Arch Gynecol Obstet
DOI 10.1007/s00404-015-3810-2
GENERAL GYNECOLOGY
Restoring vaginal microbiota: biological control of bacterial
vaginosis. A prospective case–control study using Lactobacillus
rhamnosus BMX 54 as adjuvant treatment against bacterial
vaginosis
Nadia Recine1 • Ettore Palma1 • Lavinia Domenici1 • Margherita Giorgini1 •
Ludovica Imperiale1 • Carolina Sassu1 • Angela Musella1 • Claudia Marchetti1
Ludovico Muzii1 • Pierluigi Benedetti Panici1
Received: 1 April 2015 / Accepted: 26 June 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract
Purpose Bacterial vaginosis (BV) is the most prevalent
lower genital tract infection in reproductive-age women
worldwide. BV is an ecological disorder of the vaginal
microbiota characterized microbiologically by replacement
of the lactobacilli, predominant vaginal microbiota. It is
characterized by a high rate of relapse in sexual active
women, and these patients show three or more relapses
each year. A healthy vagina is characterized by hydrogen
peroxide and acid-producing lactobacilli, which are crucial
to maintain the physiological vaginal ecosystem and their
depletion speeds up bacterial overgrowth with pH eleva-
tion, salidase and amine production, leading to the
observed signs and symptoms of BV. The aim of this study
is to evaluate the efficacy of long-term vaginal lacto-
bacilli’s implementation in restoring and maintaining
vaginal microflora and pH and to collect data about pro-
phylactic approach based on probiotics supplementation
with lactobacilli.
Methods This is a prospective case–control study, per-
formed between January 2013 and September 2014 at
Department of Gynecological Obstetrics and Urologic
Sciences of ‘‘Sapienza’’ University of Rome. 250 non-
pregnant sexually active women with diagnoses of BV
were collected. Patients selected were divided in Group A
(125 patients assigned to standard treatment for BV—
metronidazole 500 mg orally twice a day for 7 days) and
Group B (125 women undergoing the same standard
& Lavinia Domenici
lavinia.domenici@uniroma1.it
1 not completely unstated, research has confirmed that BV is
Department of Gynecological Obstetrics and Urologic
Sciences, University Sapienza of Rome, Viale del Policlinico
1555, 00161 Rome, Italy
•
antibiotic regimen followed by vaginal tablets containing
Lactobacillus rhamnosus BMX 54). Patients were evalu-
ated after 2, 6, and 9 months (T0, T2, T6, and T9) in term
of recurrences rates of BV, vaginal symptoms, re-estab-
lishment of healthy vaginal flora, vaginal pH, and treatment
tolerability.
Results Vaginal flora was significantly replaced in Group
B patients after 2 months comparing with Group A
(p = 0.014). These data were confirmed at 6 and 9 months
follow-up: patients that underwent prophylactic therapy
with NORMOGINÒ experienced significantly low rate of
recurrences comparing with patients treated with antibi-
otics only (p \ 0.001). During follow-up patients contin-
uing supplementation had significant pH decrease respect
to other patients (p \ 0.001 at 9 months follow-up visit).
Conclusions Probiotic supplementation with vaginal
Lactobacillus rhamnosus BMX54 seems to be useful in
hindering bacteria growth especially after antibiotic ther-
apy; therefore this intervention may be considered a new
prophylactic treatment for preventing recurrence of BV, in
particular in high-risk patients.
Keywords Probiotics
Lactobacillus rhamnosus
BMX54
Bacterial vaginosis
Vaginal flora
Introduction
Bacterial vaginosis (BV) is the most widespread gyneco-
logic disorder in the primary care setting. The prevalence
of BV among girls and women aged 14–49 years is about
29.2 % [1]. Although the etiology of bacterial vaginosis is
a synergistic, polymicrobial infection characterized by a
normal microbiota shift in the bacterial flora of the vagina,
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by a decline in the concentration of lactobacilli and an
overgrowth of other microorganisms.
Vaginal lactobacilli provide protection against intrusive
pathogenic bacteria. Lactobacillus spp produces a thick pro-
tective biofilm [2]. Biofilm are particularly complex biological
conglomerate structures where bacteria commonly thrive. It is
been recognized that humans coexist with complex bacterial
communities that are relatively unique to specific niches such
as the gut the oral or the vaginal cavity. The microbes asso-
ciated with humans outnumber the total number of human
cells by a ratio of more than 10:1 and that the total number of
genes in the human genome is outnumbered by those in the
bacterial microbioma by a factor greater than 1000:1 [3].
