# The Human Vaginal Bacterial Biota and Bacterial Vaginosis

Authors: Sujatha Srinivasan; David N. Fredricks
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2008)

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## Abstract

The bacterial biota of the human vagina can have a profound impact on the health of women and their neonates. Changes in the vaginal microbiota have been associated with several adverse health outcomes including premature birth, pelvic inflammatory disease, and acquisition of HIV infection. Cultivation-independent molecular methods have provided new insights regarding bacterial diversity in this important niche, particularly in women with the common condition bacterial vaginosis (BV). PCR methods have shown that women with BV have complex communities of vaginal bacteria that include many fastidious species, particularly from the phyla Bacteroidetes and Actinobacteria. Healthy women are mostly colonized with lactobacilli such asLactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners, though a variety of other bacteria may be present. The microbiology of BV is heterogeneous. The presence of Gardnerella vaginalis and Atopobium vaginae coating the vaginal epithelium in some subjects with BV suggests that biofilms may contribute to this condition.


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## Body

## 1. Introduction

The vagina is the Rodney
Dangerfield of the human body; it gets no respect.  Although frequently regarded as a mere
passageway for menses, sperm, or neonates, the human vagina is a highly
versatile organ that can profoundly affect the health of women and their
newborn infants.  The environment in the
vagina can impact the probability of conception, the ability to carry a fetus
to term, and the risk of acquiring sexually transmitted diseases such as HIV
infection.  Microbes play a critical role
in determining the biochemical and inflammatory profile of the vaginal
environment.  Although decades of studies
based on cultivation technologies have illuminated the microbiota of the human
vagina, recent studies employing cultivation-independent methods have
significantly increased our understanding of bacterial diversity in this
important niche.  This review will focus
on the bacterial biota in the human vagina, with particular attention paid to studies
using nucleic acid sequence-based approaches.
We will highlight the changes in vaginal bacterial communities that are
associated with the common condition bacterial vaginosis (BV) and will discuss
the challenges to using Koch’s postulates [1, 2]
to assess evidence of causation for fastidious bacteria in these microbial
communities.  There are many important
pathogens in the vaginal niche such as Neiserria
gonorrhea, Ureaplasma species, Mycoplasma genitalium, Streptococcus species, Escherichia coli, Chlamydia trachomatis,
and Trichomonas vaginalis which we
will not explore in this review.  Studies
of fungal, viral, archaeal, and protistan diversity in the human vagina are
important but will not be the focus of this review due to the paucity of published
molecular surveys.  Studies of the human
vaginal microbiome in 2009 are in their infancy.  Both metagenomic and whole bacterial genome
sequencing projects are underway to help define the collection of microbial
genes present in the vagina and to understand their contribution to normal host
physiology and disease.The picture that emerges from most
studies of the vaginal microbiota described here is static because it is based
on cross-sectional studies that assess the microbial constituents at discrete
and infrequent time points.  However,
microbial communities in the human vagina likely undergo shifts in the
representation and abundance of key species over time that are influenced by
factors which may include age of the woman, hormonal fluctuations (e.g., stage
of menstrual cycle, contraception), sexual activity (e.g., types of sexual
activities such as oral or anal sex followed by vaginal sex, frequency of sex,
number of sex partners, and the genitourinary tract microbiota of these
partners), underlying health conditions (e.g., diabetes, urinary tract
infection), use of medications (e.g., intravaginal and systemic antibiotics),
intravaginal washing practices and hygiene.
Future studies will benefit from the use of high throughput technologies
that will facilitate measuring fluctuations in the human vaginal microbiota
over time in longitudinal analyses with more frequent sampling.  Current data suggest that these studies will
reveal a highly dynamic human vaginal ecosystem in many women.


## 2. The Vaginal Microbiota: “Normal” versus Bacterial Vaginosis

Gram stains of vaginal fluid smears
from women without BV typically show Gram-positive rods, with cultures
revealing a predominance of lactobacilli, particularlyLactobacillus crispatus and Lactobacillus
jensenii [3–5]. Lactobacilli are believed to promote a
healthy ecosystem by producing lactic acid, hydrogen peroxide, and bacteriocins
that have antimicrobial properties thereby excluding pathogens from this niche [6]. Lactobacillus
iners is an underappreciated member of the normal vaginal biota, as it does
not grow on Rugosa agar that is typically used to isolate lactobacilli [3]. In contrast, women with the condition bacterial
vaginosis (BV) have loss of many Lactobacillus species (except L. iners) and
acquisition of a variety of anaerobic and facultative bacteria  [7, 8]. Gram stains of vaginal fluid from women with
BV show loss of Gram-positive rods and their replacement with Gram-negative and
Gram-variable cocci and rods [9]. Cultures of vaginal fluid from subjects with
BV typically yield Gardnerella vaginalis and a mixture of other bacteria that may include Prevotella, Porphyromonas, Mobiluncus, and Mycoplasma species. It is
not known whether the primary event initiating BV is the loss of key
lactobacilli or acquisition of the complex bacterial communities found in this
syndrome; these may be simultaneous processes (Figure 1).  It is also possible that some other factor is
the primary etiological agent, and that the changes in vaginal microbiota
reflect a downstream event in the pathogenesis of BV.Figure 1
Competing models for the pathogenesis of BV. At least 2 models exist to explain
the pathogenesis of BV. The
lactobacillus depletion model suggests that there is a decrease in hydrogen
peroxide producing lactobacilli as the primary event that allows for the
overgrowth of facultative anaerobes resulting in BV. The primary pathogen model suggests that the
entry of facultative anaerobes causes the displacement of lactobacilli thereby
resulting in BV.


## 3. Bacterial Vaginosis

BV is the most common cause of
vaginal discharge and a frequent reason for women to seek medical attention [10].  BV is highly prevalent, affecting ~10–30% of women in
the United States [11], with higher rates reported
in African American women and women from Sub-Saharan Africa [12–14].  Although BV is an important medical condition
itself, it is associated with several more serious adverse outcomes including
preterm birth [15], pelvic inflammatory disease [16], and acquisition of HIV
infection [17]. Women with BV may have a
malodorous vaginal discharge or local irritation, but about half of the women
with diagnosable BV have no clear symptoms [18].  Some women do not report abnormal vaginal
discharge, but discharge is nonetheless noted on examination by a clinician,
highlighting that many women with BV are not aware of their diagnosis or
consider their discharge to be within normal bounds. The high prevalence of BV
and the lack of symptoms in a substantial fraction of affected women lead to
the question whether BV should be considered a normal variant of the vaginal
microbiota or a disease entity. For
women affected by severe symptomatic BV as manifested by profuse vaginal
discharge and less frequently by local burning or itching, there is little
question that they have a disease. For women with laboratory evidence of BV but
no symptoms, the disease designation seems inappropriate, though the condition
may still impart increased risk of adverse health outcomes such as preterm
birth. Antibiotics such as metronidazole and clindamycin are usually effective
in treating BV in most subjects, leading to resolution of symptoms, though
rates of relapse are high [19, 20]. Either systemic (usually oral) or
intravaginal antibiotics can be used to treat BV.Symptomatic BV can be described as a
syndrome based on the presence of a collection of clinical features without a
specific etiologic agent defined. The
diagnosis of BV is usually made using a series of clinical criteria collected
by a clinician performing a pelvic examination, or by interpretation of vaginal
fluid Gram stains. Amsel clinical
criteria are usually employed for the diagnosis of BV in the clinical setting
because the approach is rapid, but it does require access to a microscope [18]. At least 3 of 4 Amsel criteria must be
present to establish a diagnosis of BV, including (1) elevated vaginal fluid pH>4.5; (2) a positive “whiff test” which consists of the detection of a fishy
odor upon addition of 10% potassium hydroxide to a slide containing vaginal
fluid; (3) the presence of clue cells (>20%) in vaginal fluid which are shed vaginal epithelial cells coated with bacteria creating indistinct borders;
(4) a homogeneous, milky vaginal discharge. Note that it is possible to have a
diagnosis of BV based on Amsel clinical criteria without the presence of frank
vaginal discharge.  Accordingly,
presuming that women without vaginal discharge do not have BV is not valid, and
studies of the “normal” vagina should ideally employ an objective method to
assess for BV.  Unfortunately there are
numerous studies in the field that have claimed that BV-associated bacteria are
part of the normal microbiota without having assessed for BV status, although
self-reported vaginal discharge may have been absent. It is possible, indeed probable, that many
BV-associated bacteria can be part of the normal human vaginal microbiota, but
the failure to use consensus guidelines to define BV in the research setting is
a recipe for scientific confusion that is completely avoidable with well-designed
studies.An alternative method for diagnosis
of BV relies on analysis of Gram stains performed on vaginal fluid smears. This approach is most commonly employed in
the research setting where Gram stains are used to classify subjects but is
less well suited to the clinical setting because analysis of the vaginal fluid
Gram stains requires a degree of expertise that is rarely available in real
time when the clinician is faced with the decision whether to treat for
BV.  For better or for worse, the vaginal
fluid Gram stain is considered the current diagnostic gold standard as it
offers greater reproducibility and objectivity when compared with the Amsel’s
clinical criteria. For example, there
can be variation between technicians in the evaluation of wet mounts for
vaginal clue  cells. Several scoring
systems are used to classify vaginal smears.
The method of Nugent et al. [9] assesses the presence and
relative amounts of three bacterial morphotypes, including Gram-positive rods
(lactobacilli), Gram-negative and Gram-variable rods (Gardnerella vaginalis, and Bacteroides species), and curved rods (Mobiluncus species). A Nugent score of 0–3 is considered
normal (no BV) and is marked by the presence of Gram-positive rods, or at least
no Gardnerella vaginalis or Mobiluncus morphotypes. A Nugent score of 7–10 confers the
diagnosis of BV and is marked by the absence of Gram-positive rods and the
presence of high concentrations of Gardnerella or Mobiluncus morphotypes.  A Nugent score of 4–6 is designated
intermediate flora and has Gram stain features between the two poles.  Alternative scoring systems for
interpretation of vaginal fluid Gram stains exist, such as that of Ison and Hay
[21].


