# Adhesion of Human ProbioticLactobacillus rhamnosus to Cervical and Vaginal Cells and Interaction with Vaginosis-Associated Pathogens

Authors: Sophie Coudeyras; Gwendoline Jugie; Marion Vermerie; Christiane Forestier
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2008)

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## Abstract

Objectives. The ability of a probiotic Lactobacillus rhamnosus strain (Lcr35) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis-associated pathogens, Prevotella bivia, Gardnerella vaginalis, as well as Candida albicans was investigated.
Methods. Adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix, ectocervix, and vagina. Coculture experiments were performed to count viable pathogens cells in the presence of Lcr35. Results. Lcr35 was able to specifically and rapidly adhere to the three cell lines. In coculture assays, a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation, mostly with P. bivia. Conclusion. The ability of Lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis-associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy.


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## Body

## 1. Introduction

Bacterial  vaginosis (BV) is the most frequent  vaginal
infectious disorder in women of childbearing age with prevalences ranging from
10% to 50% [1]. In addition to the physiological burden that induces BV, it can cause
serious sequelae such as preterm birth and facilitate the acquisition of
sexually transmitted diseases. The cause of BV remains poorly understood, and
no specific infectious agents have been identified. However, the disorder is
characterized by modifications of the genital tract microflora, including a
reduction in or absence of lactobacillus colonization and overgrowth of several
anaerobic bacteria [2]. The vaginal ecosystem in healthy premenopausal women harbors a
microbiota dominated by Lactobacilli [3, 4], that is, being increasingly recognized as protecting it from invading
pathogens, including those that cause urinary tract infections and sexually
transmitted diseases. Different mechanisms are potentially involved in the
activity of Lactobacilli against
pathogens, including the competitive exclusion of genitourinary pathogens from
receptors present on the surface of the epithelial cells. Under healthy
conditions, cervicovaginal cells are constantly exposed to the normal vaginal
microbiota.The recommended treatment regimens for vaginal infections
are oral or intravaginal antibiotics [5], but these conventional treatments are associated with frequent
recurrences. Alternative therapeutic agents need to be sought, and it has been
suggested that the administration of Lactobacilli can restore ecological balance in the vagina by controlling the infectivity of
pathogenic microbes [3], but the treatment is still a subject of debate. Several clinical
trials have been performed to investigate the effects of specific strains,
mainly with L. acidophilus and L. rhamnosus species [6, 7], but no definitive conclusions as to whether these probiotics represent
an effective and safe method for treating women with BV can be drawn. The
behavior of the probiotics in the vaginal tract is likely to be strain specific
and therefore, it is important to determine the characteristics of the strain
to be used as a therapeutic agent. The most relevant properties in this context
are likely to be adhesion to cervicovaginal cells and adequate pathogen growth
inhibition. In vitro studies assessing these properties might not be able to
fully simulate the in vivo behavior, but they could be reliable indicators when
selecting the probiotic strain. The purpose of this study was to determine the
in vitro adherence of a well characterized L.
rhamnosus probiotic strain, Lcr35 [8–10], and its ability to inhibit growth of three vaginosis-associated
pathogens. We used immortalized morphologically and functionally distinct
epithelial cell lines from normal endocervix, ectocervix, and vagina to
characterize Lcr35 epithelial interactions pertinent to the lower female
genital tract and determined its antimicrobial activity against Prevotella bivia, Gardnerella vaginalis, and Candida
albicans in coculture experiments.


