U.S. patent application number 15/359439 was filed with the patent office on 2017-06-15 for synergistic bacterial compositions and methods of production and use thereof.
The applicant listed for this patent is Seres Therapeutics, Inc.. Invention is credited to Noubar B. Afeyan, John Grant Aunins, David Arthur Berry, David N. Cook, Anthony Mario D'Onofrio, Matthew R. Henn, Kevin Daniel Litcofsky, Geoffrey von Maltzahn.
Application Number | 20170165302 15/359439 |
Document ID | / |
Family ID | 50773492 |
Filed Date | 2017-06-15 |
United States Patent
Application |
20170165302 |
Kind Code |
A1 |
Henn; Matthew R. ; et
al. |
June 15, 2017 |
SYNERGISTIC BACTERIAL COMPOSITIONS AND METHODS OF PRODUCTION AND
USE THEREOF
Abstract
Provided are therapeutic compositions containing ecobiotic.TM.
populations for prevention, treatment and reduction of symptoms
associated with a dysbiosis of a mammalian subject such as a
human.
Inventors: |
Henn; Matthew R.;
(Somerville, MA) ; von Maltzahn; Geoffrey;
(Boston, MA) ; D'Onofrio; Anthony Mario;
(Northborough, MA) ; Litcofsky; Kevin Daniel;
(Boston, MA) ; Berry; David Arthur; (Brookline,
MA) ; Cook; David N.; (Brooklyn, NY) ; Afeyan;
Noubar B.; (Lexington, MA) ; Aunins; John Grant;
(Doylestown, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seres Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
50773492 |
Appl. No.: |
15/359439 |
Filed: |
November 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14592481 |
Jan 8, 2015 |
9533014 |
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15359439 |
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14221190 |
Mar 20, 2014 |
9028841 |
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14592481 |
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14091201 |
Nov 26, 2013 |
8906668 |
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14221190 |
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PCT/US2013/071758 |
Nov 25, 2013 |
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14091201 |
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61729515 |
Nov 23, 2012 |
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61729517 |
Nov 23, 2012 |
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61729518 |
Nov 23, 2012 |
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61729519 |
Nov 23, 2012 |
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61729520 |
Nov 23, 2012 |
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61729521 |
Nov 23, 2012 |
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61729522 |
Nov 23, 2012 |
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61729524 |
Nov 23, 2012 |
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61729525 |
Nov 23, 2012 |
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61729526 |
Nov 23, 2012 |
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61729527 |
Nov 23, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/127 20160801;
Y02A 50/48 20180101; Y02A 50/481 20180101; A61K 9/4891 20130101;
A61K 35/74 20130101; A23V 2002/00 20130101; A61K 9/0053 20130101;
A61K 35/742 20130101; Y02A 50/475 20180101; Y02A 50/478 20180101;
A23P 10/30 20160801; Y02A 50/30 20180101; A23L 33/135 20160801;
A61K 38/13 20130101; A61K 45/06 20130101; Y02A 50/402 20180101;
A61K 38/13 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/742 20060101
A61K035/742; A61K 9/48 20060101 A61K009/48; A23P 10/30 20060101
A23P010/30; A23L 33/135 20060101 A23L033/135; A23L 33/10 20060101
A23L033/10; A61K 45/06 20060101 A61K045/06; A61K 9/00 20060101
A61K009/00 |
Claims
1. -10. (canceled)
11. A therapeutic composition comprising an effective amount of a
bacterial combination comprising a first purified bacterial
population and a second purified bacterial population, the first
purified bacterial population comprising a first 16S rDNA sequence
at least 95% identical to a first member of a binary pair selected
from Table 3 and the second purified bacterial population
comprising a second16S rDNA sequence at least 95% identical to a
second member of the binary pair selected from Table 3, wherein the
binary pair can synergistically inhibit Clostridium difficile
growth in a CivSim assay and wherein the therapeutic composition is
formulated for oral delivery.
12. The therapeutic composition of claim 11, wherein the binary
pair can synergistically inhibit Clostridium difficile growth in a
CivSim assay with a confidence interval of >99% (++++).
13. The therapeutic composition of claim 11, wherein the first
member has a 16S rDNA at least 95% identical to SEQ ID NO:705 or
SEQ ID NO:1896.
14. The therapeutic composition of claim 11, wherein the first
member is a Dorea longicatena.
15. The therapeutic composition of claim 11, wherein the first 16S
rDNA sequence is at least 97% identical to the first member of a
binary pair selected from Table 3 and the second 16S rDNA sequence
is at least 97% identical to the second member of the binary pair
selected from Table 3.
16. The therapeutic composition of claim 11, wherein the first 16S
rDNA sequence is 100% identical to the first member of a binary
pair selected from Table 3 and the second 16S rDNA sequence is 100%
identical to the second member of the binary pair selected from
Table 3.
17. The therapeutic composition of claim 11, wherein at least one
of the 16S rDNA sequences is a 16S rDNA V4 sequence.
18. The therapeutic composition of claim 11, wherein at least one
of the first purified bacterial population and the second purified
bacterial population is capable of functionally populating a
gastrointestinal tract of a human subject.
19. The therapeutic composition of claim 18, wherein the functional
populating of the gastrointestinal tract comprises reducing the
number of one or more types of pathogenic bacteria in the
gastrointestinal tract, and/or increasing the number of one or more
non-pathogenic bacteria in the gastrointestinal tract.
20. The therapeutic composition of claim 11, wherein the first
purified bacterial population or the second purified bacterial
population is a spore-forming bacteria.
21. The therapeutic composition of claim 11, wherein the first
purified bacterial population and the second purified bacterial
population are both spore-forming bacteria.
22. The therapeutic composition of claim 11, wherein the first
purified bacterial population or the second purified bacterial
population is in a spore form.
23. The therapeutic composition of claim 11, wherein the first
purified bacterial population and the second purified bacterial
population are in a spore form.
24. The therapeutic composition of claim 11, wherein the bacterial
combination consists of the first and second bacterial
populations.
25. The therapeutic composition of claim 11, wherein the effective
amount is 10 8 or 10 9 or 10 10 total viable CFU of the bacterial
combination.
26. The therapeutic composition of claim 11, further comprising an
effective amount of an anti-bacterial agent, an anti-fungal agent,
an anti-viral agent or an anti-parasitic agent.
27. The therapeutic composition of claim 11, formulated in a
capsule.
28. The therapeutic composition of claim 27, encapsulated in an
enteric coating.
29. A method of treating or preventing recurrence of Clostridium
difficile infection in a gastrointestinal tract in a mammal, the
method comprising administering the therapeutic composition of
claim 11 to the mammal.
30. The method of claim 29, wherein the mammal is a human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/592,481, filed Jan. 8, 2015 (allowed),
which is a continuation of U.S. patent application Ser. No.
