U.S. patent application number 12/148200 was filed with the patent office on 2008-08-14 for probiotic lactic acid bacterium to treat bacterial infections associated with sids.
Invention is credited to Sean Farmer, Robert J. Mikhail.
Application Number | 20080193429 12/148200 |
Document ID | / |
Family ID | 39387557 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080193429 |
Kind Code |
A1 |
Farmer; Sean ; et
al. |
August 14, 2008 |
Probiotic lactic acid bacterium to treat bacterial infections
associated with sids
Abstract
Compositions including a non-pathogenic lactic acid-producing
bacteria, such as a Bacillus species, spores or an extracellular
product of B. coagulans, formulated for oral administration to the
intestinal tract for inhibiting bacterial gastrointestinal
infections are described. Methods and systems using the
compositions for treating gastrointestinal infections, particularly
sudden infant death syndrome (SIDS) are also disclosed.
Inventors: |
Farmer; Sean; (San Diego,
CA) ; Mikhail; Robert J.; (Lakeside, CA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C;ATTN: PATENT INTAKE
CUSTOMER NO. 30623
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
39387557 |
Appl. No.: |
12/148200 |
Filed: |
April 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09424527 |
May 29, 2003 |
7374753 |
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PCT/US98/11347 |
Jun 3, 1998 |
|
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12148200 |
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60048452 |
Jun 3, 1997 |
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Current U.S.
Class: |
424/93.45 ;
424/780 |
Current CPC
Class: |
A61K 45/06 20130101;
Y02A 50/473 20180101; A61K 35/742 20130101; Y02A 50/469 20180101;
A61P 1/00 20180101; C12N 1/20 20130101; Y02A 50/30 20180101 |
Class at
Publication: |
424/93.45 ;
424/780 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 35/00 20060101 A61K035/00; A61P 1/00 20060101
A61P001/00 |
Claims
1. An oral electrolyte composition for reducing a bacterial
infection in a human comprising: i) viable colony forming units
(CFU) of a non-pathogenic lactic acid bacteria, wherein said
non-pathogenic lactic acid bacteria is Bacillus coagulans; and ii)
an oral electrolyte maintenance formulation.
2. The oral electrolyte composition of claim 1, wherein said oral
electrolyte maintenance formulation in (ii) is rehydrated with
water to produce a solution comprising 45 to 75 mEq/L of sodium, 20
mEq/L of potassium, 35 to 65 mEq/L of chloride, 30 mEq/L of
citrate, 20-25 g/l of glucose, and wherein said non-pathogenic
lactic acid bacteria in (i) comprises about 5.times.10.sup.5 to
about 5.times.10.sup.7 viable CFU of said bacteria/L.
3. The oral electrolyte composition of claim 1, wherein the oral
electrolyte maintenance formulation in (ii) comprises 45 to 75
mEq/L of sodium, 20 mEq/L of potassium, 35 to 65 mEq/L of chloride,
30 mEq/L of citrate, and 20 to 25 g/L of glucose.
4. The oral electrolyte composition of claim 1, wherein said
bacterial gastrointestinal infection is selected from the group
consisting of Clostridium perfringens, Clostridium difficile,
Clostridium botulinum, Clostridium tributrycum, Clostridium
sporogenes, Escherichia coli, Pseudomonas aeruginosa, and
Staphylococcus aureus.
5. The oral electrolyte composition of claim 1, wherein said
non-pathogenic lactic acid bacteria exhibit probiotic activity that
inhibits growth of bacteria associated with Sudden Infant Death
Syndrome (SIDS).
6. The oral electrolyte composition of claim 1, wherein said human
is an infant at risk for SIDS.
7. The oral electrolyte composition of claim 1, wherein said
non-pathogenic lactic acid bacteria is in the form of spores.
8. The oral electrolyte composition of claim 1, wherein said
non-pathogenic lactic acid bacteria is in the form of a dried cell
mass.
9. The oral electrolyte composition of claim 1, wherein said
non-pathogenic lactic acid bacteria is in the form of spores, and
wherein said spores germinate in the gastrointestinal tract of said
human.
10. The oral electrolyte composition of claim 1, wherein said
composition contains 10.sup.3 to 10.sup.12 CFU of viable
non-pathogenic lactic acid bacteria or spores per gram of
composition.
11. The oral electrolyte composition of claim 1, wherein said
composition further comprises an effective amount of a bifidogenic
oligosaccharide to promote the growth of the non-pathogenic lactic
acid bacteria.
12. The oral electrolyte composition of claim 11, wherein said
bifidogenic oligosaccharide is selected from the group consisting
of fructo-oligosaccharide (FOS), gluco-oligosaccharide (GOS),
raffinose, and long-chain oligosaccharides.
13. The oral electrolyte composition of claim 12, wherein said
bifidogenic oligosaccharide comprises a polysaccharide having a
polymer chain length of about 4 to 100 sugar units.
14. The oral electrolyte composition of claim 1, wherein said
composition further comprises about 10 milligrams to about 1 gram
of FOS per gram of composition.
15. The oral electrolyte composition of claim 1, wherein said
composition further comprises from 100 to 500 milligrams of FOS per
gram of composition.
16. The oral electrolyte composition of claim 1, wherein the
composition further comprises a food substance, flavoring, vitamin
or mineral.
17. The oral electrolyte composition of claim 1, wherein the oral
electrolyte maintenance formulation in (ii) is a powder comprising
sodium chloride, potassium citrate, citric acid, or glucose.
18. The oral electrolyte composition of claim 1, wherein said
composition further comprises an extracellular product of Bacillus
coagulans.
19. The oral electrolyte composition of claim 18, wherein said
extracellular product is a supernatant or filtrate of a culture of
an isolated Bacillus coagulans strain.
20. The oral electrolyte composition of claim 1, wherein said
non-pathogenic lactic acid bacteria comprises Bacillus coagulans
hammer strain Accession No. ATCC 31284.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of 09/424,527
filed May 29, 2003, pending, which is a national stage Application
filed under 371 based on PCT/US98/11347 filed Jun. 3, 1998, which
claims priority to provisional Application 60/048,452, filed Jun.
3, 1997.
TECHNICAL FIELD
[0002] This invention relates to utilizing a probiotic organism as
a food additive or supplement, and specifically relates to use of
Bacillus coagulans in food or as a food supplement to prevent
Sudden Infant Death Syndrome (SIDS) associated with infant gut
microbial infections.
BACKGROUND OF THE INVENTION
[0003] Probiotic agents are organisms that confer a benefit when
they grow in a particular environment, often by inhibiting the
growth of other biological organisms in the same environment.
Examples of probiotics include bacteria and bacteriophages which
can grow in the intestine, at least temporarily, to displace or
destroy pathogens and provide other benefits to the host organism
(Salminen et al, Antonie Van Leeuwenhoek, 70 (2-4): 347-358, 1996;
Elmer et al, JAMA, 275:870-876, 1996; Rafter, Scand. J.
Gastroenterol., 30:497-502, 1995; Perdigon et al, J. Dairy Sci.,
78:1597-1606, 1995; Gandi, Townsend Lett. Doctors & Patients,
pp. 108-110, January 1994; Lidbeck et al, Eur. J. Cancer Prey.
1:341-353, 1992). Probiotic preparations were systematically
evaluated for their effect on health and longevity in the early
1900's (Metchnikoff, E., Prolongation of Life, Wilham Heinemann,
London, 1910; republished by G.P. Putnam's Sons, New York, N.Y.,
1970). Since the discovery and widespread use of antibiotics in
about 1950 to treat pathological microbes, the use of probiotics
has been limited.
[0004] The widespread use of antimicrobial drugs, especially broad
spectrum antibiotics, has produced serious consequences.
Individuals taking antibiotics often suffer from gastrointestinal
upset when beneficial microorganisms in the gut are killed, thus
changing the balance of the intestinal flora. This imbalance can
result in vitamin deficiencies when vitamin-producing gut bacteria
are killed and additional illness if a pathogenic organism
overgrows and replaces the beneficial gut microorganisms. In
addition, widespread antibiotic use has produced increasing numbers
of antibiotic-resistant pathogenic microorganisms, including
vancomycin-resistant bacteria. Microorganisms that are resistant to
multiple drugs have also developed, often with multiple drug
resistance spreading between species, leading to systemic
infections that cannot be controlled by use of known antibiotics.
Thus, there is a need for preventive and therapeutic agents that
can control pathogenic microorganisms without the use of antibiotic
chemicals.
[0005] Sudden Infant Death Syndrome (SIDS) refers to the sudden and
unexpected death of an apparently healthy infant, typically between
the ages of three weeks to five months, peaking at about three
months of age. Generally, the death is due to cardiorespiratory
failure in which the child dies quietly with no symptoms that would
indicate grave illness before death, although infections in the few
weeks before death have been observed in about 85% of SIDS victims.
Although SIDS is a leading cause of infant mortality in the
developed countries of the world, its cause is not well
understood.
