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Lactobacillus crispatus M247: a possible tool to counteract CST IV

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Nutrafoods (2018) 17:169-172
DOI 10.17470/NF-018-0001-4
Correspondence to: Francesco Di Pierro -
Francesco Di Pierro1, Alexander Bertuccioli2, Daniela Cattivelli3, Sara Soldi3, Marina Elli3
Lactobacillus crispatus M247:
a possible tool to counteract CST IV
Bacterial CST (community state type) I is characterized by
Lactobacillus crispatus dominance and is associated with a
healthy vagina and a reduced risk of vaginosis, vaginitis, pre-
term birth, infertility and vaginal atrophy. On the other hand,
CST IV is characterized by the absence of lactobacillus and
is associated with unhealthy gynaecological conditions. Un-
fortunately, very few L. crispatus strains have been properly
studied and documented for human use. Here we discuss
the genetic, probiotic and vaginal colonization properties of
strain M247, isolated in 1989 from infant faeces, and de-
scribe its possible gynaecological uses especially in woman
characterized by CST IV.
Culture-independent investigation of the human vaginal
microbiota has revealed ve distinct community state types
(CSTs), four of which are mostly dominated by a single Lac-
tobacillus species (CST I: Lactobacillus crispatus; CST II: L.
gasseri; CST III: L. iners; CST V: L. jensenii), while CST IV
is characterized by a paucity of Lactobacillus species and
consists of a diverse group of facultative and strict anaer-
obes, including bacteria associated with bacterial vaginosis
(BV) [1]. CST IV has been linked to BV, candidiasis, chla-
mydia, HPV persistence, sine causa infertility and spontane-
ous preterm birth. These conditions seem to be particularly
correlated with CST IV, especially when in the presence of
increased bacterial richness and vaginal microbiota diversity
[2]. CST IV has also been associated with the menopause
and vulvovaginal atrophy [3]. Research has revealed that the
lactobacilli inhabiting the human vagina provide the rst
line of defence in the female urogenital and reproductive
tracts. However, not all lactobacilli are equally protective
and recent research indicates that of the four Lactobacillus-
based CSTs, the vaginal microbiota CST I is most closely cor-
related with a healthy status, thereby suggesting L. crispatus
as a biomarker of a healthy vaginal tract [4]. The healthy role
played by L. crispatus seems to also affect urinary infections.
As a matter of fact, eight different urotypes (UTs) have been
described in humans, all of which are found in both men and
women, except for UT 7, which occurs in healthy women
only and has a relative abundance of L. crispatus [5]. Con-
sequently, probiotics containing L. crispatus strains might be
useful for prophylactic purposes in the uro-gynaecological
context. Unfortunately, very few L. crispatus strains have
been described in the scientic literature, and fewer than 30
genomes of this species have been sequenced and depos-
ited in bacterial collections. However, one of these strains
has recently been investigated carefully for possible use in
gynaecological conditions.
Genetic features of L. crispatus M247
L. crispatus M247 (IDA: LMG-P-23257) was isolated in 1989
from the faeces of a healthy baby. Its genome is 2,112,063
bp (2.1 Mbp) long and it has 2,187 coding and 55 riboso-
mal genes [6]. The most abundant genes are those involved
in carbohydrate metabolism (236 genes), followed by pro-
1Scientic Department, Velleja Research, Milan, Italy
2D.I.S.B., Urbino, Italy
3Advanced Analytical Technologies, Piacenza, Italy
Keywords Vaginal microbiota, Lactobacilli, Preterm birth, Vaginitis, Vaginosis, Community State Type
Nutrafoods (2018) 17:169-172
tein metabolism (203), DNA (107) and RNA (84) genes, and
genes involved in cell wall and capsule biosynthesis (83).