14/221,190, filed Mar. 20, 2014, now U.S. Pat. No. 9,028,841,
issued on May 12, 2015, which is a divisional of U.S. patent
application Ser. No. 14/091,201, filed on Nov. 26, 2013, now U.S.
Pat. No. 8,906,668, issued on Dec. 9, 2014, which is a continuation
of International Application No. PCT/US2013/071758, filed on Nov.
25, 2013, titled "SYNERGISTIC BACTERIAL COMPOSITIONS AND METHODS OF
PRODUCTION AND USE THEREOF", which claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/729,518, filed Nov. 23,
2012, U.S. Provisional Patent Application Ser. No. 61/729,519,
filed Nov. 23, 2012, U.S. Provisional Patent Application Ser. No.
61/729,520, filed Nov. 23, 2012, U.S. Provisional Patent
Application Ser. No. 61/729,521, filed Nov. 23, 2012, U.S.
Provisional Patent Application Ser. No. 61/729,522, filed Nov. 23,
2012, U.S. Provisional Patent Application Ser. No. 61/729,524,
filed Nov. 23, 2012, U.S. Provisional Patent Application Ser. No.
61/729,515, filed Nov. 23, 2012, U.S. Provisional Patent
Application Ser. No. 61/729,517, filed Nov. 23, 2012, U.S.
Provisional Patent Application Ser. No. 61/729,525, filed Nov. 23,
2012, U.S. Provisional Patent Application Ser. No. 61/729,526,
filed Nov. 23, 2012, and U.S. Provisional Patent Application Ser.
No. 61/729,527, filed Nov. 23, 2012, all of which are incorporated
herein by reference in their entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing with 1926
sequences submitted electronically as a text file named
35575_US_CRF_sequence_listing.txt, created on Nov. 21, 2016, with a
size of 5,686,552 bytes. The sequence listing is incorporated by
reference.
INTRODUCTION
[0003] Mammals are colonized by microbes in the gastrointestinal
(GI) tract, on the skin, and in other epithelial and tissue niches
such as the oral cavity, eye surface and vagina. The
gastrointestinal tract harbors an abundant and diverse microbial
community. It is a complex system, providing an environment or
niche for a community of many different species or organisms,
including diverse strains of bacteria. Hundreds of different
species may form a commensal community in the GI tract in a healthy
person, and this complement of organisms evolves from the time of
birth to ultimately form a functionally mature microbial population
by about 3 years of age. Interactions between microbial strains in
these populations and between microbes and the host, e.g. the host
immune system, shape the community structure, with availability of
and competition for resources affecting the distribution of
microbes. Such resources may be food, location and the availability
of space to grow or a physical structure to which the microbe may
attach. For example, host diet is involved in shaping the GI tract
flora.
[0004] A healthy microbiota provides the host with multiple
benefits, including colonization resistance to a broad spectrum of
pathogens, essential nutrient biosynthesis and absorption, and
immune stimulation that maintains a healthy gut epithelium and an
appropriately controlled systemic immunity. In settings of
`dysbiosis` or disrupted symbiosis, microbiota functions can be
lost or deranged, resulting in increased susceptibility to
pathogens, altered metabolic profiles, or induction of
proinflammatory signals that can result in local or systemic
inflammation or autoimmunity. Thus, the intestinal microbiota plays
a significant role in the pathogenesis of many diseases and
disorders, including a variety of pathogenic infections of the
gut.
[0005] For instance, patients become more susceptible to pathogenic
infections when the normal intestinal microbiota has been disturbed
due to use of broad-spectrum antibiotics. Many of these diseases
and disorders are chronic conditions that significantly decrease a
patient's quality of life and can be ultimately fatal.
[0006] Fecal transplantation has been shown to be an effective
treatment for patients suffering from severe or refractory GI
infections by repopulating the gut with a diverse array of microbes
that control key pathogens by creating an ecological environment
inimical to their proliferation and survival. Such approaches have
demonstrated significant potential to decrease host susceptibility
to infection. Fecal transplantation, however, is considered to be a
procedure of last resort because it has the potential to transmit
infectious or allergenic agents between hosts, involves the
transmission of potentially hundreds of unknown strains from donor
to patient, and is difficult to perform on a mass scale.
Additionally, fecal transplantation is inherently nonstandardized
and different desired and/or undesired material may be transmitted
in any given donation. Fecal transplantation is not approved by the
FDA and is unlikely to gain approval since the product cannot be
standardized and characterized according to regulatory requirements
for identity, potency, purity and safety. Thus, there is a need for
defined compositions that can be used to decrease susceptibility to
infection and/or that facilitate restoration of a healthy gut
microbiota.
[0007] Thus practitioners have a need for a much safer and
reproducible treatment for disorders currently treated on an
experimental (non-FDA approved) basis using fecal transplantation.
In order to prepare a therapeutic with commercial potential, we
have designed bacterial compositions of isolated bacterial strains
with a plurality of beneficial properties based on our
understanding of those bacterial strains and our analysis of the
properties that would enhance the utility and commercialization of
a bacterial composition.
[0008] Therefore, in response to the need for durable, efficient,
and effective compositions and methods for treatment of GI
diseases, in particular serious pathogenic infections, by way of
restoring or enhancing microbiota functions, we address these and
other shortcomings of the prior art by providing compositions and
methods for treating patients.
SUMMARY
[0009] In one aspect, provided are compositions comprising an
effective amount of a bacterial composition comprising at least a
first type of isolated bacterium capable of forming a spore and a
second type of isolated bacterium capable of forming a spore,
wherein the first type and the second type are not identical, and
wherein at least one of the first type and the second type are
capable of decreasing and/or inhibiting the growth and/or
colonization of at least one type of pathogenic bacteria. In an
embodiment, the bacterial composition comprises at least about 3,
4, 5, 6, 7, 8, 9, or 10 types of isolated bacteria. In an
embodiment, the bacterial composition comprises at least about 3,
4, 5, 6, 7, 8, 9, or 10 types of isolated bacteria and at least 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 of the isolated bacteria are capable
of forming spores. In an embodiment, the bacterial composition
comprises at least about 5 types of isolated bacteria and at least
2 of the isolated bacteria are capable of forming spores. In an
embodiment, the bacterial composition comprises: i) at least about
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more types of isolated
bacteria capable of forming spores, ii) at least about 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or more types of isolated bacteria not
known to be capable of forming spores, or iii) any combination of
i) and ii). In an embodiment, the first type and the second type
are present in the composition in approximately equal
concentrations. In an embodiment, the first type and the second
type are present in the composition in not substantially equal
concentrations. In an embodiment, the first type is present in the
composition in at least about 150% the concentration of the second
type, or wherein the second type is present in the composition in
at least about 150% the concentration of the first type. In an
embodiment, the composition consists essentially of: i) between two
and about twenty types of isolated bacteria, wherein at least two
types of isolated bacteria are independently capable of spore
formation; ii) between two and about twenty types of isolated
bacteria, wherein at least two types of isolated bacteria not known
to be capable of spore formation, or iii) any combination of i) and
ii). In an embodiment, the first type of isolated bacterium and the
second type of isolated bacterium are selected from Table 1. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium comprise an operational taxonomic unit
(OTU) distinction. In an embodiment, the OTU distinction comprises
16S rRNA sequence similarity below about 95% identity. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium independently comprise bacteria that
comprise 16S rRNA sequence at least 95% identical to 16S rRNA
sequence present in a bacterium selected from SEQ ID NOs.: 1-1,864.