## 4. The Role ofGardnerella vaginalis in BV

In a sentinel paper published in
1955, Herman Gardner and Charles Dukes reported the successful isolation of a
novel bacterium from subjects with the syndrome nonspecific vaginitis, now
known as BV. The bacterium was initially
namedHaemophilus vaginalis but was
later renamed Gardnerella vaginalis.
The authors stated, “We are prepared to present evidence that the vast majority
of so-called “nonspecific” bacterial vaginitides constitute a specific
infectious entity caused by a single etiological agent [22].” These investigators believed that G. vaginalis was the sole cause of BV
and set out to fulfill Koch’s postulates for disease causation in a series of
clinical experiments. Pure cultures of G. vaginalis were inoculated into the
vaginas of 13 healthy women, which resulted in the development of BV in 1 of
the 13, with a corresponding rate of disease production of 7.7%. Based on these data, the investigators
concluded that Koch’s postulates were fulfilled, though the 92% failure rate
calls this conclusion into question. The
investigators went on to perform an additional experiment wherein whole vaginal
fluid obtained from subjects with BV was used to inoculate the vaginas of 15
women without BV. Eleven of these 15
subjects developed BV, yielding a disease induction rate of 73%. The authors felt that these data further
supported the causal role of G. vaginalis in BV because this bacterium was cultured from most of the induced cases. It is our interpretation of these studies
that whole vaginal fluid is a much more successful inoculum for the
transmission of BV than is a pure culture of G. vaginalis, suggesting that there are other factors besides G. vaginalis important in disease
induction.Other evidence suggests thatGardnerella vaginalis is not the sole
etiological agent in BV. Koch’s
postulates demand that the etiological microbe should be found in every case of
disease but should not be detected in subjects without disease [1] (see section on Koch’s
postulates). G. vaginalis fails this later test of specificity because it can be
detected in about 30–50% of women
without BV using cultivation methods  and
70% of women without BV using PCR methods [23]. After more than half a century, we are still
debating the role of G. vaginalis in
BV. Although G. vaginalis likely plays an important role in the pathogenesis of
BV, it is unlikely to be the sole instigator because it is never found as the
sole bacterium in vaginal fluid from subjects with BV. Our hypothesis is that BV is a syndrome
caused by communities of bacteria that include uncultivated species, precluding
the formal application of Koch’s postulates and necessitating new approaches
for establishing causation.


## 5. Vaginal Microbial Diversity: The Perspective from Cultivation

With the advent of molecular
techniques used to measure bacterial diversity, it is easy to discount the
contributions from studies based on cultivation because these studies may fail
to detect a large number of fastidious microbes in any given niche.  However, cultivation studies provide critical
insights about the phenotypic characteristics of microbes that are not easily
derived from molecular studies.
Furthermore, cultivated microbes allow for the experimental manipulation
of these organisms in the laboratory and the testing of hypotheses about
pathogenesis and virulence factors.
Accordingly, cultivation studies remain an important area of
investigation in vaginal microbiology, despite the limitations of the approach [24]. One reason for pursuing the combined approach
using cultivation and cultivation-independent methods is that some bacteria are
more likely to be detected by cultivation when present in low
concentrations. For example, Verhelst et
al. [25]
reported that of the 38 vaginal bacterial species identified from 8 subjects
with and without BV, 5 were detected by cultivation alone. Novel cultivation approaches may be required
to grow the many fastidious bacterial species found in the human vagina.Prior to Burton and Reid’s study in
2002 [26], almost all of our knowledge
about the bacteria in the vaginal niche came from cultivation studies which
involved isolating the organisms by culture on selective or nonselective media
and subsequent identification by phenotypic techniques. Just as use of a variety of broad range
bacterial PCR primers helps to maximize species diversity (see section on Molecular
Approaches), a number of media and growth conditions may be needed for the
optimal isolation of diverse bacterial species.
Relatively nonselective media such as MacConkey agar, mannitol salt agar,
and tryptic soy base with 5% sheep blood agar can be useful to estimate numbers
of aerobic and anaerobic bacteria in vaginal samples. Selective or semiselective media include Rogosa
[27] or de Man, Rogosa and Sharpe
media (MRS) for lactobacilli and the human bilayer Tween (HBT) agar for the
isolation of Gardnerellavaginalis [28]. It should be noted that Lactobacillusiners, present in many subjects with and without BV,
does not grow on Rogosa agar but can grow on HBT agar.Cultivation-based approaches have
identifiedGardnerellavaginalis,
anaerobic bacteria such as Prevotella, Porphyromonas, Peptostreptococcus, Mobiluncus, and Mycoplasma to be largely associated with the disturbed microbiota
in subjects with BV. Healthy women are
commonly colonized with hydrogen peroxide producing lactobacilli which are
thought to inhibit the growth of the fastidious anaerobes associated with
BV.  Specific details of cultivation
studies will not be discussed further but can be obtained from recent reviews [29, 30].


## 6. Vaginal Microbial Diversity: The Molecular Perspective

Cultivation-independent approaches
have consistently documented the high proportion of fastidious bacteria in a
variety of ecological niches [31] and these tools have recently
been applied to study the vaginal ecosystem.
Results from many different research groups confirm that the human
vagina hosts numerous bacterial species that are either not cultivated or not
easily identified using cultivation methods.
These results help to augment, but do not replace, the census data
generated using cultivation-based approaches. Indeed every method for characterizing
the human indigenous microbiota is subject to some degree of bias.  Therefore, it is our position that the most
complete picture of the human microbiota will emerge from the application and
synthesis of different technologies and approaches, including cultivation.  We highlight both the strengths and
limitations of various molecular approaches for describing the vaginal
microbiota below.The most commonly employed target
for molecular identification of bacteria is the small ribosomal subunit or 16S
rRNA gene.  The 16S rRNA gene is useful
because it is present in all bacteria and has regions of sequence conservation
that can be targeted with broad range PCR primers and areas of sequence
heterogeneity that can be used to identify bacteria or infer phylogenetic
relationships (see [32–36]). Once the 16S rRNA gene has been sequenced
from a bacterium, the variable regions can be used for species-specific PCR
either in a qualitative or quantitative manner. Quantitative PCR is especially useful for rapidly identifying bacteria
when an internal probe is employed and for measuring how levels of vaginal
bacteria change.  Nine highly variable
and therefore phylogenetically rich regions of the ~1540 base pair 16S rRNA
gene have been described and designated V1 to V9  [37].  The choice of primers targeting the conserved
regions flanking the different variable regions can profoundly affect the
diversity of bacterial species identified [38, 39].It is theoretically possible to
detect every known bacterial species if suitable broad range PCR primers or
combinations of different primer pairs are employed. Current studies focus on the extraction of
total genomic DNA from vaginal fluid on swabs or from cervicovaginal lavage
fluid and amplification of 16S rRNA genes with primers that bind to conserved
sites present in many species. The
sequences obtained are aligned and compared to large databases of 16S rRNA
sequences (http://greengenes.lbl.gov/ [40], http://rdp.cme.msu.edu/ [41, 42],
http://www.arb-home.de/ [43]
to infer phylogenetic relationships to known species.  Some studies rely on the construction of
clone libraries and direct sequencing of a particular number of clones [44, 45].  This approach allows for good phylogenetic
resolution if a suitable portion of the 16S rRNA gene is amplified. However, this method tends to be expensive,
slow, and tedious. Some investigators
try to limit the sequencing of large numbers of samples by using
electrophoretic fingerprinting techniques such as denaturing gradient gel
electrophoresis (DGGE) [46]
or terminal restriction fragment length polymorphism (T-RFLP) [47]. In the case of DGGE, as the amplification
products pass through the denaturing gel, their melting behavior depends
primarily on the length of the product and the GC content [48]. Typically one of the primers carries a 5′-GC
rich clamp, around 40 bp, which is used to detect single-base changes between
close products. This clamp tends to
lower the PCR amplification efficiency and can increase the presence of PCR
artifacts such as heteroduplexes [48].  T-RFLP involves PCR amplification of the
community DNA using primers with fluorescent tags. The resulting PCR products are digested with
restriction enzymes and the fluorescent terminal restriction products are
detected using a DNA sequencer.  The
species diversity revealed by DGGE is much less than the diversity detected by
T-RFLP [49],
and this likely reflects  greater
sensitivity of the fluorescence detection platform.  Screening clones in a library by amplified
ribosomal DNA restriction analysis (ARDRA) in order to limit the number of
clones to be sequenced is also commonly used.
ARDRA is based on the restriction digestion of 16S rRNA gene clones or
amplified DNA and electrophoretic separation on high percent agarose or
polyacrylamide gels [7].An approach complementing broad range
PCR is characterization of the vaginal bacterial community by using nucleic
acid probes, oligonucleotides complementary to rRNA gene targets.  Probes are designed using sequences generated
from broad range PCR and sequencing experiments which can have a wide range of
phylogenetic specificities ranging from domain to strain levels.  There is also a database maintaining probes
designed for many bacteria from other niches (http://www.microbial-ecology.net/probebase/)
[50]. The probes are labeled with a fluorescent tag
and hybridized to the clinical samples.
Cells are visualized using epifluorescence microscopy in a process
referred to as fluorescence in situ hybridization (FISH). Data can be collected in both quantitative
and qualitative modes.  For example, when
fluorescent probes are combined with flow cytometry, one can rapidly count and
collect cells.  With confocal scanning laser microscopy, one can visualize the spatial arrangement of cells in tissues
or body fluids.


