microbes that can be formulated into many different types of products, including foods, drugs, and dietary supplements (Species of Lactobacillus and Bifidobacterium are most commonly used as probiotics) Prebiotic is a specialized plant fiber that beneficially nourishes the good bacteria already in the large bowel or colon. Synbiotics contain prebiotics and probiotics in the same preparation.
Vaginal Microbiome
Throughout a woman's life, the vaginal microbiome undergoes dramatic shifts that coincide with hormonal and lifestyle changes. The complexity of these communities and the genetic and environmental factors that influence these changes have yet to be fully described.
History of studies of vaginal microbiome on culture and microscopy • 1892- Doderlein isolated Doderlein bacillus from a specimen of normal pregnant women which was later renamed Lactobacillus • End 19th century – Menge and Kronig first described the isolation of anaerobic organisms in addition to Lactobacillus from the vagina. • 1923 – Curtis described a vaginal discharge syndrome in women that he termed “white discharge syndrome” using culture techniques he related this syndrome with – – – –
black-pigmented anaerobes curved anaerobic motile rods anaerobic cocci and Gram-variable diphtheroidal rods. He also noted a relative dearth of Doderlein’s bacillus in women with this syndrome
OVER 90 YEARS It was known that significant shift in the vaginal microbiome in women were associated with a symptomatic vaginal discharge syndrome (name then used Curtis’s white-discharge syndrome)
many investigators interested in women’s health continued to believe that this Symptomatic vaginal discharge syndrome must in some way be caused by a single organism.
After the work of Gardner and Duke 1955 Based on their work, clinicians and investigators over the next 25 years ďƒ used Gardnerella vaginitis for this discharge, reflecting the general belief that this single organism was the cause.
Concept of shift in the vaginal microbiome seminal work of the University of Washington group of investigators redirected the attention This nonspecific vaginitis was associated with dramatic shift in the vaginal microbiome Condition was renamed “bacterial vaginitis” and later changed to “bacterial vaginosis” (BV) due to the fact that few inflammatory cells were observed microscopically in the vaginal fluid
Change in the vaginal ecosystem Or shift in the vaginal microbiome
Results a vaginal fluid with low number of inflammatory cells BACTERIAL VAGINAOSIS
1. Asymptomatic 2. Symptomatic discharge with no self reporting 3. Symptomatic discharge with self reporting
Amsel defined BV Nugent defined BV
Amsel criteria defined BV (Based on clinical criteria)
– 1. vaginal secretion with a pH level of >4.5 – 2. a fishy odor that was best elicited by mixing vaginal secretions with 10% KOH solution – 3. a rate of at least 20 percent of microscopically observed vaginal epithelial cells coated with bacteria (these cells were named “clue cells”) – 4. a typically white, skim-milk-like vaginal discharge
• In order for a clinical diagnosis of BV to be made, at least three of the four markers needed to be present
Nugent score defined BV
(Based on Gram stain evaluation of vaginal smear) • Proposed a numerical score (Nugent Score) based on the evaluation of morphology and Gram stain reactivity of vaginal secretions with semiquantization of – Gram-positive bacilli/rods (Lactobacillus spp) – Purpule/Violet – Gram-negative coccobacilli forms (G.vaginalis) and – Gram-negative curved bacilli/rods (Mobiluncus spp).
Nugent Interpret score ation
morphology and Gram stain reactivity
Gram stain reactivity of bacteria
Gram-positive rods dominant (Lactobacillus spp)
Predominantly Violet
Mixed morphotypes (Gram positive rods,
Mixed purple and Red (safranin)
Gram negative coccobacilli and Gram
Red (Safranin red)
0-3
Normal
4-6
Interme Gram negative coccobacilli, Gram negative diate curved rods)
7-10
Bacterial negative curved rods predominates with the vaginosis absence of Gram positive rods
Nugent score used for clinical research rather than routine clinical care • Because of the time it takes to read the slides and the requirement for specially trained microscopists.
Nugent score defined BV
(Gram stain evaluation of vaginal smears)
associated with abnormal pregnancy outcomes
• • • • •
Preterm birth Premature rupture of membranes Early labor Postpartum endometritis However, efforts to prevent these complications by treatment have not uniformly met with success. There is evidence that treatment of BV in women who have had a previous preterm delivery decreases the probability of a subsequent preterm birth
Sensitivity and Specificity of the two method • Nugent score compared with Amsel criteria – Sensitivity 89% – Specificity 83%
• Amsel criteria when compared with Nugent score – Sensitivity 70% – Specificity 94% Schwebke JR, Hillier SL, Sobel JD, McGregor JA, Sweet RL. Validity of the vaginal gram stain for the diagnosis of bacterial vaginosis. Obstet Gynecol. 1996;88(4 pt 1):573–576.
