Download Virulence, adherence, and growth of a Gardnerella vaginalis biofilm

Summer Research Proposal:
Virulence, adherence, and growth of a Gardnerella vaginalis biofilm and other anaerobes
associated with bacterial vaginosis
BBSI 2009
Annica Stull-Lane
VCU Mentor: Kimberly Jefferson
Home Mentor: Taylor Allen
Introduction
Bacterial vaginosis (BV) is the most prevalent vaginal infection in women of
reproductive age, with a point prevalence of 8%–23%.1 Complications associated with BV
include preterm delivery, 2 an increased risk of pelvic inflammatory disease, 3 and an increased
susceptibility to HIV acquisition and transmission.4 Treatment is generally with the antimicrobial
agent metronidazole, but drug therapy is not adequate, as there is still over a 50% recurrence
rate. 5,6 Current knowledge of the etiology and pathogenesis of BV is limited, but it is associated
with three characteristics: a rise in pH (> 4.5), an overgrowth of pathogenic anaerobic bacteria,
and a reduction in lactobacilli, the bacteria present in the healthy vagina. Lactobacilli produce
lactic acid and hydrogen peroxide, maintaining the low pH of the vagina and, ideally, preventing
BV-associated bacteria from colonizing. If we can characterize how these pathogenic anaerobes
can thrive in the vagina during BV, then perhaps we can develop methods to disrupt their
colonization and prevent recurring infections. Of these pathogenic bacteria, Gardnerella
vaginalis has been found to be the predominant species. 7,8 Virulence mechanisms of G.
vaginalis include a pore-forming toxin vaginolysin9 as well as the formation of a very resilient
biofilm.7 A biofilm is an adherent community of microorganisms surrounded by a protective
slime consisting of a complex matrix of polysaccharides, proteins, and/or nucleic acids. 3,10
Some biofilm communities, like dental plaque, involve a specific succession of bacterial
adherence: the later colonizers depend on the earlier colonizers for adherence and growth in an
otherwise unsuitable environment. 11 Other anaerobes associated with BV include Atopobium
vaginae, Prevotella bivia, Mobiluncus mulieris, Fusobacterium nucleatum, Peptostreptococcus,
and Corynebacterium.12,13,14 It is possible that the community of anaerobes present in BV
develops through species succession, and I hypothesize that the G. vaginalis biofilm may act as
an initial colonizer, creating a suitable environment for other BV-associated anaerobes to adhere
to the vaginal epithelium and proliferate.
Progress Report
I. Summer 2008
Purpose: Last summer I investigated the potential for G. vaginalis to contribute to the
growth of other BV-associated anaerobes and/or to their adherence to vaginal epithelial cells.
Methods: I used confocal microscopy to visualize adherence of bacterial species to ME180 vaginal epithelial cells. I isolated DNA and performed qRT-PCR to quantify adherence of
the different anaerobes when added in combination with G. vaginalis to the ME-180s. Finally, I
used qRT-PCR to quantify growth of the anaerobes in brain heart infusion broth alone or in
coculture with G. vaginalis biofilms under both aerobic and anaerobic conditions.
Results: G. vaginalis and M. mulieris adhered to ME-180s, while P. bivia and A. vaginae
did not. G. vaginalis did not appear to augment adherence of the other species. Under static
conditions, both aerobic (p<0.0002) and anaerobic (p<0.0003) growth of M. mulieris was
inhibited in the presence of a G. vaginalis biofilm. Growth of P. bivia under aerobic conditions
was increased in the presence of the G. vaginalis biofilm (p<0.0001) but under anaerobic
conditions growth was higher in mono-culture (p<0.0001). There was no statistically significant
effect of co-culture on growth of A. vaginae.
