U.S. patent application number 15/206950 was filed with the patent office on 2017-06-01 for compositions and methods for treating obesity and related disorders by characterizing and restoring mammalian bacterial microbiota.
This patent application is currently assigned to New York University. The applicant listed for this patent is New York University. Invention is credited to Martin J. BLASER, llseung Cho, Laura Cox.
Application Number | 20170151290 15/206950 |
Document ID | / |
Family ID | 45605700 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170151290 |
Kind Code |
A1 |
BLASER; Martin J. ; et
al. |
June 1, 2017 |
COMPOSITIONS AND METHODS FOR TREATING OBESITY AND RELATED DISORDERS
BY CHARACTERIZING AND RESTORING MAMMALIAN BACTERIAL MICROBIOTA
Abstract
The present invention relates to characterizing changes in
mammalian intestinal microbiota associated with associated with
high-fat and low-fat diets and with diets containing
hydroxypropylmethylcellulose (HPMC) and related methods for
diagnosing, preventing and treating obesity and related conditions
such as metabolic syndrome and diabetes mellitus. Therapeutic
methods of the invention involve the use of probiotics, and/or
prebiotics, and/or narrow spectrum antibiotics/anti-bacterial
agents that are capable of restoring healthy mammalian bacterial
intestinal microbiota.
Inventors: |
BLASER; Martin J.; (New
York, NY) ; Cox; Laura; (Brooklyn, NY) ; Cho;
llseung; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New York University |
New York |
NY |
US |
|
|
Assignee: |
New York University
New York
NY
|
Family ID: |
45605700 |
Appl. No.: |
15/206950 |
Filed: |
July 11, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13214034 |
Aug 19, 2011 |
9386793 |
|
|
15206950 |
|
|
|
|
61375678 |
Aug 20, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/135 20160801;
A61P 3/00 20180101; A61P 3/08 20180101; A61K 35/741 20130101; A61K
9/0031 20130101; A61K 45/06 20130101; A23L 33/10 20160801; A61P
3/04 20180101; A61K 9/0053 20130101; A61P 9/10 20180101; A61P 3/06
20180101 |
International
Class: |
A61K 35/741 20060101
A61K035/741; A61K 9/00 20060101 A61K009/00; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Research and development leading to certain aspects of the
present invention were supported, in part, by grants 1UL1RR029893
and R01DK098989 from the National Institutes of Health.
Accordingly, the U.S. government may have certain rights in the
invention.
Claims
1.-19. (canceled)
20. A method for promoting weight loss or preventing weight gain in
a mammal in need thereof comprising administering to the mammal a
therapeutically effective amount of a probiotic composition,
wherein said probiotic composition stimulates growth or metabolic
activity of at least one strain from the taxon selected from the
group consisting of Coprobacillus, Sporacetigenium, Holdemania,
Dorea, Blautia, Enterococcus, Erysipelotrichaceae, Clostridium
cocleatum, and Peptosteptococcaceae IS (PIS) in the intestinal
microbiota of the mammal.
21. A method for preventing or treating a disease in a mammal in
need thereof, wherein the disease is selected from the group
consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition
stimulates growth or metabolic activity of at least one strain from
the taxon selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae, Clostridium cocleatum, and
Peptosteptococcaceae IS (PIS) in the intestinal microbiota of the
mammal.
22-27. (canceled)
28. A method for promoting weight loss or preventing weight gain in
a mammal in need thereof comprising administering to the mammal (i)
a therapeutically effective amount of a narrow spectrum antibiotic
or (ii) a therapeutically effective amount of a probiotic
composition, wherein said narrow spectrum antibiotic or probiotic
composition inhibits growth or metabolic activity of at least one
strain from the taxon selected from the group consisting of
Johnsonella, Oscillibacter, Lachnospiraceae, Ruminococcaceae, and
Clostridiales in the intestinal microbiota of the mammal.
29. A method for preventing or treating a disease in a mammal in
need thereof, wherein the disease is selected from the group
consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal (i) a therapeutically effective amount
of a narrow spectrum antibiotic or (ii) a therapeutically effective
amount of a probiotic composition, wherein said narrow spectrum
antibiotic or probiotic composition inhibits growth or activity of
at least one strain from the taxon selected from the group
consisting of Johnsonella, Oscillibacter, Lachnospiraceae,
Ruminococcaceae, and Clostridiales in the intestinal microbiota of
the mammal.
30. (canceled)
31. The method of claim 28, wherein the probiotic composition
comprises at least one strain from the taxon selected from the
group consisting of Coprobacillus, Sporacetigenium, Holdemania,
Dorea, Blautia, Enterococcus, Erysipelotrichaceae, Clostridium
cocleatum, and Peptosteptococcaceae IS (PIS).
32-119. (canceled)
120. The method of claim 20, wherein the probiotic composition
comprises at least one strain from the taxon selected from the
group consisting of Coprobacillus, Sporacetigenium, Holdemania,
Blautia, Enterococcus, Erysipelotrichaceae, Clostridium cocleatum,
and Peptosteptococcaceae IS (PIS).
121. The method of claim 120, wherein said strain is selected from
the group consisting of live bacterial strains, spores and
conditionally lethal bacterial strains.
122. The method of claim 20, wherein the probiotic composition is
administered conjointly with a prebiotic composition which
stimulates growth and/or metabolic activity of bacteria contained
in the probiotic composition.
123. The method of claim 122, wherein the probiotic and prebiotic
compositions are administered in one composition, or simultaneously
as two separate compositions, or sequentially.
124. The method of claim 20, wherein the probiotic composition is
administered orally or rectally.
125. The method of claim 20, wherein the probiotic composition is
administered in a form of a capsule or in a form of a
suppository.
126. The method of claim 20, wherein the mammal is human.
127. The method of claim 21, wherein the probiotic composition
comprises at least one strain from the taxon selected from the
group consisting of Coprobacillus, Sporacetigenium, Holdemania,
Dorea, Blautia, Enterococcus, Erysipelotrichaceae, Clostridium
cocleatum, and Peptosteptococcaceae IS (PIS).
128. The method of claim 127, wherein said strain is selected from
the group consisting of live bacterial strains, spores and
conditionally lethal bacterial strains.
129. The method of claim 21, wherein the probiotic composition is
administered conjointly with a prebiotic composition which
stimulates growth and/or metabolic activity of bacteria contained
in the probiotic composition.
130. The method of claim 129, wherein the probiotic and prebiotic
compositions are administered in one composition, or simultaneously
as two separate compositions, or sequentially.
131. The method of claim 21, wherein the probiotic composition is
administered orally or rectally.
132. The method of claim 21, wherein the probiotic composition is
administered in a form of a capsule or in a form of a
suppository.
133. The method of claim 21, wherein the mammal is human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S. patent
application Ser. No. 13/214,034, filed on Aug. 19, 2011, now U.S.
Pat. No. 9,386,793, which claims the benefit of U.S. Provisional
Patent Application No. 61/375,678, filed on Aug. 20, 2010, both of
which applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to characterizing changes in
mammalian intestinal microbiota associated with high-fat and
low-fat diets and with diets containing
hydroxypropylmethylcellulose (HPMC) and related methods for
diagnosing, preventing and treating obesity and related conditions
such as metabolic syndrome and diabetes mellitus. Therapeutic
methods of the invention involve the use of probiotics, and/or
prebiotics, and/or narrow spectrum antibiotics/anti-bacterial
agents that are capable of restoring healthy mammalian bacterial
intestinal microbiota.
BACKGROUND OF THE INVENTION
[0004] Obesity has become widespread with increases in prevalence
across all developed nations (Bouchard, C (2000) N Engl J Med. 343,
1888-9). According to the Center for Disease Control (CDC), over
60% of the United States population is overweight, and greater than
30% are obese. For affected persons, the problem often begins in
childhood, and continues for life. Major contributors are believed
to be increased consumption of high calorie foods and a more
sedentary life style. However, neither of these alone or together
are sufficient to explain the rise in obesity and subsequent or
concomitant obesity-related disorders, such as, e.g., type II
diabetes mellitus, metabolic syndrome, hypertension, cardiac
pathology, and non-alcoholic fatty liver disease. According to the
National Institute of Diabetes, Digestive and Kidney Diseases
(NIDDK) approximately 280,000 deaths annually are directly related
to obesity. The NIDDK further estimated that the direct cost of
healthcare in the U.S. associated with obesity is $51 billion. In
addition, Americans spend $33 billion per year on weight loss
products. The prevalence of obesity continues to rise at alarming
rates.
[0005] It is estimated that between 20-25% of American adults
(about 47 million) have metabolic syndrome, a complex condition
associated with an increased risk of vascular disease. Metabolic
syndrome is also known as Syndrome X, metabolic syndrome X, insulin
resistance syndrome, or Reaven's syndrome. Metabolic syndrome is
generally believed to be a combination of disorders that affect a
large number of people in a clustered fashion. The symptoms and
features of the syndrome include at least three of the following
conditions: diabetes mellitus type II; impaired glucose tolerance
or insulin resistance; high blood pressure; central obesity and
difficulty losing weight; high cholesterol; combined
hyperlipidemia; including elevated LDL; decreased HDL; elevated
triglycerides; and fatty liver (especially in concurrent obesity).
Insulin resistance is typical of metabolic syndrome and leads to
several of its features, including glucose intolerance,
dyslipidemia, and hypertension. Obesity is commonly associated with
the syndrome as is increased abdominal girth, highlighting the fact
that abnormal lipid metabolism likely contributes to the underlying
pathophysiology of metabolic syndrome.
[0006] Metabolic syndrome was codified in the United States with
the publication of the National Cholesterol Education Program Adult
Treatment Panel III (ATP III) guidelines in 2001. On a physiologic
basis, insulin resistance appears to be responsible for the
syndrome. However, insulin resistance can be defined in a myriad of
different ways, including impaired glucose metabolism (reduced
clearance of glucose and/or the failure to suppress glucose
production), the inability to suppress lipolysis in tissues,
defective protein synthesis, altered cell differentiation, aberrant
nitric oxide synthesis affecting regional blood flow, as well as
abnormal cell cycle control and proliferation, all of which have
been implicated in the cardiovascular disease associated with
metabolic syndrome. At least at present, there is no obvious
molecular mechanism causing the syndrome, probably because the
condition represents a failure of one or more of the many
compensatory mechanisms that are activated in response to energy
excess and the accumulation of fat.
[0007] Individuals at risk for metabolic syndrome include those who
exhibit central obesity with increased abdominal girth (due to
excess visceral adiposity) of about more than 35 inches in women
and more than 40 inches in men. Individuals at risk for metabolic
syndrome also include those that have a BMI greater than or equal
to 30 kg/M2 and may also have abnormal levels of nonfasting
glucose, lipids, and blood pressure.
[0008] Although certain bacterial associations have been examined
for these and related conditions, the role of bacterial microbiota
in these conditions has not been clearly understood or appreciated.
Thus, there remains a need for methods for diagnosing, treating and
preventing conditions such as obesity, metabolic syndrome,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, diabetes mellitus, non-alcoholic fatty liver, abnormal
lipid metabolism, atherosclerosis, and related disorders.
[0009] The average human body, consisting of about 10.sup.13 cells,
has about ten times that number of microorganisms. The
.about.10.sup.14 microbes that live in and on each of our bodies
belong to all three domains of life on earth--bacteria, archaea and
eukarya. The major sites for our indigenous microbiota are the
intestinal tract, skin and mucosal surfaces such as nasal mucosa
and vagina as well as the oropharynx. By far, the largest bacterial
populations are in the colon. Bacteria make up most of the flora in
the colon and 60% of the dry mass of feces. Probably more than 1000
different species live in the gut. However, it is probable that
>90% of the bacteria come from less than 50 species. Fungi and
protozoa also make up a part of the gut flora, but little is known
about their activities. While the microbiota is highly extensive,
it is barely characterized. Consequently, the Roadmap of the
National Institutes of Health (NIH) includes the "Human Microbiome
Project" to better characterize our microbial communities and the
genes that they harbor (our microbiome) and better understand its
relation to both human health and disease. Reviewed in Dethlefsen
et al., Nature, 2007, 449:811-818; Turnbaugh et al., Nature, 2007,
449:804-810; Ley et al., Cell, 2006, 124:837-848.
[0010] Studies show that the relationship between gut flora and
humans is not merely commensal (a non-harmful coexistence), but
rather often is a mutualistic, symbiotic relationship. Although
animals can survive with no gut flora, the microorganisms perform a
host of useful functions, such as training the immune system,
preventing growth of harmful species, regulating the development of
the gut, fermenting unused energy substrates, metabolism of glycans
and amino acids, synthesis of vitamins (such as biotin and vitamin
K) and isoprenoids, biotransformation of xenobiotics, and producing
hormones to direct the host to store fats. See, e.g., Gill et al.,
Science. 2006, 312:1355-1359; Zaneveld et al., Curr. Opin. Chem.
Biol., 2008, 12(1):109-114; Guarner, Digestion, 2006, 73:5-12; Li
et al., Proc. Natl. Acad. Sci. USA, 2008, 105:2117-2122; Hooper,
Trends Microbiol., 2004, 12:129-134; Mazmanian et al., Cell, 2005,
122:107-118; Rakoff-Nahoum et al., Cell, 2004, 118:229-241. It is
therefore believed that changes in the composition of the gut
microbiota could have important health effects (Dethlefsen et al.,
PLoS Biology, 2008, 6(11):2383-2400). Indeed, a correlation between
obesity and changes in gut microbiota has been observed (Ley et
al., Proc Natl Acad Sci USA, 2005; 102:11070-11075; Baackhed et
al., Proc Natl Acad Sci USA, 2004; 101:15718-15723). Furthermore,
in certain conditions, some microbial species are thought to be
capable of directly causing disease by causing infection or
increasing cancer risk for the host (O'Keefe et al., J Nutr. 2007;
137:175S-182S; McGarr et al., J Clin Gastroenterol., 2005;
39:98-109).
[0011] Substantial number of species in vertebrate microbiota is
very hard to culture and analyze via traditional cultivation-based
studies (Turnbaugh et al., Nature, 2007, 449:804-810; Eckburg et
al., Science, 2005, 308:1635-1638). In contrast, broad-range PCR
primers targeted to highly conserved regions makes possible the
amplification of small subunit rRNA gene (16S rDNA) sequences from
all bacterial species (Zoetendal et al., (2006) Mol Microbiol 59,
1639-1650), and the extensive and rapidly growing 16S rDNA database
facilitates identification of sequences to the species or genus
level (Schloss and Handelsman, (2004) Microbiol Mol Biol Rev 68,
686-691). Such techniques can also be used for identifying
bacterial species in complex environmental niches (Smit et al.,
(2001) Appl Environ Microbiol 67, 2284-2291), including the human
mouth, esophagus, stomach, intestine, feces, skin, and vagina, and
for clinical diagnosis (Harris and Hartley, (2003) J Med Microbiol
52, 685-691; Saglani et al., (2005) Arch Dis Child 90, 70-73).
[0012] Much of the microbiota is conserved from human to human, at
least at the level of phylum and genus (for a general description
of human microbiota see, e.g., Turnbaugh et al., Nature 2007;
449:804-810; Ley et al., Nature 2006; 444:1022-1023; Gao et al.,
Proc Natl Acad Sci USA 2007; 104:2927-32; Pei et al., Proc Natl
Acad Sci USA 2004; 101:4250-4255; Eckburg et al., Science 2005;
308:1635-1638; Bik et al., Proc Natl Acad Sci USA 2006;
103:732-737). A major source of the human microbiota is from one's
mother (for a summary of typical maternal colonization patterns
see, e.g., Palmer et al., Plos Biology 2007; 5:e177; Raymond et
al., Emerg Infect Dis 2004; 10:1816-21), and to a lesser extent
from one's father and siblings (for examples of typical
colonization patterns see, e.g., Raymond et al., Emerg Infect Dis
2004; 10:1816-21; Raymond et al., Plos One 2008; 3:e2259; Goodman
et al., Am J Epidemiol 1996; 144:290-299; Goodman et al., Lancet
2000; 355:358-362). However, many of the natural mechanisms for the
transmission of these indigenous organisms across generations and
between family members have diminished with socioeconomic
development. The impediments include: childbirth by caesarian
section, reduced breast-feeding, smaller family size (fewer
siblings), reduced household crowding with shared beds, utensils,
in-door plumbing.
[0013] It has been known for more than 50 years that the
administration of low doses of antibiotics promotes the growth of
farm animals. See, e.g., Jukes, Bioscience 1972; 22: 526-534; Jukes
(1955) Antibiotics in Nutrition. New York, N.Y., USA: Medical
Encyclopedia; Feighner and Dashkevicz, Appl. Environ. Microbiol.,
1987, 53: 331-336; McEwen and Fedorka-Cray, Clin. Infect. Dis.,
2002, 34 (Suppl 3): S93-S106).
[0014] The mechanism for this widespread phenomenon has not been
established but because of the activity of anti-bacterial but not
anti-fungal agents, it can be ascertained to be anti-bacterial.
[0015] The vertebrate intestinal tract has a rich component of
cells involved in immune responses. The nature of the microbiota
colonizing experimental animals or humans affects the immune
responses of the populations of reactive host cells (see, e.g.,
Ando et al., Infection and Immunity 1998; 66:4742-4747; Goll et
al., Helicobacter. 2007; 12:185-92; Lundgren et al., Infect Immun.
2005; 73:523-531).
[0016] The vertebrate intestinal tract also is a locus in which
hormones are produced. In mammals, many of these hormones related
to energy homeostasis (including insulin, glucagon, leptin, and
ghrelin) are produced by organs of the intestinal tract (see, e.g.,
Mix et al., Gut 2000; 47:481-6; Kojima et al., Nature 1999;
402:656-60; Shak et al., Obesity Surgery 2008; 18(9):1089-96; Roper
et al., Journal of Clinical Endocrinology & Metabolism 2008;
93:2350-7; Francois et al., Gut 2008; 57:16-24; Cummings and
Overduin, J Clin Invest 2007; 117:13-23; Bado et al., Nature 1998;
394:790-793).
[0017] Changing of the microbiota of the intestinal tract appears
to affect the levels of some of these hormones (see, e.g., Breidert
et al., Scand J Gastroenterol 1999; 34:954-61; Liew et al., Obes.
Surg. 2006; 16:612-9; Nwokolo et al., Gut. 2003; 52, 637-640;
Kinkhabwala et al., Gastroenterology 132:A208). The hormones affect
immune responses (see, e.g., Matarese et al., J Immunol 2005;
174:3137-3142; Matsuda et al., J. Allergy Clin. Immunol. 2007; 119,
S174) and adiposity (see, e.g., Tschop et al., Nature 2000;
407:908-13).
[0018] Hydroxypropylmethylcellulose (HPMC) is modified cellulose
fiber that produces viscous solutions in the gastrointestinal
tract. It has been demonstrated that high viscosity (HV) HPMC
consumed as part of a meal reduced peak blood glucose
concentrations in subjects with type 2 diabetes compared with a
cellulose control (Reppas et al., Diabetes Res. Clin. Pract., 1993,
22:61-9). It has been further demonstrated that HPMC reduced weight
gain and insulin resistance in diet-induced obese mice and syrian
hamsters fed a high fat (HF) diet similar in fat content to the
American diet. (Hung et al., J Diab 2009; 1(3):194-206); Kim et
al., FASEB J., 2009, Meeting Abstracts, Abstract 212.2).
[0019] PCT Pat. Appl. Publ. Nos. WO 2008/051793 and WO 2008/051794
disclose the use of HPMC and other water-soluble and
water-insoluble cellulose derivatives for preventing or treating
metabolic syndrome and related conditions. See also U.S. Pat. Nos.
5,576,306; 5,585,366; 6,899,892; 5,721,221. PCT Pat. Appl. Publ.
No. WO 2004/022074 discloses the use of a composition comprising a
non-glucose carbohydrate and soluble fiber or a mixture of pectin
and soluble fiber for controlling metabolic syndrome, diabetes
mellitus and obesity, and for the promotion of weight loss or
maintenance of the desired body weight.
SUMMARY OF THE INVENTION
[0020] As specified in the Background section above, there is a
great need in the art to understand the impact that mammalian
bacterial microbiota has on development of obesity and related
disorders such as metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis. There is further a great need in the art to
employ such knowledge in development of new therapeutics to treat
these and related disorders.
[0021] The present invention addresses these and other needs by
characterizing specific diet-induced-obesity-associated changes in
mammalian bacterial microbiota and by providing related diagnostic
and therapeutic methods and probiotic and prebiotic compositions.
The present invention further provides novel prebiotic compositions
based on a surprising finding that cellulose ethers with a beta 1,4
linkage of anhydrous glucose units have a prebiotic effect although
they are known to be substantially non-fermentable and
non-digestible materials in the digestive tract of mammals.
[0022] In one aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0023] (a) measuring the populations of Firmicutes
and/or Bacteroidetes in the ileal microbiota of the mammal; [0024]
(b) measuring the populations of Firmicutes and/or Bacteroidetes in
the ileal microbiota of a healthy control; [0025] (c) comparing the
populations measured in steps (a) and (b), and [0026] (d)
determining that the mammal has a predisposition to the disease if
the populations of Firmicutes and/or Bacteroidetes in the ileal
microbiota of the mammal are increased as compared to the healthy
control.
