The Effect of pH on Growth and Succinate Production by Prevotella

The Effect of pH on Growth and Succinate Production by Prevotella

MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
9: 19-25 (1996)
The Effect of pH on Growth and Succinate Production
by Prevotella bivia
V. PYBUS* and A. B. ONDERDONK
Channing Laboratory, Harvard Medical School, Boston, USA
Received 10 July 1995; revised 10 October 1995
Prevotella bivia is frequently isolated as a vaginal commensal and is also one of several organisms associated with
bacterial vaginosis. In an attempt to define the role of this organism as part of the vaginal ecosystem, the influence
of pH o n viable cell density and short chain fatty acid (SCFA) production was examined. Seven strains of P. bivia
isolated from the vagina of healthy women were grown in vaginal defined medium for 3 d at pH values 6.0, 5.5, 5.0
and 4.5 using an in v i m continuous culture system. At p H 6.0, P. bivia was present at a mean maximal cell density
of log,, 7-93colony forming units per ml (CFUlml). A mean decrease in viable cell density of log,, 1-39CFUfmllday
following a pH change to either 5.5 o r 5.0 for all strains, indicated that the organism was pH-sensitive. Succinate,
the only SCFA detected during growth in this system, was produced at a mean concentration of 0.025 m M at
maximal cell density. Regression analysis of information contained in an in vivo data set of observations from
healthy, menarcheal women also correlated the concentration of P. bivia with vaginal p H (P=0.049). The apparent
sensitivity of this organism to pH suggests that the concentration of P. bivia within the vagina may serve as a
surrogate marker for vaginal pH.
KEY WORDS: Prevotella biviu; bacterial vaginosis; pH; succinate; vagina.
INTRODUCTION
Prevotella b i ~ i aformerly
,~~
Bacteroides bivizis,l 3 is
a gram-negative, obligately anaerobic bacterium,
and it is the most numerous of the vaginal Prevotella species (A. B. Onderdonk, unpublished observations). Data accumulated in this laboratory
from over 2000 subject observations characterising
the normal vaginal microbiota during the menstrual cycle, indicate that P. bivia is frequently
isolated as a commensal in healthy, non-pregnant
menarcheal women. When a statistical model
was applied to this data set the concentration of
Prevotella spp was identified as one of five major
factors, which, in combination, can be used to
calculate the likelihood that a given microbiota is
within the range of 'normal'.'6 This was unexpected given that P. biviu is usually present at a
concentration of log,, 4-6 CFU/ml, which is approximately 100-fold lower than the numericallydominant organisms."
*Author to whom correspondence should be addressed
at: Channing Laboratory, Harvard Medical School, 180
Longwood Avenue, Boston, MA 02115, USA.
CCC 0891-060W96/010019-07
0 1996 by John Wiley & Sons, Ltd.
Bacterial vaginosis is a common lower genital
tract infection, affecting women of child-bearing
age," and it has been reported as the most common vaginal disorder seen in primary health
care.14 Criteria most commonly used to diagnose
bacterial vaginosis include three out of four of the
following: an abnormally high (>4.5)vaginal pH;
a 'fishy' amine odour; a thin homogenous discharge and the presence of clue cells in the vaginal
Recent interest in this clinical syndrome
has been increased by its association with serious
upper genital tract sequelae and pregnancy
complication^.^*^*^
Bacterial vaginosis has been associated with a
constellation of microbial species and progress in
understanding this condition has been hampered
by attempts to identify a single aetiologic agent.5
Microbial populations in women with bacterial
vaginosis differ from those associated with the
normal vaginal microbiota. In contrast to the
dominant Lactobacillus populations of healthy
women,17 individuals with bacterial vaginosis
often have a microbiota dominated by anaerobic
gram-negative rods such as Prevotella spp,
20
Gardnerella vaginalis, Peptostreptococcus spp,
Mycoplasma hominis, Ureaplasma urealyticum and
Mobiluncus spp,’ in concentrations 100- to 1000fold above levels measured in asymptomatic
women.4 In one study of 67 women with bacterial
vaginosis, 94 per cent were reported to carry at
least one isolate from the genus Bacteroides.
Against this background, our aim was to understand the physiology of P. bivia in an attempt to
determine the role of this organism as both a
vaginal commensal and as a possible contributory
agent to bacterial vaginosis. As pH has been
proposed as a potent control mechanism for
microbial opulations within the vaginal environment, 18*20*‘ these studies address the influence
of pH on growth and production of SCFAs by
P. bivia.
MATERIALS AND METHODS
Bacterial strains and culture conditions
Strains of P. biviu were isolated from the vaginal
tract of healthy women using the duplicate swab
technique2 by cultivation onto pre-reduced brucellabase agar containing 5 per cent laked sheep blood,
100 pg of kanamycin and 7.5 pg of vancomycin per
ml, supplemented with 0.01 g of both haemin and
vitamin K1 (BKV, Remel, Lenexa, Kansas, USA)
within an anaerobic growth chamber (Forma Scientific, Marietta, Ohio, USA) containing 10 per cent
(vlv) hydrogen, 10 per cent carbon dioxide and 80
per cent nitrogen. Isolates were identified using the
Microbial Identification System (MIDI, Microbial
Identification Inc., Newark, Delaware, USA) and
confirmed using antibiotic susceptibility patterns
and SCFA production, as previously described.”