## 7. Diversity Studies Based on the 16S rRNA Gene: Limitations

While molecular methods have many
advantages over cultivation approaches for characterizing microbial diversity,
there are numerous limitations [51–53]. Use of some so-called “universal primers”
targeting conserved regions of the 16S rRNA gene may not detect all bacteria
present in a sample due to the presence of polymorphic nucleotides at conserved
positions. The primers are more
accurately designated as broad range. Heterogeneity of the 16S rRNA gene within
the same species can also hamper fingerprinting analysis [39]. Lowering the annealing temperature during PCR
permits mismatches when using broad range primers thereby increasing the
diversity of the PCR products formed, though this may also allow nonspecific
amplification of DNA from human tissues. Degenerate nucleotides can help in overcoming the deficiency of broad
range primers when polymorphic base positions are encountered but can lead to
lower efficiency of primer binding due to exact matches of variants being
diluted in the primer pool. If the primer concentrations are increased to
overcome this dilution problem, then there is the potential for increased
nonspecific product formation. Inosine-based
primers are an alternative to degenerate primers [54] but these cannot be
successfully used with Pfu [55], a high fidelity
polymerase.  One example of a commonly
used broad range PCR primer targeting the 16S rRNA gene is the 27f (8f) primer
at the 5′ end of the 16S rRNA gene. This primer
has multiple mismatches with many Chlamydiae and Bifidobacteria, highlighting
the fact that this primer may be highly inefficient in detecting bacteria in
these phylogenetic groups [38].  More frequently, individual species within
phylogenetic groups may have mismatches that result in reduced amplification
efficiencies [30].  Frank et
al. [38] evaluated the 27f (8f)
primers (designated as 27f-CC and 27f-CM) that are commonly used in many broad
range PCR studies and formulated a 27f primer mixture (designated as 27f-YM+3)
that included three sequences not usually accounted for in many contemporary
studies. These primers are better matches with bacteria in the Chlamydiales and Bifidobacteriales orders as well as bacteria in the Borrelia genus.  Using a combination of linear amplification
with the 27f formulation and quantitative PCR, they showed that the formulated
primer mixture performed better at detecting Gardnerella vaginalis sequences even at elevated annealing
temperatures (60°C) than the 27f primers typically used in the
literature. Several studies have
attempted to characterize the vaginal bacterial biota using the conventional
27f primer and these studies appear to underrepresent bacteria such as G. vaginalis which is a common member of
the vaginal ecosystem [25, 56]. Although use of complex primer mixtures may
increase the diversity of bacteria detected by broad range PCR, this advantage
comes at a cost. When using the primer
mixture, there is a slight decrease in amplification efficiency due to a
reduction in primer concentrations with exact matches.We have seen similar problems with
the 27f primer in our broad range bacterial PCR studies of the vaginal
niche. We amplified a region of the
ribosomal RNA operon using 27f  [57] modified with one degeneracy
(27f-CM) and 189r [58] at an annealing temperature
of 55°C.  Clone library analysis
on a model subject with BV revealed the absence of Gardnerella vaginalis (Figure 2, unpublished data). In contrast, by utilizing a different forward
primer (338f) and the same reverse primer, G. vaginalis emerged as the dominant clone in the library (Figure 2). Moreover, as can be seen in Figure 2, use of
different forward primers on the same vaginal sample results in vastly
differing rank abundance plots. For
example, a fastidious bacterium in the Clostridiales order designated BV-associated bacterium 1 (BVAB1) was detected using the 338f
primer while all three novel bacteria in the Clostridiales order associated with BV (BVAB1, BVAB2, BVAB3) were
detected with the 27f primer. The 5 most
prevalent clones detected with 338f included sequences matching G. vaginalis type 1, Atopobium vaginae type 1, BVAB1, G. vaginalis type 2, and Peptostreptococcus while the most
abundant clones seen with the 27f primer were A. vaginae type 2, BVAB2, Mobiluncus
mulieris, BVAB1, and an Eggerthella-like
bacterium.  Using both sets of primers,
we detected a total of 22 phylotypes of which 10 were represented as singleton
species (detected as a single clone).  When
this is compared with each primer pair alone, we were able to detect only 15 phylotypes
each, including 5 singletons with the 338f primer and 8 single clones with the
27f primer. We also noted that the 27f
primer in combination with 189r tended to be biased to A. vaginae, thereby not providing representative reflections of
bacterial abundance (Figure 2). However,
we found that creating two clone libraries with different forward primers
resulted in detection of more phylotypes, again highlighting the limitations
imposed by the selection of a single primer pair. Accordingly, we suggest that using
combinations of broad range primers on the same sample may maximize the
diversity of species detected, though this comes at a cost of additional time
and money expended.Comparison of vaginal
bacterial species detected by broad range 16S rRNA gene PCR using two different
forward primers and the same reverse primer in one sample. The pie charts show the percentages
of clones in each library corresponding to specific bacterial 16S rRNA gene
sequences obtained using broad range PCR followed by cloning and sequencing in
a vaginal sample from a subject diagnosed with bacterial vaginosis.  Data obtained using the 338f (a) primer
shows a balanced representation of clones while the data obtained using the 27f
(b) primer is skewed toward Atopobium vaginae.  Note the absence of Gardnerella vaginalis clones in the clone library created with the
27f primer.  BVAB denotes bacterial
vaginosis associated bacterium.
(a)
Forward primer—338f(b)
Forward primer—27fThe DNA extraction step is vital to
getting a representative pool of DNA which will then be used for PCR
amplification. Species bias for
different extraction methods is well known [59, 60].  Presence of inhibitors in the clinical
samples from blood, mucus, or vaginal products can lead to failed amplification
or a reduction in the amount of product.
Amplification controls are useful in tracking DNA quality wherein PCR of
specific target genes such as beta-globin [23] or the 18S rRNA gene [61] can indicate if the DNA
extracted from human tissues is amplifiable.
Use of internal amplification controls by adding an exogenous template
at known concentrations to the clinical samples can help in detection of subtle
PCR inhibitors [62, 63],
particularly when performing quantitative PCR analysis.Another issue with broad range PCR
targeting the 16S rRNA gene is the lack of phylogenetic resolution for some
bacteria, even at the species level. For
example, different species within theEnterobacteriaceae have very similar 16S rRNA gene sequences. Other gene targets offer improved
phylogenetic resolution for some species, such as the sigma factor rpoB present in just one copy per genome
[64–67]. A downside of using rpoB as a marker is the dearth of sequences available when compared
to the 16S rRNA gene. An alternate
option is to examine the internal transcribed spacer region by ribosomal
intergenic spacer analysis to distinguish closely related strains [68–70]. Here again, sequence and size heterogeneity
can be critical limitations, and databases (http://egg.umh.es/rissc/)
supporting this region are small in comparison to those supporting 16S rRNA
gene sequences.Correlating the number of 16S rRNA
gene copies (and hence clones) to the number of bacteria is frequently not
possible as different bacterial species can have varying numbers of rRNA gene
operons per genome (between 1 and 15) and the exact number is unknown for most
species [71–73]. Bacteria with higher rRNA operon copy numbers
will be excessively represented in a clone library when compared with bacteria
with lower copy numbers, thereby introducing a bias in the community analysis [74]. Moreover, different bacteria may have varying
susceptibilities to lysis based on the extraction methods being used thus
leading to different quantities of bacteria observed in subsequent analysis.Similarly, false positives can
impact community analysis when targeting the 16S rRNA gene using broad range
primers. Low levels of bacterial DNA may
be present in laboratory or PCR reagents and in DNA extraction kits.Taq polymerase used for PCR amplification can have contaminating 16S rRNA sequences
[75, 76]. A way to monitor this problem is to include
negative controls in every run of PCR.
No template PCR controls allow for detection of contaminants arising
from PCR reagents and the water being used in every PCR experiment.  Additionally, it is extremely useful to
include extraction controls wherein sham samples are processed and extracted in
the same manner as the experimental samples.
These extraction controls should be subjected to PCR and analysis of
products (such as cloning/sequencing) alongside samples of interest to identify
any contaminants.  Limiting the number of
amplification cycles and using high amounts of template DNA also help in
reducing amplification of low level contaminants that may have been introduced
during the different steps of sample preparation.  An important source of PCR contamination is
from previously amplified products.  This
can be managed by separating pre- and post-PCR working spaces, use of aerosol
filter pipette tips, and addition of uracil glycosylase to inactivate
previously amplified PCR products.The PCR amplification step itself
can introduce biases such as skewed representation of a sample based on the
guanosine plus cytosine (G+C) content of the bacterium [77, 78].  Bacteria with higher G+C content may result
in lower throughputs when compared with bacteria with lower G+C.  PCR enhancing additives such as betaine [79], dimethyl sulfoxide  [80], or formamide [81] are typically used to
equalize the read-through efficiencies of the different templates with varying
G+C contents while the reducing environments created by β-mercaptoethanol
or dithiothreitol [82] seem to provide unspecified
PCR enhancing effects.  PCR enhancers
that are commercially available (e.g., Q-solution from Qiagen, PCR enhancer
solution from Invitrogen) can be expensive and their composition is not
known.  Low cost in-house reagents such
as a combination of betaine, dithiothreitol and dimethyl sulfoxide have been
shown to improve both qualitative and quantitative outputs of PCRs [83].PCR artifacts are a well-known
limitation when using the broad range PCR approach. Incorporation of incorrect
nucleotides usingTaq polymersase may
lead to errors in the sequence.
Heteroduplexes may form when primers become limiting and/or there is
greater template diversity [84, 85].  Use of Pfu polymerase which possesses 3′
to 5′
exonuclease proofreading capabilities allows for the correction of
misincorporated nucleotides and hence has fewer errors when compared with Taq polymerase [86].  There are other high fidelity DNA polymerases
that are currently available such as Vent DNA polymerase isolated from Thermococcus litoralis and Phusion DNA
polymerase which is a Pyrococcus-like
enzyme with a double-stranded DNA-binding domain.  One recommended strategy to limit
heteroduplex molecules prior to cloning is to reamplify 10-fold diluted PCR
product containing mixed templates in a process referred to as “reconditioning
PCR” [85].  Formation of chimeras [87, 88]
needs careful monitoring and identification.
Chimeric sequences are PCR artifacts that arise when two or more
phylogenetically distinct sequences become combined into a single sequence when
the polymerase jumps between templates during extension.  Several online tools are available to detect
chimeras such as Bellerophon [89], Mallard [90],
or Pintail analysis [91].While the broad range 16S rRNA gene
PCR approach provides a good census of the bacteria present in the clinical
sample, no functional genomic information is obtained.  Metagenomic approaches have been applied to
environmental samples [92, 93]
but are slow to be applied to the vaginal environment due to lack of whole
genome sequence information for creation of a scaffold.  There is presently an NIH-led initiative to
sequence whole genomes from cultivable bacteria from the vaginal niche which
will provide the necessary foundation for metagenomic studies (http://nihroadmap.nih.gov/hmp/).