[0027] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition lowers the
populations of Firmicutes and/or Bacteroidetes in the ileal
microbiota of the mammal.
[0028] In another related embodiment, the invention provides a
method for preventing or treating a disease in a mammal selected
from the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising administering to the mammal a therapeutically effective
amount of a probiotic composition, wherein said probiotic
composition lowers the populations of Firmicutes and/or
Bacteroidetes in the ileal microbiota of the mammal.
[0029] In another embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition lowers the
populations of Firmicutes and/or Bacteroidetes in the ileal
microbiota of the mammal.
[0030] In a further embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition lowers
the populations of Firmicutes and/or Bacteroidetes in the ileal
microbiota of the mammal.
[0031] In a separate embodiment, the invention provides a method of
lowering populations of Firmicutes and/or Bacteroidetes in the
ileal microbiota of a mammal comprising administering to the mammal
a prebiotic composition.
[0032] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0033] (a) measuring the populations of Firmicutes in
the cecal and/or fecal microbiota of the mammal; [0034] (b)
measuring the populations of Firmicutes in the cecal and/or fecal
microbiota of a healthy control; [0035] (c) comparing the
populations measured in steps (a) and (b), and [0036] (d)
determining that the mammal has a predisposition to the disease if
the populations of Firmicutes in the cecal and/or fecal microbiota
of the mammal are increased as compared to the healthy control.
[0037] In a related aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0038] (a) measuring the ratio of the populations of
Firmicutes to the populations of Eubacteria (F/E ratio=relative
abundance of Firmicutes) in the cecal and/or fecal microbiota of
the mammal; [0039] (b) measuring the F/E ratio in the cecal and/or
fecal microbiota of a healthy control; [0040] (c) comparing the F/E
ratios measured in steps (a) and (b), and [0041] (d) determining
that the mammal has a predisposition to the disease if the F/E
ratio is increased in the cecal and/or fecal microbiota of the
mammal as compared to the healthy control.
[0042] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition lowers the
populations of Firmicutes in the cecal and/or fecal microbiota of
the mammal.
[0043] In another related embodiment, the invention provides a
method for preventing or treating a disease in a mammal selected
from the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising administering to the mammal a therapeutically effective
amount of a probiotic composition, wherein said probiotic
composition lowers the populations of Firmicutes in the cecal
and/or fecal microbiota of the mammal.
[0044] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition lowers the
populations of Firmicutes in the cecal and/or fecal microbiota of
the mammal.
[0045] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition lowers
the populations of Firmicutes in the cecal and/or fecal microbiota
of the mammal.
[0046] In a separate embodiment, the invention provides a method of
lowering the populations of Firmicutes in the cecal and/or fecal
microbiota of a mammal comprising administering to the mammal a
prebiotic composition.
[0047] In another embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition lowers the ratio of
the populations of Firmicutes to Eubacteria (F/E ratio=relative
abundance of Firmicutes) in the cecal and/or fecal microbiota of
the mammal.
[0048] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition lowers
the ratio of the populations of Firmicutes to Eubacteria (F/E
ratio=relative abundance of Firmicutes) in the cecal and/or fecal
microbiota of the mammal.
[0049] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition lowers the ratio of
the populations of Firmicutes to Eubacteria (F/E ratio=relative
abundance of Firmicutes) in the cecal and/or fecal microbiota of
the mammal.
[0050] In an additional embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition lowers
the ratio of the populations of Firmicutes to Eubacteria (F/E
ratio=relative abundance of Firmicutes) in the cecal and/or fecal
microbiota of the mammal.
[0051] In a separate embodiment, the invention provides a method of
lowering the ratio of the populations of Firmicutes to Eubacteria
(F/E ratio=relative abundance of Firmicutes) in the cecal and/or
fecal microbiota of the mammal comprising administering to the
mammal a prebiotic composition.
[0052] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0053] (a) measuring the populations of at least one
genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae IS (PIS) in the intestinal microbiota of
the mammal; [0054] (b) measuring the populations of the same genus
in the intestinal microbiota of a healthy control; [0055] (c)
comparing the populations measured in steps (a) and (b), and [0056]
(d) determining that the mammal has a predisposition to the disease
if the populations of at least one genus selected from the group
consisting of Coprobacillus, Sporacetigenium, Holdemania, Dorea,
Blautia, Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS),
Clostridium cocleatum, and Peptosteptococcaceae Incertae Sedis
(PIS) in the intestinal microbiota of the mammal are decreased as
compared to the healthy control.
[0057] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition stimulates growth
or metabolic activity of at least one strain from the genus
selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal.
[0058] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition stimulates growth
or metabolic activity of at least one strain from the genus
selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal.
[0059] The invention also provides a method for determining whether
weight loss can be achieved in a mammal by the above two methods
comprising [0060] (a) measuring the populations of at least one
genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal; [0061] (b) measuring the populations of
the same genus in the intestinal microbiota of a healthy control;
[0062] (c) comparing the populations measured in steps (a) and (b),
and [0063] (d) determining that weight loss can be achieved in the
mammal by the above two methods if the populations of at least one
genus selected from the group consisting of Coprobacillus,
Sporacetigenium Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal is decreased as compared to the healthy
control.
[0064] In another related aspect, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition
stimulates growth or metabolic activity of at least one strain from
the genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal.
[0065] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition
stimulates growth or metabolic activity of at least one strain from
the genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal.
[0066] The invention also provides a method for determining whether
a disease in a mammal selected from the group consisting of
obesity, metabolic syndrome, diabetes mellitus, insulin-deficiency
related disorders, insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis can be prevented or treated by the above two
methods comprising [0067] (a) measuring the populations of at least
one genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal; [0068] (b) measuring the populations of
the same genus in the intestinal microbiota of a healthy control;
[0069] (c) comparing the populations measured in steps (a) and (b),
and [0070] (d) determining that the disease in the mammal can be
prevented or treated by the above two methods if the populations of
at least one genus selected from the group consisting of
Coprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,
Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridium
cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in the
intestinal microbiota of the mammal is decreased as compared to the
healthy control.
[0071] In a separate embodiment, the invention provides a method of
stimulating growth or metabolic activity of at least one strain
from the genus selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of a mammal comprising administering to the mammal a
prebiotic composition.
[0072] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0073] (a) measuring the populations of at least one
taxon selected from the group consisting of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of the mammal; [0074] (b) measuring
the populations of the same taxonin the intestinal microbiota of a
healthy control; [0075] (c) comparing the populations measured in
steps (a) and (b), and [0076] (d) determining that the mammal has a
predisposition to the disease if the populations of at least one
taxon selected from the group consisting of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of the mammal is increased as compared
to the healthy control.
[0077] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a composition or a
compound, wherein said composition or compound inhibits growth or
metabolic activity of at least one strain from the taxon selected
from the group consisting of Johnsonella, Oscillibacter,
Lachnospiraceae, Ruminococcaceae, and Clostridiales in the
intestinal microbiota of the mammal.
[0078] In another aspect, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
composition or a compound, wherein said composition or compound
inhibits growth or activity of at least one strain from the taxon
selected from the group consisting of Johnsonella, Oscillibacter,
Lachnospiraceae, Ruminococcaceae, and Clostridiales in the
intestinal microbiota of the mammal.
[0079] In one specific embodiment, the compound used in the above
two methods is a narrow spectrum antibiotic. In another specific
embodiment, the composition used in the above two methods is a
probiotic composition comprising at least one strain from the genus
selected from the group consisting of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS).
[0080] In a related embodiment, the invention provides a method for
determining whether weight loss can be achieved in a mammal by the
above method comprising [0081] (a) measuring the populations of at
least one taxon selected from the group consisting of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of the mammal; [0082] (b) measuring
the populations of the same taxon in the intestinal microbiota of a
healthy control; [0083] (c) comparing the populations measured in
steps (a) and (b), and [0084] (d) determining that weight loss can
be achieved in the mammal by the above method if the populations of
at least one taxon selected from the group consisting of
Johnsonella, Oscillibacter, Lachnospiraceae, Ruminococcaceae, and
Clostridiales in the intestinal microbiota of the mammal is
increased as compared to the healthy control.
[0085] In another related embodiment, the invention provides a
method for determining whether a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis can be prevented or
treated by the above method comprising [0086] (a) measuring the
populations of at least one taxon selected from the group
consisting of Johnsonella, Oscillibacter, Lachnospiraceae,
Ruminococcaceae, and Clostridiales in the intestinal microbiota of
the mammal; [0087] (b) measuring the populations of the same taxon
in the intestinal microbiota of a healthy control; [0088] (c)
comparing the populations measured in steps (a) and (b), and [0089]
(d) determining that the disease in the mammal can be prevented or
treated by the above method if the populations of at least one
taxon selected from the group consisting of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of the mammal is increased as compared
to the healthy control.
[0090] In a separate embodiment, the invention provides a method of
inhibiting growth or metabolic activity of at least one strain from
the taxon selected from the group consisting of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of a mammal comprising administering
to the mammal a prebiotic composition.
[0091] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0092] (a) measuring the populations of at least one
genus selected from the group consisting of Coprobacillus (C),
Sporacetigenium (S), and Holdemania (H), in the intestinal
microbiota of the mammal; [0093] (b) measuring the populations of
at least one genus selected from Johnsonella (J) and Oscillibacter
(O) in the intestinal microbiota of the mammal; [0094] (c)
determining a ratio of one of C+S+H, C+H, C+S, S+H, C, S, or H as
measured in step (a) to one of J+O, J, or O as measured in step
(b), and [0095] (d) determining that the mammal has a
predisposition to the disease if the ratio in step (c) is below 1,
or determining that the mammal has no predisposition to the disease
if the ratio in step (c) is above 3.
[0096] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition increases the ratio
of populations of one of (a) Coprobacillus (C), Sporacetigenium
(S), Holdemania (H), C+H, C+S, S+H, or C+S+H to populations of one
of (b) Johnsonella (J), Oscillibacter (O), or J+O to above 3 in the
intestinal microbiota of the mammal.
[0097] In another embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition increases
the ratio of populations of one of (a) Coprobacillus (C),
Sporacetigenium (S), Holdemania (H), C+H, C+S, S+H, or C+S+H to
populations of one of (b) Johnsonella (J), Oscillibacter (O), or
J+O to above 3 in the intestinal microbiota of the mammal.
[0098] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition increases the ratio
of populations of one of (a) Coprobacillus (C), Sporacetigenium
(S), Holdemania (H), C+H, C+S, S+H, or C+S+H to populations of one
of (b) Johnsonella (J), Oscillibacter (O), or J+O to above 3 in the
intestinal microbiota of the mammal.
[0099] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition increases
the ratio of populations of one of (a) Coprobacillus (C),
Sporacetigenium (S), Holdemania (H), C+H, C+S, S+H, or C+S+H to
populations of one of (b) Johnsonella (J), Oscillibacter (O), or
J+O to above 3 in the intestinal microbiota of the mammal.
[0100] In a separate embodiment, the invention provides a method of
increasing the ratio of populations of one of (a) Coprobacillus
(C), Sporacetigenium (S), Holdemania (H), C+H, C+S, S+H, or C+S+H
to populations of one of (b) Johnsonella (J), Oscillibacter (O), or
J+O in the intestinal microbiota of a mammal comprising
administering to the mammal a prebiotic composition.
[0101] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0102] (a) measuring the populations of at least one
genus selected from the group consisting of Erysipelotrichaceae
Incertae Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
and Clostridium cocleatum (Cc) in the intestinal microbiota of the
mammal; [0103] (b) measuring the populations of Johnsonella (J) in
the intestinal microbiota of the mammal; [0104] (c) determining a
ratio of one of EIS, PIS, EIS+PIS, or Cc as measured in step (a) to
J as measured in step (b), and [0105] (d) determining that the
mammal has a predisposition to the disease if the ratio in step (c)
is below 1, or determining that the mammal has no predisposition to
the disease if the ratio in step (c) is above 1.
[0106] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition increases the ratio
of populations of one of (a) Erysipelotrichaceae Incertae Sedis
(EIS), Peptostreptococcaceae Incertae Sedis (PIS), Clostridium
cocleatum (Cc), or EIS+PIS to populations of (b) Johnsonella (J) to
above 1 in the intestinal microbiota of the mammal.
[0107] In another embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition increases
the ratio of populations of one of (a) Erysipelotrichaceae Incertae
Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
Clostridium cocleatum (Cc), or EIS+PIS to populations of (b)
Johnsonella (J) to above 1 in the intestinal microbiota of the
mammal.
[0108] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition increases the ratio
of populations of one of (a) Erysipelotrichaceae Incertae Sedis
(EIS), Peptostreptococcaceae Incertae Sedis (PIS), Clostridium
cocleatum (Cc), or EIS+PIS to populations of (b) Johnsonella (J) to
above 1 in the intestinal microbiota of the mammal.
[0109] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition increases
the ratio of populations of one of (a) Erysipelotrichaceae Incertae
Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
Clostridium cocleatum (Cc), or EIS+PIS to populations of (b)
Johnsonella (J) to above 1 in the intestinal microbiota of the
mammal.
[0110] In a separate embodiment, the invention provides a method of
increasing the ratio of populations of one of (a)
Erysipelotrichaceae Incertae Sedis (EIS), Peptostreptococcaceae
Incertae Sedis (PIS), Clostridium cocleatum (Cc), or EIS+PIS to
populations of (b) Johnsonella (J) in the intestinal microbiota of
a mammal comprising administering to the mammal a prebiotic
composition.
[0111] In an additional embodiment, the invention provides a method
for diagnosing predisposition to a disease in a mammal selected
from the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0112] (a) measuring the populations of at least one
genus selected from the group consisting of Coprobacillus (C),
Sporacetigenium (S), Holdemania (H), Erysipelotrichaceae Incertae
Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS), and
Clostridium cocleatum (Cc) in the intestinal microbiota of the
mammal; [0113] (b) measuring the populations of Firmicutes (F) in
the intestinal microbiota of the mammal; [0114] (c) determining a
ratio of one of C+S+H, C+H, C+S, S+H, C, S, H, EIS, PIS, or Cc as
measured in step (a) to F as measured in step (b), and [0115] (d)
determining that the mammal has a predisposition to the disease if
the ratio in step (c) is below 0.1, or determining that the mammal
has no predisposition to the disease if the ratio in step (c) is
above 0.1.
[0116] In a related embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition increases the ratio
of populations of one of (a) Coprobacillus (C), Sporacetigenium
(S), Holdemania (H), Erysipelotrichaceae Incertae Sedis (EIS),
Peptostreptococcaceae Incertae Sedis (PIS), Clostridium cocleatum
(Cc), C+H, C+S, S+H, or C+S+H to populations of (b) Firmicutes (F)
to above 0.1 in the intestinal microbiota of the mammal.
[0117] In another embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition increases
the ratio of populations of one of (a) Coprobacillus (C),
Sporacetigenium (S), Holdemania (H), Erysipelotrichaceae Incertae
Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H to populations
of (b) Firmicutes (F) to above 0.1 in the intestinal microbiota of
the mammal.
[0118] In yet another embodiment, the invention provides a method
for promoting weight loss in a mammal comprising administering to
the mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition increases the ratio
of populations of one of (a) Coprobacillus (C), Sporacetigenium
(S), Holdemania (H), Erysipelotrichaceae Incertae Sedis (EIS),
Peptostreptococcaceae Incertae Sedis (PIS), Clostridium cocleatum
(Cc), C+H, C+S, S+H, or C+S+H to populations of (b) Firmicutes (F)
to above 0.1 in the intestinal microbiota of the mammal.
[0119] In a further embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition increases
the ratio of populations of one of (a) Coprobacillus (C),
Sporacetigenium (S), Holdemania (H), Erysipelotrichaceae Incertae
Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H to populations
of (b) Firmicutes (F) to above 0.1 in the intestinal microbiota of
the mammal.
[0120] In a separate embodiment, the invention provides a method of
increasing the ratio of populations of one of (a) Coprobacillus
(C), Sporacetigenium (S), Holdemania (H), Erysipelotrichaceae
Incertae Sedis (EIS), Peptostreptococcaceae Incertae Sedis (PIS),
Clostridium cocleatum (Cc), C+H, C+S, S+H, or C+S+H to populations
of (b) Firmicutes (F) in the intestinal microbiota of a mammal
comprising administering to the mammal a prebiotic composition.
[0121] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0122] (a) measuring the populations of at least one
family selected from Erysipelotrichaceae and Peptostreptococcacea,
in the intestinal microbiota of the mammal; [0123] (b) measuring
the populations of at least one family selected from
Lachnospiraceae and Ruminococcaceae in the intestinal microbiota of
the mammal; [0124] (c) determining a ratio of one of
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea as measured in step (a) to one of
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae as measured in step (b), and (d) determining that
the mammal has a predisposition to the disease if the ratio in step
(c) is below 0.1, or determining that the mammal has no
predisposition to the disease if the ratio in step (c) is above
0.1.
[0125] In a related embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition increases the ratio
of populations of one of (a)
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea to populations of one of (b)
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae to above 0.1 in the intestinal microbiota of the
mammal.
[0126] In another embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition increases
the ratio of populations of one of (a)
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea to populations of one of (b)
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae to above 0.1 in the intestinal microbiota of the
mammal.
[0127] In a further embodiment, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition increases the ratio
of populations of one of (a)
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea to populations of one of (b)
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae to above 0.1 in the intestinal microbiota of the
mammal.
[0128] In yet another embodiment, the invention provides a method
for preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition increases
the ratio of populations of one of (a)
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea to populations of one of (b)
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae to above 0.1 in the intestinal microbiota of the
mammal.
[0129] In a separate embodiment, the invention provides a method of
increasing the ratio of populations of one of (a)
Erysipelotrichaceae+Peptostreptococcacea, Erysipelotrichaceae, or
Peptostreptococcacea to populations of one of (b)
Lachnospiraceae+Ruminococcaceae, Lachnospiraceae, or
Ruminococcaceae in the intestinal microbiota of a mammal comprising
administering to the mammal a prebiotic composition.
[0130] In any of the above methods, the populations of bacteria can
be determined by any method known in the art. In a preferred
embodiment, the populations of bacteria are determined by qPCR of
bacterial 16S rRNA.
[0131] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0132] (a) measuring the total number of Butyryl CoA
transferase (BCoAT)-encoding genes in the intestinal microbiota of
the mammal; [0133] (b) measuring the total number of BCoAT-encoding
genes in the intestinal microbiota of a healthy control; [0134] (c)
comparing the total number of BCoAT-encoding genes measured in
steps (a) and (b), and [0135] (d) determining that the mammal has a
predisposition to the disease if the total number of BCoAT-encoding
genes in the intestinal microbiota of the mammal is increased as
compared to the healthy control.
[0136] In another aspect, the invention provides a method for
diagnosing predisposition to a disease in a mammal selected from
the group consisting of obesity, metabolic syndrome, diabetes
mellitus, insulin-deficiency related disorders, insulin-resistance
related disorders, glucose intolerance, non-alcoholic fatty liver,
abnormal lipid metabolism, and atherosclerosis, said method
comprising [0137] (a) measuring a ratio of the total number of
Butyryl CoA transferase (BCoAT)-encoding genes to copies of
Bacteroidetes 16S rRNA in the intestinal microbiota of the mammal;
[0138] (b) measuring a ratio of the total number of BCoAT-encoding
genes to copies of Bacteroidetes 16S rRNA in the intestinal
microbiota of a healthy control; [0139] (c) comparing the ratios of
the total number of BCoAT-encoding genes to copies of Bacteroidetes
16S rRNA measured in steps (a) and (b), and [0140] (d) determining
that the mammal has a predisposition to the disease if the ratio of
the total number of BCoAT-encoding genes to copies of Bacteroidetes
16S rRNA in the intestinal microbiota of the mammal is increased as
compared to the healthy control.
[0141] In the above methods, the total number of BCoAT-encoding
genes and copies of Bacteroidetes 16S rRNA can be measured by any
method known in the art. In a preferred embodiment, the total
number of BCoAT-encoding genes and copies of Bacteroidetes 16S rRNA
are measured by qPCR.
[0142] In a related aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition lowers the levels
of Butyryl CoA transferase (BCoAT) enzyme and/or the levels of
butyrate in the intestinal microbiota of the mammal.
[0143] In another embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition lowers
the levels of Butyryl CoA transferase (BCoAT) enzyme and/or the
levels of butyrate in the intestinal microbiota of the mammal.
[0144] In yet another embodiment, the invention provides a method
for promoting weight loss in a mammal comprising administering to
the mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition lowers the levels
of Butyryl CoA transferase (BCoAT) enzyme and/or the levels of
butyrate in the intestinal microbiota of the mammal.
[0145] In a further embodiment, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition lowers
the levels of Butyryl CoA transferase (BCoAT) enzyme and/or the
levels of butyrate in the intestinal microbiota of the mammal.
[0146] In a separate embodiment, the invention provides a method of
lowering the levels of Butyryl CoA Transferase (BCoAT) enzyme
and/or the levels of butyrate in the intestinal microbiota of a
mammal comprising administering to the mammal a prebiotic
composition.