Stock cultures of each strain were prepared by the
addition of glycerol (final concentration 10 per cent
[vlv]) to 24h vaginal defined medium (VDM8)
cultures and stored at - 80’C. An incubation
temperature of 37’C was used throughout.
Continuous culture studies examining the effect of
p H on viable cell density and SCFA production
The following strains of P. bivia were selected at
random from the laboratory culture collection and
tested for the influence of pH on growth (measured
as viable cell density, CFU/ml) and production of
SCFAs: ATCC 29303, 12-7, 22-16, 71-14, 80-21,
109-19 and 1-7. The vaginal pH of the specimens
from which the strains were isolated was in the
range 4.1 to 6.0. A 1 ml frozen stock culture of
V. PYBUS AND
A. B. ONDERDONK
each was streaked onto both pre-reduced brucellabase agar containing 5 per cent laked sheep blood
and 0.01 g of both haemin and vitamin K1 per litre
(BMB, Remel) and BKV to verify purity. P. bivia
colonies were inoculated from BMB into 10 ml
VDM and incubated for 20 h anaerobically. One
ml of this culture was inoculated into a 1.5 litre
fermentation vessel (New Brunswick Scientific Co.
Inc., Edison, New Jersey, USA) containing 1 litre
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 HC1 and the culture switched from
batch to continuous culture to give a dilution rate
of 0.050-0.052/h, corresponding to a generation
time of 13-2-13.8 h. Each strain was grown for 3 d,
sequentially, at pH values of 6.0, 5.5, 5.0 and 4.5.
Five ml samples were collected daily. Growth was
determined by dilution plate count onto BKV.
Every 4 d samples were also seeded onto BMB to
ensure purity. The presence in culture supernates
of SCFAs (formic, acetic, propionic, isobutyric,
butyric, isovaleric, valeric, isocaproic, caproic,
lactic and succinic) was tested using gas-liquid
chromatography (GLC)by comparison of retention times with those of authentic standards, as
described previously.I y In preliminary experiments
using this culture system, strains 12-7 and
22-1 6 were tested in duplicate, with reproducible
results. The remaining strains were each grown in
continuous culture.
In vivo data set
Information on the concentration of P. bivia and
vaginal pH was obtained from an in vivo data base
containing over 2000 subject observations on the
vaginal microbiota of healthy, non-pregnant
menarcheal women collected in this laboratory
over 12 yrs, as outlined previously.’6 Briefly, the
duplicate swab technique was used to sample the
vaginal tract for each subject2 and processed using
standard procedures.” Up to 79 variables were
recorded for each sample, including day of sample,
pH, total concentrations of aerobes (facultative)
and anaerobes, and concentrations of the various
genera and species of bacteria identified, which
were reported as log,,, CFU/g of secretion.
Statistical analysis
Data from the in vitro continuous culture experiments were analysed by analysis of variance
21
PREVOTELLA BIVIA, EFFECT OF PH
Table 1. Summary of the main features from continuous culture experiments examining the influence of pH on
growth and succinate production for seven strains of P.biviu. Note for this system, with a generation time of 13.5 h,
a bacteriostatic effect corresponds to a decrease in viable cell density of log,, 0.53 CFUlmllday
Standard
Mean Deviation
Feature
Maximum viable cell density under steady state conditions (log,, CFUlml)
Minimum viable cell density (log,, CFU/ml)
Decrease in viable cell density over the pH decrease from 6.0 to 4.5 (log,, CFU/mI)
Initial rate of decrease in viable cell density at sensitive pH value* (log,, CFU/ml/day)
Initial rate of decrease in viable cell density for strains sensitive to pH 5.5 (log,, CFU/ml/day)
Initial rate of decrease in viable cell density for strains sensitive to pH 5.0 (log,, CFU/ml/day)
Final rate? of decrease in viable cell density (log,, CFU/ml/day)
Concentration of succinate produced under steady state conditions (mM)
Viable cell density at which succinate production no longer detected (log,, CFU/ml)
7.93
3.29
0.23
4.58
1.39
0.92
0.78
0:56
0.22
1.68
0.30
0.11
0.75
0.51
0.025 0.008
4.77
0.73
*All strains were sensitive to either pH 5.5 (strains ATCC 29303, 12-7 and 71-14) or 5.0 (strains 1-17, 22-16, 80-21 and 109-19).