[0006] Several researchers have reported that various toxigenic
bacteria and their enterotoxins are implicated in the aetiology of
SIDS (Amon S. S. et al., Lancet 1:1273-1277, 1978; Gurwith M. J. et
al., Am. J. Dis. Child. 135:1104-1106, 1981; Cooperstock M. S. et
al., Pediatr. 70:91-95, 1982; Donta S. & Myers M., J. Pediatr.
100:431-434, 1982; Amon S. S. et al., J. Pediat. 104(1):34-40,
1984; Murrell T. G. et al., Med. Hypoth. 22:401-413, 1987;
Blackwell C. C. et al., J. Clin. Pathol. 45(11 Suppl.):20-24, 1992;
Lindsay J. A. et al., Curr. Microbiol. 27:51-59, 1993; Murrell W.
G. et al., J. Med. Microbiol. 39(2):114-127, 1993; Mach A. S. &
Lindsay J. A., Curr. Microbiol. 28:261-267, 1994; Siarakas S. et
al., Toxicon 33(5):635-649, 1995). Bacterial species implicated in
SIDS include Clostridium perfringens, C. difficile, C. botulinum,
Staphylococcus aureus and Escherichia coli, although the
correlation between the presence of particular bacterial species
and SIDS has not been entirely consistent between studies (Gurwith
M. J. et al., Am. J. Dis. Child. 135:1104-1106, 1981; Blackwell C.
C. et al., J. Clin. Pathol. 45(11 Suppl.):20-24, 1992; Murrell W.
G. et al., J. Med. Microbiol. 39(2):114-127, 1993; Lindsay J. A. et
al., Curr. Microbiol. 27:51-59, 1993; Siarakas S. et al., Toxicon
33(5):635-649, 1995). Clostridium species, particularly C.
perfringens and C. difficile, are most often associated with fecal
samples obtained from children who have died of SIDS. Bacterial
toxins found in fecal matter and serum of SIDS babies may be
etiological agents of SIDS. These bacterial toxins include C.
perfringens enterotoxin and alpha-toxin, Staphylococcus enterotoxin
B, E. coli heat-stable toxin (STa), C. difficile toxins A and B,
and C. botulinum toxin (Blackwell C. C. et al., J. Clin. Pathol.
45(11 Suppl.):20-24, 1992; Murrell W. G. et al., J. Med. Microbiol.
39(2):114-127, 1993; Siarakas S. et al., Toxicon 33(5):635-649,
1995). C. perfringens Type A enterotoxin has been particularly
implicated because of its ability to modulate cytokine production
by human animal cells (Lindsay J. A., Crit. Rev. Microbiol.
22(4):257-277, 1996). Some of these toxins act synergistically
(Siarakas S. et al., Toxicon 33(5):635-649, 1995). In animals, C.
perfringens is responsible for death of several young species
(e.g., lamb, pony) and C. difficile causes pseudomembranous colitis
(Murrell T. G. C. et al. Med. Hypotheses 22:401-413, 1987; Murrell
W. G. et al, J. Med. Microbiol. 39:114-127, 1993).
[0007] Although different hypotheses have been offered to explain
how these bacteria and/or bacterial toxins may cause or contribute
to SIDS, it is generally thought that SIDS results from a series of
events in which pathogenic bacteria enter the gut, colonize and
produce cytotoxin that initiates a cascade of reactions that lead
to silent death (Lindsay J. A., Crit. Rev. Microbiol.
22(4):257-277, 1996; Murrell W. G. et al., J. Med. Microbiol.
39:114-127, 1993). The cytotoxin may damage intestinal tissue
resulting in more efficient systemic absorption of the enterotoxin,
without systemic migration of the bacteria. Moreover, intestinal
injury may result in increased production of cytokines (e.g.,
interferon-gamma, tumor necrosis factor and interleukins) that
exacerbate the effects of the toxins leading to a biochemical
cascade that alters the circuits that control cardiorespiration,
leading to irreversible shock and death (Lindsay J. A. et al.,
Curr. Microbiol. 27:51-59, 1993; Mach A. S. & Lindsay J. A.,
Curr. Microbiol. 28:261-267, 1994). For example, toxin-induced
changes in cell membrane permeability leading to abnormal levels of
intracellular ions (potassium and/or calcium) in heart tissue may
lead to cardiac failure. These explanations for SIDS are consistent
with other studies that have shown an association between
intestinal injury and the development of a septic state and distant
organ failure in the absence of systemic bacterial infection
(Deitch E. A. et al., Shock 1(2):141-145, 1994).
[0008] Because SIDS occurs generally in young infants, before the
immune system as fully developed, a vaccine against bacterial
pathogens associated with SIDS would usually not be effective to
prevent SIDS-associated infections because the infant would not
produce a sufficient immune response to the immunogen. Anti-toxin
antibodies (e.g., as disclosed in U.S. Pat. No. 5,599,539) have
limited efficacy because they do not limit growth of the
toxin-producing bacteria which can continue to produce toxin and
the antibodies may produce an allergic reaction when orally
administered. Thus, there is a need for preventive and therapeutic
agents that can control the growth of SIDS-associated pathogenic
microorganisms, without the use of antibiotics that can affect the
beneficial microflora of the infant's gut or contribute to
development of microbial drug resistance. Probiotics, which can be
taken internally because they are generally regarded as safe, can
be used replace or preclude growth of gut pathogens associated with
SIDS. Moreover, because of their mode of action, probiotics do not
produce antibiotic side effects or lead to drug-resistant
pathogens.
[0009] Lactic acid producing bacteria (e.g., Bacillus,
Lactobacillus and Streptococcus species) have been used as food
additives and there have been some claims that they provide
nutritional and therapeutic value (Gorbach S. L., Ann. Med.
22(1):37-41, 1990; Reid, G. et al., Clin. Microbiol. Rev.
3(4):335-344, 1990). Some lactic acid producing bacteria (e.g.,
those used to make yogurt) have been suggested to have
antimutagenic and anticarcinogenic properties useful for preventing
human tumors (Pool-Zobel B. L. et al., Nutr. Cancer 20(3):261-270,
1993; U.S. Pat. No. 4,347,240). Some lactic acid producing bacteria
also produce bacteriocins which are inhibitory metabolites
responsible for the bacteria's antimicrobial effects (Klaenhammer
T. R., FEMS Microbiol. Rev. 12(1-3):39-85, 1993; Barefoot S. F.
& Nettles C. G., J. Dairy Sci. 76(8):2366-2379, 1993).
[0010] The therapeutic use of probiotic bacteria, especially
Lactobacillus strains, that colonize the gut has been previously
disclosed (Winberg et al, Pediatr. Nephrol. 7:509-514, 1993; Malin
et al, Ann. Nutr. Metab. 40:137-145, 1996; and U.S. Pat. No.
5,176,911).
[0011] Selected Lactobacillus strains that produce antibiotics have
been disclosed as effective for treatment of infections, sinusitis,
hemorrhoids, dental inflammations, and other inflammatory
conditions (U.S. Pat. No. 4,314,995). L. reuteri produces
antibiotics with activity against Gram negative and Gram positive
bacteria, yeast and a protozoan (U.S. Pat. No. 5,413,960 and U.S.
Pat. No. 5,439,678). L. casei ssp. rhamnosus strain LC-705, DSM
7061, alone or in combination with a Propionibacterium species, in
a fermentation broth has been shown to inhibit yeast and molds in
food and silage (U.S. Pat. No. 5,378,458). Also, antifungal
Serratia species have been added to animal forage and/or silage to
preserve the animal feedstuffs, particularly S. rubidaea FB299,
alone or combined with an antifungal B. subtilis (strain FB260)
(U.S. Pat. No. 5,371,011).
[0012] Bacillus coagulans is a non-pathogenic gram positive
spore-forming bacteria that produces L(+) lactic acid
(dextrorotatory) in homofermentation conditions. It has been
isolated from natural sources, such as heat-treated soil samples
inoculated into nutrient medium (Bergey's Manual of Systemic
Bacteriology, Vol. 2, Sneath, P. H. A. et al., eds., Williams &
Wilkins, Baltimore, Md., 1986). Purified B. coagulans strains have
served as a source of enzymes including endonucleases (e.g., U.S.
Pat. No. 5,200,336), amylase (U.S. Pat. No. 4,980,180), lactase
(U.S. Pat. No. 4,323,651) and cyclo-malto-dextrin
glucano-transferase (U.S. Pat. No. 5,102,800). B. coagulans has
been used to produce lactic acid (U.S. Pat. No. 5,079,164). A
strain of B. coagulans (referred to as L. sporogenes Sakaguti &
Nakayama (ATCC 31284)) has been combined with other lactic acid
producing bacteria and B. natto to produce a fermented food product
from steamed soybeans (U.S. Pat. No. 4,110,477). B. coagulans
strains have also been used as animal feed additives for poultry
and livestock to reduce disease and improve feed utilization and,
therefore, to increase growth rate in the animals (International
PCT Pat. Applications No. WO 9314187 and No. WO 9411492).