[0147] In any of the above methods, the levels of BCoAT enzyme can
be measured by any method known in the art. In a preferred
embodiment, the levels of BCoAT enzyme are measured by determining
the total number of BCoAT-encoding genes. In another preferred
embodiment, the levels of BCoAT enzyme are measured by BCoAT enzyme
functional assay.
[0148] In any of the above methods, the levels of butyrate can be
measured by any method known in the art. In a preferred embodiment,
the levels of butyrate are measured using chromatographic
methods.
[0149] In another aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a probiotic
composition, wherein said probiotic composition lowers the ratio of
the total number of Butyryl CoA transferase (BCoAT)-encoding genes
to copies of Bacteroidetes 16S rRNA in the intestinal microbiota of
the mammal.
[0150] In yet another aspect, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
probiotic composition, wherein said probiotic composition lowers
the ratio of the total number of Butyryl CoA transferase
(BCoAT)-encoding genes to copies of Bacteroidetes 16S rRNA in the
intestinal microbiota of the mammal.
[0151] In a further aspect, the invention provides a method for
promoting weight loss in a mammal comprising administering to the
mammal a therapeutically effective amount of a prebiotic
composition, wherein said prebiotic composition lowers the ratio of
the total number of Butyryl CoA transferase (BCoAT)-encoding genes
to copies of Bacteroidetes 16S rRNA in the intestinal microbiota of
the mammal.
[0152] In an additional aspect, the invention provides a method for
preventing or treating a disease in a mammal selected from the
group consisting of obesity, metabolic syndrome, diabetes mellitus,
insulin-deficiency related disorders, insulin-resistance related
disorders, glucose intolerance, non-alcoholic fatty liver, abnormal
lipid metabolism, and atherosclerosis, said method comprising
administering to the mammal a therapeutically effective amount of a
prebiotic composition, wherein said prebiotic composition lowers
the ratio of the total number of Butyryl CoA transferase
(BCoAT)-encoding genes to copies of Bacteroidetes 16S rRNA in the
intestinal microbiota of the mammal.
[0153] In a separate embodiment, the invention provides a method of
lowering the ratio of the total number of Butyryl CoA Transferase
(BCoAT)-encoding genes to copies of bacteroidetes 16S rRNA in the
intestinal microbiota of a mammal comprising administering to the
mammal a prebiotic composition.
[0154] In the above methods, the total number of BCoAT-encoding
genes and copies of Bacteroidetes 16S rRNA can be measured by any
method known in the art. In a preferred embodiment, the total
number of BCoAT-encoding genes and copies of Bacteroidetes 16S rRNA
are measured by qPCR.
[0155] In conjunction with therapeutic methods, the present
invention also provides various probiotic and prebiotic
compositions which can be used in such methods. Probiotic
compositions according to the present invention can contain live
bacterial strains and/or spores and also include conditionally
lethal bacterial strains. Non-limiting examples of useful bacterial
strains include, e.g., strains from the genera Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS).
[0156] Probiotic compositions of the present invention can further
comprise a buffering agent such as, e.g., sodium bicarbonate,
juice, milk, yogurt, infant formula, etc.
[0157] Probiotic compositions of the present invention can be
administered conjointly with a prebiotic composition which
stimulates growth and/or metabolic activity of bacteria contained
in the probiotic composition. Such combinations of probiotic and
prebiotic compositions can be administered in one composition or as
two separate compositions (administered simultaneously or
sequentially).
[0158] In a specific embodiment, a probiotic composition further
comprises a compound selected from the group consisting of xylose,
arabinose, ribose, galactose, rhamnose, cellobiose, fructose,
lactose, salicin, sucrose, glucose, esculin, tween 80, trehalose,
maltose, mannose, mellibiose, raffinose, fructooligosaccharides,
galacto-oligosaccharides, amino acids, alcohols, and any
combinations thereof. In another specific embodiment, a probiotic
composition further comprises a compound selected from the group
consisting of trehalose, cellobiose, maltose, mannose, sucrose,
fructose, galactose, lactose, salicin, melibiose, raffinose, and
any combinations thereof. In yet another specific embodiment, a
probiotic composition further comprises a compound selected from
the group consisting of water-soluble cellulose derivatives,
water-insoluble cellulose derivatives, unprocessed oatmeal,
metamucil, all-bran, and any combinations thereof. In a preferred
embodiment, the water-soluble cellulose derivative is selected from
the group consisting of methylcellulose, methyl ethyl cellulose,
hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationic
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl
methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl
cellulose. In another preferred embodiment, the water-insoluble
cellulose derivative is ethyl cellulose.
[0159] In one specific embodiment, the invention provides a
probiotic composition comprising one or more strain from the genus
Holdemania and one or more compounds selected from the group
consisting of Tween 80, esculin, fructose, glucose, lactose,
maltose, salicin, and sucrose.
[0160] In another specific embodiment, the invention provides a
probiotic composition comprising one or more strain from the genus
Sporoacetigenicum and one or more compounds selected from the group
consisting of arabinose, fructose, glucose, maltose, and
xylose.
[0161] In yet another specific embodiment, the invention provides a
probiotic composition comprising one or more strain from the genus
Coprobacillus and one or more compounds selected from the group
consisting of mannose, fructose, sucrose, maltose, cellobiose,
trehalose, salicin, lactose, glucose, and galactose.
[0162] In yet another specific embodiment, the invention provides a
probiotic composition comprising one or more strain from the genus
Clostridium cocleatum and one or more compounds selected from the
group consisting of cellobiose, fructose, galactose, glucose,
inulin, lactose, maltose, mannose, mellibiose, raffinose, and
sucrose.
[0163] In one embodiment, the invention provides a prebiotic
composition useful in the methods of the present invention which
prebiotic composition comprises a compound selected from the group
consisting of trehalose, cellobiose, maltose, mannose, sucrose,
fructose, galactose, lactose, salicin, melibiose, raffinose, and
any combinations thereof. In another embodiment, the invention
provides a prebiotic composition comprising a compound selected
from the group consisting of xylose, arabinose, ribose, galactose,
rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose,
esculin, tween 80, trehalose, maltose, mannose, mellibiose,
raffinose, fructooligosaccharides, galactooligosaccharides, amino
acids, alcohols, water-soluble cellulose derivatives,
water-insoluble cellulose derivatives, unprocessed oatmeal,
metamucil, all-bran, and any combinations thereof. In one preferred
embodiment, the water-soluble cellulose derivative is selected from
the group consisting of methylcellulose, methyl ethyl cellulose,
hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationic
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl
methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl
cellulose. In another preferred embodiment, the water-insoluble
cellulose derivative is ethyl cellulose.
[0164] Probiotic and prebiotic compositions useful in the methods
of the present invention can be formulated in different forms
(e.g., as a liquid solution, powder, capsule, tablet, suppository,
etc.) and can be administered by various methods (e.g., orally,
rectally, via esophagogastroduodenoscopy, colonoscopy, nasogastric
tube, orogastric tube, etc.).
[0165] In one embodiment, the mammal in any of the above methods is
human.
[0166] In one embodiment, the mammal in any of the above methods is
on a high fat diet.
BRIEF DESCRIPTION OF THE DRAWINGS
[0167] FIGS. 1A-1D are plots showing the effect of diet on host
metabolism. Adult C57BL/6 mice were fed high fat diet (HFD, 60%
kcal from fat) for two months prior to the study, then continued on
HFD, or switched to either low fat diet (LFD, 10% kcal from fat) or
to HFD with 10% HPMC supplementation (HPMC). Panels: (FIG. 1A)
weight for the mice over the 4-week study; (FIG. 1B) total energy
intake (kcal) for the duration of the 4-week experiment (bar at
median); (FIG. 1C) correlation by linear regression between 4-week
weight change and total energy intake; (FIG. 1D) Difference (mean
.+-.SE) between actual and predicted weight change, based on energy
intake and the best fit line for LFD and HFD mice. *p <0.05, **p
<0.01, ***p <0.001, Mann-Whitney U test for FIG. 1B and FIG.
1D, non-zero slope for FIG. 1C.
[0168] FIGS. 2A-2T are scatter plots showing changes in murine
metabolic phenotypes in response to 4 weeks of dietary
intervention. Panels represent fasting plasma: FIG. 2A)
cholesterol, FIG. 2B) HDL, FIG. 2C) LDL, FIG. 2D) VLDL, FIG. 2E)
free fatty acids, FIG. 2F) triglycerides, FIG. 2G) glucose, FIG.
2H) insulin, FIG. 21) leptin, FIG. 2J) adiponectin, FIG. 2K) liver
adiposity (% lipids), FIG. 2L) liver triglycerides, FIG. 2M) fecal
saturated fat, FIG. 2N) fecal unsaturated fat, FIG. 20) fecal
transunsaturated fats, FIG. 2P) fecal bile acid, FIG. 2Q) fecal
sterols, FIG. 2R) fecal monoacyglycerides, FIG. 2S) fecal
diacylgycerridess, and FIG. 2T) fecal triacylglycerides. *p
<0.05, **p <0.01, ***p <0.001, FDR-corrected Mann-Whitney
U test.
[0169] FIGS. 3A-3H are plots of quantitative PCR analysis of the
intestinal microbiota. Analysis of fecal, cecal, and ileal samples
measuring population copy number per gram of sample of FIG. 3A)
total bacteria, FIG. 3B) Bactoidetes, FIG. 3C) Firmicutes, FIG. 3D)
BCoAT, FIG. 3E) ratio of Bacteroidetes/Firmicutes, and relative
abundance (%) of FIG. 3F) Bacteroidetes, FIG. 3G) Firmicutes, FIG.
3H) BCoAT, *p<0.05, **p <0.01, ***p <0.001, Mann-Whitney U
test.
[0170] FIG. 4A is a bar diagram demonstrating that HPMC exposure
significantly alters the composition of the cecal microbiome
compared to mice maintained on a 60% diet or those switched to a
10% diet. Mice exposed to a 60% fat diet, then switched to a diet
of 60% fat +HPMC had significant changes in cecal microbiota
compared to the control mice maintained on the 60% fat diet alone.
Notably, there were marked increases in populations of
Coprobacillus, Sporacetigenium, Holdemania, Dorea, Enterococcus,
and Blautia, as well as marked decreases in Johnsonella, and
Oscillibacter. Little change in cecal microbiota was observed in
mice switched from a 60% fat diet to a 10% fat diet, compared to
the control mice maintained on a 60% fat diet. Observed changes
include an increase in Dorea, and a decrease in Coprobacillus and
Papillibacter. Note that at each level of taxa, there are two
scales for abundances.
[0171] FIG. 4B is a summary table of significant changes in the
cecal microbiome seen in FIG. 6 at different taxonomic levels.
[0172] FIG. 5 represents scatterplots showing relative abundance of
Coprobacillus, Sporacetigenium and Holdemania in fecal, cecal, and
ileal samples from C57B6 mice on diets consisting of 60% fat 10%
fat, or 60% fat+HPMC. The figure demonstrates that HPMC exposure
significantly increases Coprobacillus, Sporacetigenium, and
Holdemania abundance. The effects of HPMC on Coprobacillus and
Holdemania are seen primarily in the cecum and are noted in the 2
and 4 fecal specimens as well. Sporacetigenium census was higher in
the ileum than in the cecum or in fecal pellets for all groups. *P
<0.05, **P <0.01, ***P<0.001.
[0173] FIGS. 6A-6C are scatter plots of ratios that represent
diagnostic criteria for predicting predisposition to weight gain on
a high fat diet and effectiveness of fiber treatment for weight
loss or weight gain prevention. C57B6 mice were maintained on a 60%
fat diet for 2 months, then 1 group was switched to a 10% fat diet,
another switched to a 60% fat+HPMC diet, and a 3.sup.rd group was
maintained on the 60% fat diet. FIG. 6A. Ratios at the genus level
are calculated by dividing the sum of any combination of
Coprobacillus, Sporacetigenium, and/or Holdemania (CSH) by any
combination of Johnsonella and/or Oscillibacter. A ratio below 1
indicates a state that is predisposed to weight gain while a ratio
above 3 indicates a state that has a high propensity to prevent
weight gain. A ratio between 1 and 3 is intermediate. FIG. 6B.
Additional ratios for CSH are calculated by dividing the sum of any
combination of Coprobacillus, Sporacetigenium, and/or Holdemania by
the phylum Firmicutes to measure the relative abundance. A ratio
below 0.1 indicates a state that is predisposed to weight gain
while a ratio above 0.1 indicates a state that has a high
propensity to prevent weight gain. FIG. 6C. Ratios at the family
level are calculated by dividing the sum of any combination of
Erysipelotrichaceae and/or Peptostreptococcacea by Lachnospiraceae
and/or Ruminococcaceae. A ratio below 0.1 indicates a state that is
predisposed to weight gain while a ratio above 0.1 indicates a
state that has a high propensity to prevent weight gain.
[0174] FIGS. 7A-7C are graphs showing the relative abundance (%) of
454-pyrosequencing reads classified at the phylum level for FIG.
7A) Firmicutes, FIG. 7B) Bacteroidetes, and FIG. 7C) the ratio of
Bacteroidetes to Firmicutes.
[0175] FIGS. 8A-8D are graphs showing the relative abundance (%) of
454-pyrosequencing reads classified at the class level for FIG. 8A)
Clostridia, FIG. 8B) Erysipelotrichi, FIG. 8C) Bacteroidia, and
FIG. 8D) Bacilli. *P <0.05, **P <0.01, ***P<0.001 for
FDR-corrected Mann-Whitney-U.
[0176] FIGS. 9A-9D are graphs showing the relative abundance (%) of
454-pyrosequencing reads classified at the order level for FIG. 9A)
Clostridiales, FIG. 9B) Erysipelotrichales, FIG. 9C) Bacteroidales,
and FIG. 9D) Lactobacillales, ***P<0.001 for FDR-corrected
Mann-Whitney-U.
[0177] FIGS. 10A-10G are graphs showing the relative abundance (%)
of 454-pyrosequencing reads classified at the family level for FIG.
10A) Lachnospiraceae, FIG. 10B) Ruminococcaceae, FIG. 10C)
Erysipelotrichaeceae, FIG. 10D) Peptostreptococcaceae, FIG. 10E)
Lactobacillaceae, FIG. 10F) Porphyromonadaceae, FIG. 10G)
Clostridiaceae. ***P<0.001 for FDR-corrected Mann-Whitney-U.
[0178] FIGS. 11A-11E are graphs showing the relative abundance (%)
of 454-pyrosequencing reads classified at the genus level for FIG.
11A) Johnsonella, FIG. 11B) Erysipelotrichaceae incertae sedis,
FIG. 11C) Peptostreptococcaceae incertae sedis, FIG. 11D)
Clostridium, FIG. 11E) Lactobacillus. ***P<0.001 for
FDR-corrected Mann-Whitney-U.
[0179] FIG. 12 shows mean relative abundance of the combined 59
OTUs with a closest match to Clostridium cocleatum in fecal 0 week,
2 week, 4 week, and 4-week cecal and ileal samples. *p <0.05,
**p <0.01, ***p <0.001. Mann-Whitney U Test.
[0180] FIGS. 13A-13F is a scatter plot of ratios based on Qiime
bioinformatic pipeline that represent diagnostic criteria for
predicting predisposition to weight gain on a high fat diet and
effectiveness of fiber treatment for weight loss or weight gain
prevention for 4-week cecal (FIGS. 13A, 13C, 13E) and 4-week fecal
(FIGS. 13B, 13D, 13F). FIGS. 13A-13B). Ratios at the genus level
are calculated by dividing the sum of any combination of
Erysipelotrichaceae Incertae Sedis, Peptostreptococcaceae Incertae
Sedis, and/or Clostridium cocleatum by Johnsonella. A ratio below 1
indicates a state that is predisposed to weight gain while a ratio
above 1 indicates a state that has a high propensity to prevent
weight gain. FIGS. 13C-13D). Additional ratios are calculated by
dividing the sum of any combination of Erysipelotrichaceae Incertae
Sedis, Peptostreptococcaceae Incertae Sedis, and/or Clostridium
cocleatum by the phylum Firmicutes to measure the relative
abundance. A ratio below 0.1 indicates a state that is predisposed
to weight gain while a ratio above 0.1 indicates a state that has a
high propensity to prevent weight gain. FIGS. 13E-13F). Ratios at
the family level are calculated by dividing the sum of any
combination of Erysipelotrichaceae and/or Peptostreptococcacea by
Lachnospiraceae and/or Ruminococcaceae. A ratio below 0.1 indicates
a state that is predisposed to weight gain while a ratio above 0.1
indicates a state that has a high propensity to prevent weight
gain.
[0181] FIGS. 14A and 14B are graphs showing diversity of the
bacterial populations in the fecal and cecal microbiota at the
class level. Rarefaction curves for class richness and Shannon
diversity index for evenness are shown at the class level.
[0182] FIGS. 15A and 15B demonstrate assessment of microbial
diversity in relation to treatments. Top. Rarefaction curves at the
OTU level for FIG. 15A) taxonomic richness and FIG. 15B) Shannon
index for evenness of the intestinal microbiome in fecal (week 0,
2, and 4) and cecal (week 4) microbiota, according to dietary
treatment.
[0183] FIGS. 16A-16G show the effect of diet and fiber on microbial
community structure. PCA analysis of the unweighted UniFrac
distances of microbial 16S rDNA sequences from the V3-5 region in
fecal samples at week 0 (baseline) (FIG. 16A), week 2 (FIG. 16B),
and week 4 (FIG. 16C), cecal samples at sacrifice (FIG. 16F), and
ileal samples at sacrifice (FIG. 16G). Unweighted UniFrac distances
in LFD, HFD, and HPMC mouse fecal samples comparing community
distance from 0 weeks (FIG. 16D) and from 2 weeks (FIG. 16E), *p
<0.001. Three principal components were plotted by KiNG Kinetic
Image, Next Generation version 2.16 with each sample represented as
a circle.
[0184] FIGS. 17A and 17B show weighted UniFrac distance of the
fecal microbiome at the OTU level. Distance (mean .+-.95% CI) are
shown from baseline (FIG. 17A), or from week 2 (FIG. 17B). ***p
<0.001. As with unweighted UniFrac distances, there were no
differences for the HFD mice over the course of the experiment, as
expected. In the HPMC mice, there were progressive difference in
the community structure at weeks 2 and 4, whereas for the LFD mice,
the communities stabilized after week 2.
[0185] FIGS. 18A-18E show phylogenetic differences between
treatment groups. Heat map of intestinal microbiome. Representation
of relative abundance of predominant taxa classified at the family
level (columns) for 30 individual mice on the three different diets
(rows) in (FIG. 18A) week-0 fecal, (FIG. 18B) week-2 fecal, (FIG.
18C) 4-week fecal, (FIG. 18D) cecal, and (FIG. 18E) ileal
samples.
[0186] FIG. 19 shows clustering of intestinal microbiota by heat
map analysis. Number of samples falling within either the top or
bottom major branch for mice fed HFD, LFD, or HPMC, P-values for
.sub..chi..sup.2 and Fisher's exact test for contingency.
[0187] FIG. 20 shows associations between predominant taxa in fecal
specimens. Specimens were obtained at baseline (week 0) and at
weeks 2 and 4 from the three experimental groups of mice (LFD, HFD,
and HPMC). A circle indicates that Order level taxon is present at
.gtoreq.1% in all specimens and the circle size corresponds to
relative abundance. Taxa classified at the Order level are: 1,
Bacteroidales; 2, Bacteroidetes: unclassified; 3, Lactobacillales;
4, Clostridiales; 5, Erysipelotrichales; 6, Firmicutes:
unclassified; 7, Bacteria: unclassified. A solid line indicates a
significant (p<0.05) correlation between two Orders, whereas a
dashed line is not significant (p>0.05). The numerical values
indicate the strength of the correlation and the directionality
(positive or negative).
[0188] FIGS. 21A-21F show significant relationships between taxa
and host phenotype, conditioned on dietary intervention. Metabolic
parameters for mice on the three different diets, LFD (triangle),
HFD (square), and HPMC (circle) were examined with respect to
relative abundance of taxa, and the correlation constant (R) from
linear regression analysis shown. Top panels represent weight
change vs. cecal Firmicutes (FIG. 21A), cecal Bacteroidetes (FIG.
21B), and cecal Erysipelotrichaceae Incertae Sedis (FIG. 21C).
Bottom panels represent energy intake vs. 4 week fecal
Lachnospiraceae (FIG. 21D), liver free cholesterol vs. cecal
Porphyromonadaceae (FIG. 21E), and fecal saturated fat vs. cecal
Erysipelotrichaceae (FIG. 21F). *p<0.05, **p<0.01,
***p<0.001 for a non-zero slope.
[0189] FIG. 22 is an outline of experiments directed to comparing
the effect of diet-induced obesity (DIO) diet on control mice, mice
exposed to at least one of Coprobacillus, Sporacetigenium,
Holdemania, Erysipelotrichaceae Incertae Sedis,
Peptostreptococaceae Incertae Sedis, and Clostridium cocleatum
(CSHEPCc) or prebiotics alone, or mice exposed to CSHEPCc and
prebiotics simultaneously.
[0190] FIG. 23 is an outline of experiments directed to evaluating
the effects of prophylactic exposure to Coprobacillus,
Sporacetigenium, Holdemania, Erysipelotrichaceae Incertae Sedis,
Peptostreptococaceae Incertae Sedis, and Clostridium cocleatum
(CSHEPCc), and/or prebiotics. In this experiment, the mice are
exposed to nothing (controls), CSHEPCc or prebiotics alone, or
CSHEPCc and prebiotics upon weaning. After 4 weeks, they are given
either high fat or regular chow to see the effects of the
prophylactic exposure.
