Eighty-four patients with bacterial vaginosis diagnosed according to Amsel&apos;s criteria were randomized to receive either oral metronidazole 500 mg twice a day for seven days, or one vaginal tablet containing freeze-dried Lactobacillus rhamnosus once a week at bedtime for two months starting one week after the last antibiotic administration. Followup was performed at days 30, 90 and 180. Chi-squared analysis showed a significant difference between the two treatment groups at day 90 (P=0.05). Safe and effective long-term vaginal administration of Lactobacillus rhamnosus appears to be a useful complementary approach in the management of bacterial vaginosis

Dr Valentina Marcone

Ettore Calzolari

Marco Bertini

Valentina Marcone

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NEW MICROBIOLOGICA, 31, 429-433, 2008
Effectiveness of vaginal administration 
of Lactobacillus rhamnosus following conventional
metronidazole therapy: how to lower the rate 
of bacterial vaginosis recurrences
Valentina Marcone1, Ettore Calzolari1, Marco Bertini2
1Department of Gynaecology, Perinatology and Child Health-Gynaecologic Infections and Cervicovaginal Pathology Unit,
University “La Sapienza”, Rome, Italy;
2Pharmacology and Chemoterapy Division, University of Pisa, Italy
Eighty-four patients with bacterial vaginosis diagnosed according to Amsel’s criteria were randomized to receive ei-
ther oral metronidazole 500 mg twice a day for seven days, or one vaginal tablet containing freeze-dried Lactobacillus
rhamnosus once a week at bedtime for two months starting one week after the last antibiotic administration. Follow-
up was performed at days 30, 90 and 180. Chi-squared analysis showed a significant difference between the two treat-
ment groups at day 90 (P=0.05). Safe and effective long-term vaginal administration of Lactobacillus rhamnosus ap-
pears to be a useful complementary approach in the management of bacterial vaginosis.
KEY WORDS: Vaginosis, Lactobacilli, Probiotics
SUMMARY
Received February 24, 2007 Accepted March 07, 2008
Corresponding author
Dr. Valentina Marcone
Dipartimento di Scienze Ginecologiche
Perinatologia e Puericultura
Università “La Sapienza”
Viale Regina Elena, 285 - 00161 Roma, Italy
E-mail: valentinamarcone@libero.it
In healthy women, the vaginal environment is a
balanced ecosystem characterized by various
species of Lactobacillus. These bacteria inhibit
the growth of other microorganisms through dif-
ferent mechanisms including production of or-
ganic acids such as lactic acid and other antimi-
crobial substances (hydrogen peroxide and bac-
teriocins), competition for mannose and glyco-
protein receptors and adhesion to the epithelium
(Reid and Burton, 2002).
The depletion of vaginal Lactobacilli is known to
be associated with bacterial vaginosis (BV)
(Spiegel 1991; Wang, 2000), a disorder repre-
sented by a complex change in vaginal flora and
characterized by a reduction in the prevalence
and concentration of Lactobacilli and an increase
in some vaginal anaerobic or facultative anaero-
bic bacteria, accompanied by loss of the usual
vaginal acidity (Hay, 2005). 
Since a pool of pathogens is involved and classi-
cal signs of inflammation are absent, the older
term “Gardnerella vaginitis” was definitively re-
placed by the new one “ bacterial vaginosis” more
than twenty years ago (Holmes et al., 1981;
Blackwell et al., 1983).
The aetiology of BV is probably multifactorial
and the factor initiating the shift is still unknown
(Donders et al., 2000). 
A body of evidence indicates that a dysregulation
in vaginal microecology of lactic acid producing
bacteria and an impaired mucosal vaginal im-
mune response play a crucial role in the patho-
genesis of the disorder (Cauci et al., 1997). BV is
not regarded as a sexually transmitted disease
(STD), though it is sexually associated (Larsson
et al., 1991).
In women of reproductive age the prevalence of
BV varies between 9% and 29% according to the
epidemiologic characteristics of the population
subgroups enrolled in the studies conducted
worldwide (Lamont et al. 2000; Allsworth et al.
