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Arch Gynecol Obstet

DOI 10.1007/s00404-013-3064-9

REVIEW

The role of lactobacilli and probiotics in maintaining vaginal


health
Sandra Borges • Joana Silva • Paula Teixeira

Received: 9 June 2013 / Accepted: 15 October 2013


Ó Springer-Verlag Berlin Heidelberg 2013

Abstract particularly as better information (including that obtained


Background The vaginal microbiota of healthy women through developed molecular methodologies that are not
consists typically of a diversity of anaerobic and aerobic dependent on culture) is changing the present paradigm. It
microorganisms. Lactobacilli are the most prevalent and is recognized that a spectrum of microbial profiles can
often numerically dominant microorganisms and are rele- produce a stable vaginal ecosystem with the ability to
vant as a barrier to infection. The capacity of lactobacilli to maintain vaginal health without succumbing to disease [1].
adhere and compete for adhesion sites in the vaginal epi- Thus, the microbial species that inhabit the vaginal tract
thelium and the capacity to produce antimicrobial com- play an important role in the maintenance of health and
pounds (hydrogen peroxide, lactic acid, bacteriocin-like prevention of infection. In general, the presence of high
substances), are important in the impairment of coloniza- numbers of lactic acid bacteria (LAB) in the vagina is often
tion by pathogens. equated with ‘‘healthy’’ and low numbers or absence, as
Objective This review summarizes the role of lactic acid being ‘‘abnormal’’ [2].
bacteria in preventing illness of the host, including bacte- It appears evident that the balance between a healthy
rial vaginosis, yeast vaginitis, urinary tract infection and and diseased state involves some type of equilibrium or
sexually transmitted diseases. see-saw effect, which can swing in either direction
Conclusions The administration of probiotics that colo- depending on a number of factors, such as hormone levels,
nize the vaginal tract can be important in maintaining a douching, sexual practices, as well as bacterial interactions
normal urogenital health and also to prevent or treat and host defenses [3].
infections. Urogenital infections can be treated by administration of
antibiotics with the undesirable effects of decreasing the
Keywords Urogenital tract  Lactobacilli  number of lactobacilli, do not restore the urinary tract
Vaginal microbiota  Probiotics natural barrier to infections and raise drug resistance [4].
Since the introduction of antibiotics around 1940, many
pathogens have developed antimicrobial drug resistance
Introduction mechanisms. Different mechanisms of resistance in clinical
isolates have been identified, including alterations of drug
The concept of ‘‘normal microbiota’’ as a static and well- targets, modification of the drugs and reduced access of the
defined microbial population is in need of revision, drug to the intracellular target. In many microorganisms
the reduced access of a variety of drugs was found to be the
result of active drug efflux systems. These so-called mul-
S. Borges  J. Silva  P. Teixeira (&) tidrug resistance systems (MDRs) are mainly responsible
CBQF, Centro de Biotecnologia e Quı́mica Fina, Escola Superior for the intrinsic or acquired resistance of microorganisms
de Biotecnologia, Centro Regional do Porto da Universidade
to antimicrobial drugs [5]. Furthermore, drugs can have
Católica Portuguesa, Rua Dr. António Bernardino Almeida,
4200-072 Porto, Portugal side effects including disruption of the protective vaginal
e-mail: pcteixeira@porto.ucp.pt microbiota which creates an increased risk of recurrent