[0191] FIG. 24 is an outline of experiments directed to determining
the effects of specific colonizations in germ-free mice. There are
three potential types of studies summarized here, comparing
different control and study groups. JO is at least one of
Johnsonella and Oscillibacter; CSHEPCc is at least one of
Coprobacillus, Sporacetigenium, Holdemania, Erysipelotrichaceae
Incertae Sedis, Peptostreptococaceae Incertae Sedis, and
Clostridium cocleatum.
DETAILED DESCRIPTION OF THE INVENTION
[0192] The present invention is based on an unexpected experimental
observation that prevention of weight gain associated with adding
HPMC to a high-fat diet in mice is associated with changes in the
population size and composition of an intestinal microbiota with
(i) reductions in total bacterial populations, primarily reflecting
reduction in phylum Firmicutes (the effect being significant in
both the cecum and the ileum), (ii) significant decreases in the
populations of genera Johnsonella and Oscillibacter, family
Lachnospiraceae and Ruminococcaceae, order Clostridiales, class
Clostridia, and phylum Firmicutes, (iii) marked increases in the
populations of genera Coprobacillus, Sporacetigenium, Holdemania,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS), moderate increases
in genera Dorea, Blautia, and Enterococcus, increases in family
Erysipelotrichaceae, Peptostreptococcaceae, Clostridiales Insertae
Sedis XIV, and Enterococcaceae, order Erysipelotrichales, and
Lactobacillales, and class Erysipelotrichi and Bacilli, especially
in relation to the total numbers of Firmicutes, and (iv)
significant decreases in BCoAT gene levels, in a manner predicted
to lower butyrate availability and energy production. The present
invention is further based on a surprising observation that
cellulose ethers with a beta 1,4 linkage of anhydrous glucose units
have a prebiotic effect although they are known to be substantially
non-fermentable and non-digestible materials in the digestive tract
of mammals.
[0193] The present invention provides novel probiotic and prebiotic
compositions and methods for diagnosing predisposition to and
methods for treating obesity, metabolic syndrome,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, diabetes mellitus, non-alcoholic fatty liver, abnormal
lipid metabolism, atherosclerosis, and related disorders based on
the above-identified changes in mammalian bacterial intestinal
microbiota.
[0194] Definitions and Abbreviations
[0195] The term "Eubacteria" refers to all bacteria and excludes
archaea. In mammals, >90% of all colonic bacteria are in the
phyla Firmicutes or Bacteroidetes (Ley et al., Nat Rev Microbiol
2008; 6:776-88).
[0196] The term "intestinal microbiota" refer to bacteria in the
digestive tract.
[0197] The term "cecal microbiota" refers to microbiota derived
from cecum, which in mammals is the beginning region of the large
intestine in the form of a pouch connecting the ileum with the
ascending colon of the large intestine; it is separated from the
ileum by the ileocecal valve (ICV), and joins the colon at the
cecocolic junction.
[0198] The term "ileal microbiota" refers to microbiota derived
from ileum, which in mammals is the final section of the small
intestine and follows the duodenum and jejunum; ileum is separated
from the cecum by the ileocecal valve (ICV).
[0199] As used herein, the term "probiotic" refers to a
substantially pure bacteria (i.e., a single isolate), or a mixture
of desired bacteria, and may also include any additional components
that can be administered to a mammal for restoring microbiota. Such
compositions are also referred to herein as a "bacterial
inoculant." Probiotics or bacterial inoculant compositions of the
invention are preferably administered with a buffering agent to
allow the bacteria to survive in the acidic environment of the
stomach, i.e., to resist low pH and to grow in the intestinal
environment. Such buffering agents include sodium bicarbonate,
juice, milk, yogurt, infant formula, and other dairy products.
[0200] As used herein, the term "prebiotic" refers to an agent that
increases the number of one or more desired bacteria and/or desired
metabolic activity. The term "metabolic activity of bacteria"
broadly refers to any aspect of microbial catabolism and/or
anabolism (including, e.g., the breakdown of carbohydrates,
proteins, and lipids, secretion of small molecules such as, e.g.,
short chain fatty acids and proteins, synthesis or modifications of
large molecular weight bioactive molecules that involve energy
generation, building of cell walls, capsules, and internal
structures) as well as to any pathway affecting the ability of
bacteria to move and/or reproduce. The metabolic activity need not
relate to the desired bacteria but can be general (e.g., BCoAT
activity).
[0201] Non-limiting examples of prebiotics useful in the methods of
the present invention include xylose, arabinose, ribose, galactose,
rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose,
esculin, tween 80 (e.g., 0.2%), trehalose, maltose, mannose,
mellibiose, raffinose, fructooligosaccharides (e.g., oligofructose,
inulin, inulin-type fructans), galactooligosaccharides, amino
acids, alcohols, water-soluble cellulose derivatives (most
preferably, methylcellulose, methyl ethyl cellulose, hydroxyethyl
cellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose,
hydroxypropyl methylcellulose, and carboxymethyl cellulose),
water-insoluble cellulose derivatives (most preferably, ethyl
cellulose), unprocessed oatmeal, metamucil, all-bran, and any
combinations thereof. See, e.g., Ramirez-Farias et al., Br J Nutr
(2008) 4:1-10; Pool-Zobel and Sauer, J Nutr (2007),
137:2580S-2584S.
[0202] The term "water-soluble cellulose derivative" as used herein
means that the cellulose derivative has a solubility in water of at
least 2 grams, preferably at least 3 grams, more preferably at
least 5 grams in 100 grams of distilled water at 25.degree. C. and
1 atmosphere. The term "water-soluble cellulose derivative" does
not include unmodified cellulose itself which tends to be
water-insoluble.
[0203] The term "water-insoluble cellulose derivative" as used
herein does not include unmodified cellulose and means that the
cellulose derivative has a solubility in water of less than 2
grams, preferably less than 1 gram, in 100 grams of distilled water
at 25.degree. C. and 1 atmosphere.
[0204] The terms "treat" or "treatment" of a state, disorder or
condition include:
[0205] (1) preventing or delaying the appearance of at least one
clinical or sub-clinical symptom of the state, disorder or
condition developing in a subject that may be afflicted with or
predisposed to the state, disorder or condition but does not yet
experience or display clinical or subclinical symptoms of the
state, disorder or condition; or
[0206] (2) inhibiting the state, disorder or condition, i.e.,
arresting, reducing or delaying the development of the disease or a
relapse thereof (in case of maintenance treatment) or at least one
clinical or sub-clinical symptom thereof; or
[0207] (3) relieving the disease, i.e., causing regression of the
state, disorder or condition or at least one of its clinical or
sub-clinical symptoms.
[0208] The benefit to a subject to be treated is either
statistically significant or at least perceptible to the patient or
to the physician.
[0209] A "therapeutically effective amount" means the amount of a
bacterial inoculant or a compound (e.g., a prebiotic or a narrow
spectrum antibiotic or anti-bacterial agent) that, when
administered to a subject for treating a state, disorder or
condition, is sufficient to effect such treatment. The
"therapeutically effective amount" will vary depending on the
compound, bacteria or analogue administered as well as the disease
and its severity and the age, weight, physical condition and
responsiveness of the subject to be treated.
[0210] The term "narrow spectrum antibiotic" is an antibiotic which
can selectively inhibit growth and/or activity of one or few
bacterial species or taxa.
[0211] The terms "diet-induced obesity (DIO) diet" and "high fat
diet" are used herein interchangeably to refer to a high-fat diet,
typically of 45% or 60% in total fat content, that leads to
obesity, hyperglycemia, hyperinsulinemia, and hypertension in a
mouse model. The composition of the diet was designed to
approximate the typical Western diet. See, e.g., Surwit et al.,
Metabolism, 1995, 44:645-651.
[0212] The term "Butyryl CoA transferase (BCoAT)-encoding genes" as
used herein refers to genes encoding an enzyme involved in the
regulation of metabolism of short chain fatty acids and,
preferably, butyrate synthesis.
[0213] As used herein, the term "metagenome" refers to genomic
material obtained directly from a subject, instead of from culture.
Metagenome is thus composed of microbial and host components.
[0214] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are generally
regarded as physiologically tolerable.
[0215] As used herein, the term "combination" of a bacterial
inoculant, probiotic, analogue, or prebiotic compound and at least
a second pharmaceutically active ingredient means at least two, but
any desired combination of compounds can be delivered
simultaneously or sequentially (preferably, within a 24 hour
period).
[0216] Within the meaning of the present invention, the term
"conjoint administration" is used to refer to administration of a
probiotic and a prebiotic simultaneously in one composition, or
simultaneously in different compositions, or sequentially
(preferably, within a 24 hour period).
[0217] "Patient" or "subject" as used herein refers to all mammals
and includes human and veterinary animals. The term "healthy
control" refers to a mammal of the same species (and preferably
same sex and age group) which does not have a disease or condition
that is being treated.
[0218] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water or aqueous solution saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Alternatively, the
carrier can be a solid dosage form carrier, including but not
limited to one or more of a binder (for compressed pills), a
glidant, an encapsulating agent, a flavorant, and a colorant.
Suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0219] The abbreviations used in the nucleotide sequences
throughout this application are as follows: A=adenine, G=guanine,
C=cytosine, T=thymine, U=uracil, R=purine (G or A), Y=pyrimidine (T
or U or C), M=amino (A or C), S=strong interactions 3H-bonds (G or
C), V=(A or C or G), K=(G or T), W=weak interactions 2H-bonds (A or
T or U), N=any (A or G or C or T or U), I=inosine.
[0220] Diagnostic Methods of the Invention
[0221] In one embodiment, the present invention provides a method
for diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by comparing the populations of
Firmicutes and/or Eubacteria and/or Bacteroidetes in the ileal
microbiota of the mammal and in healthy controls, wherein the
increased populations of Firmicutes and/or Eubacteria and/or
Bacteroidetes in the ileal microbiota as compared to healthy
controls are indicative of predisposition to obesity and associated
conditions.
[0222] In another embodiment, the invention provides a method for
diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by comparing the levels populations of
Firmicutes in the cecal and/or fecal microbiota of the mammal and
in healthy controls, wherein the increased level populations of
Firmicutes in the cecal and/or fecal microbiota as compared to
healthy controls are indicative of predisposition to obesity and
associated conditions.
[0223] In yet another embodiment, the invention provides a method
for diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by comparing the ratio of Firmicutes
to Eubacteria (F/E ratio=relative abundance of Firmicutes) in the
cecal and/or fecal microbiota of the mammal and in healthy
controls, wherein the increased F/E ratio in the cecal and/or fecal
microbiota as compared to healthy controls is indicative of
predisposition to obesity and associated conditions.
[0224] In a further embodiment, the invention provides a method for
diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by determining the level of at least
one of Coprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,
Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridium
cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in the
intestinal microbiota of the mammal, comparing the level to the
level of the same bacteria in the intestinal microbiota of healthy
controls, and identifying as a mammal predisposed to obesity etc.
any mammal in which the level of at least one of said bacteria is
lower than in healthy controls.
[0225] In a separate embodiment, the invention provides a method
for diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by determining the level of at least
one of Johnsonella, Oscillibacter, Lachnospiraceae,
Ruminococcaceae, and Clostridiales in the intestinal microbiota of
the mammal, comparing the level to the level of the same bacteria
in the intestinal microbiota of healthy controls, and identifying
as a mammal predisposed to obesity etc. any mammal in which the
level of at least one of said bacteria is higher than in healthy
controls.
[0226] In another embodiment, the invention provides a method for
diagnosing predisposition to obesity and associated conditions
(e.g., metabolic syndrome, diabetes mellitus, insulin-deficiency or
insulin-resistance related disorders, glucose intolerance,
non-alcoholic fatty liver, abnormal lipid metabolism, and
atherosclerosis) in a mammal by comparing the total number of
Butyryl CoA transferase (BCoAT)-encoding genes (or the ratio of the
total number of BCoAT-encoding genes to copies of Bacteroidetes 16S
rRNA) in the intestinal microbiota of the mammal and in healthy
controls, wherein the increased levels of BCoAT genes (or the
increased ratio of BCoAT-encoding genes to copies of Bacteroidetes
16S rRNA) as compared to healthy controls are indicative of
predisposition to obesity and associated conditions.
[0227] Specific changes in microbiota can be detected using various
methods, including without limitation quantitative PCR (qPCR) or
high-throughput sequencing methods which detect over- and
under-represented genes in the total bacterial population (e.g.,
454-sequencing for community analysis), or transcriptomic or
proteomic studies that identify lost or gained microbial
transcripts or proteins within total bacterial populations. See,
e.g., Eckburg et al., Science, 2005, 308:1635-8; Costello et al.,
Science, 2009, 326:1694-7; Grice et al., Science, 2009, 324:1190-2;
Li et al., Nature, 2010, 464: 59-65; Bjursell et al., Journal of
Biological Chemistry, 2006, 281:36269-36279; Mahowald et al., PNAS,
2009, 14:5859-5864; Wikoff et al., PNAS, 2009, 10:3698-3703. While
any number of suitable molecular techniques may be utilized,
particularly useful molecular techniques for the purposes of the
present invention include (i) screening of microbial 16S ribosomal
RNAs (16S rRNA) using PCR and (ii) high-throughput "metagenome"
sequencing methods, which detect over- and under-represented genes
in the total bacterial population. Screening of 16S rRNA genes
permits characterizing microorganisms present in the microbiota at
the species, genus, family, order, class, or phylum level. Such
screening can be performed, e.g., by conducting PCR using universal
primers to the
[0228] V2, V3, V4, V6 (or V2-V4) region of the 16S rRNA gene
followed by high-throughput sequencing and taxonomic analysis. See
e.g., Gao et al. Proc. Natl. Acad. Sci. USA, 2007; 104:2927-32;
Zoetendal et al., Mol. Microbiol., 2006, 59:1639-1650; Schloss and
Handelsman, Microbiol. Mol. Biol. Rev., 2004, 68:686-691; Smit et
al., Appl. Environ. Microbiol., 2001, 67:2284-2291; Harris and
Hartley, J. Med. Microbiol., 2003, 52:685-691; Saglani et al., Arch
Dis Child, 2005, 90:70-73. The high-throughput "metagenome"
sequencing methods involve obtaining multiple parallel short
sequencing reads looking for under- and over-represented genes in a
total mixed sample population. Such sequencing is usually followed
by determining the G+C content or tetranucleotide content (Pride et
al., Genome Res., 2003, 13;145) of the genes to characterize the
specific bacterial species in the sample. Additional techniques
include those involving cultivation of individual microorganisms
from mixed samples. See, e.g., Manual of Clinical Microbiology, 8th
edition; American Society of Microbiology, Washington DC, 2003.
[0229] Therapeutic Methods of the Invention
[0230] In conjunction with the diagnostic methods, the present
invention also provides therapeutic methods for treating obesity,
metabolic syndrome, insulin-deficiency or insulin-resistance
related disorders, glucose intolerance, diabetes mellitus,
non-alcoholic fatty liver, abnormal lipid metabolism,
atherosclerosis, and related disorders by restoring mammalian
bacterial intestinal microbiota to the composition observed in
healthy subjects.
[0231] In certain specific embodiments, restoring of microbiota is
achieved by administering to a mammal in need thereof a
therapeutically effective amount of a probiotic composition
comprising an effective amount of at least one bacterial strain, or
a combinations of several strains, or a prebiotic composition, or a
mixture thereof, wherein the composition (i) stimulates or inhibits
specific metabolic pathways involved in host energy homeostasis
and/or (ii) stimulates growth and/or activity of bacteria which are
under-represented in a disease and/or (iii) inhibits growth and/or
activity of bacteria which are over-represented in a disease.
[0232] In one embodiment, the present invention provides a method
for promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by administering a probiotic or a
prebiotic composition or a combination thereof, that stimulates
growth or activity of at least one of Coprobacillus,
Sporacetigenium, Holdemania, Dorea, Blautia, Enterococcus,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum,
and Peptosteptococcaceae Incertae Sedis (PIS) in the intestinal
microbiota of the mammal. In a related embodiment, the invention
provides a method for determining whether weight loss can be
achieved or obesity and associated conditions can be treated in a
mammal by the latter method by determining the level of at least
one of Coprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,
Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridium
cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) in the
intestinal microbiota of the mammal and comparing said level to the
level of the same bacteria in the intestinal microbiota of healthy
controls, and identifying as a mammal treatable by the latter
method any mammal in which the level of at least one of said
bacteria is lower than in healthy controls.
[0233] In another embodiment, the invention provides a method for
promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by inhibiting growth or activity
of at least one of Johnsonella, Oscillibacter, Lachnospiraceae,
Ruminococcaceae, and Clostridiales in the intestinal microbiota of
the mammal (e.g., by administering a narrow spectrum antibiotic, or
another anti-bacterial agent, including a probiotic [e.g., at least
one of Coprobacillus, Sporacetigenium, Holdemania, Dorea, Blautia,
Enterococcus, Erysipelotrichaceae Incertae Sedis (EIS), Clostridium
cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS)] which
competes with at least one of Johnsonella, Oscillibacter,
Lachnospiraceae, Ruminococcaceae, and Clostridiales for metabolic
substrates, physical niches, or produces relevant antibiotic(s)).
In a related embodiment, the invention provides a method for
determining whether weight loss can be achieved or obesity and
associated conditions can be treated in a mammal by the latter
method by determining the level of at least one of Johnsonella,
Oscillibacter, Lachnospiraceae, Ruminococcaceae, and Clostridiales
in the intestinal microbiota of the mammal and comparing said level
to the level of the same bacteria in the intestinal microbiota of a
healthy control, and identifying as a mammal treatable by the
latter method any mammal in which the level of at least one of said
bacteria is higher than in healthy controls.
[0234] In yet another embodiment, the invention provides a method
for promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by administering a probiotic or a
prebiotic composition or a combination thereof, that lowers the
populations of Firmicutes and/or Eubacteria and/or Bacteroidetes in
the ileal microbiota of the mammal.
[0235] In a further embodiment, the invention provides a method for
promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by administering a probiotic or a
prebiotic composition or a combination thereof, that lowers the
populations of Firmicutes in the cecal and/or fecal microbiota of
the mammal.
[0236] In a separate embodiment, the invention provides a method
for promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by administering a probiotic or a
prebiotic composition or a combination thereof, that lowers the
ratio of Firmicutes to Eubacteria (F/E ratio=relative abundance of
Firmicutes) in the cecal and/or fecal microbiota of the mammal.
[0237] In another embodiment, the invention provides a method for
promoting weight loss, preventing or treating obesity and
associated conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis) in a mammal by administering a probiotic or a
prebiotic composition or a combination thereof, that lowers the
levels of Butyryl CoA transferase (BCoAT) enzyme in the intestinal
microbiota of the mammal. In a related embodiment, the invention
provides a method for promoting weight loss, preventing or treating
obesity and associated conditions in a mammal by administering a
probiotic or a prebiotic composition or a combination thereof, that
lowers the ratio of the total number of Butyryl CoA transferase
(BCoAT)-encoding genes to copies of Bacteroidetes 16S rRNA in the
intestinal microbiota of the mammal. In another related embodiment,
the invention provides a method for promoting weight loss,
preventing or treating obesity and associated conditions in a
mammal by administering a probiotic or a prebiotic composition or a
combination thereof, that lowers the levels of butyrate (e.g.,
measured using chromatographic methods [see, e.g., Renom et al.,
Clin. Chem. Lab. Med., 2001, 39(1): 15-19]) in the intestinal
microbiota of the mammal.
[0238] Probiotic and Prebiotic Compositions, Dosages and
Administration
[0239] In conjunction with the above-identified therapeutic
methods, the present invention provides probiotic and prebiotic
compositions or combinations of prebiotics and probiotics useful
for promoting weight loss and/or treating obesity and associated
conditions (e.g., metabolic syndrome, diabetes mellitus,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, non-alcoholic fatty liver, abnormal lipid metabolism,
and atherosclerosis).
[0240] Probiotics useful in the methods of the present invention
can comprise live bacterial strains and/or spores. In a preferred
embodiment, such live bacterial strains and/or spores are from the
genus Coprobacillus, Sporacetigenium, Holdemania,
Erysipelotrichaceae Incertae Sedis (EIS), Clostridium cocleatum, or
Peptosteptococcaceae Incertae Sedis (PIS). In certain embodiments,
the bacteria administered in the therapeutic methods of the
invention comprise one or more of Coprobacillus, Sporacetigenium,
Holdemania, Erysipelotrichaceae Incertae Sedis (EIS), Clostridium
cocleatum, and Peptosteptococcaceae Incertae Sedis (PIS) and one or
more additional bacterial strains (such as, e.g., Oxalobacter
species, Lactobacillus species, etc.).
[0241] One or several different bacterial inoculants can be
administered simultaneously or sequentially (including
administering at different times). Such bacteria can be isolated
from microbiota and grown in culture using known techniques.