The strain appears to be safe as it does not demonstrate viru-
lence factors and is unlikely to contain plasmids. From a
phenotypic perspective, data conrm that the strain is re-
sponsive to all tested antibiotics, according to EFSA guid-
ance. Analysis performed using CRISPRFinder identied
four possible CRISPR (clustered regularly interspaced short
palindromic repeats) in its genome; two of these CRISPR
have been conrmed, while the other two are still under
investigation. Genetic analysis also identied a gene encod-
ing for a Lactobacillus epithelium adesin (LEA). This gene,
previously characterized in L. crispatus ST1, was shown to
play a major role in vaginal epithelium colonization and in
determining competition with Gardnerella vaginalis [7, 8].
Moreover, two bronectin-encoding genes (bronectin type
III domain and bronectin-binding protein A N-terminus
FbpA) were also identied in strain M247. These genes have
been associated with the ability to colonize the vaginal mu-
cosa and also contribute to controlling Gardnerella vaginalis
through a competitive colonization mechanism [8].
The production of bacteriocins is an important feature of ben-
ecial bacteria in helping combat pathogenic bacteria. At
the gene level, a number of bacteriocins have been identi-
ed in strain M247: two helveticins, one penocin, two enter-
olysins and one bacteriocin of the LS2 group. These results
are in agreement with previous analyses of other L. crispatus
genomes and suggest possible exploitation of the strain for
the control of microbial pathogens [8]. Although the pheno-
typic presence of bacteriocins released by M247 has not yet
been demonstrated, antagonism against some uro-pathogenic
strains of Escherichia coli has been observed as well as against
Staphylococcus epidermidis but not against Enterococcus fae-
cium [6]. As is well-known, Lactobacillus strains can produce
different types of exopolysaccharides (EPS), facilitating bac-
terial adhesion and protecting against antibiotics and drying
[9]. As regards strain M247, 18 genes encoding for enzymes
of the glycosyltransferase group, which are involved in the
biosynthesis of EPS, were identied [6].
This suggests that, in suitable environmental
conditions, strain M247 can produce EPS.
Probiotic and colonizing
properties of M247
Regarding its probiotic features, strain
M247 survives in gastric juice (pH 3; 90
minutes), bile salts (0.5%; 48 hours) and
when subject to the pancreatin tolerance test (about 0.2%;
3 hours) losing 3 logs in acid conditions, 1 or 2 logs in bile
medium (depending on the bile salts used) and increasing
by 1 log in experimental pancreatic juice. Regarding its
adhesion properties, strain M247 adhered, eight times bet-
ter, to human ileostomy glycoproteins and to Caco2 cells
(model of colonic epithelium) than did its spontaneous iso-
genic non-aggregating mutant (called MU5). Administered
in double-blind conditions to healthy volunteers at a dose
of at least 10 billion live cells for 8 consecutive days, the
strain was demonstrated to be a strong colonizer as it was
found in all faecal samples and in most biopsy samples [10].
These results (Table 1) were not observed when the strain
was administered in poor/non-viable conditions (as shown
by trial 2 in Table 1).
Biosynthesis and role of hydrogen peroxide
produced by strain M247
Hydrogen peroxide production by vaginal lactobacilli was
long thought to be an important defence mechanism against
vaginal colonization by undesirable microorganisms. Its pro-
duction and release by L. jensenii strains is especially strong,
while varying among L. crispatus and L. gasseri strains [11].
M247 has been shown experimentally to be a very good
producer of hydrogen peroxide (Table 2). However, recent
papers [12, 13] have shown that, in contrast to lactic acid,
hydrogen peroxide does not have a very strong killing ability,
which has prompted some authors to consider as implausi-
ble the in vivo role of hydrogen peroxide as an antimicrobial
agent produced by vaginal microbiota [14]. It has been re-
cently observed that L. crispatus M247 instead uses hydrogen
peroxide as a signal transducing molecule to induce PPAR-γ
activation in intestinal epithelial cells, directly modulating
epithelial cell responsiveness to inammatory stimuli [15].
