U.S. patent application number 11/738102 was filed with the patent office on 2008-01-24 for lactic acid bacteria strains useful against urogenital pathogens and compositions containing same.
Invention is credited to Dominique Brassart, Federico Graf, Philipp Grob.
Application Number | 20080019954 11/738102 |
Document ID | / |
Family ID | 35534903 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080019954 |
Kind Code |
A1 |
Graf; Federico ; et
al. |
January 24, 2008 |
LACTIC ACID BACTERIA STRAINS USEFUL AGAINST UROGENITAL PATHOGENS
AND COMPOSITIONS CONTAINING SAME
Abstract
The invention relates to a method for preventing or treating
urogenital infections in females, which comprises administering a
pharmaceutical preparation comprising, in combination with a
suitable galenical carrier, a therapeutically effective amount of
at least one lactic acid bacteria strain of the genus L.
acidophilus, L. crispatus, L. gasseri, L. helveticus and L.
jensenii selected for their ability to kill urogenital and/or
gastrointestinal pathogens and their ability to inhibit
internalization of urogenital and/or gastrointestinal pathogens
within vaginal epithelial cells, as well as for their ability to
modulate a humoral and/or cellular immune response at the vaginal
and/or gastrointestinal level, in particular to inhibit the
inflammatory syndrome as well as the infectious syndrome.
Inventors: |
Graf; Federico; (Zurich,
CH) ; Grob; Philipp; (Zurich, CH) ; Brassart;
Dominique; (Chavannes/Renens, CH) |
Correspondence
Address: |
WINSTON & STRAWN LLP;PATENT DEPARTMENT
1700 K STREET, N.W.
WASHINGTON
DC
20006
US
|
Family ID: |
35534903 |
Appl. No.: |
11/738102 |
Filed: |
April 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP05/11150 |
Oct 17, 2005 |
|
|
|
11738102 |
Apr 20, 2007 |
|
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Current U.S.
Class: |
424/93.45 ;
435/252.9 |
Current CPC
Class: |
A61K 9/48 20130101; A61P
15/02 20180101; A61K 2035/115 20130101; A61K 35/747 20130101; A23L
33/135 20160801; A61K 9/0053 20130101; A61P 13/10 20180101; A61K
9/0034 20130101; A23L 2/382 20130101; A61K 9/02 20130101; A61P
15/00 20180101; A61P 29/00 20180101; A61P 31/00 20180101; A61P
37/04 20180101; Y02A 50/473 20180101; A61P 31/10 20180101; A61P
13/02 20180101; A23V 2002/00 20130101; A61P 1/00 20180101; C12R
1/225 20130101; C12R 1/23 20130101; A61P 37/02 20180101; Y02A 50/30
20180101; A61P 31/04 20180101; A61P 13/00 20180101; A61K 35/747
20130101; A61K 2300/00 20130101; A23V 2002/00 20130101; A23V
2200/32 20130101; A23V 2200/3204 20130101 |
Class at
Publication: |
424/093.45 ;
435/252.9 |
International
Class: |
A01N 63/00 20060101
A01N063/00; A01P 15/00 20060101 A01P015/00; C12N 1/20 20060101
C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 22, 2004 |
EP |
PCT/EP04/11980 |
Claims
1. A method for treating urogenital infections in females which
comprises administering to a female in need of such treatment a
pharmaceutical preparation comprising a therapeutically effective
amount of at least one lactic acid bacteria strain selected from
the group consisting of L. crispatus KS 116.1 (CNCM 1-3483), L.
crispatus KS 119.4 (CNCM 1-3484), L. crispatus 127.1 (CNCM 1-3486),
L. gasseri KS 114.1 (CNCM 1-3482), L. gasseri KS 120.1 (CNCM
1-3218), L. gasseri KS 123.1 (CNCM 1-3485), L. gasseri KS 124.3
(CNCM 1-3220), L. helveticus KS 300 (CNCM 1-3360), L. jensenii KS
119.1 (CNCM 1-3217) and L. jensenii KS 121.1 (CNCM 1-3219) in
combination with a pharmaceutically acceptable galenical
carrier.
2. The method of claim 1 wherein the galenical carrier is designed
for local administration.
3. The method of claim 1 wherein the galenical carrier is designed
for oral administration.
4. A method for inhibiting adhesion, colonization and/or growth of
pathogeris in the urogenital tract of females which comprises
administering to a female in need of such treatment a
pharmaceutical preparation comprising a therapeutically effective
amount of at least one lactic acid bacteria strain selected from
the group consisting of L. crispatus KS 116.1 (CNCM 1-3483), L.
crispatus KS 119.4 (CNCM 1-3484), L. crispatus 127.1 (CNCM 1-3486),
L. gasseri KS 114.1 (CNCM 1-3482), L. gasseri KS 120.1 (CNCM
1-3218), L. gasseri KS 123.1 (NCM 1-3485), L. gasseri KS 124.3 (NCM
1-3220), L. helveticus KS 300 (CNCM 1-3360), L. jensenii KS 119.1
(CNCM 1-3217) and L. jensenii KS 121.1 (CNCM 1-3219) in combination
with a pharmaceutically acceptable galenical carrier.
5. The method of claim 4 wherein the galenical carrier is designed
for local administration.
6. The method of claim 4 wherein the galenical carrier is designed
for oral administration.
7. Lactic acid bacteria strains selected from-the group consisting
of L. crispatus KS 116.1 (CNCM 1-3483), L. crispatus KS 119.4 (CNCM
1-3484), L. crispatus 127.1 (CNCM 1-3486), L. gasseri KS 114.1
(CNCM 1-3482), L. gasseri KS 120.1 (CNCM 1-3218), L. gasseri KS
123.1 (CNCM 1-3485), L. gasseri KS 124.3 (CNCM 1-3220), L.
helveticus KS 300 (CNCM 1-3360), L. jensenii KS 119.1 (CNCM 1-3217)
and L. jensenii KS 121. 1 (CNCM 1-3219).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of international
application PCT/EP2005/011150 filed October 17, 2005, the entire
content of which is expressly incorporated herein.
FIELD OF THE INVENTION
[0002] This invention refers to the treatment of infectious
troubles caused by various pathogens in humans, more specifically
to the prevention and/or the treatment of urogenital infections in
females.
BACKGROUND OF THE INVENTION
[0003] Several experimental and clinical studies have assessed
already the potential of certain lactobacilli in the prevention or
treatment of certain genito-urinary tract infections such as
bacterial vaginitis, vaginosis, or urinary tract infections and
relevant therapy is applied for many decades already.
[0004] Urogenital infections remain a common problem, particularly
in the female population. Bacterial adherence to the urogenital
epithelium is recognized as an important mechanism in the
initiation and pathogenesis of urinary tract infections (UTI) and,
in particular, of vaginal infections. The urogenital pathogens
originate predominantly in the intestinal tract and initially
colonize the per-urethral region and ascend into the bladder,
resulting in symptomatic or asymptomatic bacterial uria.