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urinary tract infections (UTI) and yeast vaginitis. In fact, The vaginal vault is colonized within 24 h of a female
yeast infections are extremely common, with an estimated child’s birth and remains colonized until death [1]. Lac-
500 million cases per year globally [6]. tobacilli become the predominant inhabitants of the vagina
Therefore, vaginal probiotics could be a good alternative at the time of puberty, presumably because of the effect of
to antibiotic therapy due to their quality to adhere to uro- estrogens on the glycogen content of vaginal epithelial
epithelial cells and produce inhibitors of pathogenic growth cells [19]. Menopause is marked by a dramatic reduction in
and biosurfactant secretion [4]. estrogen production, resulting in drying and atrophy of the
vaginal epithelium. When estrogen levels drop, glycogen
content in the vaginal epithelium drops as well, leading to
The microbiota of the vaginal tract depletion of lactobacilli. Diminishing numbers of lactoba-
cilli result in a subsequent rise in vaginal pH, since glucose
The species that occupy sites in the human body can is not converted to lactic acid. High pH values promote
change based on intrinsic host factors such as stage of life growth of pathogenic bacteria, particularly colonization by
cycle, hormone levels, immune responses, nutritional status enteric bacteria [1]. Burton and Reid [20] performed a
and disease states. The vaginal microbiota can also be study with 20 postmenopausal women (range 44–72 years)
altered by external factors such as environmental expo- and 70 % had either intermediate-grade bacterial coloni-
sures, microbial interspecies competition or commensal- zation or bacterial vaginosis (BV).
ism, and hygiene behaviors [7]. Therefore, the vaginal microbial ecosystem suffers sig-
The first extensive study of human vaginal microbiota nificant structural changes at various stages in a woman’s
was published by Döderlein in 1892. Döderlein [8] con- life that are directly associated to the level of estrogen in
sidered the vaginal microbiota to be homogenous, consist- the body [2].
ing only of gram-positive bacilli. These Döderlein’s bacilli The complex interactions of the various members of the
are now known to be members of the genus Lactobacillus vaginal microbiota are not well understood; however, it is
spp. This concept has been modified by researchers who recognized that the composition of the microbiota can vary
have found the microbiota of asymptomatic women to from day to day, even in women without indication of
consist usually of a diversity of anaerobic and aerobic infections. Studies using sequential vaginal cultures or
microorganisms. Knowledge on the composition and vaginal smears have shown that in some women, there are
structure of the vaginal microbiome has significantly significant, transient changes in the microbiota. The most
broadened as a result of using cultivation-independent unstable time appears to be around the time of menses, but
methods based on the analysis of 16S ribosomal RNA the exact factors responsible for these changes are
(rRNA) gene sequences [9]. unknown [19].
Lactobacilli are the most prevalent and often numeri- The microorganisms that inhabit the vaginal environ-
cally dominant microorganisms, at 107–108 CFU/g of ment play a major role in preventing illnesses of the host,
vaginal fluid in healthy premenopausal women [1, 10]. including BV, UTI, yeast vaginitis, and sexually trans-
Among the lactobacilli species found in the vaginal mic- mitted diseases including human immunodeficiency virus
robiota, L. iners, L. crispatus, L. gasseri, L. jensenii, fol- (HIV) [21].
lowed by L. acidophilus, L. fermentum, L. plantarum, L. There seems to be an association between absence (or
brevis, L. casei, L. vaginalis, L. delbrueckii, L. salivarius, low concentrations) of vaginal lactobacilli and the devel-
L. reuteri, and L. rhamnosus are the most frequently iso- opment of BV. Compared with that of normal women, the
lated from healthy women [3]. vaginal microbiota of women with BV consists more
Individual differences in Lactobacillus spp. composition commonly, and in higher numbers, of Gardnerella vagi-
of the vaginal tract among women of different geographic nalis, Mycoplasma hominis, Prevotella spp., Peptostrep-
locations, races and ethnicities have been noted across tococcus spp., Mobiluncus spp., Bacteroides spp., Atopium
multiple studies (Table 1). In a study by Ravel et al. [11], vaginae and Megasphera spp. [3, 22]. Bacterial vaginosis
the vaginal bacterial communities of asymptomatic North is characterized by the presence of three of five criteria of
American women who represented four ethnic groups Amsel criteria [21]. Nugent et al. [23] suggested that BV
(white, black, Hispanic and Asian) were characterized by should be diagnosed by vaginal smears examined following
pyrosequencing of barcoded 16S rRNA genes and the Gram’s stain.
vaginal microbiota varied among the four ethnic groups. Bacterial vaginosis is associated with high pH, a
In addition to multiple species of Lactobacillus spp., decrease in antimicrobial activity of the vaginal fluid
other LAB genera have been found in the vaginal tract, compared to healthy women, and local impairment of the
such as Pediococcus spp., Weisella spp., Streptococcus multiple innate immune pathways [24]. A typical feature of
spp. and Leuconostoc spp. [12]. BV is the absence of inflammation. In BV, there is only a

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Table 1 Prevalence of Lactobacillus spp. in vaginal tract of different women (expressed in percentage values)
Lactobacillus Antonio et al. [13] Anukam et al. [14] Aslim and Kilic Tamrakar Vitali et al. Brolazo
spp. (Seatle, n = 302) (Nigeria, n = 24) [15] (Turkey, et al. [16] [17] et al. [18]
n = 10)a (Japan, (Belgium, (Brazil,
n = 98) n = 26) n = 135)b

L. crispatus 32 3.0 14 61.2 30.1


L. jensenii 23 3 29.6 26.5
L. gasseri 5 7.3 21 33.7 Present 22.9
L. ruminis 0.3
L. reuteri 0.3 2.4
L. fermentum 0.3 1.3 2.4
L. oris 0.3 2
L. vaginalis 0.3 2.7 16 Present 8.4
L. iners 64.4 39.8 Present
L. plantarum 6.0 5
L. suntoryeus 6.0
L. rhamnosus 2.7 2.4
L. helveticus 1.3
L. johnsonii 1.3
L. salivarius 1.3 3 1.2
L. acidophilus 16 Present
L. delbrueckii 14 2.4
L. cellobiosus 3
L. curvatus 2
L. brevis 2
L. mucosae 1.2
No homology 4
No lactobacilli 29
Lactobacillus spp. 15 2.7
Methodology Whole-chromosomal Processed by denaturing Biochemical PCR with DGGE and Multiplex
DNA probes to 20 gradient gel tests primers for real-time PCR
Lactobacillus strains electrophoresis (DGGE) 16S ribosomal PCR analysis
and identified by DNA DNA
sequencing
a
Percentage relative to the number of isolates (58 isolates of Lactobacillus spp.)
b
Percentage relative to the number of isolates (83 isolates of Lactobacillus spp.)