## 8. Molecular Studies in the Vaginal Niche: A Critical Examination

With advancing technologies
and decreasing costs of sequencing, there have been many recent additions to
our knowledge regarding the human vaginal microbiota.  As conditions in the vagina may be transient
and dependent on numerous factors, most molecular studies offer a snapshot of
the vaginal microbiota under specific conditions.  Moreover, with differing definitions of
“normal,” it can be difficult to compare the data across many studies.  We present here a survey of key molecular
investigations in the vaginal niche, highlighting the important contributions
and the limitations of each approach.Burton and Reid [26] were the first investigators
to analyze the microbiota of the vaginal niche using broad range molecular
methods.  They applied a combination of
broad range bacterial PCR using primers HDA-1-GC (338f with a GC clamp) and
HDA-2 (515r) and DGGE to vaginal samples obtained from 20 asymptomatic
postmenopausal women and used Nugent scores to distinguish between healthy and
diseased states.  Interestingly, 70% of
the women had intermediate flora or BV as indicated by Nugent score, suggesting
that women with abnormal vaginal flora were overrepresented in their study
compared to the general population.
Broad range PCR targeting about 200 bp of the V2-V3 variable regions of
the 16S rRNA gene and DGGE analysis showed that subjects with low Nugent scores
had only one to two bands, mainly derived from Lactobacillus species, while subjects with intermediate flora or
high Nugent scores had zero to four bands representing Gardnerella, Prevotella, Peptostreptococcus, Bacteroides, Lactobacillus, Streptococcus, and Slackia species.  The detection of Lactobacillus iners in subjects with
normal flora by Gram stain was a novel finding.
Genus specific PCR was also used to monitor the bacterial species
detected by broad range PCR.  The
strength of this study is the utilization of both broad range and
taxon-specific PCR approaches, but the DGGE method may have limited the
diversity detected.An important observation from this
study [26] is that different subjects
with BV had different DGGE profiles indicating heterogeneity in the composition
of bacterial taxa in subjects with BV.
We have observed similar results using different methods. For example,
Figure 3 illustrates the differences observed in the composition and number of
bacterial phylotypes in two subjects with BV. Vaginal samples were subjected to
broad range 16S rRNA gene PCR using primers 338f and 1407r followed by cloning,
sequencing, alignment, and phylogenetic analysis. The most prevalent phylotypes
in Subject A include Gardnerella
vaginalis, Prevotella sp. type 1,
BVAB2, Prevotella sp. type 2, and Leptotrichia amnionii.  In contrast, the most prevalent bacterial
clones in Subject B include BVAB1, Sneathia
sanguinegens, Prevotella sp. type
1, Candidate division TM7, and Prevotella sp. type 2, thereby illustrating the differences in bacterial phylotypes
between two subjects with BV.The microbiology of BV
is heterogeneous. Comparison of rank abundance plots
from 2 subjects diagnosed with BV.  The
charts show the percentages of clones in each library corresponding to specific
bacterial 16S rRNA gene sequences obtained using broad range PCR followed by
cloning and sequencing.  The most
prevalent bacterial clones in Subject A include those matching Gardnerella vaginalis, Prevotella sp. type 1, BVAB2, Prevotella sp. type 2, and Leptotrichia amnionii.  In contrast, the most prevalent clones in
Subject B include BVAB1, Sneathia sanguinegens, Prevotella sp. type 1, candidate
division TM7, and Prevotella sp. type
2.
(a)
Subject A(b)
Subject BIn a subsequent study from these
investigators, the same primers HDA-1-GC and HDA-2 with the same PCR conditions
were applied to 6 samples obtained weekly from a 51-year-old woman with
recurrent BV (determined by Nugent score).
Overall, 7 bacterial species were detected includingKlebsiella oxytoca, Serratia fonticola,
Citrobacter freundii, Morganella morganii, Kluyvera ascorbata, Escherichia coli, and Staphylococcus epidermidis [94].  None of the bacteria typically associated with
the vaginal niche were detected in this study.
Similarly, when the primers HDA-1-GC and HDA-2 were applied to vaginal
samples from a cohort of 34 HIV-seronegative Nigerian women with BV, atypical
BV-associated bacteria were detected by broad range PCR and DGGE [95].  Surprisingly, of the 34 samples, 10 had only
4 bands, 16 had 3 bands, 6 had 2 bands, and 2 had one band.  If each band corresponds to a single
bacterial phylotype, the bacterial diversity associated with BV in this study is substantially lower
than the diversity detected in other studies and likely reflects the limits of
the DGGE method employed.  The dominant
organism in 35% of subjects was found to be Mycoplasma
hominis. An uncultured Streptococcus sp. was found in 24% of
the subjects and a bacterium related to a rainbow trout intestinal bacterium
was found in 26% of subjects.  The
absence of several prominent BV-associated bacteria may be related to the
choice of primers, although the authors used the same primers to detect Gardnerella, Prevotella, Mobiluncus, and Atopobium sp. in a previous study [26]. The different results observed in this study
could also be due to differences in annealing temperatures: 56°C in
the earlier study [26] and 60°C in the
later study [96], or due to differences in
subject populations. The primers used in
these studies have a 40-mer GC clamp that has been included for DGGE analysis
resulting in primers that are 60 bases long, which may contribute to
inefficient amplification.  Based on the
data presented, the authors suggest that the bacteria associated with BV in
Nigerian women are different from those bacteria associated with BV in other
populations of women studied. Additional
molecular studies evaluating the bacterial community associated with BV from a
variety of women representing different demographic groups are required to
assess the degree of heterogeneity in vaginal microbiota among women.Zhou et al. [45] investigated the bacterial
community in 5 “apparently healthy” women.
The women were classified as healthy using a combination of
gynecological exams and self-reported symptoms, but data on Amsel’s clinical
criteria or vaginal fluid Gram stains were not obtained or provided.  This is a major limitation of this study as
many women with BV are asymptomatic. A
920 bp fragment of the 16S rRNA gene was amplified using primers 8f, also known
as 27f (actual primer sequence not specified), and 926r and the products were
cloned and sequenced. Between 176 and
250 clones were sequenced from each subject resulting in 2 to 7 bacterial
phylotypes per subject. Two subjects had
vaginal bacterial biotas dominated by Lactobacillus
crispatus, while Lactobacillus iners was detected in 3 subjects.  These
investigators suggest that three novel taxa were associated with the healthy
vagina including Atopobium vaginae, a Megasphaera species, and a Leptotrichia species. However, these bacteria have been associated
with BV by other investigators [7, 25, 26, 97]. As standard objective criteria were not used
for the diagnosis of BV, it is difficult to draw conclusions from this study
about the constituents of the normal vaginal bacterial biota.Hyman et al. [44] surveyed the bacteria on the
vaginal epithelium by broad range PCR, clone library construction, and
sequencing approximately 1400 bp of the 16S rRNA gene in 20 premenopausal women
who were presumably healthy. While
physical exams were conducted in the clinic and the women were reported to be
asymptomatic, the authors did not report data on BV status using Amsel’s
clinical criteria or vaginal fluid Gram stains; this is a significant
limitation of the study. PCR
amplification of the genomic DNA was conducted using the conventional 8f
(27f-CM) and 1492r primers. The forward
primer has one mismatch to Atopobium spp. and the reverse primer also has poor homology possibly leading to poor
representation of Atopobium spp. in
the libraries. One thousand clones were
selected for each subject and sequenced from both ends using conventional
sequencing.  Four of the 20 subjects had
only Lactobacillus species with very
high sequence diversity indicating that these vaginal bacteria were not
clonal. Nine subjects had a combination
of Lactobacillus spp. and other
bacteria including Bifidobacterium, Gardnerella, and Atopobium. The remaining
group of 7 women did not have any lactobacilli but were colonized with mixed
bacterial populations that include bacteria that have been associated with BV
by other investigators. This study
provides a rich resource of vaginal bacterial 16S rRNA gene sequences in GenBank,
but would have been more useful if additional clinical and microbiological data
had been collected to exclude women with BV or define those with the
condition. These investigators detected
sequences from some bacteria such as Pseudomonas and Stenotrophomonas species in clone
libraries that are known PCR contaminants.
It would have been helpful to describe PCR and extraction controls to
prove that these bacteria are arising from the vaginal epithelium and are not
spuriously detected by broad range PCR.Verhelst et al. [25]
used a combination of cultivation and molecular techniques to identify vaginal
bacteria in 8 subjects of whom 3 had normal flora, 2 had intermediate flora,
and 3 had BV as determined by the Gram stain method of Ison and Hay [21].  Isolates from culture studies were identified
using either 16S rRNA gene sequencing or by evaluating the fingerprinting
patterns of the spacer regions between transfer RNA genes.  Broad range PCR with primers 10f (27f-CC, not
including the first two bases of the 27f primer) and 534r was used to amplify a ~500 bp fragment of
the 16S rRNA gene resulting in 854 clones from the 8 subjects.  The clones were analyzed using ARDRA and
clones with unique ARDRA patterns were sequenced for identification of the
bacteria.  A total of 38 species were
identified using both approaches, of which 18 were detected by cloning only, 5
were detected by culture alone.  Healthy
subjects had vaginal bacterial biotas dominated by lactobacilli whereas
subjects with intermediate flora or BV flora had greater bacterial
diversity. Atopobium vaginae and several BV-associated bacteria were detected
in a large number of clones generated from subjects with abnormal flora.  The primers selected for broad range PCR
proved to be a poor match for detecting Gardnerella
vaginalis, which was isolated by cultivation.  However, G.
vaginalis specific PCR showed that this bacterium was associated with
BV.  This study underscores the
importance of using a combination of approaches to attain a complete picture of
vaginal bacterial diversity and the need to optimize primers for broad range
PCR. The use of Gram stain analysis to evaluate BV status is commendable.Fredricks et al. [7] evaluated the bacterial
community in the vaginal niche using broad range PCR with primers 338f and
1407r amplifying a
~1000 bp fragment from the 16S rRNA gene.
This approach was applied to vaginal samples from 9 subjects with BV and
8 without BV using Amsel’s clinical criteria to define BV in a cross-sectional
analysis. In addition, serial vaginal
samples were also obtained from a limited number of subjects to study the
change in bacterial composition associated with incident, cured, relapsing, and
persistent BV. One hundred clones from
each subject were selected and screened using ARDRA with two restriction
enzymes. Inserts with unique patterns
were sequenced. Women with BV showed a
high level of species diversity with a mean of 12.6 bacterial phylotypes versus
women without BV who had a mean of 3.3 phylotypes per clone library. Lactobacillus species, particularly Lactobacillus
crispatus and Lactobacillus iners were predominant in women without BV. L. crispatus was not detected in
subjects with BV, although L. iners was widely prevalent. Other bacteria