SUMMARY OF THE INVENTION
[0013] It has now been discovered that lactic acid bacteria possess
the ability to exhibit probiotic activity in preventing
gastrointestinal bacterial infections, particularly Sudden Infant
Death Syndrome (SIDS). Non-pathogenic lactic acid bacteria are
preferably used, with spore-forming Bacillus species, particularly
B. coagulans, being a preferred embodiment. The invention describes
therapeutic compositions, therapeutic systems, and methods of use
for treating and/or preventing various bacterial gastrointestional
infections, particularly infections associated with SIDS.
[0014] According to one aspect of the invention, there is provided
a composition comprising viable non-pathogenic lactic acid
bacterium in a pharmaceutically acceptable carrier suitable for
oral administration to the digestive tract of a human. In one
embodiment, a Bacillus coagulans strain is included in the
composition in the form of spores. In another embodiment, a
Bacillus coagulans strain is included in the composition in the
form of a dried cell mass. In one embodiment, the Bacillus
coagulans strain is present in the composition at a concentration
of 10.sup.3-10.sup.12 colony forming units/g, whereas in other
preferred embodiments the concentrations are 10.sup.9-10.sup.13
colony forming units/g, 10.sup.5-10.sup.7 colony forming units/g,
or 10.sup.8-10.sup.9 colony forming units/g. In one embodiment, the
Bacillus coagulans strain is in a pharmaceutically acceptable
carrier suitable for oral administration to a human infant,
preferably, a powdered food supplement, a infant formula or an oral
electrolyte maintenance formulation.
[0015] In another aspect of the invention, there is provided a
composition comprising an extracellular product of a Bacillus
coagulans strain in a pharmaceutically acceptable carrier suitable
for oral administration to a human. In one embodiment, the
extracellular product is a supernatant or filtrate of a culture of
an isolated Bacillus coagulans strain.
[0016] Another aspect of the invention is a method of preventing or
treating a bacterial gastrointestinal infection in a human,
comprising the steps of orally administering to a human subject a
food or drink formulation containing viable colony forming units of
a non-pathogenic lactic acid bacterium, preferably a Bacillus
species and more preferably an isolated Bacillus coagulans strain,
and allowing the bacteria to grow in the human subject's
gastrointestinal tract. In one embodiment, the human subject is an
infant at risk for Sudden Infant Death Syndrome. In another
embodiment, the viable colony forming units are spores of Bacillus
coagulans.
[0017] In one embodiment of the method, the step of allowing the
non-pathogenic bacteria to grow further includes inhibiting growth
of Staphylococcus species, Streptococcus species, Pseudomonas
species, Escherichia coli, Gardnerella vaginalis, Propionibacterium
acnes, Aeromonas hydrophilia, Aspergillus species, Proteus species,
Aeromonas species, Clostridium species, Klebsiella species, Candida
species and Trichophyton species. In a preferred embodiment, the
method inhibits Staphylococcus aureus, Staphylococcus pyrogenes,
Clostridium perfringens, C. difficile, C. botulinum, C.
tributrycum, C. sporogenes, or combinations thereof.
[0018] One aspect of the invention is a probiotic composition
comprising an isolated Bacillus species strain, combined with a
pharmaceutically acceptable carrier suitable for oral
administration to a human infant, wherein the isolated Bacillus
species strain is capable of growing at temperatures of about
30.degree. C. to about 65.degree. C., produces L(+) dextrorotatory
lactic acid, produces spores resistant to heat up to 90.degree. C.,
and exhibits probiotic activity that inhibits growth of bacteria
associated with Sudden Infant Death Syndrome. In one embodiment,
the bacteria associated with Sudden Infant Death Syndrome are
Staphylococcus aureus and Clostridium species. In another
embodiment, the probiotic activity results from vegetative growth
of the isolated Bacillus species strain in the gastrointestinal
tract of a human infant. In yet another embodiment, the probiotic
activity results from an extracellular product of the isolated
Bacillus species strain produced in the gastrointestinal tract of a
human infant.
[0019] The invention also describes a therapeutic system for
treating, reducing or controlling gastrointestinal bacterial
infections, particularly infections associated with SIDS,
comprising a container comprising a label and a therapeutic
composition as described herein, wherein said label comprises
instructions for use of the composition for treating infection.
[0020] The invention provides several advantages. In particular,
insofar as there is a detrimental effect to the use of antibiotics
because of the potential to produce antibiotic-resistant microbial
species, it is desirable to have an antimicrobial therapy which
does not utilize conventional antimicrobial reagents. The present
invention does not contribute to the production of future
generation of antibiotic resistant pathogens.
[0021] It should be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention is directed to the discovery that
lactic acid bacteria, particularly Bacillus species, can be used in
therapeutic compositions as a probiotic for preventing or
controlling gastrointestinal bacterial infections. As discussed
further, the compositions can be formulated in many configurations
because the bacterium is presented as a viable organism, e.g., as a
vegetative cell or as a spore depending on the species and form of
probiotic organism, and colonize tissues of the gastrointestinal
tract. The cells/spores can be presented in a variety of
compositions suited for oral administration to the gastrointestinal
tract, directed at the objective of introducing the bacteria to
tissues of the gastrointestinal tract.
[0023] As used herein, "probiotic" refers to bacteria that form at
least a part of the transient or endogenous flora and thereby
exhibit a beneficial prophylactic and/or therapeutic effect on the
host organism. Probiotics are generally known to be safe by those
skilled in the art. Although not wishing to be bound by any
particular mechanism, the prophylactic and/or therapeutic effect of
a lactic acid bacterium of this invention results from competitive
inhibition of growth of pathogens due to superior colonization,
parasitism of undesirable microorganisms, lactic acid production
and/or other extracellular products having antimicrobial activity,
or combinations thereof. These products and activities of a lactic
acid bacterium of this invention act synergistically to produce the
beneficial probiotic effect.
[0024] A lactic acid bacterium suitable for use in the methods and
compositions of the invention, as defined for use in the present
invention, produces L(+) lactic acid, and does not substantially
produce D(-) lactic acid. There are many L(+) lactic acid producing
bacteria currently identified as described herein. The property of
L(+) lactic acid production is key to the effectiveness of the
probiotic lactic acid producing bacteria of this invention because
the acid production increases acidity in the local microfloral
environment, which does not support growth of deleterious and
undesirable bacteria. By the mechanism of lactic acid production,
the probiotic inhibits growth of competing and deleterious
bacteria. In addition, whereas L(+) lactic acid is absorbed and
metabolised in the glycogen synthesis pathway, D(-) lactic acid is
metabolised very slowly, and can lead to metabolic disturbances
such as acidosis.
[0025] Typical lactic acid producing bacteria useful as a probiotic
of this invention which are L(+) lactic acid producers include
Lactobacillus acidophilus, L. salivarius, L. gg., L. planterum, L.
delbrukeii, L. sporegenes (aka B. coagulans), L. rhamnosus, L.
casei, Bifidobacterium longum, B. bifidum, B. infantus, Bacillus
species, and the like.
[0026] There are several Bacillus species particularly useful
according to the present invention, including Bacillus coagulans,
Bacillus subtilis, Bacillus laterosporus and Bacillus
laevolacticus. Although exemplary of the invention, Bacillus
coagulans is only a model for the other lactic acid producing
species of probiotic bacteria useful in the invention, and
therefore the invention is not to be considered as limiting and it
is intended that any of the lactic acid producing species of
probiotic bacteria can be used in the compositions, therapeutic
systems and methods of the present invention.
[0027] A Bacillus species is particularly suited for the present
invention due to the properties in common between species of the
Bacillus genus, including in particular the ability to form spores
which are relatively resistant to heat and other conditions, making
them ideal for storage (shelf-life) in product formulations, and
ideal for survival and colonization of tissues under conditions of
pH, salinity, and the like on tissues subjected to microbial
infection. Additional useful properties include non-pathogenic,
aerobic, facultative and heterotrophic, rendering these species
safe, and able to colonize gastrointestinal tissue, including
intestinal villi.
[0028] Because Bacillus spores are heat-resistant and additionally
can be stored as a dry power, they are particularly useful as a
prophylactic or for treatment of infection by bacteria associated
with SIDS by including the spores in infant formula, infant foods
and food supplements, infant rehydration and electrolyte
maintenance compositions and the like, which are generally
rehydrated and heated before feeding them to an infant. These
pressure-resistant spores are also suitable for use in
pressure-treated compositions such as pressed wafers and chewable
tablets.
[0029] It will be appreciated that B. coagulans is also useful as a
probiotic gastrointestinal treatment for children over the age of
one year who exhibit symptoms of gastrointestinal infection or
adults at risk of complications from intestinal infections (e.g.,
the elderly or immunocompromised individuals). For older children
and adults, B. coagulans is orally administered as a food
supplement mixed with food or drinks, a pressed wafer or chewable
tablet or similar well-known compositions suitable for oral
administration.