The biofilm is responsible for maintaining an ecological
unit in a stable condition, in which bacteria are balanced
and we have a mutually beneficial interaction. The vaginal
lactobacilli seem to provide protection against intrusive
pathogenic bacteria [4].
The BV is characterized by a high rate of recurrence in
sexual active women and these patients show three or more
relapses each year [5–7].
Lactobacilli have a critical role in maintaining the balance
in vaginal ecosystem. Through the production of lactic acid,
lactobacilli lower vaginal pH and help avoid the growth of
potentially pathogenic microorganisms [8]. Other mechanisms
of lactobacilli action are production of oxygen peroxide,
bacteriocins, and surface binding proteins that inhibit adhesion
to uroepithelial cells [9]. Altogether, these actions contribute to
inhibit the increase of pathogenic microorganisms. Interfer-
ence of this fragile ecosystem by hormonal changes, sexual
behavior, pregnancy or antibiotic therapy can shrink the levels
and activity of the lactobacilli, leading the vagina more sus-
ceptible to infection by pathogens, in particular to BV [10].
Alteration of normal vaginal flora sometimes remains
untreated, predisposing to recurrence of vaginal infections (for
example, E. Coli, Gardnerella spp, Chlamydia, Ureaplasma
spp, and Streptococcus spp.) [9, 10]. In fact, exogenous
vaginal lactobacilli can produce lactate and lower the vaginal
pH, inhibiting the growth of pathogens, while concomitantly
favoring reconstruction of biofilm and the development of new
lactobacilli reinforcing the natural ecosystem [4].
The aim of this case–control study was to assess the
potential value of vaginal tablets containing viable Lacto-
bacillus rhamnosus BMX 54 (NORMOGINÒ) in the
treatment of abnormal vaginal flora (AVF), preventing new
vaginal infections or their recurrences.
Materials and methods
This prospective case–control study was performed from
January 2013 through September 2014 at Department of
Gynecological Obstetrics and Urologic Sciences of
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Arch Gynecol Obstet
‘‘Sapienza’’ University of Rome, in 250 non-pregnant
sexually active women with diagnoses of BV.
The inclusion criteria included sexually active women in
reproductive age (18–45-year old) with diagnosis of BV
defined as meeting at least three of Amsel’s criteria [11]
and with normal gynecological status.
Amsel’s criteria are represented of homogeneous, thin,
grayish-white vaginal discharge, vaginal pH [ 4.5, posi-
tive whiff-amine/sniff test defined as the presence of ‘‘fishy
odor’’ when a drop of 10 % potassium hydroxide (KOH)
solution is added, ‘‘clue cells’’ on saline wet mount on
microscopy [11].
Clue cells are vaginal epithelial cells studded with
adherent coccobacilli that are best appreciated at the edge
of the cell [11, 12]. For a positive result, at least 20 % of
the epithelial cells on wet mount should be clue cells. The
presence of clue cells diagnosed by an experienced
microscopist is the single most reliable predictor of BV.
Bacterioscopic exam was performed every time. All
slides were scored by the same investigator. Each vaginal
sample was rehydrated with normal saline solution and
surveyed under a phase contrast microscope with 4009
magnification and an area of 0.016 mm2. The presence of
bacteria and clue cells together with number of vaginal
leucocytes was assessed. BV was graded as predominantly
bacteria compared to lactobacilli per field [7]. Evaluation
of pH was made with Litmus paper test.
The exclusion criteria included urogenital infections or
sexual transmitted disease, pregnancy or breastfeeding,
smoking, exogenous hormone treatments, and other gyne-
cological conditions that could cause bleeding (polyps,
endometrial hyperplasia, etc.).
The study was approved by the Ethic Committee and
written informed consent was obtained from all patients.
Patients were subdivided in Group A (125 patients
subjected to standard treatment for BV—metronidazole
500 mg orally twice a day for 7 days) and Group B (125
women undergoing the same standard antibiotic regimen
followed by vaginal tablets containing [104 CFU/tablet
freeze-dried Lactobacillus rhamnosus BMX 54—
NORMOGINÒ, Baldacci Laboratories). Probiotic therapy
was administered according a previously used schedule:
once a day for 10 days, twice a week for 15 days, and once
every 5 days for 7 months as maintenance therapy.
The patients were evaluated at follow-up after 2, 6, and
9 months (T0, T2, T6, and T9) and returned to our
department during these months only in the case of wors-
ening of the BV symptoms.
Outcomes were measured after 2, 6, and 9 months later
by vaginal examination and specimens of vaginal fluid to
evaluate restored vaginal flora and pH.