In an embodiment, a combination of the first type and the second
type are: i) cytotoxic, ii) cytostatic, iii) capable of decreasing
the growth of the pathogenic bacterium, iv) capable of inhibiting
the growth of the pathogenic bacterium, v) capable of decreasing
the colonization of the pathogenic bacterium, vi) capable of
inhibiting the colonization of the pathogenic bacterium, or vii)
any combination of i)-vi). In an embodiment, the combination is
capable of inhibiting proliferation of the pathogenic bacteria
present at a concentration at least equal to the concentration of
the combination of the first type and the second type. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacterial present at a concentration at least
about twice the concentration of the combination of the first type
and the second type. In an embodiment, the combination is capable
of inhibiting proliferation of the pathogenic bacterial present at
a concentration at least about ten times the concentration of the
combination of the first type and the second type. In an
embodiment, the pathogenic bacterium is selected from the group
consisting of Yersinia, Vibrio, Treponema, Streptococcus,
Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia,
Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,
Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,
Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,
Corynebacterium, Clostridium, Chlamydia, Chlamydophila,
Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella,
Bifidobacterium, Bacillus, multi-drug resistant bacteria,
Carbapenem-resistent Enterobacteriaceae (CRE), extended spectrum
beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant
Enterococci (VRE). In an embodiment, the first type and the second
type synergistically interact. In an embodiment, the first type and
the second type synergistically interact to inhibit the pathogenic
bacterium.
[0010] In another aspect, provided are compositions comprising an
effective amount of a bacterial composition comprising at least a
first type of isolated bacterium and a second type of isolated
bacterium, wherein only one of the first type and the second type
are capable of forming a spore, and wherein at least one of the
first type and the second type are capable of decreasing the growth
and/or colonization of at least one type of pathogenic
bacteria.
[0011] In another aspect, provided are compositions comprising an
effective amount of a bacterial composition comprising at least a
first type of isolated bacterium and a second type of isolated
bacterium, wherein the first type and the second type are not
spores or known to be capable of forming a spore, and wherein at
least one of the first type and the second type are capable of
decreasing the growth and/or colonization of at least one type of
pathogenic bacteria.
[0012] In an embodiment, at least one of the first type and the
second type are capable of reducing the growth rate of at least one
type of pathogenic bacteria. In an embodiment, at least one of the
first type and the second type are cytotoxic to at least one type
of pathogenic bacteria. In an embodiment, at least one of the first
type and the second type are cytostatic to at least one type of
pathogenic bacteria. In an embodiment, the first type and the
second type are selected from Table 1. In an embodiment, the first
type and the second type comprise different species. In an
embodiment, the first type and the second type comprise different
genera. In an embodiment, the first type and the second type
comprise different families. In an embodiment, the first type and
the second type comprise different orders.
[0013] In another aspect, provided are compositions comprising an
effective amount of a bacterial composition comprising at least a
first type of isolated bacterium and a second type of isolated
bacterium, wherein: i) the first type and the second type are
independently capable of forming a spore; ii) only one of the first
type and the second type are capable of forming a spore or iii)
neither the first type nor the second type are capable of forming a
spore, wherein the first type and the second type are not
identical, wherein the first type and the second type are capable
of functionally populating the gastrointestinal tract of a human
subject to whom the composition is administered. In an embodiment,
the functional populating of the gastrointestinal tract comprises
preventing a dysbiosis of the gastrointestinal tract. In an
embodiment, the functional populating of the gastrointestinal tract
comprises treating a dysbiosis of the gastrointestinal tract. In an
embodiment, the functional populating of the gastrointestinal tract
comprises reducing the severity of a dysbiosis of the
gastrointestinal tract. In an embodiment, the functional populating
of the gastrointestinal tract comprises reducing one or more
symptoms of a dysbiosis of the gastrointestinal tract. In an
embodiment, the functional populating of the gastrointestinal tract
comprises preventing growth and/or colonization of the
gastrointestinal tract by a pathogenic bacterium. In an embodiment,
the functional populating of the gastrointestinal tract comprises
reducing growth and/or colonization of the gastrointestinal tract
by a pathogenic bacterium. In an embodiment, the functional
populating of the gastrointestinal tract comprises reducing the
number of one or more types of pathogenic bacteria in the
gastrointestinal tract. In an embodiment, the functional populating
of the gastrointestinal tract comprises increasing the number of
one or more non-pathogenic bacteria in the gastrointestinal tract.
In an embodiment, the bacterial composition comprises 0, 1, 2, 3 or
greater than 3 types of isolated bacteria capable of forming
spores. In an embodiment, the bacterial composition comprises at
least about 5 types of isolated bacteria capable of forming spores.
In an embodiment, the bacterial composition comprises at least
about 7 types of isolated bacteria capable of forming spores. In an
embodiment, the first type and the second type are present in the
composition in not substantially equal concentrations. In an
embodiment, the first type and the second type are present in the
composition in approximately equal concentrations. In an
embodiment, the first type is present in the composition in at
least about 150% the concentration of the second type. In an
embodiment, the second type is present in the composition in at
least about 150% the concentration of the first type. In an
embodiment, the composition consists essentially of between two and
about ten types of isolated bacteria, wherein at least one type of
isolated bacteria are independently capable of spore formation. In
an embodiment, the first type of isolated bacterium and the second
type of isolated bacterium are selected from Table 1. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium comprise an operational taxonomic unit
(OTU) distinction. In an embodiment, the OTU distinction comprises
16S rRNA sequence similarity below about 95% identity. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium independently comprise bacteria that
comprise 16S rRNA sequence at least 95% identical to 16S rRNA
sequence present in a bacterium selected from Table 3. In an
embodiment, a combination of the first type and the second type are
cytotoxic or cytostatic to the pathogenic bacterium. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacteria present at a concentration at least
equal to the concentration of the combination of the first type and
the second type. In an embodiment, the combination is capable of
inhibiting proliferation of the pathogenic bacterial present at a
concentration at least about twice the concentration of the
combination of the first type and the second type. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacteria present at a concentration at least
about ten times the concentration of the combination of the first
type and the second type. In an embodiment, the pathogenic
bacterium is selected from the group consisting of Yersinia,
Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella,
Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria,
Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,
Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,
Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,
Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,
Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug
resistant bacteria, Carbapenem-resistent Enterobacteriaceae (CRE),
extended spectrum beta-lactam resistant Enterococci (ESBL), and
vancomycin-resistant Enterococci (VRE). In an embodiment, the first
type and the second type synergistically interact to be cytotoxic
to the pathogenic bacterium. In an embodiment, wherein the first
type and the second type synergistically interact to be cytostatic
to the pathogenic bacterium.