2007; Evans et al. 2007). BV seems to be more
common in women with STDs and in those who
have recently changed sex partner, but it has been
reported even in virgin women. Furthermore, in
many studies it is associated with smoking, black
race and use of an intrauterine device (IUD)
(Schmid 1999; Calzolari et al., 2000).
Some differences in the prevalence of BV may al-
so be explained by the use of different diagnostic
criteria. 
Furthermore, in about 50% of cases BV appears
to be asymptomatic. Although it was previously
regarded as a harmless condition, studies in the
past decade have highlighted the association of
BV with various gynaecologic and obstetric
pathologies such as pelvic inflammatory disease,
increased risk of infection after gynaecologic sur-
gery, human papilloma virus (HPV) infection and
progression of squamous intraepithelial lesion
(SIL), chorioamnionitis, postpartum and
postabortion endometritis, preterm labour and
delivery and spontaneous abortion. BV may also
be associated with an increased risk for human
immunodeficiency virus (HIV) transmission be-
tween adults (Taha et al., 1998; Rauth et al., 2000;
Morris et al., 2001; Romero et al., 2002; Beverly et
al., 2005; Tolosa et al., 2006).
The social, clinical and economic implications of
adverse outcomes associated with BV have led to
increased attention to diagnosis and treatment.
Attempts to correct the imbalance in vaginal mi-
croecology only with the use of antibiotics have
been substantially unsuccessful, especially in the
management of recurrences, while recent ad-
vances in our understanding of microbiological,
immune and metabolic alterations in the vaginal
microenvironment of women with BV have raised
further interest in the therapeutic use of probi-
otics (Famularo et al., 2001).
The concept of probiotics dates back more than
100 years and treatment of vaginitis and vaginosis
with Lactobacillus replacement therapy was first
described in the USA in 1933 by Molher and
Brown. Since then the attention of the investiga-
tors has focused on the role that probiotics may
play in the treatment and prevention of various
urogenital infections (Butler and Beakley, 1960;
Reid et al., 1990; Bruce et al., 1992). One current
trend is to add or even prefer “natural” products
to the usual therapy: the Food and Agriculture
Organization of the United Nations and the World
Health Organization Working Group recently de-
fined probiotics as “live microorganisms which
when administered in adequate amounts confer a
health benefit on the host” so developing guidelines
for what constitutes a true probiotic. (FAO/ WHO
2001; FAO/WHO 2002). 
According to these criteria Lactobacillus rhamno-
sus can be considered a probiotic. Open clinical
studies showed vaginal colonization and beneficial
effect on the host following oral intake or vaginal
application of the probiotic. Lactobacillus rham-
nosus was found 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 uro-
genital pathogen growth (Reid et al., 1994; Mc
Lean and Rosenstein, 2000; Reid and Burton,
2002; Gardiner et al., 2002).
Various clinical studies seem to provide evidence
that in many cases the use of selected
Lactobacillus strains can effectively recolonize the
vagina and lower the rate of BV recurrences,
(Rossi et al., 1999; Cadieux et al., 2002; Rossi et al.,
2003; Marcone et al., 2003; Anukam et al., 2006).
We carried out a prospective randomized con-
trolled trial to test the effectiveness of vaginal ap-
plication of Lactobacillus rhamnosus in improv-
ing the long-term outcome of BV treatment with
conventional metronidazole therapy.
We conducted our study at “La Sapienza”
University of Rome, Institute of Gynecology,
Perinatology and Child Health - Gynecologic
Infection and Cervicovaginal Pathology Unit
starting in 2004. 
Eligible patients were women of childbearing age
with a diagnosis of BV defined as meeting all of
Amsel’s criteria (>20% clue cells, off-white thin
homogeneous vaginal discharge, vaginal pH >4.5
and a positive “ whiff test”) (Amsel et al., 1983).
The exclusion criteria were urogenital infections
not falling in the above diagnostic criteria or an
STD, pregnancy, subjects younger than 18 or old-
er than 40 years, smoking, contraception other
than natural methods. 
