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REVIEW
published: 02 June 2015
doi: 10.3389/fphys.2015.00164
Frontiers in Physiology | www.frontiersin.org 1June 2015 | Volume 6 | Article 164
Edited by:
Rita Verhelst,
Ghent University, Belgium
Reviewed by:
Sarah Lebeer,
UAntwerpen, Belgium
Deborah Jean Anderson,
Boston University School of Medicine,
USA
*Correspondence:
Gilda Tachedjian,
Centre for Biomedical Research,
Burnet Institute, 85 Commercial Rd.,
Melbourne, VIC 3004, Australia
gildat@burnet.edu.au
Specialty section:
This article was submitted to
Clinical and Translational Physiology,
a section of the journal
Frontiers in Physiology
Received: 18 February 2015
Accepted: 12 May 2015
Published: 02 June 2015
Citation:
Aldunate M, Srbinovski D, Hearps AC,
Latham CF, Ramsland PA, Gugasyan
R, Cone RA and Tachedjian G (2015)
Antimicrobial and immune modulatory
effects of lactic acid and short chain
fatty acids produced by vaginal
microbiota associated with eubiosis
and bacterial vaginosis.
Front. Physiol. 6:164.
doi: 10.3389/fphys.2015.00164
Antimicrobial and immune
modulatory effects of lactic acid and
short chain fatty acids produced by
vaginal microbiota associated with
eubiosis and bacterial vaginosis
Muriel Aldunate 1, 2, Daniela Srbinovski 1, 2, Anna C. Hearps 1, 3, Catherine F. Latham 1,
Paul A. Ramsland 1, 4, 5, 6, Raffi Gugasyan 1, 4, Richard A. Cone 7and Gilda Tachedjian 1, 2, 3, 8*
1Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia, 2Department of Microbiology, Nursing and
Health, Faculty of Medicine, Monash University, Clayton, VIC, Australia, 3Department of Infectious Disease, Monash
University, Melbourne, VIC, Australia, 4Department of Immunology, Monash University, Melbourne, VIC, Australia,
5Department of Surgery Austin Health, The University of Melbourne, Heidelberg, VIC, Australia, 6School of Biomedical
Sciences, CHIRI Biosciences, Curtin University, Perth, WA, Australia, 7Department of Biophysics, Johns Hopkins University,
Baltimore, MD, USA, 8Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty
Institute for Infection and Immunity, Parkville, VIC, Australia
Lactic acid and short chain fatty acids (SCFAs) produced by vaginal microbiota
have reported antimicrobial and immune modulatory activities indicating their potential
as biomarkers of disease and/or disease susceptibility. In asymptomatic women of
reproductive-age the vaginal microbiota is comprised of lactic acid-producing bacteria
that are primarily responsible for the production of lactic acid present at ∼110 mM and
acidifying the vaginal milieu to pH ∼3.5. In contrast, bacterial vaginosis (BV), a dysbiosis
of the vaginal microbiota, is characterized by decreased lactic acid-producing microbiota
and increased diverse anaerobic bacteria accompanied by an elevated pH>4.5. BV
is also characterized by a dramatic loss of lactic acid and greater concentrations of
mixed SCFAs including acetate, propionate, butyrate, and succinate. Notably women
with lactic acid-producing microbiota have more favorable reproductive and sexual health
outcomes compared to women with BV. Regarding the latter, BV is associated with
increased susceptibility to sexually transmitted infections (STIs) including HIV. In vitro
studies demonstrate that lactic acid produced by vaginal microbiota has microbicidal
and virucidal activities that may protect against STIs and endogenous opportunistic
bacteria as well as immune modulatory properties that require further characterization
with regard to their effects on the vaginal mucosa. In contrast, BV-associated SCFAs
have far less antimicrobial activity with the potential to contribute to a pro-inflammatory
vaginal environment. Here we review the composition of lactic acid and SCFAs in
respective states of eubiosis (non-BV) or dysbiosis (BV), their effects on susceptibility
to bacterial/viral STIs and whether they have inherent microbicidal/virucidal and immune
modulatory properties. We also explore their potential as biomarkers for the presence
and/or increased susceptibility to STIs.
Keywords: lactic acid, short chain fatty acids, bacterial vaginosis, lactobacilli, vaginal microbiota, metabolites,
microbiome
Aldunate et al. Activity of vaginal microbiota SCFAs
Introduction
The lower female reproductive tract, specifically the vagina and
ectocervix, is considered a formidable chemical and physical
barrier to exogenous invading organisms, in part due to the
structure of the stratified vaginal epithelium and the presence
of cervicovaginal fluid (CVF). Eubiotic viscoelastic CVF acts
as an effective lubricant, facilitates the trapping of exogenous
organisms and, acts as an acidified medium in which there
is an arsenal of antimicrobial molecules (antibodies, defensins
etc.). Importantly, this mucosal layer (mucus and layers of
dead epithelial cells) also enables the adhesion of mutualistic
vaginal microbiota (Boris et al., 1998). In asymptomatic women,
microbiota acidify the vagina through lactic acid-production
whilst bacterial vaginosis-associated bacteria (BVAB) produce
many short chain fatty acids (SCFAs) that contribute the
development of a dysbiotic vaginal environment (Aroutcheva
et al., 2001a,b; Valore et al., 2002; Yeoman et al., 2013). The
study of asymptomatic reproductive-age women has revealed
that vaginal microbiota are unique to each woman but are largely
dominated by lactic acid-producing bacteria, most often from
the Lactobacillus genus (Verhelst et al., 2004; Fredricks et al.,
2005; Ravel et al., 2011). Despite the differences in bacterial
composition of vaginal communities, it seems that acidifying
the vaginal milieu via generation of organic acid metabolites,
predominantly lactic acid, is a conserved function (Ravel et al.,
2011; Gajer et al., 2012). Humans are unique among primates in
supporting lactic acid-producing vaginal microbiota (Mirmonsef
et al., 2012a; Yildirim et al., 2014), particularly lactobacilli, which
are largely responsible for acidifying the vaginal milieu with
∼110 mM of lactic acid by anaerobic glycolysis of glycogen
released from shed and shedding vaginal epithelial cells (Stanek
et al., 1992; Boskey et al., 1999, 2001). This ultimately acidifies the
vagina to an average pH ∼3.5 (Fox et al., 1973; O’Hanlon et al.,
2013), with both D and L optical isomers of lactic acid that differ
in their arrangement of the same chemical components around
a central carbon. The D/L ratio of lactic acid isomers matches
the ratio produced by lactobacilli cultured from the same vaginal
sample of CVF (Boskey et al., 2001)strongly indicating that lactic
acid produced by lactobacilli is responsible for vaginal acidity. In
women with lactobacillus-dominated flora, the vaginal pH tightly
correlates with lactic acid concentration in CVF (O’Hanlon
et al., 2013) suggesting that lactic acid is likely responsible for
generating the protective acidic environment considered to be so
important for antimicrobial activity.