## 2. Materials and Methods

### 2.1. Adhesion Assay

Adhesion assays were performed with epithelial cells from
normal human vagina (VK2/E6E7 ATCC-CRL-2616), ectocervix (Ect1/E6E7
ATCC-CRL-2614), and endocervix (End1/E6E7 ATCC-CRL-2615), immortalized by
expression of the E6 and E7 genes of human papillomavirus type 16 [11]. The morphological and immunocytochemical characteristics of the
immortalized lines closely resembled those of their tissues of origin and
primary cultures and are likely to represent the different compartments of the
vaginal tract.The cell lines were maintained in keratinocyte serum-free
medium (Gifco BRL 17005-042) supplemented with human recombinant EGF (0.1 ng/mL),
bovine pituitary extract (0.05 mg/mL), and calcium chloride (0.4 mM) at37°C
with a 5%CO2 in air atmosphere.Adhesion of the Lcr35 was assayed by seeding cell lines in
24-well tissue culture plates at2.5×105epithelial cells/well and
allowing them to grow to complete confluence (105cells/well). After gentle washing of the cell monolayer,
the adhesion capacity of Lcr35 was determined by adding 105 multiplicity of infection
(MOI,1), 106 (MOI, 10), and 107 (MOI, 100) bacteria from
an overnight culture in de Man, Rogosa, Sharpe (MRS) agar medium. Bacterial cells were previously washed in
phosphate buffered saline and resuspended in the cell culture medium. Adhesion
was monitored after 1 and 3 hours of incubation carried out at 37°C under 5%CO2.
The monolayers were washed three times with 1 mL of Dulbecco’s phosphate
buffered saline, detached by addition of 0.1% TritonX-100 solution and the number
of viable bacteria determined by plating serial dilutions of the suspensions
onto MRS agar plates. For qualitative analysis, the cell monolayers and the
bacteria were methanol fixed and stained by addition of a 10% Giemsa solution.


### 2.2. Growth Inhibition of Vaginosis-Associated Pathogens

The effect of Lcr35 on the growth of three pathogens was
investigated using the following strains: Candida albicans ATCC10231,Prevotella bivia ATCC29303, and Gardnerella vaginalis ATCC14018.
Coculture assays were performed in either Sabouraud broth (Candida) or brain
heart infusion supplemented with yeast extract (1%), maltose (0.1%), glucose
(0.1%), and horse serum (10%) (Prevotella and Gardnerella). Each pathogen (108UFC) was incubated alone (control) and with the Lcr35 (108UFC) at
37°C under anaerobic conditions for the two vaginosis-associated bacteria
(AnaeroGen, Oxoïd). Aliquots were removed after 4, 8, and 24 hours of
incubation, serially diluted and plated on appropriate media (Sabouraud,
Gardnerella, or MRS) to determine the bacterial colony counts of both the
pathogens and Lcr35. Statistical analyses of the data were performed using the Mann-Whitney test.


## 2.1. Adhesion Assay

Adhesion assays were performed with epithelial cells from
normal human vagina (VK2/E6E7 ATCC-CRL-2616), ectocervix (Ect1/E6E7
ATCC-CRL-2614), and endocervix (End1/E6E7 ATCC-CRL-2615), immortalized by
expression of the E6 and E7 genes of human papillomavirus type 16 [11]. The morphological and immunocytochemical characteristics of the
immortalized lines closely resembled those of their tissues of origin and
primary cultures and are likely to represent the different compartments of the
vaginal tract.The cell lines were maintained in keratinocyte serum-free
medium (Gifco BRL 17005-042) supplemented with human recombinant EGF (0.1 ng/mL),
bovine pituitary extract (0.05 mg/mL), and calcium chloride (0.4 mM) at37°C
with a 5%CO2 in air atmosphere.Adhesion of the Lcr35 was assayed by seeding cell lines in
24-well tissue culture plates at2.5×105epithelial cells/well and
allowing them to grow to complete confluence (105cells/well). After gentle washing of the cell monolayer,
the adhesion capacity of Lcr35 was determined by adding 105 multiplicity of infection
(MOI,1), 106 (MOI, 10), and 107 (MOI, 100) bacteria from
an overnight culture in de Man, Rogosa, Sharpe (MRS) agar medium. Bacterial cells were previously washed in
phosphate buffered saline and resuspended in the cell culture medium. Adhesion
was monitored after 1 and 3 hours of incubation carried out at 37°C under 5%CO2.
The monolayers were washed three times with 1 mL of Dulbecco’s phosphate
buffered saline, detached by addition of 0.1% TritonX-100 solution and the number
of viable bacteria determined by plating serial dilutions of the suspensions
onto MRS agar plates. For qualitative analysis, the cell monolayers and the
bacteria were methanol fixed and stained by addition of a 10% Giemsa solution.