430 V. Marcone, E. Calzolari, M. Bertini
Eighty-four subjects fulfilling the entry criteria
were treated either with oral metronidazole
(Group A, n=42) or with oral metronidazole and
vaginal Lactobacillus rhamnosus (Group B, n=42)
following a randomization scheme. Study sub-
jects were not blinded to the treatment they re-
ceived. Group A received oral metronidazole 500
mg twice a day for seven days according to CDC
guidelines. In Group B the treatment with
metronidazole was followed by vaginal applica-
tion (one tablet containing 40 mg , i.e. >40000
CFU) of freeze-dried Lactobacillus rhamnosus
once a week at bedtime for two months starting
one week after the last antibiotic administration.
In addition to the study entry criteria, all patients
included in statistical analyses met the following
requirements:
1) had no antimicrobial therapy for conditions
other than BV during the study;
2) started study medication within 48 hours of
the baseline visit. 
At days 30, 90 and 180, clinical assessment, pH
measurement and KOH testing of each patient
subject were carried out by a single physician
who also collected vaginal swabs. Microscopic
evaluation of wet mount was carried out blindly
by a skilled physician. Chi square test was applied
for the statistical comparison of the outcome in
the two groups. 
All patients enrolled in this study returned for fol-
low-up visits scheduled 30, 90 and 180 days after
the end of metronidazole treatment. At the first re-
examination 37 (88%) patients in group B showed
a complete remission and none of them had a re-
currence at 90 day follow-up. 
After 180 days 35 (83%) patients in group B still
had a balanced vaginal ecosystem while just 28
(67%) patients in group A had not been treated for
recurrences and still had a satisfactory assessment
(Table 1).
Chi-squared analyses showed a significant differ-
ence between the two treatment groups at day 90
(P=0.05). 
The results obtained in the present trial appear to
confirm that CDC-recommended therapy of BV
with metronidazole was effective in healing BV,
and that subsequent vaginal administration of
Lactobacillus rhamnosus further increased the rate
of therapeutic success and prolonged the period
without BV relapses. Interestingly, the significant
(P=0.05) protective effect of Lactobacillus rham-
nosus vaginal application persisted for at least 30
days after the end of treatment with the probiot-
ic (follow-up day 90). Consistently, a higher per-
centage of treated patients showed no BV recur-
rencies at follow-up day 180 compared to con-
trols.
Although the difference was not statistically sig-
nificant (P=0.07), this indicates that vaginal colo-
nization by Lactobacillus rhamnosus greatly out-
lasts the supplementation of probiotic and sug-
gests that treatment with Lactobacillus rhamno-
sus for longer than two months after metronida-
zole therapy could result in better control of BV
recurrencies in patients at risk for BV relapse. 
Our data show that vaginal application of
Lactobacillus rhamnosus significantly prolongs
the effectiveness of antibiotic treatment in pre-
venting BV recurrence. 
Therefore longer administration of probiotic may
be useful for long-term control of BV relapses af-
ter conventional therapy with metronidazole. This
safe and effective complementary treatment may
represent a valuable approach in the management
of BV for maintenance of healthy vaginal ecosys-
tem. 
Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy 431
TABLE 1
Therapy day 0 Healing Healing Healing 
follow-up day 30 follow-up day 90 follow-up day 180
42 34 30 28
Oral Metronidazole 81% 71% 67%
42 37 37 35
Oral metronidazole followed 88% 88% 83%
by vaginal lactobacillus rhamnosus
P=0.36 P=0.05 P=0.07
REFERENCES
ALLSWORT J.E., PEIPERT J.F. (2007). Prevalence of bac-
terial vaginosis: 2001-2004 National Health and
Nutrition Examination Survey data. Obstet.
Gynecol. 109, 114-120.
AMSEL R., TOTTEN P.A., SPIEGEL C.A., CHEN K.C.,
ESCHENBACH D., HOLMES K.K. (1983). Non-specific
vaginitis. Diagnostic criteria and microbial and epi-
demiologic associations. Am. J. Med. 74, 14-22.
ANUKAM K., BRUCE A., REID G. (2006). Clinical study
comparing probiotic Lactobacillus GR-1 an RC-14
with metronidazole vaginal gel to treat sympto-
matic bacterial vaginosis. Microbes and Infection.
