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Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic
Interaction with Cervicovaginal Epithelial Cells and Antagonistic
Activity to Genitourinary Pathogens
Vyacheslav Abramov •Valentin Khlebnikov •Igor Kosarev •Guldana Bairamova •
Raisa Vasilenko •Natalia Suzina •Andrey Machulin •Vadim Sakulin •
Natalia Kulikova •Nadezhda Vasilenko •Andrey Karlyshev •Vladimir Uversky •
Michael L. Chikindas •Vyacheslav Melnikov
Published online: 16 July 2014
ÓSpringer Science+Business Media New York 2014
Abstract Lactobacillus crispatus 2029 isolated upon
investigation of vaginal lactobacilli of healthy women of
reproductive age was selected as a probiotic candidate. The
aim of the present study was elucidation of the role of L.
crispatus 2029 in resistance of the female reproductive
tract to genitourinary pathogens using cervicovaginal epi-
thelial model. Lactobacillus crispatus 2029 has surface
layers (S-layers), which completely surround cells as the
outermost component of their envelope. S-layers are
responsible for the adhesion of lactobacilli on the surface
of cervicovaginal epithelial cells. Study of interactions
between L. crispatus 2029 and a type IV collagen, a major
molecular component of epithelial cell extracellular matrix,
showed that 125I-labeled type IV collagen binds to lacto-
bacilli with high affinity (Kd =(8.0 ±0.7) 910
-10
M).
Lactobacillus crispatus 2029 consistently colonized epi-
thelial cells. There were no toxicity, epithelial damage and
apoptosis after 24 h of colonization. Electronic microscope
images demonstrated intimate association between L.
crispatus 2029 and epithelial cells. Upon binding to epi-
thelial cells, lactobacilli were recognized by toll-like 2/6
receptors. Lactobacillus crispatus induced NF-jB activa-
tion in epithelial cells and did not induce expression of
innate immunity mediators IL-8, IL-1b, IL-1aand TNF-a.
Lactobacillus crispatus 2029 inhibited IL-8 production in
epithelial cells induced by MALP-2 and increased pro-
duction of anti-inflammatory cytokine IL-6, maintaining
the homeostasis of female reproductive tract. Lactobacillus
crispatus 2029 produced H
2
O
2
and provided wide spectrum
of antagonistic activity increasing colonization resistance
to urinary tract infections by bacterial vaginosis and vul-
vovaginal candidiasis associated agents.
Keywords Lactobacillus crispatus
Immunomodulation Antagonistic activity Homeostasis
Introduction
The dominance of the lactobacilli in the vagina suggests
that they play a crucial role in the protection of genito-
urinary tract against the generation of pathological condi-
tions [1]. Among the latter are those caused by the
excessive proliferation of indigenous microorganisms, such
V. Abramov V. Khlebnikov I. Kosarev R. Vasilenko
V. Sakulin N. Kulikova N. Vasilenko
Institute of Immunological Engineering, Lyubuchany 142380,
Russia
G. Bairamova
Research Center for Obstetrics, Gynecology and Perinatology of
the Ministry of Health, Moscow 117997, Russia
N. Suzina A. Machulin
G.K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Sciences,
Pushchino 142290, Russia
A. Karlyshev
Kingston University, 93 Grand Avenue, Surbiton,
Surrey KT5 9HY, UK
V. Uversky
Institute for Biological Instrumentation, Russian Academy of
Sciences, Pushchino 142290, Russia
M. L. Chikindas
School of Environmental and Biological Sciences, Rutgers State
University, New Brunswick, NJ 08901, USA
V. Melnikov (&)
International Science and Technology Center, Moscow 127473,
Russia
e-mail: Melnikov@istc.ru
123
Probiotics & Antimicro. Prot. (2014) 6:165–176
DOI 10.1007/s12602-014-9164-4
as Gardnerella vaginalis, whose prevalence may lead to
bacterial vaginosis (BV) [2–4], colonization by pathogens,
such as Candida spp. [5,6] and Trichomonas vaginalis [7,
8], which may induce vulvovaginal candidiasis (VVC) [9,
10] or, less frequently, cervicitis [11] and other regional
and systemic diseases [12,13]. The healthy vaginal mic-
robiota is typically dominated by Lactobacillus species,
such as L. iners,L. crispatus,L. jensenii and L. gasseri [3,
14–17]. Lactobacillus crispatus appears to be substantially
prevailing over the other hydrogen peroxide producing
Lactobacillus species [15,18–20]. Lactobacillus crispatus
has been reported to promote the stability of the normal
vaginal microbiota [21]. This bacterium is depleted in BV
patients [22,23], and in a recent study, L. crispatus was the
only species negatively associated with all four Amsel
criteria [4]. The basic method for correction of the
impaired vaginal microbiocenosis is the use of probiotic
preparations. Reduced effectiveness of antibiotic-based
treatments calls for novel approaches such as use of pro-
biotics for prophylaxis and treatment of genitourinary
conditions in women. Vaginal colonization with human-
derived L. crispatus strain CTV-05 has been successful
[24]. Lactobacillus crispatus CTV-05 was used as a vagi-
nal suppository for healthy women with a history of
recurrent urinary tract infections (UTIs) [25,26] and for the
treatment of BV [27]. It should be mentioned that health-
promoting effects of probiotics are usually strain specific.