Bacterial vaginosis (BV) is largely defined as a dysbiosis
of the vaginal microbiome and is the most common and
enigmatic vaginal condition in reproductive-age women. BV is
characterized by a loss of lactic acid-producing bacteria and
an increase in the number and diversity of anaerobic bacteria.
Symptoms include an elevated vaginal pH >4.5 and malodorous
abnormal discharge (Spiegel et al., 1980; Ling et al., 2010).
BV is commonly diagnosed using two methods, the Amsel
criteria or the Nugent Gram stain scoring method. A positive
diagnosis of BV requires that three of the four Amsel criteria be
satisfied; thin homogenous discharge, pH >4.5, amine odor and
shed epithelial cells covered with bacteria (clue cells), whereas
the Nugent Gram stain scoring method enumerates bacterial
morphotypes associated with health (Gram-positive lactobacilli)
and BV-associated morphotypes (Gram-variable rods). A low
Nugent score (0–3) is indicative of healthy microbiota and a
high Nugent score (7–10) is a positive diagnosis for BV (Nugent
et al., 1991). Using this method, women diagnosed with BV
may have a high Nugent score in the presence (symptomatic
BV) or the absence (asymptomatic BV) of the aforementioned
symptoms. BV risk factors commonly include menses, sexual
activities and hygiene practices (Schwebke et al., 1999; Jespers
et al., 2012; Srinivasan et al., 2012; Yeoman et al., 2013). BV
is estimated to affect 5–15% Caucasian women, 20–30% Asian
women, ∼30% of Hispanic women, and 45–55% of Black women
(Sewankambo et al., 1997; Schneider et al., 1998; Allsworth and
Peipert, 2007; Bhalla et al., 2007; Fang et al., 2007; Koumans
et al., 2007; Oliveira et al., 2007; Akinbiyi et al., 2008; Thoma
et al., 2011). This is mirrored by global BV prevalence estimates
showing a general trend toward high prevalence in Africa and
low prevalence in Europe and Asia (Kenyon et al., 2013). BV
plays a significant role in public health due to is association
with various reproductive sequelae (Hillier et al., 1995; Donders
et al., 2000; Ness et al., 2005; Leitich and Kiss, 2007) and
increased susceptibility to sexually transmitted infections (STIs)
including the human immunodeficiency virus (HIV) (Taha et al.,
1998; Cu-Uvin et al., 2001; Cherpes et al., 2003; Wiesenfeld
et al., 2003; Allsworth and Peipert, 2007; Coleman et al.,
2007; Kaul et al., 2007; Atashili et al., 2008; Cohen et al.,
2012).
Studies investigating the metabolites produced by
lactobacillus-dominated microbiota and the polymicrobial
BV states have observed a striking loss of lactic acid and a
shift toward mixed SCFA (SCFA) production during BV (Al-
Mushrif et al., 2000; Mirmonsef et al., 2011, 2012c; Gajer et al.,
2012; O’Hanlon et al., 2013; Yeoman et al., 2013). Generally,
acetate is the predominant metabolite in the CVF of women
with symptomatic BV in addition to increased concentrations
of propionate, butyrate and succinate (Stanek et al., 1992;
Chaudry et al., 2004). Lactic acid and SCFA metabolites found
in CVF have reported microbicidal, virucidal, and immune
modulatory activities indicating their potential as biomarkers
of disease and/or disease susceptibility (Al-Mushrif et al., 2000;
O’Hanlon et al., 2011; Mirmonsef et al., 2012c; Aldunate et al.,
2013).
The Vaginal Microbiota
Albert Döderlein first described the presence of Gram-positive
bacilli and low vaginal pH in the CVF of healthy reproductive-
age women in 1892 (Döderlein, 1892). Later known as the
Lactobacillus acidophilus complex (Thomas, 1928), lactobacilli
came to be regarded as the foundation of healthy vaginal
microbiota. In contrast, a paucity of lactobacilli and an
increase in other bacterial morphotypes could be associated
with symptomatic vaginal discharge (Döderlein, 1892; Curtis,
1914; Spiegel et al., 1980; Amsel et al., 1983; Jokipii et al.,
1986). Many of the concepts of what could be considered
Frontiers in Physiology | www.frontiersin.org 2June 2015 | Volume 6 | Article 164
Aldunate et al. Activity of vaginal microbiota SCFAs
healthy or unhealthy were based on distinctions made from
microscopy and culture-dependent techniques; however, many
also acknowledged that fastidious microbiota members were
unlikely to be cultured. Nonetheless, the paradigm that
lactobacilli equate to a healthy vaginal ecosystem became
established and the true complexity of the microbiome
would remain obscure until the advent of culture-independent
molecular techniques.
Cross-sectional Studies of the Vaginal
Microbiome in Healthy Asymptomatic Women
Early cross-sectional studies revealed that the microbiota of
asymptomatic reproductive-age women are indeed dominated by
lactic acid-producing bacteria, primarily Lactobacillus (Verhelst
et al., 2004; Zhou et al., 2004, 2007; Fredricks et al., 2005; Oakley
et al., 2008). New members of the vaginal microbiome were also
identified including Atopobium and Lactobacillus iners (Burton
and Reid, 2002; Zhou et al., 2004). Later, Ravel et al. (2011)
employed next generation sequencing in their landmark study
to characterize the vaginal microbiomes of 396 ethnically diverse
women of reproductive-age, revealing five core microbiomes
termed community state types (CSTs). Four CSTs were described
according to the dominant Lactobacillus species; L. crispatus,L.