?The final rate is that observed over the remainder of the experiment. after the initial rate of decrease.
(INSTAT‘@ GraphPad Software, San Diego,
California, USA) and by regression analysis
(QUATTRO(@PRO, Borland International Inc.,
Scotts Valley, California, USA). The relationship
between vaginal pH and the concentration of
either the numerically-dominant Prevotella spp or
P. bivia was determined by regression analysis of
information from the in vivo data base (see
above), using the Complete Statistical System:
STATISTICA@ (Statsoft, Inc., Tulsa, Oklahoma,
USA).
RESULTS
The inpuence of p H on growth and SCFA
production for P. bivia, in vitro studies
To examine the influence of pH on growth
and production of SCFAs for P. bivia, seven
strains were grown in continuous culture for 3 d at
successive pH values of 6.0, 5.5, 5.0 and 4.5, the
range generally found over a single menstrual
cycle.” As a preliminary experiment, to ensure
that time did not influence either growth or SCFA
production during continuous culture, strain 1-17
was grown at pH 6.0 for 13 d (Figure la). The
viable cell density was maintained at steady state
conditions of log,, 8 CFU/ml over the entire
experiment. Succinate was the only SCFA detected
by GLC analysis, and was maintained at a
consistent concentration of 0.025 mM.
As time did not appear to influence either
growth or succinate production for P. bivia 1-17
at pH 6.0, the influence of pH on these growth
parameters was tested for seven P. bivia strains
(Table 1). At pH 6.0 P. bivia was present at a mean
steady state population of log,, 7.93 CFU/ml.
However, a mean decrease in viable cell density of
log,, 1-39 CFU/mYday was noted for all strains,
following a shift in pH to either 5.5 or 5.0,
depending on the strain. The decrease in viable cell
density of log,, 1-68 CFU/ml/day was greater for
strains sensitive at pH 5-0 (strains 22-16, 80-21,
109-19 and 1-17) than for strains sensitive at
pH 5.5 (strains 12-7, 71-14 and ATCC 29303),
which demonstrated a rate of decrease of log,, 0.92
CFU/mYday. As the rates of change in viable cell
density were greater than those calculated for a
bacteriostatic effect in this system (log,, 0.53 CFUI
mYday), a decrease in pH to the strain-sensitive pH
value was bactericidal for P,bivia.
The initial sharp decrease (log,, 1.39 CFU/ml/
day) in viable cell density associated with the
change in pH to either 5.5 or 5.0, eased to a
mean value of log,, 0.30 CFU/mYday, over the
remainder of the experiment (Table 1 and Figures
l b and lc). This value was greater than that defined
for a bactericidal effect in this system, but less
than zero (steady state conditions), indicating that
the rate of multiplication for P. bivia was less than
that required to maintain a consistent concentration. Overall, a mean decrease in viable cell
density of log,, 4.58 CFU/ml was associated with
the pH decrease from 6.0 to 4.5. A mean minimum
viable cell density of log,, 3.29 CFU/ml was
recorded on day 13, at the completion of the
experiment.
22
V. PYBUS A N D A. B. O N D E R D O N K
a)
1
020
10
-1
0
I
2
4
8
8
10
12
c0.m
14
12
14
T
m
i (days)
b)
g
~
. .0
2
4
8
8
10
Tbne (days)
Figure 1. Effect of pH (-U-)
on growth (-+-,
CFU/ml) and succinate
for P. bivin grown in VDM, in continuous culture. (a) A
production (-*-)
preliminary experiment was carried out to determine the influence of time on
growth and succinate production for strain 1-17. As both parameters
appeared constant over time, the influence of pH was tested. @) Strain 12-7
showed a sharp decrease in viable cell density at pH 5.5, which coincided
with the decrease in succinate production. (c) A similar result was recorded
for strain 109-19, which was sensitive to pH 5.0
23
PREVOTELLA BIVIA. EFFECT OF PH
PH
Figure 2. Scattergram from an in vivo data set containing 791 observations, showing the relationship between the concentration
of the numerically-dominant Prevofriiu spp (PREV) and vaginal pH. This was defined by regression analysis as
PREV=0.0680 x pH+2.352 ( P ~ O ~ O O O I )
For all strains, succinate was the only SCFA
produced during growth (Figures la-c and Table
l), with a mean concentration of 0.025 mM detected at maximal cell density (pH 6.0). Succinate,
which had a lower detection limit of 0.004mM,
was no longer detected below a mean viable cell
density of log,, 4.77 CFU/ml, which coincided
with the strain-dependent sensitive pH value.
(Pc0.0001). Using a more recent data set of 44
observations with known levels of P. bivia, the
concentration of P. bivia (PREVB) was also correlated with vaginal pH, and was defined by
PREVBz0.602 x pH+3.183 (PzO.049).