Conclusions: Under static conditions in vitro, it did not seem that G. vaginalis biofilms
aided adherence or growth, except for P. bivia growth under aerobic conditions. This, however,
was not an accurate reconstruction of the vaginal environment. The original hypothesis may still
stand, if more physiologically relevant experiments are conducted. In the static growth
environment, it is possible that G. vaginalis used up all the nutrients and/or produced acid
metabolic byproducts, bacteriocins, or other compounds that harmed the other species. In the
vaginal environment, there is a continual flow of mucosal fluids over the epithelium,
replenishing nutrients and removing metabolic waste. For the adherence assays, it is possible that
other bridging bacteria are required for adherence succession.
II. Academic Year 2008-2009
Over the academic school year, I accomplished two aims: 1) To correlate qRT-PCR data with
bacterial concentration, and 2) To research characteristics of vaginal physiology.
1) Correlating qRT-PCR data with bacterial concentration
Purpose: I used quantitative real-time polymerase chain reaction (qRT-PCR)
experiments to correlate cycle threshold value (Ct) data gathered from the qRT-PCR experiment
with actual bacterial cell counts. The purpose was to develop a strategy to convert my qRT-PCR
data into meaningful, quantitative numbers that represent bacterial counts.
Methods: The VCU Jefferson Lab isolated DNA from samples with known bacterial cell
concentrations: G. vaginalis as 4.39 * 109 CFU/mL, P. bivia as 2.2 * 108 CFU/mL, M. mulieris
as 1.47 * 109 CFU/mL, and A. vaginae as 2.28 * 109 CFU/mL. These samples were sent to my
home institution. I then standardized the DNA at a concentration of 0.5 * 106 CFU/mL and did
10-fold serial dilutions. Using 2µL DNA in the qRT-PCR reaction made the analyzed
concentrations 106, 105, 104, and 103 CFU/mL. Since P. bivia had a lower initial concentration,
serial dilutions were done with the provided sample and then 4.5µL DNA were added to the
qRT-PCR reaction mix. In qRT-PCR, the reaction is measured after each amplification cycle by
monitoring fluorescence. SYBR Green is a fluorescent dye that binds to double-stranded DNA,
and the measure of its fluorescence corresponds to the amount of DNA amplified. Eventually
the DNA amount and fluorescence will reach a threshold after a given number of cycles, and a
cycle threshold (Ct) value is designated with each reaction well. The lower the Ct value, the
faster it takes to reach the threshold point, and the more DNA there was in the initial reaction.
From this experiment, I generated a standard plot for each bacterial species. I plot my Ct values
and log(CFU).
Results:
Standard Plot, Threshold bar at 100.0
40
y = -4.12x + 46.24
R2 = 0.942
35
30
y = -4.02x + 39.34
R2 = 0.9386
Ct
25
Gv
Av
Mm
Pb
Linear (Mm)
Linear (Av)
Linear (Pb)
Linear (Gv)
20
y = -2.64x + 34.08
R2 = 0.9794
15
10
y = -3.272x + 30.809
R2 = 0.989
5
0
0
1
2
3
4
5
6
7
log(CFU)
Conclusions:
These graphs can be used to correlate obtained cycle threshold values with bacterial counts, and
will help to make my observations more quantitative in future experiments.
2) Research on characteristics of vaginal physiology
Even though the adherence and growth assays from last summer overall seemed not to
support the initial hypothesis, there are still other factors to consider. The assays used were static,
in vitro experiments, and they certainly did not emulate all of the physiological vaginal
conditions. In other words, the hypothesis that G. vaginalis is an initial colonizer that creates a
more suitable environment for anaerobe growth and adherence may be true in other, more
physiologically relevant conditions.
The human vagina is not a static environment; indeed, it is constantly changing on many
levels: over years, months, and even minutes. As a newborn, lactic acid-producing microbes
colonize the vagina shortly after birth.15 In early childhood, there is a decrease in these bacteria,
as the vaginal epithelium is more thin, the pH is neutral or basic, there is less glycogen content
available, and there is a lack of hormonal influence. However, once a woman reaches
reproductive-age, the vaginal epithelium thickens, hormones influence a cyclical menses, and the
lactic acid-producing microbes proliferate. In suggesting ways to more accurately represent
physiological conditions, I would like to focus on vaginal conditions including physical
epithelial cell characteristics, vaginal fluid chemical composition, pH, pO2, and the cyclical
hormonal influences.