gasseri,L. iners, and L. jensenii corresponding to CST I, II, III, and
V (Table 1). Collectively, Lactobacillus was the dominant species
in 73% of the vaginal communities in this study group of healthy
women. These four Lactobacillus species appear to represent the
most prevalent bacteria in reproductive-age women, consistent
with many previous investigations (Table 1) (Zhou et al., 2009,
2010; Forney et al., 2010; Ravel et al., 2011; Yoshimura et al.,
2011; Martin et al., 2012; Smith et al., 2012). Importantly, this
study also highlighted that 27% of women had communities
characterized by microbial diversity assigned to CST IV (Ravel
et al., 2011). Here, two subgroups IV-A or IV-B contained
modest or no lactobacilli, respectively, in addition to several
anaerobic bacteria known to produce lactic acid (Table 1). These
community members are primarily strict or facultative anaerobes
some of which are traditionally associated with dysbiosis, as in
BV and include; Atopobium, Gardnerella Prevotella, Mobiluncus,
Megasphaera, Dialister, Sneathia, Streptococcus, Pseudomonas,
Leptotrichia, and Aerococcus amongst others. Although there is
some debate as to whether these women are actually healthy
and not asymptomatic for BV, it is worth noting a multitude of
previous studies using various molecular methods also identified
at least one group in their phylogenetic analyses that is analogous
to the microbially diverse CST IV (Kim et al., 2009; Yamamoto
et al., 2009; Forney et al., 2010; Zhou et al., 2010; Jespers et al.,
2012). Therefore, it seems that bacteria traditionally associated
with BV due to a high bacterial load, are likely members of
eubiotic vaginal communities that have been overlooked in the
past (Antonio et al., 1999; Pavlova et al., 2002; Zhou et al.,
2004; Biagi et al., 2009; Yamamoto et al., 2009). Recent studies
now show that 20–30% of women have diverse microbiomes not
dominated by Lactobacillus that are not classically considered
normal or healthy (Zhou et al., 2004, 2007, 2010; Srinivasan and
Fredricks, 2008; Schellenberg et al., 2009; Ravel et al., 2011, 2013;
Gajer et al., 2012).
Temporal Studies of the Vaginal Microbiome and
Community Dynamics using Molecular
Characterization
Numerous cross-sectional studies have helped revise our
understanding of bacterial communities in the vaginal
ecosystem. However, this ecosystem is not static, there are
many disturbances through continual efflux of CVF containing
bacteria, antimicrobials, and sloughed epithelial cells from
constant renewal of the vaginal epithelium. There are also
cyclical changes in estrogen, glycogen content, pH and menses
TABLE 1 | Microbial communities and metabolite spectrum in the vaginal ecosystem and their proposed relationship to risks of adverse sexual and
reproductive outcomes.
Community State Type (CSTs) pH Metabolite profile
IL. crispatus 4.0a
II L. gasseri 5.0a
VL. jensensii 4.7a
III L. iners (ubiquitous regardless of BV status) 4.4a
IV-A Modest levels of L. crispatus, L. iners or other
Lactobacillus sp. with low proportions of
Streptococcus, Anaerococcus,
Corynebacterium and Finegoldia 5.3a
IV-B Relatively high levels of Atopobium,
Gardnerella, Mobiluncus, Peptoniphilus,
Sneathia, Prevotella and several other taxa of
BVAB
BV Polymicrobial
Increased diversity and bacterial load of BVAB
>4.5
apH was measured using a VpH glove test strip with pH determined according to color chart (Ravel et al., 2011) pH is likely to be lower if measured under vaginal hypoxic conditions
with a pH electrode (O’Hanlon et al., 2013).
Frontiers in Physiology | www.frontiersin.org 3June 2015 | Volume 6 | Article 164
Aldunate et al. Activity of vaginal microbiota SCFAs
as well as the introduction of exogenous bacteria due to human
activities such as sex and hygiene practices (Srinivasan et al.,
2010; Gajer et al., 2012; Yeoman et al., 2013). Therefore, studies
that analyze samples from a single time point merely offer a
glimpse of the microbiome as it is affected by and responds to
these intrinsic and extrinsic disturbances. The dynamic nature of
the vaginal ecosystem can be assessed with temporal longitudinal
studies that complement cross-sectional findings by evaluating
shifts in community composition and structure, and their
stability over time.
Overall, longitudinal studies reveal that the temporal
dynamics of vaginal microbiota is unique to each reproductive-
age woman (Santiago et al., 2011; Gajer et al., 2012; Hickey
et al., 2013), with some possessing relatively stable communities
(Wertz et al., 2008; Biagi et al., 2009; Santiago et al., 2011; Gajer
et al., 2012; Jespers et al., 2012) and others experiencing brief
albeit dramatic shifts (Brotman et al., 2010b; Gajer et al., 2012;
Ravel et al., 2013). Altered community dynamics were commonly
associated with menses (Srinivasan et al., 2010; Santiago et al.,
2011; Gajer et al., 2012; Jespers et al., 2012; Hickey et al., 2013),
sex (Srinivasan et al., 2010; Jespers et al., 2012; Santiago et al.,
2012), and bacterial composition within a CST (Verstraelen et al.,
2009; Gajer et al., 2012). Conversely, a high level of estrogen
alone or estrogen and progesterone were associated with greater
community stability (Gajer et al., 2012); this effect was also seen in
pregnant women with high estrogen levels (Romero et al., 2014).
Recent studies have highlighted that shifts in vaginal
community composition seem to be preferential (Verstraelen
et al., 2009; Gajer et al., 2012; Smith et al., 2012; Romero
et al., 2014). L. crispatus (CST I, Table 1) has been reported to
experience few transitions to other CSTs and seems to be quite
stable and promote vaginal community stability (Verstraelen
et al., 2009; Gajer et al., 2012). L. crispatus is common in
Caucasian and Asian women and less so in Black and Hispanic
women (Zhou et al., 2004; Ravel et al., 2011; Jespers et al., 2012),
and is present primarily in healthy asymptomatic women whilst
L. iners is ubiquitous, even in women with dysbiosis such as
BV (Verhelst et al., 2004; Zozaya-Hinchliffe et al., 2008; Biagi
et al., 2009). It is therefore not surprising that microbiomes
dominated by L. iners (CST III, Table 1) exhibit the greatest
variation in composition and stability. One study reported
that L. iners was twice as likely to transition to the diverse
CST IV-B than IV-A (Gajer et al., 2012) meanwhile another
concluded that L. gasseri/L. iners signify a predisposition to the
occurrence of dysbiotic vaginal bacteria (Verstraelen et al., 2009).