detected in subjects with BV included Gardnerella
vaginalis, Megasphaera, Leptotrichia, Dialister, Atopobium, and several bacterial vaginosis
associated bacteria (BVABs) from the Clostridiales order. Three novel bacteria from the Clostridiales order were highly specific indicators of BV [7]. BVAB1, BVAB2, and BVAB3 belong to the phylum
Clostridium but are not closely related to any bacteria with known 16S rRNA
gene sequences. A subject with incident
BV had a shift from a biota dominated by lactobacilli to one with increased
diversity including many putative anaerobes.
A subject with cured BV had an increase in lactobacilli clones and a
contraction in species diversity.  A
subject with relapsed BV had great diversity on day 0 with BV, followed by a
contraction to predominantly L. iners on day 28 with cure, and then an expansion of phylogenetically rich microbiota on
day 100 with relapse. A subject with
persistent BV had a consistently diverse vaginal biota on days 0, 24, and 64,
though there were some changes in species representation over time. A limitation of this study is that use of
ARDRA to screen clones for sequencing could have underrepresented the bacterial
diversity observed, as this approach tends to lump together different
phylotypes with similar sequences.
Moreover, only 100 clones were analyzed per library (or vaginal sample)
and this limited the detection of minority
species. In order to visualize the
bacteria, FISH was performed on vaginal smears targeting each of the novel
BVABs.  BVAB1 was shown to be a thin
curved rod (Figure 4); BVAB2 appears as a short, fat rod and BVAB3 is a long,
lancet-shaped rod. We have performed
transmission electron microscopy on a vaginal sample containing high levels of
BVAB1 as determined by broad range PCR with clone library analysis,
species-specific PCR, and FISH experiments.
The electron micrographs show long curved bacteria with a translucent
zone in the outer edge of the cells which we presume to be BVAB1 (Figure
5).  This is in contrast to the larger,
wider, and homogeneously electron dense cells observed in a transmission
electron micrograph of Mobiluncus
curtisii obtained from a pure culture.Figure 4
Fluorescence image of
vaginal fluid from a subject with BV. Bacteria are shown hybridizing with
probes targeting BVAB1 (green) and Mobiluncus (red) and visualized by fluorescence in situ hybridization (FISH).  Other bacteria (blue) are seen with 4′,6-diamidine-2-phenylindole, dihydrochloride, (DAPI), which stains DNA.  The inset shows that Mobiluncus (green) is larger than BVAB1 (red) but has the same curved
morphology. (With permission from D. N. Fredricks, T. L. Fiedler, and J. M.
Marrazzo, “Molecular identification of bacteria associated with bacterial
vaginosis,” New England Journal of
Medicine, vol. 353, pp. 1899–1911, 2005.)Transmission electron
micrographs. (a) Electron micrograph of
vaginal fluid from a woman with bacterial vaginosis and high concentrations of
bacterial vaginosis associated bacterium 1 (BVAB1) shows many curved rods with
an electron translucent zone at the outer edge of the cell. (b) These cells are different from the larger, wider, and more electron dense curved rods observed in a pure culture of Mobiluncus curtisii.   Both images are at
20 000x magnification.
(a)(b)We have further compiled clone
library data from subjects with and without BV (Figure6).  Using broad range bacterial PCR with 16S rRNA
gene primers 338f and 1407r, 1327 clones were sequenced from 13 subjects without
BV (Figure 6(a)).  Of the 1327 clones analyzed, 65.4% of the
sequences were Lactobacillus crispatus and 28.8% represented Lactobacillus iners clones.  The remaining 5.8% of clones
included other bacteria such as Gardnerella
vaginalis and other lactobacilli (Figure 6(a)).  These data further validate that subjects
without BV have vaginal bacterial biotas dominated by lactobacilli.  In contrast, analysis of 23 clone libraries
from 17 subjects with BV produced 2577 clones and demonstrated a very high
degree of bacterial diversity (Figure 6(b)).
Each subject with BV had an average of 14 species and the top 12
phylotypes accounted for 89% of clones sequenced.  The remaining 11% of sequences represented 32
phylotypes.  Currently, we do not
appreciate the role of the “long tail” of less prevalent bacteria though it is
likely that they contribute to metabolic and functional diversity in this
niche.  Moreover, the diversity of
bacteria observed in women with BV suggests that this may be a polymicrobial
syndrome.Summary data of rank
abundance plots depicting the bacterial species detected in clone libraries
from subjects without BV (A) and with BV (B) in our studies. Broad range PCR using primers 338f
and 1407r along with clone library analysis of 1327 clones from 13 subjects
without BV resulted in 16 phylotypes being detected.
Similar analysis of 2577 clones from 23 clone libraries from 17 subjects
with BV resulted in the detection of 44 different bacterial species.  Vaginal bacterial species are indicated on
the x-axis and the numbers of clones are indicated on the y-axis and above every
bar.  Subjects without BV have bacterial
biotas dominated by lactobacilli while subjects with BV have a diverse
bacterial biota.  BVAB denotes bacterial
vaginosis associated bacterium.
(a)(b)The use of broad range bacterial
PCR combined with cloning and sequencing provides a reasonable estimate of the
diversity of the most abundant bacteria but is an expensive approach with low
throughput. Thies et al. [98] used a combination of broad
range PCR amplification of the 16S rRNA gene in combination with T-RFLP
fingerprinting to characterize the vaginal bacterial communities in vaginal
swabs from 50 women with BV and 20 healthy women as determined by Nugent
scoring.  The authors propose that PCR
combined with T-RFLP is useful to rapidly assess the most abundant bacteria and
hence can be used as a tool to screen for BV.
Primers for amplification included 27f (27f-CC) and 926r and were
labeled at the 5′
using 6-carboxyfluorescein (6-FAM) and 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein (HEX),
respectively.  The restriction fragment
lengths were determined using an automated sequencer and the fragments were
analyzed using an in-house software program.
Identification of the fragments was verified by sequencing of the PCR
products. A total of 23 phylotypes were detected in the samples from subjects
with BV, with a mean of 6.3 phylotypes per subject (range 2–14) including Atopobium vaginae, Gardnerella vaginalis, Megasphaera sp., Lactobacillus iners, Eggerthella sp. and BVAB1, BVAB2, and
BVAB3.  Note that the species richness
detected in subjects with BV in this study was less than that reported by
investigators using different molecular approaches.  In concordance with the results obtained in
other studies [7, 44], Mobiluncus sp. was detected in only 2 of the 50 subjects with
BV.  Only lactobacilli including Lactobacillus iners, Lactobacillus crispatus group, and Lactobacillus gasseri group were
detected in samples from subjects without BV.
One of the limitations of this fingerprinting approach is the inability
to distinguish between closely related species.
For example, the study authors were unable to differentiate between Mobiluncus curtisii and Mobiluncus mulieris and also between the
different Prevotella phylotypes.  This resolution problem could account for the
low numbers of phylotypes per subject that was observed in this study.  A key strength of the study is the large
number of samples processed from subjects with/without BV defined by Gram
stain.Ferris et al. [97] PCR amplified a 300 bp portion of the
16S rRNA gene with broad range primers 1055f and 1392r from vaginal samples
obtained from subjects with and without BV as determined from vaginal fluid Gram
stains. The DNA was subjected to DGGE
and bands confirmed as Atopobium vaginae were identified in 12 of the 22 subjects with BV and only in 2 of the 24
control subjects. A. vaginae was also isolated by cultivation from 2 subjects and was
shown to be metronidazole resistant.  In
a separate study, A. vaginae-specific
PCR primers amplifying a 155 bp amplicon were applied to the same study cohort [99].  The specific primers further enhanced the
detection of A. vaginae in subjects
with BV while this bacterium was not detected in BV negative subjects leading
to the suggestion that A. vaginae is
highly specific for BV.  PCR
amplification using universal bacterial primers and T-RFLP studies also showed
a correlation of A. vaginae to BV by
Verstraelen et al. [100].Fredricks et al. [23] used a targeted PCR approach
to detect 17 key vaginal bacteria in a more sensitive fashion than is possible
with broad range PCR.  The PCR results
were compared with the current consensus diagnostic methods for BV in order to
determine if a qualitative PCR approach could be used for the molecular
diagnosis of BV.  Specific primers
targeting various regions of the 16S rRNA gene that are specific to the
bacterial species were designed.  The
bacteria were chosen based on clone library data previously generated [7], their apparent specificity
for BV, or their novelty.  All PCR
products were sequenced to confirm their similarity to the intended
target.  The primers were applied to 264
vaginal samples obtained from 81 subjects with BV and 183 subjects without
BV.  Bacteria from the Clostridiales order, Atopobium, an Eggerthella-like bacterium, Sneathia/Leptotrichia, Megasphaera types 1 and 2, and a
bacterium from the TM7 division were highly specific for BV. Lactobacillus
crispatus was inversely associated with BV with an odds ratio of 0.02
confirming that it is largely associated with healthy vaginal flora. Gardnerella
vaginalis, typically associated with BV, was found to have poor specificity
for BV. G. vaginalis was found in 96% of subjects with BV but was also
detected in 70% of the subjects without BV.
The combination of detecting one of the Clostridiales bacteria (BVAB2) or Megasphaera type 1 produced the best sensitivity and specificity
for PCR diagnosis of BV, regardless of the gold standard diagnostic criteria
employed (sensitivity 99% and specificity 89%).
This suggests that PCR amplification of key vaginal bacteria can indeed
be used for the molecular diagnosis of BV.
However, the approach used here requires electrophoresis to detect the
amplification products which may not be optimal in clinical settings.  A better approach would be to use
quantitative PCR that offers real-time results and the ability to quantify
bacteria.  Levels of the bacteria may be
a better indicator of disease than the presence/absence of particular species.Some studies have investigated the
utility of quantitative PCR (qPCR) as a diagnostic tool for BV.  Sha et al. [101] were the first group to
examine the use of qPCR for the diagnosis of BV, targeting Gardnerella vaginalis, Mycoplasma
hominis, and Lactobacillus species using 203 samples
from women with BV (Nugent score 7–10) and 203
samples from women without BV (Nugent score 0–3).  Only 75 of the 203 women with BV by Nugent
score were positive by Amsel criteria.
Increasing levels of G. vaginalis and M. hominis and decreasing levels
of lactobacilli were shown to be significantly associated with BV with a
sensitivity and specificity of 83% and 78% when compared with Nugent
score. The study did not evaluate women
with intermediate flora.In a subsequent study, Menard et
al. [102] also investigated the
association of Gardnerella vaginalis as well as Atopobium vaginae loads by
quantitative PCR and assessed their utility as a diagnostic tool in 231 samples
from 204 women.  Nugent criteria were
used to assess BV status, classifying 167 samples as normal flora, 20 samples
as BV, and 44 samples as intermediate flora.
They showed that the combination of the presence of A. vaginae at the DNA level  ≥108 copies/mL and G. vaginalis at  ≥109 copies/mL had a sensitivity and specificity of 95% and 99%,
respectively. However, subjects with intermediate flora were excluded from this