The same mechanism increases the amount of TLR-2 while
reducing the amount of TLR-4 [16], thereby improving the
strength of the tight junctions and reducing the ability of
Table 1 - Results of human colonization trials with Lactobacillus crispatus strain M247
and its non-aggregating isogenic mutant MU5
Trial Number of subjects
in the tested group
Number of subjects with L. crispatus
identied on day 8
Faeces Biopsies
M247 MU5 M247 MU5 M247 MU5
1 5 5 5 ND 2 ND
2 3 3 ND ND ND ND
3 2 01 1
ND not detected
Nutrafoods (2018) 17:169-172
Gram-negative LPS-endowed
bacteria to determine TNF-α-
mediated inammatory pro-
cesses (Fig. 1).
This anti-inammatory ac-
tion exerted by M247 after
colonization is evident when
tested in a model of murine
experimental colitis [17]
where the strain can reduce
most signs and symptoms of
the condition (Fig. 2).
Vaginal presence af-
ter oral use of M247
As lactobacillus can be trans-
ferred from the gut to the va-
gina, it is thought this is also
possible following probiotic
administration of a mixture
of lactobacilli. The literature
indicates that oral adminis-
tration of lactobacillus is re-
quired for 4–5 weeks before
any effects on the vagina are
seen [18]. We therefore eval-
uated the ability of M247 to colonize the va-
gina after oral administration of 10 billion live
cells daily for 2 weeks to healthy women. Pre-
liminary results from PCR analysis showed that
M247 was present in the colon of 70% of sub-
jects and in the vagina of 40% of subjects (Fig.
3). Other tests have further demonstrated that
strain M247 can be recovered alive from the
vagina of almost 100% of subjects following
topical application for only 3 days by vaginal
douche, suggesting M247 can colonize the va-
Intestinal epithelial cells
+ L. crispatus M247
+ L. crispatus MU5
TLR-2 TLR-4 H2O2
Figure 1 - Lactobacillus crispatus strain M247 releases hydrogen peroxide which induces up-modula-
tion of PPAR-γ in intestinal epithelial cells. As a direct consequence, TLR-2 increases, TLR-4 decreases
and tight junctions become more structured. These events together produce an anti-inammatory
Figure 2 - Benecial effect of Lactobacillus crispatus strain M247 on a murine experimental model of
colitis induced by dextran sodium sulfate (DSS).
Reproduced from Gastroenterology 2008;135(4):1216-1227
Figure 3 - Lactobacillus crispatus strain M247 in the vagina after oral adminis-
tration. Green arrows indicate positive sampling, while red arrows indicate the
negative sampling
Table 2 - Lactobacillus crispatus strain M247 released hydrogen peroxide in nal concentrations higher than those measured for other pro-
ducing strains
Incubation time (hours) H2O2 released (mg/103 CFUs)
L. johnsonii
ATCC 33200T
L. gasseri
DSM 20243T
L. fermentum
DSM 20052T
L. plantarum
ATCC 21028
L. paracasei
L. crispatus
M247 L. crispatus
122.10 15.67 0.18 0 0.00 32.02 47.44
224.50 17.04 0.17 0.06 0.03 32.15 35.33
430.16 22.56 0.15 0.06 0.06 37.39 8.33
6 35.22 25.98 0.15 0.07 0.06 42.44 9.27
837.40 28.55 0.13 0.13 0.08 41.69 7.98
Nutrafoods (2018) 17:169-172
gina very quickly using this method. A pilot trial evaluating
vaginal colonization after 3 and 4 weeks of oral use of M247
is currently ongoing and results will soon be available.
L. crispatus M247 is one of the few L. crispatus strains which
have been properly documented for human use. A good
colonizer of the human gut, where it exerts strong anti-in-
ammatory effects, and the human vagina, where it combats
dysbiosis, the strain could be used orally and/or topically to
restore the normal vaginal microbiota. The current literature
suggests strain M247 can be used in:
1) women with CST IV;
2) women with CST II, CST III or CST V and recurrent in-
fections, a risk of preterm birth or sine causa infertility;
3) women with persistent HPV;
4) women with an HIV-positive partner or engaging in
risky sexual behaviour;
5) women undergoing the menopause and/or with initial
signs of vaginal atrophy;
6) women in whom other therapies against recurrent uri-
nary infections have failed.