However, little is known about L. gasseri and L. jensenii and non
lactobacillus-dominated microbiomes (CST IV, Table 1), because
they are less prevalent in reproductive-age women. The observed
shifts in vaginal communities has led to the proposition of an
ecological hypothesis describing the maintenance of equilibrium
in the vagina by transitioning between alternative communities
whereby not all are favorable or conducive to maintaining
community function (Ravel et al., 2011; Gajer et al., 2012).
Role of Lactic Acid Producing Vaginal Microbiota
in the Maintenance of Community Function
Previous studies have cited the production of lactic acid
as a major conserved characteristic maintained through the
functional redundancy exhibited by members of a transitioning
community (Zhou et al., 2004; Linhares et al., 2010; Ravel
et al., 2011; Gajer et al., 2012). This finding lends credence to
the notion that several lactic acid-producing microbiota act to
conserve community function and that variability in community
composition is not necessarily a precursor to dysbiosis that
leads to pathology. The vagina is subject to many disturbances;
it is therefore not surprising that the ecosystem has evolved
to accommodate changes in community composition through
divergent organisms that perform a common function since
vaginal homeostasis is more likely to be maintained. The range
of lactic acid-producing bacteria commonly found in the vagina
may therefore represent a group of particularly adept allelopathic
organisms that use acid fermentation products, especially lactic
acid, to suppress or otherwise outcompete other organisms
and interact with the host in a manner that favors their
competition. It follows that different community types may
have varying levels of functionally redundant microbiota that
maintain community performance; hence may exhibit varying
degrees of resilience in the face of disturbances that could
influence susceptibility to disease when combined with host
determinants (Table 1). The incredibly complex and dynamic
nature of the microbiome brings to light the numerous
obstacles that must be surmounted by researchers who seek
to uncover factors that elicit changes in the microbiome
and consequently maintain eubiosis or lead to a dysbiotic
state associated with risks of disease. It also highlights the
need to move away from merely identifying what organisms
are present toward determining their metabolic capacity,
antimicrobial, immune modulatory effects, and function in the
microbiome.
Immune Modulation by the Vaginal Microbiome
The vaginal mucosal epithelium acts both as a physical
barrier and immunological mediator providing the first defense
against potential infections (Kaushic, 2011; Anderson et al.,
2014). Mucosal epithelia generally comprise multiple layers
of rarely keratinized stratified squamous epithelium that rests
on a lamina propria where the uppermost apical layers lack
tight junctions (Blaskewicz et al., 2011; Anderson et al.,
2014). These layers are permeable to water, soluble proteins,
viruses, and penetrable by vaginal microbiota as well as
cellular (e.g., CD4+T cells and macrophages) and molecular
mediators of the immune system (e.g., cytokines) (Blaskewicz
et al., 2011; Carias et al., 2013; Politch et al., 2014).
The vagina and endocervix provide immunological defenses
by conferring tolerance to microbes, maintaining epithelial
integrity, and recruiting and supporting immune cells (Fichorova
and Anderson, 1999). As the front line of immune defense,
epithelial cells express pattern recognition receptors (PRRs)
including Toll-like receptors (TLRs) that respond to microbe-
or pathogen-associated molecular patterns (MAMPs/PAMPs)
(Table 2) by secreting cytokines and chemokines, antimicrobial
peptides and other immune factors (Fichorova and Anderson,
1999; Herbst-Kralovetz et al., 2008; Kaushic, 2011; Rose et al.,
2012). The pro-inflammatory response elicited by pathogens
is normally required to control infections (Sandler et al.,
2014). However, vaginal mucosal inflammation can promote
Frontiers in Physiology | www.frontiersin.org 4June 2015 | Volume 6 | Article 164
Aldunate et al. Activity of vaginal microbiota SCFAs
TABLE 2 | The effects of lactic acid and BV-associated SCFAs on TLR responses.
PRR Common MAMPs/PAMPs Organism
recognized
Effect of lactic acid or SCFAs
TLR1a,b
Lipopeptides, lipopolysaccharide, peptidoglycan,
flagellin
Bacteria
ND
TLR2a,b
Acetate and butyrate induced production of pro-inflammatory
cytokines IL-8, TNFαand IL-1βfrom human PBMCs and potentiated
pro-inflammatory responses to TLR2 ligandsc
TLR6a,bND
TLR3a,bDouble stranded ribonucleic acid (ds RNA) Virus In the VK2/E6E7 human vaginal epithelial cell line, L-lactic acid and poly
I:C treatment led to the production of the pro-inflammatory immune
mediators IL-8 and IL-1βin a standard tissue culture plate setupd.
TLR4a,bLipopolysaccharide
Lipoteichoic acid
Viral envelope proteins
Protozoal phospholipids
Gram negative bacteria
Gram positive bacteria
Virus
Trichomonas vaginalis
L-lactic acid and lipopolysaccharide increased IL-23 production from
human PBMCs, may lead to preferential stimulation of Th17
T-lymphocytes that protect mucosa from extracellular bacteriae
TLR5a,bFlagellin Bacteria ND
TLR7a,b
Single stranded ribonucleic acid (ss RNA) Virus
Acetate and butyrate induced the production of pro-inflammatory
cytokines of IL-8, TNFαand IL-1βfrom human PBMCs and potentiated
pro-inflammatory responses to TLR7 ligandsc
TLR8a,bND
TLR9a,bUn-methylated components of nucleic acid Bacteria
Virus
ND
ND-Not determined.
aNasu and Narahara (2010).
bHerbst-Kralovetz et al. (2008).
cMirmonsef et al. (2012c).
dMossop et al. (2011).
eWitkin et al. (2011).
transmission of viral STIs, such as HIV, by compromising
epithelium integrity (Nazli et al., 2010) and by recruiting and
activating HIV target cells (Li et al., 2009). Epithelial cell-
derived immune mediators have pivotal roles in cell recruitment,
immune regulation and tissue repair (Fichorova and Anderson,
1999). Given the intimate contact between the microbiota
and their acid metabolites with the vaginal epithelium it is
important to study how these interactions modulate mucosal
immunity.
Lactic acid-producing bacteria are known to maintain
homeostasis by attenuating inflammation in the gut and by
preserving gut barrier function (Zeuthen et al., 2008; Donato
et al., 2010; Santos Rocha et al., 2012; Castillo et al., 2013; Perez-
Santiago et al., 2013). Vaginal microbiota are reported to mediate
immune modulatory effects on the vaginal mucosa (Al-Mushrif
et al., 2000; Sakai et al., 2004; Nikolaitchouk et al., 2008; Joo
et al., 2011; Kyongo et al., 2012). Studies in women from distinct
geographical regions colonized with lactobacillus-dominated
microbiota suggest an absence of vaginal inflammation, which
may in part depend on the dominant Lactobacillus species.