analysis.  Unfortunately, the promising results
from this study do not reflect how these assays would perform in a clinical
setting where all women are being screened for BV, including those with
intermediate flora on Nugent score. It would have been helpful to collect data
on Amsel clinical criteria in these women to assess BV status using an
alternative standard to determine the reliability of the molecular approach in
all women.  Another limitation is the
relatively small number of women with BV (20) in the study.  A smaller validation cohort of 56 women was
assessed, of which 7 were considered to have BV by Gram stain and 10
intermediate flora.  Eleven of these 56
women had molecular criteria for BV.  It
is not clear if the authors are proposing to treat all women with intermediate
flora for BV when they have molecular evidence of BV-associated bacteria.Zozaya-Hinchliffe et al. [103] assessed the prevalence and
abundance of uncultivated Megasphaera-like
bacteria in the vaginal niche using quantitative PCR targeting two Megasphaera phylotypes in a cohort of 41
women.  The subjects were diagnosed by
vaginal Gram stains and Amsel’s criteria.
Primers specifically targeting each type were tested for cross-reactivity
using vaginal clones. Megasphaera type 1 was detected in 76%
of the subjects while Megasphaera type 2 was found in 52% of the subjects.
Moreover, Megasphaera type 1
concentrations were higher in subjects with BV (up to 5 orders of magnitude)
than subjects without BV, and this bacterium was significantly associated with
BV (P=.0072), as was Megasphaera type 2 (P=.0366).  Phylogenetic
analysis of sequence data indicated that the Megasphaera phylotypes form two well-supported clades that do not
match sequences originating from the rumen, gut, or oral environments,
suggesting that these two phylotypes may be specific to the vaginal niche.Current treatment strategies for BV
include the administration of antibiotics either orally or topically.  The use of oral metronidazole for 7 days or
vaginal metronidazole for 5 days results in an improvement of symptoms in 83%–87% of women within 2 to 3 weeks [104, 105].  Similar response rates are observed with the
use of vaginal clindamycin.  Vaginal
recolonization rates with lactobacilli are similar with both antibiotics, as
defined by detection of lactobacilli on Gram stain 21–30 days after
start of antibiotic treatment [106, 107].  Although there is response to antibiotics in
many women, persistence or recurrence of the condition occurs in 11%–29% of women at 1 month [104, 108, 109].  Moreover, long-term recurrence rates have
been shown to be greater than 70% [19, 110, 111].  Marrazzo et al. [63] investigated several risk
factors for BV persistence one month after treatment, including the detection
of key vaginal bacteria by species-specific PCR.  Persistent
BV
was present in 25.8% of women at the 1-month
follow-up visit as determined by Amsel’s clinical criteria, also confirmed by
vaginal fluid Gram stains.
Taxon-specific PCRs targeting bacterial 16S rRNA genes were used to
detect BVAB1, BVAB2, BVAB3, Peptoniphilus
lacrimalis, Megasphaera type 2,
and Mobiluncus curtisii at baseline
and 1-month follow-up visits.  Data were
analyzed by presence or absence of the bacteria. Atopobium, Gardnerella vaginalis, Megasphaera type 1, and Lactobacillus iners were found in ≥96%
of subjects at baseline and therefore, these bacteria were not included in the
assessment of risk factors for persistence.
Women with BVAB1, BVAB2, or BVAB3 at baseline were shown to have a 2–8-fold increased
risk of persistent BV. Likewise,
presence of P. lacrimalis or Megasphaera type 2 at baseline imparted
a >3-fold increased risk of persistent BV.
Other risk factors such as sexual behaviors commonly linked with
persistence were also examined but were not associated with persistent BV in
this study.  A limitation of this
approach is that the persistence data was based on qualitative detection of
bacteria rather than quantitative analyses.
Quantitative PCR would help determine if the bacterial levels remain
unchanged during antibiotic treatment (antibiotic resistance), or if the levels
decline but bacteria are not eradicated, allowing for a future relapse.  Another limitation of this study is the
focus on women who have sex with women.
It is not clear if the same patterns will hold in heterosexual women
with BV.Oakley et al. [112] performed a systematic
analysis of bacterial diversity in women with and without defined BV,
incorporating data from Genbank that included publicly available 16S rRNA gene
sequence data obtained from the vaginal niche.
A total of 969 sequences were aligned and assigned taxonomic
classifications using the Greengenes 16S rRNA gene database [113].  The sequences were further analyzed based on
self-similarities rather than in comparison with an external database and
classified into operational taxonomic units (OTUs) using the DOTUR software
package [114] at a 97% sequence similarity
cutoff, which is commonly used for species definition [115].  Indeed, subjects with BV had a much greater
diversity of bacteria; at the 97% cutoff, women with BV had three times the
number of OTUs (15 OTUs) when compared with subjects without BV (5 OTUs). An interesting observation made
in this study was that even though there was quite a bit of variability in the
bacterial species between different subjects with BV, at the phylum level, the
presence of bacteria from Bacteroidetes and Actinobacteria was strongly
associated with BV. The authors point out that studies assessing bacterial
diversity in the vaginal niche might be underestimating the true diversity by
labeling bacteria with the NCBI-based designations that lump bacteria with
known species.  For example, sequences
classified as Prevotella using the
NCBI classification scheme of the Greengenes classification tool actually
represented 21 OTUs based on the 97% cutoff using the DOTUR analytical tool,
revealing an unexpectedly high number of vaginal phylotypes or species in this
genus.  These different vaginal
phylotypes may have different functional, metabolic, and inflammatory
properties.  A limitation of the study by
Oakley et al. [112] is that the Greengenes NCBI
classification tool used may assign different identities to the same sequence
simply based on sequence length.  For
instance, two sequences of 100% identity but different lengths can be
designated as either Gardnerella or Bifidobacterium. Similarly, two identical Atopobium sequences different only in sequence length can either be Atopobium or Olsenella. Sequences classified as Bifidobacterium in the NCBI classification scheme of the Greengenes
database were classified as Gardnerella in the RDP database.  This discrepancy
highlights the larger problem of defining bacterial nomenclature, which is a
continuing challenge for microbial ecologists.
One way of addressing this problem is to create a database of reference
sequences to which all new sequences from the same niche are submitted.  This would also allow rigorous tracking of
novel sequences.  As we develop greater
understanding of the ecology of the vaginal ecosystem, we hope that all
researchers will be able to use the same taxonomic nomenclature to facilitate
comparisons across studies. For example, there is a human oral microbiome
database that provides cross-referenced taxonomic and genomic information for
approximately 600 species (http://www.homd.org/) [116].Zhou et al. [56]
studied vaginal bacterial communities in Caucasian and African American women
in the United States
.  They applied T-RFLP analysis to 144 women
ranging equally in ages and racial groups from various locations in the US.  The subjects were classified as healthy based
on examinations by medical personnel, but again BV status was not reported
using either Amsel clinical criteria or Gram stain assessment of vaginal fluid.  Restriction fragment pattern analysis
resulted in the identification of 12 bacterial communities present in at least
2 women, and 8 communities present in single subjects.  Using broad range 16S rRNA gene PCR primers
8f (27f-CC) and RD1r [117], 57 clone libraries were
analyzed and ~6000 clones were sequenced.
Phylogenetic analysis of the 16S rRNA gene sequences obtained led to the
classification of the bacterial biota into 8 “supergroups.”  Five of the 8 supergroups were dominated by
lactobacilli, representing 80% of the women sampled.  Supergroup III, accounting for 16.5% of women
sampled, had low levels of lactobacilli and a diversity of bacteria that
multiple other groups have associated with BV, such as Atopobium vaginae, bacteria from the Clostridiales order, Megasphaera, Dialister, Anaerococcus, Finegoldia, Peptostreptcoccus, and Eubacterium.  Since objective criteria were not used to
assess for BV status (or were not reported), it is unclear if these subjects
had BV or whether BV-associated bacteria colonized women without BV in this
study.  The study authors analyzed
whether the bacterial community “supergroups” were associated specifically with
race.  Statistical analysis showed that
supergroups III and VIII (containing a single clade of Lachnospiraceae) were found more often in African American women.
Vaginal bacterial communities not dominated by lactobacilli were found in 33%
of African American women and 7% of Caucasian women.  It is known that African American women have
a higher rate of BV than Caucasian women [11].  The racial differences in vaginal microbiota
of “healthy” women noted in this study may simply reflect the failure to assess
for BV. A substantial fraction of women with BV are asymptomatic, therefore assessing
for BV status based on self-report of symptoms, as done in this study, is
unreliable. Nevertheless, the fact that African American women have a higher
prevalence of BV and therefore tend to have more diverse vaginal bacterial
communities begs for an explanation. Strengths of the paper include the large
number of samples processed, the use of T-RFLP to screen for community types,
and rigorous statistical analyses applied.
Limitations are the lack of objective diagnostic criteria for BV and use
of a 27f primer for broad range analysis with poor homology to some vaginal
bacteria that may account for the almost negligible abundance of Gardnerella vaginalis detected.  The meaning of bacterial community
“supergroups” is diminished when key members of the vaginal bacterial community
are underrepresented, though this is a problem that is shared by all studies
using broad range PCR to some degree.One study assessed the vaginal
microbiota from 16 women without BV (assessed by Nugent score<4), using a
PCR-based approach targeting the chaperonin-60 gene (cpn60) [118].  Chaperonin-60 is present in all bacteria and
is required for the folding and assembly of proteins and protein complexes.  Most subjects were colonized largely with
lactobacilli including Lactobacillus
crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners.  Other
sequences identified included those with similarity to Gardnerella vaginalis, Porphyromonas spp., Megasphaera spp, and Chlamydophila psittaci.  This is the only study that has examined the
diversity of bacteria in the vaginal niche using a different target gene.  This study provides a nice corroboration of
results from studies using the 16S rRNA gene as a target, wherein lactobacilli
have been shown to dominate the bacterial biota in subjects without BV.  The detection of C. psittaci as part of the normal vaginal flora is interesting and
rather surprising since this Chlamydia species is considered a respiratory and
zoonotic pathogen and has not been previously detected in the human vagina,
though it has been detected in the ovine vagina.  Using a different target gene offers a
different perspective on the constituents of a microbial community.  However, the limited database of cpn60 gene sequences may hinder accurate
bacterial identification and the generation of phylogenetic inferences.