Factors associated with BV Behavioural • New sexual partner • Vaginal douching • Receptive oral sex • Lack of condom use • Menstruation • Multiple sexual partners • Early sexual debut • Concordance of BV status in monogamous lesbian couples range up to 95% (main suggesting fact that BV is sexually transmitted. • Increased rate of acquisition of STIs including HIV
Microbiological • Polymicrobial condition • Caused by shift in vaginal microbime/ microbiota • (Microscopy and conventional culture methods, Molecular techniques (16s r RNA))
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Biochemical • High pH (reduced lactic acids – produced by Lactobacillus • Low Bacteriocins (due to low lactobacili) • Low Hydrogen Peroxide (due to low lactobacili) • Production of sialidases, prolidases, Proteases (produced by BV associated bacteria)
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Molecular
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We do not know • How protective vaginal microbiota are maintained? • Why frequent shift in microbial composition occur? • How these shift induce changes in the vaginal environment that relate to adverse health outcomes? • How vaginal ecosystem restrict the growth of nonindigenous organisms?
We need to advance our understanding of the microbiological, biochemical, molecular and behavioural contributors to the aetiology of BV
Microbiome and Microbiota • Earlier these two words used synonymously • Microbiome - entire habitat including microorganisms (composed of bacteria, bacteriophage, fungi, protozoa and viruses) their genomes and environment • The term “microbiota” will be used primarily to refer to human site-specific bacterial communities as determined by 16sRNA sequencing studies.
Concept of Microbiome, Microbiota, Metagenome Microbiome
Conventional methods culture and microscopy
Microbiome
(entire habitat including microorganisms their genomes and environment) Microbiota
Microbiota
Metagenome (collection of genomes from the members of a microbiota
Bacterial census established by using molecular methods relying on the analysis of 16rRNA gene sequence amplified from a given environment
How to identify unknown microorganisms on a location by Molecular microbiologic investigations •
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Ribosomal RNA detection (16S rRNA)- is the most conserved (least variable) DNA in all cells. Proportions of the rDNA sequence from distantly related organisms are remarkably similar. For this reason, genes that encode the rRNA (rDNA) have been used extensively to determine taxonomy, phylogeny (evolutionary relationships) and to estimate rates of species divergence among bacteria Powerful tool to reveal the phylogenic diversity of the microorganisms found within the vaginal ecosystem
Taxonomic scale (systematic classification) Kingdom Phylum Class Order Family Genus Species
• The highest taxon in the taxonomy of organisms is a Kingdom and lowest a species (Singular-taxon, Pleural-taxa)
• Measurement of biodiversity – taxonomic richness of an area • BV is associated with increased taxonomic richness and diversity • The microbiota composition is highly variable among subjects at a fine taxonomic scale (genus or species level)
BV is associated with a shift in the relative abundance of various species of Lactobacillus to greater abundance of strictly anaerobic bacteria Traditional culture and microscopy based methods
16S rRNA gene sequenced-based analysis
• Lactobacillus spp Anaerobic bacteria • Gardnerella vaginalis • Prevotella spp • Mobiluncus spp • Ureaplasma urealyticum • Mycoplasma hominis
• • • •
Atophobium vaginae Megasphaera Eggerthella Clostridiales (3 new members)
Lactobacillus spp Over 20 species of Lactobacillus
L.crispatus, L.iners, L.jensenii and L.gasseri
However, majority of healthy vagina contains one or two Lactobacillus spp from this four species
Over 20 species of Lactobacillus have been detected in the vaginaďƒ However, majority of healthy vagina contains one or two Lactobacillus spp from a range of three or four spp mainly L.crispatus, L.iners, L.jensenii and L.gasseri
A diverse array of other vaginal bacteria • Staphylococcus, • Ureaplasma, Corynebacterium, • Strepto coccus , • Pept o s treptococcus, • Gardnerella , • Prevotella,
• • • • • • •
Clostridium, Bacteroides, Mycoplasma, Enterococcus, Escherichia, Veillonella, Bifidobacterium
Issues • Lactobacillus spp were not the dominant species in 27% of women (Ravel et al) • Half of women (50%) with high Nugent scores are asymptomatic (Klebanoff et al) • The term BV, based on Amsel criteria or Nugent scores dos not necessarily distingush a women’s self-reported symptomatic or asymptomatic status. • The factors leading to and the adverse outcomes resulting from symptomatic BV and asymptomatic BV are not fully understood and are an active area of research
Issues • Healthy vaginal communities are always colonized with high number of lactobacilli. This fact is disputed by Forney et al.ďƒ communities that lack lactobacillus sp. May have high number of other lactic acid-producing bacteria such as Atopobium spp, Megasphaera spp or Leptotrichia spp. • Are all women in the low-lactobacillus state with diverse microbiota, at risk for reproductive track sequelae? Or are there other factors, specific communities of bacteria or host response that elevate the risk
Conclusion • Healthy vaginal microbiota is important for maintaining vaginal health and preventing infections • The complexities of the vaginal microbiota has begun to emerge based on the techniques of molecular microbiology • Many previously unknown bacteria are important components of the vaginal microbiota. • It is more and more apparent that none of these organisms alone is likely to be of great significance. • We need to advance our understanding of the microbiological, biochemical, molecular and behavioural contributors to the aetiology of BV • Healthy vaginal flora not only protects a women from the acquisition of STIs, but also exert a beneficial effect on her partner and sibling.