The Complexity of

Bacterial Vaginosis:
Do Gardnerella vaginalis
biofilms contribute to
colonization by other
anaerobes in bacterial
August 5, 2008
vaginosis?
Annica Stull-Lane VCU Mentor: Kimberly Jefferson
BBSI Summer 2008 Home Mentor: Taylor Allen
Bacterial Vaginosis
Most common vaginal disorder in
reproductive-age women, world-wide
Symptoms: abnormal homogeneous
discharge, sometimes “fishy” odor
Many cases are asymptomatic, >50%
Imbalance of vaginal flora,
characterized by:
 Raised pH
 Overgrowth of unhealthy anaerobes
 Reduction of lactobacilli

http://www.yamagiku.co.jp/pathology/photo/photo138-1.htm
Complications with BV
Preterm delivery
Increased risk of PID
Increased acquisition
& transmission of HIV
Recurring infections!
 Treatment

http://the-half-decent-pharmaceutical-chemistry-blog.chemblogs.org/archives/2006/11/23/drug-of-the-week-metronidazole
Etiology of BV: Causes?
Etiology unknown
Sexually transmitted? Reduction of lactobacilli
Reduction in H2O2
production
Important connection between
the characterization factors
Overgrowth of BV
associated bacteria Raised pH

Generally considered a polymicrobial

disorder with a variety of anaerobes;
however, is the presence of a certain species
necessary for BV??
Wilson, J. Managing recurrent bacterial vaginosis. Sex Transm Infect 2004;80:8-11.
Bacteria Associated with
BV
Gardnerella vaginalis --- most prevalent
 Gram-positive or variable rod-shaped anaerobe
 Present in up to 95% of BV cases, but not sole factor, as
does not fulfill Koch’s Postulates
 Adheres to vaginal epithelium as a biofilm---perhaps
allows other anaerobes to colonize

Many other
anaerobes
 Strict anaerobes
Prevotella bivia
Mobiluncus mulieris
Atopobium vaginae Gardnerella vaginalis biofilm
Swidsinski A, Mendling W, Loening-Baucke V, et al. Adherent biofilms in bacterial vaginosis. Obstet
Gynecol 2005;106.5;1012-23.
 Bacterial Biofilms
Complex matrix of polysaccharides, proteins and/or nucleic
acids

Resistance to chemicals, pH extremes,

host immune defenses, and antibiotics
 Related to recurrence of BV?

S. aureus biofilm on a catheter

http://phil.cdc.gov/phil/details.asp
 Succession in Biofilms
initial colonizers and
secondary colonizers
 For example, dental plaque
 Streptococcus and Actinomyces
 Fusobacterium

Later colonizers depend

on earlier colonizers for
adherence and growth in
an otherwise unsuitable
environment

Succession in
BV? http://www.nature.com.proxy.library.vcu.edu/nrmicro/journal/v2/n7/fig_tab/nrmicro928_F3.h
Initial Hypothesis:

Gardnerella vaginalis biofilm acts as

an initial colonizer
 This allows for other BV-associated

anaerobes to adhere to the vaginal

epithelium and survive in the
presence of O2, lactic acid, H2O2
etc…
 Experiments
Adherence
 Do the anaerobes stick better to
vaginal epithelial cells in the presence
of Gv?
Growth
 Does the presence of a Gv biofilm
facilitate the growth of strict
anaerobes?
• For the three anaerobes, strict anaerobic
conditions required.
 Maintaining Anaerobic
Conditions

Anaerobic air mix:

• 5% CO2
• 5% H2
• 90% N2
Adherence Assay: • Single bacterial
Confocal Microscopy strain on ME180
vaginal epithelial
BacLight Green Bacterial cells
Stain • Multi-species?
Red Vibrant Cell Membrane
Stain
• How can
one measure
adherence?
Control G. vaginalis

P. bivia A. vaginae M. mulieris

 What is qPCR?
• SYBR Green as a fluorescent dye
Repeat 40X,
DNA template measuring
fluorescence
after each
Denaturation
cycle
Primers anneal

Extension
 What is qPCR?
• SYBR Green as a fluorescent dye
DNA amount and fluorescence will reach a
threshold value after a number of cycles
Cycle threshold value:
lower number = reached threshold in fewer
cycles
= started with more DNA
• Logarithmic scale,
so use equation:
(- cycle threshold value)
2^

Acquiring DNA…
 Adherence in Co-culture:
Methods
Gardnerella vaginalis (Gv)
 Mobiluncus mulieris (Mm)
 Prevotella bivia (Pb)
 Atopobium vaginae (Av) not tested • Centrifuge
yet
bacteria onto
epithelial cells and
allow toagain
adhere 30
ME180 Vaginal Epithelial Cells min