However, many bacterial species are very difficult to culture and
administration of others may lead to various undesirable
side-effects. The present invention therefore also comprises
administering "bacterial analogues", such as recombinant carrier
strains expressing one or more heterologous genes derived from the
bacteria affected in a disease. The use of such recombinant
bacteria may allow the use of lower therapeutic amounts due to
higher protein expression and may simultaneously minimize any
potential harmful side-effects associated with reintroduction of
specific bacterial strains. Non-limiting examples of recombinant
carrier strains useful in the methods of the present invention
include E. coli and Lactobacillus, Bacteroides and Oxalobacter.
Methods describing the use of bacteria for heterologous protein
delivery are described, e.g., in U.S. Pat. No. 6,803,231.
[0242] In certain embodiments, a conditional lethal bacterial
strain can be utilized as the inoculant or to deliver a recombinant
construct. Such a conditional lethal bacteria survives for a
limited time typically when provided certain nutritional
supplements. It is contemplated that such a supplement could be a
liquid, formulated to contain the nutritional component necessary
to keep the bacteria alive. It is further contemplated that a
patient/subject would drink such a supplement in intervals to keep
the bacteria alive. Once the supplement is depleted, the
conditional lethal bacteria dies. Methods relating to conditional
lethal strains of H. pylori are described in U.S. Pat. No.
6,570,004.
[0243] In certain embodiments, the bacterial inoculant used in the
methods of the invention further comprises a buffering agent.
Examples of useful buffering agents include sodium bicarbonate,
juice, milk, yogurt, infant formula, and other dairy products.
[0244] Administration of a bacterial inoculant can be accomplished
by any method likely to introduce the organisms into the desired
location. In a preferred embodiment, bacteria are administered
orally. Alternatively, bacteria can be administered rectally, by
enema, by esophagogastroduodenoscopy, colonoscopy, nasogastric
tube, or orogastric tube.
[0245] The bacteria can be mixed with an excipient, diluent or
carrier selected with regard to the intended route of
administration and standard pharmaceutical practice. For easier
delivery to the digestive tract, bacteria can be applied to liquid
or solid food, or feed or to drinking water. For oral
administration, bacteria can be also formulated in a capsule. The
excipient, diluent and/or carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and should be non-toxic to the bacteria and the subject/patient.
Preferably, the excipient, diluent and/or carrier contains an
ingredient that promotes viability of the bacteria during storage.
The formulation can include added ingredients to improve
palatability, improve shelf-life, impart nutritional benefits, and
the like. Acceptable excipients, diluents, and carriers for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington: The Science and Practice of
Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit.
2005). The choice of pharmaceutical excipient, diluent, and carrier
can be selected with regard to the intended route of administration
and standard pharmaceutical practice.
[0246] The dosage of the bacterial inoculant or compound of the
invention will vary widely, depending upon the nature of the
disease, the patient's medical history, the frequency of
administration, the manner of administration, the clearance of the
agent from the host, and the like. The initial dose may be larger,
followed by smaller maintenance doses. The dose may be administered
as infrequently as weekly or biweekly, or fractionated into smaller
doses and administered daily, semi-weekly, etc., to maintain an
effective dosage level. It is contemplated that a variety of doses
will be effective to achieve colonization of the intestinal tract
with the desired bacterial inoculant, e.g. 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, and 10.sup.10CFU for example, can be
administered in a single dose. Lower doses can also be effective,
e.g., 10.sup.4, and 10.sup.5 CFU.
[0247] Non-limiting examples of prebiotics useful in the methods of
the present invention include xylose, arabinose, ribose, galactose,
rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose,
esculin, tween 80, trehalose, maltose, mannose, mellibiose,
raffinose, fructooligosaccharides (e.g., oligofructose, inulin,
inulin-type fructans), galactooligosaccharides, amino acids,
alcohols, water-soluble cellulose derivatives (most preferably,
methylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose,
ethyl hydroxyethyl cellulose, cationic hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxyethyl methylcellulose,
hydroxypropyl methylcellulose, and carboxymethyl cellulose),
water-insoluble cellulose derivatives (most preferably, ethyl
cellulose), unprocessed oatmeal, metamucil, all-bran, and any
combinations thereof.
[0248] Table 1 provides a chart of prebiotics useful for
stimulating growth and metabolic activity (by acting as substrate
for fermentation) of Coprobacillus, Sporacetigenium, Holdemania, or
Clostridium cocleatum based on information from Kageyama et al.,
Microbiol. Immunol., 2000, 44:23-28; Chen et al., Int J Syst Evol
Microbiol, 2006, 56:721-725; Moore et al., Int J Syst Bact, 1997,
47(4):1201-1204, Willems et al., Int J Syst Bact, 1995, 45:855-857;
Lino et al., Int J Syst Evol Microbiol, 2007, 57:1840-1845;
Kaneuchi et al., Int J Syst Bact, 1979, 29, 1. As follows from
Table 1, trehalose, cellobiose, lactose, maltose, mannose, sucrose,
fructose, galactose, salicin, mellibiose, and raffinose stimulate
growth and metabolic activity of two or more genera selected from
Coprobacillus, Sporacetigenium, Holdemania, or Clostridium
cocleatum, but not of Johnsonella and Oscillibacter.
TABLE-US-00001 TABLE 1 Coprobacillus Holdemania Sporacetegenium
Clostridium cocleatum Johnsonella Oscillibacter Arabinose - w + - -
+/-.sup.a Cellobiose + w w + - - Esculin - + - Fructose + + + + - -
Galactose + w + - Glucose + + + + + + Inulin + Lactose + + w + - -
Maltose + + + + - - Mannose + w w + - - Mellibiose w w + - -
Raffinose - w w + - - Rhamnose - w w - - - Ribose - w +/- - - +
Salicin + + - w - - Sucrose + + w + - - Trehalose + w w - - -
Xylose - w + - - + + facilitates growth, w weak growth promotion,
+/- growth variable dependent on strain, - no growth effect. Bold
facilitates growth in >2 CSH organisms. .sup.a+ for L-arabinose;
- for D-arabinose.
[0249] Preferred water-soluble cellulose derivatives for use in the
present invention are water-soluble cellulose esters and cellulose
ethers. Preferred cellulose ethers are water-soluble
carboxy-C.sub.1-C.sub.3-alkyl celluloses, such as carboxymethyl
celluloses; water-soluble carboxy-C.sub.1-C.sub.3-alkyl
hydroxy-C.sub.1-C.sub.3-alkyl celluloses, such as carboxymethyl
hydroxyethyl celluloses; water-soluble C.sub.1-C.sub.3-alkyl
celluloses, such as methylcelluloses; water-soluble
C.sub.1-C.sub.3-alkyl hydroxy-C.sub.1-3-alkyl celluloses, such as
hydroxyethyl methylcelluloses, hydroxypropyl methylcelluloses or
ethyl hydroxyethyl celluloses; water-soluble
hydroxy-C.sub.1-3-alkyl celluloses, such as hydroxyethyl celluloses
or hydroxypropyl celluloses; water-soluble mixed
hydroxy-C.sub.1-C.sub.3-alkyl celluloses, such as hydroxyethyl
hydroxypropyl celluloses, water-soluble mixed C.sub.1-C.sub.3-alkyl
celluloses, such as methyl ethyl celluloses, or water-soluble
alkoxy hydroxyethyl hydroxypropyl celluloses, the alkoxy group
being straight-chain or branched and containing 2 to 8 carbon
atoms. The more preferred cellulose ethers are methylcellulose,
methyl ethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl
cellulose, cationic hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxyethyl methylcellulose, hydroxypropyl
methylcellulose, and carboxymethyl cellulose, which are classified
as water-soluble cellulose ethers by the skilled artisans. The most
preferred water-soluble cellulose ethers are methylcelluloses with
a methyl molar substitution DS.sub.methoxyl of from 0.5 to 3.0,
preferably from 1 to 2.5, and hydroxypropyl methylcelluloses with a
DS.sub.methoxyl of from 0.9 to 2.2, preferably from 1.1 to 2.0, and
a MS.sub.hydroxypropoxyl of from 0.02 to 2.0, preferably from 0.1
to 1.2. The methoxyl content of methyl cellulose can be determined
according to ASTM method D 1347-72 (reapproved 1995). The methoxyl
and hydroxypropoxyl content of hydroxypropyl methylcellulose can be
determined by ASTM method D-2363-79 (reapproved 1989). Methyl
celluloses and hydroxypropyl methylcelluloses, such as K250M,
K100M, K4M, K1M, F220M, F4M and J4M hydroxypropyl methylcellulose
are commercially available from The Dow Chemical Company).
[0250] Preferred cationic hydroxyethyl celluloses are those
described in U.S. Pat. No. 3,472,840. Preferably the cationic
hydroxyethyl celluloses have groups of the formula
[R.sup.1R.sup.2R.sup.3R.sup.4N.sup.+] (A.sub.z-).sub.l/z, (II),
wherein R.sup.1, R.sup.2 and R.sup.3 each independently are
C.sub.1-6-alkyl, preferably --CH.sub.3 or --C.sub.2H.sub.5, R.sup.4
is --CH.sub.2--CHOH--CH.sub.2-- or --CH.sub.2CH(OH)--, A.sup.z-is
an anion, and z is 1, 2 or 3. The cationic degree of substitution
(often referred to as the CS orcationic substitution) of the
cationic hydroxyethyl cellulose is in a range from about 0.075 to
about 0.8, preferably about 0.15 to about 0.60. A range of about
0.15 to about 0.60 corresponds to a Kjeldahl nitrogen content of
about 0.8% to about 2.5%. More preferably, the cationic
hydroxyethyl cellulose has a Kjeldahl nitrogen content between 1.5
and 2.2%, which corresponds to a CS of about 0.3 to about 0.5. In
one embodiment, the cationic hydroxyethylcellulose has a Brookfield
LVT determined solution viscosity of from about 5 cP (=mPa.s) to
about 10,000 cP, preferably from about 5 cP to about 3,000 cP,
measured as a one weight percent aqueous solution at 25.degree.
C.
[0251] Combinations of two or more water-soluble cellulose
derivatives are also useful. The water-soluble cellulose derivative
generally has a viscosity of from 5 to 2,000,000 cps (=mPa.s),
preferably from 50 cps to 1,000,000 cps, more preferably from 1,000
to 300,000 cps, measured as a two weight percent aqueous solution
at 20.degree. C. The viscosity can be measured in a rotational
viscometer.
[0252] Preferred water-insoluble cellulose derivatives for use in
the present invention are water-insoluble cellulose ethers,
particularly ethyl cellulose, propyl cellulose or butyl cellulose.
Other useful water-insoluble cellulose derivatives are cellulose
derivatives which have been chemically, preferably hydrophobically,
modified to provide water insolubility. Chemical modification can
be achieved with hydrophobic long chain branched or non-branched
alkyl, arylalkyl or alkylaryl groups. "Long chain" typically means
at least 5, more typically at least 10, particularly at least 12
carbon atoms. Others type of water-insoluble cellulose are
crosslinked cellulose, when various crosslinking agents are used.
Chemically modified, including the hydrophobically modified,
water-insoluble cellulose derivatives are known in the art. They
are useful provided that they have a solubility in water of less
than 2 grams, preferably less than 1 gram, in 100 grams of
distilled water at 25.degree. C. and 1 atmosphere. The most
preferred cellulose derivative is ethyl cellulose. The ethyl
cellulose preferably has an ethoxyl substitution of from 40 to 55
percent, more preferably from 43 to 53 percent, most preferably
from 44 to 51 percent. The percent ethoxyl substitution is based on
the weight of the substituted product and determined according to a
Zeisel gas chromatographic technique as described in ASTM
D4794-94(2003). The molecular weight of the ethyl cellulose is
expressed as the viscosity of a 5 weight percent solution of the
ethyl cellulose measured at 25.degree. C. in a mixture of 80 volume
percent toluene and 20 volume percent ethanol. The ethyl cellulose
concentration is based on the total weight of toluene, ethanol and
ethyl cellulose. The viscosity is measured using Ubbelohde tubes as
outlined in ASTM D914-00 and as further described in ASTM D446-04,
which is referenced in ASTM D914-00. The ethyl cellulose generally
has a viscosity of up to 400 mPa's, preferably up to 300 mPa's,
more preferably up to 100 mPa's, measured as a 5 weight percent
solution at 25.degree. C. in a mixture of 80 volume percent toluene
and 20 volume percent ethanol. The preferred ethyl celluloses are
premium grades ETHOCEL ethyl cellulose which are commercially
available from The Dow Chemical Company of Midland, Mich.
Combinations of two or more water-insoluble cellulose derivatives
are also useful. Preferably the water-insoluble cellulose
derivative has an average particle size of less than 0.1
millimeter, more preferably less than 0.05 millimeter, most
preferably less than 0.02 millimeter. Preferably the
water-insoluble cellulose derivative is exposed to an edible fat or
oil before being administered to an individual so that the
cellulose derivative imbibes the fat or oil. Advantageously the
water-insoluble cellulose derivative is exposed to an excess of the
fat or oil at about 40 to 60.degree. C.
[0253] In certain other specific embodiments, the therapeutic
methods of the invention rely on the administration of a
therapeutically effective amount of a naturally or recombinantly
produced bacterial protein or a combination of such proteins which
(i) increase the number and/or activity of one or more bacteria
which are under-represented in a disease and/or (ii) decrease the
number and/or activity of one or more bacteria which are
over-represented in a disease. The proteins according to this
embodiment may be produced by the same strain of bacteria which is
intended to be regulated or by a different strain.
[0254] Prior to administering to humans, the effectiveness of the
novel therapeutic compositions of the present invention can be
studied in animal models of obesity, such as, e.g., sub-therapeutic
antibiotic treatment (STAT) mice (Cho et al., Gastroenterology,
2009, 136(5) Supplement 1: A-102), ob/ob mice (Ley et al., Proc.
Natl. Acad. Sci. USA 2005; 102:11070-5; Turnbaugh et al., Nature
2006; 444:1027-31), db/db mice (Kobayashi et al., Metabolism, 2000,
48(1):22-31), diet-induced obesity (DIO) mice (Petro et al.,
Metabolism, 2004, 53(4):454-457), NOD mice (Wen et al., Nature,
2008; 455(7216):1109-1113), etc.
[0255] Combination Treatments
[0256] For an enhanced therapeutic effect, the probiotics and/or
prebiotics as described herein can be administered in combination
with other therapeutic agents or regimes as discussed. The choice
of therapeutic agents that can be co-administered with the
probiotics and/or prebiotics of the invention depends, in part, on
the condition being treated.
[0257] Non-limiting examples of additional pharmaceutically active
compounds useful for treatment of obesity, metabolic syndrome,
insulin-deficiency or insulin-resistance related disorders, glucose
intolerance, diabetes mellitus, non-alcoholic fatty liver, abnormal
lipid metabolism, atherosclerosis, and related disorders include
anti-inflammatory agents, antioxidants, antiarrhythmics, cytokines,
analgesics, vasodilators, antihypertensive agents including
beta-blockers, angiotensin converting enzyme inhibitors (ACE
inhibitors), and calcium channel blockers, inhibitors of
cholesterol synthesis, cholesterol binding agents, antithrombotic
agents, central modulators of appetite, and diabetes drugs.
Examples of inhibitors of cholesterol synthesis or absorption which
are useful in the combination therapies of the present invention
include Hmg-CoA reductase inhibitors and their bio-active
metabolites, such as, e.g., simvastatin, lovastatin, pravastatin,
compactin, fluvastatin, dalvastatin, atorvastatin, HR-780,
GR-95030, CI-981, BMY 22089, and BMY 22566. See, e.g., U.S. Pat.
Nos. 4,346,227; 4,444,784; 4,857,522; 5,190,970; 5,316,765, and
5,461,039; PCT Publ. No. W084/02131; GB Pat. No. 2,202,846. As used
in the methods or compositions of the present invention, any one or
several of the Hmg-CoA reductase inhibitor compounds may be mixed
with L-arginine or a substrate precursor to endogenous nitric
oxide, as described in U.S. Pat. Nos. 6,425,881 and 6,239,172, and
5,968,983, to provide a therapeutically effective mixture for use
in conjunction with probiotics and/or prebiotics of the present
invention.
[0258] Non-limiting examples of diabetes drugs useful in the
combination therapies of the present invention include insulin,
proinsulin, insulin analogs, activin, glucagon, somatostatin,
amylin, actos (pioglitazone), amaryl (glimepiride), glipizide,
avandia (rosiglitazone), glucophage, glucotrol, glucovance (a
combination of glyburide and metformin), and the like. See, e.g.,
U.S. Pat. No. 6,610,272. The term "insulin" encompasses natural
extracted human insulin, recombinantly produced human insulin,
insulin extracted from bovine and/or porcine sources, recombinantly
produced porcine and bovine insulin and mixtures of any of these
insulin products. In accordance with the present invention,
administering probiotics and/or prebiotics of the present invention
in combination with insulin is expected to lower the dose of
insulin required to manage the diabetic patient, while also
alleviating the symptoms of metabolic syndrome.
[0259] In accordance with the present invention there may be
numerous tools and techniques within the skill of the art, such as
those commonly used in molecular immunology, cellular immunology,
pharmacology, and microbiology. Such tools and techniques are
describe in detail in e.g., Sambrook et al. (2001) Molecular
Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current
Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken,
N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell
Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al.
eds. (2005) Current Protocols in Immunology, John Wiley and Sons,
Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in
Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et
al. eds. (2005) Current Protocols in Protein Science, John Wiley
and Sons, Inc. Hoboken, N.J.; and Enna et al. eds. (2005) Current
Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken,
N.J.
EXAMPLES
[0260] The present invention is also described and demonstrated by
way of the following examples. However, the use of these and other
examples anywhere in the specification is illustrative only and in
no way limits the scope and meaning of the invention or of any
exemplified term. Likewise, the invention is not limited to any
particular preferred embodiments described here. Indeed, many
modifications and variations of the invention may be apparent to
those skilled in the art upon reading this specification, and such
variations can be made without departing from the invention in
spirit or in scope. The invention is therefore to be limited only
by the terms of the appended claims along with the full scope of
equivalents to which those claims are entitled.
Example 1
Analysis of Diet-Associated Changes in Intestinal Microbiota of
Mice
Materials and Methods
1. Animals and Diets
[0261] Thirty (30) obese male C57/B16J mice (from Jackson
Laboratories, Bar Harbor, Me.) were studied. All mice were fed a
high fat (60% fat) diet (also termed diet-induced obesity [DIO]
diet; supplied by Research Diets Inc., New Brunswick, N.J.) and
water ad libitum for at least two months. Then baseline fecal
samples were obtained and animals were divided in three groups of
ten (10) mice each. One group was maintained on the high-fat (60%
fat) diet, one group was converted to a low fat (10% fat) diet
(also supplied by Research Diets Inc., New Brunswick, N.J.), and
the third group was fed a 60% fat diet+HPMC. Hydroxypropyl
methylcellulose (HPMC) was present at 8 percent weight level in the
treatment diet. It was mixed with the powdered components of the
diet. The HPMC had a methoxyl content of 19-24 percent, a
hydroxypropoxyl content of 7-12 percent and a viscosity of about
250,000 mPa's, measured as a 2 wt. % aqueous solution at 20.degree.
C., and is commercially available from The Dow Chemical Company
under the Trademark METHOCEL K250M hypromellose. Animals were
weighted periodically. A fresh fecal pellet was collected from each
individual mouse at baseline, after 2 and 4 weeks, and shortly
after sacrifice and frozen at -80.degree. C. At the time of
sacrifice, the cecal and ileal contents were also frozen and stored
at -80.degree. C. for future study.
2. DNA extraction
[0262] Approximately 10 mg each of the fecal, cecal, and ileal
samples were extracted using the MoBio Powersoil 2 DNA Isolation
kit (MoBio Laboratories, Carlsbad, Calif. ) as per the
manufacturer's instructions. This extraction method uses a
combination of mechanical disruption using bead-beating and spin
filtration using silica filter tubes to extract bacterial genomic
DNA from each of the samples.
3. qPCR, sequencing and taxonomic analysis
[0263] DNA extracted from cecal, ileal, and fecal specimens was
subjected to PCR using barcoded universal primers interrogating
regions V3-V5 of the 16S rRNA gene followed by 454 sequencing and
taxonomic analysis.
[0264] Total eubacterial levels were determined by a standardized
quantitative PCR (qPCR) using primers Eub519F:
5'-CAGCAGCCGCGGTRATA-3' (SEQ ID NO: 1) and Eu785R:
5'-GGACTACCVGGGTATCTAAKCC-3' (SEQ ID NO: 2) directed to conserved
16S rRNA fragments.
PCR reaction mixture and program (Power SYBR Green) were as
follows:
TABLE-US-00002 Reagents Stock Vol/Reac (.mu.l) Final PCR Master 2x
12.5 1x Mix F. primer 10 .mu.M 1 0.4 .mu.M R. primer 10 .mu.M 1 0.4
.mu.M BSA 20 ng/.mu.l 0.125 0.1 ng/.mu.l Template 1 Total 25
[0265] Program: 50.degree. C. 2 min, 95.degree. C. 10 min
[0266] 95.degree. C. 15 Seconds and
[0267] 56.degree. C. 60 Seconds 40 cycles
[0268] Firmicutes levels were determined by qPCR using primers
Firm934F: 5'-GGAGYATGTGGTTTAATTCGAAGCA-3' (SEQ ID NO: 3) and
Firm1060R: 5'-AGCTGACGACAACCATGCAC-3' (SEQ ID NO: 4) directed to
conserved 16S rRNA fragments.