Conict of interest
Francesco Di Pierro is the owner of Velleja Research.
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... For Lactobacillus crispatus M247, they have been characterized as having 18 genes encoding enzymes involved in the production of EPS [16]. This set of characteristics and the data published in the literature indicate that Lactobacillus crispatus M247 has suitable characteristics for the colonization of vaginal tissue following oral or local administration [19,20] if the appropriate environmental conditions occur. Furthermore, in line with what was observed with the gene analysis of other Lactobacilli, suitable characteristics for the production of bacteriocins were also found for the M247 strain, including two elveticins, a penocin, two enterolysins, and a bacteriocin of the LS2 group [21]. ...
... Although this characteristic has not yet been demonstrated at the phenotypic level, antagonistic action against certain strains of uropathogenic Staphylococcus epidermidis and Escherichia coli was documented [16]. In light of the current knowledge, the strain is considered to be safe, it does not demonstrate elements of virulence, and a plasmid presence is unlikely, showing itself to be safe from a phenotypic point of view according to the criteria established by EFSA [20,22]. The combination of these characteristics render Lactobacillus crispatus M247 a candidate strain for microbial control at the intestinal level and, above all, at the urogynecological level. ...
... The scientific literature has documented the ability of transferring Lactobacilli from the intestinal to the vaginal environment following continuous administration for 4-5 weeks [21]. An evaluation of the intestinal and vaginal colonization capacity of Lactobacillus crispatus M247, following the oral administration of 10 billion live cells per day for 2 weeks in healthy women, documented the presence of the M247 strain in the colon of 70% of the subjects, and in the vagina of 40% of the subjects [20]. Further evaluations describe that, following topical application for 3 days by vaginal douche, the M247 strain was recovered alive from almost 100% of the subjects, suggesting a rapid colonization capacity with this method [20]. ...
Full-text available
The altered patterns of a microbial population colonizing an organ are increasingly recognized as a relevant item in human disease pathogenesis. The female urogenital tract is no exception, as some vaginal microbiota patterns, named community state types (CSTs), and urinary tract microbiota patterns, named urotypes (UTs), have been linked to viral, inflammatory, and gestational diseases. Treating these conditions is an issue, as antibiotic therapies alone are not always effective. Lactobacillus crispatus M247 is a strain with good intestinal and vaginal adhesion capability, combined with local antibacterial and anti-inflammatory properties; this strain also has proven nontransferable resistance to antibiotics commonly used in female genital tract infections, such as metronidazole. Lactobacillus crispatus M247 could, therefore, be considered as a potential add-on therapy to antibiotics in vaginal tract infections, with the aim to restore a favorable microbiota pattern.
... A trial on HPV-infected women and a very recent clinical case report hasin fact demonstrated the capability of the strain M247 to effectively restore a CST I [47,48]. Moreover, a study performed using a probe to specifically detect the strain M247 has shown that following oral treatment, the strain M247 was indeed found first in the gut and then the vaginal environment of a treated volunteer [50]. ...
Full-text available
The aim of our study was to retrospectively evaluate whether the oral administration of L. crispatus (M247) could increase pregnancy and live birth rates in women undergoing assisted reproductive technology procedures. Enrolled women (N = 160) were divided into two groups: treated (N = 80) or untreated (N = 80) with the probiotic strain. The odds ratio (OR) for a treated woman to have a clinical pregnancy (CP) was 1.56. In women aged 30-40 years, M247 increased the probability of a CP in correlation with the progressive rise in BMI, reaching 47% (35% in controls) with a BMI of 35 (OR: 2.00). The CAID statistics showed that in a woman of the blastocyst subgroup, below 43 years, with a BMI over 18.6, treatment with M247 increased the chance of a CP from 28.4% to 44.5% (OR: 2.08; p < 0.05). Considering live births, the rate of the probiotic group was 12.5% versus 7.5% (OR: 1.76). Considering only the blastocyst subgroup, the treatment increased the number of live births by 200% (OR: 3.64; p = 0.05). As confirmed also by statistical indices NNT, NNH, and LHH, the use of M247 demonstrated a risk-benefit ratio to the full advantage of the benefits.