DISCUSSION
Relationship between vaginal p H and the
Concentration of P. bivia, in vivo studies
Regression analysis of an in vivo data set containing 791 observations was camed out to determine any relationship between the concentration of
the numerically-dominant Prevotella spp and vaginal pH. Each data entry was from a vaginal swab
sample from healthy non-pregnant, menarcheal
women harbouring Prevotella spp as part of the
normal vaginal microbiota (Figure 2). A significant
correlation (P<O.OOOl) was found between the
Prevotella spp concentration (PREV, expressed as
log,,) and vaginal pH, which was defined by the
following equation, PREV=0-680 x pH+2.352
The aim of the present study was to examine
the influence of pH on the growth of P. bivia, in
an attempt to understand better the role of this
organism as both a vaginal commensal and as a
contributing organism to bacterial vaginosis.
Using a continuous culture growth system, all P.
bivia strains were present in high concentrations
(c. log,, 8 CFU/ml) at pH 6.0, the highest pH
tested. A decrease in pH to either pH 5.5 or 5.0
had a bactericidal effect on cell growth for approximately three generations, after which the
rate of multiplication was below that needed to
maintain steady state conditions. The apparent
preference of conditions of high pH for the growth
of P. bivia demonstrated in these in vitro studies
24
was also supported by in vivo observations. Regression analysis of data collected from healthy
subjects over 12 years indicated a significant correlation (P<O.OOOl) between pH and the concentration of the numerically-dominant Prevotellu spp
(which is generally P. biviu) within the vaginal
tract. This was supported by more recent data
which include subjects with known concentrations
of P. biviu (P=O-049).
The observation that P. biviu is present at high
concentration when the pH value is 5.5 or greater,
seen in both the in vivo and in vitro studies,
correlated well with one of the characteristic
features of bacterial vaginosis, an abnormally high
vaginal
In addition, previous studies have
recorded an increase in the vaginal concentration
of P. biviu from log,, 4-6 CFU/ml for healthy
subjects,18 to levels of c. log,, 7-9 CFU/ml for
subjects with bacterial vaginosis.’ Thus, conditions
of elevated pH appear to be favoured by P. biviu
for growth and could be a major factor accounting
for their presence in high concentrations, during
bacterial vaginosis.
These results may also explain observations
made in this laboratory, on the negative correlation between the concentration of Bucteroides spp
(the most numerous of which is P. biviu) and the
concentration of lactobacilli, observed during the
normal menstrual cycle.’* On the second day of
menstrual flow, when the pH is in the range
5-5-6.0, P. bivia is present at a concentration range
of 5-6 log,, CFUlml. By day 21, when the pH has
decreased to c. 4.5, the P. biviu concentration has
decreased to around 4-5 log,, CFU/ml. In contrast, lactobacilli, which prefer conditions of low
pH,” are most numerous on day 21 and are
present in lowest concentration on day two. This
observation also supports pH as a major controlling factor for the growth of microbial populations
in the vagina. However, factors other than pH
could contribute to the negative correlation
between the concentrations of Bucteroides spp and
lactobacilli in the vaginal ecosystem. For example,
the production of H,Oz by some strains of vaginal
lactobacilli has been postulated to regulate the
growth of H,O,-sensitive organisms,6 such as P.
biviu. Moreover, H,O,-generating strains of lactobacilli were shown to be bactericidal in vitro to P.
bivia.15 However, the extent to which this mechanism operates in vivo has yet to be determined.
Concentrations of succinate (0.025 mM) produced at maximal cell density during growth in
continuous culture, when the P. biviu concentra-
V. PYBUS AND A. B. ONDERDONK
tions were similar to those recorded in vivo during
bacterial vaginosis,’ were considered too low to
influence the overall vaginal pH. However, these
levels could contribute to the presence of succinate
within the vagina which has been re orted to be
characteristic of bacterial vaginosis.2 P
Host- or bacterial-derived factors which lead to
the development and maintenance of the high
vaginal pH characteristic of bacterial vaginosis,
remain a fundamental unresolved issue for this
~yndrorne.~=
In’ ~addition,
-~~
their relationship to
the changes from the normal vaginal microbiota
to that characteristic of bacterial vaginosis, remain
to be fully described. Studies on the physiology
of the microorganisms associated with bacterial
vaginosis may provide insight into the
processes which lead to the development of this
condition, which has been aptly described as
‘microbiologically indeterminate’.
ACKNOWLEDGEMENTS
This work was funded by grants from Smith Kline
Beecham and Tambrands, Inc. The authors gratefully
acknowledge Dr Robin Ross for discussions on the
in vitro continuous culture system, Mary Delaney for
discussions on the in vivo studies and Andrea DuBois
and Cheryl Fay for kind provision of bacterial isolates.
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