Specifically, vaginal lactobacilli are associated with lower levels
of pro-inflammatory cytokines in CVF (e.g., IL-1αand/or IL-
8) (Sakai et al., 2004; Nikolaitchouk et al., 2008; Kyongo et al.,
2012), and in the case of L. iners, higher levels of secretory
leukocyte peptidase inhibitor (SLPI) (Nikolaitchouk et al., 2008),
an antimicrobial peptide normally depleted in women with
dysbiosis such as BV (Draper et al., 2000; Mitchell et al.,
2009; Balkus et al., 2010; Dezzutti et al., 2011). The presence
of L. crispatus and L. jensenii were negatively associated with
cellular inflammatory markers (Kyongo et al., 2012). In contrast,
a study in adolescents failed to show differences for most
cytokines in CVF from young women with dysbiosis compared
to women with lactobacillus-dominated microbiota (Alvarez-
Olmos et al., 2004), although further studies in distinct cohorts
are required. Largely consistent with in vivo observations, in vitro
studies demonstrate that vaginal lactobacilli are generally non-
inflammatory when cultured on vaginal epithelium. L. jensenii,
and L. crispatus do not elicit a pro-inflammatory response
from vaginal epithelial cells (Figure 1A) (Libby et al., 2008;
Fichorova et al., 2011; Eade et al., 2012; Rose et al., 2012;
Yamamoto et al., 2013; Doerflinger et al., 2014). L. iners and
L. jensenii induce PRR-signaling but this does not lead to
an increase in secretion of pro-inflammatory mediators IL-
6 and IL-8 (Yamamoto et al., 2013; Doerflinger et al., 2014).
Although lactobacilli contain MAMPs that could potentially
activate TLRs, L. crispatus and L. jensenii significantly dampen
cytokine expression of IL-6, IL-8, and TNFαfrom vaginal
epithelial cells following addition of exogenous TLR agonists
(Rose et al., 2012). This suggests a role for commensal bacteria
in maintaining vaginal homeostasis through as yet unknown
Frontiers in Physiology | www.frontiersin.org 5June 2015 | Volume 6 | Article 164
Aldunate et al. Activity of vaginal microbiota SCFAs
FIGURE 1 | The vaginal environment during alternative states of
eubiosis and BV. (A) During eubiosis, lactic acid-producing bacteria
acidify the vaginal milieu pH <4.5 (average ∼3.5) with lactic acid as the
predominant metabolite. Lactic acid potently inactivates STIs while lactic
acid-producers, such as Lactobacillus, generate a non-inflammatory
environment. (B) During BV, the vaginal environment has a lower redox
potential conducive to the growth of diverse anaerobic bacteria and
higher bacterial load. The concentration of mixed SCFAs and amines
also increase and is accompanied by loss of vaginal acidity pH >4.5.
The diverse anaerobic bacteria generate virulence factors which
compromise epithelial barrier integrity, degrade mucin and generate a
pro-inflammatory environment.
mechanism(s) for modulating cytokine responses. Since these
studies have largely focused on individual bacterial species,
further investigations are required to fully elucidate the mucosal
immune responses associated with the more complex CSTs
dominated by distinct lactobacilli including the specific effects
of lactic acid, as well as longitudinal studies to account for
the dynamic nature of the vaginal microbiota (Gajer et al.,
2012).
Bacterial Vaginosis, an Ecological Disorder
BV is a dysbiosis of the vaginal microbiota in reproductive-
age women with unknown etiology and poorly understood
pathogenesis. It is not known whether the bacteria commonly
associated with the syndrome are a cause or a consequence of
dysbiosis and unclear whether abnormal colonization is due to an
exogenous pathogen, opportunistic endogenous microbiota, or if
dysbiosis is triggered by vaginal microbiota unable to maintain
community function in the face of particular disturbances.
For women with lactobacillus-dominated microbiota, there is
a depletion of lactobacilli and an overgrowth of anaerobic
BV-associated bacteria (BVAB) (Figure 1B). The roles of different
lactobacilli in preventing or promoting the transition to dysbiosis
are an increasing area of investigation. As previously mentioned,
an L. crispatus-dominated microbiota rarely transitions to CST
IV-B (Table 1) containing BVAB, is not commonly found in
women with BV and is associated with a more acidic vaginal
milieu (Ravel et al., 2011; Gajer et al., 2012; O’Hanlon et al., 2013).
Conversely, an L. iners-dominated microbiota is encountered in
women regardless of BV status and may offer less protection from
progression to BV. In this respect, a recent study by Macklaim
et al. (2013) found that L. iners differentially expressed 10% of
its genome when the vaginal ecosystem transitioned to dysbiosis.
Further investigation is required to determine if L. iners is not
antagonistic toward BVAB or less so than other lactobacilli,
why L. iners seems tolerant of dysbiosis and if it contributes to
dysbiosis.
BV-associated Bacteria (BVAB)
As well as a paucity of lactobacilli, BV is accompanied by
increased diversity and bacterial load of Gardnerella, Atopobium,
Prevotella, Megasphaera, Dialister, Sneathia, Leptotrichia,
Mobiluncus, Streptococcus, Bacteroides, Mycoplasma,
Clostridiales BVAB 1, 2, 3, and, several other bacteria (Fredricks
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Aldunate et al. Activity of vaginal microbiota SCFAs
et al., 2005; Wertz et al., 2008; Kim et al., 2009; Verstraelen et al.,
2009; Ling et al., 2010; Zozaya-Hinchliffe et al., 2010; Jespers
et al., 2012; Santiago et al., 2012; Srinivasan et al., 2012). BVAB
increase vaginal pH by generating mixed SCFAs and amines,
and are also capable of utilizing lactic acid as an energy source
(Figure 1B) (Spiegel et al., 1980; Wolrath et al., 2001; Macklaim
et al., 2013; Yeoman et al., 2013). Many of these BVAB are
common inhabitants of a eubiotic vaginal ecosystem but little is
known of synergistic or antagonistic effects between them, with
only Gardnerella and Prevotella displaying a synergism noted
by several studies (Biagi et al., 2009; Ling et al., 2010; Zozaya-
Hinchliffe et al., 2010; Ravel et al., 2011; Jespers et al., 2012;
Datcu et al., 2013). Furthermore, different isolates may represent
different strains/sub-strains that might be associated with BV.