8, 2772-2776.
BLACKWELL A.L., FOX A.R., PHILLIPS I., BARLOW D.
(1983). Anaerobic vaginosis (non-specific vagini-
tis): clinical, microbiological, and therapeutic find-
ings. Lancet. 2, 1379-1382.
BRUCE A.W., REID G., MC GROARTY J.A. (1992).
Preliminary study on the prevention of recurrent
urinary tract infection in adult women using in-
travaginal Lactobacilli. Int. Urogynecol. J. 3, 22-25.
BUTLER B.C., BEAKLEY J.W. (1960). Bacterial flora in
vaginitis: a study before and after treatment with
pure cultures of Doderlein bacillus. Am. J. Obstet.
Gynecol. 79, 432-440.
CADIEUX P., REID G.P., BURTON J., GARDINER G.,
BRAUNSTEIN I., BRUCE A.W., KANG C.Y., REID G.
(2002). Lactobacillus strains and vaginal ecology.
JAMA. 278, 1940-1941.
CALZOLARI E., MASCIANGELO R., MILITE V., VERTERAMO
R. (2000). Bacterial Vaginosis and contraceptive
methods. Int. J. Gynecol. Obstet. 70, 341-346.
CAUCI S., DRIUSSI S., MONTE R., LANZAFAME P., PITZUS
E., QUADRIFOGLIO F. (1997). Immunoglobulin A re-
sponse against Gardnerella vaginalis hemolysin and
sialidase activity in bacterial vaginosis. Am. J.
Obstet. Gynecol. 177, 1184-1187.
CDC - CENTERS FOR DISEASE CONTROL AND PREVENTION.
(2006). STD treatment guidelines. Donders G.,
Gilbert G.G., Bosmans E., Dekeersmaecker A.,
Vereecken A., Pharm B., Van Bulck B., Spitz B.
(2000). Pathogenesis of abnormal vaginal flora. Am.
J. Obstet. Gynecol. 182, 872-878.
EVANS A.L., SCALLY A.J., WELLARD S.J., WILSON J.D.
(2007). Prevalence of bacterial vaginosis in lesbians
and heterosexual women in a community setting.
Sex. Transm. Infect. Published Online First: 4 July
2007.
FAMULARO G., PIELUIGI M., COCCIA R., MASTROIACOVO P.,
DE SIMONE C. (2001). Microecology, bacterial vagi-
nosis and probiotics: perspectives for bacteriother-
apy. Medical Hypotheses. 56, 421-430.
FAO/WHO. (2002). Guidelines for the evaluation of
probiotics in food. Report of a joint FAO/WHO
working group 2002. www.fao.org./ag/agn/food/
food_probiocons_en.stm.
GARDINER G.E., HEINEMANN C., BRUCE A.W., BEUERMAN
D., REID G. (2002). Persistence of Lactobacillus fer-
mentum RC-14 and Lactobacillus rhamnosus GR-
1 but not L. rhamnosus GG in the human vagina as
demonstrated by randomly amplified polymorphic
DNA. Clin. Diagn. Lab. Immunol. 9, 92-96.
HAY P. (2005). Bacterial vaginosis. Medicine. 33, 58-61.
HOLMES K.K., SPIEGEL C., AMSEL R., ESCHENBACH D.A.,
CHEN K.C.S., TOTTEN P. (1981). Nonspecific vagi-
nosis. Scan. J. Infect. Dis. 26, 110-114.
LAMONT R.F., MORGAN D.J., WILDEN S.D., TAYLOR-
ROBINSON D. (2000). Prevalence of bacterial vagi-
nosis in women attending one of three general prac-
tices for routine cervical cytology. Int. J. STD &
AIDS. 11, 495-498.
LARSSON P.G., PLATZ-CHRISTENSEN J.J., SUNDSTROM E.
(1991). Is bacterial vaginosis a sexually transmit-
ted disease? Int. J. STD & AIDS. 2, 362-364.
