Lactobacillus crispatus M247: a possible tool to counteract CST IV

Full text

Nutrafoods (2018) 17:169-172
DOI 10.17470/NF-018-0001-4
169
www.ceceditore.com
SHORT REPORT
Correspondence to: Francesco Di Pierro - f.dipierro@vellejaresearch.com
Francesco Di Pierro1, Alexander Bertuccioli2, Daniela Cattivelli3, Sara Soldi3, Marina Elli3
Lactobacillus crispatus M247:
a possible tool to counteract CST IV
Abstract
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.
Introduction
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

170 www.ceceditore.com
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

www.ceceditore.com
Nutrafoods (2018) 17:169-172
171
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-
GRAM- TJ
M247
Intestinal epithelial cells
DSS DSS
+ L. crispatus M247
DSS
+ L. crispatus MU5
???
TLR-2 TLR-4 H2O2
PPAR-γTNF-α
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
response
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
NCDO 151T
L. crispatus
M247 L. crispatus
MU5
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

172 www.ceceditore.com
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.
Conclusions
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.
REFERENCES
1. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL,
Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC,
Ault K, Peralta L, Forney LJ (2011) Vaginal microbiome of reproduc-
tive-age women. Proc Natl Acad Sci U S A 108(Suppl 1):4680–4687
2. Freitas AC, Bocking A, Hill JE, Money DM; VOGUE Research Group
(2018) Increased richness and diversity of the vaginal microbiota
and spontaneous preterm birth. Microbiome 6(1):117
3. Brotman RM, Shardell MD, Gajer P, Fadrosh D, Chang K, Silver MI,
Viscidi RP, Burke AE, Ravel J, Gravitt PE (2018) Association between
the vaginal microbiota, menopause status, and signs of vulvovaginal
atrophy. Menopause 25(11):1321–1330
4. Lepargneur JP (2016) Lactobacillus crispatus as biomarker of the
healthy vaginal tract. Ann Biol Clin (Paris) 74(4):421–427
5. Gottschick C, Deng ZL, Vital M, Masur C, Abels C, Pieper DH,
Wagner-Döbler I (2017) The urinary microbiota of men and women
and its changes in women during bacterial vaginosis and antibiotic
treatment. Microbiome 5(1):99
6. AAT-Advanced Analytical Technologies (2017) Lactobacillus crispa-
tus LMG-P23257 in the prevention of recurrent urinary tract infec-
tions (RUTI). Internal document
7. Edelman SM, Lehti TA, Kainulainen V, Antikainen J, Kylväjä R, Bau-
mann M, Westerlund-Wikström B, Korhonen TK (2012) Identica-
tion of a high-molecular-mass Lactobacillus epithelium adhesin
(LEA) of Lactobacillus crispatus ST1 that binds to stratied squamous
epithelium. Microbiology 158(Pt 7):1713–1722
8. Ojala T, Kankainen M, Castro J, Cerca N, Edelman S, Westerlund-
Wikström B, Paulin L, Holm L, Auvinen P (2014) Comparative genom-
ics of Lactobacillus crispatus suggests novel mechanisms for the com-
petitive exclusion of Gardnerella vaginalis. BMC Genomics 15:1070
9. Badel S, Bernardi T, Michaud P (2011) New perspectives for Lacto-
bacilli exopolysaccharides. Biotechnol Adv 29(1):54–66
10. Cesena C, Morelli L, Alander M, Siljander T, Tuomola E, Salminen
S, Mattila-Sandholm T, Vilpponen-Salmela T, von Wright A (2001)
Lactobacillus crispatus and its nonaggregating mutant in human
colonization trials. J Dairy Sci 84:1001–1010
11. Martín R, Suárez JE (2010) Biosynthesis and degradation of H2O2 by
vaginal lactobacilli. Appl Environ Microbiol 76(2):400–405
12. Strus M, Brzychczy-Włoch M, Kochan P, Heczko P (2004) Hydro-
gen peroxide produced by Lactobacillus species as a regulatory mol-
ecule for vaginal microora. Med Dosw Mikrobiol 56(1):67–77
13. Gong Z, Luna Y, Yu P, Fan H (2014) Lactobacilli inactivate Chla-
mydia trachomatis through lactic acid but not H2O2. PLoS One
9(9):e107758
14. Tachedjian G, O’Hanlon DE, Ravel J (2018) The implausible “in
vivo” role of hydrogen peroxide as an antimicrobial factor produced
by vaginal microbiota. Microbiome 6(1):29
15. Voltan S, Martines D, Elli M, Brun P, Longo S, Porzionato A, Macchi
V, D’Incà R, Scarpa M, Palù G, Sturniolo GC, Morelli L, Castagliu-
olo I (2008) Lactobacillus crispatus M247-derived H2O2 acts as a
signal transducing molecule activating peroxisome proliferator ac-
tivated receptor-gamma in the intestinal mucosa. Gastroenterology
135(4):1216–1227
16. Voltan S, Castagliuolo I, Elli M, Longo S, Brun P, D’Incà R, Porzi-
onato A, Macchi V, Palù G, Sturniolo GC, Morelli L, Martines D
(2007) Aggregating phenotype in Lactobacillus crispatus determines
intestinal colonization and TLR2 and TLR4 modulation in murine
colonic mucosa. Clin Vaccine Immunol 14(9):1138–1148
17. Castagliuolo I, Galeazzi F, Ferrari S, Elli M, Brun P, Cavaggioni A,
Tormen D, Sturniolo GC, Morelli L, Palù G (2005) Benecial ef-
fect of auto-aggregating Lactobacillus crispatus on experimentally
induced colitis in mice. FEMS Immunol Med Microbiol 43(2):197–
204
18. Strus M, Chmielarczyk A, Kochan P, Adamski P, Chełmicki Z,
Chełmicki A, Pałucha A, Heczko PB (2012) Studies on the effects
of probiotic Lactobacillus mixture given orally on vaginal and rec-
tal colonization and on parameters of vaginal health in women
with intermediate vaginal ora. Eur J Obstet Gynecol Reprod Biol
163(2):210–215