Growth Characteristics of Lactobacillus iners that Promote
Persistence in Bacterial Vaginosis Margot Oliver, Emily Diekema, & Amy Wilstermann, Ph.D.
Bacterial Species: Bacteria selected for testing were
Gardnerella vaginalis and four common vaginal
Lactobacillus species: L. gasseri, L. jensenii, L.
crispatus, and L. iners. All bacteria were grown in
NYCIII media under anaerobic conditions.
Inhibition Assay: Each Lactobacillus species was
grown in pH 5.51 NYC III media and prepared G.
vaginalis supernatant in 1:3, 1:1, and 3:1 ratios.
Head-to-Head Competition: Preparation of Bacteria:
Co-cultures at pH 4.27 or 5.51 were created by
combining Lactobacillus culture with G. vaginalis
culture. Pure culture was grown in parallel with the
co-cultures.
Experimental procedure: Immediately after
preparation of co-cultures (t=0), both pure and cocultures were plated. Plates containing 2 ug/ml of
metronidazole were used to inhibit G. vaginalis
growth. Bacteria were plated when the G. vaginalis
pure culture reached an O.D. of 0.6 (t=1), and again
one time interval later (approximately 4 hours).
*
600
500
*
400
600
400
Supernatant Inhibition: Neither the generation time
nor the lag time of L. iners is inhibited by factors
secreted by G. vaginalis (Figure not shown) as no
significant differences are observed between the
samples grown in L. iners supernatant (control) and
G. vaginalis supernatant. The generation time of L.
jensenii was increased with exposure to
L. jensenii
L. gasseri
L. iners
L. crispatus
G. vaginalis
200
0
4.59
5.15
5.62
Media pH
6.41
7.12
Figure 1. Effect of media pH on generation times of Lactobacillus species
and Gardnerella vaginalis.
* indicates significant difference from all other data points.
L. jensenii, pH 4.3
L. jensenii, pH 5.5
100
100
80
60
40
20
0
80
60
40
20
0
t=0
t=1
t=2
t=0
t=1
t=2
L. gasseri, pH 4.3
100
100
Percent of Total
80
60
40
20
0
t=0
t=1
t=2
80
60
40
20
t=0
L. iners, pH 5.5
t=1
6.0
80
60
40
t=2
G. vaginalis
L. iners
L. jensenii
L. gasseri
6.5
100
5.5
5.0
4.5
4.0
20
3.5
0
0
t=0
t=1
t=2
• L. jensenii and L. gasseri have a growth advantage
in the healthy pH range (< 4.5).
• Growth rates of L. iners and L. crispatus increase
as environmental pH shifts from healthy to BV
conditions.
• G. vaginalis has the ability to directly inhibit the
growth of several Lactobacillus species (L. gasseri,
L. crispatus, L. jensenii)
• In mixed culture, L. iners is able to become
dominant under BV conditions, but does not
inhibit G. vaginalsis at healthy conditions while
the inverse is true for L. jensenii and L. gasseri .
The pH-dependent growth characteristics of L iners
may contribute to its persistence in BV
communities, which is not observed among other
common vaginal Lactobacilli. Clearly, however, this
factor alone cannot account for L. iners persistence
since L. crispatus, shares the same growth
characteristics and is not found in BV communities.
Growth inhibition of Lactobacillus species could
account for the inability of probiotic Lactobacilli to
successfully recolonize a reproductive tract that is
inhabited by G. vaginalis.
0
Culture pH
Analysis of Bacterial Growht: L. iners and L. crispatus
have significantly longer generation times than the
other Lactobacilli studied when grown within a
normal vaginal pH range (Fig. 1). As shown in the
figure inset, growth rates of L. iners and L. crispatus
increase dramatically as pH is shifted from normal
(pH<4.5) to BV (pH>4.5) conditions. The
generation time of G. vaginalis was less than that of
L. iners and L. crispatus, but greater than that of L.
jensenii and L. gasseri at pH <4.5 (Fig. 1).