[0030] One aspect of the invention thus relates to inhibition of
growth of SIDS-associated bacteria in an infant. This inhibition
has value in promoting a healthy population of intestinal flora,
whether or not the inhibited organisms are ultimately the cause of
SIDS.
[0031] There are a variety of different Bacillus species useful in
the present invention, including, but not limited to many different
strains available through commercial and public sources, such as
the American Tissue Culture Collection (ATCC). For example,
Bacillus coagulans strains are available as ATCC Accession Numbers
15949, 8038, 35670, 11369, 23498, 51232, 11014, 31284, 12245, 10545
and 7050. Bacillus subtilis strains are available as ATCC Accession
Numbers 10783, 15818, 15819, 27505, 13542, 15575, 33234, 9943,
6051a, 25369, 11838, 15811, 27370, 7003, 15563, 4944, 27689, 43223,
55033, 49822, 15561, 15562, 49760, 13933, 29056, 6537, 21359,
23160, 7067, 21394, 15244, 7060, 14593, 9799, 31002, 31003, 31004,
7480, 9858, 13407, 21554, 21555, 27328 and 31524. Bacillus
laterosporus strains are available as ATCC Accession Numbers 6456,
6457, 29653, 9141, 533694, 31932 and 64, including Bacillus
laterosporus BOD. Bacillus laevolacticus strains are available as
ATCC Accession Numbers 23495, 23493, 23494, 23549 and 23492.
[0032] The growth of these various Bacillus species to form cell
cultures, cell pastes and spore preparations is generally well
known in the art. Exemplary culture and preparative methods are
described herein for Bacillus coagulans and can readily be used
and/or modified for growth of the other lactic acid producing
bacteria of this invention.
[0033] Exemplary methods and compositions are described herein
using Bacillus coagulans as a probiotic for controlling, treating
or reducing gastrointestinal bacterial infections.
[0034] A. Bacillus coagulans Compositions
[0035] The present invention describes the use of purified Bacillus
coagulans as an exemplary and preferred probiotic for biological
control of various bacterial infections in the intestinal
tract.
[0036] Because B. coagulans forms heat-resistant spores, this
species is particularly useful for making pharmaceutical
compositions for treating microbial infections. Formulations that
include viable B. coagulans spores cells in a pharmaceutically
acceptable carrier are particularly preferred for making and using
both preventive and therapeutic compositions.
[0037] B. coagulans is non-pathogenic and is generally regarded as
safe (i.e., GRAS. classification by the U.S. Food and Drug
Administration). The Gram positive rods have a cell diameter of
greater than 1.0 .mu.m with variable swelling of the sporangium,
without parasporal crystal production.
[0038] 1. Growth of B. coagulans
[0039] B. coagulans is aerobic and facultative, grown typically in
nutrient broth, pH 5.7 to 6.8, containing up to 2% (by wt) NaCl,
although neither NaCl nor KCl are required for growth. A pH of
about 4 to about 6 is optimum for initiation of growth from spores.
It is optimally grown at about 30.degree. C. to about 55.degree.
C., and the spores can withstand pasteurization. It exhibits
facultative and heterotrophic growth by utilizing a nitrate or
sulphate source. Additional metabolic characteristics of B.
coagulans are summarized in Table 1.
TABLE-US-00001 TABLE 1 Characteristic B. coagulans Response
Catalase production Yes Acid from D-Glucose Yes Acid from
L-Arabinose Variable Acid from D-Xylose Variable Acid from
D-Mannitol Variable Gas from Glucose Yes Hydrolysis of Casein
Variable Hydrolysis of Gelatin No Hydrolysis of Starch Yes
Utilization of Citrate Variable Utilization of Propionate No
Degradation of Tyrosine No Degradation of Phenylalanine No Nitrate
reduced to Nitrite Variable Allatoin or Urate Required No
[0040] B. coagulans can be grown in a variety of media, although it
has been found that certain growth conditions produce a culture
which yields a high level of sporulation. For example, sporulation
is enhanced if the culture medium includes 10 milligrams per liter
of manganese sulfate, yielding a ratio of spores to vegetative
cells of about 80:20. In addition, certain growth conditions
produce a bacterial spore which contains a spectrum of metabolic
enzymes particularly suited for the present invention, i.e.,
control of microbial infections. Although spores produced by these
particular growth conditions are preferred, spores produced by any
compatible growth conditions are suitable for producing a B.
coagulans useful in the present invention.
[0041] Suitable media for growth of B. coagulans include Nutristart
701, PDB (potato dextrose broth), TSB (tryptic soy broth) and NB
(nutrient broth), all well known and available from a variety of
sources. Media supplements containing enzymatic digests of poultry
and fish tissue, and containing food yeast are particularly
preferred. A preferred supplement produces a media containing at
least 60% protein, and about 20% complex carbohydrates and 6%
lipids. Media can be obtained from a variety of commercial sources,
notably DIFCO (Detroit, Mich.), Oxoid Newark, N.J.), BBL
(Cockeyesville, Md.) and Troy Biologicals (Troy, Mich.).
[0042] A preferred procedure for preparation of B. coagulans is
described in the Examples.
[0043] 2. Extracellular Products having Antimicrobial Activity.
[0044] B. coagulans cultures contain secreted products which have
antimicrobial activity. These secreted products are useful in
therapeutic compositions according to the present invention. Cell
cultures are harvested as described above, and the culture
supernatants are collected, by filtration or centrifugation, or
both, and the resulting supernatant contains antimicrobial activity
useful in a therapeutic composition. The preparation of a B.
coagulans extracellular product is described in the Examples.
[0045] Extracellular products of B. coagulans may be included in
compositions such as foods and liquids to be fed to infants.
[0046] 3. Sources of B. coagulans
[0047] Purified B. coagulans bacteria are available from the
American Type Culture Collection (Rockville, Md.) using the
following accession numbers: B. coagulans Hammer NRS T27
(ATCC#11014), B. coagulans Hammer strain C (ATCC#11369), B.
coagulans Hammer (ATCC#31284), and B. coagulans Hammer NCA 4259
(ATCC#15949). Purified B. coagulans bacteria are also available
from the Deutsche Sammlung von Mikroorganismen und Zellkuturen GmbH
(Braunschweig, Germany) using the following accession numbers: B.
coagulans Hammer 1915.sup.AL (DSM#2356), B. coagulans Hammer
1915.sup.AL (DSM#2383, corresponds to ATCC#11014), B. coagulans
Hammer.sup.AL (DSM#2384, corresponds to ATCC#11369), and B.
coagulans Hammer.sup.AL (DSM#2385, corresponds to ATCC#15949). B.
coagulans bacteria can also be obtained from commercial suppliers
such as Sabinsa Corporation (Piscataway, N.J.).
[0048] These B. coagulans strains and their growth requirements
have been described previously (Baker et al, Can. J. Microbiol.
6:557-563, 1960; Blumenstock, "Bacillus coagulans Hammer 1915 und
andere thermophile oder mesophile, sauretolerante
Bacillus-Arten-eine taxonomische Untersuchung", Doctoral thesis,
Univ. Gottingen, 1984; Nakamura et al, Int. J. Syst. Bacteriol.,
38:63-73, 1988). Strains of B. coagulans can also be isolated from
natural sources (e.g., heat-treated soil samples) using well known
procedures (Bergey's Manual of Systemic Bacteriology, Vol. 2, p.
1117, Sneath, P. H. A. et al., eds., Williams & Wilkins,
Baltimore, Md., 1986). The results described herein were obtained
with B. coagulans Hammer obtained from the American Type Culture
Collection (ATCC#31284) which was grown as described herein and
stored in lyophilized aliquots at -20.degree. C. All B. coagulans
that exhibit the properties described herein are considered
equivalents of this strain.
[0049] B. coagulans had previously been mischaracterized as a
Lactobacillus in view of the fact that as originally described,
this bacterium was labeled as Lactobacillus sporogenes (See
Nakamura et al, cited above). However, this was incorrect because
the bacterium of this invention produces spores and through
metabolism excretes L(+)-lactic acid, both aspects which provide
key features to its utility. Instead, these developmental and
metabolic aspects required that the bacterium be classified as a
lactic acid bacillus, and therefore it was renamed.
[0050] 4. Probiotic Antimicrobial Activity of B. coagulans
[0051] Pathogenic enteric bacteria inhibited by B. coagulans
activity include Staphylococcus aureus, S. epidermidis,
Streptococcus pyogenes, S. spp., Pseudomonas aeruginosa,
Escherichia coli (enterohemorragic species), Clostridium species
including C. perfingens, C. difficile, C. difficile, C. botulinum,
C. tributrycum, and C. sporogenes, Gardnerella vaginalis,
Propionibacterium acnes, Aeromonas hydrophilia, Aspergilha species,
Proteus species and Klebsiella species. These pathogens can cause a
variety of gastrointesinal disorders, including SIDS, and the like
conditions as are well known in the art. Therefore, use of
compositions containing a probiotic that inhibits these pathogens
are useful in preventing or treating conditions associated with
infection by these pathogens.