## 2.2. Growth Inhibition of Vaginosis-Associated Pathogens

The effect of Lcr35 on the growth of three pathogens was
investigated using the following strains: Candida albicans ATCC10231,Prevotella bivia ATCC29303, and Gardnerella vaginalis ATCC14018.
Coculture assays were performed in either Sabouraud broth (Candida) or brain
heart infusion supplemented with yeast extract (1%), maltose (0.1%), glucose
(0.1%), and horse serum (10%) (Prevotella and Gardnerella). Each pathogen (108UFC) was incubated alone (control) and with the Lcr35 (108UFC) at
37°C under anaerobic conditions for the two vaginosis-associated bacteria
(AnaeroGen, Oxoïd). Aliquots were removed after 4, 8, and 24 hours of
incubation, serially diluted and plated on appropriate media (Sabouraud,
Gardnerella, or MRS) to determine the bacterial colony counts of both the
pathogens and Lcr35. Statistical analyses of the data were performed using the Mann-Whitney test.


## 3. Results

### 3.1. Adhesion of Lcr35 to Cervical and Vaginal Cells

The ability of Lcr35 to adhere to vaginal and cervical
cells is shown in Figure1. Whatever the MOI and the cell line, the probiotic
strain was able to adhere to the cell surface monolayer. The highest number of
adherent bacteria was observed with the vaginal cell line, with an average of
4.75 10e5 CFU per cm2 after 1 hour of incubation. No major
difference was observed between the levels of adhesion obtained after 1 hour
and 3 hours of incubation (data not shown), suggesting that adhesion occurs
rapidly after the initial contact between the cells and the bacteria.
Microscopical observations of Giemsa-stained preparation showed typical chains
of Lcr35 randomly dispersed on the cell surface (Figure 2).Figure 1
Adherence ofL. rhamnosus Lcr35 to vaginal, ecto-
and endocervical cells. Epithelial cells were incubated with three different
bacterial inocula and incubated for 1 hour. The number of viable Lcr35 adhering
to the cell monolayer surface was determined by plating onto appropriate media.
The data are averages of three independent experiments performed in triplicate.
Error bars indicate standard deviations.Giemsa-stains from adherence assays performed for
the experiment shown in Figure1 with vaginal (a) and ectocervical (b) cells.
(a)(b)


### 3.2. Growth Inhibition of Vaginosis-Associated Pathogens

The antagonist effect of Lcr35 against three main
pathogens,P. bivia, G. vaginalis, and C. albicans, was assessed in coculture assays and compared with the
growth ability of each pathogen in the same culture medium (Figure 3). A
decrease in the cell division rate of the three microorganisms tested was
observed from 4 hours of coincubation. When the viable bacteria in the mixed
suspension were counted over a longer period of time, bactericidal activity was
detected between 8 and 24 hours of incubation for all pathogens with the Prevotella strain being the most
susceptible (4-log10 units decrease in the number of viable cells). In no case
there was a bactericidal effect against Lactobacilli;
the number of viable Lcr35 cells was either constant over the incubation period
(coculture with C. albicans) or
increased (coculture with G. vaginalis and P. bivia) (data not shown).Effect of Lcr35 on the viability ofPrevotella bivia (a), Gardnerella vaginalis (b), and Candida albicans (c) as a function of
the time of coculture. The pathogen was incubated without (filled square) or
with (empty squares) Lcr35 at 37°C for 24 hours and the colony forming unit mL−1 was determined after 4, 8, and 24 hours of incubation by plating onto
appropriate media. Each value shown is the mean ± SD from three experiments. :*
statistically significant differences (P=.050, Mann-Whitney test).
(a)(b)(c)


