The vaginal microbiome: new information about genital tract flora using molecular based techniques

Normal flora vagina

Protection Mechanisms

predispose

colonisation

Bacterial vaginosis (BV) Variable symptoms Phenotypical outcomes Different responses to antibiotic

Sepsis Recurrent abortion Preterm birth.ect Upper genital tract Sexually transmitted infections Acquisition of HIV

Qualitative and semi-quantitative descriptive studies Superorganism (human genes plus the microorganisms) The core microbiome, as well as the variable microbiome Cultivation-independent techniques Taxonomy

The 16S rRNA : present all bacteria heterogeneity Universal/specific primers

Bacterial DNA extracted Amplified(PCR) Identification uncertainty

universal primers specific primers

specific organisms

Cultivation-based studies disadvantage

Lack of phenotypic tools Lower relative titers Inappropriate media

isolate or detect fastidious microorganisms

Cultivation-independent techniques

Limited - tendency to sample - prevalent bacteria combination

Normal vaginal flora

Lactic acid Hydrogen peroxide (H2O2) Antibiotic toxic hydroxyl Radicals Bacteriocins Probiotics 1892, Prof.Albert Dderlein source of lactic acid that could inhibit the growth of pathogens in vitro and in vivo Redondo-Lopez

1999 L. iners

Rogosa/MRS

two women were colonised by the blood agar same two Lactobacillus species

2002 L. iners in a woman with a normal Nugent score May be better adapted to the conditions associated with BV L. iners most frequently contrast to L.crispatus

healthy vaginal microflora does not contain high numbers of many different species of Lactobacillus

one or two lactobacilli from a range of three or four species

202 vaginal isolates 26 type 18 out of 23 women

5 women had 2 different species of the same species of Lactobacillus competitive exclusion superior ability L. iners and L. crispatus pre-emptive colonisation by a particular species Racial variation and geographical area have significant differences in what is the dominant vaginal organism

302 women

32 % 23%
5% 15%

L. crispatus L. jensenii
7% 9%

95 % 94%
H2O 2

H2 O 2

L. gasseri L. iners 9% 7%

L. crispatus L. jensenii

BV( -) BV( +)

16%

L. gasseri L. iners

43% 36%

L. gasseri

higher rectal colonisation

sexual practices

Culture-independent techniques Lack appreciable numbers of Lactobacillus species in the vagina, may be replaced by other lactic acid-producing bacteria (Atopobium vaginae, Megasphaera, and Leptotrichia species) Structure of the communities may differ between populations

Transition, asymptomatic BV,no symptoms,Genetic/other factors

Stanley Thomas in 1928 ,lactobacilli absent in presence gonococci L.fermentum and L.rhamnosi -poor results in urogenital infection

Abnormal vaginal flora may occur because of a sexually transmitted infection BV is a polymicrobial condition, characterised by a decrease in the quality or quantity of lactobacilli Healthy lactic acid-producing vaginal flora acts as a barrier against the acquisition of HIV bacteria function as mono-etiological agents require a change in the micro-environment

Gardner and Dukes

induced vaginal colonisation in 1 of 13 volunteers.In contrast, 11 of 15 volunteers (inoculated vaginal secretions of donors _

The genus Atopobium lies within the family Coriobacteriaceae, forms a distinct branch within the phylum Actinomycetes.Following sequence analysis, exhibit peptidyl peptidase activity, and produce significant quantities of ammonia(sugars) Prevotella bivia produces ammonia, substrate (G. vaginalis.)

Atopobium vaginae (anaerobic)very sensitive to clindamycin in vitro, but is highly resistant to nitroimidazoles
High diversity in women with BV compared with normal flora (the presence of novel bacterial species) Different subjects with BV have different microbial profiles, indicating heterogeneity

BV+ - Clinically, composite clinical criteria, microscopically, enzymatically, chromatographically, or using qualitative or semiquantative culture methods, the gold standard (Nugent score) Fluorescence in situ hybridization (FISH) technology has demonstrated that BV-associated bacterium 1 (BVAB1) has morphology similar to Mobiluncus Urea produced by Atopobium sp(Megasphaera (beer spoilage by turbidity, off-flavours and off-colours),malodorous metabolites can be found in the vagina of healthy women, and amines can be found in women without BV, diagnostic techniques to diagnose BV, based on upon amine production and odour formation,may need to be amended(Zhou)

morphology of Atopobium renders it well camouflaged AMONG other species present in bacterial communities (Nugent score is 4)
Atopobium vaginae is fastidious, grows anaerobically, and forms small pin-head colonies on cultures that are easily missed.

Although phylogenetically different from other lactic acidproducing bacteria, not phenotypically exceptional
The combination of an A. vaginae DNA level 108 copies/ml and a G. vaginalis DNA level 109 copies/ml demonstrated the best predictive criteria for the diagnosis of BV with excellent sensitivity (95%), specificity (99%), negative predictive value (NPV) (99%), and positive predictive value (PPV) (95%).

