406
Evidence for a Commensal, Symbiotic Relationship between Gardnerella vaginalis
and Prevotella bivia Involving Ammonia: Potential Significance for
Bacterial Vaginosis
Vivien Pybus and Andrew B. Onderdonk
Channing Laboratory, Harvard Medical School, Brigham and Women's
Hospital, Boston, Massachusetts
Six strains of Prevotella bivia and 4 of Gardnerella vaginalis were examined for nutrient substrate
utilization as part of ongoing studies on the pathogenesis of bacterial vaginosis. Addition of single
amino acids to vaginal defined medium (VDM) was stimulatory to the growth of P. bivia but not
to G. vaginalis. However, peptides significantly promoted the growth of both organisms. Growth of
P. bivia in VDM and VDM supplemented with either amino acids or peptone was accompanied by
net ammonia production, while growth of G. vaginalis under the same conditions resulted in net
ammonia utilization. Ammonia-enriched supernatants from the growth of P. bivia in peptonesupplemented VDM were stimulatory to G. vaginalis growth. However, ammonia-reduced supernatants from G. vaginalis growth in peptone-supplemented VDM had a neutral effect on P. bivia
growth. A commensal relationship between P. bivia to G. vaginalis is proposed, with ammonia flow
as a mechanism to support this hypothesis.
Bacterial vaginosis (BV) is the most common vaginal tract
infection seen in women of reproductive age in primary health
care [1, 2]. Clinical diagnosis of BV is based on the presence
of three of the following four criteria: an elevated (>4.5) vaginal pH, release of a fishy odor on addition of 10% KOH to
the vaginal fluid, an abnormal discharge that is thin and homogenous, and clue cells in the vaginal fluid [1]. During BV, clinical signs of inflammation are not apparent [3, 4]. While BV is a
distinct clinical entity, coinfection with other vaginal (Candida
albicans or Trichomonas vaginalis) or cervical (Neisseria gonorrhoeae, Chlamydia trachomatis, or herpes simplex virus)
pathogens can occur [5].
Bacterial vaginosis has a complex microbiology. Lactobacillus populations, which are usually dominant in healthy women,
are replaced by a polymicrobial group of organisms that includes Gardnerella vaginalis, anaerobic gram-negative rods
such as Prevotella species, Peptostreptococcus species, Mycoplasma hominis, Ureaplasma urealyticum, and often Mobiluncus species [6]. Overall, concentrations are 100- and IOOO-foid
greater for aerobes and anaerobes, respectively, than levels
measured in women without BV [7]. However, the factor(s),
either endogenous or exogenous, that initiate the shift in the
ecology of the vagina and result in the massive overgrowth of
these microbial populations are incompletely understood
[5, 6].
Received 21 May 1996; revised 21 August 1996.
Presented in part: World Congress on Anaerobic Bacteria and Infections,
San Juan, Puerto Rico, November 1995.
Grant support: SmithK1ine Beecham; Tambrands.
Reprints or correspondence: Dr. Vivien Pybus, Channing Laboratory, Harvard Medical School, Brigham and Women's Hospital, 181 Longwood Ave.,
Boston, MA 02115.
The Journal of Infectious Diseases 1997; 175:406-13
© 1997 by The University of Chicago. All rights reserved.
0022-1899/97/7502-0020$01.00
Results from epidemiologic studies have associated BV with
serious upper genital tract infections and adverse pregnancy
outcome (see [8, 9]). In particular, the presence of BV in pregnant women increases the risk of preterm delivery, and evidence is now compelling that BV is a cause of preterm delivery
[8]. In addition, the state of the vaginal microflora has a significant impact on a woman's overall health [10], and the abnormal
and often foul-smelling discharge that can accompany BV is
of great concern to many women. These observations, combined with the high frequency of occurrence, prompt a greater
understanding of the pathogenesis of BV.
Knowledge of the physiologic capabilities of the organisms
isolated from a particular environment is fundamental to an
understanding of their ecology. We studied the influence of
nutrients on the growth of two organisms associated in high
concentration with BV, Prevotella bivia [11] and G. vagina lis
[12], and investigated whether a symbiotic relationship could
exist between these two organisms.