PCR reaction and Program (Power SYBR Green) were as follows:
TABLE-US-00003 Reagents Stock Vol/Reac (.mu.l) Final PCR Master 2x
12.5 1x Mix F. primer 10 .mu.M 1 0.4 .mu.M R. primer 10 .mu.M 1 0.4
.mu.M BSA 20 ng/.mu.l 0.125 0.1 ng/.mu.l Template 1 Total 25
[0269] Program: 50.degree. C. 2 min, 95.degree. C. 10 min
[0270] 95.degree. C. 15 Seconds and
[0271] 60.degree. C. 60 Seconds 40 cycles
[0272] Bacteroidetes levels were determined by qPCR using primers
Bact934F: 5'-GGARCATGTGGTTTAATTCGATGAT -3' (SEQ ID NO: 5) and
Bact1060R: 5'-AGCTGACGACAACCATGCAG -3' (SEQ ID NO: 6) directed to
conserved 16S rRNA fragments.
PCR reaction and Program (Power SYBR Green) were as follows:
TABLE-US-00004 Reagents Stock Vol/Reac (.mu.l) Final PCR Master 2x
12.5 1x Mix F. primer 10 .mu.M 1 0.4 .mu.M R. primer 10 .mu.M 1 0.4
.mu.M BSA 20 ng/.mu.l 0.125 0.1 ng/.mu.l Template 1 Total 25
[0273] Program: 50.degree. C. 2 min, 95.degree. C. 10 min
[0274] 95.degree. C. 15 Seconds and
[0275] 60.degree. C. 60 seconds 40 cycles
[0276] The samples then underwent 454 pyrosequencing (Roche) using
barcoded primers designed to interrogate the 16S rRNA regions
V3-V5. The average number of sequence reads obtained from fecal
pellets were 5671.+-.1981 reads, while the average number of
sequence reads obtained from cecal and ileal samples were
4901.+-.2271 and 6662.+-.2438, respectively. The total amount of
data generated in the sequencing experiment was about 0.18 Gb (180
Mb). Sequence data were summated to the phylum, class, order,
family and genus levels and analyzed.
[0277] Total fungal levels were determined by qPCR using primers
directed to the conserved ITS2 region in the fungal rrn operon
ITS1F CTYGGTCATTTAGAGGAAGTAA (SEQ ID NO: 7) and ITS2
RCTGCGTTCTTCATCGWTG (SEQ ID NO: 8) and probe TCYGTAGGTGAACCTGCRG
(SEQ ID NO: 9).
[0278] The total number of genes encoding Butyryl CoA transferase
(BCoAT), regardless of the taxonomic origin of the gene, were
determined by qPCR using primers BCoATscrF
GCIGAICATTTCACITGGAAYWSITGGCAYATG (SEQ ID NO: 10) and BCoATscrR
CCTGCCTTTGCAATRTCIACRAANGC (SEQ ID NO: 11). BCoAT gene number was
determined by quantitative PCR using FastStart SYBR Green Master
Mix (Roche) with primer concentration at 500 nM. Samples were run
at the following temperature profile: 50.degree. C. for 2 minutes,
95.degree. C. for 10 minutes, then cycle 40 times at 95.degree. C.
for 15 seconds, 53.degree. C. for 30 seconds, and 72.degree. C. for
30 seconds. Positive samples were confirmed by melting curve
analysis.
[0279] Bioinformatic Pipeline 1. After the completion of
sequencing, a read processing pipeline consisting of a set of
modular scripts designed at the JCVI were employed for
deconvolution, trimming, and quality filtering. First reads were
deconvoluted or assigned to samples based on their unique 10 nt
barcode allowing no more than a one nt mismatch to the barcode.
After deconvolution, barcode and 16S primer sequences were removed
allowing a maximum of 6 mismatches to the 16S primer and a maximum
primer to barcode distance of 3 nt. Reads with an average length of
<100 nt, and reads with `Ns` were removed. A Blastn quality
check was performed against an internal data set of 16S reads to
remove any sample reads not consistent with 16S gene sequences in
which at least 30% of the query must be covered by the alignment
(60 nt minimum). Passing reads were subsequently checked for
chimeras using a modified version of the RDP Chimera Check, using a
reference data set maintained in-house. Remaining reads were then
classified to lowest taxonomic level possible using the RDP
Classifier with 80% confidence. Taxonomic results were then
converted to relative abundance or ratios for each sample, and the
difference between was calculated with the Mann-Whitney U test
(means .+-. and ratios depicted in FIGS. 4-6).
Results
Correlations Between Diets and Weight Gain
[0280] In the group which was continuously fed high fat (60% fat)
diet, animals gained weight. In the group where HPMC was added to
the high fat (60% fat) diet, animals stopped gaining weight as soon
as HPMC was added. And animals switched to the low fat (10% fat)
diet lost weight. These observations were consistent with prior
observations of the role of HPMC in controlling metabolic syndrome,
diabetes mellitus and obesity, and in promotion of weight loss or
maintenance of the desired body weight (see the Background section,
above).
Intestinal Populations of Microorganisms
[0281] The present inventors set to investigate whether weight gain
associated with high fat (60% fat) diet and the absence of such
weight gain upon the addition of HPMC to the same diet correlates
with changes in intestinal microbiota.
[0282] Eubacteria
[0283] As demonstrated in FIG. 3A, at baseline, mice had 9-10
log16S copies/g of fecal pellet without significant differences
between the three groups. At 2 weeks, Eubacterial (total bacteria)
counts rose slightly in the 10% fat and 60% fat groups, and fell in
the 60% fat+HPMC group, but none of the changes were statistically
significant. By 4 weeks, levels in the 10% fat and 60% fat groups
were unchanged, but the 60% fat+HPMC group was significantly lower.
The levels in the cecum at sacrifice were very similar to the 4
week results, as expected, with the same lower trends for the 60%
fat+HPMC group. The ileal samples at sacrifice were lower,
especially in the 60% fat+HPMC group. Thus, there is consistency in
the decrease observed in the 60% fat+HPMC group with respect to the
baseline, in the 2-week, 4-week, cecal, and ileal samples. These
data provide evidence that adding HPMC to the diet lowers total
Eubacterial populations with reference to the other two groups.
[0284] The Eubacterial populations within each group of 10 mice
were also analyzed over the study period. For the mice maintained
on the 60% fat diet, and the mice converted to the low fat (10%
fat) diet, there were no significant differences over time
(comparing Basline, 2-week, and 4-week samples). However, for the
mice converted from the 60% fat diet to the 60% fat+HPMC diet,
there was a progressive and significant decline between Basline and
4-week samples. In all 3 diet groups, ileal levels were 0.5-1.0
log.sub.10 lower than in cecum.
[0285] Firmicutes
[0286] As demonstrated in FIG. 3C, results for Firmicutes were
generally similar to those for all Eubacteria. This internal
consistency is not surprising since Firmicutes represent the major
population within Eubacteria in the mammalian intestinal tract. At
baseline, the Firmicutes populations of all three groups were
similar, as expected, but by the second week, the population in the
60% fat+HPMC group was trending lower, and, in the 4-week sample,
cecal and ileal samples were significantly lower than in the two
other diet groups.
[0287] There were no differences over time in the group of mice
maintained on the 60% fat diet, or changed to the 10% fat diet, but
there was a progressive and significant decrease in the 60%
fat+HPMC group. Ileal populations also were significantly lower
(0.5-1.0) than in the cecal samples.
[0288] Bacteroidetes
[0289] As demonstrated in FIG. 3B, Bacteroidetes populations were
not substantially different in mice on the 3 diets at baseline or
at 2 weeks, however, by 4 weeks, and in the ileal samples, levels
were lower in the 60% fat+HPMC group than in the other two groups.
After mice were switched from the 60% fat diet to the 10% fat diet,
Bacteroidetes levels rose significantly. Thus, changing from the
60% fat diet to a low fat (10%) diet or adding HPMC to the 60% fat
diet perturbed the Bacteroidetes numbers, but in apparently
opposite directions.
[0290] Fungal populations
[0291] Fungal concentrations were much lower than were measures for
Eubacteria, with a median of 4.0-5.0 log.sub.10 16S copies/g. There
were no significant differences between the groups fed different
diets or over time. Ileal concentrations were higher than cecal,
opposite to the Eubacterial concentrations, but the differences
were not significant.
Ratios Between Populations
[0292] Firmicutes/Eubacteria (F/E)
[0293] Firmicutes represented a median of 40% to 60% of the total
bacterial population in the fecal specimens. As shown in FIG. 3G,
no trends over time in the Firmicutes/Eubacteria (F/E) ratios were
present comparing the three groups of mice put on different diets.
However, the mice who switched to the low (10%) fat diet had ratios
that were significantly lower in the cecal samples than mice fed
the 60% fat+HPMC diet, and higher in the ileal samples than mice
fed the 60% fat diet. The intragroup comparisons did not show any
significant differences for the fecal specimens over time.
[0294] Bacteroidetes/Eubacteria (B/E)
[0295] Inversely from the Firmicutes/Eubacteria (F/E) ratio, the
Bacteroidetes/Eubacteria (B/E) ratios in the cecal specimens were
significantly higher for animals fed the 60% fat+HPMC diet than for
mice fed either the 10% fat or 60% fat diet (FIG. 3F). Changing the
diet from 60% fat at baseline to 10% fat also was accompanied by a
significant increase in the B/E ratio over 4 weeks, with the major
increase occurring by 2 weeks.
BCoAT Studies
[0296] Microbes can contribute to obesity through fermentation of
non-digestible carbohydrates in the colon to short chain fatty
acids, such as acetate, butyrate, and propionate. See Bergman,
Physiol Rev 1990; 70:567-90; Wong et al., J Clin Gastroenterol
2006; 40:235-43; Pryde et al., FEMS Microbiol Lett 2002; 217:133-9;
Wolfe, Microbiol Mol Biol Rev 2005; 69:12-50. This process
represents a 75% energy conversion to a product that is readily
absorbed in the intestine, contributing 10% of host caloric intake.
Butyrate is the preferred energy source for colonocytes. Butyryl
CoA transferase (BCoAT) is critical for butyrate synthesis. See,
e.g., Charrier et al., Microbiol., 2006,152:179-85; Duncan et al.,
Appl. Environ. Microbiol., 2002, 68:5186-90; Louis and Flint, Appl.
Environ. Microbiol., 2007, 73:2009-12. The BCoAT-encoding gene is
widely conserved in intestinal bacteria.
[0297] The present inventors have hypothesized that the change in
diet, and, specifically, the addition of HPMC, affects intestinal
energy metabolism. This hypothesis was tested by examining the
number of copies of BCoAT genes, as well as the ratio of BCoAT
genes, relative to major taxonomic groups (FIGS. 3D and 3H).
[0298] Inter-Group Comparisons of BCoAT Copy Number
[0299] As shown in FIGS. 3D and 3H, at baseline, most fecal samples
had between 7 and 8 log.sub.10 BCoAT copies detected, and, as
expected, there were no significant differences between the three
diet groups. After 2 weeks, the number of BCoAT copies in feces was
significantly lower in the 60% fat+HPMC group versus the 60% fat
group. After 4 weeks, the BCoAT numbers in feces were significantly
lower than in both of the other groups, which also was found in the
cecal samples at sacrifice. Thus, in 3 different groups of
specimens, BCoAT populations were significantly different after
HPMC was added to the diet. In the ileal samples, the highest BCoAT
levels were in the 60% fat group, with significantly lower levels
in the 10% fat and 60% fat+HPMC groups. Thus, these studies confirm
the hypothesis that addition of HPMC lowers BCoAT levels in
relation to the other groups, in a manner predicted to lower
butyrate availability and energy production.
[0300] Intra-Group Comparisons of BCoAT Copy Number
[0301] There were no significant differences from baseline over 4
weeks in the 10% fat and 60% fat groups. However, in the 60%
fat+HPMC group, there was a progressive and significant decline in
BCoAT levels of about 1 log.sub.10 (90% reduction). This is both
statistically and biologically significant.
[0302] Relationship of BCoAT Copy Number to Taxonomic
Findings-Inter-Group Analyses
[0303] There were no significant differences in the ratio of BCoAT
genes to numbers of total bacteria, Firmicutes, or Bacteroidetes,
with three exceptions. In the ileal samples, the BCoAT/total
bacteria ratios and the BCoAT/Firmicutes ratios were significantly
higher in the 60% fat+HPMC group compared with the 10% fat group.
In both cases, the 60% fat group was intermediate, but the
differences were not significant. The BCoAT/Bacteroidetes ratios
rose significantly from the 10% fat group to the 60% fat+HPMC
group. These results provide evidence that the energy metabolism in
proportion to the numbers of Firmicutes and Bacteroidetes changed
in the ileum depending on diet. No other changes were
significant.
[0304] Relationship of BCoAT Copy Number to Taxonomic
Findings-Intra-Group Analyses
[0305] There were no significant differences between the baseline,
2 week, or 4 week samples with only a single exception. Mice fed
the 10% fat diet had a progressive and significant decrease in the
BCoAT/Bacteroidetes ratios over four weeks.
Comparison of Sequence Data at Baseline, 2 Weeks, and 4 Weeks
[0306] Sequence data extracted from fecal pellets obtained from
study mice at baseline, 2 weeks, and 4 weeks was compared using
heat map (FIG. 18) and principal component analysis (PCA) plots
(FIG. 16). At baseline, the distribution of the mice based on the
genus level microbial composition of their fecal pellets was
random. This was corroborated by unsupervised hierarchical
clustering analysis at the same taxonomic level in an NMDS
analysis. This was an expected result because all mice to this
point had been raised and fed under identical conditions (60% fat
diet). After 2 weeks of intervention, mice in each of the 3 study
groups began to cluster, although the clustering was not
statistically significant at this intermediate time point. By 4
weeks, there was significant clustering of the mice into their
respective study groups. In a heat map analysis, there were three
distinct deep branch points (termed I, II, and III). In branch I, 7
of the 10 mice were from the 60% fat+HPMC group. In branch II, 9 of
the 10 mice were from the 60% fat group. In branch III, 7 of the 10
mice were from the 10% fat group. The clustering was corroborated
in NMDS plots, in which all the 60% fat+HPMC mice were adjacent,
and most of the 60% fat mice also were contiguous. These data
demonstrate that there are significant and consistent effects of
the diets on the intestinal microbiota of mice that are observable
within 4 weeks of initiation.
Comparison of Cecal and Ileal Samples
[0307] Comparison of the sequencing data obtained from ileal and
cecal samples also was accomplished by heat map analysis. Ileal
samples generated 2 deep branch points containing 18 and 10 mice.
In the larger branch of 18 mice, 14 were exposed to the 60% fat
diet (9 on 60% fat and 5 mice on 60% fat+HPMC) while the branch of
10 mice was primarily composed of mice exposed to the 10% fat diet.
The data obtained from the cecal samples generated three branch
points (termed I, II, and III). The most notable finding is in
branch III, in which 10 of 10 mice were exposed to the 60% fat+HPMC
diet. Branch I consisted primarily of the 10% fat group (6/10 mice)
and branch II consisted of the 60% fat group (5/9 mice). These
findings suggest that HPMC exposure has a more significant effect
on the microbiome found in the cecum than in the ileum. This is
consistent with the fact that the microbial numbers in the cecum
and thus their metabolic contributions are likely greater than
anywhere else in the gastrointestinal tract (see, e.g., Turnbaugh
et al., Nature 2006, 444(7122):1027; Qu et al., PLoS One 2008,
3(38):e2945).
Alterations of the Taxonomic Composition Caused by Exposure to
HPMC
[0308] Comparison of microbial abundance in the cecal samples
obtained from 10% fat, 60% fat, and 60% fat+HPMC groups is shown in
FIG. 4A and is summarized in FIG. 4B. In comparing the three study
groups, the differences between the 10% fat and 60% fat groups were
relatively modest, with the most notable changes at the genus
level, an increase in Dorea, and decrease in Coprobacillus and
Papillibacter was observed. No changes were found at higher
taxonomic levels. However, when comparing the 60% fat+HPMC group to
the other two diets, there were marked and significant differences
at multiple taxonomic levels. Notable changes in genera are seen in
the decrease of Oscillibacter (lino et al., Int. J. Syst. Evol.
Microbiol., 2007, 57:1840-1845; Walker at al., ISME J., 2010, 1-11)
and Johnsonella (Moore and Moore, Int. J. Syst. Bacteriol., 1994,
44(2):187-192) as well as in the marked increase in Coprobacillus
(Kageyama and Benno, Microbiol. Immunol., 2000, 44(1):23-28),
Sporacetigenium (Chen et al., Int. J. Systematic Evol. Microbiol.,
2006, 56:721-725), and Holdemania (Willems et al., Int. J.
Systematic Bacteriol., 1997, 47(4):1201-1204) (FIG. 5) and slight
increase in Dorea, Blautia, and Enterococcus.
[0309] By 454-pyrosequencing, at the family level, 60% fat+HPMC
groups showed a marked increase in Erysipelotrichaeceae and
Peptostreptococcaceae, a slight increase in Clostridiales Insertae
Sedis XIV, and Enterococcaceae, and a decrease in Lachnospiraceae
and Ruminococcaceae when compared to the 60% fat control mice. At
the order level, there was an increase in Erysipelotrichales and
Lactobacillales, and a decrease in Clostridiales. At the Class
level, there was an increase in Erysipelotriche and Bacilli, and a
decrease in Clostridia. There was a downward trend in Firmicutes
relative abundance (P=0.065 by Mann-Whitney U) and an upward trend
in Bacteroidetes relative abundance (P=0.076). There were no
statistically significant changes between 60% fat+HPMC and the 60%
treatment groups at the phylum level, although the decrease in
Firmicutes neared significance (p=0.065).
[0310] When cecal microbiota abundance in mice on 60% fat+HPMC diet
is compared to mice on the 10% fat diet, the changes in taxa are
similar to the comparison between 60% fat+HPMC and 60% fat with the
following exceptions. There is a significant increase in
Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order),
and Anaerotruncus (genus) in the 60%+HPMC group. There is no
significant difference in Clostridiales Insertae Sedis XIV,
Enterococcaceae (family), Blautia, or Enterococcus (genus).
Summary of 454 Sequencing Data
[0311] These data provide strong evidence that adding HPMC to a 60%
diet changes the population composition of the intestinal
microbiota. Clustering of the groups based on sequence data
demonstrates that the dietary interventions, both of 60% fat diet
and 60% fat+HPMC diet, caused consistent and significant changes at
the genus level in the composition of the gut microbiome. The data
also show that the primary effect of the HPMC may be in the cecum,
rather than in the ileum. Finally, the addition of HPMC to the diet
caused significant shifts in the composition of the gut microbiome,
more than simply diet alone.
Conclusions
[0312] The above data provide evidence that adding HPMC to a 60%
fat diet changes the population size and composition of the
intestinal microbiota. The data are internally consistent and show
reductions in total bacterial populations, primarily reflecting
reduction in Firmicutes. Diet change also affects Bacteroidetes
populations. The fact that the changes were in the same direction
and to a similar degree supports the hypothesis that adding HPMC
affects the microbiota in ways equivalent to lowering dietary fat
content. The 454 sequencing data confirmed the consistency of the
findings.
[0313] The data provided herein with respect to specific changes
also allows to develop various diagnostic methods for predicting
predisposition to weight gain on a high fat diet and effectiveness
of fiber (e.g., HPMC) treatment for weight loss or weight gain
prevention. As shown in FIGS. 6A-6B, a useful diagnostic ratio at
the genus level can be developed by dividing the sum of any
combination of Coprobacillus, Sporacetigenium, and/or Holdemania
(i.e., C+S+H or C+H or C+S or S+H or C or S or H) by any
combination of Johnsonella and/or Oscillibacter (i.e., J+O or J or
O), wherein a ratio below 1 indicates a state that is predisposed
to weight gain while a ratio above 3 indicates a state that has a
high propensity to prevent weight gain. Additional diagnostic
ratios can be developed by dividing the sum of any combination of
Coprobacillus, Sporacetigenium, and/or Holdemania (i.e., C+S+H or
C+H or C+S or S+H or C or S or H) by the phylum Firmicutes (F),
wherein a ratio below 0.1 indicates a state that is predisposed to
weight gain while a ratio above 0.1 indicates a state that has a
high propensity to prevent weight gain. As shown in FIG. 6C, useful
diagnostic ratios can be also developed by dividing the sum of any
combination of Erysipelotrichaceae and/or Peptostreptococcacea by
Lachnospiraceae and/or Ruminococcaceae, wherein a ratio below 0.1
indicates a state that is predisposed to weight gain while a ratio
above 0.1 indicates a state that has a high propensity to prevent
weight gain.
Example 2
A Cholesterol-Lowering Dietary Fiber Perturbs the Murine Intestinal
Microbiota
Materials and Methods
[0314] Animals and Diets were the same as in Example 1, supra.
[0315] Hepatic lipid analysis. Lyophilized liver samples were
extracted using an accelerated solvent extractor (Dionex ASE,
Sunnyvale, Calif.) at 100.degree. C., .about.13.8 MPa with 75/25
hexane/2-propanol, dried and weighed to determine the percentage of
total hepatic lipids, and hepatic total cholesterol, free
cholesterol, and triglyceride levels (Roche Diagnostic/Hitachi 914
clinical analyzer).