The superficial or inner layer of the vaginal wall consists of glycogen-containing
squamous cells that are constantly exfoliated.16 The epithelium is also lubricated by mucous from
the cervical glands. How does this relate to microbes? The glycogen is necessary for production
of lactic acid. One example of replicating the natural vaginal environment is to produce
conditions in which there is a continuous turnover of the surrounding milieu. In vivo, nutrients
are replenished as vaginal secretions bathe the vaginal epithelium and in vitro it is feasible to
remove “spent” media and replace it with new media. One possible method is the use of a flow
cell, which would provide a constant stream of fresh media. The vaginal epithelium is also a
multi-cell thick tissue. In one article17, Levin describes his research on the electrical potential
difference across the vaginal epithelium, using an isolated everted vagina18 as well as with
human vaginal tissue incubated in vitro as a sheet19. The latter study may be a potential method
to test BV microbe growth and adherence on multi-layer vaginal tissue cells.
Genital tract secretions are fluids found on the vaginal epithelium, and they are mixture
of Bartholin’s glands secretions, oviductal fluids, follicular fluids, and uterine fluids.20 The
mixture contains proteins, immunoglobulins, enzymes including proteases, carbohydrates
including glucose and glycogen, acids produced from microbe metabolism, amino acids, as well
as ion components. One study formulated a growth medium to simulate these conditions and it
was able to support the growth of certain vaginal microflora. The pH of the medium is made at
7.2, and it contains a buffering component of potassium phosphate. This would be a useful
medium to use to grow the microbial species. It seems more physiologically relevant, however,
to test growth of BV-associated species at other buffered pH values. For example, another
vaginal fluid simulant has been formulated with a pH of 4.2, but it is not specifically for growing
microbes.21
The vaginal fluid of a healthy, nonmenstruating, premenopausal woman is around 4.2.21
However, the pH can vary over the menstrual cycle. One study showed that the vaginal pH was
on average 6.5+0.3 on day 2 of the menstrual cycle, 5.3+0.2 on day 4, and 4.15+0.2 on day 14.22
The days correspond with day 1 being the first day of menstruation. The observed trend is a
higher vaginal pH during menstruation and a decrease in pH over the menstrual cycle. Another
study observed a pH of 4.6 during days 1-5, 4.4 during days 7-12, and 4.4 during days 19-24.23
Again, the vaginal pH increased when there was menstrual blood in the vagina. How does this
relate to microbes? The first study reported that a raised pH favors a lower redox potential, as the
reduction in hydrogen concentration is accompanied by a release of elections.22 The environment
is therefore more reducing and more favorable for anaerobic organisms.25
The oxygen concentration of the vagina is dependent on many factors: both blood flow to
the area and exogenous oxygen. In one study, pO2 was found to vary extremely widely, with 0 to
77 mm Hg on day 2 of the menstrual cycle, 0 to 76 mm Hg on day 4, and 0 to 53 mm Hg on day
14.22 However, a more recent study looked at oxygen concentration of a basal (nonsexually
aroused state) vaginal surface versus that during sexual arousal.17 They found that basal pO2 = 9
± 11mmHg, which is almost anaerobic, yet sexual arousal caused the pO2 to increase to 50
mmHg. The pO2 of normal air is usually around 150 mmHg. Fluxation of vaginal oxygen
concentrations is also seen in cyclical REM cycles during sleep.24 Another study found that
inserting a tampon increased the pO2 to from 3 ± 11mmHg to atmospheric air pO2.27 Oxygen
concentration averaged at 112 ± 18 mmHg during the following 90 minutes, and it took 8 hrs to
return to preinsertion levels. The pCO2, on the other hand, was initially 64 ± 13 mmHg and rose
rapidly back to preinsertion levels, with a mean value of 50 ± 12 mmHg during the 90-minute
period. Vaginal pCO2 over the menstrual cycle seemed less variable, as it ranged from 50-60
mmHg.22 From these observations, I might suggest using a low oxygen concentration in vitro, as
that seemed to be the basal level.