Cure of BV or improvement in symptoms (metronidazole or clindamycin)reaches 8394% by 721 days of treatment Whereas the short-term treatment response is acceptable, BV persists or recurs in 1129% of women at 1 month, 30% of patients relapse within 3 months, and recurrence rates may be more than 50% within a year. 48% of women colonised by H2O2-producing lactobacilli 70 90 days after treatment with either clindamycin or metronidazole
the problems of recurrent or persistent BV why some women with BV resistant to cure Stability of vaginal flora requiring few phylotypes, Lactobacillus dominance with L.crispatus and/or L.jensenii in remaining/become BVIn contrast, those who become BV+ colonised by L.iners, with many other non-Lactobacillus species being present

using species-specific 16S rDNA, PCR assays targeting 17 different BV-associated bacteria in 131 women with BV, the vaginal microbiome was sampled before treatment and 1 month after for a test of the cure. Treatment was with a 5-day course of intravaginal metronidazole gel At 1 month after treatment, BV was still present in 26% of women. The baseline detection of BVAB1, BVAB2, and BVAB3, Peptoniphilus lacrimalis, or Megasphaera phylotype was significantly associated with persistence of BV at the test of the cure

pretreatment vaginal microbiology at diagnosis might define the risk of antibiotic failure. We anticipate that these correlations will become clearer and more meaningful when studies are repeated with quantitative PCR, instead of simply using detection/incidence.

Information of the vaginal microbiome in pregnant women is limited Using species-specific primers, Wilks quantified the production of H2O2 by lactobacilli from swabs taken at 20 weeks of gestation from the vagina of 73 women considered to be at high risk of preterm birth The presence of lactobacilli producing high levels of H2O2 was associated with a reduced incidence of BV at 20 weeks of gestation, and subsequent chorioamnionitis
In pregnant Japanese women, Tamrakar(confirmed the Fredricks) in non-pregnant women. The prevalence of L.crispatus, L.jensenii, and L.gasseri was higher, whereas that of BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium were lower in women (normal Nugent score), compared with those who had BV.

The prevalence of L.iners did not differ between these groups, and women with L.iners were more likely to be colonised by BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium. In a longitudinal study of 100 pregnant women, vaginal swabs were obtained at mean gestational ages of 8.6, 21.2, and 32.4 weeks, respectively. In the first trimester 77 women had normal or Lactobacillusdominated flora; 13 developed abnormal flora in the second or third trimester. When first-trimester normal flora was dominated by L. gasseri or L. iners, there was a ten-fold risk of conversion to abnormal flora. In contrast, normal flora comprising L. crispatus had a five-fold decreased risk of conversion to abnormal flora. This may be because L. gasseri and L. iners only produce H2O2 in a small percentage of strains

Conclusion Molecular-based techniques provide important new information about vaginal microbial flora, and permit the identification of previously under-detected and hence under-appreciated organisms, such as L. iners and A. vaginae. Molecular-based techniques are not without their problems, and will not replace culture-based techniques, but, when used in combination, add greatly to our understanding of vaginal flora. In the majority of circumstances, normal vaginal flora is dominated by Lactobacillus species.In the absence of lactobacilli, normality can be maintained by other, more fastidious lactic acid-producing bacteria. In keeping with the theories of competitive exclusion and bacterial interference, when lactobacilli dominate the vaginal flora, cultureindependent techniques have demonstrated that the healthy vagina is usually colonised by only one or two dominant.

The dominant Lactobacillus species may differ racially or geographically, but the principle of numerical dominance persists, and may be an important defense mechanism, without molecularbased techniques, phenotypic identification of lactobacilli is difficult, and so is normally only carried out to the genus level. the ability to identify lactobacilli to species level (enable us to gain a better understanding of the role of different species of Lactobacillus)
Molecular-based techniques indicate that there is a far greater diversity of micro-organisms associated with BV than has been evident from cultivation-dependant techniques. These diverse organisms accumulate to form different communities or profiles, which make it likely that BV is not a single entity, but a syndrome of variable composition that causes a variety of symptoms, different phenotypical outcomes, and may result in variable responses to different antibiotic regimens.

Some organisms or combinations of organisms have a high specificity for BV, such that in the future, by using molecular quantification, we may better diagnose each subtype of BV, and be able to tailor treatment appropriately.
The information to the vaginal microbiome in pregnant women is limited, particularly with respect to preterm birth. Given the importance of the preterm birth problem worldwide, and must become a research and funding priority.

Better understanding the vaginal microbiome during pregnancy, we may be able to predict and prevent some of the great obstetric syndromes like PPROM, preterm labor, and preterm birth, which is associated with infection and significant infant mortality and morbidity.