Materials and Methods
Bacterial strains. P. bivia strains 12-7, 22-16, 71-14, 80-21,
and 109-19 and G. vaginalis strains 007, 219-5, S06, and S17
were isolated from the vaginal vaults of healthy women using the
duplicate swab technique [13], and samples were processed as
described [14]. P. bivia ATCC 29303 was included as a type
strain. P. bivia was cultivated onto prereduced brucella-base agar
containing 5% laked sheep blood, supplemented with hemin and
vitamin KI (BMB; Remel, Lenexa, KS) within an anaerobic
growth chamber (Forma Scientific, Marietta, OH) containing 10%
(vol/vol) hydrogen, 10% carbon dioxide, and 80% nitrogen. G.
vaginalis was cultured on chocolate agar (CROC; Remel) and
incubated anaerobically. Isolates were identified using a microbial
identification system (MIDI; Microbial Identification, Newark,
DE). Stock cultures of each strain were prepared by the addition
JlD 1997; 175 (February)
Symbiosis in Bacterial Vaginosis
of glycerol (final concentration, 10% [vol/vol]) to 20-h vaginal
defined medium (VDM [15]) cultures and stored at - 80°C. Routine strain maintenance was by weekly subculture on BMB for P.
bivia or CHOC for G. vaginalis for up to 6 weeks. An incubation
temperature of 37°C was used throughout.
Growth in different nutrient conditions. Growth of the 6 strains
of P. bivia and the 4 strains of G. vaginalis was tested in the
following media: VDM, CAS (VDM supplemented with 1% [wtl
vol] vitamin assay casamino acids [Difco, Detroit] as a source of
amino acids), PEP (VDM supplemented with 1% [wt/vol] Difco
proteose peptone [Difco]) as a source of peptides, and VDM minus
glucose (VDM-glc). Each strain was inoculated from a 24- to 48h plate culture into 10 mL of VDM and incubated anaerobically
for ~ 20 h. Organisms (100 J-lL) were inoculated into 20 mL of
each medium in 125-mL shake flasks and incubated anaerobically
with orbital shaking at 20 rpm. The following parameters were
measured as determinants of growth: viable cell density (determined by dilution plate count on BMB for P. bivia and CHOC
for G. vaginalis) expressed as 10glO cfu/mL, A 450 relative to uninoculated controls incubated in parallel, pH, and concentrations of
both short-chain fatty acids (SCFAs, see below) and ammonia (see
below) over 72 h for P. bivia and 96 h for G. vaginalis. The pH
of each medium was adjusted to 6.0 for P. bivia and to 7.0 for G.
vaginalis. It was previously established that under these conditions,
growth is more optimal than at lower pH values.
Growth in continuous culture. A 1-mL frozen stock culture of
each G. vaginalis culture was streaked onto CHOC to verify purity,
then inoculated into 10 mL ofVDM and incubated for 20 h anaerobically. Next, 1 mL of this culture was inoculated into a 1.5L fermentation vessel (New Brunswick Scientific, Edison, NJ)
containing 1.1 L of VDM and agitated at a rate of 200 rpm,
maintained under anaerobic conditions by sparging with mixed
anaerobic gases (as above), and held in batch culture for 24 h. The
pH was adjusted to 6.0 using 0.5 M HCI, and the culture was
switched from batch to continuous culture to give a dilution rate
of 0.050-0.052/h, corresponding to a generation time of 13.213.8 h. Each strain was grown for 3 days, sequentially, at pH of
6.0, 6.5, 7.0, and 7.5. Ten-milliliter samples were collected daily.
Growth was determined by dilution plate count onto CHOC. Ammonia and SCFA concentration were determined as below.
Effect of PEP culture supernatants from P. bivia and G. vaginalis on reciprocal growth. Twenty-hour VDM cultures of P.
bivia strains 12-7 and 80-21 were inoculated into 35 mL of PEP,
pH 6.0, as above, and incubated for 40 h in parallel with 35 mL
of uninoculated PEP (control). The supernatants were harvested
by centrifugation, analyzed for ammonia concentration, pH-adjusted to 7.0, then sterilized by passage through a 0.22-J-lm filter
(Falcon, Lincoln Park, NJ). Ten-milliliter volumes were dispensed
into three 125-mL sterile shake flasks. The uninoculated PEP was
similarly prepared. One hundred microliters of 20-h G. vaginalis
cultures from strains 007 and S06 grown in VDM were each
inoculated into the two P. bivia culture supernatants and the uninoculated control. These six test flasks plus three uninoculated controls-10 mL each of the two P. bivia culture supernatants and
the uninoculated PEP- were incubated anaerobically for 46 h,
with shaking (20 rpm). Growth of G. vaginalis was monitored
over time by plate count on BMB. The concentration of ammonia
and SCFAs was measured at the completion of the experiment.