slight increase in interleukin I and a low production of vagina, different nutrient availability and competition with
interleukin 8, preventing the attraction of inflammatory indigenous organisms can be the reasons for this lower
cells such as macrophages and neutrophils [25]. diversity in vaginal tract. Some microbes found in the gut
A second major abnormality of the vaginal microbiota, can also be found in the vagina, demonstrating the proper
denominated as aerobic vaginitis (AV), has been descri- receptors, nutrients, and oxygen tension are present for
bed. In this disorder, the normally present Lactobacillus these organisms to grow [3].
spp. are replaced with aerobic organisms, predominantly Accessible data about types and numbers of bacteria
enteric commensals or pathogens. Such a community was present in the vagina have not correlated well with clinical
most often classified as ‘‘intermediate’’. Group B strep- symptoms, an observation which may well be the result of
tococci (GBS), Escherichia coli, and Staphylococcus the incomplete analysis of the vaginal ecosystem available
aureus are the organisms most frequently associated with through traditional methods.
AV [26]. Development of molecular techniques that can identify
Despite the proximity of the vagina to the anus, the (without the need for laborious microscopy or prior cul-
variety of microorganisms present in the vagina is much ture) the organisms comprising the vaginal microbial
lower than in the gut. The reduced receptivity of the community is proving useful in defining organisms that

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have defied identification through traditional cultural there is an interest in developing alternative treatments
methods [1]. against BV. Local treatment of BV with no systemic
effects would be safer for pregnant women [24].
Vaginal microbiota and pregnancy Prospective analyses have linked AV with several
pregnancy-related complications including late miscar-
Numerous types of microorganisms which can be found in riage, chorioamnionitis, and preterm birth. The precise role
association with preterm labor and delivery, such as of AV during pregnancy, the utility of screening, and
Neisseria gonorrhoeae, Chlamydia trachomatis and potential therapies require further study [26].
Trichomonas vaginalis, Haemophilus influenza only occa-
sionally acts like a pathogen, being associated with preterm
labor. GBS can have devastating effects on the preterm or Role of Lactobacillus spp. in maintaining vaginal health
low birth weight infant. A number of genital microorgan-
isms such as E. coli, Listeria monocytogenes and viridians Antimicrobial activity
streptococci may be involved in chorioamnionitis. Bacte-
rial vaginosis is a polymicrobial condition increasingly The role that the vaginal ecosystem plays in the mainte-
associated with adverse perinatal sequelae [25]. nance of vaginal health is important in a functional equi-
The importance of LAB in life is perhaps best seen in librium. This healthy equilibrium acts to supply a barrier to
relation to the health of women and babies [27]. Lactoba- new colonization by pathogenic organisms and overgrowth
cilli are able to reduce the incidence of ascending infec- of organisms that are otherwise commensal. The mecha-
tions of the uterus and the subsequent production of nisms by which lactobacilli stabilize the vaginal microbiota
proinflammatory molecules [28]. are the production of antimicrobial compounds (hydrogen
Once pregnant, the loss of lactobacilli from the vagina peroxide, lactic acid, bacteriocin-like substances) and the
and subsequent development of BV is associated with capability to adhere and compete for adhesion sites in the
serious complications include preterm delivery of low birth vagina.
weight infants, spontaneous abortion, premature rupture of
membranes, preterm birth, amniotic fluid infections, post- pH
partum endometritis and endometritis following Caesarian
section [2]. Toxins from BV-associated microorganisms The role that vaginal LAB plays in inhibiting pathogens
(such as lipopolysaccharides) may cross the placenta and could be linked to their ability to acidify the vaginal tract
cause brain injuries in fetuses. The toxins may cause per- [29]. The pH may become more alkaline as the microbiota
manent neurological brain damage such as cerebral palsy, a shifts toward BV and may be affected by other factors,
risk of developing Parkinson’s disease and schizophrenia such as menses, douching, and semen [19]. The vaginal pH
[24]. varies from 6.6 (±0.3) to 4.2 (±0.2) between day 2 and day
In terms of pregnancy, BV has been associated with a 14 of the menstrual cycle. The production of lactic acid by
40 % increased risk of preterm labor and it occurs in LAB can contribute to this acidification; nevertheless, this
approximately 16 % of the untreated female population. low pH is also maintained by the secretion of organic acids
Yet, the treatment of this common vaginal infection with by the vaginal epithelial cells themselves [29].
antibiotics may or may not decrease the incidence of pre- Acid production has long been known to be detrimental
mature labor. Standard antibiotic therapy for BV with to some microorganisms, not only killing viruses such as
metronidazole is quite ineffective in that more than 30 % HIV, rotavirus and even influenza virus, but also displacing
of women have yeast vaginitis after therapy and more than some pathogens from surfaces. Some microorganisms in
50 % get a recurrent BV infection within 3–6 months. The the vagina, such as yeast and enterococci, can tolerate acids
failure of antibiotic therapy for BV to prevent preterm and resist hydrogen peroxide action. This may be due to
delivery may reflect organisms already having ascended cell wall structures and biofilm formation. The end result is
the uterus or antibiotics being unable to eradicate BV that very few probiotic strains are effective against Can-
biofilms and negate their sialidase activity [21]. dida and enterococci in the vagina [30].
Moreover, the healthy vaginal microbiota is disturbed There are evidences to suggest that the presence of
by antibiotics and the risk of developing antimicrobial drug Lactobacillus spp., the production of lactic acid, and the
resistance increases dramatically with overall increased use resulting low pH are important for preventing the coloni-
of antimicrobial preparations (feminine hygiene and treat- zation and proliferation of non-indigenous organisms in the
ment). In vitro studies have shown that clindamycin and vagina. However, these observations have been over-
metronidazole inhibit Lactobacillus spp. at concentrations interpreted and have led to the statement that Lactobacillus
lower than doses topically applied for treatment. Therefore, spp. must be present for maintaining vaginal health. When