During each visit, patients were asked to assess the
impact of the treatment on BV-related symptoms, and the
Arch Gynecol Obstet
intensity of symptoms (itching, vaginal discharge, burning
sensation, and their recurrence 24 h after sexual inter-
course) was scored by the patient on a subjective scale
from 0 no symptoms, to 1 mild symptoms, 2 moderate, and
3 severe symptoms intensity.
Statistical analysis was performed by running SPSS
software package (SPSS Inc., Chicago, IL, USA) given as
mean (SD) or number (%) of cases, with 95 % confidence
intervals (CIs), when appropriate. Comparison of quanti-
tative variables between the study groups was performed
using the 2-sample Chi-square and Fisher’s test. Compar-
ison for repeated measures was performed using ANOVA
test. 80. The values in ANOVA had normal distribution
and the variances of assumed variables were equal. The
measures were reported as mean ± SD. All tests used were
2-tailed. A p value of \0.05 was considered statistically
significant.
Results
Out of 250 non-pregnant sexually active women with
diagnosis of BV following all inclusion criteria were
collected.
Mean patient age in Group A was 29.5 (4.5 SD; 22–38,
95 % CI) years, while in Group B group was 29.3 (4.0 SD;
19–36, 95 % CI). A complete medical history was col-
lected. Patients were asked to avoid sexual intercourses
during for the first 10 days of treatment. Tolerability of the
local therapy was good.
The first follow-up visit was performed after 2 months:
in this occasion, 113 of Group B patients (90.4 %) showed
a restore of physiological vaginal microflora and 99 of
Group A (79.2 %) a complete clinical remission of BV
(p = 0.014). Figure 1a shows changes in vaginal micro-
flora between two groups after 2 months.
Vaginal physiological flora was branded in 106 patients
of Group B (74.6 %) and only 36 of Group A (25.4 %)
after 6 months (p \ 0.0001—Fig. 1b). Patients with
recurrence of BV were treated with antibiotic therapy.
The third follow-up visit has been done after 9 months:
79.7 % of the patients (n = 118) that had gone on main-
tenance therapy with probiotics revealed a physiological
microflora in the vagina while restoring was observed only
20.3 % (n = 30) of the other antibiotics plus probiotics
group (p \ 0.001—Fig. 1c).
Figure 2 shows changes in pH values of vaginal swabs
measured after 2, 6, and 9 months of therapy in patients
applying or not Lactobacillus rhamnosus BMX 54-con-
taining vaginal tablets.
Vaginal pH was above the physiological value of 4.5 in
88 % of all patients at the first visit.
Mean pH value, valuated with ANOVA test, in Group A
at 2 months of follow-up was 4.272 ± 0.54 SD (3–6, 95 %
CI) and in Group B was 4.232 ± 0.44 SD (3–6, 95 % CI)
with a no significant difference between two groups
(p = 0.524). At 6 months average, pH resulted
4.476 ± 0.57 SD (4–6, 95 % CI) and 4.344 ± 0.38 SD
(4–5.5, 95 % CI). The difference between two groups
proved statistically significant (p = 0.034).
During the last visit (after 9 months), median pH was
5.052 ± 0.54 SD (4–6, 95 % CI) in patients belong to
Group A and 4.228 ± 0.33 SD (4–5.5, 95 % CI) to Group
B. The difference among two groups was significant too
(p \ 0.001).
The gradual return to a vaginal physiological pH was
associated with a reduction of the intensity of symptoms as
shown by the decrease in the symptoms score in both groups
that were related with the restoring of the vaginal flora. We
detected symptoms improvement in 92 % of patients that
had carried on probiotics vaginal tablets comparing to 79 %
other patients (p \ 0.001) after 9 months.
Conclusions
BV is the most frequent vaginal syndrome distressing
fertile, premenopausal, and pregnant women. The disease
has been identified in 12–25 % of women in routine clinic
population [13–15].
Furthermore, BV is often asymptomatic, as about 50 %
of women with this condition have no symptoms at all, and
the prevalence in apparently healthy women is around
10 % [16].
The first-line therapy for BV suggested by international
guidelines [1] includes antimicrobial agents. However,
relapses often crop up, due to persisting of pathogens
protected by microbial biofilm within a changed vaginal
flora [15–18].
The overall long-term cure rate is low, as BV recurs in
up to 40 % of women within 3 months [18].
In cases of BV, beneficial lactic acid-producing bacteria
(lactobacilli) are substituted by amine-producing anaerobic
bacteria [17, 19, 20]. This condition is responsible of the
positivity of whiff test, one of the Amsel’s criteria neces-
sary to the diagnosis of BV.