407
The reciprocal experiment was tested by growing P. bivia strains
12-7 and 80-21 for 30 h, in 40-h supernatants from G. vaginalis
strains 007 and S06. The pH of PEP was adjusted to 7.0 for G.
vaginalis growth and to 6.0 before inoculation of P. bivia into the
G. vaginalis culture supernatants.
SCFA detection. Volatile (acetate, propionate, isobutyrate, butyrate, isova1erate, valerate, isocaproate, caproate) and nonvolatile
(lactate, oxalacetate, oxalate, methyl malonic, malonic, fumaric,
succinate) SCFA concentrations were determined using gas-liquid
chromatography, using a Perkin-Elmer (Norwalk, CT) Sigma 300
gas chromatograph fitted with a flame ionization detector. Briefly,
culture supernatants were prepared for analysis of volatile or nonvolatile fatty acids using standard procedures [16]; detection was
by comparison of peak areas and retention times with those of
authentic standards (Matreya, Pleasant Gap, PA). The active phase,
10% Carbowax 20M on a solid support of 80/100 Chromosorb
WAW (Supelco, Bellefonte, PA), was packed in a 6.35-mm (internal diameter) X 3.68-m glass column (Perkin-Elmer), and peak
areas were calculated by computing integrator (model LCI 100;
Perkin-Elmer). The flow rate ofthe carrier gas, air, was 50 mLimin.
The injection port and detector were both maintained at 225°C.
Ammonia detection. The presence of ammonia in culture supernatants was determined using the method of Chaney and Marbach [17], using 0.05 mM and 0.5 mM solutions of (N~)2S04 as
positive control solutions. Briefly, 1 mL of solutions 1 and 2 were
added to 0.1 mL of culture supernatant (or control) and the A 625
was read after 1 h relative to distilled water (A 625 = 0).
Statistical analysis. Data analysis was by analysis of variance
(INSTAT GraphPad Software, San Diego).
Results
Six strains of P. bivia were grown under different nutrient
conditions for 72 h, during which viable cell density, A 450 , pH,
and SCFAs were measured as parameters ofgrowth. The results
were compared with those obtained for VDM alone (table 1).
Growth of P. bivia in the absence of glucose (VDM-glc) did
not result in a statistically significant difference between any
of the growth parameters measured. However, growth in VDM
supplemented with amino acids (CAS) did stimulate growth of
P. bivia to some extent. In particular, there was a significant
(P < .005) increase in maximum A 450 and in the production
of the SCFAs succinate and isovalerate. Although mean maximal cell density and pH were also elevated and depressed,
respectively, relative to VDM, these results were not statistically significant. The increased growth rate of P. bivia in CAS
compared to VDM (data not shown) also indicated that amino
acids promoted growth in VDM.
The addition of peptides to VDM (PEP) was highly stimulatory to the growth of P. bivia, with a statistically significant
(P < .005) difference recorded for all growth parameters relative to those obtained in VDM alone (table 1). Of particular
note was the increase in maximum viable cell density (8.36
10glO cfu/mL), which approached 1 order of magnitude greater
than that obtained in either VDM (7.52 10glO cfu/mL) or CAS
(7.71 10glO cfu/mL). The high viable cell density obtained dur-
408
Pybus and Onderdonk
JID 1997; 175 (February)
Table 1. Growth, as maximum detected viable cell density and A 450 , final pH, and maximum shortchain fatty acid production, recorded for 6 strains of P. bivia after growth in different media for 72 h.