In this work, we report on the ability of vaginal L. crispatus
2029 to colonize and regulate the innate immune response
in cervicovaginal epithelial cells and to inhibit growth of
selected pathogens.
Materials and Methods
Bacterial Strains and Growth Conditions
Lactobacillus crispatus 2029 was originally isolated in
2011 from a vaginal smear of a healthy woman of repro-
ductive age. Lactobacillus crispatus 2029 was deposited at
the All-Russian Collection of Microorganisms at the G.K.
Skryabin Institute of Biochemistry and Physiology of
Microorganisms under the registration number VKM
B-2727D. Lactobacillus crispatus MD IIE-1385 was orig-
inally isolated in 2011 from vaginal smear of a woman of
reproductive age with clinical diagnosis: recurrent VVC,
had history of BV. A complete list of microorganisms used
in this study is provided in Table 1.
Epithelial Models
Human immortalized cervical epithelial HeLa cells (HC)
were obtained from Institute of Cytology (RAS, St
Table 1 Lactobacilli strains and strains of indicator microorganisms
used in this study
Microorganism Strain Growth
conditions
Comments
Lactobacillus
crispatus
2029
(VKM
B-2729D)
MRS* 37
°
Cin
5%CO
2
or
anaerobically
48 h
IBPM RAS
a
.
Vaginal strain
was originally
isolated in
healthy woman
Lactobacillus
crispatus
MD IIE-
1385
The same IIE
b
. Clinical
isolate
(recurrent
VVC, had
history of
BV)
Gardnerella
vaginalis
14018 MCB** 37
°
C
in 5 % CO
2
or
anaerobically
48 h
ATCC
c
Gardnerella
vaginalis
CI-24-Gv The same IIE. Clinical
isolate (BV)
Atopobium
vaginae
CI-2-Av The same IIE. Clinical
isolate (BV)
Escherichia coli 11775 BHI*** 37
°
C
in 5 % CO
2
24–48 h
ATCC
Escherichia coli CI-25-Ec The same IIE. Clinical
isolate
(recurrent
VVC)
Proteus
vulgaris
13315 The same ATCC
Enterococcus
faecalis
19433 The same ATCC
Enterococcus
faecalis
CI-12-Ef The same IIE. Clinical
isolate (mixed
genital
infections)
Streptococcus
agalactiae
CI-5-Stra The same IIE. Clinical
isolate (mixed
genital
infections)
Staphylococcus
aureus
29P The same FDA
d
Staphylococcus
aureus
CI-36-Sa The same IIE. Clinical
isolate (mixed
genital
infections)
Candida
albicans
10231 SD**** 37
°
C
in 5 % CO
2
24–48 h
ATCC
Candida
albicans
CI-8-Ca The same IIE. Clinical
isolate
(recurrent
VVC)
Candida
tropicalis
750 The same ATCC
Candida
glabrata
15126 The same ATCC
166 Probiotics & Antimicro. Prot. (2014) 6:165–176
123
Petersburg, Russia) and grown in DMEM (Gibco, Paisley,
UK) at 37
°
Cin5%CO
2
atmosphere. Human immortal-
ized Vk2/E6E7 vaginal epithelial cells (VK2) (ATCC
CRL-2616, Rockville, MD) were grown in keratinocyte
serum-free medium (KSFM) (Invitrogen, Carlsbad, CA)
supplemented with 0.1 ng/ml epithelial growth factor and
25 lg/ml bovine pituitary extract and calcium chloride at
37
°
Cin5%CO
2
atmosphere as described in [28]. All cell
lines used tested negative for mycoplasma.