Pb Pb Mm Mm • Wash with PBS

to remove
Pb Pb Mm Mm bacteria that
have not adhered
Adherence Assay: Data
Extract bacterial DNA from wells with
Qiagen DNeasy Kit
qPCR to determine relative amounts of
DNA, i.e. relative amount of adherence
Use different 16s primers to differentiate
between bacteria strains
DNA amount corresponds to cell amount,
but qPCR does not discriminate if cell is
dead or alive
 CFU’s to determine initial amount of
inoculated cells
Adherence Assay: Data
Adherence of M. mulieris to epithelial cells
with and without G. vaginalis
0.9
p=0.027
Relative amount of

0.8

0.7
MmDNA

0.6

0.5

0.4

0.3

0.2

0.1

Mm GvMm
Bacteria
Adherence Assay: Data
Adherence of P. bivia to epithelial cells
with and without G. vaginalis
Relative amount of Pb DNA

p=0.022
5

Pb only GvPb
 Conclusion: Data suggests that G.
vaginalis biofilms decrease adherence.
 Anaerobes adhere better without biofilm
 Also, no detectable co-aggregation between Gv
and each anaerobe
 Implication: Perhaps competing for a receptor on
the cell?

Next Question: Does a G. vaginalis

biofilm aid growth?
 Growth
Assay
Experiment both in aerobic and anaerobic
conditions

Pb Incubate 3-5 days

Pb

Pb Gv BHIG
 Qiagen DNeasy Kit
to extract DNA for
Pb Gv BHIG qPCR

Gv biofilm:
b anaerobe: Pb (repeated for all 3 anaerobe
Growth Assay: qPCR Data
Preliminary results for Mobiluncus mulieris

G ro w th A s s a y: a e ro b ic a n d a n a e ro b ic gMro . w th o f
m u lie risw ith a n d w ith o uGt . av a g in a lis
b io film
1 .2

p=0.0003
1

p=0.0002
Relative amount of

0 .8
Mm DNA

0 .6

0 .4

0 .2

M m o n ly G vM m M m o n ly G
A e ro b ic A n a e ro b ic
 Growth Assay: qPCR Data
Preliminary results for Prevotella bivia

G row th A ssay: aerobic and anaerobic grow P.th of

bivia w ith and w ithoutGa. vaginalisbiofilm
Relative amount of DNAPb DNA

0.45 p=0.0001
0.4

0.35

0.3
p=0.0001
0.25

0.2

0.15

0.1

0.05

Pb only G vPb Pb only G vPb

A erobic A naerobic
• Av and Mm grow much slower than Pb
Growth Assay: qPCR Data
Preliminary results for Atopobium vaginae

Growth Assay: aerobic and anaerobic growth A. of

vaginae with and without G.
a vaginalisbiofilm
1.6
p=0.62
1.4
p=0.44
Relative amount of

1.2

1
Av DNA

0.8

0.6

0.4

0.2

Av only GvAv Av only GvAv

Aerobic Anaerobic
Implications
Growth was condition-specific for Pb,
so next step is to find oxygen level of
vagina
Seems that some anaerobes may
actually grow worse in the presence of
a Gv biofilm
In that case,
 Competition with Gardnerella?
 Gardnerella produces acid
 Does Gardnerella produce bacteriocins?
 Preliminary inhibition assay
Disk Diffusion Assay
• a filter disk was
soaked in sterile
Gv culture
supernatant and
placed on a lawn
of anaerobic
bacteria
• no zone of
inhibition

Av Pb • suggests that
inhibition is
nonspecific, or the
concentration was
Conclusions
Adherence assay
 Confocal: Gv and Mm adhere strongly to epithelial
cells, Pb and Av don’t adhere as well
 Gv negatively affects adherence of Mm and Pb
Growth assay
 Av may not be affected by Gv biofilm
 Mm may grow better alone
 Anaerobically, Pb may grow better alone,
aerobically, Pb grows better with a Gv biofilm
Unclear whether Gv contributes to growth of
anaerobes in BV. Other conditions? Other
factors?
 Host factors
 Loss of lactobacillus
 Future Directions…
Academic Year: qPCR analysis

DNA from additional adherence and growth assays

Correlate cycle threshold value with CFU counts

Also, literature search for physiological conditions of the vagina

BBSI Summer 2009:

Repeat preliminary experiments, gather more
data
qPCR analysis for adherence and growth assays using
CFU’s
Growth assays, in presence of H2O2, lactic acid,
as well as other vaginal conditions
Acknowledgements
The Jefferson Lab
 Dr. Kimberly Jefferson
 Jennifer Patterson
Dr. Philippe Girerd
VCU BBSI and Jeff Elhai
BBSI Friends
NSF & NIH!
Questions?
References
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