[0014] In another aspect, provided are single dose units comprising
the compositions of the present invention. In an embodiment, the
dose unit comprises at least 1.times.10.sup.4, 1.times.10.sup.5,
1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8,
1.times.10.sup.9, 1.times.10.sup.10, 1.times.10.sup.11 or greater
than 1.times.10.sup.11 colony forming units (CFUs) of either spores
or vegetative bacterial cells. In an embodiment, the dose unit
comprises a pharmaceutically acceptable excipient, an enteric
coating or a combination thereof. In an embodiment, the dose unit
further comprises a drug selected from corticosteroids, mesalazine,
mesalamine, sulfasalazine, sulfasalazine derivatives,
immunosuppressive drugs, cyclosporin A, mercaptopurine,
azathiopurine, prednisone, methotrexate, antihistamines,
glucocorticoids, epinephrine, theophylline, cromolyn sodium,
anti-leukotrienes, anti-cholinergic drugs for rhinitis,
anti-cholinergic decongestants, mast-cell stabilizers, monoclonal
anti-IgE antibodies, vaccines, and combinations thereof. In an
embodiment, the dose unit is formulated for oral administration,
rectal administration, or the combination of oral and rectal
administration, or is formulated for topical, nasal or inhalation
administration.
[0015] In another aspect, provided are kits comprising in one or
more containers: a first purified population of a first type of
bacterial spores substantially free of viable vegetal bacterial
cells; and a second purified population of a second type of
bacterial spores substantially free of viable vegetal bacterial
cells, wherein the first type and the second type of bacterial
spores are not identical, and wherein the first type and the second
type of bacterial spores, when co-localized in a target region of a
gastrointestinal tract of a human subject in need thereof, are
capable of functionally populating the gastrointestinal tract. In
an embodiment, the first purified population and the second
purified population are present in a single container. In an
embodiment, the first purified population and the second purified
population are present in two containers. In an embodiment, the
first purified population and the second purified population are
lyophilized or substantially dehydrated. In an embodiment, the kit
further comprises in one or more containers an effective amount of
an anti-bacterial agent, an effective amount of an anti-viral
agent, an effective amount of an anti-fungal agent, an effective
amount of an anti-parasitic agent, or a combination thereof in one
or more containers. In an embodiment, the kit further comprises a
pharmaceutically acceptable excipient or diluent.
[0016] Also provided are pharmaceutical formulations comprising an
effective amount of the compositions of the invention, and further
comprising an effective amount of an anti-bacterial agent, an
effective amount of an anti-fungal agent, an effective amount of an
anti-viral agent, an effective amount of an anti-parasitic
agent.
[0017] Also provided are comestible products comprising a first
purified population of a first type of bacterial spores and a
second purified population of a second type of bacterial spores,
wherein the first type and the second type of bacterial spores are
not identical, wherein the comestible product is substantially free
of viable vegetal bacterial cells, and wherein the first type and
the second type of bacterial spores, when administered to a human
subject in need thereof, are capable of functionally populating the
gastrointestinal tract of the human subject. In an embodiment, the
comestible product comprises a food or food additive, a beverage or
beverage additive, or a medical food. In an embodiment, the
comestible product comprises at least 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11 or greater than 1.times.10.sup.11 colony forming
units (CFUs) of viable spores. In an embodiment, the comestible
product comprises a first type of bacterial spores and a second
type of bacterial spores selected from Table 1, or where the first
type of bacterial spores and the second type of bacterial spores
independently comprise bacterial spores that comprise 16S rRNA
sequence at least 95% identical to 16S rRNA sequence present in a
bacterium selected from SEQ ID NOs.: 1-1,864.
[0018] Also provided are methods comprising administering to a
human subject in need thereof an effective amount of a bacterial
composition comprising at least a first type of isolated bacterium
and a second type of isolated bacterium, wherein: the first type
and the second type are independently capable of forming a spore;
only one of the first type and the second type are capable of
forming a spore or neither the first type nor the second type are
capable of forming a spore, wherein the first type and the second
type are not identical, and wherein at least one of the first type
and the second type exert an inhibitory-effect on a pathogenic
bacterium present in the gastrointestinal tract of the human
subject, such that the number of pathogenic bacteria present in the
gastrointestinal tract is not detectably increased or is detectably
decreased over a period of time. In an embodiment, the human
subject is diagnosed as having a dysbiosis of the gastrointestinal
tract. In an embodiment, wherein the human subject is diagnosed as
infected with a pathogenic bacterium selected from the group
consisting of Yersinia, Vibrio, Treponema, Streptococcus,
Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia,
Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,
Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,
Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,
Corynebacterium, Clostridium, Chlamydia, Chlamydophila,
Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella,
Bifidobacterium, Bacillus, multi-drug resistant bacteria,
Carbapenem-resistent Enterobacteriaceae (CRE), extended spectrum
beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant
Enterococci (VRE). In an embodiment, the bacterial composition is
administered simultaneously with i) an antibiotic, ii) a prebiotic,
or iii) a combination of i) and ii). In an embodiment, the
bacterial composition is administered prior to administration of i)
an antibiotic, ii) a prebiotic, or iii) a combination of i) and
ii). In an embodiment, the bacterial composition is administered
subsequent to administration of i) an antibiotic, ii) a prebiotic,
or iii) a combination of i) and ii). In an embodiment, the number
of pathogenic bacterium present in or excreted from the
gastrointestinal tract of the human subject is detectably reduced
within one month, within two weeks, or within one week of
administration of the bacterial composition. In an embodiment, the
number of pathogenic bacterium present in or excreted from the
gastrointestinal tract of the human subject is detectably reduced
within three days, two days or one day of administration of the
bacterial composition. In an embodiment, the human subject is
detectably free of the pathogenic bacterium within one month, two
weeks, one week, three days or one day of administration of the
bacterial composition. In an embodiment, the bacterial composition
comprises at least about 3, 4, 5, 6, 7, 8, 9, or 10 types of
isolated bacteria. In an embodiment, the bacterial composition
comprises at least about 3, 4, 5, 6, 7, 8, 9, or 10 types of
isolated bacteria and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of
the isolated bacteria are capable of forming spores. In an
embodiment, the bacterial composition comprises at least about 5
types of isolated bacteria and at least 2 of the isolated bacteria
are capable of forming spores. In an embodiment, the bacterial
composition comprises: i) at least about 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30 or more types of isolated bacteria capable of forming
spores, ii) at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or
more types of isolated bacteria not known to be capable of forming
spores, or iii) any combination of i) and ii). In an embodiment,
the bacterial composition comprises at least about 5 types of
isolated bacteria and at least 1 of the isolated bacteria are
capable of forming spores. In an embodiment, the bacterial
composition comprises at least about 5 types of isolated bacteria
and at least 1 of the isolated bacteria is not capable of forming
spores. In an embodiment, the bacterial composition comprises at
least about 3, 4, 5, 6, 7, 8, 9 or 10 types of isolated bacteria,
wherein i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types of
isolated bacteria are capable of forming spores, ii) at least 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 types of isolated bacteria are not
capable of forming spores, or iii) any combination of i) and ii).