Epithelial Colonization
For epithelial colonization, suspensions of lactobacilli in
antibiotic-free KSFM (Invitrogen, Carlsbad, CA) at
2910
6
CFU/ml or 2 910
7
CFU/ml were added to cer-
vicovaginal epithelial cells at a 10:1 or 100:1 bacterial cell/
epithelial cell ratio. The epithelium-bacteria co-cultures
were incubated for 24–72 h at 37
°
Cin5%CO
2
atmo-
sphere as described in [28]. Cell culture supernatants were
collected during the period of incubation for the assessment
of soluble immune mediators. When necessary, epithelial
cells were lysed and apoptosis or NF-kB activation was
measured (see sections below). After 24 h, epithelium-
bacteria co-cultures were washed five times with sterile
PBS to remove unbound bacteria, fixed with methanol,
stained with azure-eosin (PanEco, Russia) according to
Ved’mina et al. [29] and examined under the light micro-
scope (NICON ECLIPSE E 400, Japan). Adhesion of lac-
tobacilli to epithelial cells was expressed as a percentage of
100 randomly selected epithelial cells with adhering bac-
teria and as average number of adhering bacteria per cell.
Transmission Electron Microscopy of Thin Sections
The cell biomass of bacterial pure culture was prefixed
with 1.5 % (v/v) glutaraldehyde solution in 0.05 M caco-
dylate buffer (pH 7.2) at 4 °C for 1 h. After three washings
with the same buffer, the material was additionally fixed
with 1 % OsO
4
in 0.05 M cacodylate buffer at 20
°
C for
3 h. Cell wall polysaccharides were additionally contrasted
by glutaraldehyde–osmium fixation in the presence of
ruthenium red [30]. After dehydration, the material was
embedded into epoxy resin Epon 812. Ultrathin sections
were made on an 8800 ULTROTOME III (Sweden). The
sections were mounted on copper grids covered with a
Formvar film, contrasted with uranyl acetate (3 % solution
in 70 % ethanol) for 30 min and then stained with lead
citrate [31]at20
°
C for 4–5 min. The sections were
examined in a JEM-100B (JEOL, Japan) electron micro-
scope at an 80 kV accelerating voltage.
Scanning Electron Microscopy
The samples for scanning electron microscopy (SEM) were
fixed in glutaraldehyde vapor for 24 h at ?4
°
C and then
fixed in osmium tetroxide vapor for 24 h at room temper-
ature. After that the samples were dried and coated with
gold using sputter coater Q150T S (Quorum Technologies,
UK). Observation of prepared samples was made using
JSM 6510LV (JEOL, Japan) scanning electron microscope
operated at 10 kV.
Epithelial Cell Viability Tests
Epithelial cell viability was elucidated as described in [28].
The CellTiter 96 MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide] assay (Promega, Madi-
son, WI) was used to assess lactobacilli cytotoxicity for the
HC and the VK2. In addition, the trypan blue assay was
used for the enumeration of viable epithelial cells in the
bacteria colonized epithelial cultures and for a quantitative
assessment of cleaved versus total caspase-3 amounts as a
marker of epithelial cell apoptosis (Meso Scale Discovery
[MSD], Gaithersburg, MD).