Alternatively, these bacteria invade and then colonize the vagina
causing there various types of symptomatic as well as asymptomatic
vaginal infections. Thereafter, depending on host factors and
bacterial virulence factors, the organisms may further ascend and
give rise to pyelonephritis, respectively ascending infections of
the genital tract in women. Urogenital pathogens express virulence
characteristics that enable them to resist the normally efficient
host defense mechanisms.
[0005] The use of bacteria of the autochthonous flora, such as
lactobacilli, to exclude urogenital pathogens from colonizing the
urogenital tract is an established concept studied rather
extensively since years ago (see e.g. Cadieux et al.--Lactobacillus
strains and vaginal ecology--Jama. 287:1940-41/2002; Butler B C,
Beakley J W. Bacterial flora in vaginitis. Am J Obstet Gynaecol
1960;78:432-40; Eschenbach D A, Davick P R, Williams B L, Klebanoff
S J, Young-Smith K, Critchlow C M et al. Prevalence of hydrogen
peroxide-producing Lactobacillus species in normal women and women
with bacterial vaginosis. J Clin Microbiol 1989;27:251-6; Sobel J
D, Cook R L, Redondo-Lopez V. Lactobacilli: a role in controlling
the vaginal microflora? in Horowitz B J, Mardh P-A, eds. Vaginitis
and Vaginosis, pp 47-53. New York: Wiley-Liss, 1991; Lauritzen C,
Graf F, Mucha M. Restoration of the physiological vaginal
environment with Doederlein bacteria and estriol. Frauenarzt 1984;
4). Lactobacilli, when used in this context, are believed to
contribute to the control of the urogenital micro flora by
different mechanisms such as the production of lactic acid,
antagonistic substances like bacteriocins, and H.sub.2O.sub.2.
[0006] The main goal of therapy with bio therapeutic agents should
be to prevent overgrowth of pathogens until such a time that the
normal microflora can be re-established. In addition, bacterial
therapy is considered as "natural" and without side effects in
contrast with conventional pharmaceutical treatments. Nowadays,
despite of the progresses which have already been made concerning
the intimate knowledge of lactic acid bacteria (LAB) strains, their
properties and their potential use in the pharmaceutical area,
there still remains the need to propose more convenient and more
efficient LAB strains to the medical community.
[0007] The purpose of this invention is to provide new and useful
LAB strains particularly efficient in the treatment of infections
caused by various pathogens, namely infections of the urogenital
tract in females, or in the restoration of a balanced and healthy
urogenital flora after e.g. severe medical treatments like those
performed with antibiotics. This invention provides as well new
methods of prophylactic or therapeutic treatment of such infections
which involve specifically selected LAB strains.
SUMMARY OF THE INVENTION
[0008] The present invention now provides lactic acid bacteria
(LAB) strains of the genus L. acidophilus, L. crispatus, L.
gasseri, L. helveticus and L. jensenii selected for their ability
to kill urogenital and/or gastrointestinal pathogens and their
ability to inhibit internalization of urogenital and/or
gastrointestinal pathogens within vaginal epithelial cells.
[0009] These bacteria are further selected for their ability to
modulate a humoral and/or cellular immune response in the
urogenital and/or gastrointestinal mucous membrane cells and to
inhibit the inflammatory syndrome within said mucous membrane
cells.
[0010] The invention also provides a method for establishing,
maintaining or restoring a healthy urogenital flora in females
throughout life, which comprises administering a pharmaceutical
preparation comprising, in combination with a suitable galenical
carrier, a therapeutically effective amount of at least one of the
selected LAB strains.
[0011] The invention further refers to a method for preventing or
treating urogenital infections in females which comprises
administering a pharmaceutical preparation comprising, in
combination with a suitable galenical carrier, a therapeutically
effective amount of at least one of said LAB strains.
[0012] The invention still further refers to a method for
preventing or inhibiting adhesion, colonization and/or growth of
pathogens in the urogenital tract of females which comprises
administering a pharmaceutical preparation comprising, in
combination with a suitable galenical carrier, a therapeutically
effective amount of at least one of said LAB strains.
[0013] The invention also refers to compositions, in particular
pharmaceutical compositions useful within the above mentioned
frame, which comprise a therapeutically effective amount of at
least one of the selected LAB strain in combination with a suitable
galenical carrier.
[0014] Eventually, this invention refers to the use of one of said
LAB strains in the preparation of compositions, more particularly
pharmaceutical compositions mentioned here above.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The LAB strains of this invention have been first selected
for their ability to adhere to epithelial cells such as cervix HeLa
or Caco-2 which were chosen as models. Cell adhesion is indeed a
prerequisite selection feature as it conditions the capacity of the
said LAB strains to colonize epithelial tissues, e.g. that of the
urogenital tract, and then to compete with, inhibit or exclude
pathogens adhesion from that specific location.
[0016] The said LAB strains have been further selected for their
additional ability to inhibit adhesion, growth and even survival of
pathogens, namely urogenital and gastrointestinal pathogens from
epithelial cells. Gram-negative as well as Gram-positive pathogens,
such as those mentioned here after are representative of those
which are significantly affected by the LAB strains of this
invention in terms of adhesion, growth or pathogenic activity:
Salmonella species, like S. enterica serovar Typhimurium, E. coli,
Streptococcus species, e.g. S. agalactiae, Staphylococcus species
like S. aureus, Gardnerella species, e.g. G. vaginalis, Prevotella
species, e.g. P. bivia; this enumeration is of course not
exhaustive.
[0017] The LAB strains of the present invention have also the
ability to ability to kill and/or to inhibit internalization of
urogenital and/or gastrointestinal pathogens within vaginal
epithelial cells, in particular urogenital or gastrointestinal
gram-negative or gram-positive pathogens such as those referred to
here above. The most efficient LAB within that frame are those
which express high amounts of both hydrogen peroxide
(H.sub.2O.sub.2) and lactic acid in situ where both factors act
synergistically. The latter have indeed proved highly efficient
against anaerobic urogenital pathogens like e.g. Gardnerella and
Prevotella species.
[0018] The LAB strains of this invention, eventually, exhibit a
further important feature, i.e. the ability to modulate the immune
response of immune competent cells, namely cells of the
gastrointestinal or urogenital mucous membrane as well as
epithelial cells, in other words the ability to initiate, stimulate
or reinforce the immune response of said cells when infected by
Gram-negative or Gram-positive pathogens like those mentioned here
above, in particular urogenital pathogenic E. coli. Due to their
specific feature the said LAB strains have consequently the
capacity to inhibit the inflammatory syndrome of the
gastrointestinal or the urogenital mucous membrane, or of the
vaginal epithelial cells when exposed to pathogen
contamination.
[0019] Quite interestingly that specific feature performs the
modulation of the immune response referred to here above using two
distinct routes, i.e. via the induction of either pro- or
anti-inflammatory cytokines like IL10, respectively, IL12, TNF or
IFN. It has been further observed that some LAB strains of this
invention exhibit a high IFN.gamma. induction potential, namely L.
crispatus KS 116.1 and L. gasseri KS 124.3, a feature which favors
the use of same as anti-infectious agents.
[0020] This strain specificity provides consequently to the man
skilled in the art the possibility to select the most appropriate
strains or combination of strains for performing the medical
treatment which is at stake.