MARCONE V., MANCINI E., VERTERAMO R., RECINE N.,
CALZOLARI E. (2003). Results of treatment with
Lactobacillus casei sub-rhamnosus after antibiotic
therapy of bacterial vaginosis. 8th World congress
for infectious and immunological dieseases in ob-
stetrics and gynecology. Venice. 166-167.
MC LEAN N., ROSENSTEIN I.J. (2000). Characterisation
and selection of a Lactobacillus species to re-
colonise the vagina of women with recurrent bac-
terial vaginosis. J. Med. Microbiol. 49, 543-552.
MOLHER R.W., BROWN C.P. (1933). Doderlein’s bacillus
in the treatment of vaginitis. Am. J. Obstet. Gynecol.
25, 718-723.
MORRIS M., NICOLL A., SIMMS I., WILSON J., CATCHPOLE
M. (2001). Bacterial vaginosis: a public health re-
view. Br. J. Obstet. Gynecol. 108, 439-450.
RAUTH V.A., CULHANE J.F., HOGAN V.K. (2000). Bacterial
Vaginosis: a public health problem for women. J.
Am. Wom. Assoc. 55, 220-224.
REID G., BURTON J. (2002). Use of lactobacillus to pre-
vent infection by pathogenic bacteria. Microbes and
Infection. 4, 319-324.
REID G.K., MILLS A.P., BRUCE A.W. (1994). Implantation
of Lactobacilli Casei var-rhamnosus into the vagi-
na. The Lancet. 344, 1229.
REID G., BRUCE A.W., MC GROARTY J.A., CHENG KJ.,
COSTERTON J.W. (1990). Is there a role for lacto-
bacilli in prevention of urogenital and intestinal in-
fections? Clin. Microbiol. Rev. 3, 335-344.
ROMERO R., ESPINOZA J., CHAIWORAPONGSA T., KALACHE
K. (2002). Infections and prematurity: the role of
preventive strategies. Semin. Neonat. 7, 259-274.
ROSSI A., CATTADORI F., SCIENZA G., CORTI BOLLA E.,
CHIANTERA V., BERTINI M. (1999). Therapy of recur-
rent perimenarcheal vaginitis using exogenously-
applied Lactobacilli Casei sub-rhamnosus. J. Clin.
Res. 2, 173-178.
SHA B.E., ZARIFFARD M.R., WANG Q.J., CHEN H.Y.,
BREMER J., COHEN M.H., SPEA G.T. (2005). Female
genital-tract HIV load correlates inversely with
432 V. Marcone, E. Calzolari, M. Bertini
Lactobacillus species but positively with Bacterial
Vaginosis and Mycoplasma hominis. The Journal of
Infectious Diseases. 191, 25-32.
SCHMID G.P. (1999). The epidemiology of bacterial vagi-
nosis. Int. J. Obstet. Gynecol. 67, S17-S20.
SPIEGEL C.A. (1991). Bacterial Vaginosis. Clin. Microb.
Rev. 4, 485.
TAHA T.E., HOOVER D.R., DALLABETTA G.A., KUMWENDA
N.I., MTIMAVALYE L.A.R., YANG L.P., LIOMBA G.N.,
BROADHEAD R.L., CHIPHANGWI J.D., MIOTTI P.G.
(1998). Bacterial vaginosis and disturbance of vagi-
nal flora: associated with incresed acquisition of
HIV. AIDS. 12, 1699-1706.
TOLOSA J.S., CHAITHONGWONGWATTHANA S.D., MAW W.,
GAITÀN H., LUMBIGANON P., FESTIN M., CHIPATO T.,
SAUVARIN J., GOLDENBERG R. (2006). The international
infection in pregnancy (IIP) study: variations in the
prevalence of bacterial vaginosis and distribution of
morphotypes in vaginal smears among pregnant
women; Am. J. Obst. Gynecol. 195, 1198-1204.
WANG J. (2000) Bacterial vaginosis. Primary care up-
date for OB/GYNS 7, 181-185.
Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy 433



Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy: how to lower the rate of bacterial vaginosis recurrences.

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Effectiveness of vaginal administration of Lactobacillus rhamnosus following conventional metronidazole therapy: how to lower the rate of bacterial vaginosis recurrences.

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