Caspase 3 Assay
Determination of caspase 3 was performed as described in
[28]. Monolayers of HC were co-cultured with lactobacilli
or incubated with 1 lM proapoptotic agent staurosporine
(Sigma) as a positive control. At the end of incubation
period (24 h), the epithelial monolayers were lysed in Tris
Table 1 continued
Microorganism Strain Growth
conditions
Comments
Candida krusei 24408 The same ATCC
a
Russian Collection of Microorganisms at the G.K. Skryabin Insti-
tute of Biochemistry and Physiology of Microorganisms, Pushchino,
Moscow Region, Russia
b
Collection of Microorganisms at the Institute of Immunological
Engineering (IIE), Department of Biochemistry of Immunity and
Biodefense, Lyubuchany, Moscow Region, Russia
c
American Type Culture Collection, Manassas, VA, USA
d
Collection of Microorganisms at the Food and Drug Administra-
tion, USA
Media
* Man-Rogosa-Sharpe (MRS) Broth or agar containing MRS plates
** Modified Columbia Broth (MCB) or agar containing MCB plates
(Himedia, India)
*** Brain Heart Infusion (BHI) broth supplemented with 0.5 % yeast
extract or agar containing BHI plates (Sigma-Aldrich, St. Louis,
MO,USA)
**** Sabouraud-dextrose (SD) broth or agar containing SD plates
(Himedia, India)
Probiotics & Antimicro. Prot. (2014) 6:165–176 167
123
lysis buffer containing protease inhibitor cocktail provided
by MSD, per manufacturer’s protocol. Levels of cleaved
and total caspase-3 were measured simultaneously in each
cell lysate using an MSD electrochemiluminescence (ECL)
multiplex assay and Sector Imager 2400 with Workbench
software (MSD).
NF-jB Luciferase Assay
Human immortalized cervical epithelial HeLa cells (HC)
were transfected with pHTS-NFjB firefly luciferase
reporter vector (Biomyx Technology, San Diego, CA) and
colonized with L. crispatus 2029 as described above.
Supernatant was removed at the end of the treatment per-
iod, and the cells were lysed with GloLysis buffer. The
luciferase activity was determined using the Bright-Glo
Luciferase Assay System according to the manufacturer’s
protocol (Promega, Madison, WI). Luminescence signal
was measured using a Victor 2 1420 multilabel microplate
counter with Wallac 2.01 software (Perkin Elmer Life
Sciences, Boston, MA).
Radiolabeling and Binding of Radioactive Type IV
Collagen to Lactobacilli
Radioligand analysis of binding
125
I-labeled type IV col-
lagen to L. crispatus 2029 was performed as previously
described [32].
125
I-labeled type IV collagen (0.1 mCu/lg)
samples were prepared with Iodo-Gen (Sigma) and Na
125
I
and then separated by chromatography on Sephadex G-25.
Lactobacillus crispatus 2029 cells were collected, washed
three times with RPMI-1640 medium and then adjusted to
a concentration of 10
7
per ml of the same medium.
Radioactive ligands were added to individual cultures (total
volume of 300 ll), which were then incubated for 1 h at
4
°
C. Thereafter, 50 ll of the cell culture was layered on
250 ll of an n-dibutylphthalale/bis (2-ethylhexyl)-phthal-
ate mixture (1:1 v/v) and centrifuged for 2 min at
14,0009g. Radioactivity in the resulting precipitate was
measured using a model 1275 MINI GAMMA counter
(LKB WALLAC, Sweden). Nonspecific binding of radio-
active ligands to cells or plastic plates was determined by
incubation in 10,000-fold excess of corresponding unla-
beled ligand and was equal to 25–27 % of the total (spe-
cific plus nonspecific) binding. Results were expressed as
the (mean ±SEM, n=3) in molar concentration, from
which nonspecific binding was subtracted.
Determination of Cytokines in Culture Supernatants
Production of IL-8 and IL-6 was measured with an ELISA
kits (Biosource International, Carlsbad, CA). TNF-a, IL-1b
and IL-1awere similarly determined according to the
protocol specified for a specific ELISA kits provided by
State Research Institute of Highly Pure Biopreparations
(St.-Petersburg, Russia). The sensitivity of all ELISA kits
was 1 pg/ml.
Antibacterial Activity Assay
For detection of antibacterial activity, an agar spot test as
described by Schillinger and Lucke [33] with modifications
was used. Overnight cultures of lactobacilli (3 ll
5910
9
CFU/ml) were spotted on the surface of MRS agar
containing 0.2 % glucose and 1.2 % agar and incubated
anaerobically (anaerostate Himedia, India, containing Hi-
AnaeroGas Pac Himedia) for 24 h at 30
°
C to form colo-
nies. One hundred microliter of an overnight culture of the
indicator bacteria (approximately 10
8
cells) was mixed
with 7 ml of soft agar (0.7 %), using brain heart infusion
(BHI, Sigma) agar, and poured over the plate. The plates
were incubated at 37
°
C. After 24–48 h of incubation, the
plates were examined for the presence of the zones of
inhibition. A clear zone of more than 1 mm around a spot
was scored as positive.