The relationship between vaginal microbiology, menses, and estrogen level is complex.
One study looked at numbers of Lactobacillus and non-Lactobacillus species over the menstrual
cycle in both healthy women and women with BV.23 In women with BV, any present
Lactobacillus sp. tended to increase over the menstrual cycle, and this was mainly due to H2O2positive Lactobacillus. However, there was no significant change in non-Lactobacillus flora. In
healthy women, Lactobacillus sp. changed little over the menstrual cycle, but non-Lactobacillus
species (including Prevotella and Gardnerella vaginalis,23 Fusobacterium and
Peptostreptococcus25) shifted from high to low levels over the cycle. This means that more
anaerobes proliferated during menses. One idea here is that hemoglobin during menses may be a
source of iron for growth of microorganisms that are otherwise iron-limited.20 It is also true that
lower estrogen levels are present during menses. The peak in oestrogen roughly occurs around
day 10-16 of the menstrual cycle, just before ovulation. High levels of oestrogen increase the
glycogen concentration in vaginal epithelial cells,16,23 which feeds the lactic acid-producing
Lactobacilli. A low vaginal pH is usually accredited to lactic acid-producing Lactobacillus;
however, lactic acid is also produced by the conversion of glycogen to lactic acid by the vaginal
epithelial cells themselves.23 It has been observed that estrogen lowers vaginal pH before an
increase in Lactobacillus.25 An acidic pH increases redox potential, making the environment less
favorable for anaerobic organisms since it is less reducing. Lactobacillus adherence to epithelial
cells is also stimulated by the increase in oestrogen levels, which generates movement of ions
across the vaginal epithelium and alters the vaginal cell charge. It is thought that this electrical
potential difference might change the bacterial adherence properties.16 It seems that a rise in
estrogen is associated with a proliferation of Lactobacillus sp. This correlates to another study
that notes that when postmenopausal women were treated with estrogen, there were less
anaerobic species and more Lactobacillus species.25
With all of this in mind, it seems that the vaginal microenvironment is least
Lactobacillus-stable during menses, as this is when a peak in microorganism diversity appears.
Perhaps it is during the time of menses when BV-associated bacteria are most likely to adhere
and grow. In one study, it was within the first 9 days of the menstrual cycle when 5 of 7 women
with normal flora changed to either an intermediate flora or fully developed BV.26 In studying
the pathogenesis of bacterial vaginosis, perhaps it would be insightful to cater experiments to as
many of these physiological characteristics as possible: low levels of estrogen, less Lactobacilli,
low pO2 around 10 mmHg, pCO2 around 50-60 mmHg, a raised pH around 4.5-6, a vaginal
chemically defined medium, a thick vaginal cell layer, and a constant flow of fluids across the
vaginal cells. There are other factors that influence the composition of the vaginal flora, such as
host immune factors16 and contraceptive drugs23 (through hormone pathways), but it seems that
the above factors may be easier to emulate than other physiological factors of the vaginal
microenvironment.