[0021] Among the LAB strains which exhibit these properties
preferred species according to this invention are listed here
after: L. jenseii KS 109, L. gasseri KS 114.1, L. crispatus KS
116.1, L. jensenii KS 119.1, L. crispatus KS 119.4, L. gasseri KS
120.1, L. jensenii KS 121.1, L. jensenii KS 122.1, L. gasseri KS
123.1, L. gasseri 124.3, L. gasseri KS 126.2, L. crispatus 127.1,
L. jensenii KS 129.1, L. jensenii KS 130.1, L. helveticus KS 300
and L. acidophilus KS 400. Most of these strains are representative
of the healthy human vaginal micro flora.
[0022] As particularly preferred species, one can further cite the
following strains: [0023] L. gasseri KS 114.1 (CNCM1-3482): gram
positive--catalase negative--oxidase negative--lactic acid
production 10.5 g/l--H.sub.2O.sub.2 production 10 mg/I [0024] API
50 CHI test: positive for GAL, GLU, FRU, MNE, NAG, ESC, SAL, CEL,
MAL, SAC, TRE and GEN [0025] negative for: KON, GLY, ERY, DARA,
LARA, RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM,
MDG, AMY, ARE, LAC, MEL, INU, MLZ, RAF, AMD, GLYG, XLT, TUR, LYX,
TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0026] L. crispatus
KS 116.1 (CNCM 1-3483): gram positive--catalase negative--oxidase
positive--lactic acid production 9.6 g/l--H.sub.2O.sub.2 production
2 mg/l [0027] API 50 CHI test: positive for GAL, FRU, MNE, NAG,
ESC, SAL, MAL and SAC [0028] negative for: KON, GLY, ERY, DARA,
LARA, RIB, DXYL, LXYL, ADO, MDX, GAL, SBE, RHA, DUL, INO, MAN, SOR,
MDM, MDG, AMY, ARB, CEL, LAC, MEL, TRE, INU, MLZ, RAF, AMD, GLYG,
XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG
[0029] L. jensenii KS 119.1 (CNCM 1-3217): gram positive--catalase
negative--oxidase negative--lactic acid production 7.4
g/l--H.sub.2O.sub.2 production 20 mg/l [0030] API 50 CHI test:
positive for GLU, FRU, MNE, NAG, AMY, ESC, SAL, CEL, MAL, MEL, SAC,
GEN and TAG--variable for: RIB [0031] negative for: KON, GLY, ERY,
DARA, LARA, DXYL, LXYL, ADO, MDX, GAL, SBE, RHA, DUL, INO, MAN,
SOR, MDM, MDG, ARB, LAC, TRE, INU, MLZ, RAP, AMD, GLYG, XLT, TUR,
LYX, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0032] L. crispatus
KS 119.4 (CNCM 1-3484): gram positive--catalase negative--oxidase
positive--acid production 10.3 g/l--H.sub.2O.sub.2 production
negative [0033] API 50 CHI test: positive for GAL, GLU, FRU, MNE,
NAG, ESC, MAL, LAC, SAC and AMD [0034] negative for: KON, GLY, ERY,
DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN,
SOR, MDM, MDG, AMY, ARB, SAL, CEL, MEL, TRE, INU, MLZ, RAF, GLYG,
XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG
[0035] L. gasseri KS 120.1 (CNCM 1-3218): gram positive--catalase
negative--oxidase negative--lactic acid production 10.6
g/l--H.sub.2O.sub.2 production 1 mg/l [0036] API 50 CHI test:
positive for: GAL, GLU, FRU, MNE, AMY, ESC, SAL, CEL, MAL, LAC,
SAC, THE and AMD [0037] negative for: KON, GLY, ERY, DARA, LARA,
RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DDL, INO, MAN, SOR, MDM, MDG,
NAG, ARE, MEL, INU, MLZ, RAF, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC,
LFUC, DARL, LARL, GNT, 2KG and 5KG [0038] L. jensenii KS 121.1
(CNCM1-3219): gram positive--catalase negative--oxidase
negative--lactic acid production 10.6 g/l--H.sub.2O.sub.2
production 1 mg/l [0039] API 50 CHI test: positive for: GAL, GLU,
FRU, MNE, AMY, ARE, ESC, SAL, CEL, MAL, SAC and AMD--variable for:
RIB, NAG, LAC, RAF and LFUC [0040] negative for: KON, GLY, ERY,
DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR,
MDM, MDG, MEL, TRE, INU, MLZ, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC,
DARL, LARL, GNT, 2KG and 5KG [0041] L. gasseri KS 123.1 (CNCM
1-3485): gram positive--catalase negative--oxidase negative--lactic
acid production 8.5 g/l--H202 production 10 mg/l [0042] API 50 CHI
test: positive for: GLU, MNE, NAG, ESC, MAL and SAC--variable for
RIB and 5KG [0043] negative for: KON, GLY, ERY, DARA, LARA, DXYL,
LXYL, ADO, MDX, GAL, FRU, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG,
AMY, ARB, SAL, CEL, LAC, MEL, TRE, INU, MLZ, RAF, AMD, GLYG, XLT,
GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT and 2KG [0044] L.
gasseri KS 124.3 (CNCM 1-3220): gram positive--catalase
negative--oxidase negative--lactic acid production 17.0
g/l--H.sub.2O.sub.2 production 20 mg/l [0045] API 50 CHI test:
positive for: GAL, GLU, FRU, MNE, NAG, ESC, SAL, MAL, SAC and
TRE--variable for: RIB, AMD, GEN and 5KG [0046] negative for: KON,
GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO,
MAN, SOR, MDM, MDG, AMY, ARB, CEL, LAC, MEL, INU, MLZ, RAF, GLYG,
XLT, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT and 2KG [0047] L.
crispatus KS 127.1 (CNCM 1-3486): gram positive--catalase
negative--oxidase positive--lactic acid production 16.7
g/l--H.sub.2O.sub.2 production negative [0048] API 50 CHI test:
positive for RIB, GAL, GLU, FRU, MNE, MAN, SOR, NAG, AMY, ESC, SAL,
CEL, MAL LAC, SAC, TRE, MLZ, AMD, GLYG, GEN, TAG and GNT--variable
for GLY and DXYL [0049] negative for: KON, ERY, DARA, LARA, LXYL,
ADO, MDX, SBE, RHA, DUL, INO, MDM, MDG, ARB, MEL, INU, MLZ, RAF,
XLT, TUR, LYX, DFUC, LFUC, DARL, LARL, 2KG and 5KG [0050] L.
helveticus KS 300 (CNCM1-3360): gram positive--lactic acid
production 10.45 g/kg--H.sub.2O.sub.2 production 1 mg/l [0051] API
50 CHI test--positive for: GAL, GLU, FRU, MNE, AMY, ARE, ESC, SAL,
GEL, MAL, LAC, SAC, TRE and AMD [0052] negative for: RIB, MAN, GLY,
SOR, KON, ERY, MLZ, DARA, LARA, LXYL, ADO, MDX, SBE, RHA, DUL, INO,
MDM, MDG, MEL, INU, RAF, TAG, GNT, XLT, TUR, LYX, DFUC, LFUC, DARL,
LARL, 2KG and 5KG.