... A trial on HPV infected women and a very recent clinical case report have in fact demonstrated the capability of the strain M247 to effectively restore a CST I [40,41]. Moreover, a study performed using a probe to 13 specifically detect the strain M247, has shown that following oral treatment, the strain M247 is indeed found first in the gut and then the vaginal environment of treated volunteers [43]. ...
Full-text available
Aim of our study was to retrospectively evaluate whether the oral administration of L. crispatus (M247) could increase pregnancy and live birth rates in women undergoing assisted reproductive technology procedures. Enrolled women (N=160) were divided into two groups: treated (N=80) or untreated (N=80) with the probiotic strain. The odds ratio for a treated woman to get a positive pregnancy test (PPT) was 1.56. In women aged 30–40 years, M247 increased the probability of a PPT in correlation with the progressive rise in BMI, reaching 47% (35% in controls) with a BMI of 35 (odds ratio: 2.00). The CAID statistics showed that in a woman of the blastocyst subgroup, below 43 years, with a BMI over 18.6, treatment with M247 increased the chance of a PPT from 28.4% to 44.5% (odds ratio: 2.08; p<0.05). Considering live births, rate of probiotic group was 12.5% versus 7.5% (odds ratio=1.76). Considering only the blastocyst subgroup, the treatment increased the number of live births by 200% (odds ratio: 3.64; p=0.05). As confirmed also by statistical indices like NNT, NNH and LHH, the use of M247 demonstrated a risk-benefit ratio to the full advantage of the benefits.
... In vivo murine models it was also evaluated for its ability to colonize the colonic mucosa upmodulating Toll-like receptors 2 and 4 [29] and for its protective effect exerted during experimentally induced colitis [30]. The strain was also genetically described for its aggregation features [31] and it was also investigated for its intestinal mucus-binding properties and in terms of ability to restore the normal vaginal microbiota [32][33][34]. ...
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In the last decade, many authors have reported low viability for probiotic products. Investigators commonly find they are not meeting claimed active counts and/or incorrect species and/or strains have been identified. We have therefore decided to verify viability, the real dose and species correspondence in nine probiotic products (seven nutritional supplements and two medical devices) collected from the Italian and French markets claiming to contain at least one strain of L. crispatus among the different species/strain included in the formulation. In fact, the medical relevance of L. crispatus strains has recently grown., as evaluating the possible dominance clusters typical of the vaginal microbiota, the Community State Type I, the one dominated by L. crispatus, appears to be "protective" in terms of infections, fertility and gestational duration of pregnancy. The results obtained demonstrate the generally poor quality of probiotics. Out of nine products, only two definitely contained viable Lactobacilluscrispatus cells with a daily dose of at least 1 × 109 CFU/g and with an acceptable correspondence with what is declared on the label. Among these two, only one was found to be formulated with a strain (M247) that has been scientifically documented.
... Recent studies have also shown its capability to colonize vaginal tissue after local and/or oral administration [9] . considered not dangerous but useless, if not a "no-sense" practice [10] . ...
Full-text available
In an attempt to better characterize the strain M247, a strain of Lactobacillus crispatus isolated in 1989 from the faeces of a healthy newborn baby, we have further investigated its antibiotic resistance profile, evaluating also antibiotics not recommended by the EFSA, and analyzing in addition its viability in the presence of a high concentration of boric acid, a molecule commonly use to eradicate antimycotic-resistant fungi from the vagina. At the same time, we have evaluated the ability of M247 to adhere to vaginal epithelial cells to get a better understanding of its vaginal colonizing properties. According to the obtained results, M247 appears to be insensitive to metronidazole, sulfamethoxazole and boric acid. Such resistance is not transferrable due to the total absence of mobile elements inside its genome. Moreover, the M247 strain has been shown to adhere by more than 70% to vaginal epithelial cells, thereby providing a mechanistic explanation of its vaginal colonizing capabilities observed both after local and oral use.