[0316] Fecal lipid analysis. Fecal lipids were extracted on a
Dionex ASE system using a mixture of hexane and 2-propanol (3:2,
v/v, 2% acetic acid) at 15 MPa and 60.degree. C. for 30 min, then
divided into two aliquots. The first sample was analyzed for
saturated and unsaturated fatty acid composition by GC separation.
Briefly, the fatty acids in the lipid extract were methylated using
boron trifluoride methanol127, and the derivatized samples analyzed
by gas chromatography Agilent 6890 series GC, with a flame
ionization detector and a DB-23 analytical column (Agilent, Santa
Clara, Calif.). The initial oven temperature was 200.degree. C. for
5 min, then increased to 250.degree. C. at 5.degree. C./min and
held for 5 min. A calibration solution was prepared to contain 2
mg/mL of methyl palmitate (C16:0), methyl stearate (C18:0),
methyloleate (C18:1), methyl linoleate (C18:2), and methyl
linolenate (C18:3), and 11 .mu.g/mL methyl erucate (Nu-chek,
Elysian, Minn.) in heptane. The second aliquot was analyzed for
total bile acids and sterols using a modified chromatographic
method28. Briefly, using a 1200RR HPLC system (Agilent) with an
Acquity BEH C18 column [1.7 .mu.m, 2.1.times.100 mm; (Waters)], a
reversed-phase separation was performed with a gradient of two
mobile solvent phases: (A) methanol/acetonitrile/water (53:23:24,
v/v/v) and (B) 2-propanol (100%). Crystalline ammonium acetate was
added to each phase to form a 30 mM solution. Solvent A was
acidified by adding 2.4% (v/v) glacial acetic acid. A linear
gradient at a flow rate of 0.25 mL/min was performed as follows:
0-6 min, 4-36% B; 6-8 min, 36-48% B; 8-17 min, 48-51% B; 17-18 min,
51-73% B; 18-31 min, 73-85% B; and 31-34 min, 85-96% B. In all
experiments, the columns were re-equilibrated between injections
with the initial mobile phase (10 mL). The LC effluent was
monitored using a Corona Plus charged aerosol detection apparatus
(CAD; ESA Biosciences, Chelmsford, Mass.) with a nebulizer
temperature at 30.degree. C.
[0317] Plasma biomarker analysis. Total cholesterol, free
cholesterol, and triglycerides in plasma were determined by
enzymatic colorimetric assays using a Roche Diagnostics/Hitachi 914
Clinical Analyzer with assay kits from Roche Diagnostics
(Indianapolis, Ind.) and Diagnostic Chemicals, Ltd. (Oxford,
Conn.). The concentrations of plasma, LDL-cholesterol and
HDL-cholesterol were determined using L-type LDL-cholesterol (Roche
Diagnostics) and L-type HDL-cholesterol [Wako Chemicals (Richmond,
Va.)] assay kits. The VLDL-cholesterol levels were calculated by
subtracting HDL-cholesterol and LDL-cholesterol from total
cholesterol levels. Plasma concentrations of adiponectin, leptin,
and insulin of 12 h-fasted mice were determined using mouse
adiponectin (B-Bridge International, Sunnyvale, Calif.), leptin
(Assay Designs, Ann Arbor, Mich.), and insulin (Mercodia Inc.,
Winston Salem, N.C.) immunoassay kits, as described15. Fasting
glucose levels were measured by collecting blood from each mouse by
the tail-prick approach. A drop of blood collected by a sterile
needle was analyzed using a OneTouch.RTM.Ultra.RTM. meter with
FastDraw.TM. test strips (Johnson & Johnson, Milpitas,
Calif.).
[0318] PCR Amplifcation. After genomic DNA extraction and
quantification, samples were prepared for amplification and
sequencing at the JCVI Joint Technology Center (JTC). Genomic DNA
sample concentrations were normalized to .about.2-6 ng/.mu.l. The
V3-V5 region of the 16S rRNA gene was amplified using forward
primer 341F (5'-CCTACGGGAGGCAGCAG-3' (SEQ ID NO: 12)) and reverse
primer 926R (5'-CCGTCAATTCMTTTRAGT-3' (SEQ ID NO: 13)). A barcoded
primer design was completed using a set of algorithms developed at
the JCVI. The `A` and `B` adapters for 454 library construction
were included as a part of the PCR primers. To the 926R primer, 10
nt barcodes were included as part of the primer design
(5'-A-adapter-N(10)+165 primer-3'). This design allowed for the
inclusion of a unique barcode to each sample at the time of PCR so
that the tagged samples could be multiplexed for sequencing. Every
effort was made to prevent contamination of PCR reactions with
exogenous DNA including a set of reactions in a laminar flow hood.
PCR reactions were completed as follows (per reaction): 2 .mu.L of
gDNA, 1.times. final concentration of Accuprime PCR Buffer II
(Invitrogen, Carlsbad, Calif., USA), 200 nM forward and reverse
primers, 0.75 units of Accuprime Taq DNA Polymerase High Fidelity
(Invitrogen, Carlsbad, Calif., USA), and nuclease-free water to
bring the final volume to 20 .mu.L. PCR cycling conditions were:
initial denaturation of 2 minutes at 95.degree. C. followed by 30
cycles of 20 seconds at 95.degree. C., 30 seconds at 50.degree. C.,
and 5 minutes at 72.degree. C. A negative control (water blank)
reaction also was included and examined after 35 cycles. PCR
reactions were visualized on 1% agarose gels and quantified using a
Tecan SpectraFluor Plus (Tecan Group Ltd., Mannedorf, Switzerland).
Each reaction was cleaned individually using the Agencourt AMPure
system (Beckman Coulter Genomics, Danvers Mass., USA) prior to
normalization and pooling of samples for sequencing.
[0319] Sequencing. The pooled samples were further cleaned using
the Agencourt AMPure system (Beckman Coulter Genomics, Danvers
Mass., USA) prior to emulsification (em)PCR. Steps for emPCR,
enrichment and 454 sequencing were performed by following the
vendor's standard operating procedures with some modifications.
Specifically, qPCR was used to accurately estimate the number of
molecules needed for emPCR. We also utilized automation (BioMek FX)
to "break" the emulsions after emPCR, and we used butanol to enable
easier sample handling during the breaking process.
[0320] Bioinformatic pipeline 2. The Qiime pipeline (Caporaso et
al., Nat Methods 7, 335-336, 2010) was used to further process the
sequences. The sequences were first grouped into operational
taxonomic units (OTUs) with a sequence similarity threshold of 97%,
then taxonomic assignment was generated using the RDP database and
Qiime algorithm. This data was used to produce the operational
taxonomic unit (OTU) absolute abundance table and weighted UniFrac
beta-diversity matrix (Lozupone et al., UniFrac: an effective
distance metric for microbial community comparison. ISME J 2010;
Lozupone et al., Appl Environ Microbiol 71, 8228-8235, 2005).
Principle component analysis (PCA) plots were produced based on
unweighted UniFrac distances. The rarefactions for richness and
Shannon diversity indices were calculated in R statistical
programming environment (R: A Language and Environment for
Statistical Computing, in R Foundation for Statistical Computing,
Vol. 1, 2009; Gentleman et al., Genome Biol 5, R80, 2004) using
Community Ecology Package vegan. Comparison of unweighted and
weighted UniFrac distances was performed using two-sided t-test.
The OTU absolute abundances were converted to relative abundances
by normalizing to total sequence count per sample analyzed. The
resulting relative abundance matrix was used to produce heatmaps
for major (relative abundance>1%) taxa.
[0321] Comparison of bioinformatic pipeline 2 to bioinformatic
pipeline 1 used in Example 1. The taxonomic data in Example 1 was
derived from a bioinformatic pipeline that matched the 16S sequence
reads to sequences in the Ribosomal Database Project (RDP) (Wang et
al., Applied and Environmental Microbiology 73, 5261-5267, 2007)
using the RDP classifier with an 80% confidence. In bioinformatics
pipeline 2 analysis (FIGS. 7-12), the same sequence data was first
grouped into OTU's at a 97% sequence similarity, then assigned a
taxonomic name using the Qiime pipeline and RDP database. The
taxonomic data varies between the two data sets generated by the
automated RDP classifier (Example 1, FIGS. 4-6) and generated by
the automated Qiime taxonomic assignment tool, because they
represent two independent algorithms that aim to assign microbial
taxonomic names to sequence data. Diet-mediated alterations varied
by pipeline at the genus level, but were conserved at the family
and higher levels of classification. Significance testing was done
by the Mann Whitney U test, however the second data set was
corrected for false discovery rate. This more stringent analysis
explains why fewer taxa are revealed as significant changes.
[0322] Statistical analysis. All biochemical data are expressed as
Scatter plots with means. Differences between dietary groups were
evaluated using Mann-Whitney U test. Significance was corrected for
false discovery (Benjamini et al., Journal of the Royal Statistical
Society. Series B (Methodological), 289-300, 1995).
[0323] Bioinformatic analysis. The Qiime pipeline29 was used to
further process the sequences and produce the operational taxonomic
unit (OTU) absolute abundance table and weighted UniFrac
beta-diversity matrix 30,31. Principle component analysis (PCA)
plots were produced based on weighted UniFrac distances. The
rarefactions for richness and Shannon diversity indices were
calculated in R statistical programming environment 32,33 using
Community Ecology Package vegan. Comparison of weighted UniFrac
distances was performed using two-sided t-test. The OTU absolute
abundances were converted to relative abundances by normalizing to
total sequence count per sample analyzed. The resulting relative
abundance matrix was used to produce heatmaps for major (relative
abundance >1%) taxa.
Results
[0324] All C57BL/6J mice were fed a high fat diet (HFD, 60% kcal
from fat) for 2 weeks prior to the study and then randomized to
continue the HFD, to receive the HFD supplemented with HPMC (HPMC
diet), or to receive a low fat diet (LFD, 10% kcal from fat). Over
the course of the 4-week study, the mice on the HFD continued to
gain weight, while HPMC supplementation reduced weight gain (FIG.
1A), despite isocaloric food intake (FIG. 1B). Mice receiving the
LFD had significantly lower energy intake and lost weight (FIGS.
1A-1B). Weight change and energy intake were correlated in both the
individual HFD and LFD mice (R=0.84 and 0.65, p=0.003 and 0.042,
respectively); when combined, the correlation strengthened (R=0.98)
(FIG. 1C). Based on this best-fit line, the actual weight of the
HPMC mice was 4.7 g .+-.2.2 lower than predicted (FIG. 1D), with no
significant correlation with energy intake. Thus, the HPMC diet
disrupted the energy intake/weight gain relationships observed for
the two other dietary groups.
[0325] After 4 weeks, mice on the LFD and HPMC diets had
significantly reduced total cholesterol, HDL, LDL, and VLDL,
compared to the HFD mice (FIGS. 2A-2D). The LFD mice also had
decreased fasting blood glucose, and free-fatty acids (FIGS. 2E,
2G). Leptin levels were decreased in both the LFD and HPMC mice,
consistent with the lowered weights (FIG. 21), as were liver
triglycerides (FIG. 2L). Insulin was decreased in HPMC mice (FIG.
2H). Compared to both the LFD and HFD mice, HPMC supplementation
increased fecal fat excretion, including saturated, unsaturated,
and trans-unsaturated fats, and bile acids, but decreased sterol
excretion (FIGS. 2M-2Q). HPMC increased fecal excretion of mono-
but not di- or tri-acylglycerides (FIGS. 2R-2T). Thus, in the
setting of a high-fat, high-calorie diet, HPMC improved metabolic
biomarkers to an extent similar to that of the LFD, but with
increased fecal loss of specific metabolites.
[0326] Microbial population sizes. To assess quantitative changes
in microbial populations, total bacteria were enumerated and the
two predominant phyla: Bacteroidetes and Firmicutes, using
quantitative PCR of 16S rRNA genes. At baseline, as expected, there
were no significant differences in total fecal bacteria or B/F
ratio between the mice about to be randomized into the three
treatment groups (FIG. 7). Total bacteria levels were significantly
decreased in fecal, cecal, and ileal samples from HPMC mice
compared to the two other groups. The Bacteroidetes/Firmicutes
(B/F) ratio in fecal samples was unchanged as expected in the HFD
mice, but increased in LFD and HPMC mice and in cecal samples from
HPMC mice. In contrast, in ileal samples, the B/F ratios were
decreased in LFD and HPMC mice (FIG. 7C). These data indicate that
the dietary changes, especially adding HPMC, affected the size and
composition of the intestinal microbiota.
[0327] Microbial community composition. To assess changes in murine
intestinal microbial community structure induced by the dietary
changes, measures of richness and diversity were calculated for
microbial 16S rDNA V3-V5 sequences in fecal and cecal samples. At
the class level, richness in the fecal and cecal communities from
the three groups were similar (FIG. 14A), but at the OTU level, the
LFD and HPMC mice had decreased fecal community richness compared
to HFD, and the HPMC mice had reduced richness in cecal samples as
well (FIG. 15A). Thus, although population structure was conserved
at the higher (class) taxonomic level, the dietary changes
diminished diversity at the more specific (OTU) taxonomic level.
Evenness at the class level as measured by Shannon score, increased
in the LFD and HPMC mouse fecal samples, and in cecal samples for
HPMC mice (FIG. 14B), but decreased in the same groups at the OTU
level (FIG. 15B). These findings suggest that the dietary
interventions favored a relatively small number of specialist
organisms within larger taxonomic units that became more
balanced.
[0328] The phylogenetic differences between the three treatment
groups also were assessed by principal component analysis (PCA)
based on unweighted Unifrac values (FIG. 16). At week 0 (baseline),
all samples cluster together (FIG. 16A), while at week 2 (FIG. 16B)
and week 4 (FIG. 16C), the HFD samples were little different from
baseline, whereas the LFD and HPMC communities had shifted in
separate directions forming distinct clusters (FIGS. 16D, 16E).
There was no significant change in pairwise UniFrac distances over
time for any of the HFD fecal samples. For the LFD samples,
pairwise distances increased from weeks 0 to 2, but not from 2 to
4. For the HPMC fecal samples, pairwise distances increased from
weeks 0 to 2, and from 2 to 4. Analysis of the weighted UniFrac
distances showed similar trends (FIG. 17). The PCA of the cecal
specimens obtained at sacrifice is similar to the week 4 fecal
specimens, but the LFD and HFD samples are well-mixed, with HPMC
forming a distinct cluster (FIG. 16F). The ileal specimens at
sacrifice show no distinctive clustering by treatment group (FIG.
16G). In total, the data indicate stability of the microbial
community structure in the mice continued on HFD, as expected,
whereas there was an early and persisting shift for the mice given
the LFD, and progressive changes for the microbiota of the mice
given HPMC.
[0329] Population changes induced by dietary intervention.
Hierarchical clustering by heat map analysis (FIG. 18) for the most
abundant taxons (>1%) at the family level in cecal specimens
showed clear separation (p<0.001) of the HPMC samples from the
HFD and LFD, which were not distinguishable (FIG. 18A). The fecal
samples at week 0 (baseline) were well-mixed between the three
groups, as expected, (FIG. 18B), but composition gradually (FIGS.
18C, 18D) moved toward three clusters, with each treatment group
individuating.
[0330] To assess specific changes in intestinal microbiota, the raw
pyrosequencing counts were converted to relative abundance, and
false discovery rate (FDR)-corrected pair-wise comparisons for each
predominant (>1%) taxon from the phylum through genus level were
made for HFD vs LFD (to assess for the effects of fat% dietary
change) and HFD vs. HPMC (to assess for the effects of fiber
addition) (Table 2). No significant differences were seen in week 0
fecal samples, as expected, since at baseline all mice were
receiving the same (HFD) diet. Over the course of the experiment,
there were no significant differences at the phylum level. All
differences at lower taxonomic levels were within the phylum
Firmicutes, involving members of class Bacilli, Clostridia, and
Erysipelotrichi (and order Lactobacillates, Clostridiates, and
Erysipelotrichales).
[0331] At the family level, there were seven predominant taxa (FIG.
10. Lachnospiraceae populations in LFD and HPMC mice became
decreased (FIG. 10A) and Ruminococcaceae levels became reduced in
HPMC mice (FIG. 10B), while Erysipelotrichaceae levels increased in
both LFD and HPMC treated mice (FIG. 10C), and
Peptostreptococcaceae levels were consistently increased in HPMC
mice (FIG. 10D). Differences in cecal and ileal populations were
limited to HPMC and LFD mice, respectively. At the genus level,
there also were significant changes in community structure (FIG.
11). The HPMC treatment led to significant decreases in Johnsonella
and Lactobacillus, and significant increases in Erysipelotrichaceae
Insertae Sedis and Peptostreptococcus Insertae Sedis. Thus, the
dietary changes selected for different compositions within
Firmicutes, with reproducible compositional effects at the family
level.
TABLE-US-00005 TABLE 2 Composition of the three experimental diets
Low-Fat High-Fat High-Fat/10% HPMC Component gm % kcal % gm % kcal
% gm % kcal % Protein 19.2 20 26.2 20 23.6 20 Carbohydrate 67.3 70
26.3 20 23.6 20 Fat 4.3 10 34.9 60 31.3 60 Total 100 100 100
kcal/gm 3.85 5.24 4.71 Ingredient gm kcal gm kcal gm kcal Casein,
200 800 200 800 200 800 80 Mesh L-Cystine 3 12 3 12 3 12 Corn
starch 315 1260 0 0 0 0 Maltodextrin 35 140 125 500 125 500 10
Sucrose 350 1400 68.8 275.2 68.8 275.2 Cellulose, 50 0 50 0 50 0
BW200 Soybean Oil 25 225 25 225 25 225 Lard 20 180 245 2205 245
2205 Mineral Mix 10 0 10 0 10 0 S10026 Dicalcium 13 0 13 0 13 0
Phosphate Calcium 5.5 0 5.5 0 5.5 0 Carbonate Potassium 16.5 0 16.5
0 16.5 0 Citrate, 1 H2O Vitamin 10 40 10 40 10 40 Mix V10001
Choline 2 0 2 0 2 0 Bitartrate HPMC.sup.a 0 0 0 0 88 0 FD&C
0.05 0 0 0 0.025 0 Yellow Dye #5 FD&C 0 0 0.05 0 0.025 0 Blue
Dye #1 .sup.aHPMC, hydroxyl-methyl cellulose (K250 M)
[0332] Correlation Network Analysis. To understand the dynamic
relationships between intestinal microbiota under differing dietary
conditions, a correlation network was constructed at the order
level (FIG. 20). At baseline (week 0), Clostridiales significantly
negatively correlated with Lactobacillales in all three groups of
mice and with Bacteroidales in two of the three groups. The
negative correlations between these three taxa persisted in the HFD
mice over the 4-week study. However, both dietary interventions
(LFD and HPMC) eliminated the stable link between
Bacteroidales-Clostridiales-Lactobacillales. LFD mice showed a
stable negative correlation between Clostridiales and
Erysipelotrichales, whereas no significant correlations emerged in
the HPMC mice. Thus, the major bacterial networks established under
HFD conditions were not conserved after dietary change.
[0333] Conditional Correlation Analysis. The results show that HPMC
or LFD treatments altered both host metabolism and intestinal
microbiota. To detect individual taxa that may be responsible for
metabolic effects, conditional correlation analysis was performed,
to remove the effect of the treatment condition (either presence of
fiber or fat percent) from the two linked variables: host phenotype
and taxa. Significant FDR-corrected p-values for each pairwise
comparison of taxa in particular samples with each metabolic
variable are indicated (Tables 3-4) and regression analysis was
used to avoid reporting correlations influenced by outlier samples
(FIG. 21. Weight change in HFD mice was positively correlated with
cecal Firmicutes and cecal Erysipelotrichaceae Insertae Sedis (I.
S.) and negatively associated with cecal Bacteroidetes (FIGS.
21A-21C). For the HPMC mice, weight changes and fecal saturated fat
were positively associated with cecal Erysipelotrichaceae I.S. and
cecal Erisipelotrichales, respectively (FIGS. 21C, 21F). Four-week
fecal abundances of Lachnospiraceae negatively correlated with
energy intake in the HFD and HPMC mice (FIG. 21D and FIG. 23) and
cecal Porphyromonadaceae correlated positively with liver free
cholesterol in HFD and HPMC mice (FIG. 21E). In total, there were
no significant correlations between taxa and host metabolic
phenotype for the LFD group, however, the HPMC treatment-induced
host phenotypes correlated with altered representation of
particular taxa.
[0334] Table 3 shows p-values calculated for false-discovery
corrections from Mann Whitney U-pair comparisons of relative
abundance. Sample types included week 0 fecal (baseline, 0F), week
2 fecal (2F), week 4 fecal (4F), wee 4 cecal (4C), week 4 ileal
(41I).
[0335] p-values <0.05 are shaded in grey.
Analysis of Erysipelotrichaceae Incertae Sedis
[0336] Sequences that are categorized at the genus level within
Erysipelotrichaceae incertae sedis (EIS) account for 4.5% of all
microbial 16S rRNA sequences in the present study. Incertae sedis
in Latin means uncertain seat and notates a placeholder for a
potential new genus in which defined species and sequences of
uncultivated organisms are placed. A list of cultured and named
species in unclassified Erysipelotrichaceae, according to NCBI
taxonomy is shown below:.