[0053] These strains have been duly registered at the Pasteur
Institute, Paris (France) in accordance with the Budapest
Treaty.
[0054] According to the present invention, due to their specific
antipathogen activity, the LAB strains can be used advantageously
for preventing or treating urogenital infections in females and for
preventing or inhibiting the colonization and/or growth of
pathogens in the urogenital tract of females as well, i.e. in a
context wherein said LAB strains proved quite efficient.
[0055] Also, the said LAB strains can be used in a quite efficient
way for maintaining or restoring a healthy urogenital flora in
females throughout life, more particularly for establishing,
maintaining or restoring a healthy urogenital flora in females
prior to, during and after pregnancy, or for restoring a balanced
and healthy flora after severe medical treatments like those
performed with antibiotics.
[0056] The corresponding therapeutic or prophylactic treatments are
performed by administering either orally or locally a
pharmaceutical preparation comprising, in combination with a
suitable galenical excipient, support or carrier a therapeutically
effective amount of LAB strains of this invention.
[0057] Pharmaceutical compositions or preparations suitable to
perform the above treatments can further comprise the usual LAB
growth factors. When designed for an oral administration said
compositions can be provided as e.g. food supplements, preferably
in the form of ingestible capsules comprising lyophilized
microorganisms, or in the form of edible milk based powders,
suspensions or emulsions or in the form of edible fermented milk
products, e.g. yogurts.
[0058] Compositions as those mentioned here above can comprise
mixtures of LAB strains of this invention and mixtures of at least
one of these strains together with one or several strains of the
prior art as well. Also, these compositions may contain additional
pharmaceutically active ingredients like chemical compounds
specifically selected.
[0059] The compositions or preparations referred to here above may
contain the selected microorganisms in amounts which can range from
about 106 cfu (colony forming units), preferably from about
10.sup.8 to about 10.sup.11 cfu per g or dose or unit, usually in a
dehydrated form that keeps their viability and their specificity
intact, e.g. in a lyophilized form. The ultimate details of said
compositions as well as their dosage shall depend eventually on the
specific application and the target organ they are intended for,
the age or health status of the patients or persons to be treated,
the nature of the pathogen contamination. It is within current
skills and expertise of the medical community to adjust all the
relevant parameters.
[0060] When compared to prior known reference strains (see examples
below) the LAB strains of the present invention have shown either
similar or higher antipathogens activity depending on the
experimental model which has been selected therefore.
[0061] The following examples illustrate only some of the
embodiments of this invention and so are not intended to constitute
any limitation or restriction thereof.
EXAMPLES
[0062] TABLE-US-00001 Material and Methods Tested Strain Code L.
jensenii 109 KS 109 L. crispatus 116.1 KS 116.1 L. jensenii 119.1
KS 119.1 L. gasseri 120.1 KS 120.1 L. jensenii 121.1 KS 121.1 L.
jensenii 122.1 KS 122.1 L. gasseri 124.3 KS 124.3 L. gasseri 126.2
KS 126.2 L. jensenii 129.1 KS 129.1 L. jensenii 130.1 KS 130.1 L.
helveticus 300 KS 300 L. acidophilus 400 KS 400
[0063] The control adhering lactobacilli strain are the L. casei
rhamnosus strain GG (ATCC Accession no 53103), the L. rhamnosus
strain GR-1 (ATCC Accession no 55826) and the L. fermentum strain
RC-14 (ATCC Accession no 55845).
[0064] All the lactobacilli strains were grown in De Man, Rogosa,
Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for 18 h
at 37.degree. C.
[0065] Bacterial pathogens. Salmonella enterica serovar Typhimurium
strain SL 1344 was a gift of B.A.D. Stocker (Stanford, Calif.).
Bacteria were grown overnight for 18 h at 37.degree. C. in Luria
broth (Difco Laboratories).
[0066] Uropathogenic diffusely-adhering Escherichia coli strains IH
11128 and 7372, and diarrheagenic strain C1845 were gifts from B.
Nowicki (Texas University, Galvestone) and S. Moseley (Seattle
University). Strain 7372 carries the class II papG allele, the hly
gene (haemolysin) and the Dr operon. Strain IH11128 carries the Dr
operon. Strain C1845 carries the Daa operon. All bacterial strains
were maintained on LB plates and prior to infection, bacteria were
grown in LB broth at 37.degree. C. for 18 h.
[0067] Strains of Streptococcus agalactiae DSM 2134, Gardnerella
vaginalis DSM 4944, Prevotella bivia Cl-I (DSM 20514) and Candida
albicans DSM 1386 were from Medinova Ltd, Zurich, Switzerland).
Bacteria were grown overnight at 37.degree. C. in TSA broth (Difco
Laboratories). Streptococcus agalactiae strain was grown overnight
at 37.degree. C. in BHI broth (Difco Laboratories).
[0068] Candida albicans strain was grown overnight at 37.degree. C.
in Sabouraud broth. Gardnerella vaginalis and Prevotella bivia were
grown on Gardnerella agar plates purchased from Bio Merieux
France.
[0069] Bacteria were suspended in buffered sodium chloride-peptone
solution pH 7.0 to about 10.sup.6 colony forming unit (CFU/ml). 500
ul or the prepared suspensions were spread out on the agar plate.
The inoculated plates were dried under sterile laminar air flow
conditions. The agar plates were then incubated under anaerobic
conditions using a sealed anaerobic jar (Becton Dickinson, USA) at
37.degree. C. for 36 h in maximum. Before use, the Gardnerella
vaginalis strain was sub-cultured in BHI supplemented with yeast
extract, maltose and horse serum, under anaerobic conditions using
a sealed anaerobic jar at 37.degree. C. for 36 h in maximum.
[0070] Before use, bacterial cultures were centrifuged at
5.500.times.g for 5 mm at 4.degree. C. The culture medium was
discarded and the bacteria were washed once with phosphate-buffered
saline (PBS) and re suspended in PBS.
[0071] Cell lines and cultures. Human cervical HeLa cells were
cultured at 37.degree. C. in a 5% CO.sub.2-95% air atmosphere in
RPMI 1640 with L-glutamine (Life Technologies) supplemented with
10% heat-inactivated (30 min, 56.degree. C.) foetal calf serum
(FCS; Boehringer, Mannheim, Germany), as previously described.
Cells were used for infection assays before confluence, i.e., after
5 days in culture.
[0072] The human intestinal cell line used was the TC7 clone
(Caco-2/TC7), established from the parental Caco-2 cell line. Cells
were routinely grown in Dulbecco modified Eagle's minimal essential
medium (DMEM) (25 mM glucose) (Invitrogen, Cergy, France),
supplemented with 15% heat-inactivated (30 min, 56.degree. C.)
foetal calf serum (Invitrogene) and 1% non-essential amino acids
(Invitrogene) as previously described. For maintenance purposes,
cells were passaged weekly using 0.02% trypsin in
Ca.sup.2+Mg.sup.2+-free PBS containing 3 mM EDTA. Experiments and
maintenance of the cells were carried out at 37.degree. C. in a 10%
CO.sub.2/90% air atmosphere. The culture medium was changed daily.