Background: Vaginal microbiotas can be clustered into five different possible categories (CST I to V), according to their bacterial dominance. In CST I, the dominance of Lactobacillus crispatus seems to correlate with better vaginal health and with a lower incidence of sine causa infertility, preterm delivery, bacterial vaginosis, and viral (including human papillomavirus; HPV) infection. According to the same method of classifying the vaginal microbiome, CST IV (non-Lactobacillusdominated) demonstrates a higher incidence of disorders. Methods: In an open, non-controlled study, we enrolled 35 HPV-positive women who mostly (N=24) demonstrated CST IV status, with the other individuals categorized as having either CST III (N=10) or CST II (N=1) microbiotas. Results: After 90 days of oral treatment with a probiotic (L. crispatus M247) we observed a reduction of approximately 70% in HPV positivity and a significant change in CST status with 94% of women now classified as CST I. Conclusions: Despite the limitations of our study, it is the first demonstration that it is possible to intervene orally with an L. crispatus probiotic to bring about a change in CST status and, in parallel, increased HPV clearance.
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Abstract Background The bacterial community present in the female lower genital tract plays an important role in maternal and neonatal health. Imbalances in this microbiota have been associated with negative reproductive outcomes, such as spontaneous preterm birth (sPTB), but the mechanisms underlying the association between a disturbed microbiota and sPTB remain poorly understood. An intrauterine infection ascending from the vagina is thought to be an important contributor to the onset of preterm labour. Our objective was to characterize the vaginal microbiota of pregnant women who had sPTB (n = 46) and compare to those of pregnant women who delivered at term (n = 170). Vaginal swabs were collected from women at 11–16 weeks of gestational age. Microbiota profiles were created by PCR amplification and pyrosequencing of the cpn60 universal target region. Results Profiles clustered into seven community state types: I (Lactobacillus crispatus dominated), II (Lactobacillus gasseri dominated), III (Lactobacillus iners dominated), IVA (Gardnerella vaginalis subgroup B or mix of species), IVC (G. vaginalis subgroup A dominated), IVD (G. vaginalis subgroup C dominated) and V (Lactobacillus jensenii dominated). The microbiota of women who experienced preterm birth (
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In the cervicovaginal environment, the production of hydrogen peroxide (H2O2) by vaginal Lactobacillus spp. is often mentioned as a critical factor to the in vivo vaginal microbiota antimicrobial properties. We present several lines of evidence that support the implausibility of H2O2 as an "in vivo" contributor to the cervicovaginal milieu antimicrobial properties. An alternative explanation is proposed, supported by previous reports ascribing protective and antimicrobial properties to other factors produced by Lactobacillus spp. capable of generating H2O2. Under this proposal, lactic acid rather than H2O2 plays an important role in the antimicrobial properties of protective vaginal Lactobacillus spp. We hope this commentary will help future research focus on more plausible mechanisms by which vaginal Lactobacillus spp. exert their antimicrobial and beneficial properties, and which have in vivo and translational relevance.