[0052] Although B. coagulans is exemplary, by virture of the common
properties of the lactic acid producing bacteria, a therapeutic
composition comprising a lactic acid bacterium of this invention
can be used against many of the above-described pathogens. In
addition, it is contemplated that the present therapeutic
compositions can be used, when formulated for oral administration
to the intestinal tissue, to treat infections by bacteria
associated with SIDS.
[0053] B. Bifidogenic Oligosaccharides
[0054] Bifidogenic oligosaccharides, as used in the context of the
present invention, are a class of sugars particularly useful for
preferentially promoting the growth of a lactic acid bacteria of
this invention. These oligosaccharides include
fructo-oligosaccharides (FOS), gluco-oligosaccharides (GOS), and
other long-chain oligosaccharide polymers that are not readily
digested by pathogenic bacteria. The preferential growth is
promoted due to the nutrient requirements of this class of lactic
acid bacterium as compared to pathogenic bacteria. Bifidogenic
oligosaccharides are long chain polymers that are utilized almost
exclusively by the indigenous Bifidobacteria and Lactobacillus in
the intestinal tract and can be similarly utilized by Bacillus.
Deleterious bacteria such as Clostridium, Staphylococcus,
Salmonella and E. Coli cannot metabolize FOS or other bifidogenic
oligosaccharides, and therefor use of these bifidogenic
oligosaccharides in combination with a lactic acid bacteria of this
invention, particularly Bacillus, allows the beneficial and
probiotic bacteria to grow and to replace any undesirable or
pathogenic microorganisms.
[0055] The use of bifidogenic oligosaccharides in therapeutic
compositions of the present invention provides a synergistic effect
thereby increasing the effectiveness of the probiotic-containing
compositions of this invention. This synergy is manifest at least
by increasing the ability of the bacterium to grow by increasing
the food supplement for probiotic bacteria which preferentially
selects for growth of the probiotic bacteria over many other
bacterial species in the infected tissue. Thus, the presence of the
bifidogenic oligosaccharides in the formulation allows for more
effective microbial inhibition by increasing the ability of the
probiotic bacteria to grow and therefore provide its benefit.
[0056] The bifidogenic oligosaccharide can be used either alone or
in combination with a lactic acid bacterium in a therapeutic
composition. That is, due to the growth promoting activity of
bifidogenic oligosaccharides, the invention contemplates a
composition comprising a bifidogenic oligosaccharide of this
invention in a lactic acid bacterium growth-promoting amount. As
shown herein, these amounts can vary widely since the probiotic
will respond to any metabolic amount of nutrient oligosaccharide,
and therefore the invention need not be so limited.
[0057] A preferred and exemplary bifidogenic oligosaccharide is
FOS, although the other sugars can also be utilized, either alone
or in combination.
[0058] FOS can be obtained from a variety of natural sources,
including commercial suppliers. As a product isolated from natural
sources, the components can vary widely and still provide the
beneficial agent, namely FOS. FOS typically has a polymer chain
length of from about 4 to 200 sugar units, with the longer lengths
being preferred. For example, the degree of purity can vary widely
so long as functional FOS is present in the formulation. Preferred
FOS formulations contain at least 50% by weight of
fructooligosaccharides compared to simple (mono or disaccharide)
sugars such as glucose, fructose or sucrose, preferably at least
80% fructooligosaccharides, more preferably at least 90% and most
preferably at least 95% fructooligosaccharides. Sugar content and
composition can be determined by any of a variety of complex
carbohydrate analytical detection methods as is well known.
[0059] Preferred sources of FOS include insulin, Frutafit IQ.TM.
from Imperial Suiker Unie (Sugar Land, Tex.), NutraFlora.TM. from
Americal Ingredients, Inc., (Anaheim, Calif.), Fabrchem, Inc.,
(Fairfield, Conn.), and Fruittrimfat Replacers and Sweeteners
(Emeryville, Calif.). Bifidogenic oligosaccharides such as GOS, and
other long chain oligosaccharides are also available from
commercial vendors.
[0060] C. Therapeutic Compositions
[0061] Compositions of this invention suitable for use in
preventing, treating or controlling gastrointestinal bacterial
infections, particularly infant bacterial infections, by organisms
capable of producing enterotoxin and infections associated with
SIDS include live probiotic lactic acid producing bacteria
according to the present invention, provided in the form of colony
forming units (CFU's) of vegetative cells and/or spores,
extracellular antibiotic metabolites of B. coagulans, or
combinations thereof.
[0062] The active ingredients, i.e., live bacteria or extracellular
components, comprise about 0.1% to about 50% by weight of the final
composition, preferably 1% to 10% by weight, in a formulation
suitable for use in making infant formula, added to food, or used
directly as a food supplement for infants (e.g. as a powder mixed
with infant formula or in a flavored buffered solution administered
with a dropper applicator, similar to that used for liquid infant
vitamins).
[0063] The formulation for a therapeutic composition of this
invention may include other probiotic agents or nutrients for
promoting spore germination and/or bacterial growth. A particularly
preferred material is a bialogenic factor which promotes growth of
beneficial probiotic bacteria as described herein. The compositions
may also include known antimicrobial agents, known antiviral
agents, known antifungal agents, all of which must be compatible
with maintaining viability of the Bacillus active agent when
Bacillus organisms or spores are the active agent. The other agents
in the compositions can be either synergists or active agents.
Preferably, the known antimicrobial, antiviral and/or antifungal
agents are probiotic agents compatible with Bacillus. The
compositions may also include known antioxidants, buffering agents,
and other agents such as coloring agents, flavorings, vitamins or
minerals. Thickening agents may be added to the compositions such
as polyvinylpyrrolidone, polyethylene glycol or
carboxymethylcellulose.
[0064] Preferred additional components of a therapeutic composition
of this invention can include assorted colorings or flavorings well
known in the art, vitamins, fiber, enzymes and other nutrients.
Preferred vitamins include vitamins B, C, D, E, folic acid, K,
niacin, and the like vitamins. Preferred sources of fiber include
any of a variety of sources of fiber including psyllium, rice bran,
oat bran, corn bran, wheat bran, fruit fiber and the like fibers.
Dietary or supplementary enzymes such as lactase, amylase,
glucanase, catalase, and the like enzymes can also be included.
[0065] Exemplary vitamins are used in the composition as follows:
choline (160 mg/lb), B-6 (10 mg/lb), B-12 (2 ug/lb), niacin (120
mg/lb), pantothenic acid (4 mg/lb), riboflavin (12 mg/lb), inositol
(1 gm/lb), thiamine (1.5 mg/lb), folic acid (0.5 mg/lb), and the
like.
[0066] Chemicals used in the present compositions can be obtained
from a variety of commercial sources, including Spectrum Quality
Products, Inc (Gardena, Calif.), Seltzer Chemicals, Inc.,
(Carlsbad, Calif.) and Jarchem Industries, Inc., (Newark,
N.J.).
[0067] The active agents are combined with a carrier that is
physiologically compatible with oral administration. That is, the
carrier is preferably substantially inactive except for surfactant
properties used in making a suspension of the active ingredients.
The compositions may include other physiologically active
constituents that do not interfere with the efficacy of the active
agents in the composition.
[0068] Specifically, probiotic lactic acid bacterium include viable
bacteria or spores (cumulatively referred to as "colony forming
units") that can be ingested to form part of the gut microflora of
an infant (generally two week to six month old).
[0069] A typical therapeutic composition will contain in a one gram
dosage formulation from 10.sup.3 to 10.sup.12, preferably
2.times.10.sup.5 to 10.sup.10, colony forming units (CFU) of viable
lactic acid bacterium (i.e., vegetative cell) or bacterial spore.
In one preferred embodiment a therapeutic composition may include
from about 10 milligrams (mg) to one gram of a bifidogenic
oligosaccharide, preferably a fructooligosaccharide. The
formulation may be completed in weight using any of a variety of
carriers and/or binders. A preferred carrier is micro-crystalline
cellose (MCC) added in an amount sufficient to complete the one
gram dosage total weight. Particularly preferred formulations for a
therapeutic composition of this invention are described in the
Examples.
[0070] In a related embodiment, the invention contemplates a
therapeutic composition comprising a bifidogenic oligosaccharide.
The composition typically contains a lactic acid bacterium
growth-promoting amount of the bifidogenic oligosaccharide, which
growth-promoting amount can vary widely and be readily measured by
growth assays as described herein. The composition will typically
contain 10 mg to 1 gm of bifidogenic oligosaccharide per gram of
composition depending on the dosage, route of administration and
intended usage.
[0071] Carriers can be solid-based dry materials for formulations
in powdered form, and can be liquid or gel-based materials for
formulations in liquid or gel forms, which forms depend, in part,
upon the routes or modes of administration.
[0072] Typical carriers for dry formulations include trehalose,
malto-dextrin, rice flour, micro-crystalline cellulose (MCC),
magnesium sterate, inositol, FOS, glucooligosaccharides (GOS),
dextrose, sucrose, talc, and the like carriers.