Antifungal Activity Assay
To assess the antifungal activity of lactobacilli, a double-
layer agar method was used [34]. The Minimum Inhibitory
Concentration of Antagonist (MICA), causing total inhi-
bition of the growth of Candida spp, was determined.
Cultivation of lactobacilli in different concentrations was
carried out in MRS agar bottom layer. Cultivation of
Candida spp (5 ll10
4
CFU/ml) was performed in the
upper layer of Sabourand
’
s agar.
Quantitative Hydrogen Peroxide Determination Test
Hydrogen peroxide production by lactobacilli was tested
colorimetrically through detection of the color developed
by o-dianisidine in the presence of horseradish peroxidase
(Sigma) as previously described [35]. Absorbance reading
(A
405
nm) was measured, and peroxide content was quan-
tified by comparing the values obtained with those of an
H
2
O
2
standard curve.
Production of Lactic acid
Lactic acid production was determined in grams per liter,
by acid–base titration, according to Edema and Sanni [36].
Statistical Analysis
Results were analyzed using ANOVA. Values were rep-
resented means and standard errors of the means.
168 Probiotics & Antimicro. Prot. (2014) 6:165–176
123
Statistically significant differences in cytokine production
between nonstimulated cells (control) and cells stimulated
with bacteria or proapoptotic and proinflammatory ligands,
H
2
O
2
and lactic acid production by lactobacilli strains were
accepted at p\0.05.
Results and Discussion
The adherence of L. crispatus 2029 to HC was visualized
by light and scanning electron microscopy (SEM) (Fig. 1).
Specifically, Fig. 1d depicts the intimate contact of the
epithelial cells and bacteria. These data are consistent with
the result of Fichorova et al. [28]. Using the transmission
electron microscopy (TEM), Fichorova et al. [28] have
discovered the intimate contact of vaginal L. crispatus
strain originally isolated from healthy woman (cited in
[37]) with Vk2 vaginal epithelial cells, displaying a lack of
morphological signs of apoptosis. Adhesion to epithelial
tissue and biofilm formation are the essential steps toward
the formation of a barrier by lactobacilli that will prevent
colonization by pathogenic microorganisms [38–42]. Lac-
tobacillus crispatus 2029 isolated from healthy woman
adheres to HC or Vk2 (Table 2), thereby allowing it to
colonize the epithelial cells more successfully than L.
crispatus MD IIE-1385 isolated from a woman with
recurrent VVC (Fig. 1b, d).
The presence and ultrastructural organization of S-layers
on the surface of L. crispatus 2029 were studied by electron
microscopy of thin sections. S-layers were arranged on the
outer surface of the bacterial cell wall, which had atypical
structure for Gram-positive bacteria (Fig. 2A.a). Fragments
of mono- or multiS-layers were observed on the cell sur-
face L. crispatus 2029. Number of S-layers varied from 5
to 8 (Fig. 2A. b, c). Several functions have been attributed
to the regular glycoprotein S-layers. The role of S-layers as
Fig. 1 Colonization of HC by L. crispatus 2029. a,bLight microscopy after azure-eosin staining (x1512), acontrol—intact HC, badhesion of
lactobacilli to HC, c,dscanning electron microscopy: ccontrol—intact HC, dinteraction of HC with lactobacilli
Table 2 Adhesion of lactobacilli to cervicovaginal epithelial cells
Epithelial
cells
Strain % Cells with
adhering
bacteria
No. of
adhering
bacteria/cell
HC Lactobacillus
crispatus 2029
100** 37 ±5*
Lactobacillus
crispatus MD
IIE-1385
24 ±86±2
VK2 Lactobacillus
crispatus 2029
100** 32 ±4*
Lactobacillus
crispatus MD
IIE-1385
26 ±55±2
*p\0.01; ** p\0.001 differences between L. crispatus 2029 and
L. crispatus MD IIE-1385. The adhesion experiment was performed
three times, tested in triplicate. Values represent means and SEM
Probiotics & Antimicro. Prot. (2014) 6:165–176 169
123
protective coats, cell shape determinants, molecule and ion
traps, molecular sieves, adhesion sites for exo-enzymes as
well as structures involved in cell adhesion and surface
recognition have been reported [43–47]. Many of these
functions are still hypothetical and need further study to be
confirmed [44]. The diversity of the S-layers, even within
microorganisms of the same species, may contribute to the
challenge in proposing structure–function relations [48].