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Background The urinary microbiota is similarly complex as the vaginal and penile microbiota, yet its role as a reservoir for pathogens and for recurrent polymicrobial biofilm diseases like bacterial vaginosis (BV) is not clear. Results Here, we analysed the urinary microbiota of healthy men and women and compared it with that of women during BV and after antibiotic treatment using next-generation sequencing of the 16S rRNA gene V1-V2 regions. Eight different community types, so called urotypes (UT), were identified in healthy humans, all of which were shared between men and women, except UT 7, dominated in relative abundance by Lactobacillus crispatus, which was found in healthy women only. Orally applied metronidazole significantly reduced Shannon diversity and the mean relative abundance of Gardnerella vaginalis, Atopobium vaginae, and Sneathia amnii, while L. iners increased to levels twofold higher than those found in healthy women. Although individual urine microbial profiles strongly responded to the antibiotic, the healthy community could not be restored. The correlation between urinary and vaginal fluid microbiota was generally weak and depending on UT and BV status. It was highest in UT 1 in acute BV (59% of samples), but after metronidazole treatment, only 3 out of 35 women showed a significant correlation between their urinary and vaginal microbiota composition. Conclusions Urethra and bladder thus harbor microbial communities distinct from the vagina. The high abundance of BV related species in the urine of both men and women suggests that urine may act as a reservoir of pathogens and contribute to recurrence. Trial registration, NCT02687789 Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0305-3) contains supplementary material, which is available to authorized users.
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Lactobacillus crispatus is a ubiquitous micro-organism encountered in a wide range of host-associated habitats. It can be recovered from the gastrointestinal tract of animals and it is a common constituent of the vaginal microbiota of humans. Moreover, L. crispatus can contribute to the urogenital health of the host through competitive exclusion and the production of antimicrobial agents. In order to investigate the genetic diversity of this important urogenital species, we performed a comparative genomic analysis of L. crispatus. Utilizing the completed genome sequence of a strain ST1 and the draft genome sequences of nine other L. crispatus isolates, we defined the scale and scope of the pan- and core genomic potential of L. crispatus. Our comparative analysis identified 1,224 and 2,705 ortholog groups present in all or only some of the ten trains, respectively. Based on mathematical modeling, sequencing of additional L. crispatus isolates would result in the identification of new genes and functions, whereas the conserved core of the ten strains was a good representation of the final L. crispatus core genome, estimated to level at about 1,116 ortholog groups. Importantly, the current core was observed to encode bacterial components potentially promoting urogenital health. Using antibody fragments specific for one of the conserved L. crispatus adhesins, we demonstrated that the L. crispatus core proteins have a potential to reduce the ability of Gardnerella. vaginalis to adhere to epithelial cells. These findings thereby suggest that L. crispatus core proteins could protect the vagina from G. vaginalis and bacterial vaginosis. Our pan-genome analysis provides insights into the intraspecific genome variability and the collective molecular mechanisms of the species L. crispatus. Using this approach, we described the differences and similarities between the genomes and identified features likely to be important for urogenital health. Notably, the conserved genetic backbone of L. crispatus accounted for close to 60% of the ortholog groups of an average L. crispatus strain and included factors for the competitive exclusion of G. vaginalis, providing an explanation on how this urogenital species could improve vaginal health.
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Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H2O2, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H2O2 concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H2O2 production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H2O2, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection.
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Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.
Decades of research have shown that the lactobacilli inhabiting the human vagina are the first line of defense in the female urogenital and reproductive tracts. In healthy cervicovaginal microbiota, Lactobacillus crispatus is prevalent and beneficial with production of copious amounts of lactic acid potent broad spectrum bactericide virucide and immunomodulator. Future and preventic approaches may need to include probiotics, prebiotics also have the potential to optimize and restore the vaginal ecosystem.