For example, different isolates of Atopobium have been shown
to be resistant to metronidazole, the antibiotic used to treat BV
(Ferris et al., 2004a,b; De Backer et al., 2006). BV isolates of G.
vaginalis also have significant differences in genome content and
gene order that have variable metabolic and virulence capacity
(Harwich et al., 2010; Yeoman et al., 2010; Macklaim et al., 2013).
Interestingly, dispersed and cohesive forms of G. vaginalis have
also been identified, with the latter cohesive form associated
with the initiation and formation of biofilms hypothesized to
contribute to BV pathogenesis (Patterson et al., 2010; Swidsinski
et al., 2010). Atopobium and L. iners have also been implicated
in the formation of these biofilms (Swidsinski et al., 2005, 2008,
2010; Machado et al., 2013). However, further investigations
are required to elucidate the contribution of various
organisms in BV.
Altered Immune Modulation during BV
While BV does not cause overt clinical inflammation
characterized by redness and pain, it is nevertheless characterized
by a pro-inflammatory response in the vaginal mucosa
(Schwebke and Weiss, 2002; Mitchell and Marrazzo, 2014),
which is likely to have distinct characteristics based on the
bacterial communities responsible for BV. The cervicovaginal
immune response in women with BV has been examined in
several studies, which are reviewed in Mitchell and Marrazzo
(2014). Overall pro- and anti-inflammatory cytokine levels
tend to be elevated in women with BV (Cherpes et al., 2008).
However, the levels of specific cytokines and antimicrobial
peptides vary among studies, which have been proposed to be
due to several factors including differences in study population,
the bacterial communities responsible for BV, and the cross-
sectional design and small sample sizes. Nevertheless, in most
studies the pro-inflammatory cytokine, IL-1βis elevated and
the SLPI antimicrobial peptide is decreased in women with
BV while elevation of IL-6 and IL-8 is inconsistent among
studies (Mitchell and Marrazzo, 2014). In addition, vaginal
bacteria can modulate the innate immune response elicited by
epithelial cells with species-specific immune signatures (Libby
et al., 2008; Rose et al., 2012; Doerflinger et al., 2014). The
BVAB G. vaginalis and A. vaginae, when cultured on vaginal
or cervical epithelial cell monolayers and tissue models, elicit
a pro-inflammatory response by up-regulating the production
of cytokines (IL-6, IL-1β, TNFα, IL-8) and chemokines (e.g.,
RANTES, MIP-1β), some antimicrobial peptides (i.e., hBD-2,
defensins) and membrane-associated mucins (Figure 1B) (Libby
et al., 2008; Eade et al., 2012; Rose et al., 2012; Doerflinger et al.,
2014). These responses are similar to what is observed in women
with BV (Schwebke and Weiss, 2002; Hedges et al., 2006; Valore
et al., 2006; Mitchell and Marrazzo, 2014). In contrast, Prevotella
bivia, another BVAB, failed to elicit a pro-inflammatory response
when cultured on vaginal epithelium (Doerflinger et al., 2014)
indicating distinct pathogenicity of BV bacteria.
The Need to Improve Diagnosis of BV
The Amsel criteria and the Nugent score are the two main
methods for BV diagnosis. The Amsel criteria, contains four
criteria, at least three of which must be satisfied for a diagnosis
of BV; a vaginal pH >4.5, thin grayish homogenous discharge,
an amine odor upon addition of 10% potassium hydroxide
to vaginal discharge and the presence of sloughed epithelial
cells coated with BVAB (clue cells) in a Gram stained wet
mount (Amsel et al., 1983). In contrast, the Nugent score is
based on a Gram stain scoring method and is determined
by assessing Lactobacillus morphotypes present as large Gram-
positive bacilli, Gram-variable bacilli indicative of G. vaginalis
morphotypes and curved Gram-variable bacilli indicative of
Mobiluncus morphotypes (Nugent et al., 1991). A Nugent score
of 7–10 is a positive diagnosis of BV. The Nugent scoring method
is reported to have superior reproducibility and sensitivity
relative to the Amsel criteria (Sha et al., 2005a; Modak et al.,
2011); however, current diagnosis methods are biased in that
they rely on the simplistic concept that lactobacilli equate to
healthy vaginal microbiota and assessment occurs at a single
time point. Our current understanding of the microbiome
reveals that 20–30% of reproductive-age women have diverse
lactic acid-producing microbiota that are not lactobacilli and
that vaginal microbiota can experience dramatic transitions in
community composition over time without transitioning to
dysbiosis (Zhou et al., 2004, 2007, 2010; Ravel et al., 2011;
Gajer et al., 2012). Consequently, current diagnostics may have
confounded our current understanding of BV. Nonetheless, these
are the best currently available diagnostic methods and in the
future they are likely to be replaced by molecular quantification
and characterization as technology advances.
BV Treatment and Recurrence
In most women, BV will spontaneously resolve without
treatment (Hay et al., 1997; Koumans et al., 2007; Laghi et al.,
2014) however, treatment is recommended for symptomatic
women due to potential BV-associated complications. Current
recommended regimens include oral metronidazole (500 mg)
twice a day for 7 days, metronidazole gel (0.75%) once a day
for 5 days or clindamycin cream (2%) for 7 days, in conjunction
with abstinence from sex or the use of condoms (CDC, 2010).
Antibiotic treatment is often efficacious but to varying degrees
in different women (Srinivasan et al., 2010). Recurrence is
frequently inevitable and because the etiology of BV is unknown,
it is unclear whether recurrence is due to antibiotic resistance,
re-inoculation, re-emergence of endogenous bacteria, or some
other disturbance leading to the loss of microbiota community
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Aldunate et al. Activity of vaginal microbiota SCFAs
function. Estimates indicate that BV will recur in 30% of women
within 3 months of antibiotic treatment and >50% of women
within 6 months (Hanson et al., 2000; Sanchez et al., 2004; Sobel
et al., 2006; Bradshaw et al., 2006a,b; Nyirjesy et al., 2007).