Extracellular matrix of human vaginal, cervical and
urethral epithelial cells contains a type IV collagen [49,
50]. Earlier, it has been shown that S-layer-expressing
strain L. crispatus JCM 5810 adhered efficiently to
immobilized type IV collagens [51]. Here, we reported on
125
I-labeled type IV collagen binding to L. crispatus 2029
cells carrying the S-layers. The specific high affinity
binding of radiolabeled type IV collagen to L. crispatus
2029 was characterized by Kd =(8.0 ±0.7) 910
-10
M.
Bacterial adherence to cervicovaginal cell surfaces is
considered a pathogenicity-related process for the sexually
transmitted diseases. Therefore, highly adhesive Lactoba-
cillus strains could be used to develop anti-adhesion agents
to prevent invasion by genitourinary pathogens and con-
sequent development of infectious diseases [52]. In any
case, in addition to the beneficial effects on the cervico-
vaginal mucoses of the strain, it is necessary to demon-
strate the lack of any cytotoxic or proinflammatory effect
Fig. 2 Microscopic study of the interaction of Lactobacillus crisp-
atus 2029 with HeLa cells and the effects on caspase and NF-kB
levels. aTransmission electron microscopic image showing S-layers
on the bacterial surface. bScatchard analysis for specific binding of
125
I-labeled type IV collagen to L. crispatus 2029 cells: molar
concentration of specifically bound radioactive collagen is plotted as
the abscissa, ratios of the bound and free labeled collagen (B/F)
constitute the ordinate. Points—means ±standard errors of the
means (SEM) represent duplicate determinations from each of three
assays. cCaspase-3 cleavage is presented as percentages of cleaved
total caspase-3 measured in HC lysates at 24 h after bacterial
colonization or treatment with 1 lM staurosporine as a positive
control. Bars represent means and SEM of the results determined with
duplicate cultures used in two experiments. **p\0,01 different from
medium control, L. crispatus 2029. dInduction of NF-kB expres-
sion. 24-h lysates and supernatants harvested from HC cultured with
2910
6
L. crispatus 2029 or MALP-2 (50 nM) as a positive control.
Luciferase activity measured in lysates from triplicate cultures in one
representative of five experiments. Bars represent means and SEM
***p\0.001 different from medium control
170 Probiotics & Antimicro. Prot. (2014) 6:165–176
123
of the putative probiotic strain. In this regard, L. crispatus
2029 consistently colonized HC with no signs of apoptosis
(Fig. 2c). Lactobacillus crispatus 2029 adhered to HC and
VK2 with no evidence of cytotoxity (Fig. 3a, c). The
studied microorganism promoted NF-jB driven luciferase
activity in HC similar to that induced by the TLR2/6 ligand
MALP-2 (Fig. 2d) at significantly higher levels than the
medium control (p\0.001). However, only MALP-2, in
contrast to L. crispatus 2029, induced a significant IL-8
upregulation in HC and VK2 (Fig. 3b, d). Lactobacilli
reduced IL-8 production induced by the TLR2/6 ligand
MALP-2 in HC and VK2 (Fig. 3b, d). These data suggest
that L. crispatus 2029 was recognized by epithelial cells as
TLR2/6 ligand. Lactobacillus crispatus 2029 recognition
by TLRs is required for cervicovaginal homeostasis, and
IL-8 is a powerful chemoattractant and activator of neu-
trophils [53]. Our data suggest that L. crispatus 2029 tune
in the host innate immune response to avoid production of
proinflammatory cytokines in the presence of a potent NF-
jB activation. MALP-2 induced significant increase in the
level of IL-1b(p\0.01) and of TNF-a(p\0.001) in HC
and VK2, while L. crispatus 2029 had no effect (Figs. 4a,
b, 5a, b). Level of IL-1ain L. crispatus 2029 colonized HC
and VK2 did not change either (Fig. 4c or d). At the same
time, level of IL-6 in colonized HC (p\0.05) and
VK2 (p\0.05) was increased (Fig. 5c, d). IL-6 is an
Fig. 3 Effect of the interaction
of Lactobacillus crispatus 2029
on the viability and IL-8
production of HeLa and VK2
cells. a,cViability of HC
(a) and VK2 (b) assessed by
trypan blue inclusion test at
48-h postcolonization. IL-8
levels measured in supernatants
cultured for 24 h with L.
crispatus 2029 or MALP-2
(50 nM) as a positive control.