TABLE-US-00006 Clostridium cocleatum Clostridium innocuum
Clostridium ramosum Clostridium spiraforme Eubacterium bioforme
Eubacterium cylindroides Eubacterium dolichum Eubacterium tortuosum
Lactobacillus catenaformis Lactobacillus vitulinus Streptococcus
pleomorphus
[0337] Erysipelotricaceae Incertae Sedis (EIS) is a taxon of
interest because it is increased significantly in the HPMC-treated
mice (FIG. 12). To investigate the potential identity of members of
EIS, the 109 unique sequences assigned to EIS at the OTU level were
aligned using Greengenes to find the nearest neighbor. Minimum
length was set at 100 nucleotides and minimum percent identity was
set at 75%. There were 24 unique organisms identified as closest
matches.
[0338] 59 of the 109 EIS OTUs had a closest match to Clostridium
cocleatum str. DSM 1551, accounting for 62.1% of the EIS sequences
and 2.8% of the total taxonomic sequences from the
454-pyrosequencing run. C. cocleatum had an average sequence
identity of 95.5% (.+-.0.28% S.E., min 89.4%, max 98.6%) over an
average sequence length of 367 nucleotides (.+-.11.3 S.E, min 108,
max 489). One OTU comprised 20.0% of the EIS sequences, and 0.9% of
the total taxonomic sequences, and had a closest match to
uranium-contaminated aquifer clone 1013-28-CG45 (UCAC), classified
in RDP as Firmicutes; Clostridia; Clostridiales; Peptococcaceae;
Peptococcaceae 1; Desulfosporosinus, with a sequence identity of
93.7% over a length of 270 nucleotides. All other closest matches
accounted for less than 0.2% of the total taxonomic sequences, and
less than 4% of the EIS sequences (Table 5). Due to the high
relative abundance (FIG. 24) and high percent identity of OTUs to
Clostridium cocleatum, this organism is a promising candidate.
TABLE-US-00007 TABLE 6 Identity of closest matches of OTUs
classified as Erysipelotrichaceae incertae sedis (EIS) determined
by sequence BLAST using GreenGenes. The first column indicates
relative abundance in all 147 samples from the Diet/Fiber study and
the second column is % of sequences classified as EIS. % of Average
BLAST % of total Erysipelotrichaceae percent identity to sequences
incertae sedis template #OTUs Closest Match 2.77 62.14 95.5 59
Clostridium cocleatum str. DSM 1551 0.89 20.00 93.7 1
uranium-contaminated aquifer clone 1013-28- CG45 0.14 3.16 81.6 1
marine bone clone boneC3C9 0.12 2.68 86.3 5 human fecal clone
RL305aal86g01 0.12 2.65 88.9 7 culturable kangaroo Macropus
giganteus Eastern grey Kangaroo forestomach contents 0.10 2.35 84.6
2 Caminicella sporogenes str. AM1114 0.10 2.16 90.5 5 Coprobacillus
cateniformis str. JCM 10605 0.08 1.73 83.8 1 mouse cecum clone
SWPT19_aaa04g11 0.03 0.74 82.7 2 human fecal clone RL197_aah85c11
0.02 0.50 88.3 2 swine intestine clone p-2772-24E5 0.02 0.38 80.4 1
Eubacterium cylindroides str. JCM 7786 0.01 0.26 87.1 2 Spiroplasma
insolitum str. ATCC 33502; M55 0.01 0.25 89.2 3 human fecal clone
RL117_aae92d08 0.01 0.21 94.0 7 Eubacterium sp. Pei061 0.01 0.18
82.0 1 sediment-free PCB-dechlorinating enrichment culture clone
JN18_A14_H 0.01 0.16 88.2 2 mesophilic anaerobic digester clone
G35_D8_H_B_E07 0.004 0.10 84.7 1 human fecal clone RL179aan75d04
0.004 0.09 86.0 1 Catenibacterium mitsuokai str. JCM 10609 0.003
0.08 87.0 1 human fecal clone RL200_aai60f02 0.003 0.07 84.2 1
Electricigen Enrichment MFC full-scale anaerobic bioreactor sludge
treating brewery waste clone 31a07 0.003 0.06 86.2 1 Inhibition
nitrate reduction chromium (VI) microcosms heavy metal contaminated
anaerobic soil microcosm isolate GNCr-2GNCr-2 str. GNCr-2 0.002
0.04 87.2 1 Enterococcus mundtii str. NFRI 7393 0.001 0.02 91.7 1
human fecal clone RL176_aan58b04 0.004 0.02 88.8 1 mouse cecum
clone M2_d06 4.46 100.00 109 .rarw. Total
Conclusions
[0339] The present study demonstrates that reducing dietary fat, or
adding dietary fiber improves markers of metabolic health in mice
receiving a high-calorie/high-fat diet. In particular, this study
shows that adding HPMC disrupts a strong relationship between
energy intake and weight change. One explanation for the observed
deviation may be the increased excretion of bile salts and fats in
the feces induced by HPMC, representing a form of energy wasting.
As shown herein, changing from HFD to a LFD, or adding 10% HPMC to
HFD resulted in marked shifts in the intestinal microbiota over a
4-week period, providing evidence that HPMC alters the intestinal
microbiota. The results are highly consistent within the
experimental groups and show progressive changes in the microbiota
sampled in the feces, with consistent shifts in the cecal and ileal
samples collected at sacrifice.
[0340] HPMC and LFD both improve metabolic health and shape the
microbiome, however linkages between specific microbial taxa and
host metabolic phenotypes could only be identified in HPMC treated
mice and not in LFD mice. This dichotomy suggests that the
improvement in health by HPMC is mediated by the intestinal
microbiota, while health benefits achieved by LFD are independent
of gut microbes. Since HPMC is assumed to be inert, and not acted
on by intestinal microbiota, how could this be achieved?
[0341] One possible mechanism of action related to HPMC effects on
the gut microbiome could be the reduction in microbial load.
Although changing from HFD to LFD did not affect total bacterial
densities, adding HPMC clearly had a lowering effect and modulated
overall composition as observed in essentially every analysis.
Although ecological richness showed no changes at higher taxonomic
orders, richness of the HPMC-impacted intestinal community and
evenness at the OTU (species) level declined. Such dynamics are
consistent with HPMC selection for a small group of specialist
organisms that are over-represented at the expense of the usual
community members.
[0342] The extent of the dynamics swings, as detailed in studies of
community structure that are based on phylogenetic relationships
among the taxa and shown by PCA presentations of the Unifrac
analyses (FIGS. 16-17) and by heat map (FIG. 18) indicate
HPMC-induced progressive shifts in community structure in fecal, as
well as in cecal, populations. Changing from HFD to LFD also
produces shifts in community composition, but in ways that differ
from the HPMC-induced shifts.
[0343] All of the major HPMC-induced shifts involve families within
Firmicutes, with major decreases in Lachnospiraceae and
Ruminococcaceae, and increases in Erysipelotrichaceae and
Peptostreptococcaceae (FIG. 10). That Firmicutes but not
Bacteroidetes are affected indicates differences in their
functional or anatomic niches worth future exploration; in this
sense, HPMC treatment is a probe of intestinal microbiome
population structure. Based on the multiple specimens obtained from
mice receiving the HFD, there are conserved numerical relationships
among order-level taxa (FIG. 20) that are disrupted after HPMC is
added. Informatic analysis indicated conserved patterns of
co-variance among major taxa in the baseline and continuing HFD
mice. Importantly, change to HMPC and LFD diet ablated the prior
co-variances, and a new relationship was established in LFD
mice.
[0344] Importantly from this work, we now identify specific taxa
that are associated with weight gain (e.g. Erysipelotrichaceae
Incertae Sedis (EIS)), and with other metabolic phenotypes (FIG. 21
and Tables 4-5).
[0345] Without wishing to be bound by a specific theory, HPMC could
alter the microbial ecosystem in several ways: (i) HPMC may bind to
mucus glycoproteins 22-24 and swell in the large intestine,
potentially displacing mucosa-associated microbiota. (ii) HPMC may
sequester glycoside hydrolases or other catalytic enzymes, by
mimicking the carbohydrate structures to which they bind,
decreasing microbiome capacity for energy harvest from complex
carbohydrates (for example, HPMC, resembling the structural subunit
of amylase, has been used to trap and recover (.beta.-amylase from
Clostridium thermosulfurongenes 25), and (iii) such sequestration
may affect the balance between bile salt excretion and reabsorption
affecting cholesterol and fat excretion. (iv) HPMC may be partially
fermented in the intestine, stimulating the growth of organisms
that digest cellulose or secondary metabolites from HPMC.
Example 3
Testing of Probiotic Compositions of the Invention in Mouse
Models
Methods and Abbreviations
[0346] Abbreviations. All abbreviations also apply to FIGS. 22-24.
CSHEPCc=at least one of Coprobacillus, Sporacetigenium, Holdemania.
JO=at least one of Johnsonella, Oscillibacter. Prebiotic=at least
one of trehalose, cellobose, maltose, mannose, sucrose, lactose,
salicin, mellibiose, raffinose, galactose, and fructose. C57B6
mice=C57/B16J mice (approximately 2 months old in the beginning of
the experiment).
3.1 Determination of the Effect of CSHEPCc and/or Prebiotic
Treatment on Weight Gain in a DIO Mouse Model
[0347] As schematically shown in FIG. 22, four groups of C57/B16J
mice (at least 10 mice in each group) are fed 60% fat (DIO) diet
from two months to four months of age. At four months of age, group
1 is kept untreated (control), group 2 gets CSHEPCc delivered by
gastric gavage (Coprobacillus, Holdemania, and Peptostreptococcacea
Incertae Sedis are delivered as live cells, Sporacetigenium,
Erysipelotrichaceae Incertae Sedis, and Clostridium cocleatum are
delivered as spores; at a dose of 10.sup.9 cells each week), group
3 gets prebiotic only (at a dose of 10 g/L in the drinking water
every day), group 4 gets CSHEPCc+Prebiotic.
[0348] Measurements: [0349] Genotype: qPCRs for Eubacteria,
Firmicutes, Bacteroidetes, BCoAT, and individual qPCRs for C, S, H,
E, P, Cc, J, O [0350] Phenotype: weight every week, fasting
glucose, oral glucose tolerance test, insulin tolerance test, total
cholesterol, LDL, HDL, triglycerides (TGA), dual emission X-ray
absorptiometry (DEXA) every 2 weeks to examine total weight, lean
composition, bone mineral density, and fat composition (and percent
fat); fecal short-chain fatty acids (SCFA), including butyrate and
acetate); fasting serum leptin, ghrelin, insulin, GIP
(glucose-dependent insulinotropic peptide), CRP (C-Reactive
Protein).
3.2 Determination of the Effect of CSHEPCc and/or Prebiotic
Treatment on Preventing Obesity in a DIO Mouse Model
[0351] As shown in FIG. 23, in this experiment, eighty (80)
C57/B16J mice are fed 10% fat diet until they are two months old.
At four weeks of age (while on 10% diet), mice are divided into
four treatment groups: group 1 is kept untreated (control), group 2
gets CSH delivered by gastric gavage (Coprobacillus, Holdemania,
and Peptostreptococcaceae are delivered as live cells,
Sporacetigenium, Erysipelotrichaceae Incertae Sedis, and
Clostridium cocleatum are delivered as spores; at a dose of
10.sup.9 cells of each every week), group 3 gets prebiotic only (at
a dose of 10 g/L in the drinking water every day), group 4 gets
CSHEPCc+Prebiotic. At two months of life, half of the mice (10
mice) in each treatment group are switched to 60% DIO diet, and
half of the mice (10 mice) are kept on 10% diet. The same genotypes
and phenotype parameters are measured as specified in section 3.1,
above.
3.3 Determination of the Effect of JO and/or CSHEPCc Treatment on
Weight Gain and Prevention of Obesity in a Germ-Free Mouse
Model
[0352] As shown in FIG. 24, in this experiment, germ-free (GF)
C57/Black 6 mice are generated and treated with: (1) CSHEPCc as
monocultures (one, two, three, or four organisms at a time) or with
JO as monocultures (J or O); (2) altered Schaedler flora (ASF)
which consists of eight common mouse intestinal commensal bacteria
which were developed to colonize germ-free mice and to establish a
uniform baseline of conventionally colonized mice (Dewhirst et at.,
Appl. Environ. Microbiol., 1999, 65:3287-3292) followed by
introduction of CSHEPCc as monocultures or JO (J or O), or nothing
(control); (3) feces from normal mice treated with CSHEPCc or
control untreated normal mice. From two months of age to four
months of age, mice are fed a 10% fat diet. At four months of age,
half of the mice (10 mice) in each treatment group are switched to
the 60% DIO diet, and half of the mice (10 mice) are kept on the
10% diet. The same genotypes and phenotype parameters are measured
as specified in section 3.2, above.
REFERENCES
[0353] 1. Reppas, C., Swidan, S. Z., Tobey, S. W., Turowski, M.
& Dressman, J. B.
[0354] Hydroxypropylmethylcellulose significantly lowers blood
cholesterol in mildly hypercholesterolemic human subjects. Europ J
Clin Nutri 63, 71-77 (2009). (Need better ref) [0355] 2. Carr, T.,
Gallaher, D., Yang, C. & Hassel, C. Increased intestinal
contents viscosity reduces cholesterol absorption efficiency in
hamsters fed hydroxypropyl methylcellulose. J of Nutri 126, 1463
(1996). (Need better ref) [0356] 3. Gallaher, D., Hassel, C. &
Lee, K. Relationships between viscosity of hydroxypropyl
methylcellulose and plasma cholesterol in hamsters. J of Nutri 123,
1732 (1993). [0357] 4. Maki, K. C., et al. High-viscosity
hydroxypropylmethylcellulose blunts postprandial glucose and
insulin responses. Diabetes Care 30, 1039-1043 (2007). [0358] 5.
Maki, K. C., et al. Dose-response characteristics of high-viscosity
hydroxypropylmethylcellulose in subjects at risk for the
development of type 2 diabetes mellitus. Diabetes Technol Ther 11,
119-125 (2009). [0359] 6. Maki, K. C., et al.
Hydroxypropylmethylcellulose and methylcellulose consumption reduce
postprandial insulinemia in overweight and obese men and women. J
Nutr 138, 292-296 (2008). [0360] 7. Qin, J., et al. A human gut
microbial gene catalogue established by metagenomic sequencing.
Nature 464, 59-65 (2010). [0361] 8. Wong, J. M. W., de Souza, R.,
Kendall, C. W. C., Emam, A. & Jenkins, D. J. A. Colonic health:
fermentation and short chain fatty acids. Journal of Clinical
Gastroenterology 40, 235-243 (2006). [0362] 9. Louis, P., et al.
Restricted distribution of the butyrate kinase pathway among
butyrate-producing bacteria from the human colon. Journal of
Bacteriology 186, 2099-2106 (2004). [0363] 10. Savage, D. C.
Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol
31, 107-133 (1977). [0364] 11. Turnbaugh, P. J., et al. An
obesity-associated gut microbiome with increased capacity for
energy harvest. Nature 444, 1027-1131 (2006). [0365] 12. Savage, D.
C. Gastrointestinal microflora in mammalian nutrition. Annu Rev
Nutr 6, 155-178 (1986). [0366] 13. Siepmann, J. & Peppas, N. A.
Modeling of drug release from delivery systems based on
hydroxypropyl methylcellulose (HPMC). Adv Drug Deliv Rev 48,
139-157 (2001). [0367] 14. Gallaher, D. D., Hassel, C. A., Lee, K.
J. & Gallaher, C. M. Viscosity and fermentability as attributes
of dietary fiber responsible for the hypocholesterolemic effect in
hamsters. J Nutr 123, 244-252 (1993). [0368] 15. Hung, S.-C., et
al. Dietary fiber improves lipid homeostasis and modulates
adipocytokines in hamsters. J Diabetes 1, 194-206 (2009). [0369]
16. Topping, D. Hydroxypropylmethylcellulose, viscosity, and plasma
cholesterol control. Nutr Rev 52, 176-178 (1994). [0370] 17. Cook,
S. I. & Sellin, J. H. Review article: short chain fatty acids
in health and disease. Aliment Pharmacol Ther 12, 499-507 (1998).
[0371] 18. Braun, W. H., Ramsey, J. C. & Gehring, P. J. The
lack of significant absorption of methylcellulose, viscosity 3300
CP, from the gastrointestinal tract following single and multiple
oral doses to the rat. Food Cosmet Toxicol 12, 373-376 (1974).
[0372] 19. Machle, W., Heyroth, F. & Witherup, S. The Fate of
Methylcellulose in the Human Digestive Tract. 1-9 (1944). [0373]
20. Yokoyama, W., Knuckles, B., Davis, P. & Daggy, B. Stability
of ingested methylcellulose in the rat determined by polymer molar
mass measurements by light scattering. Journal of agricultural and
food chemistry 50, 7726-7730 (2002). [0374] 21. Ferguson, M. &
Jones, G. Production of short chain fatty acids following in vitro
fermentation of saccharides, saccharide esters, fructo
oligosaccharides, starches, modified starches and non starch
polysaccharides. Journal of the Science of Food and Agriculture 80,
166-170 (2000). [0375] 22. Cai, X., Yang, L., Zhang, L.-M. &
Wu, Q. Synthesis and anaerobic biodegradation of
indomethacin-conjugated cellulose ethers used for colon-specific
drug delivery. Bioresource Technology 100, 4164-4170 (2009). [0376]
23. Haupt, S. & Rubinstein, A. The colon as a possible target
for orally administered peptide and protein drugs. Critical reviews
in therapeutic drug carrier systems 19, 499 (2002). [0377] 24.
Hamman, J. H., Enslin, G. M. & Kotze, A. F. Oral delivery of
peptide drugs: barriers and developments. BioDrugs 19, 165-177
(2005). [0378] 25. Miranda, E. & Berglund, K. Recovery of
Clostridium thermosulfurogenes produced.
[0379] beta.-amylase by hydroxypropyl methylcellulose partition.
Biotechnology Progress 6, 214-219 (1990). [0380] 26. Clark, J., et
al. Guide for the care and use of laboratory animals. Institute of
Laboratory Animal Resources, National Research Council, Washington,
DC (1996). [0381] 27. AOCS. Preparation of methyl esters of
long-chain fatty acids from sampling analysis of commercial fats
and oils. Official method, AOCS, Champaign, Ill. Ce 2-66 (1997).
[0382] 28. Hong, Y. J., Turowski, M., Lin, J. T. & Yokoyama, W.
H. Simultaneous characterization of bile acid, sterols, and
determination of acylglycerides in feces from soluble cellulose-fed
hamsters using HPLC with evaporative light-scattering detection and
APCInMS. Journal of agricultural and food chemistry 55, 9750-9757
(2007). [0383] 29. Caporaso, J. G., et al. QIIME allows analysis of
high-throughput community sequencing data. Nat Methods 7, 335-336
(2010). 30. Lozupone, C., Lladser, M. E., Knights, D., Stombaugh,
J. & Knight, R. UniFrac: an effective distance metric for
microbial community comparison. ISME J(2010). [0384] 31. Lozupone,
C. & Knight, R. UniFrac: a new phylogenetic method for
comparing microbial communities. Appl Environ Microbiol 71,
8228-8235 (2005). [0385] 32. R: A Language and Environment for
Statistical Computing. in R Foundation for Statistical Computing,
Vol. 1 (R Foundation for Statistical Computing, 2009). [0386] 33.
Gentleman, R. C., et al. Bioconductor: open software development
for computational biology and bioinformatics. Genome Biol 5, R80
(2004). [0387] 34. Arumugam M, Raes J, Pelletier E, Le Pastier D,
et al. Enterotypes of the human gut microbiome. Nature
2011;473:174-80. [0388] 35. Muegge B D, Kuczynski J, Knights D,
Clemente J C, Gonzalez A, Fontana L, Henrissat B, Knight R, Gordon
J I. Diet drives convergence in gut microbiome functions across
mammalian phylogeny and within humans. Science 2011; 332:970-4.
[0389] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims. It is further to be understood that all values are
approximate, and are provided for description.
[0390] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
Sequence CWU 1
1
13117DNAArtificial SequencePCR primer 1cagcagccgc ggtrata
17222DNAArtificial SequencePCR primer 2ggactaccvg ggtatctaak cc
22325DNAArtificial SequencePCR primer 3ggagyatgtg gtttaattcg aagca
25420DNAArtificial SequencePCR primer 4agctgacgac aaccatgcac
20525DNAArtificial SequencePCR primer 5ggarcatgtg gtttaattcg atgat
25620DNAArtificial SequencePCR primer 6agctgacgac aaccatgcag
20722DNAArtificial SequencePCR primer 7ctyggtcatt tagaggaagt aa
22819DNAArtificial SequencePCR primer 8rctgcgttct tcatcgwtg
19919DNAArtificial SequenceProbe 9tcygtaggtg aacctgcrg
191029DNAArtificial SequencePCR primer 10gcgacatttc actggaayws
tggcayatg 291125DNAArtificial SequencePCR primer 11cctgcctttg
caatrtcacr aangc 251217DNAArtificial SequencePCR primer
12cctacgggag gcagcag 171318DNAArtificial SequencePCR primer
13ccgtcaattc mtttragt 18
* * * * *