[0073] Where the composition is dry and includes evaporated oils
that produce a tendency for the composition to cake (adherence of
the component spores, salts, powders and oils), it is preferred to
include dry fillers which distribute the components and prevent
caking. Exemplary anti-caking agents include MCC, talc,
diatomaceous earth, amorphous silica and the like, typically added
in an amout of from about 1 to 95% by weight.
[0074] Dry formulations that are rehydrated (e.g., infant formula,
fruit flavored drink mix) or given to the infant in the dry state
(e.g., chewable wafers, teething tablets) are preferred to hydrated
formulations. Dry formulations (e.g., powders) may be added to
supplement commercially available foods (e.g., infant formulas,
strained prepared foods, ice cream or ice milk). The type of
formulation appropriate for the infant will be readily determined
by the parent or care-giver, but generally liquid formulations
(e.g, electrolyte compositions and infant formula) are suitable for
younger infants (about four months of age or less) and solid
formulations are suitable for older infants (about four to six
months or older). For compositions that are given to an infant in
liquid form, the B. coagulans spores are preferably included in
infant formula, infant food or food supplement, infant rehydration
and electrolyte maintenance compositions and similar types of
compositions that are rehydrated before use. These may be heated
(up to about 55.degree. C.) and cooled before use.
[0075] The carrier is preferably a formulation in which, for
example, B. coagulans can be suspended, more preferably for
hydration by the user before it is fed to the infant. For example,
the formulation may be any standard powdered infant formula in
which B. coagulans spores are mixed and suspended, which is then
prepared (hydrated) before use. Similarly, B. coagulans spores may
be suspended in a powdered rehydration formulation that includes
glucose, potassium citrate, sodium chloride and/or sodium citrate
to which water is added before use to produce a solution
containing, for example, about 5.times.10.sup.5 to 5.times.10.sup.7
CFU of bacteria/145 to 75 mEq/l of sodium, 20 mEq/l of potassium,
35 to 65 mEq/l of chloride, 30 mEq/l of citrate and 25 g/l of
glucose.
[0076] Suitable liquid or gel-based carriers are well known in the
art, such as water and physiological salt solutions, urea, alcohols
and glycols such as methanol, ethanol, propanol, butanol, ethylene
glycol and propylene glycol, and the like. Preferably, water-based
carriers are about neutral pH.
[0077] Suitable liquid carriers are well known in the art, such as
water, fruit juice, glucose or fructose solutions, physiological
electrolyte solutions, and the like, which may be stored
refrigerated or frozen (e.g., as frozen popsicles). Preferably,
water-based carriers are about neutral pH. The compositions may
also include natural or synthetic flavorings and food-quality
coloring agents, all of which must be compatible with maintaining
viability of the lactic acid bacterium. Well known thickening
agents may be added to the compositions such as corn starch, guar
gum, xanthan gum and the like.
[0078] Where a liquid-based composition containing spores is
provided, it is desirable to include a spore germination inhibitor
to promote long term storage. Any inhibitor can be used, and
therefore the invention is not to be construed as limiting. Typical
and preferred inhibitors include hyper-saline carriers,
methylparaben, guargum, polysorbates, preservatives, and the like
germination inhibitors well known in the art.
[0079] Suitable carriers include aqueous and oleaginous carries
such as, for example, white petrolatum, isopropyl myristate,
lanolin or lanolin alcohols, mineral oil, fragrant or essential
oil, nasturtium extract oil, sorbitan mono-oleate, propylene
glycol, cetylstearyl alcohol (together or in various combinations),
hydroxypropyl cellulose (MW=100,000 to 1,000,000), detergents
(e.g., polyoxyl stearate or sodium lauryl sulfate) and mixed with
water to form a lotion, gel, cream or semi-solid composition. Other
suitable carriers comprise water-in-oil or oil-in-water emulsions
and mixtures of emulsifiers and emollients with solvents such as
sucrose stearate, sucrose cocoate, sucrose distearate, mineral oil,
propylene glycol, 2-ethyl-1,3-hexanediol,
polyoxypropylene-15-stearyl ether and water. For example, emulsions
containing water, glycerol stearate, glycerin, mineral oil,
synthetic spermaceti, cetyl alcohol, butylparaben, propylparaben
and methylparaben are commercially available. Preservatives may
also be included in the carrier including methylparaben,
propylparaben, benzyl alcohol and ethylene diamine tetraacetate
salts. Well-known flavorings and/or colorants may also be included
in the carrier. The composition may also include a plasticizer such
as glycerol or polyethylene glycol (MW=800 to 20,000). The
composition of the carrier can be varied so long as it does not
interfere significantly with the pharmacological activity of the
active ingredients or the viability of the lactic acid bacterium or
Bacillus spores.
[0080] A therapeutic composition can be formulated to be suitable
for oral administration in a variety of ways, for example in a
liquid, a powdered food supplement, a solid food, a packaged food,
a wafer, and the like as described in more detail in the Examples.
Other formulations will be readily apparent to one skilled in the
art.
[0081] D. Therapeutic Methods for Treating Bacterial Infections
[0082] The present invention contemplates a method for treating,
reducing or controlling gastrointestinal bacterial infections using
a therapeutic composition or therapeutic system of this invention.
The disclosed methods of treatment inhibit pathogenic bacterial
growth associated with gastrointestinal infections and also reduce
symptoms of these pathogenic infections.
[0083] Probiotic lactic acid bacterium, particularly B. coagulans,
are generally regarded as safe by those skilled in the art and,
therefore, suitable for ingestion in food stuffs or as a food
supplement.
[0084] The method of the present invention comprises administration
of a composition containing a viable lactic acid bacteria to the
gastrointestinal tract of a human or animal to treat or prevent
bacterial infection. Administration is preferably made using a
liquid, powder, solid food and the like formulation compatible with
oral administration, all formulated to contain a therapeutic
composition of this invention using methods well known in the
art.
[0085] The method of the present invention includes administration
of a composition containing lactic acid bacterium cells and/or
spores or isolated extracellular B. coagulans antibiotic metabolite
to a human or animal to treat or prevent symptoms associated with
enterotoxin production in the gut. In particular, for human
infants, the method includes administering to the infant, for
example, B. coagulans in food or as a food supplement. Oral
administration is preferably in an aqueous suspension, emulsion,
powder or solid, either already formulated into a food or as a
composition which is added to food by the user. Administration to
the gut may also be in the form of an anal suppository (e.g., in a
gel or semi-solid formulation). All such formulations are made
using standard methods.
[0086] Administration of a therapeutic composition is preferably to
the gut using a gel, suspension, aerosol spray, capsule, tablet,
powder or semi-solid formulation (e.g., a suppository) containing a
therapeutic composition of this invention, all formulated using
methods well known in the art.
[0087] Administration of the compositions containing the active
probiotic lactic acid bacterium effective in preventing or treating
a bacterial infection generally consist of one to ten dosages of 10
mg to 10 g of a composition per dosage for one day up to one month.
Administrations are generally once every twelve hours and up to
once every four hours. Preferably two to four administrations of
the composition per day, of about 0.1 g to 5 g per dose, for one to
seven days are sufficient to prevent or treat a bacterial
infection. Of course, the specific route, dosage and timing of the
administration will depend, in part, on the particular pathogen
and/or condition being treated and the extent of the condition.
[0088] A preferred method involves the administration of from
10.sup.3 to 10.sup.12 viable bacteria or spore per day, preferably
from 10.sup.5 to 10.sup.10, and more preferably about from
5.times.10.sup.8 to 10.sup.9 viable bacteria or spores per day.
Where the condition to be treated is SIDS and the patient is an
infant under 6 months old, the dosage is typically 10.sup.3 to
10.sup.6, preferably about 5,000 to 105, and more preferably about
10,000 to 50,000 viable CFU of bacteria or spores per day. Where
the condition to be treated is SIDS and the patient is an infant
over 6 months old, the dosage is typically 10.sup.6 to 10.sup.9,
preferably about 50,000 to 250,000 and more preferably about
150,000 to 200,000 viable CFU of bacteria or spores per day.
[0089] In addition, the invention contemplates a method that
comprises oral administration of a composition that contains from
10 mgs to 20 gms of a bifidogenic oligosaccharide, preferably a
fructooligosaccharide, per day, preferably about 50 mg-10 gm, and
more preferably about from 150 mgs to 5 gms per day, to promote
growth of the probiotic lactic acid bacterium preferentially over
the growth of the pathogen. The method can be combined with
treatment methods using a probiotic lactic acid bacterium as
described herein.
[0090] Specific methods for treating a bacterial infection are
described in the Examples, and include sudden infant distress
syndrome (SIDS), and the like.
[0091] E. Therapeutic Systems for Treating Bacterial Infections
[0092] The invention further contemplates a therapeutic system for
treating, reducing and/or controlling bacterial infections
comprising a container comprising a label and a therapeutic
composition according to the present invention, wherein said label
comprises instructions for use of the composition for treating said
infection.