A number of studies have attempted to address the cause of
recurrent BV and suggest that antibiotic resistance and biofilms
may play a role (Beigi et al., 2004; Verstraelen et al., 2004;
Austin et al., 2005; Swidsinski et al., 2005, 2008, 2010)as well
as extravaginal reservoirs (El Aila et al., 2009; Marrazzo et al.,
2012; Petricevic et al., 2012; Santiago et al., 2012). Also, antibiotic
treatment of BV can severely disrupt the microbiota (Santiago
et al., 2012) and does not ensure that the microbiota will
return to its pre-BV state (Agnew and Hillier, 1995; Nyirjesy
et al., 2007). This suggests that whilst treatment may reduce
the abundance of BVAB the consequent ecological void may
allow for the re-emergence of opportunistic endogenous bacteria
or unimpeded re-inoculation with exogenous pathogens. The
antibiotic rifaximin is reported to overcome the limitations of
existing antibiotic therapy (Cruciani et al., 2012, 2013; Donders
et al., 2013) by restoring the vaginal community as well as
the metabolome (Laghi et al., 2014). Alternative strategies for
BV treatment such as oral and topical probiotics, which also
aim to restore vaginal community function, are undergoing
evaluation. However, the data are currently inconclusive, likely
due to heterogeneity in clinical trial methodology and study
endpoints, the administration route (oral/intravaginal), dosage,
and strains of probiotic bacteria used and if they were provided
as a cocktail or a single strain as well as the characteristics of the
study populations (Senok et al., 2009; Mastromarino et al., 2013;
Huang et al., 2014).
It is also important to consider that due to the nature
of BV diagnosis methods, such as the Nugent score, women
with non lactobacillus-dominated microbiota (CST IV) may
have been misdiagnosed with BV and this may contribute to
apparent BV recurrence and explain spontaneous BV resolution.
Furthermore, the high rates of BV recurrence are not surprising
when considering that the composition and abundance of BVAB
in each woman exhibits great variation (Fredricks et al., 2005;
Ravel et al., 2013; Yeoman et al., 2013) yet treatments are
not targeted to accommodate these differences. With current
technological advances it should be possible to stratify women
according to the profile of BVAB and tailor therapy for better
treatment outcomes.
BV-associated Complications and Sequelae
BV is of clinical significance due to the multitude of adverse
reproductive outcomes and increased risk of acquiring STIs. BV
is associated with preterm birth (Eschenbach et al., 1984; Gravett
et al., 1986; Kurki et al., 1992; Riduan et al., 1993; Hay et al.,
1994; Holst et al., 1994; McGregor et al., 1994; Hillier et al.,
1995; Wennerholm et al., 1997), pelvic inflammatory disease
(Ness et al., 2005), spontaneous abortion (Donders et al., 2000),
post-abortal infection (Larsson et al., 2000), and miscarriage
(Hay et al., 1994; Llahi-Camp et al., 1996; Ralph et al., 1999;
Oakeshott et al., 2002; Nelson et al., 2007); indicating that BV may
predispose women to abnormal ascending colonization of the
upper reproductive tract. BV is also associated with an elevated
risk of acquiring STIs including Neisseria gonorrhea (Morris et al.,
2001; Wiesenfeld et al., 2003; Brotman et al., 2010a; Gallo et al.,
2012), Chlamydia trachomatis (Morris et al., 2001; Wiesenfeld
et al., 2003; Schwebke and Desmond, 2007; Brotman et al., 2010a;
Gallo et al., 2012), Trichomonas vaginalis (Martin et al., 1999;
Brotman et al., 2010a, 2012), herpes simplex virus type-2 (HSV-2)
(Cherpes et al., 2003; Kaul et al., 2007; Nagot et al., 2007), human
papilloma virus (HPV) (Watts et al., 2005; Gillet et al., 2011) as
well as HIV (Sewankambo et al., 1997; Taha et al., 1998; Cu-Uvin
et al., 2001; Coleman et al., 2007; Atashili et al., 2008; Cohen et al.,
2012).
BV may facilitate the acquisition of STIs, through a variety
of mechanisms stemming from the production of factors and
metabolites that create a favorable environment for the growth
other organisms (Hedges et al., 2006; Beigi et al., 2007; Rose
et al., 2012; Yeoman et al., 2013; Doerflinger et al., 2014) whilst
reducing the capacity to inactivate exogenous pathogens due
to a loss of lactic acid and lactic acid-producing microbiota
(Figure 1B). In this regard, BVAB have been shown to increase
vaginal pH which would enable the survival of acid-labile
exogenous pathogens including most of the aforementioned STIs
a woman with BV is at increased risk of contracting (McGrory
et al., 1994; Brotman et al., 2010a; Graver and Wade, 2011;
Aldunate et al., 2013; Gong et al., 2014). In addition to increased
acquisition risks, the altered dynamics of the vaginal ecosystem
during BV (Figure 1B) may also facilitate the sexual transmission
of STIs, with several studies noting increased viral replication
and viral load in the CVF of women with HIV-1 and HSV-2
(Cu-Uvin et al., 2001; Cherpes et al., 2005; Cohn et al., 2005;
Sha et al., 2005b; Coleman et al., 2007; Mitchell et al., 2013) and
transmission of HIV to their male partners (Cohen et al., 2012).
In contrast, women with vaginal microbiota dominated by lactic
acid-producing bacteria negatively correlate with BV acquisition
(Skarin and Sylwan, 1986; Tamrakar et al., 2007; Srinivasan
and Fredricks, 2008; Zozaya-Hinchliffe et al., 2010) and have a
reduced risk of acquiring STIs including N. gonorrhea (Martin
et al., 1999; Wiesenfeld et al., 2003), C. trachomatis (Wiesenfeld
et al., 2003), T. vaginalis (Martin et al., 1999; Brotman et al., 2012),
and HIV (Martin et al., 1999).
Overall, several studies denote that acidic vaginal conditions
and lactic acid-producing microbiota are associated with reduced
susceptibility to STIs whereas BV, with an elevated pH and
reduction in lactic acid-producing bacteria, appears to increase
susceptibility (Figure 1). However, the maintenance of the
vaginal ecosystem and the development of dysbiosis remain a
mystery and the degree of protection afforded by distinct vaginal
communities requires further investigation. Furthermore, BV
symptoms are varied amongst women and these may reflect
different etiologies and or different pathogenesis mechanisms
leading to BV in the context of a woman’s unique genetic
determinants and microbiota. The characterization of microbiota
members has certainly enhanced our understanding of the
vaginal ecosystem but supplementary information regarding
microbiota metabolic activities and their impact on the
function and performance of the vaginal ecosystem is required
to gain a complete understanding of vaginal eubiosis and
dysbiosis.