Bars represent means and SEM
of the results from triplicate
cultures. b,dIL-8 production
analyzed in HC supernatants
(b) and VK2 supernatants (d),
bars are means and SEM from
duplicate cultures in one of
three experiments
??
p\0.01
different from L. crispatus
2029, *p\0.05 different
between MALP-2 and MALP-
2?L. crispatus 2029
Probiotics & Antimicro. Prot. (2014) 6:165–176 171
123
anti-inflammatory cytokine [54]. IL-6 is also a repair and
cytoprotective factor. It plays direct role in the protection
of various cell types, such as hepatocytes [55], renal [56]
and lung epithelia [57] from injury. IL-6 reduces the level
of apoptosis among Ag-stimulated cells [58]. It is crucial
for the protection of endothelial cells against H
2
O
2
-induced
cell death [59]. Lactobacillus crispatus 2029 produces high
level H
2
O
2
(Table 5), and IL-6 is needed to support
homeostatic balance in female reproductive tract. Thus, L.
crispatus 2029 induces NF-jB activation and at the same
time maintains low levels of inflammation-associated
cytokines, which is important for the microorganism’s
potential use as a vaginal probiotic. NF-jB is a major
transcription factor that plays a key role in inflammatory
diseases; it upregulates many inflammation-associated
genes [60]. At the same time, NF-jB participates in its own
negative feedback loop promoting the downregulation of
inflammation in vivo [61]. The net effect of NF-jB acti-
vation depends on the cell and tissue properties, the
interaction of intra- and extra-cellular factors, and the
nature of the activating signal [60]. It has been previously
shown that vaginal Lactobacillus species (L. crispatus, L.
acidophilus and L. jensenii) can cause NF-jB activation
and yet maintain low levels of IL-8 and RANTES [28,62].
L. jensenii can suppress IL-8 induced by TLR ligands [63].
A nonvaginal Lactobacillus species induced production of
proinflammatory protein MIP-3a[64]. Vaginal pathogens,
associated with BV such as Atopobium vaginae and Pre-
votella bivia induced simultaneous NF-jB activation and
upregulation of proinflammatory proteins in contrast to
vaginal strains L. crispatus, L. acidophilus and L. jensenii
[28,62]. BV is the most common vaginal infection among
women of reproductive age. The pathogenesis of BV is still
poorly understood, but it is defined by a transition in the
vaginal flora from the predominant Lactobacillus species to
other bacterial species such as G. vagnalis [65] and A.
vaginae [20]. Inflammation is the most frequent primary
manifestation of VVC, caused by Candida spp [5,9]. VVC
is associated with an aggressive neutrophil migration into
the vagina, and subsequent inflammatory response initiated
Fig. 4 IL-1band IL-1a
responses in L. crispatus 2029
colonized epithelial cells. aIL-
1bin HC, bIL-1bin VK2, cIL-
1ain HC and dIL-1ain VK2.
Cytokine levels measured in
supernatants cultured for 8 h
with L. crispatus 2029 or
MALP-2 (50 nM) as a positive
control. Bars represent mean
and SEM from triplicate
cultures in two independent
experiments.
??
p\0.01
different from L. crispatus 2029
and **p\0.01 different from
medium control
172 Probiotics & Antimicro. Prot. (2014) 6:165–176
123
Fig. 5 Proinflammatory TNF-a
response and induction of anti-
inflammatory IL-6 cytokine
response in L. crispatus 2029
colonized epithelial cells.
aTNF-ain HC, bTNF-ain
VK2, cIL-6 in HC and dIL-6 in
VK2. Cytokine levels measured
in supernatants cultured for 8 h
with L. crispatus 2029 or
MALP-2 (50 nM) as a positive
control. Bars represent mean
and SEM from triplicate
cultures in two independent
experiments.