## 9. Pyrosequencing: A High Throughput Sequencing Approach

While conventional sequencing
techniques have provided us with a framework, the true extent of bacterial
diversity in the vaginal niche is poorly understood.  Analysis of the sequence data from 100 even
1000 clones results in a library with a long tail of many phylotypes detected
as singlet clones when the data is represented in rank abundance plots.  Based on culture techniques, it is estimated
that the density of vaginal bacteria per gram of vaginal fluid ranges up to108 colony forming units [119].  If a subject has 108 bacteria/gm
of vaginal fluid and 100 clones are characterized, bacteria present at  106 CFU or below are less likely to be included in the analysis.  Moreover, classical clone library analysis
tends to provide less emphasis to the long tail of minority species [51].  In fact, the census of bacteria present at
low concentrations may provide important details about genetic and functional
diversity in this niche [51, 120, 121].  This is especially relevant in a syndrome
such as BV where we still do not understand the pathogenesis of infection.An alternate approach for obtaining
large numbers of sequences is by using pyrosequencing technology.  Pyrosequencing is a “sequencing by synthesis”
method which involves taking a single strand of DNA to be sequenced and
sequencing the complementary strand enzymatically while monitoring the photons
generated with the addition of each base [122].  The technology was applied on a small scale
level to identify isolates by analyzing the signature sequences of the V1 and
V3 regions of the 16S rRNA gene in 96 well plates [123, 124].  A disadvantage of the early approach was the
very short read lengths obtained (25 to 100 nucleotides long) limiting accurate
phylogenetic classification.Currently, pyrosequencing
technology has been further developed and it is now possible to achieve longer
reads of 250 to 300 bps in a throughput of 400 000 reads per 7.5 hour run which
can generate over 100 million bases (Genome Sequencer FLX System—454 Life Sciences).  The extracted DNA from the vaginal sample can
be amplified using fusion broad range primers (modified with adaptor sequences)
targeting the variable regions of the 16S rRNA gene.  The PCR products with the adaptor sequence
are attached to microscopic capture beads.
Emulsion-based clonal amplification (emPCR) can create several copies of
the target 16S rRNA gene sequence per bead without the need for cloning the
sequences into bacteria.  The beads are
then transferred to a picotitre plate for sequencing.  Pyrosequencing technology has been used for
microbial community analysis in a variety of environments [125–130].Sundquist et al. examined the
bacterial biota in vaginal samples from 6 pregnant women in all three
trimesters of pregnancy using broad range bacterial PCR with deep
pyrosequencing [129].  Most of the bacterial 16S rRNA gene was
amplified by PCR and portions of the gene were then subjected to
pyrosequencing.  A total of 100 000 to
200 000 sequence reads of about 100 bp average length were obtained for each of
the 6 samples.  Each read was processed
using the BLAT tool, a BLAST-like alignment tool [131], and a database of bacterial
sequences obtained from RDP and archaeal sequences from prokMSA. Two major
roadblocks were faced by the study investigators.  First, the short read lengths made it
challenging to assign phylogeny to the sequence reads.  For example, while 90% of the reads were
identified to the domain level, less than 10% were identified to the species
level.  Only about 50% of sequences were
unambiguously assigned to the class level, and this was likely due to the
amplification of both conserved and variable regions of the 16S rRNA gene,
limiting phylogenetic resolution.  The
authors also performed simulation calculations and showed that increasing
sequence read lengths up to 800 bp had significant impacts on phylogenetic
assignments.  Current 454 technology allows
a read length of 250 bp with 400 bp reads on the horizon or in place at the
time of this review.  The second
challenge encountered by the investigators was the lack of sequences in public
databases resulting in many bacteria being classified as “unknown.”  This problem will improve with time as more
sequences are added to the databases.  In
accordance with data obtained from conventional cloning and sequencing
experiments, the Sundquist study showed that subjects were largely colonized
with lactobacilli, with a variety of other bacteria at lower concentrations
including some such as Comamonas.  In our hands, Comamonas spp. are common PCR contaminants that are typically
present in water samples. As the study did not report results from negative
controls such as sham DNA extractions with PCR and subsequent pyrosequencing,
it is difficult to evaluate if Comamonas is indeed a part of the vaginal bacterial
biota. It is imperative to conduct
appropriate negative controls especially for pyrosequencing studies as the
technique involves deep sequencing and can therefore easily pick up
contaminating sequences even at low concentrations.