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women have a lack of Lactobacillus spp. in the vaginal lactobacilli, using fluorescence and in vitro bacterial-
community, this situation is considered abnormal. This exposure experiments, and this activity was blocked with
fallacy is the premise of the Nugent criteria used for the cervicovaginal fluid and semen. According to this study, it
diagnosis of BV in which the degree of ‘‘healthiness’’ is is improbable that H2O2-production by vaginal lactobacilli
assessed by the abundance of Lactobacillus morphotypes, is a significant mechanism of protection in vivo.
ignoring the possibility that their ecological function could Therefore, clinical studies concerning the role of H2O2-
be surpassed by bacteria with other morphotypes. It might producing lactobacilli are controversial.
be more logical to assume that an ecological function of
vaginal communities, specifically the production of lactic Bacteriocins
acid, might be accomplished by a variety of taxa capable of
homolactic and heterolactic fermentation of substrates [2]. Bacteriocins (antimicrobial peptides or proteins) are pro-
duced by almost all genera of LAB, and can be divided into
H2O2 different classes based on their biochemical properties.
Bacteriocins have a variety of killing mechanisms,
Hydrogen peroxide (H2O2) is an oxidizing agent, toxic to including cytoplasmic membrane pore formation, interfer-
catalase-negative bacteria such as most anaerobic micro- ence with cellular enzymatic reactions (such as cell wall
organisms. It has been suggested that H2O2 may also help synthesis) and nuclease activity [39].
prevent invasions since some vaginal species of Lactoba- Nisin (a lantibiotic produced by certain strains of Lac-
cillus produce H2O2. Antonio et al. [13] found that H2O2 tococcus lactis) is the only bacteriocin that has been
was produced by 95 % of L. crispatus and 94 % of approved by the World Health Organization for use as a
L. jensenii vaginal isolates. Aroutcheva et al. [31] reported food preservative. However, nisin may not be a good
that approximately 80 % of the strains of vaginal origin choice for vaginal application as it is strongly bactericidal
produced H2O2. for the healthy vaginal Lactobacillus spp. [24].
There were striking differences between the amounts of LAB strains have also been screened for bacteriocins
H2O2 production. Aslim and Kilic [15] reported a range of production, to develop vaginal probiotics [29].
1.01–15.50 lg/mL H2O2. A few bacteriocins from vaginal isolates of Lactoba-
Some studies in vitro suggest that women with H2O2- cillus spp. have been identified.
producing lactobacilli are less likely to be infected with Ocaña et al. [40] found a vaginal L. salivarius subsp.
HIV-1, herpes simplex virus type 2 [32] and pathogens salivarius CRL 1328 that produced a bacteriocin with
associated with BV [33]. However, hydrogen peroxide- activity against Enterococcus faecalis, Enterococcus fae-
producing strains, deemed by some researchers to be crit- cium and N. gonorrhoeae.
ical for vaginal defense, can activate HIV-1 and increase Aroutcheva et al. [31] studied 22 vaginal isolates of
production of intact virions [34]. Lactobacillus spp., of which approximately 73 % exhibited
The H2O2-producing microorganisms that are present bacteriocin activity against G. vaginalis. In that study, it
in the vagina of healthy women have been suggested as was observed that not all strains of G. vaginalis responded
the bacterial group that is responsible for the mainte- identically to the bacteriocinogenic activity of lactobacilli.
nance of the ecologic balance, mainly in pregnant The authors believe that this difference in the ability of G.
women. Wasiela et al. [35] demonstrated that the pre- vaginalis to resist the action of bacteriocin may be
sence of H2O2-producing lactobacilli (L. acidophilus, L. responsible for the more resistant cases of BV.
fermentum, L. minutus, L. jenseni and L. fermentum) Pascual et al. [41] studied a L. fermentum strain L23 that
seems to protect against the development of BV during produced a small bacteriocin which displayed a wide
pregnancy. Pregnant women with BV lack lactobacilli, inhibitory spectrum including both Gram-negative and
especially H2O2-producing strains. Kim et al. [36] Gram-positive pathogenic strains and two species of Can-
observed that the distribution of H2O2-producing lacto- dida spp.
bacilli in vaginal microbiota, as defense factors for Vera Pingitore et al. [42] described a bacteriocin pro-
infection, may have an important role in the patho- duced by vaginal L. salivarius CRL 1328 that exhibited
physiology of preterm labor. activity against E. faecalis by dissipating membrane
Some studies have shown this is not likely to be the case potential and transmembrane proton gradient (both com-
in vivo, since dissolved oxygen levels in the vagina are ponents of proton motive force).
exceptionally low and may limit the amount of hydrogen However, some studies reported that the bacteriocino-
peroxide produced by lactobacilli to sub-inhibitory levels genic activity of vaginal isolates that inhibited the healthy
[2, 37]. Furthermore, O’Hanlon et al. [38] measured the vaginal microbiota led to the disruption of the ecology of
H2O2 concentration from women with H2O2-producing the healthy vagina and paved the way for the establishment