Growth medium
Growth parameter
VDM
CAS
PEP
VDM-glc
Mean maximum viable cell density (JOglO cfu/mL)
SE
p
7.52
0.08
Mean final pH
SE
p
5.71
0.02
Mean maximum A 450
SE
p
0.08
0.011
Mean maximum succinate concentration (mM)
SE
p
0.036
0.004
Mean maximum isovalerate concentration (mM)
SE
p
0
7.71
0.10
.184
5.58
0.09
.095
0.18
0.034
.002
0.177
0.043
<.001
0.055
0.008
<.005*
8.36
0.21
<.001
4.58
0.06
<.001
0.74
0.050
<.001
0.691
0.113
<.001
0.182
0.054
<.005*
7.62
0.09
.457
5.72
0.02
.814
0.07
0.006
.574
0.044
0.004
.289
NT
NOTE. VDM, vaginal defined medium; CAS, VDM supplemented with amino acids; PEP, VDM supplemented
with peptone; VDM-glc, VDM without glucose. P is calculated relative to VDM. - , data point not relevant; NT,
not tested.
* P calculated considering mean and SD of 0, recorded for VDM.
ing growth in PEP was followed by elevated A 450 readings and
by a dramatic decrease in pH from 6.0 to 4.58, which coincided
with a decrease in viable cell density to 0 (data not shown).
The pH decrease seen during growth in PEP contrasted with
pH measurements obtained after growth in the presence of
other nutrient conditions, in which the pH changed little from
the starting value of 6.0 and a decrease in viable cell density
to 0 was not observed (data not shown).
Succinate was the dominant SCFA detected during growth
by P. bivia and was produced under all nutrient conditions
tested (table 1). Concentrations appeared cell density-dependent, ranging from ~0.04 mM after growth in VDM or
VDM -glc to ~5 times this level (0.177 mM) after growth in
CAS and ~ 19 times this concentration after growth in PEP
(0.691). Isovalerate was detected only when amino acids or
peptides were added to VDM, with mean levels of 0.055 mM
and 0.182 mM recorded, respectively. Isovalerate concentrations were also related to cell density.
Four strains of G. vaginalis were also tested for growth
under the same nutrient conditions (table 2). When the growth
parameters were statistically compared, there were no significant differences between growth in VDM, CAS, or VDM-glc.
However, the addition of peptides to VDM stimulated the
growth of all strains of G. vaginalis, and differences for all
growth parameters were significant (P < .005) relative to those
recorded during growth in VDM. The maximum mean viable
cell density (8.56 10glO cfu/mL) was similar to that recorded
for P. bivia in the presence of peptides and notably higher than
that observed for G. vaginalis in VDM (5.87 10glO cfu/mL),
CAS (6.22 10glO cfu/ml.), and VDM-glc (6.71 log., cfu/mL).
Similar to P. bivia, detection of maximum viable cell density
was followed by an increase in A450 and a dramatic decrease
in pH, from the starting value of 7.0 to 4.25, and coincided
with a decline in cell density to 0 (data not shown). Absorbance
(A450 ) was measurable only when the viable cell density reached
a minimum of 7 10glO cfu/ml.. As concentrations remained
below this level, it would account for why the A 450 remained
essentially undetectable during growth in CAS, PEP, and
VDM-glc.
Acetate was the main SCFA detected during growth by G.
vaginalis. Similar to A 450 readings, acetate was produced within
a detectable range when cell numbers reached ~ 7 Iog., cfu/
mL. Consequently, acetate concentrations were highest after
growth in PEP, when they were produced by all strains at a
mean concentration of 4.02 mM, but were lower after growth
in CAS (0.24 mM), when production was detectable for only
2 of the 4 strains present in sufficient viable cell density. Lactate, mean maximal concentration 2.56 mM, was also produced
by all strains of G. vaginalis but was only detected after growth
in PEP. Similar to acetate, lactate detection in this system was
dependent on a viable cell density of ~8 10glO cfu/ml. or
greater. Succinate production was detected for 2 ofthe 4 strains
after growth in PEP only, in a relatively low concentration
(0.006 mM).
All culture supernatants were tested for a net change in
ammonia concentration relative to the uninoculated controls,
which had been incubated in parallel (figure 1). Growth under
all nutrient conditions was accompanied by ammonia production by P. bivia and ammonia utilization by G. vaginalis. The
exception was for G. vaginalis after growth in VDM - glc,
JlD 1997; 175 (February)
Symbiosis in Bacterial Vaginosis
409
Table 2. Growth, as maximum detected viable cell density and A450 , final pH, and maximum shortchain fatty acid production, recorded for 4 strains of G. vaginalis after growth in different media for
96 h.