Cells were used at post-confluence after 15 days of culture (fully
differentiated cells) for infection assay of S. enterica serovar
Typhimurium.
[0073] Adhesion assays. The adhesion of lactobacilli strains onto
cervix HeLa cells and intestinal Caco-2/TC7 cells was examined
according to the following steps: the cells monolayers were washed
twice with phosphate-buffered saline (PBS). For each adhesion
assay, 0.5 ml of the Lactobacillus suspension (bacteria with spent
broth culture supernatant) was mixed with DMEM (0.5 ml), and then
added to each well of the tissue culture plate (24 wells) which was
then incubated at 37.degree. C. in 10% CO.sub.2/90% air. The final
concentrations of bacteria examined were 1.times.10.sup.8,
2.times.10.sup.8, 1.times.10.sup.9, and 2.times.10.sup.9 bacteria
per ml. After 1 h incubation, the monolayers were washed five times
with sterile PBS, fixed with methanol, stained with Gram stain, and
then examined microscopically under oil immersion. Each adhesion
assay was conducted in duplicate with cells from three successive
passages. For each assay, the number of adherent bacteria was
determined in 20 random microscopic areas (adhesion score: 0 to 5).
Moreover, the level of viable adhering lactobacilli was determined
by quantitative determination of bacteria associated with the
infected cell monolayers. After being infected, cells were washed
twice with sterile PBS and lysed with sterilized H.sub.2O.
Appropriate dilutions were plated on tryptic soy agar (TSA) to
determine the number of viable cell-associated bacteria by
bacterial colony counts.
[0074] Each cell-association assay was conducted at least in
triplicate, with three successive cell passages. Results were
expressed as CPU/ml of cell-associated bacteria.
[0075] Activity against the growth of pathogens. A culture medium
containing MRS (5 ml) and specific pathogen culture medium (5 ml)
was inoculated with 0.1 ml of a cultivated pathogen and 0.1 ml of
cultured Lactobacillus strain. Control was a culture medium
inoculated with 0.1 ml of a cultivated pathogen and 0.1 ml of
non-cultivated MRS adjusted to pH 4.5. At indicated time-points,
aliquots were removed, serially diluted and plated on tryptic soy
agar to determine bacterial colony counts of pathogen. Each assay
was conducted at least in triplicate. Results were expressed as
CFU/ml.
[0076] Activity against the viability of pathogens. Colony count
assays were performed by incubating 10.sup.8 CFU/ml pathogen (0.5
ml) with the lactobacilli culture (10.sup.8 CFU/ml, 0.5 ml) at
37.degree. C. Control was non-cultivated MRS adjusted to pH 4.5.
Initially and at predetermined 15 intervals, aliquots were removed,
serially diluted and plated on tryptic soy agar to determine
bacterial colony counts of pathogen. Each assay was conducted at
least in triplicate. Results were expressed as CFU/ml.
[0077] Inhibition of uropathogenic E. coli adhesion onto epithelial
HeLa cells. For cell monolayer infection, pathogens were cultured
at 37.degree. C. for 18 h in appropriate culture media as described
above. Prior to infection, the cell monolayers, prepared in twenty
four-well TPP tissue culture plates (ATGC, Paris, France), were
washed twice with PBS. Infecting bacteria were suspended in the
culture medium and a total of 0.5 ml DMEM+0.25 ml culture pathogen
(1.times.10.sup.8 CFU/ml)+0.25 ml lactobacilli culture
(1.5.times.10.sup.9 CFU/ml) were added to each well of the tissue
culture plate. The plates were incubated at 37.degree. C. in 10%
CO.sub.2/90% air for different time of infection as indicated and
then were washed three times with sterile PBS and lysed with
sterilized H.sub.2O. Appropriate dilutions were plated on tryptic
soy agar to determine the number of viable cell-associated bacteria
by bacterial colony counts. Each assay was conducted hi triplicate
with three successive passages of HeLa cells.
[0078] Analysis. Results are expressed as means.+-.standard error
to the mean. For statistical comparisons, Student's t test was
performed.
Results
Example 1
1. Adhesion Capacity of L. Jensenii KS 119.1 and KS 130.1, L.
Crispatus KS 116.1 and L. Gasseri KS 124.3 onto HeLa and Caco-2/TC7
Cells.
[0079] The level of adhesion of the above strains was determined
after the cells were inoculated with four concentrations of
lactobacilli (5.times.10.sup.7; 1.times.10.sup.8; 5.times.10.sup.8;
1.times.10.sup.9 CPU/well). Generally, a concentration-dependent
adhesion was observed.
[0080] In cervix HeLa cells, adhesion levels observed show that all
the tested strains are adhering. The L. jensenii KS 119.1 and KS
130.1 strains appeared the best adhering strains (7.5 log CFU/ml at
5.times.10.sup.8 CPU/well) as compared with the control adhering
strains, L. casei rhamnosus GG and L. rhamnosus GR1 strains.
[0081] In intestinal Caco-2/TC7 cells, adhesion levels observed
show that all the Medinova strains are adhering. The L. crispatus
KS 116,1, L. jensenii 119.1,129.1 and KS 130.1, L. gasseri 124.3
strains appeared the best adhering strains (7.5-8 logs CFU/ml at
5.times.10.sup.8 CPU/well) as compared with the control adhering
strains, L. casei rhamnosus GG and L. rhamnosus GR1 strains.
[0082] As observed by scanning electron microscopy, all the
"invention lactobacilli strains" appeared adhering in close contact
with the HeLa and Caco-2/TC7 cells.
[0083] On the basis of their adhering properties, the L. crispatus
KS 116.1 and L. jensenii 119.1 have been selected for the following
studies of antibacterial activities against urovaginal and
intestinal pathogens.
2. Activity of KS 116.1 and KS 119.1 on the growth of Urogenital
and Intestinal Pathogens.
[0084] It has been examined whether the above mentioned strains are
active on the growth of Staphylococcus aureus, Streptococcus
agalactiae, uropathogenic and diarrheagenic E. coli, and
diarrheagenic Salmonella enterica serovar Typhimurium. The growth
of pathogens was measured at 5, 8, 18 and 24 h.
[0085] For Staphylococcus aureus, the control L. rhamnosus strain
GR-1 and L. fermentum strain RC-14 inhibited the growth of
bacteria. Similarly, L. crispatus KS 116.1 and L. jensenii 119.1
inhibited the growth of Staphylococcus aureus and showed a decrease
in the viable bacteria number. When activities of lactobacilli
strains were compared, the L. jensenii 119.1 appeared the most
active strain.
[0086] For uropathogenic E. coli strains IH11128 and 7372, the
control L. rhamnosus strain GR-1 and L. fermentum strain RC-14
inhibited the growth of the bacteria. Similarly, L. crispatus KS
116.1 and L. jensenii KS 119.1 inhibited the growth of E. coli.
When activities of lactobacilli strains were compared, the L.
jensenii 119.1 appeared the most active strain.