In an embodiment, the first type and the second type are present in
the composition in approximately equal concentrations. In an
embodiment, the first type and the second type are present in the
composition in not substantially equal concentrations. In an
embodiment, the first type is present in the composition in at
least about 150% the concentration of the second type, or wherein
the second type is present in the composition in at least about
150% the concentration of the first type. In an embodiment, the
composition consists essentially of between two and about ten types
of isolated bacteria, wherein at least two types of isolated
bacteria are independently capable of spore formation. In an
embodiment, the composition consists essentially of between two and
about ten types of isolated bacteria, wherein at least two types of
isolated bacteria are not capable of spore formation. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium are selected from Table 1. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium comprise an operational taxonomic unit
(OTU) distinction. In an embodiment, the OTU distinction comprises
16S rRNA sequence similarity below about 95% identity. In an
embodiment, the first type of isolated bacterium and the second
type of isolated bacterium independently comprise bacteria that
comprise 16S rRNA sequence at least 95% identical to 16S rRNA
sequence present in a bacterium selected from SEQ ID NOs: 1-1,864.
In an embodiment, a combination of the first type and the second
type are cytotoxic or cytostatic to the pathogenic bacterium. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacterial present at a concentration at least
equal to the concentration of the combination of the first type and
the second type. In an embodiment, the combination is capable of
inhibiting proliferation of the pathogenic bacterial present at a
concentration at least about twice the concentration of the
combination of the first type and the second type. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacterial present at a concentration at least
about ten times the concentration of the combination of the first
type and the second type. In an embodiment, the pathogenic
bacterium is selected from the group consisting of Yersinia,
Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella,
Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria,
Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,
Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,
Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,
Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,
Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug
resistant bacteria, Carbapenem-resistent Enterobacteriaceae (CRE),
extended spectrum beta-lactam resistant Enterococci (ESBL), and
vancomycin-resistant Enterococci (VRE). In an embodiment, the first
type and the second type synergistically interact to be cytotoxic
to the pathogenic bacterium. In an embodiment, the first type and
the second type synergistically interact to be cytostatic to the
pathogenic bacterium.
[0019] Also provided are methods of functionally populating the
gastrointestinal tract of a human subject, comprising administering
to the subject an effective amount of a bacterial composition
comprising at least a first type of isolated bacterium and a second
type of isolated bacterium, wherein i) the first type and the
second type are independently capable of forming a spore; ii) only
one of the first type and the second type are capable of forming a
spore or iii) neither the first type nor the second type are
capable of forming a spore, wherein the first type and the second
type are not identical, under conditions such that the first type
and the second type functionally populate the gastrointestinal
tract of the human subject. In an embodiment, the bacterial
composition is orally administered, rectally administered, or the
combination of orally and rectally administered. In an embodiment,
the bacterial composition is topically or nasally administered or
inhaled. In an embodiment, the first type of isolated bacteria and
the second type of isolated bacteria are selected from Table 1. In
an embodiment, the bacterial composition consists essentially of
spores, wherein the spores comprise spores of the first type of
isolated bacteria and spores of the second type of isolated
bacteria. In an embodiment, the first type of isolated bacteria and
the second type of isolated bacteria independently comprise
bacterial spores that comprise 16S rRNA sequence at least 95%
identical to 16S rRNA sequence present in a bacterium selected from
SEQ ID NOs. 1-1,864. In an embodiment, the functional populating of
the gastrointestinal tract comprises preventing a dysbiosis of the
gastrointestinal tract. In an embodiment, the functional populating
of the gastrointestinal tract comprises treating a dysbiosis of the
gastrointestinal tract. In an embodiment, the functional populating
of the gastrointestinal tract comprises reducing the severity of a
dysbiosis of the gastrointestinal tract. In an embodiment, the
functional populating of the gastrointestinal tract comprises
reducing one or more symptoms of a dysbiosis of the
gastrointestinal tract. In an embodiment, the functional populating
of the gastrointestinal tract comprises preventing colonization of
the gastrointestinal tract by a pathogenic bacterium. In an
embodiment, the functional populating of the gastrointestinal tract
comprises reducing colonization of the gastrointestinal tract
and/or growth by a pathogenic bacterium. In an embodiment, wherein
the functional populating of the gastrointestinal tract comprises
reducing the number of one or more types of pathogenic bacteria in
the gastrointestinal tract. In an embodiment, the functional
populating of the gastrointestinal tract comprises increasing the
number of one or more non-pathogenic bacteria in the
gastrointestinal tract. In an embodiment, the bacterial composition
comprises at least about 3, 5, 7 or 9 types of isolated bacteria
capable of forming spores. In an embodiment, the bacterial
composition comprises at least about 5 types of isolated bacteria
and at least 20% of the isolated bacteria are capable of forming
spores. In an embodiment, the bacterial composition comprises at
least about 5 types of isolated bacteria and at least 2 of the
isolated bacteria are capable of forming spores. In an embodiment,
the bacterial composition comprises at least about 3, 4, 5, 6, 7,
8, 9 or 10 types of isolated bacteria, wherein i) at least 1, 2, 3,
4, 5, 6, 7, 8, 9 or 10 types of isolated bacteria are capable of
forming spores, ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types
of isolated bacteria are not capable of forming spores, or iii) any
combination of i) and ii). In an embodiment, the first type and the
second type are present in the composition in approximately equal
concentrations. In an embodiment, the first type and the second
type are present in the composition in not substantially equal
concentrations. In an embodiment, the first type is present in the
composition in at least about 150% the concentration of the second
type, or wherein the second type is present in the composition in
at least about 150% the concentration of the first type. In an
embodiment, the composition consists essentially of between two and
about ten types of isolated bacteria, wherein i) at least one type
of isolated bacteria is capable of spore formation, ii) at least
one type of isolated bacteria is not capable of spore formation, or
iii) a combination of i) and ii). In an embodiment, a combination
of the first type and the second type are inhibitory to the
pathogenic bacterium. In an embodiment, the combination reduces the
growth rate of the pathogenic bacterium. In an embodiment, the
combination is cytostatic or cytotoxic to the pathogenic bacterium.