Growth medium
Growth parameter
VDM
CAS
PEP
VDM-glc
Mean maximum viable cell density (log.; cfu/mL)
SE
p
5.87
0.27
0
0
0
0
8.56
0.25
<.001
4.25
0.03
<.001
1.007
0.027
<.001
4.02
0.17
<.005 1
2.56
0.33
<.005 1
0.006*
0.003
<.005 1
6.71
0.31
.083
6.53
0.03
.390
0.009
0.004
.538
0
0
0
0
6.22
0.40
.487
6.35
0.05
.121
0.008
0.006
.690
0.24*
0.21
.05-.101
0
0
Mean final pH
SE
p
6.48
0.05
Mean maximum A 450
SE
p
0.005
0.003
Mean maximum acetate concentration (ruM)
SE
p
0
0
Mean maximum lactate concentration (ruM)
SE
p
Mean maximum succinate concentration (ruM)
SE
p
0
0
0
0
NOTE. VDM, vaginal defined medium; CAS, VDM supplemented with amino acids; PEP, VDM supplemented
with peptone; VDM - glc, VDM without glucose. P is calculated relative to VDM. - , data point not relevant.
* Only 2 of the 4 strains produced detectable levels of fatty acid.
1 P calculated considering mean and SD of 0, recorded for VDM.
219-5 and S06, in VDM over the pH range 6.0-7.5. For all
strains, the ammonia concentration decreased from "'-'0.75 mM
to almost 0, coinciding with an increase in G. vaginalis concentration to ~7 10glO cfulmL. This appeared to be pH-independent, occurring at pH 6.0, 6.5, and 7.0 for strains S06, 219-5,
and 007, respectively. For all strains, the trend for increasing
which showed a small net production of ammonia (0.023 mM),
which could suggest that substrate utilization pathways differ
for G. vaginalis in the presence or absence of glucose.
Apparent ammonia utilization by G. vaginalis was also demonstrated during continuous culture (data not shown), in triplicate experiments for G. vagina lis strain 007 and then for strains
0.25--,--------------------------,
0.2
0.15
Figure 1. Net change in ammonia concentration
after growth of 6 strains of P. bivia and 4 strains
of G. vagina/is in vaginal defined medium (VDM),
VDM supplemented with amino acids (CAS),
VDM supplemented with peptone (PEP), and
VDM without glucose (no glc) for 72 and 96 h,
respectively. Error bars represent SEs. Concentrations of ammonia in uninoculated media were as
follows: VDM (0.9 mM), CAS (0.02 mM), PEP
(0.5 mM), and VDM-glc (0.9 mM).
§
0.1
E
CD
0.05
~
o
§
0+---
o
.~
o
~
as
-0.05
-0.1
a> -0.15
z
-0.2
-0.25+-------,------,---r----,--------,---r--,----1
I
VDM
CAS
PEP no glc VDM
P. bivia
----'
l--
CAS
PEP no glc
G. vaginalis ----'
410
Pybus and Onderdonk
concentration from -6 10glO cfu/mL to -8 10gIO cfu/mL with
increasing pH was observed. As seen in the batch culture experiments, acetate detection in this system seemed cell densitydependent, coinciding with a viable cell density of ~7 10glO
cfu/mL.
To evaluate whether a symbiotic relationship could exist
between these two organisms, the influence on G. vaginalis
growth of PEP supernatants previously grown with P. bivia
was tested and vice versa. Results from replicate experiments
showed a reduced lag phase for G. vaginalis strains 007 and
S06 grown in supernatants from P. bivia strains 12-7 and 8021 relative to growth in uninoculated media (figure 2A). This
suggested that prior growth of P. bivia in PEP stimulated the
growth of G. vaginalis. Analysis of ammonia concentrations
in the P. bivia supernatants indicated increased levels of -0.1
mM relative to the uninoculated media, which were reduced
by -0.25 mM after G. vaginalis growth. When the reciprocal
experiment was carried out (figure 2B), results from replicate
experiments indicated no apparent difference in lag phase between growth of P. bivia in PEP supernatants previously grown
with G. vaginalis and in control media. The levels of ammonia
depleted during G. vaginalis growth (-0.2 mM) were enhanced
by -0.15 mM after the growth of P. bivia. Analysis of SCFAs
in the PEP supernatants before inoculation of the reciprocal
strain showed the presence of the following in the approximate
concentrations indicated: succinate (0.4 mM) and isovalerate
(0.08 mM) from P. bivia growth and acetate (5 mM) and lactate
(1.5 mM) from growth of G. vaginalis. Growth of P. bivia and
G. vaginalis did not appear to be inhibited by the presence of
each other's SCFAs in the culture supernatants.