[0087] For diarrheagenic E. coli strain C1845, the control L.
rhamnosus strain GR-1 and L. fermentum strain RC-14 inhibited the
growth of the bacteria. Similarly, L. crispatus KS 116.1 and L.
jensenii KS 119.1 inhibited the growth of E. coli. When activities
of lactobacilli strains were compared, the same activity was found
for all the lactobacilli strains examined.
[0088] For diarrheagenic S. enterica serovar Typhimurium strain
SL1344, the control L. rhamnosus strain GR-1 and L. fermentuin
strain RC-14 inhibited the growth of the bacteria. Similarly, L.
jensenii 119.1 inhibited the growth of S. enterica serovar
Typhimurium. When activities of lactobacilli strains were compared,
the same activity was found for the control L. rhamnosus strain
GR-1 and L. fermentum strain RC-14 and L. jensenii KS 119.1. In
contrast, the L. crispatus KS 1116.1 showed a lower activity.
[0089] For Candida albicans no activity was found for the control
L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L.
crispatus KS 116.1 and L. jensenii KS 119.1.
3. Killing Activity of KS 116.1 and KS 119.1 Against Urogenital and
Intestinal Pathogens.
[0090] It has been examined whether said lactobacilli are active on
the viability of Staphylococcus aureus, Streptococcus agalactiae,
uropathogenic and diarrheagenic E. coli, and diarrheagenic
Salmonella enterica serovar Typhimurium. The effect on viability of
pathogens was measured at 2, 3, and 4 h.
[0091] For Staphylococcus aureus, the control L. rhamnosus strain
GR-1 and L. fermentum strain RC-14, and L. jensenii 1 19.1
decreased for 2 logs the viability of bacteria. In contrast, the L.
crispatus KS 116.1 showed no activity.
[0092] For Streptococcus agalactiae, the control L. rhamnosus
strain GR-1 and L. fermentum strain RC-14, and L. jensenii 119.1
and L. crispatus KS 116.1 showed no activity.
[0093] For uropathogenic E. coli strains IH11128 and 7372, the
control L. rhamnosus strain GR-1 and L. fermentum strain RC-14
showed 4 logs of decrease in viability of bacteria. L. crispatus KS
116.1 and L. jensenii 119.1 were not active showing only one log of
decrease in viability of the bacteria.
[0094] For diarrheagenic E. coli strain C 1845, both of the control
L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L.
crispatus KS 116.1 and L. jensenii 119.1 showed a low activity on
the viability of C1845 bacteria (2 logs of decrease).
[0095] For diarrheagenic S. enterica serovar Typhimurium strain
SL1344, both of the control L. rhamnosus strain GR-1 and L.
fermentum strain RC-14, and L. crispatus KS 116.1 and L. jensenii
119.1 showed a great activity by decreasing the viability of SL1344
bacteria (5 logs of decrease).
[0096] For Gardnerella vaginalis, the control L. rhamnosus strain
GR-1 and L. fermentum strain RC-14, and L. fermentum strain RC-14,
and L. jensenii I 19.1 decreased for 2 logs the viability of
Gardnerella. In contrast, the L. crispatus KS 116.1 showed no
activity.
[0097] For Candida albicans no activity was found for the control
L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L.
crispatus KS 116.1 and L. jensenii KS 119.1.
4. Inhibition of the Adhesion of Uropathogenic E. coli Strain
IH11128 Strain onto HeLa Cells by KS 116.1 and KS 119.1.
[0098] It has been examined whether said lactobacilli are able to
inhibit the adhesion of uropathogenic E. coli strain IH11128 onto
HeLa cells. The effect of the control L. rhamnosus strain GR-1 and
L. fermentum strain RC-14, and L. jensenii 119.1 and L. crispatus
KS 116.1 was measured at three concentrations: 1.times.10,
5.times.10.sup.8, and 1.times.10.sup.9 bacteria per well.
[0099] A 30 to 40% of inhibition of IH11128 adhesion was found at a
concentration of 1.times.10.sup.8 bacteria per well for the control
L. rhamnosus strain GR-1 and L. fermentum strain RC-14. At this
concentration, the L. jensenii KS 119.1 and L. crispatus KS 116.1
were inactive. Inhibition of IH11128 adhesion was effective at a
concentration of 5.times.10.sup.8 bacteria per well for L. jensenii
119.1 and L. crispatus KS 116.1 and a similar inhibition that those
obtained with the control L. rhamnosus strain GR-1 and L. fermentum
strain RC-14 was observed. A similar high inhibition level of
IH11128 adhesion was observed with the control L. rhamnosus strain
GR-1 and L. fermentum strain RC-14, and L. jensenii KS 119.1 and L.
crispatus KS 116.1 at the concentration of 1.times.10.sup.9
bacteria per well.
Example 2
1. Activity of L. gasseri KS 124.3, L. helveticus KS 300 and L.
acidophilus KS 400 on the Growth of Urogenital and Intestinal
Pathogens.
[0100] It has been examined whether the strains referred to here
above are active against the growth of Staphylococcus aureus,
Streptococcus agalactiae, Candida albicans and uropathogenic and
diarrheagenic E. coli strains IH11128 and 7372. The growth of
pathogens was measured at 5, 8, 18 and 24 h.
[0101] No activity was developed against Streptococcus agalactiae
and Candida albicans by L. gasseri KS 124.3, L. helveticus KS 300
and L. acidophilus KS 400 as well as by the control strains GR-1
and RC-14.
[0102] Concerning Staphylococcus aureus, the control L. rhamnosus
strain GR-1 and L. fermentum strain RC-14 efficiently inhibited the
growth of the bacteria. Similarly, L. gasseri KS 124.3, L.
helveticus KS 300 and L. acidophilus KS 400 inhibited the growth of
Staphylococcus aureus and showed a decrease in the viable bacteria
number. When activities of lactobacilli strains were compared, the
L. helveticus KS 300 appeared the most active strain.
[0103] For uropathogenic E. coli strains IH11128, the control
strains L. rhamnosus GR-1 and L. fermentum RC-14 efficiently
inhibited the growth of the bacteria. Similarly, L. helveticus KS
300 efficiently inhibited the growth of E. coli. When activities of
lactobacilli strains were compared, a lower activity appeared for
L. gasseri KS 124.3 and L. acidophilus KS 400.
[0104] For uropatho genie E. coli strain 7372, both control strains
L. rhamnosus GR-1 and L. fermentum RC-14 strains inhibited the
growth of bacteria. Similarly L. helveticus KS 300 inhibited the
growth of said bacteria whereas L. acidophilus KS 400, however, was
active only at 25 hours.
2. Killing Activity of KS 124.3, KS 300 and KS 400 against
Urogenital and Intestinal Pathogens.
[0105] It has been examined whether said lactobacilli are active on
the viability of Staphylococcus aureus, Strteptococcus agalactiae,
Candida albicans, uropathogenic E. coli IH11128 and 7372,
diarrheagenic E. coli C1845 and Gardnerella vaginalis. The effect
on viability of pathogens was measured at 2, 3, and 4 h.
[0106] For Staphylococcus aureus, the control strains L. rhamnosus
GR-1 and L. fermentum RC-14, and L. gasseri KS 124.3, L. helveticus
KS 300 and L. acidophilus KS 400 decreased for 2-3 logs the
viability of bacteria.