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of the abnormal microbiota associated with the BV. Dez- residence times. Therefore, both self-aggregation and
waan et al. [43] characterized a bacteriocin produced by a adhesion may favor the colonization of the vaginal epi-
vaginal isolate of E. faecium strain 62-6; this bacteriocin thelium through the formation of a bacterial film [47].
had a wide spectrum of growth inhibition against Gram-
positive bacteria (corynebacteria, streptococci, entero-
cocci) of vaginal origin including lactobacilli. In a study Potential of LAB for use as vaginal probiotics
performed by Karaoğlu et al. [44], 100 Lactobacillus spp.
strains were isolated from vaginal samples of 75 repro- The term probiotic derives from the Greek/Latin word
ductive women and six had bacteriocin activity against ‘‘pro’’ and the Greek word ‘‘bios’’, meaning for life. The
some common pathogenic bacteria colonized in the human concept of probiotic was possibly firstly introduced by the
intestine and vagina (G. vaginalis and Pseudomonas Russian Nobel laureate Elie Metchnikoff in 1907 (‘‘The
aeruginosa) but also inhibited vaginal lactobacilli. There- Prolongation of Life: Optimistic Studies’’) where he pro-
fore, the natural inhibition of vaginal lactobacilli by bac- posed the idea that ingesting microbes could have bene-
teriocins may be important in understanding the initiation ficial effects for human beings, especially to treat
of vaginal infections or BV associated with an unexplained digestive diseases. The term ‘‘probiotic’’ was first used in
decrease of vaginal lactobacilli. 1965, by Lilly and Stillwell, to describe substances
secreted by one organism which stimulates the growth of
Adherence another. The World Health Organization and the Food and
Agriculture Organization of the United Nations have
The capacity that lactobacilli have to adhere and compete defined probiotics as ‘‘live microorganisms, which, when
for adhesion sites in the vaginal epithelium can also be administered in adequate amounts, confer a health benefit
involved in the inhibition of colonization by a pathogen. on the host’’ [4].
The renewal of the superficial epithelium of the vagina can A remarkable interest from women was shown on the
affect the equilibrium of the vaginal microbiota [29]. potential use of LAB for maintaining normal urogenital
Factors such as hormonal changes (particularly estrogen), health [48]. A probiotic may act indirectly through treating
vaginal pH, and glycogen content can all affect the ability and preventing recurrent BV or directly by secreting sub-
of lactobacilli to adhere to epithelial cells and colonize the stances (e.g., hydrogen peroxide, bacteriocins, lactic acid)
vagina. The menstrual cycle can also cause changes in the that block sexually transmitted infection [7] (Fig. 1).
vaginal microbiota, with high concentrations of estrogen As antimicrobial treatment of urogenital infections is not
increasing adherence of lactobacilli to vaginal epithelial always effective, and problems remain due to bacterial and
cells [3]. yeast resistance, recurrent infections, as well as side
In the healthy urogenital tract, it is believed that effects, alternative drugs are of interest to patients and their
indigenous lactobacilli exclude the colonization of
pathogenic bacteria by occupying or masking (by steric
hindrance) their potential binding sites in the mucosa.
However, in a depleted lactobacilli environment such as
adhere to cells
an infected urogenital tract, it should be supposed that
exogenous probiotic lactobacilli have the capacity to
compete for the same receptors and displace previously
attached pathogens. The blockage of urogenital patho- safe, non
exclude or
invasive, non
gens adherence by lactobacilli may be by exclusion, carcinogenic reduce
pathogenic
competition for receptor sites and displacement of and non
adherence
pathogenic
adhered pathogens. Several works have shown the
Probiotic
ability of Lactobacillus spp. to adhere to epithelial
vaginal cells to form a biological barrier against colo-
nization by pathogenic bacteria [45, 46]. It has been
considered that multiple components of the bacterial
cell surface participate in this process. Boris et al. [47] able to persist,
multiply and
reported that the factors responsible for adherence to able to produce
coaggregate
epithelial vaginal cells seemed to be glycoproteins and antagonistic
substances
carbohydrates.
Self-aggregation may substantially increase the coloni-
zation potential of lactobacilli in environments with short Fig. 1 Requirements in the choice of a probiotic [5]