In an embodiment, the combination is capable of inhibiting growth
of the pathogenic bacterial present at a concentration at least
equal to the concentration of the combination of the first type and
the second type. In an embodiment, the combination is capable of
inhibiting growth of the pathogenic bacterial present at a
concentration at least about twice the concentration of the
combination of the first type and the second type. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacterial present at a concentration at least
about ten times the concentration of the combination of the first
type and the second type. In an embodiment, the pathogenic
bacterium is selected from the group consisting of Yersinia,
Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella,
Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria,
Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,
Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,
Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,
Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,
Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug
resistant bacteria, Carbapenem-resistent Enterobacteriaceae (CRE),
extended spectrum beta-lactam resistant Enterococci (ESBL), and
vancomycin-resistant Enterococci (VRE). In an embodiment, the first
type and the second type synergistically interact to reduce or
inhibit the growth of the pathogenic bacterium. In an embodiment,
the first type and the second type synergistically interact to
reduce or inhibit the colonization of the pathogenic bacterium. In
an embodiment, the method comprises administering to the human
subject a single dose unit comprising at least 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10,
1.times.10.sup.11 or greater than 1.times.10.sup.11 colony forming
units (CFUs) of viable bacteria. In an embodiment, the dose unit
comprises a bacterial population substantially in the form of
spores. In an embodiment, the dose unit comprises a
pharmaceutically acceptable excipient and/or an enteric coating. In
an embodiment, the unit dose is formulated for oral administration,
rectal administration, or the combination of oral and rectal
administration. In an embodiment, the unit dose is formulated for
topical or nasal administration or for inhalation.
[0020] In another aspect, provided are methods of reducing the
number of pathogenic bacteria present in the gastrointestinal tract
of a human subject, comprising administering to the subject an
effective amount of a pharmaceutical formulation comprising an
effective amount of the composition of claim 1 and further
comprising an effective amount of an anti-microbial agent, under
conditions such that the number of pathogenic bacteria present in
the gastrointestinal tract of the human subject is reduced within
about one month of administration of the pharmaceutical
formulation. In an embodiment, the number of pathogenic bacteria
present in the gastrointestinal tract of the human subject is
reduced within about two weeks of administration of the
pharmaceutical formulation. In an embodiment, the number of
pathogenic bacteria present in the gastrointestinal tract of the
human subject is reduced within about one week of administration of
the pharmaceutical formulation. In an embodiment, the number of
pathogenic bacteria present in the gastrointestinal tract of the
human subject is reduced within about three days of administration
of the pharmaceutical formulation. In an embodiment, the number of
pathogenic bacteria present in the gastrointestinal tract of the
human subject is reduced within about one day of administration of
the pharmaceutical formulation. In an embodiment, the
anti-microbial agent comprises anti-bacterial agent. In an
embodiment, the anti-microbial agent comprises anti-fungal agent.
In an embodiment, the anti-microbial agent comprises anti-viral
agent. In an embodiment, the anti-microbial agent comprises
anti-parasitic agent.
[0021] In another aspect, provided are methods of preparing a
comestible product, comprising combining with a comestible carrier
a first purified population comprising at least a first type of
isolated bacterium and a second purified population comprising at
least a second type of isolated bacterium, wherein: i) the first
type and the second type are independently capable of forming a
spore; ii) only one of the first type and the second type are
capable of forming a spore or iii) neither the first type nor the
second type are capable of forming a spore, wherein the first type
and the second type of bacteria are not identical, wherein the
comestible product is substantially free of non-comestible
materials. In an embodiment, at least one of the first purified
population and the second purified population consist essentially
of viable spores. In an embodiment, the first purified population
and the second purified population consist essentially of viable
spores. In an embodiment, the comestible product is substantially
free of viable vegetal bacterial cells. In an embodiment, the
viable spores, when the comestible product is consumed by a human
subject in need thereof, are capable of functionally populating the
gastrointestinal tract of the human subject. In an embodiment, the
comestible product comprises a food or food additive. In an
embodiment, the comestible product comprises a beverage or beverage
additive. In an embodiment, the comestible product comprises a
medical food. In an embodiment, the comestible product comprises at
least 1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11 or greater than
1.times.10.sup.11 colony forming units (CFUs) of viable spores. In
an embodiment, spores are of a bacterium selected from Table 1. In
an embodiment, the first purified population and the second
purified population independently comprise bacterial spores that
comprise 16S rRNA sequence at least 95% identical to 16S rRNA
sequence present in a bacterium selected from SEQ ID NOs:
1-1,864.
[0022] Also provided are methods of reducing the abundance of a
pathogen in the gastrointestinal tract of a patient comprising
administering the composition of the invention in a therapeutically
effective amount and allowing the bacterial composition to compete
with the pathogen in the gastrointestinal tract of a patient.
[0023] Further provided are methods of treating diarrhea comprising
administering the composition of the invention in a therapeutically
effective amount and allowing the bacterial composition to reduce
the diarrheal effect of a pathogen in the gastrointestinal tract of
a patient. In an embodiment, the pathogen is Aeromonas hydrophila,
Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus,
Campylobacter jejuni, Clostridium botulinum, Clostridium difficile,
Clostridium perfringens, enteroaggregative Escherichia coli,
enterohemorrhagic Escherichia coli, enteroinvasive Escherichia
coli, enterotoxigenic Escherichia coli (LT or ST), Escherichia coli
0157:H7, Helicobacter pylori, Lysteria monocytogenes, Plesiomonas
shigelloides, Salmonella spp., Salmonella typhi, Shigella spp.,
Staphylococcus spp., Staphylococcus aureus, Vibrio spp., Vibrio
cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia
enterocolitica, multi-drug resistant bacteria, Carbapenem-resistent
Enterobacteriaceae (CRE), and vancomycin-resistant Enterococci
(VRE). In an embodiment, the pathogen is Clostridium difficile,
Salmonella spp., pathogenic Escherichia coli, or
vancomycin-resistant Enterococcus spp. In an embodiment, the
pathogen is Clostridium difficile. In an embodiment, the
composition is administered orally.
[0024] Also provided are therapeutic compositions comprising a
first purified bacterial population capable forming spores
consisting of Collinsella aerofaciens and a second purified
bacterial population consisting of a species selected from Table 1
or SEQ ID NOs: 1,865-1,915, wherein at least one of the first type
and the second type are cytotoxic or cytostatic to a pathogenic
bacterium. In an embodiment, a synergistic combination of the first
bacterial spore population and the second bacterial spore
population are cytotoxic or cytostatic to the pathogenic bacterium.