[0107] Concerning Streptococcus agalactiae and Candida albicans the
two control strains and L. gasseri KS 124.3, L. helveticus KS 300
and L. acidophilus KS 400 showed no activity.
[0108] For uropathogenic E. coli strains IH11128, the control
strains L. rhamnosus strain GR-1 and L. fermentum RC-14 and L.
helveticus KS 300 as well showed 3 logs of decrease in viability of
the bacteria. L. acidophilus KS 400 and L. gasseri KS 124.3 were
not active.
[0109] Concerning uropathogenic E. coli strains 7372, the control
strains showed 2 logs of decrease in viability of the bacteria. L.
helveticus KS 300 showed 3 logs of decrease whereas L. acidophilus
KS 400 and L. gasseri KS 124.3 were not active within the same
conditions.
[0110] For Gardnerella vaginalis, both control strains, L.
acidophilus KS 400 and L. gasseri KS 124.3 showed 3 logs of
decrease in viability of the bacteria. A rapid and efficient
activity was observed for L. helveticus KS 300, higher than that
found for the above control strains.
[0111] For diarrheagenic E. coli strain C 1845, both of the control
strains L. rhamnosus GR-1 and L. fermentum RC-14 killed the
bacteria showing a 3 log decrease in the viability of same. Similar
effect was observed for L. gasseri KS 124.3 whereas no activity was
detected concerning L. acidophilus KS 400. L. helveticus KS 300
exhibits a killing which is definitely higher that that observed
for the above control strains.
Example 3
1. Killing Activity of L. jensenii KS 121.1 and KS 122.1, L.
gasseri KS 120.1 and L. helveticus KS 300 Against Urogenital and
Intestinal Pathogens.
[0112] It has been examined whether said lactobacillus strains are
active on the viability of Streptococcus agalactiae, Candida
albicans, uropatho genie E. coli H11128, Gardnerella vaginalis,
Prevotella bivia and Salmonella enterica Typhimurium. The effect on
viability of pathogens was measured at 4 h of contact. Concerning
both Streptococcus agalactiae and Candida albicans none of the
tested lactobacilli showed an activity.
[0113] For uropathogenic E. coli strains IH11128, L. jensenii KS
121.1 and KS 122.1 showed no activity whereas, in contrast, L.
gasseri KS 120.1 decreased efficiently (4 logs) the viability of E.
coli in unshaken conditions. L. helveticus KS 300 and the L.
fermentum RC-14 control strain decreased of 2 logs the viability of
E. coli in unshaked conditions only.
[0114] Concerning Gardnerella vaginalis, both L. jensenii KS 121.1
and KS 122.1 showed no activity. In contrast L. gasseri KS 120.1
decreased efficiently (6 logs) the viability of Gardnerella
vaginalis in unshaken conditions; L. helveticus KS 300 showed
similar efficiency (4 logs of decrease) in unshaken conditions
also, whereas control strain showed a 3 log of decrease only.
[0115] For Prevotella bivia, L. gasseri KS 120.1, L. jensenii
122.1, L. helveticus KS 300 and the control strain L. fermentum
RC-14 decreased viability of the bacteria for 2 logs, in unshaken
conditions. L. jensenii KS 121.1 which was highly active against
Prevotella bivia in unshaken conditions had lost its activity when
tested in shaken conditions.
[0116] Concerning Salmonella Typhimurium, L. gasseri KS 120.1 (3
logs), L. jensenii KS 121.1 and KS 122.1, L. helveticus KS 300 and
the control strain L. fermentum RC-14 were quite active (6 logs of
decrease) in unshaken conditions. L. gasseri KS 120.1 remained
active even in shaken conditions.
2. Activity of KS 120, KS 121.1, KS 122.1 and KS 300 on the growth
of Gardnerella Vaginalis and Prevotella Bivia
[0117] The tests have been perforrned in both shaken and unshaken
conditions.
[0118] In unshaken conditions L. jensenii KS 121.1 and KS 122.1
inhibited the growth of Gardnerella vaginalis whereas L. gasseri
120.1, L. helveticus and the control strain L. fermentum RC-14
inhibited said activity at still a higher level.
[0119] In shaked conditions L. jensenii KS 121.1 and KS 122.1 and
L. helveticus as well have lost their activity, whereas L. gasseri
120.1 remains active (2 logs of decrease) against Gardneralla
vaginalis.
[0120] In both shaken and unshaken conditions L. gasseri KS 120.1,
L. jensenii KS 121.1 and KS 122.1, L. helveticus KS 300 and the
control strain inhibited the growth of Prevotella bivia at a high
level.
3. Inhibition of Adhesion of Gardnerella Vaginalis and Prevotella
bivia onto HeLa Cells KS 120.1, KS 121.1 and KS 300
[0121] The effect of L. gasseri KS 120.1, L. helveticus KS 300 of
the control strains L. fennentum RC-14 as well as L. casei
rhamnosus GG was measured at the concentration of 1.times.10.sup.9
bacteria per well.
[0122] The control L. fermentum RC-14 strain and L. jensenii KS
121.1 decreased for 2 logs the level of adhesion of Gardnerella
vaginalis on the tested cells. L. gasseri KS 120.1 and L.
helveticus KS 300 decreased said adhesion for 4 logs.
[0123] L. jensenii KS 121.1 decreased adhesion of Prevotella bivia
for 1 log only, whereas L. gasseri KS 120.1, L. helveticus KS 300
as well as the control strain RC-14 decreased said adhesion for 2
logs.
4. Inhibition of Adhesion and Internalization of Uropathogenic E.
coli strain IH11128 Strain onto HeLa Cells by KS 120.1, KS 121.1
and KS 300
[0124] A strategy often used by extra-intestinal pathogens like E.
coli to evade host defense mechanism is to establish a local
reservoir within epithelial cells (M. A. Muvlea in Eschrichia coli.
Cell. Microbiol. 4, 257-271-2002) and cell entry by IH1128 strain
appears to be an effective mechanism for promoting prolonged
persistence these pathogens in the urinary tract.
[0125] The effect of L. gasseri KS 120.1, L. helveticus KS 300 and
of the control strains RC-14 and GG strain was examined concerning
the above uropathogenic E. coli.: L. jensenii 121.1 decreased for 2
logs the level of viable internalized E. coli, whereas L. gasseri
120.1, L. helveticus and both the control strains have shown a 4
logs of decrease of the internalized E. coli.
Example 4
Modulation of the Immune Response (in vivo Test Using Human
PMBC)
[0126] The following strains have been tested within the conditions
set hereafter concerning their ability to induce or modulate or
affect an immune response, more specifically their ability to
induce the secretion of cytokines and the like: L. crispatus KS
116.1, L. jensenii 119.1, L. jensenii KS 121.1 and KS 122.1, L.
gasseri KS 120.1, L. gasseri KS 124.3, L. helveticus KS 300 and L.
acidophilus KS 400.
[0127] The detection of the induction of cytokines was made by
means of a test for in vitro stimulation of isolated peripheral
blood mononuclear cells (PBMC). Among the cytokines induced during
these tests, there are interleukins 10 and 12 (IL10 & IL12),
.gamma.-interferon (.gamma.-IFN) and tumor necrosis factor .alpha.