Discussion
Chen et al. [18] proposed a symbiotic relationship between
G. vaginalis and anaerobes during BV. This centered, in part,
on their observation that G. vaginalis produces amino acids
during growth, which could be utilized by BV-associated microorganisms. Pheifer et al. [19] and Spiegel et al. [7] also
postulated that G. vaginalis and anaerobes, such as P. bivia,
act synergistically to cause BV. Results obtained during the
current study substantiate these hypotheses by providing a nutritional basis for a commensal relationship between P. bivia
and G. vaginalis. Specifically, we demonstrate that ammonia
is produced during growth by P. bivia and that it appears to
be utilized by G. vaginalis during growth. We also show that
P. bivia culture supernatants containing increased concentrations of ammonia relative to uninoculated controls stimulate
G. vaginalis growth. While this may be considered further
evidence that provision of ammonia by P. bivia forms the
basis of the commensal relationship, other nutrients or media
conditioning could also be involved. Irrespective of this, there
is a clear commensal relationship between P. bivia and G.
vaginalis demonstrated in this in vitro growth system.
JID 1997; 175 (February)
Chen et al. [18] demonstrated that amino acids are produced
during G. vaginalis growth. We demonstrate that growth of P.
bivia in VDM is enhanced in the presence of amino acids.
In combination, these observations could extend the proposed
commensal symbiotic relationship to one of mutualism, involving the cycling of ammonia and amino acids between these two
organisms (figure 3). However, more detailed studies showing
utilization by P. bivia of the specific amino acids produced by
G. vaginalis would be required to confirm this.
An abnormally high vaginal pH, >4.5, is a characteristic
feature of BV [2, 3, 12]. In in vitro studies, pH values (6.08.0) greater than those found during BV have been shown to
be favorable for the growth of both P. bivia [20] and G. vaginalis [21, 22] (this study), and the trend for increasing concentration with increasing pH has generally been observed. While
the pathogenesis of BV remains speculative, it is possible that
factors as yet undescribed lead to an elevated vaginal pH,
which causes increased concentrations of both P. bivia and G.
vaginalis. The growth of G. vaginalis becomes limited by the
shortage of available ammonia, which is provided for by P.
bivia. Amino acids produced by G. vaginalis further stimulate
the growth of P. bivia, which in tum produces more ammonia.
The cycle continues, leading to high concentrations of both
organisms, until other nutrients become growth-limiting. If this
mutualistic relationship exists in vivo, it could be one mechanism, in addition to pH, that accounts for the presence of these
two organisms in high concentration during BV.
The in vitro production of inhibitory substances of potential
significance in the control of microbial populations in the vagina has been the subject of several investigations [23-25]. In
particular, antagonistic substances produced by lactobacilli in
vitro have been postulated to play a role in regulating the
growth of bacterial populations in the vagina [26]. Specific
examples include production of H20 2 [27-29] and of acidic
conditions [30]. However, positive interactions between microbial populations are less frequently described. This is the first
report, to our knowledge, of a commensal (or mutualistic) relationship between populations of vaginal microorganisms with
possible implications for BV.
During BV, the vaginal environment is characterized by a
preponderance of proteolytic organisms [31], and P. bivia has
been shown to produce strong proteolytic activity in vitro [32].
Peptidases, such as proline aminopeptidase, from BV-associated organisms such as Mobiluncus species and G. vaginalis
[33, 34] are also reported to be present. This suggests that both
peptides and amino acids would be made available for bacterial
growth from an environment known to be protein-rich [35].