In an embodiment, the combination is capable of inhibiting
proliferation of the pathogenic bacterial present at a
concentration at least equal to the concentration of the
combination of the first type and the second type. In an
embodiment, the combination is capable of inhibiting proliferation
of the pathogenic bacterial present at a concentration at least
about twice the concentration of the combination of the first type
and the second type. In an embodiment, the combination is capable
of inhibiting proliferation of the pathogenic bacterial present at
a concentration at least about ten times the concentration of the
combination of the first type and the second type. In an
embodiment, the pathogenic bacterium is selected from the group
consisting of Yersinia, Vibrio, Treponema, Streptococcus,
Staphylococcus, Shigella, Salmonella, Rickettsia, Pseudomonas,
Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira,
Legionella, Helicobacter, Haemophilus, Francisella, Escherichia,
Enterococcus, Corynebacterium, Clostridium, Chlamydia,
Chlamydophila, Campylobacter, Brucella, Borrelia, and Bordetella.
In an embodiment, the pathogenic bacterium is Clostridium dificile.
In an embodiment, the first bacterial spore population and the
second bacterial spore population synergistically interact to be
cytotoxic to the pathogenic bacterium. In an embodiment, the first
bacterial spore population and the second bacterial spore
population synergistically interact to be cytostatic to the
pathogenic bacterium. In an embodiment, the bacterial composition
comprises at least about 3 types of isolated bacteria capable of
forming spores. In an embodiment, the bacterial composition
comprises at least about 5 types of isolated bacteria capable of
forming spores. In an embodiment, the first bacterial spore
population and the second bacterial spore population are present in
the composition in approximately equal concentrations. In an
embodiment, the composition consists essentially of between two and
about ten bacterial spore populations of isolated bacteria.
[0025] In another aspect, provided are therapeutic compositions
comprising a first purified bacterial spore population consisting
of bacteria comprising 16S rRNA sequence at least about 97%
identical to a 16S rRNA sequence present in a reference Collinsella
aerofaciens OTU, and a second purified bacterial spore population
consisting of bacteria comprising 16S rRNA sequence at least about
97% identical to a 16S rRNA sequence present in a reference
bacterium listed in Table 1 or SEQ ID NOs: 1,865-1,915, wherein at
least one of the first type and the second type are cytotoxic or
cytostatic to a pathogenic bacterium. In an embodiment, a
synergistic combination of the first type and the second type are
cytotoxic or cytostatic to the pathogenic bacterium. In an
embodiment, wherein the combination is capable of inhibiting
proliferation of the pathogenic bacterial present at a
concentration at least equal to the concentration of the
combination of the first type and the second type. In an
embodiment, the first type and the second type synergistically
interact to be cytotoxic or cytostatic to the pathogenic bacterium.
In an embodiment, the first purified bacterial spore population and
the second purified bacterial spore population are capable of
functionally populating the gastrointestinal tract of a human
subject to whom the composition is administered. In an embodiment,
the functional populating of the gastrointestinal tract comprises
preventing, treating, reducing the severity of or reducing a
symptom of a dysbiosis of the gastrointestinal tract. In an
embodiment, the functional populating of the gastrointestinal tract
comprises i) reducing the number of one or more types of pathogenic
bacteria in the gastrointestinal tract; or ii) increasing the
number of one or more non-pathogenic bacteria in the
gastrointestinal tract. In an embodiment, the composition further
comprises an effective amount of an anti-bacterial agent, an
anti-fungal agent, an anti-viral agent or an anti-parasitic
agent.
[0026] Also provided are methods of treating or preventing a
recurrence of a Clostridium difficile infection, comprising
administering to a human subject in need thereof an effective
amount of the therapeutic composition of the invention under
conditions such that the first purified bacterial spore population
and the second purified bacterial spore population exert a
cytotoxic or cytostatic effect on a pathogenic bacterium present in
the gastrointestinal tract of the human subject, such that the
number of Clostridium difficile bacteria present in the
gastrointestinal tract is not detectably increased or is detectably
decreased over a period of time. In an embodiment, the number of
Clostridium difficile bacteria present in or excreted from the
gastrointestinal tract of the human subject is detectably reduced
within one month of administration of the bacterial composition. In
an embodiment, the first purified bacterial spore population and
the second purified bacterial spore population synergistically
interact to be cytotoxic and/or cytostatic to the Clostridium
difficile bacteria. In an embodiment, the therapeutic composition
is orally administered. In an embodiment, the therapeutic
composition comprises a medical food.
[0027] In another aspect, provided are kits comprising in one or
more containers: a first purified population of a first type of
bacteria capable of forming spores; and a second purified
population of a second type of bacteria capable of forming spores,
wherein the first type and the second type are not identical, and
wherein the first type and the second type, when co-localized in a
target region of a gastrointestinal tract of a human subject in
need thereof, are capable of functionally populating the
gastrointestinal tract. In an embodiment, the first purified
population and the second purified population are present in a
single container. In an embodiment, the kit is formulated for use
as a nutritional supplement and optionally comprising a prebiotic
material.
[0028] Additional objects and advantages will be set forth in part
in the description which follows, and in part will be obvious from
the description, or may be learned by practice of the embodiments.
The objects and advantages will be realized and attained by means
of the elements and combinations particularly pointed out in the
appended claims.
[0029] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the claims.
[0030] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments and together with the description, serve to further
explain the embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1A provides a schematic of 16S rRNA gene and denotes
the coordinates of hypervariable regions 1-9 (V1-V9). Coordinates
of V1-V9 are 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043,
1117-1173, 1243-1294, and 1435-1465 respectively, based on
numbering using E. coli system of nomenclature defined by Brosius
et al., Complete nucleotide sequence of a 16S ribosomal RNA gene
(16S rRNA) from Escherichia coli, PNAS 75(10):4801-4805 (1978).
FIG. 1B highlights in bold the nucleotide sequences for each
hypervariable region in the exemplary reference E. coli 16S
sequence described by Brosius et al. FIG. 1B discloses SEQ ID NO:
1926.
BRIEF DESCRIPTION OF TABLES
[0032] Table 1 provides bacterial species and Operational Taxonomic
Units (OTUs) of the bacterial compositions of the present
invention, including taxonometric status and the ability of the OTU
to form a viable spore as provided herein.
[0033] Table 2 provides representative combinations of the
bacterial compositions of the present invention.
[0034] 16S rRNA sequences of the bacterial species and Operational
Taxonomic Units (OTUs) of the bacterial compositions of the present
invention are provided in the CRF version of the Sequence Listing
as SEQ ID NOS 1-1,864.
[0035] The taxonometric status, exemplary phylogenetic surrogacy
and 16S rRNA sequences of exemplary bacterial compositions of the
present invention are provided in the CRF version of the Sequence
Listing as SEQ ID NOS 1,865-1,915.