[0093] Typically, the system is present in the form of a package
containing a therapeutic composition of this invention, or in
combination with packaging material. The packaging material
includes a label or instructions for use of the components of the
package. The instructions indicate the contemplated use of the
packaged component as described herein for the methods or
compositions of the invention.
[0094] For example, a system can comprise one or more unit dosages
of a therapeutic composition according to the invention.
Alternatively, the system can contain bulk quantities of a
therapeutic composition. The label contains instructions for using
the therapeutic composition in either unit dose or in bulk forms as
appropriate, and may include information regarding storage of the
composition, disease indications, dosages, routes and modes of
administration and the like information.
[0095] Furthermore, depending upon the particular contemplated use,
the system may optionally contain either combined or in separate
packages one or more of the following components: bifidogenic
oligosaccharides, flavorings, carriers, and the like components.
One particularly preferred system comprises unit dose packages of
Bacillus spores for use in combination with a conventional infant
liquid formula product, together with instructions for combining
the probiotic with the formula for use in a therapeutic method.
[0096] Unless defined otherwise, all scientific and technical terms
used herein have the same meaning as commonly understood by those
skilled in the relevant art. Unless mentioned otherwise, the
techniques employed or contemplated herein are standard
methodologies well known to one of ordinary skill in the art. The
examples of embodiments are for illustration only.
[0097] Throughout the specification and the claims that follow,
unless the context requires otherwise, the term "comprise" and its
variations, will be understood to have an inclusive meaning of any
stated element, but not the exclusion of unstated elements.
EXAMPLES
[0098] The following examples relating to this invention are
illustrative and should not, of course, be construed as
specifically limiting the invention. Moreover, such variations of
the invention, now known or later developed, which would be within
the purview of one skilled in the art are to be considered to fall
within the scope of the present invention hereinafter claimed.
Example 1
Preparation of B. coagulans Cultures
[0099] B. coagulans Hammer bacteria (ATCC#31284) was inoculated and
grown to a cell density of about 10.sup.8*-10.sup.9 cells/ml in
nutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg
MnSO.sub.4 and 1,000 ml distilled water, adjusted to pH 7.0, using
a standard airlift fermentation vessel at 30.degree. C. The range
of MnSO.sub.4 acceptable for sporulation is 1 mg/l to 1 gl. The
vegetative cells can actively reproduce up to 65.degree. C., and
the spores are stable up to 90.degree. C. After fermentation, the
B. coagulans Hammer bacterial cells or spores are collected using
standard methods (e.g., filtration, centrifugation) and the
collected cells and spores can be lyophilized, spray dried, air
dried or frozen. As described herein, the supernatant from the cell
culture can be collected and used as an extracellular agent
secreted by B. coagulans which has antimicrobial activity useful in
a formulation of this invention.
[0100] A typical yield from the above culture is in the range of
about 10.sup.9-10.sup.13 viable spores and more typically about 100
to 150 billion cells/spores per gram before drying. Spores maintain
at least 90% viability after drying when stored at room temperature
for up to seven years, and thus the effective shelf life of a
composition containing B. coagulans Hammer spores at room
temperature is about 10 years.
Example 2
Preparation of B. coagulans Spores
[0101] A culture of dried B. coagulans spores was alternately
prepared as follows. Ten million spores were innoculated into a one
liter culture containing 24 gms potato dextrose broth, 10 gms of
enzymic digest of poultry and fish tissue, 5 gms of FOS and 10 gms
MnSO.sub.4. The culture was maintained for 72 hours under a high
oxygen environment at 37 degrees Centigrade to produce culture
having about 150 billion cells per gram of culture. Thereafter, the
culture was filtered to remove culture medium liquid, and the
bacterial pellet was resuspended in water and freeze-dried. The
freeze-dried powder is then ground to a fine powder using standard
good manufacturing practice (GMP).
Example 3
Preparation of B. Coagulans Extracellular Products
[0102] A one liter culture of B. coagulans was prepared as
described in Example 1. The culture was maintained for 5 days as
described, at which time FOS was added at 5 gm/liter, and the
culture was continued. 20 ml of carrot pulp was then added at day
7, and the culture was harvested when the culture became saturated
(no substantial cell division). The culture was first autoclaved
for 30 minutes at 250 degrees Farenheight, and then centrifuged at
4000 rpm for 15 min. The resulting supernatant was collected and
filtered in a Buchner funnel through a 0.8 micron (u) filter, and
the filtrate (pass through) was collected and further filtered
through a 0.2 u Nalge vacuum filter. The resulting pass-through was
collected (about 900 milliliters) to form a liquid containing an
extracellular product, and used in inhibition studies.
[0103] Following the assay described in Example 4, except using
Candida albicans, one milliliter of the above-produced
extracellular product was added to the test plate in place of live
B. coagulans. After the same culturing time, a zone of inhibition
of about 10 to 25 millimeters was observed, indicating a potent
antimicrobial activity of "excellent" quality, using the
terminology of Example 4.
Example 4
Antimicrobial Activity of B. coagulans
[0104] The ability of B. coagulans to inhibit bacterial pathogens
was demonstrated using an in vitro assay. The assay is part of a
standard bacterial pathogen screen (U.S. Food and Drug
Administration) and is commercially available on solid support
disks (DIFCO.RTM. BACTROL.RTM. disk set). In the assay,
potato-dextrose plates (DIFO.RTM.) were prepared using standard
procedures and were inoculated individually with a confluent bed
1.5.times.10.sup.6 of each species of bacteria tested. Inhibition
by B. coagulans was tested by placing on the plate about
1.5.times.10.sup.6 CFU in 10 .mu.l of broth or buffer, plated
directly in the center of the potato-dextrose plate with one test
locus of about 8 mm in diameter per plate. A minimum of three test
loci were used for each assay. The negative control was a 10 .mu.l
drop of a sterile saline solution and the positive control was a 10
.mu.l volume of glutaraldehyde. The plates were then incubated for
about 18 hr at 30.degree. C. when the zone of inhibition was
measured. As used herein, "excellent inhibition" means the zone was
10 mm or greater in diameter; and "good inhibition" means the zone
was greater than 2 mm in diameter but less than 10 mm in
diameter.
[0105] No inhibition was seen with the negative control and
excellent inhibition (about 16.2 mm diameter, average of three
tests) was seen with the positive control. For the enteric
organisms tested, Clostridium species and E. coli, excellent
inhibition by B. coagulans was seen. For the Clostridium species,
C. perfringens, C. difficile, C. botulinum, C. tributlycum and C.
sporogenes, the zone of inhibition was consistently greater than 15
mm in diameter. Similarly, excellent inhibition was also seen for
the opportunistic pathogens Pseudomonas aeruginosa and
Staphylococcus aereus.
Example 5
B. coagulans in Oral Electrolyte Maintenance Solution
[0106] An oral electrolyte maintenance powder is formulated to
contain sodium chloride, potassium citrate, citric acid, glucose
and powdered B. coagulans spores (prepared substantially as
described in Example 2) to be rehydrated with sterile or boiled
(and cooled) water. After rehydration, the final concentrations
are: 45 to 75 mEq/l of sodium, 20 mEq/l of potassium, 35 to 65
mEq/l of chloride, 30 tnEq/l of citrate, 20-25 g/l of glucose and
5.times.10.sup.5 to 5.times.10.sup.7 spores/l. Flavoring (e.g.,
cherry, orange, grape or bubble gum flavor) may be included using
standard commercially available flavorings. The powdered
formulation is packaged preferably for rehydration to one fluid
liter or in individual aliquots (e.g., individual packets for
rehydration to 100 ml). The powdered formula is stored dry at room
temperature until it is rehydrated. The rehydrated solution is
stored refrigerated for up to one week. The rehydrated solution may
also be frozen into cubes or popsicles and stored at -5.degree. C.
to -20.degree. C. for up to six months.
Example 6
B. coagulans in an Inert Carrier as a Food Supplement
[0107] Freeze dried B. coagulans (prepared substantially as
described in Example 1) is mixed thoroughly with an inert carrier
in powdered form (e.g., rice maltodextrin, sorbitol, gelatin,
powdered rolled oats, corn starch and the like, or a combination of
carriers) to form a suspension having a final concentration of
about 10.sup.5 to 10.sup.8 spores/g. The powdered suspension is
added to water, milk, infant formula, fruit juice or similar
liquids at about 0.1-0.5 g/100 ml and mixed before providing to the
infant orally.
Example 7
B. coagulans in a Solid Wafer Formulation
[0108] Freeze dried B. coagulans (prepared substantially as
described in Example 1) was mixed thoroughly with a wheat or
oat-based mixture (wheat or oat flour containing water and
optionally sodium chloride, glucose and/or sodium bicarbonate and
preservatives) to a final concentration of about 10.sup.6 to
10.sup.9 spores/g. The mixture is pressed into thin wafers of about
0.1 g each and dried or baked at about 50.degree. C. for about 1-10
min to produce a relatively dry wafer that is stored at room
temperature for up to one year. In an alternative formulation, the
above mixture further contains 150 international units (IU) of
lactase per wafer. Flavorings such as raspberry or orange are added
to taste.