## 10. Bacterial Vaginosis: A Biofilm Syndrome?

Biofilms are
strongly associated with human infections and up to 65% of infections treated
by physicians in the developed world have been attributed to biofilms [132, 133].  There is emerging new evidence that biofilms
are associated with BV [134] and it has been suggested
that this biofilm may be critical in pathogenesis.  Swidsinski et al. [134] demonstated the presence of
adherent bacterial biofilms in 90% of subjects with BV while only 10% of
subjects without BV exhibited a similar biofilm.  Adherent biofilms were defined as lawns of
bacteria that were tightly attached to the vaginal epithelial surface and
contained specific bacterial groups.
Biopsies collected from women with and without BV were sectioned and
fixed for FISH and hybridized with a variety of bacterial rRNA-targeted
probes.  Typically, subjects with BV had
an adherent biofilm that was primarily composed with 3 bacterial groups: Gardnerella vaginalis was present in 60
to 90% of the biofilm mass, Atopobium accounted for 1 to 40% of the biofilm mass, and lactobacilli were present
between 1 to 5% in only 20% of the biopsy samples.  Subjects without BV either had no biofilms
with only a few lactobacilli scattered sporadically or had a loose bacterial
biofilm which did not have any particular structure and was mainly composed of Lactobacillus species.Preliminary data from our
laboratory also indicates the presence of adherent biofilms in subjects with BV
(Figure7).  Biopsies obtained from women
with and without BV were fixed in alcoholic formalin, sectioned and examined
using FISH with a suite of bacterial rRNA-targeted probes and 4′,6-diamidino-2-phenylindole
(DAPI), a DNA binding fluorescent stain. Our data also suggests the presence of a G. vaginalis biofilm in women with BV (Figure 7) while subjects
without BV did not have a biofilm but had scattered Lactobacillus species.Vaginal biopsy from a
subject with BV. A Gardnerella vaginalis biofilm (yellow) is detected at the edge of
the vaginal epithelium (bottom) by fluorescence in situ hybridization
(FISH).  The yellow color is the result
of using a combination of probes targeting G.
vaginalis (Red), all bacteria (Eub338, green), and 4′,6-diamidine-2-phenylindole, dihydrochloride
(DAPI, blue) which stains DNA.  Note
human cell nuclei in blue.  The image on
the right shows a vaginal epithelial cell with a cluster of G. vaginalis breaking off the epithelium
and likely forming a clue cell.
(a)(b)More recently, Swidsinski et al.
evaluated the effect of oral metronidazole on the BV biofilm [135].  A cohort of 18 subjects with BV, diagnosed by
Gram stains and Amsel criteria, were treated with oral metronidazole for 1
week.  Subsequently, follow up
assessments were conducted at 1-week intervals for 5 weeks, with 3 subjects
representing each point in time.  Vaginal
biopsies were examined using FISH probes targeting all bacteria or specific
bacteria such as Gardnerella vaginalis, Atopobium, Lactobacillus spp., Bacteroides/Prevotella, and Enterobacteriaceae.
Although, all subjects studied were considered cured of BV at the end of
the antibiotic therapy, vaginal biopsies revealed a persistent biofilm.  During antibiotic therapy, the biofilm could
be visualized with DAPI (a DNA stain) but had poor uptake of FISH probes
targeting rRNA suggesting that the bacteria were not actively
metabolizing.  However, at the end of 5
weeks, an actively metabolizing adherent bacterial biofilm was detected which
primarily consisted of G. vaginalis and Atopobium sp. [135]. Clinically, recurrence of BV was not
documented due to the limited follow-up time in the study.  Important limitations of this study, also
noted by the authors, include the small sample size and lack of baseline data.  Furthermore, the dataset was treated as
a longitudinal cohort but each time point represented a group of 3 different
subjects.  Despite these limitations,
this study represents a novel approach to understanding the pathogenesis of BV.Bacteria in biofilms respond
differently to antibiotic treatment when compared with their planktonic
counterparts [132, 136–138],
and antibiotic resistance is postulated as one of the reasons for persistent
and recurrent BV.  A study has shown
that planktonic Gardnerella vaginalis are more sensitive
to hydrogen peroxide (5-fold) and lactic acid (4–8-fold) than G. vaginalis biofilm bacteria,
highlighting the physiological differences that exist in the same organism
under different growth conditions [139].  Several explanations are provided in the
literature for the tolerance to antimicrobials by biofilm bacteria including
reduced penetration of the antimicrobials within the biofilm and alterations in
the stress physiology of the biofilm bacteria (reviewed in [140]).  In order to circumvent issues of antibiotic
resistance in bacterial biofilms, one study has used a probiotic approach to attempt
clearance of the G. vaginalis biofilm
[141]. G.
vaginalis biofilms grown in vitro
were displaced with Lactobacillus reuteri RC-14 and to a limited extent with Lactobacillus
iners, commonly found in the vaginal niche. Future studies evaluating the structure and composition of biofilms in BV will become critical in understanding the pathogenesis of this common condition.


## 11. Beyond Koch’s Postulates: Molecular Guidelines for Causation

Robert Koch and his students elaborated a series of postulates to determine which microbes caused diseases
and which microbes were colonizers without a direct etiological role (Table1,
Koch’s postulates).  The birth of modern
microbiology in the latter half of the 19th century necessitated a system to
gauge evidence of causation concordant with the discovery of numerous human and
animal associated microbes through laboratory propagation. These guidelines,
later called Koch’s postulates, are elaborated in Koch’s paper “On the Etiology
of Tuberculosis” where he beautifully lays out the foundation for his
thinking.  Robert Koch was a prescient
giant of microbiology whose thinking has served us well through more than a
century of use.  However, the power of
Koch’s postulates arises not from their rigid application, but from the spirit
of critical judgment that they foster.Table 1
Koch’s postulates [1].
The etiologic microbe should be found in every case of the diseaseThe etiologic microbe should not be found in subjects without disease (specificity)The etiologic microbe should be isolated in pure culture on lifeless media and be capable of causing the characteristic disease anew upon inoculation in a susceptible hostThe etiologic microbe should be reisolated from the experimentally inoculated host.The esteemed researcher Edward
Rosenow provided evidence that a streptococcus was the cause of poliomyelitis
by fulfilling Koch’s postulates [142–144],
only to have this theory overturned with the discovery of poliovirus decades
later. The lack of specificity
demonstrated by Rosenow’s false attribution of causation to streptococci in the
case of polio highlights only one of many possible limitations of Koch’s
postulates that have emerged after more than a century of reflection (Table 2).
These limitations do not seriously undermine the generally highly specific
ability of Koch’s postulates to identify true pathogens. If a pathogen fulfills Koch’s postulates
then it is most likely the cause of the disease, though these results need to
be reproducible and consistent. In the
case of Gardnerella vaginalis and BV,
the ability of a pure culture of G.
vaginalis to produce BV in 1 of 13 inoculated subjects is not a very
compelling argument for causation without a better explanation for the 92%
failure rate (see Section 4).
Taken to its logical extreme, the successful induction of AIDS in 1 of
1000 subjects inoculated with Mycoplasma would also not “fulfill” Koch’s third postulate for the role of Mycoplasma in AIDS in any meaningful or
rigorous fashion. Nevertheless, the
experimental reproduction of disease using pure cultures of microbes is the
most powerful single approach for establishing a causal connection between a
microbe and a disease. On the other
hand, the failure to fulfill Koch’s third postulate does not mean that a
microbe is not the cause of a disease.
Koch’s postulates have excellent specificity for causation, but poor
sensitivity. For example, many microbes
have not been successfully propagated in pure culture in the laboratory; these
microbes cannot fulfill Koch’s postulates as originally defined. The historical evolution in thinking about
causation and Koch’s postulates is described elsewhere [1, 2].Table 2
Limitations of Koch’s postulates.
Ignore the
contribution of host, vector, and environment to disease susceptibility/responseColonization
state (e.g., +PPD skin test for tuberculosis in the absence of disease)
violates Koch’s second postulateMany pathogens cannot be
propagated on lifeless (cell-free) medium in the lab; these pathogens cannot
fulfill Koch’s third postulateViruses, parasites, uncultivated
bacteria may not grow in pure cultureHost range restriction of
pathogensDo not consider the possibility
of disease produced by a microbial community rather than a single pathogenNot completely specific


## 12. A Molecular Version of Koch’s Postulates

A major limitation of Koch’s
postulates is the failure to account for the possibility that uncultivated
microbes play a role in disease.  The use
of molecular methods to characterize microbial diversity in many niches has
revealed that cultivated species constitute a minority of microbes in many
ecosystems, including in the human body. Many potential pathogens can be
readily detected using molecular methods such as PCR.  Koch’s postulates can be directly translated
into molecular versions, as follows.(1)
The etiologic microbe or its
nucleic acid sequences should be found in every case of disease. This implies
that the microbe (or its products) is a sensitive indicator of disease.(2)
The etiologic microbe or its
nucleic acid sequences should not be found in subjects without disease. This
implies that the microbe is a specific indicator of disease.(3)
Experimental manipulation of
infection through factors such as antimicrobial agents or induction of immune
responses should demonstrate that changes in levels of an etiologic microbe
correlate with disease state in the host.


## 13. Disease by Microbial Community

There are some disease syndromes that may be
caused by consortia of microbes rather than single pathogens.  Examples of these polymicrobial syndromes are
gingivitis, periodontitis, and BV.  Proving
that a single cultivated or uncultivated microbe is the cause of a disease can
be challenging.  Proving that a microbial
consortium is the cause of a disease is even more daunting.Microbes probably exist in
communities in order to take advantage of syntrophic relationships wherein the
metabolic end product of one species is the energy source for a second
species.  If critical members of the
community are lost, then the metabolic networks collapse and all members of the
community may suffer.  However,
functional redundancy among microbes may mean that bacterium A is not necessary
for community health as long as bacterium B is present with its overlapping
metabolic capacity.  What does this mean
if bacteria A and B
are part of a pathogenic community?  It
means that neither bacterium will be deemed necessary for disease, because
subjects may have disease when lacking bacterium A or B, though subjects will
not have disease if lacking both bacteria.
Bacteria A and B are considered sufficient when part of the larger community, but not individually necessary
for establishing the community and producing condition. To address this issue, we
will need to assess not only the species composition of pathogenic microbial
communities, but also the metabolic capabilities and interdependencies of these
communities. Studies of the human microbiome will be vital in filling this knowledge
gap.


## 14. Conclusions

In the last two decades, there has
been a dramatic increase in our understanding of the bacterial biota in a variety
of ecological environments using cultivation-independent molecular
methods.  These methods have recently
been applied to the human vaginal microbial ecosystem, adding substantial data
on bacterial diversity in this niche.
Subjects without BV have bacterial biotas that are less complex and are
dominated byLactobacillus species.  Subjects with BV have loss of Lactobacillus crispatus and acquisition
of more complex vaginal bacterial communities that include many
heretofore-uncultivated species.  Data emerging
from molecular investigations suggest that it is possible to develop a
PCR-based strategy for the diagnosis for BV.
BV may be an example of a condition produced by a pathogenic microbial
community rather than a single pathogen, presenting many challenges for
understanding the etiology and pathogenesis of this syndrome.  A molecular version of Koch’s postulates is
presented for collecting evidence of causation for uncultivated microbes such
as those linked to BV.  There is new
evidence suggesting that BV may be a biofilm condition in some women, which may
contribute to poor treatment responses and high relapse rates.  Understanding the bacterial biota of the
human vagina is critical for optimizing reproductive health, and although many
advances have been made, there is much that is unknown about how bacterial
communities in the human vagina promote health and facilitate disease.



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