Summer 2009: Future Experiments
Purpose: I wish to look more in depth at the virulence potential and interactions between BVassociated anaerobes and the G. vaginalis biofilm. I will be using the BV-associated species G.
vaginalis, M. mulieris, A. vaginae, P. bivia, Corynebacterium, Peptostreptococcus, and F.
nucleatum. This summer, my goals are three-fold:
1) To test adherence of co-cultures and mixed cultures to vaginal cells
2) To test the cytotoxicity and biofilm-forming capability of BV-associated anaerobes
3) To test anaerobe growth with a G. vaginalis biofilm in a flow cell
Methods:
1) I will analyze adherence of anaerobes in a mono-culture, in a co-culture with G. vaginalis,
and in a mixed culture with G. vaginalis and F. nucleatum. First G. vaginalis will be added
to ME-180 vaginal epithelial cells and incubated 15 minutes in a 5% CO2 incubator. Nonadherent bacteria will be removed by washing with phosphate buffered saline (PBS) and
subsequent species will be added and incubated 15 minutes, again followed by washing. I
will analyze the experiment both qualitatively through confocal microscopy and
quantitatively by qRT-PCR via the use of species-specific primers to amplify the bacterial
16S rRNA gene of isolated DNA samples from the experiment. There will be thirteen
samples: no bacteria control, G. vaginalis alone, M. mulieris alone, A. vaginae alone, P.
bivia alone, F. nucleatum alone, G. vaginalis/M. mulieris, G. vaginalis/A. vaginae, G.
vaginalis/P. bivia, G. vaginalis/F. nucleatum, G. vaginalis/F. nucleatum/M. mulieris, G.
vaginalis/F. nucleatum/A. vaginae, and G. vaginalis/F. nucleatum/P. bivia.
2) I will test the cytotoxicity of each bacterial species on ME-180 vaginal epithelial cells by
adding bacterial culture to the cells and observing every hour for four hours. The scoring
system is 0 = normal cells, 1 = almost normal cells with <25% rounded, 2 = 50% rounded,
no breaks in the monolayer, 3 = >50% rounded, with possible breaks in the monolayer, 4 =
all rounded and large breaks in the monolayer, and 5 = complete lysis of monolayer, cells
floating. I will use confocal microscopy to visually capture the interaction, and I will use a
CellTiter Viability Assay to measure viability of the epithelial cells. To test biofilmforming capability, I will add each species to a 96-well plate, incubate for 24-48 hours, and
remove non-adherent cells by washing with PBS. The biofilms will be stained with
safranin, washed to remove excess stain, and qualitatively assessed visually. For
quantitative analysis, acetic acid will be added to solubilize the safranin and the absorbance
will be read with spectrometry. A higher absorbance reading corresponds with more
bacteria cells represented and indicates that the species is a better biofilm-producer.
3) A G. vaginalis biofilm will be grown for 24-48 hours in a flow cell, which will maintain
chemostat conditions. Then, other anaerobes (see the same 13 samples above) will be
added and left to adhere and grow for 24-48 additional hours. This will potentially be in a
chemically defined medium (CDM) that resembles vaginal secretions,20 which would more
closely resemble in vivo conditions. First I will do some pilot assays, growing anaerobes
both aerobically and anaerobically with and without biofilms in 6-well plates, replenishing
media often. Then I will utilize the flow cell apparatus. I will use confocal microscopy to
visualize the biofilm, and I will use qRT-PCR to quantify the amount of bacteria.
Possible Results and Implications:
1) If the hypothesis is correct then I would expect to find that G. vaginalis behaves as an
initial colonizer and promotes adherence of other BV-associated anaerobes. I would also
expect to find that F. nucleatum acts as a potential bridging species in multi-species biofilm
formation, as it does in dental biofilms. It is possible, though, that G. vaginalis could
negatively affect adherence in this static in vitro environment.
2) Preliminary results have shown that G. vaginalis is extremely cytotoxic while the other
anaerobes are not. Repeat experiments should verify this initial observation. I expect to
find that G. vaginalis is the best biofilm-producer and that the other species have little or no
single-species biofilm-forming capability.
3) If the hypothesis is correct then I expect to find that in chemostat conditions, anaerobes
grow better in the presence of the G. vaginalis biofilm. However, if results do show the
opposite then it may be useful to look into how G. vaginalis competes with other anaerobe
species.
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