## 3.1. Adhesion of Lcr35 to Cervical and Vaginal Cells

The ability of Lcr35 to adhere to vaginal and cervical
cells is shown in Figure1. Whatever the MOI and the cell line, the probiotic
strain was able to adhere to the cell surface monolayer. The highest number of
adherent bacteria was observed with the vaginal cell line, with an average of
4.75 10e5 CFU per cm2 after 1 hour of incubation. No major
difference was observed between the levels of adhesion obtained after 1 hour
and 3 hours of incubation (data not shown), suggesting that adhesion occurs
rapidly after the initial contact between the cells and the bacteria.
Microscopical observations of Giemsa-stained preparation showed typical chains
of Lcr35 randomly dispersed on the cell surface (Figure 2).Figure 1
Adherence ofL. rhamnosus Lcr35 to vaginal, ecto-
and endocervical cells. Epithelial cells were incubated with three different
bacterial inocula and incubated for 1 hour. The number of viable Lcr35 adhering
to the cell monolayer surface was determined by plating onto appropriate media.
The data are averages of three independent experiments performed in triplicate.
Error bars indicate standard deviations.Giemsa-stains from adherence assays performed for
the experiment shown in Figure1 with vaginal (a) and ectocervical (b) cells.
(a)(b)


## 3.2. Growth Inhibition of Vaginosis-Associated Pathogens

The antagonist effect of Lcr35 against three main
pathogens,P. bivia, G. vaginalis, and C. albicans, was assessed in coculture assays and compared with the
growth ability of each pathogen in the same culture medium (Figure 3). A
decrease in the cell division rate of the three microorganisms tested was
observed from 4 hours of coincubation. When the viable bacteria in the mixed
suspension were counted over a longer period of time, bactericidal activity was
detected between 8 and 24 hours of incubation for all pathogens with the Prevotella strain being the most
susceptible (4-log10 units decrease in the number of viable cells). In no case
there was a bactericidal effect against Lactobacilli;
the number of viable Lcr35 cells was either constant over the incubation period
(coculture with C. albicans) or
increased (coculture with G. vaginalis and P. bivia) (data not shown).Effect of Lcr35 on the viability ofPrevotella bivia (a), Gardnerella vaginalis (b), and Candida albicans (c) as a function of
the time of coculture. The pathogen was incubated without (filled square) or
with (empty squares) Lcr35 at 37°C for 24 hours and the colony forming unit mL−1 was determined after 4, 8, and 24 hours of incubation by plating onto
appropriate media. Each value shown is the mean ± SD from three experiments. :*
statistically significant differences (P=.050, Mann-Whitney test).
(a)(b)(c)