4.87 5.15
300
L. gasseri, pH 5.5
Results
Conclusions
0
4.08 4.32 4.59 4.7
Media pH
4.08
Head-to-Head Competition : Quantification of colony
forming units (Fig. 2) indicated that L. jensenii and
L. gasseri were only able to establish dominance
over the G. vaginalis species once the environmental
pH dropped to a level associated with a healthy
vaginal environment (< 4.5). By contrast, L. iners
became the dominant species while the media pH
was still consistent with BV conditions (> 4.5), but
once the pH dropped (< 4.5), the mixed culture
composition remained constant.
200
100
Percent of Total
Control wells contained supernatant obtained from
the same Lactobacillus species.
800
700
Percent of Total
Healthy vaginal environments are often dominated
by one or two species from the gram-positive genus
Lactobacillus, which are thought to play a
protective role against infection by producing lactic
acid, hydrogen peroxide, and bacteriocins
(proteinaceous toxins). In BV conditions, most
Lactobacilli are replaced with rod-type, gramvariable anaerobes and vaginal pH increases. L.
iners, however, remains in large numbers . L. iners
has been shown to produce less lactic acid and
hydrogen peroxide than other common vaginal
Lactobacilli – factors which help explain its inability
to protect against BV bacteria despite its
persistance. The ability of L. iners to persist in BV
has been the subject of several studies; it was
recently demonstrated that the binding mechanism
used by L. iners allows stronger adhesion to vaginal
mucosa than mechanisms used by other vaginal
Lactobacilli at pH conditions consistent with BV.
(McMillan, Reprod Sci, 2012) This study attempts
to uncover additional differences between L. iners
and other vaginal Lactobacillus species that may
contribute to its unique ability to persist in BV
communities. Specifically, this study explores
growth rates under various pH conditions,
susceptibility to factors secreted by BV organisms,
and competitiveness in a mixed culture containing
G. vaginalis, the most prevalent BV bacterium.
Supernatant Inhibition: Preparation of Supernatant
from G. vaginalis and all Lactobacillus species
cultures: cultures in were grown to late log phase
and supernatant was removed.
*
Percent of Total
Introduction
Analysis of Bacterial Growth: Bacteria were grown in
NYC III media, pH 4.0-7.0. Absorbance at 600nm
was measured for 8 hours using a microplate reader.
800
Percent of Total
Bacterial Vaginosis (BV) is the most common
vaginal infection presented in medical practice. It is
associated with a variety of negative effects
regarding reproduction including premature
rupture of fetal membranes (PROM). Our research
focuses on the shift between healthy and BV
conditions. This shift is characterized by an
increase in pH and a loss of the typically dominant
Lactobacilli, with the exception of L. iners. Growth
characteristics of the four most common
Lactobacillus species, L. gasseri, L. jensenii, L.
crispatus, and L. iners, were analyzed. Factors
tested included environmental pH and interaction
with the BV-associated bacterium Gardnerella
vaginalis. The growth of L. iners was inhibited by
the low pH associated with healthy environments
but not by the presence of G. vaginalis. The inverse
was found to be true for L. gasseri and L. jensenii. L.
crispatus was found to be inhibited by both a low
pH and factors produced by G. vaginalis.
Generation Time (min)
Methods
Generaiton Time (minutes)
Abstract
increasing volumes of G. vaginalis supernatant. In
addition, the period before log phase of growth of
the Lactobacillus species crispatus, and gasseri
was increased due to exposure to supernatant
collected from a G. vaginalis culture.
1
Mixed culture results are consistent with microbial
community composition observed in samples
collected from women with BV.
Remaining Questions:
Is L. iners more resistant than other Lactobacilli to
the competitive antagonists G. vaginalis produces?
What is the role of the pH change in the shift from
healthy to BV conditions?
OD (600nm)
Figure 2. Percent of total culture composed of G.
vaginalis and Lactobacillus sp. when grown together
at pH 4.3 or 5.5. Due to acid production during
growth, the pH of the cultures started at pH 5.5 (BV
conditions) were near pH 4 (healthy conditions)
between the first and second time points. Neither G.
vaginalis nor L.iners were able to grow at pH 4.32
(graph was not shown).
References
Gardnerella
vaginalis
Lactobacillus sp.
McMillan A, Macklaim JM, Burton JP, Reid G (2012) Adhesion
of Lactobacillus inersAB-1 to Human Fibronectin: A Key
Mediator for Persistence in the Vagina? Reprod Sci. Nov 30
[Epub ahead of print].