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caregivers. It is assumed that recurrences are due to anti- is evidently longer than direct vaginal instillation, and will
microbials failing to eradicate the pathogens, may be depend on viability of the strains as they pass through the
because of biofilm resistance, or that the virulent organisms stomach and gut. In addition, the load of lactobacilli that
come back from their source (the person’s gut or a sex can be delivered this way is obviously lower than via vag-
partner) and attack a host whose defenses are suboptimal inal administration. However, an advantage of the oral
[3]. approach may be the ability of the lactobacilli to reduce the
Several clinical trials have been performed to investigate transfer of yeast and pathogenic bacteria from the rectum to
whether specific strains of lactobacilli, administered either the vagina, which could potentially lower the risk of
orally [49] or intra-vaginally [50, 51], are able to colonize infection [3].
the vaginas of women with symptomatic or asymptomatic Vaginal dosage forms available around the world
BV, to reduce the colonization of pathogens, and to improve include creams, gels, tablets, capsules, pessaries, foams,
symptoms and/or signs of BV when they are present [22]. ointments, films, tampons, rings, and douches (Table 2).
Probiotics can be administered vaginally or orally because While the majority of vaginal drugs so far have been in
lactobacilli can ascend passively from the rectum to the the form of gels, there is a growing interest in alternative
vagina, which can be a significant breakthrough in being dosage forms such as rings, tablets, and films. The
able to deliver probiotics in foods and dietary supplements majority of issues with the product development and scale
[27]. The time for this intervention to affect the vaginal tract up are similar to other pharmaceutical products, although

Table 2 Several studies with different dosage forms for vaginal probiotics
Vaginal drug Microorganism Quantity Other compounds References
delivery
systems

Tablet L. brevis or L. salivarius or The initial quantity in each Fast-release layer: lactose, maize starch, Maggi et al. [53]
L. ciispaius or L. gasseri tablet is not described. adipic acid, sodium bicarbonate,
But the maximum found ascorbic acid, stearic acid, magnesium
after 1 month of storage stearate and colloidal silicon dioxide
at 4 °C is 109 cells/g Slow-release layer: ascorbic acid,
mannitol, retarding polymer, talc,
magnesium stearate and colloidal
silicon dioxide
Capsule L. fermentum RC-14 and 1 9 109 total cfu per Burton et al. [54]
L. rhamnosus GR-1 capsule
Tampons L. gasseri 108 living bacteria per Eriksson et al.
L. casei var rhamnosus tampon [55]
L. fermentum
Suppository L. crispatus CTV-05 5 9 108 cfu for suppository Preservation matrix and maltodextrin Czaja et al. [56]
Douche L. acidophilus 1 9 109 cfu/mL (final 10 % hydroxypropyl guar gum and 9.5 % Drago et al. [57]
vaginal bacterial concentration NaCl
after suspension in
100 mL of water)
Tablet Lactobacillus spp. Not described Citric acid, sodium bicarbonate and Kale et al. [58]
lactose
Gelatin L. gasseri 108–109 cfu/capsule Lactitol monohydrate, gelatine, Larsson et al.
capsules L. rhamnosus cornstarch, xanthan gum, glucose [59]
anhydrous, titanium dioxide and
magnesium stearate
Suppositories L. paracasei HL32 108 cfu for suppository Witepsol H-15 or mixed polyethylene Kaewnopparat
glycols and
Kaewnopparat
[60]
Capsule L. gasseri LN40, Between 108 and 1010 Maltodextrin and magnesium stearate Ehrström et al.
L. fermentum LN99, viable cells per capsule [50]
L. casei subsp. rhamnosus
LN113 and Pediococcus
acidilactici LN23