(TNF.alpha.).
Experimental Procedures
[0128] PMBC preparation: Fresh human blood obtained for healthy
subjects (four donors) was diluted at a 1:2 ratio with PBS-Ca
(GIBCO) and purified on a Ficoll gradient (GIBCO). After 5
centrifugation at 400.times.g for 30 min at 20.degree. C. the
peripheral blood monocular cellular cells (PMBC's) formed an
interphase ring layer in the serum. PMBC's were aspired carefully,
suspended to a final volume of 50 ml using PBD-Ca and washed three
times in the same buffer with centrifugation steps at 350.times.g
for 10 min at 20.degree. C.
[0129] PMBC's were subsequently resuspended using complete RPMI
medium (GIBCOP), supplemented with 10% w/v L-glutamine (GIBCO) and
gentamycin (150 jig/ml) (GIBCO). PBMC's were counted under the
microscope and adjusted at a concentration of 2.times.10.sup.6
cells/ml and distributed (in 1 ml aliquots) in 24-well tissues
culture plates (Corning, Inc.).
[0130] Bacteria preparation: overnight LAB cultures were washed
twice with PBS buffer, pH 7.2 before being resuspended in PBS at
concentration of 2.times.10.sup.9 cfu/ml.
[0131] PMBC incubation: from these suspensions 10 .mu.l was
transferred into wells of the PMBC plates which were incubated at
37.degree. C. in a 5% CO.sub.2/95% air atmosphere. After 24 hours
incubation the supernatant was aspirated, centrifuged at 2000 rpm
and the supernatant removed and stored at -20.degree. C. The
control consisted of bacteria-free buffer.
[0132] Cytokine quantification: cytokine expression levels have
been determined by ELISA tests (<<Enzyme linked immuno
sorbent assay>>). ELISA plates are coated with anti-cytokine
antibody (overnight procedure) and the antibody is blocked with
PBS/BSA 1%. A proper standard was prepared with known
concentrations of cytokines, covering the detection range of 15.62
to 2000 pg/ml (incubated overnight).
[0133] The anti-cytokine detection and quantification was performed
with the streptavidine reaction on substrate (TMB Pharmigen). The
commercial kits Pharmigen have been used according to the
manufacturer's description. Four cytokines were determined: the
pro-inflammatory/Th 1 cytokines TNF.alpha., IFN.gamma., IL 12 and
the anti-inflammatory/Th 2 cytokine IL10. TABLE-US-00002 TABLE I IL
10 IL 12 TNF.alpha. IFN.gamma. IL10/IL12 Control 31.25 31.25 31.25
31.25 1 KS 120.1 1228.67 176.32 17698.83 3513.36 6.96840971 KS
121.1 2297.87 47.66 14180.66 897.65 48.2138061 KS 116.1 2856.26
167.6 33569.91 7209.33 17.0540573 KS 400 3177.49 103.85 26799
6949.13 30.5969186 KS 300 2290.47 59.7 18703.66 10047.75 38.3663317
KS 119.1 307.13 198.47 6693.3 9192.74 1.54748829 KS 124.3 2969.02
660.98 31307.71 16985.56 4.49184544
Observations [0134] a high level of TNF.alpha. induction for all
the tested LAB strains [0135] a relatively low level of IFN.gamma.
concerning L. jensenii KS 121.1 [0136] the highest IL10 induction
potential concerning L. crispatus KS 116.1 and KS 400 [0137] in
contrast to the two L. jensenii strains the two L. gasseri strains
have shown a similar profile, especially when considering the
ratio's in IL10/IL12 and in TNF.alpha./IFN.gamma..
[0138] Within the above testing frame it is clear that the cytokine
induction profile is strain specific.
Example 5
Determination of the Anti-Inflammatory Activity (in vivo Test Using
an Animal Model)
[0139] An acute model of mice has been adapted from Camoglio et al.
(see Eur. J. Immunol. 2000) where the animals have been fed from
day-5 to day +2 with selected lactic acid bacteria strains, at a
rate of 10.sup.8 bacteria per mouse per day. TNBS was then injected
on day zero, at a rate of 120 mg/kg mice in order to induce acute
colitis and the animals have been sacrificed at day +2 and
eventually subjected to both macroscopic (Wallace score--Table II)
and histological (Ameho score--Table III) scoring.
[0140] These tables clearly show that the selected lactic acid
bacteria strains exhibit a significant anti-inflammatory effect
when compared to reference strains.
Example 6
6.1 Composition for Local Administration (Vaginal Capsules)
[0141] Samples of the LAB strains of this invention (see above)
have been cultured for min. 24 hours in conditions similar to those
mentioned here above. The cultured strains have been isolated,
washed and lyophilized individually, individually suspended in a
lactose/MSK powder mixture and eventually divided into unit doses
each of them containing about 10.sup.8-10.sup.9 cfu (colony forming
units).
[0142] Said unit doses have been then poured into gelatin vaginal
capsules each of them comprising about 10.sup.8-10.sup.9 cfu of
selected LAB strains of this invention.
6.2 Composition for Local Administration (Vaginal
Suppositories)
[0143] Soft vaginal suppositories have been prepared using the
following ingredients: [0144] buffered lactic acid solution [0145]
lactose [0146] PEG 4000 [0147] PEG 600
[0148] The adequate amount of selected lyophilized LAB strains of
this invention has been then added to unit doses to afford vaginal
suppositories each comprising about 10.sup.8-10.sup.9 cfu.
Example 7
7.1 Composition for Oral Administration (Edible Capsules)
[0149] Edible cellulose capsules (hydroxypropyl methyl cellulose)
each comprising about 10.sup.8-10.sup.9 cfu of selected LAB strains
of this invention have been manufactured using filler comprising
the following ingredients: [0150] dehydrated yogurt powder [0151]
anhydrous dextrose [0152] potato starch microcrystalline cellulose
[0153] selected lyophilized LAB strain 7.2 Composition for Oral
Administration (Yogurt)
[0154] Portions of a so called "Yogurt Nature Light" have been
prepared using the following process: to a batch of standardized
1.5% fat milk there was added 3% of skimmed milk powder (MSK) and
the whole was then pasteurized at 90.degree. C. for 30 minutes. 1%
volume of commercial starter cultures of L. bulgaricus and S.
thermophilus have been added to the pasteurized milk; then the
whole was gently stirred at room temperature, disposed in 100 ml
containers which were all eventually incubated at 40.degree. C.
during around 4 hours to afford the desired pH.
[0155] Then portions of selected lyophilized LAB strains of this
invention were added to the yogurt cans in such an amount to have
about 10.sup.8-10.sup.9 cfu per yogurt can and a further incubation
was carried out for about 30 min. until to afford a pH of about 4.5
to 4.7. These yogurt portions can be stored at 4.degree. C. before
consumption. TABLE-US-00003 TABLE II TNBS colitis induced at day
zero Wallace score ##STR1##
[0156] TABLE-US-00004 TABLE III TNBS colitis induced at day zero
Ameho score ##STR2##
* * * * *