The vaginal microbiota helps protect the female genital tract from disease. We sought to describe the composition of the vaginal microbiota in premenopausal, perimenopausal, and postmenopausal women and to explore the association between the microbiota and vulvovaginal atrophy (VVA). Eighty-seven women (aged 35-60 y) were classified as premenopausal (n = 30), perimenopausal (n = 29), or postmenopausal (n = 28) according to Stages of Reproductive Aging Workshop guidelines. Midvaginal bacterial community composition was characterized by 16S ribosomal RNA gene analysis. Bacterial communities clustered into six community state types (CSTs), of which four were dominated by Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, or Lactobacillus jensenii, and two (CST IV-A and CST IV-B) had low relative abundance of Lactobacillus. CST IV-A was characterized by Streptococcus and Prevotella, whereas CST IV-B was characterized by Atopobium. There were significant associations between menopause stage and CST (P = 0.004) and between VVA and CST (P = 0.002). Perimenopausal women were more likely to be classified as CST IV-A or L. gasseri CST, whereas postmenopausal women were often classified as CST IV-A. CSTs dominated by L. crispatus and L. iners were more prevalent in premenopausal women. Nineteen participants had signs of mild or moderate VVA. Compared with women with no VVA, the vaginal microbiota of women with mild or moderate atrophy had 25-fold greater odds of being classified as CST IV-A versus L. crispatus CST (adjusted odds ratio, 25.89; 95% credible interval, 2.98-406.79). A distinct bacterial community state (CST IV-A) with a low relative abundance of Lactobacillus is associated with VVA. Future studies recruiting a larger number of women are needed to replicate the findings. This study provides an impetus for future longitudinal studies designed to manage, modulate, and restore vaginal microbiota homeostasis, which would provide stronger evidence for a causal relationship with VVA and ultimately improve the treatment and prevention of atrophic vaginitis in menopause.
Objectives: The vaginal microflora is composed of many bacterial species and plays a major role in maintaining the balance of this complex environment. This study was conducted in order to assess the degree and persistence of the colonization of vaginal epithelium by strains from an orally administered mixture of lactobacilli, containing Lactobacillus fermentum 57A, Lactobacillus plantarum 57B and Lactobacillus gasseri 57C. We also monitored its effects on parameters of vaginal health, especially total lactobacilli counts, vaginal pH and Nugent score. Study design: The patient group in this open study consisted of clinically healthy women with intermediate vaginal flora. Altogether 37 women were included in the study; 25 finished the full cycle consisting of 8 visits during 70 days. Lactobacillus mixture was administered as 1×10(8) c.f.u. once a day for 60 days. Lactobacillus isolates collected from vaginal and rectal samples from studied women during all visits were typed using molecular methods (PFGE for L. fermentum and L. gasseri and MLST for L. plantarum). Total lactobacilli counts, vaginal pH and Nugent score were also determined during the visits. Results: We confirmed that the ingested strains were able to reach and colonize both sites within the third and eighth visits, i.e. between the 20th and 70th days of the study. Maximal colonization was recorded between the fifth and seventh visits (31st-60th days). Moreover, ingestion of the Lactobacillus mixture was related to normalization of vaginal parameters (within 28-60 days after the initiation of the treatment). This was demonstrated by a decrease of vaginal pH and Nugent score together with an increase of total numbers of lactobacilli in the vagina and rectum. No adverse events were noted during the course of the study. Conclusions: Oral application of the combination of the three probiotic strains derived from vaginal microbiota of healthy woman with high adherence abilities to both vaginal and colonic epithelium in vitro shows that both individual strains and their mixture can colonize vagina for some weeks, the effect of which is correlated with significant improvement of such parameters like pH and Nugent score values and total numbers of vaginal lactobacilli. This indicates that the mixture may be a good candidate for the planned double-blind, placebo-controlled randomized studies involving larger numbers of women.
Lactobacilli have the ability to produce different kinds of exopolysaccharides (EPS) exhibiting a wide diversity of structures. EPS are classified, according to their composition into homopolysaccharides and heteropolysaccharides. One of their most described applications is their utilization as texturing agents naturally synthesized in the fermented food products. Nowadays, in regard to demand of modern consumers focusing towards safe and healthy food without additives, new perspectives of development appear for these biopolymers. The GRAS (Generally Recognized As Safe) and probiotic status of some lactobacilli give to them more preference for consumable EPS production. The main drawbacks limiting their industrial expansion are their low yields of production and the validation of their healthy allegations. Moreover, the texturing role of these exopolysaccharides, notably in dairy products, is actually a controversial issue. This review focuses on the novel ways of EPS production employing Lactobacillus spp. and their potential as nutraceuticals.