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there are some unique challenges because of site of indicating BV in 52.5 and in 7.5 % of the patients before
delivery, prophylactic nature of product application, and treatment and at follow-up, respectively. After treatment,
diversity in sex and hygiene practices across the devel- significant decreases in vaginal pH were observed, to less
oping world [52]. than pH 4.5 in 34/40 women, and the odor test became
negative in all of the patients.
Ehrström et al. [50] reported a randomized double-blind
Clinical trials placebo-controlled study to assess the vaginal colonization
of LAB and clinical outcomes. Vaginal capsules containing
Some randomized controlled trials reported enhanced cure L. gasseri LN40, L. fermentum LN99, L. casei subsp.
rates or a reduced recurrence of BV among premenopausal rhamnosus LN113 and P. acidilactici LN23 (108 and 109
women treated with a vaginal probiotic (Table 3). viable cells for capsule) were administered for 5 days to 95
Marcone et al. [51] investigated the advantages of long- women after conventional treatment of BV and/or vulvo-
term vaginal administration of L. rhamnosus after oral vaginal candidiasis. Probiotic strains were present
treatment with metronidazole to prevent the recurrence of 2–3 days after administration in 89 % of the women. After
BV. A total of 49 women were diagnosed with BV, some one menstruation, 53 % were colonized by at least one LN
of them were treated with oral metronidazole for 7 days strain. Nine percent were still colonized 6 months after
followed by a once-weekly vaginal application of 40 mg of administration. The probiotic supplementation resulted in
L. rhamnosus for 6 months. During the first 6 month of less malodorous discharge, and a trend towards higher
follow-up, 96 % of patients had a balanced vaginal eco- clinical cure rate, compared with the placebo group.
system. The vaginal probiotic allowed stabilization of the However, some trials found no beneficial effects of
vaginal ecosystem and reduced the recurrence of BV. vaginal probiotic in the treatment of BV. Eriksson et al.
Drago et al. [57] treated 40 women with BV for 6 days [55] performed a double-blind placebo-controlled study
with a douche containing L. acidophilus. The Nugent score (187 patients) of adjuvant lactobacilli treatment after an
decreased significantly from BV, and remained low during open treatment with vaginal clindamycin ovules. There was
the follow-up period for almost all of the patients, no improvement in the cure rate after treatment with

Table 3 Studies of vaginal probiotics in the treatment or prevention of bacterial vaginosis and urinary tract infections
No. of Intervention Results References
patients

49 women Oral metronidazole for 7 days followed by a once- 96 % of patients had a balanced vaginal ecosystem. Marcone et al.
with BV weekly vaginal application of 40 mg of L. rhamnosus In contrast, 74 % of patients treated only with [51]
for 6 months metronidazole, showed a balanced vaginal
ecosystem
40 women Douche containing L. acidophilus for 6 days The Nugent score decreased significantly from BV, Drago et al.
and remained low during the follow-up period for [57]
almost all of the patients, indicating BV in 52.5 %
and in 7.5 % of the patients before treatment and
at follow-up, respectively
95 women Vaginal capsules containing L. gasseri LN40, L. Less malodorous discharge and a trend towards Ehrstrom et al.
with BV fermentum LN99, L. casei subsp. rhamnosus LN113 higher clinical cure rate. Ninety-three percent of [50]
and P. acidilactici LN23 were administered for 5 days the women receiving probiotic strains were cured
after conventional treatment 2–3 days after administration (placebo 83 %)
9 women The patients were inserted with vaginal suppositories A significant reduction in the number of recurrences Uehara et al.
with containing the strain L. crispatus GAI 98322 every was noted (from an average of 5.0 ± 1.6 episodes [61]
recurrent 2 days for 1 year per year to 1.3 ± 1.2), without any adverse
UTI complication
30 women Women were randomized to use suppositories of L. crispatus CTV-05 can be given as a vaginal Czaja et al.
with L. crispatus CTV-05 for 5 days suppository with minimal side effects to healthy [56]
recurrent women with a history of recurrent UTI. This study
UTI was a phase I trial to assess the safety and
tolerance of a Lactobacillus vaginal suppository
for prevention of recurrent UTI, this study was not
designed to evaluate the effect of L. crispatus
CTV-05