Example 8
Efficacy of B. coagulans Spores in Animal Model of SIDS
[0109] Experimental New Zealand white rabbits (1-3 kg) are provided
with B. coagulans spores in their water supply at a concentration
of 10.sup.3 spores/ml for one week under standard laboratory animal
conditions (food and water at will for days -7 to -1). Positive
control animals receive food and water (without B. coagulans
spores) for the same period. At day 0, experimental rabbits are
injected i.p. with 5 ml of a physiological buffered salt solution
containing 10.sup.8-10.sup.9 C. perfringens Type A cells (Group I)
or 10.sup.8-10.sup.9 C. difficile cells (Group II), and
experimental control rabbits (Group III) are mock injected i.p.
with 5 ml of a sterile physiological buffered salt solution.
Positive control animals are similarly injected i.p.: Group IV with
10.sup.8-10.sup.9 C. perfringens Type A cells, Group V with
10.sup.8-10.sup.9 C. difficile cells, and Group VI are mock
injected. Each group contains 10 rabbits. All of the rabbits
continue to receive normal laboratory care and water containing
10.sup.3 B. coagulans spores/ml (for Groups I-III) or without
spores (Groups IV-VI). After injection at day 0, the animals are
monitored hourly for behavior (lethargy), breathing and heart rate
for the next three days (days 1-3). The Group III control animals
all remain normal for all parameters for the entire period. Group I
animals generally appear to be lethargic beginning about 2-3 hr
after injection. Some of the Group I animals exhibit shallow
breathing and decreased heart by 4-6 hr post-injection and quietly
die at 6 hr and 7 hr post-injection. Group II animals appear to be
lethargic beginning about 2-3 hr after injection but recover and
appear to be normal for all parameters by 4-6 hr post-injection
until the end of the monitoring period at day 3. Group III and
group VI animals appear to be normal for all of days 1-3. Group IV
and V animals all appear to be lethargic about 1-3 hr
post-injection, with decreasing breathing and heart rate until
death at 2-6 hr post-injection.
[0110] Thus, in this animal model, oral administration of B.
coagulans spores significantly prevents SIDS symptoms and death of
the animals injected with C. perfringens or C. difficile.
Example 9
Treatment of Infant Botulism with Orally Administered B.
coagulans
[0111] Infants aged 3 weeks to 6 months who are admitted to a
medical facility with intestinal disorders having any of a variety
of symptoms (vomiting, diarrhea, lethargy or flaccid paralysis,
poor appetite, shallow breathing, fever) are tested for presence of
botulinum toxin using the mouse toxin neutralization test (Amon S.
S. et al., Lancet i:1273-1277, 1978). The infants are treated with
oral rehydration using an oral electrolyte maintenance powder
dissolved in sterile water substantially as described in Example 5.
Upon admittance, samples from the infants are tested to determine
if a heat-labile substance that can be neutralized with antitoxin
specific for C. botulinum Type A toxin is present. Briefly,
undiluted serum or a buffer extract of colon contents obtained from
each infant are divided into aliquots and one aliquot is heated to
100.degree. C. for 10 min, one aliquot is untreated, and a third
aliquot is treated with trypsin to increase toxicity. The three
aliquot (about 0.5 ml each) are injected i.p. into mice, which died
within 24 hr if C. botulinum Type A toxin is present. For those
samples which tested positive for heat-labile toxin, the presence
of C. botulinum Type A toxin is confirmed by repeating the assay
using antitoxin-neutralized samples (which do not kill the mice).
At days two and three of treatment, fecal or colon contents samples
are tested for C. botulinum Type A toxin using the same assay.
[0112] Infants are given the oral electrolyte maintenance solution
containing B. coagulans at about 5.times.10.sup.5 spores/l as soon
as possible after admittance and during the first 4-6 hr of
admittance. Infants are provided with the oral electrolyte
maintenance solution as follows. Infants up to 5 kg (11-12 lb) are
given about 200-250 ml of the oral electrolyte maintenance
solution; infants of about 6 kg (12-15 lb) are given about 300-350
ml; infants of about 8 kg (15-20 lb) are given about 400-450 ml;
and infants of about 10 kg (20-25 lb) or more are given about 500
ml. Thereafter, during the first 24 hr of admittance infants are
orally rehydrated as needed as determined by the treating
physician. During the 2-7 days following admittance, the infants
are given sufficient oral electrolyte maintenance solution
containing B. coagulans spores to administer about 5.times.10.sup.5
spores/day.
[0113] Infants having confirmed infant botulism upon admittance
respond positively to oral rehydration and none show evidence of C.
botulinum Type A toxin in fecal or colon contents samples collected
after one or two days of treatment.
Example 10
Efficacy of B. coagulans in Preventing SIDS in Human Infants
[0114] Because SIDS does not present symptoms in advance, a human
study of prevention of SIDS relies on statistical analysis of human
infants. At the beginning of the study, two groups of 500 infants
each, at risk of SIDS because of maternal smoking, are followed by
regular medical checkups from the ages of two weeks to eight
months. Group I is given a daily dose of B. coagulans spores in
water or infant formula (10.sup.5 spores for infants of two weeks
to two months old, 10.sup.6 spores for infants of nine weeks to six
months old, and a weekly dose of 10.sup.7 spores for infants over
six months old to eight months old). Group II, the age-matched
controls, are given substantially the same amounts of water and
infant formula (I.e., normal nutritional requirements, without B.
coagulans spores). Another control group (Group III) includes any
infants who are not included in Groups I or II but, during the
course of the study, die with SIDS and are necropsied at the same
medical facility. This third group is age-matched to the infants
initially included in the study, but includes infants between 1-5
months of age.
[0115] During the course of the study, fecal samples are collected
and analyzed weekly and serum samples are collected and analyzed
monthly for Groups I and II. For Group III, fecal and serum samples
are obtained as soon as possible during necropsy and analyzed
thereafter. All samples are stored at -20.degree. C. until analyzed
if they are not analyzed within one hr of collection and are stored
on ice (0.degree. C.) if not frozen upon collection. Serum samples
are analyzed for the presence of heat labile toxin (substantially
as described in Example 6), and for toxins from C. perfringens, C.
difficile, C. botulinum and S. aureus using immunoassays
substantially as previously described (Murrell W. G et al., J. Med.
Microbiol. 39:114-127, 1993). Bacterial detection and enumeration
of fecal samples are performed using standard methods (Bergey's
Manual of Systemic Bacteriology, Vol. 1-2, Sneath, P. H. A. et al.,
eds., Williams & Wilkins, Baltimore, Md., 1986) and
substantially as previously described (Murrell W. G et al., J. Med.
Microbiol. 39:114-127, 1993). Heat labile toxin is fecal samples is
determined substantially as described in Example 9.
[0116] Infants in Group II correspond roughly to the age-matched
controls reported by Murrell W. G et al. (J. Med. Microbiol.
39:114-127, 1993) and have similar incidence of SIDS-associated
bacterial infections and toxins. The Group II infants are a larger
sample size than the age-matched controls reported by Murrell W. G
et al. (J. Med. Microbiol. 39:114-127, 1993) and are at higher risk
of SIDS due to maternal smoking and, therefore, are expected to
have a somewhat higher incidence of SIDS-associated bacterial
infections and toxins. Infants in Group II that show symptoms of
gastrointestinal infection and have confirmed presence of
SIDS-associated bacteria in fecal or colon-contents samples (C.
perfringens, C. difficile, C. botulinum or S. aureus) are
immediately withdrawn from the control Group II and administered an
oral electrolyte maintenance solution containing B. coagulans
spores, substantially as described in Example 9. Thereafter, these
treated infants continue to be administered foods or liquids
containing B. coagulans spores and are included in Group I
infants.
[0117] Infants in Group I survive the entire testing period and
have significantly fewer symptoms of gastrointestinal infections
compared to Group II. Bacterial counts in fecal samples from Group
I infants are significantly fewer for C. perfringens, C. docile, C.
botulinum and S. aureus compared to group II.
[0118] The Group III infants (SIDS victims) show significantly
higher frequency of gastrointestinal infection with C. perfringens,
C. difficile, C. botulinum or S. aureus and significantly higher
frequency of serum toxins than infants in either Group I or Group
II. Thus, although Group I infants would be expected to have at
least one death due to SIDS during the test period, the B.
coagulans probiotic appears to have effectively prevented SIDS and
to have significantly reduced the frequency at which
SIDS-associated bacteria or their toxins are detected.
[0119] The invention has been described in the above examples using
a variety of formulations, although it should be apparent that
various other carrier agents that are compatible with the probiotic
compositions may be substituted in the examples to give similar
results. Accordingly, the invention may be embodied in other
specific forms without departing from it in spirit. The examples
are to be considered in all respects only as illustrative and not
as restrictive, and the scope of the invention is indicated by the
claims that follow. All modifications which come within the meaning
and range of the lawful equivalency of the claims are to be
embraced within their scope.
* * * * *