## 4. Discussion

Lactobacillus species in the female urogenital system act as a barrier
to infection and  contribute to the control of the vaginal microbiota by
competing with other microorganisms for adherence to epithelial cells,
displacing pathogen biofilm [12, 13], and/or inhibiting the growth of potential pathogens [14–16]. Hence the use of probiotic strains of Lactobacilli is potentially interesting both as preventive and
curative agents.Unlike the use of vaginal epithelial cells
collected from healthy premenopausal women, assays
performed  with immortalized  epithelial cell lines, which closely resemble the
epithelial differentiation patterns of normal human tissues, are more accurate
for standardizing tested bacterial adherence and allow comparison of different
research approaches. The three epithelial cell lines tested in this study were
developed from normal human vagina, ectocervix, and endocervix tissue, and
their characteristics closely resembled those of their tissues of origin and
primary cultures [11]. We can thus speculate that adhesion assays performed with this
material reproduce more faithfully the in vivo situation than experiments
performed with any cell line derived from human carcinoma of the lower genital
tract mucosa. This is particularly
important when comparing bacterial strains belonging to the complex Lactobacillus genus that includes
bacterial strains with highly specific characteristics. Using these cell lines,
we observed specific adhesion of an L.
rhamnosus strain, Lcr35, previously selected for its probiotic features [8–10]. Adhesion occurred even at a low MOI (1:1) and within less than 1 hour
of contact, which corresponds to a highly dynamic process.Adhesion of Lcr35 to vaginal epithelial cells
would allow colonization of the vaginal mucosa and therefore could limit the
overgrowth of pathogens, but the second main property of a potential probiotic
used as a therapeutic agent against pathogenic microorganisms is direct impairment
of their growth. In this study, we demonstrated that Lcr35 showed bactericidal
activity against bothP. bivia and G. vaginalis in the range of killing
stipulated for the bactericidal activity of antimicrobial activity (>2
log-unit). In a previous study, Atassi et al. demonstrated that the
bactericidal activity of Lactobacilli toward these two vaginal bacterial pathogens was strain dependent and occurred
within the first hours of coculture [14]. In our experiments, a longer incubation time was required to observe
bactericidal activity, probably because of the different experimental
parameters used in the two assays. We previously showed that the Lcr35
probiotic strain was also able to kill several pathogens [10]. The mechanism(s) underlying this activity has not been elucidated but
is likely to be multifaceted and probably includes the production of hydrogen
peroxide, lactic acid, and antibacterial compounds. It has been recently shown
that G. vaginalis organized in
biofilms is more
resistant to H2O2 and lactic acid than planktonic
cultures [17]. G. vaginalis is the
predominant species observed within biofilms present on the vaginal epithelium
in bacterial vaginosis [18], and Saunders et al. recently showed that strains of Lactobacilli were able to disrupt G. vaginalis preformed biofilms [13]. It would therefore be interesting to test the biofilm activity of
Lcr35 against G. vaginalis and to
determine if the sessile form of Lcr35 also exhibits antibacterial activity
against G. vaginalis in mixed biofilm
assays.The antagonist activity of Lcr35 was not limited to
bacterial pathogens since the strain was also able to reduce the viability ofC. albicans. Several strains of Lactobacilli have shown inhibitory
effects against C. albicans [16], which is the species most often associated with candidiasis. By
interfering with Candida overgrowth
in the patients’ intestinal or vaginal tract, Lactobacilli could provide colonization resistance and maintain low
numbers of yeasts, especially when administered together with antibiotics.Relevant clinical trials have suggested that
intravaginal or oral administration ofLactobacillus strains is able to
increase the numbers of vaginal Lactobacilli and restore the vaginal microbiota to normal [7]. Lcr35 was isolated from a human intestinal and not vaginal microbiota
and does not belong to the four main species of Lactobacilli considered to be predominantly linked to the vaginal
microflora, L. crispatus, L. jensenii, L. gasseri, and L. iners [4, 19, 20], but it has been shown to survive within the human gastrointestinal
tract [9]. Furthermore, Petricevic and Witt recently showed in a clinical study
that topical  administration of Lcr35 enhances the restoration of the vaginal
flora after antibiotic treatment of BV [21].
Thus, it might be an excellent candidate for use as a prophylactic
agent, taken orally or applied topically. In vivo studies to evaluate its
feasibility as such are in progress.


## 5. Conclusion

Maintenance or reconstruction of the normal composition of
the vaginal microflora by applying properly selectedLactobacilli may be of prophylactic value in preventing or curing
genitourinary system infections in women. In the light of our experiments, it
seems that the probiotic strain L.
rhamnosus Lcr35 would be a good candidate as a protective agent against
both bacterial vaginosis and Candida vaginitis since it was able to adhere to vaginal and cervical cells and to
antagonist the growth of vaginosis-associated pathogens. Clinical studies are
now required to assess the in vivo efficacy of such a therapy.



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