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lactobacilli-containing tampons compared to placebo tam- with a mixture of three Lactobacillus spp. and a P. acidi-
pons; the cure rates as defined by Amsel’s criteria were 56 lactici. VagiforteÒ (Bioflora, South Africa) is distributed in
and 62 %, respectively, and 55 and 63 %, as defined by South Africa with L. acidophilus, Bifidobacterium bifidum
Nugent’s criteria. and Bifidobacterium longum in vaginal suppositories. Gy-
In a study by Larsson et al. [59], 100 women with BV noflorÒ (Gynoflor, Switzerland) is vaginal tablet which
diagnosed by Amsel criteria, after informed consent were contains L. acidophilus and estriol.
offered vaginal clindamycin therapy followed by vaginal
gelatine capsules containing either 109 freeze-dried lacto-
bacilli or identical placebo capsules for 10 days during Negative effects of probiotics
three menstrual cycles in a double-blind, randomized,
placebo-controlled trial. The study showed that supple- Annually, over one billion doses of probiotics are admin-
mentary treatment combining two different strains of pro- istered worldwide, and those administered for urogenital
biotic lactobacilli did not improve the efficacy of BV health have been well tolerated. Endocarditis and bacter-
therapy during the first month of treatment, but for women aemia caused by lactobacilli are extremely rare; most cases
initially cured, adjunct treatment of lactobacilli during occur in patients with chronic diseases or debilitating
three menstrual cycles lengthened the time to relapse sig- conditions, and any association with exposure to probiotic-
nificantly in that more women remained BV-free at the end containing foods or supplements has been questioned
of the 6-month follow-up. [3, 62].
Some studies evaluated the use of lactobacilli for the Nevertheless, safety of probiotic use must continually be
prevention of recurrent UTI (Table 3). Uehara et al. [61] monitored and considered when performing clinical stud-
reported a study with nine patients with recurrent UTI. The ies. The potential for transfer of antibiotic resistance is one
patients were inserted with vaginal suppositories contain- factor to consider, although it remains to be proven that
ing the strain L. crispatus GAI 98322 (1.08 cfu per sup- probiotics have contributed in any way to drug resistance
pository) every 2 days for 1 year. A significant reduction in or disease [3].
the number of recurrences was noted, without any adverse
complication. Czaja et al. [56] performed a phase I trial to
assess the safety and tolerance of a Lactobacillus vaginal
Conclusion
suppository for prevention of recurrent UTI. Thirty pre-
menopausal women with a history of recurrent UTI were
The factors that affect and control the vaginal microbiota
randomized to use L. crispatus CTV-05 or placebo vaginal
are still incomplete, although we should analyze the vagi-
suppositories daily for 5 days. No severe adverse events
nal tract as a microbial ecosystem to understand the full
occurred. They concluded that L. crispatus CTV-05 can be
range of factors that affect risk to disease.
given as a vaginal suppository with minimal side effects to
The use of probiotics to colonize the vaginal tract and
healthy women with a history of recurrent UTI, but seven
prevent or treat infection has been considered for some
women randomized to L. crispatus CTV-05 developed
time and has an excellent safety record. It is recognized
pyuria without associated symptoms.
that LAB have the ability to maintain the vaginal health of
Beyond the vaginal application, probiotics can be
women. Natural antimicrobials produced by probiotics, at
administered orally to restore the vaginal microbiota.
the same time that inhibit the growth of pathogens, will
Reid et al. [49] performed a randomized, placebo-con-
improve the survival and promote the growth of the native
trolled trial; in that study, 59 premenopausal women
vaginal microbiota, thus supporting the natural defense
received either freeze-dried capsules ([109 cfu per cap-
against pathogenic microorganisms.
sule) containing L. rhamnosus GR-1 and L. fermentum RC-
To obtain evidence of the preventive and curative role of
14 or calcium carbonate placebo by mouth, once daily for
vaginal probiotics, several studies involving large numbers
60 days. After this time, the lactobacilli-dominant micro-
of patients will be necessary, to develop correct formula-
biota was restored in subjects with BV but not in controls.
tions relative to the amount of bacteria, viability, admin-
The mode of action is likely to be several-fold, in that in
istration schedule and efficacy. It is particularly important
some subjects the probiotic strains ascend from the rectum
that probiotic strains be characterized and tested clinically
to the vagina, while in other cases there is a reduction in
using the delivery system of choice (oral or vaginal, dried
ascension of pathogens, or an immune modulation that
or in solution).
somehow displaces the BV organisms.
In the market, there are vaginal probiotic products that Acknowledgments This work was supported by National Funds
claim to restore or maintain the vaginal microbiota. For from FCT—Fundação para a Ciência e a Tecnologia through project
example, the tampon EllenÒ (Ellen AB, Sweden) is sold PEst-OE/EQB/LA0016/2011. Financial support for author S. Borges

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was provided by PhD fellowship, SFRH/BD/45496/2008 (FCT— 18. Brolazo EM, Simões JA, Nader MEF, Tomás MSJ, Gregoracci
Fundação para a Ciência e a Tecnologia). Editing of this manuscript GB, Marconi C (2009) Prevalência e caracterização de espécies
by Dr P.A. Gibbs is gratefully acknowledged. de lactobacilos vaginais em mulheres em idade reprodutiva sem
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