We have demonstrated that the addition of peptides to VDM
stimulates the growth of both G. vaginalis and P. bivia. Thus,
the increased availability of peptides during BV could be a
further factor contributing to the high concentration of these
organisms during BV. Peptides have previously been shown
to be stimulatory to the growth of G. vaginalis [22] and to
other Prevotella species or closely related organisms [36-39].
JID 1997;175 (February)
Symbiosis in Bacterial Vaginosis
411
10 - , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,
A
8
::J
.§
::J
'0 6
0
......
C>
g
.J::.
j
e
o
4
----
uninoculated medium
----A-
2
P. bivia 12-7 SN
----P. bivia 80-21 SN
Figure 2. A, Growth of G. vagina lis 007 in
supernatants (SN) from P. bivia strains 12-7
and 80-21 and in uninoculated medium (control) over 46 h. Similar results were obtained
for G. vaginalis S06 grown under same conditions. B, Growth of P. bivia 12-7 in supernatants from G. vaginalis strains 007 and S06
and in uninoculated medium (control) over 30
h. Similar results were obtained for P. bivia
80-21 grown under same conditions.
0
0
10
20
30
50
40
Time (hour)
10
B
8
::J
E
'3
'0 6
0
......
C>
s
.J::.
j
e
o
4
----
uninoculated medium
----A-
2
G. vaginal is 007 SN
-----
G. vaginalis 806 8N
o
--------T ----,---------,--------,----------.---------1
o
5
10
15
20
25
30
35
Time (hour)
In the current study, amino acids also stimulated P. bivia
growth, but to a lesser extent than peptides. Overall, these
results suggest the potential importance of nitrogenous substrates for BV-associated organisms and in the ecology of the
vaginal ecosystem.
Addition of amino acids and peptides to VDM was accompanied by increased production (relative to VDM) of SCFAs and
ammonia by P. bivia. These results are preliminary evidence
that amino acids can be used as an energy source via reductive
deamination pathways, rather than by decarboxylation [40].
Utilization of amino acids as energy sources accompanied by
the formation of ammonia has been previously demonstrated by
Shah and Williams [41] for Prevotella intermedia. Ammonia
production is thought to contribute to highly alkaline pH, for
example in the periodontal pocket, where the pH has been
reported as high as 9.0 [41]. However, results from batch culture experiments showed that despite ammonia production, the
pH decreased after growth in all nutrient conditions, perhaps
due in part to SCFA production. Acid production by P. bivia
was particularly pronounced in PEP; in this case the low pH
Pybus and Onderdonk
412
ammonia
utilizes(
\
P. bivia
G. vagina/is
produces \
produces
~aminO
/utilizes
acids
Figure 3. Postulated cycling of ammonia and amino acids between
P. bivia and G. vagina lis.
of 4.58 may have contributed to the death of the organism
(seen also for G. vaginalis), which is known to be particularly
pH-sensitive [20]. In the vaginal tract during BV, ammonia
utilization by G. vaginalis (or other organisms) may keep levels
low. In addition, the concentrations produced may be insufficient to elevate the overall vaginal pH. Host factors influencing
the vaginal pH, such as hormonal activity [26], racial differences [42], and exogenous factors such as the alkaline nature
of semen [43], should not be overlooked and indeed remain a
fundamental unknown in the pathogenesis of BV.
It is becoming increasingly clear that microbial interactions
in the vaginal ecosystem, during both health and disease, are
complex. Synergistic interaction with aerobes has been described as a feature of polymicrobial anaerobic infections [44,
45]. The proposed symbioses between the anaerobe P. bivia
and the facultative aerobe G. vaginalis, described in this study,
are of potential significance to the pathogenesis of BV. Interbacterial cooperation has been previously reported as being
important in mixed infections containing Bacteroides-related
organisms [46]. In this regard, the recent finding that the highest
incidence of preterm delivery of a low-birth-weight infant was
in BV subjects with both Bacteroides species and M. hominis
[8] is of considerable interest. Undoubtedly many microbial
interactions exist in the vaginal ecosystem, and our ability to
uncover them has been proposed to provide some of the answers to the ecologic mysteries surrounding BV [5].
Acknowledgments
We gratefully acknowledge Robin Ross for discussions on the
continuous culture growth system and Mary Delaney, Andrea DuBois, Cheryl Fay, and James Christian for provision of bacterial
isolates.
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