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Aldunate et al. Activity of vaginal microbiota SCFAs
The Metabolome in Eubiosis and Dysbiosis
During the reproductive years the vaginal microbiome provides
a multifaceted primary defense mechanism against infections
whilst promoting favorable reproductive outcomes. One manner
in which this is achieved is the production of acid fermentation
products, primarily lactic acid, which acidifies the vagina through
the concerted effort of lactic acid-producing bacteria. Thus, the
production of lactic acid, together with host factors, deeply
impacts the resulting microbiota by excluding or selecting
community members (Figure 1A). Collectively, these members
generate the functional output of that particular community
thereby influencing resilience in the face of intrinsic and
extrinsic disturbances. A representation of both host and
microbial factors in the vaginal ecosystem is captured within
CVF. The metabolome, in particular SCFAs and amines, are
the most extensively studied components of this complex
medium.
Early Studies of CVF Metabolites Associated with
Eubiosis and BV
Early studies investigated SCFAs and amines in CVF through
a variety of chromatographic techniques in an effort to identify
metabolites associated with dysbiosis that could be used for
the diagnosis of a polymicrobial syndrome, then known as
non-specific vaginitis (NSV) (Gardner and Dukes, 1955; Spiegel
et al., 1980; Piot et al., 1982; Ison et al., 1983). The criteria later
established by Spiegel (Spiegel et al., 1983), Amsel (Amsel et al.,
1983), and Nugent (Nugent et al., 1991), determined that the vast
majority of NSV cases were actually BV. Overall, early studies in
healthy asymptomatic reproductive-age women found that lactic
acid was the predominant metabolite in CVF with relatively
small amounts of the SCFAs acetate, propionate, butyrate,
isobutyrate, succinate, formate, fumarate, valerate, and caproate
(Preti and Huggins, 1975; Spiegel et al., 1980; Piot et al., 1982;
Ison et al., 1983; Krohn et al., 1989; Stanek et al., 1992) as well
as trace amounts, if any, of the amines putrescine, cadaverine,
and spermidine (Chen et al., 1979, 1982; Sanderson et al., 1983;
Jones et al., 1994; Kubota et al., 1995; Wolrath et al., 2001). In
contrast, BV-associated metabolite profiles showed that lactic
acid was often dramatically decreased along with an increased
concentration and variety of SCFAs and amines that were more
prevalent in women with BV (Spiegel et al., 1980; Piot et al.,
1982; Ison et al., 1983; Jokipii et al., 1986; Krohn et al., 1989;
Stanek et al., 1992; Chaudry et al., 2004) as reviewed in Wolrath
et al. (2001).
The BV-associated SCFA profiles obtained using gas liquid
chromatography (GLC) prompted the development of a BV
marker based on the correlation of a high succinic:lactic acid
ratio and dysbiosis determined by fulfilling two of the following
criteria; pH >4.5, homogenous discharge, presence of clue cells
and amine odor (Spiegel et al., 1980). A peak ratio of =0.4
showed 86% sensitivity, 97% specificity for BV and 90% positive
predictive value, where the sensitivity could be increased to 90%
if propionate and butyrate peaks were detected. This method
was used with varying success to diagnose BV, with most studies
reporting 80–90% sensitivity and 80–97% specificity (Piot et al.,
1982; Ison et al., 1983; Gravett et al., 1986; Krohn et al., 1989).
Early studies also sought to assess changes in metabolite profiles
associated with the antibiotic treatment of BV and consistently
found a return to increased lactic acid with a concomitant
decrease in BV-associated SCFAs acetate, propionate, butyrate,
isobutyrate, succinate and caproate and, other organic acids
(Spiegel et al., 1980; Stanek et al., 1992; Chaudry et al., 2004) as
well as BV-associated amines (Chen et al., 1979, 1982; Sanderson
et al., 1983). Thus, the relative levels of lactic acid and SCFAs
in CVF often reflected the success or failure of antibiotic
treatment.
A paucity of lactobacilli in conjunction with increased
bacterial diversity and load was associated with altered
metabolite profiles of women with BV vs. those of healthy
asymptomatic women (Piot et al., 1982; Jokipii et al., 1986).
Many BVAB are known to produce several acid metabolites;
for example: Bacteroides (succinate-producers), Peptococcus
(butyrate- and acetate-producers), Clostridium and Gram-
positive cocci (caproate-producers) and, Dialister (propionate-
producer) (Spiegel et al., 1980; Debrueres and Sedallian, 1985;
Downes et al., 2003; Chaudry et al., 2004). Particular attention
was paid to G. vaginalis (acetate and succinate-producer),
because of the work of Gardner and Dukes (1955). However, G.
vaginalis was frequently cultured from women regardless of BV
status and the detection and quantity of acetate- or succinate was
not associated with G. vaginalis (Spiegel et al., 1980; Piot et al.,
1982). Holmes et al. (1985) employed an alternative approach
to ascertain whether the altered metabolic profile was due to
an unknown host determinant or microbial metabolism. Here,
the redox potential (Eh) at the vaginal epithelium was measured
in healthy asymptomatic women and women with BV, it was
hypothesized that vaginal microbiota would influence the redox
potential due to altered concentrations of organics acids that
form redox pairs (e.g., lactate/pyruvate and succinate/fumarate)
(Holmes et al., 1985). Women with BV were reported to have
a more reduced vaginal environment compared to healthy
asymptomatic women. Typically, redox potential varies by ±
60 mV per pH unit but the difference between the two groups
was 262 mV for 1 pH unit difference, indicating that pH alone
could not account for the altered redox potential. The difference
in redox potential was attributable to microbial metabolism
rather than a host determinant because antibiotic treatment
successfully increased the redox potential, similar to that of
healthy asymptomatic women. Notably, a more reduced vaginal
environment is conducive to the growth of anaerobes, which
increase in number and diversity during BV. However, as with
other BV-associated changes, this study could not conclude
whether the reduced environment was a cause or consequence
of dysbiosis.
It was hoped that important insights would be obtained into
microbial and host interactions during BV by assessing CVF in
eubiosis and dysbiosis. However, no clear association could be
made between specific BVAB and ensuing changes in metabolite
profiles. Nonetheless, these studies contributed to revealing BV
as a multifaceted syndrome associated with gross metabolic
changes in addition to dysbiotic microbiota and altered immune
regulation.
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