A healthful vagina is populated by lactobacilli pre-
dominantly. The species differ in relation to their ability to
maintain a stable population through modifications of
vaginal milieu such as pH changes due to sexual inter-
course or menstruation and with respect to their ability to
hinder the growth of other bacteria species [21–23].
Many studies have evidenced the valuable functions of a
healthy microbiota and have suggested the selection of
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Fig. 1 Vaginal flora modification during 9 months follow-up

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Arch Gynecol Obstet
Fig. 2 Variations in pH values after 2, 6, and 9 months in two groups
bacterial strains, recognized as probiotics, with beneficial
effects for the prevention or treatment of conditions
involving a disruption of endogenous microbiota.
Several clinical studies seem to provide evidence that in
lots of cases using of selected Lactobacillus strains the
vagina could be effectively recolonize obtaining also a
lower rate of BV recurrences [22–25].
Lactobacillus casei sub-rhamnosus (nowadays simply
called Lactobacillus rhamnosus) is the unique lactobacillus
strain that has been able to colonize human vaginal
microbiota once exogenously applied [26]. Taking into
account this consideration, Lactobacillus rhamnosus BMX
54—a specific selected lactobacillus strain deposited to
Pasteur Institute under Budapest Treaty—has several
clinical evidences of effectiveness (and, obviously, and
colonization) once vaginal exogenously applied for long-
term use for BV recurrences [22–27].
Lactobacilli’s use and especially Lactobacillus rham-
nosus BMX 54 used in BV is hold by positive results
obtained in some clinical trials [25–27].
This study shows how the administration of probiotics
ad adjuvant treatment to standard antibiotic therapy can
decrease the incidence of BV recurrences at 2 months
follow-up, re-establishing a physiological vaginal pH.
Furthermore a long-term therapy seems to be proficient in
preventing BV symptoms and relapses.
In fact, Lactobacillus rhamnosus BMX 54
(NORMOGINÒ) is able to restore vaginal microflora sig-
nificantly, applying a maintenance therapy too (p \ 0.001).
Results are very encouraging not only after 2 months
follow-up but also in patients long time with vaginal tablets
of Lactobacillus rhamnosus BMX 54 and the treatment
seems to be safe. The overall compliance and tolerability
were absolutely good, and no adverse events were
collected.
Considering pH values, a long-term treatment based on
probiotics seems to allow a re-establishment of the physi-
ological acid vaginal milieu significantly (p \ 0.001). This
evidence is clearer pursuant to long-term administration of
probiotics.
This evidence may be at least partially attributed to the
characteristics of Lactobacillus rhamnosus, which founded
to be one of the most effective strains with regard to
adhesion to squamous and uroepithelial cells, competitive
exclusion of pathogens and production of inhibitors of
urogenital pathogen growth [26, 28–33].
The very limited percentage of recurrences recorded in
the active group during the ninth month of the protocol
suggests a long-term physiological protection thanks to the
properties of Lactobacillus rhamnosus BMX 54.
In conclusion, the dominance of lactobacilli in healthy
vaginal microbiota and its depletion in BV has given rise to
the concept of treatment of topical probiotic Lactobacillus
for the management of this condition.
We clearly demonstrated that reestablishment of vaginal
strains’ equilibrium seems to have a crucial role in pre-
venting various infections, not only BV.
Lactobacilli support and maintain a vigorous ecosystem
by producing lactic acid, hydrogen peroxide, and bacteri-
ocins. These products have antibacterial properties against
vaginal pathogens and also produce a thick and protective
biofilm useful to protect them from harmful microbe pro-
liferation. More randomized clinical trial to confirm these
hypotheses are needed.
Surely this study has various limitations, and further
multicentric randomized clinical trial is needed to confirm
123

our data. Despite this, our data about prophylactic approach
based on probiotics supplementation with Lactobacillus
rhamnosus BMX 54 are comforting pointing out the need
for a long-term use in order to obtain good results.
In conclusion, the administration of vaginal tablets
containing Lactobacillus rhamnosus BMX 54
(NORMOGINÒ) represents an encouraging, outwardly
effective and safe long-term treatment for restoring the
physiological vaginal pH, controlling symptoms, reducing
recurrences of BV, preventing adverse sequelae associated
with this highly prevalent disruption of the vaginal
microbiome.
Conflict of interest All authors have no financial relationship with
the organization that sponsored the research and they have had full
control of all primary data and that they agree to allow the Journal to
review their data if requested.
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