???
p\0.001,
?
p\0.005 different from L.
crispatus 2029, ***p\0.001,
**p\0.01 and *p\0.05
different from medium control
Table 3 Antagonistic activity
of L. crispatus 2029 to
opportunistic and pathogenic
bacteria, expressed as inhibition
zone values
The antibacterial activity
experiment was performed three
times, tested in triplicate
Target bacteria Inhibition zone values (mm) mean ±SD
Gardnerella vaginalis ATCC 14018 9.8 ±1.1
Gardnerella vaginalis CI-24-Gv 10.1 ±1.2
Atopobium vaginae CI-2-Av 7.5 ±0.6
Escherichia coli ATCC11775 6.1 ±0.4
Escherichia coli CI-25-Ec 6.4 ±0.5
Proteus vulgaris ATCC 13315 4.8 ±0.4
Enterococcus faecalis ATCC 19433 5.9 ±0.5
Enterococcus faecalis CI-12-Ef 5.6 ±0.7
Streptococcus agalactiae CI-5-Stra 7.4 ±1.8
Staphylococcus aureus FDA 29P 4.5 ±0.2
Staphylococcus aureus CI-36-Sa 5.7 ±0.3
Probiotics & Antimicro. Prot. (2014) 6:165–176 173
123
by the interaction of Candida spp with vaginal epithelial
cells. This inflammatory response is not protective; apart, it
appears to be a major cause of the symptoms associated
with infection [66]. Lactobacillus crispatus 2029 could
downregulate the inflammation. It inhibited the production
of chemokine IL-8 in cervical and vaginal epithelial cells
induced by MALP-2 ligand of TLR2/6 (Fig. 3b, d).
As shown in Table 3,L. crispatus 2029 was able to inhibit
the growth of G. vaginalis, A. vaginae, E. coli, P. vulgaris,
Ent. faecalis, Str. agalactiae and S. aureus. Lactobacillus
crispatus 2029 is also likely to provide the urogenital tract
with protection against C. albicans, C. tropicalis, C. glab-
rata and C. krusei (Table 4). Unlike L. crispatus 2029, L.
crispatus MD IIE-1385 did not possess antifungal activity
toward studied species of Candida (Table 4). The observed
antifungal activity of L. crispatus 2029 is likely due to the
sufficient production of H
2
O
2
and lactic acid. Both studied
lactobacilli strains produced H
2
O
2
(Table 5) and lactic acid
(Table 6), but higher level H
2
O
2
(120 mg/l) and lactic acid
(2.38 ±0.14 g/l) were observed for L. crispatus 2029,
which was isolated from a healthy woman. Lactobacillus
crispatus MD IIE-1385 produced low levels H
2
O
2
(10 mg/l)
and lactic acid (1.05 ±0.12 g/l). This strain was isolated
from a woman with recurrent VVC and a history of BV.
Hydrogen peroxide production by vaginal lactobacilli rep-
resents one of the most important defense mechanisms
against vaginal colonization by pathogenic and opportunis-
tic microorganisms and maintenance of genitourinary
homeostasis [67]. The most frequently isolated vaginal
Lactobacillus species in healthy women L. crispatus could
also be found in BV patients [20]. We speculate that L.
crispatus strains detected in the human subjects with BV
display a weak adhesive properties to cervicovaginal epi-
thelial cells, produce low levels of lactic acid and H
2
O
2
;
these strains are incapable of preventing vaginal coloniza-
tion by pathogenic and opportunistic microorganisms.
According to our data, L. crispatus 2029 demonstrates a
wide spectrum of antibacterial and antifungal activity, modu-
lates production of cytokines by cervical and vaginal epithelial
cells and may divert the innate immune response in a regulatory
mode, thus maintaining the homeostasis of female reproductive
tract. Therefore, this strain might be considered as a candidate
probiotic for treatment and prevention of UTI, BV and VVC.
Acknowledgments We thank Dr. Yulia Romanova and Dr. Boris
Naroditsky at N.F. Gamaleya Institute of Epidemiology and Micro-
biology (Moscow, Russia) for their helpful suggestions and discus-
sions. This work was supported by a RSF Grant, Russia.
Conflict of interest The authors declare that there is no conflict of
interest.
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