[0036] Table 3 demonstrates the efficacy of exemplary bacterial
compositions of the present invention in inhibiting a pathogenic
bacterium.
[0037] Table 4 demonstrates the efficacy of exemplary bacterial
compositions of the present invention in inhibiting a pathogenic
bacterium.
[0038] Table 5 provides representative bacterial pathogens.
[0039] Table 6 provides representative human diseases, disorders
and conditions for which the provided bacterial compositions are
useful.
[0040] Table 7 provides representative human diseases, disorders
and conditions for which the provided bacterial compositions are
useful.
Definitions
[0041] "Microbiota" refers to the communities of microbes that live
in or on the patient's body, both sustainably and transiently,
including eukaryotes, archaea, bacteria, and viruses (including
bacterial viruses (i.e., phage)).
[0042] "Dysbiosis" refers to a state of the microbiota or
microbiome of the gut or other body area, including mucosal or skin
surfaces in which the normal diversity and/or function of the
ecological network is disrupted. Any disruption from the preferred
(e.g., ideal) state of the microbiota can be considered a
dysbiosis, even if such dysbiosis does not result in a detectable
decrease in health. This state of dysbiosis may be unhealthy, it
may be unhealthy under only certain conditions, or it may prevent a
subject from becoming healthier. Dysbiosis may be due to a decrease
in diversity, the overgrowth of one or more pathogens or
pathobionts, symbiotic organisms able to cause disease only when
certain genetic and/or environmental conditions are present in a
patient, or the shift to an ecological network that no longer
provides a beneficial function to the host and therefore no longer
promotes health.
[0043] A "spore" or a population of "spores" includes bacteria (or
other single-celled organisms) that are generally viable, more
resistant to environmental influences such as heat and
bacteriocidal agents than vegetative forms of the same bacteria,
and typically capable of germination and out-growth.
"Spore-formers" or bacteria "capable of forming spores" are those
bacteria containing the genes and other necessary abilities to
produce spores under suitable environmental conditions.
[0044] The terms "pathogen", "pathobiont" and "pathogenic" in
reference to a bacterium or any other organism or entity includes
any such organism or entity that is capable of causing or affecting
a disease, disorder or condition of a host organism containing the
organism or entity.
[0045] The term "isolated" encompasses a bacterium or other entity
or substance that has been (1) separated from at least some of the
components with which it was associated when initially produced
(whether in nature or in an experimental setting), and/or (2)
produced, prepared, purified, and/or manufactured by the hand of
man. Isolated bacteria may be separated from at least about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90%, or more of the other components with which
they were initially associated. In some embodiments, isolated
bacteria are more than about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, or more than about 99% pure. As used herein,
a substance is "pure" if it is substantially free of other
components. The terms "purify," "purifying" and "purified" refer to
a bacterium or other material that has been separated from at least
some of the components with which it was associated either when
initially produced or generated (e.g., whether in nature or in an
experimental setting), or during any time after its initial
production. A bacterium or a bacterial population may be considered
purified if it is isolated at or after production, such as from a
material or environment containing the bacterium or bacterial
population, and a purified bacterium or bacterial population may
contain other materials up to about 10%, about 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
or above about 90% and still be considered "isolated." In some
embodiments, purified bacteria and bacterial populations are more
than about 80%, about 85%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, about
99%, or more than about 99% pure. In the instance of bacterial
compositions provided herein, the one or more bacterial types
present in the composition can be independently purified from one
or more other bacteria produced and/or present in the material or
environment containing the bacterial type. Bacterial compositions
and the bacterial components thereof are generally purified from
residual habitat products.
[0046] "Inhibition" of a pathogen encompasses the inhibition of any
desired function or activity of the bacterial compositions of the
present invention.
[0047] Demonstrations of pathogen inhibition, such as decrease in
the growth of a pathogenic bacterium or reduction in the level of
colonization of a pathogenic bacterium are provided herein and
otherwise recognized by one of ordinary skill in the art.
Inhibition of a pathogenic bacterium's "growth" may include
inhibiting the increase in size of the pathogenic bacterium and/or
inhibiting the proliferation (or multiplication) of the pathogenic
bacterium. Inhibition of colonization of a pathogenic bacterium may
be demonstrated by measuring the amount or burden of a pathogen
before and after a treatment. An "inhibition" or the act of
"inhibiting" includes the total cessation and partial reduction of
one or more activities of a pathogen, such as growth,
proliferation, colonization, and function.
[0048] The "colonization" of a host organism includes the
non-transitory residence of a bacterium or other microscopic
organism. As used herein, "reducing colonization" of a host
subject's gastrointestinal tract (or any other microbiotal niche)
by a pathogenic bacterium includes a reduction in the residence
time of the pathogen in the gastrointestinal tract as well as a
reduction in the number (or concentration) of the pathogen in the
gastrointestinal tract or adhered to the luminal surface of the
gastrointestinal tract. Measuring reductions of adherent pathogens
may be demonstrated, e.g., by a biopsy sample, or reductions may be
measured indirectly, e.g., by measuring the pathogenic burden in
the stool of a mammalian host.
[0049] A "combination" of two or more bacteria includes the
physical co-existence of the two bacteria, either in the same
material or product or in physically connected products, as well as
the temporal co-administration or co-localization of the two
bacteria.
[0050] A "cytotoxic" activity or bacterium includes the ability to
kill a bacterial cell, such as a pathogenic bacterial cell. A
"cytostatic" activity or bacterium includes the ability to inhibit,
partially or fully, growth, metabolism, and/or proliferation of a
bacterial cell, such as a pathogenic bacterial cell.
[0051] To be free of "non-comestible products" means that a
bacterial composition or other material provided herein does not
have a substantial amount of a non-comestible product, e.g., a
product or material that is inedible, harmful or otherwise
undesired in a product suitable for administration, e.g., oral
administration, to a human subject. Non-comestible products are
often found in preparations of bacteria from the prior art.
[0052] "Microbiome" refers to the genetic content of the
communities of microbes that live in and on the human body, both
sustainably and transiently, including eukaryotes, archaea,
bacteria, and viruses (including bacterial viruses (i.e., phage)),
wherein "genetic content" includes genomic DNA, RNA such as micro
RNA and ribosomal RNA, the epigenome, plasmids, and all other types
of genetic information.
[0053] "Residual habitat products" refers to material derived from
the habitat for microbiota within or on a human or animal. For
example, microbiota live in feces in the gastrointestinal tract, on
the skin itself, in saliva, mucus of the respiratory tract, or
secretions of the genitourinary tract (i.e., biological matter
associated with the microbial community). Substantially free of
residual habitat products means that the bacterial composition no
longer contains the biological matter associated with the microbial
environment on or in the human or animal subject and is 100% free,
99% free, 98% free, 97% free, 96% free, or 95% free of any
contaminating biological matter associated with the microbial
community. Residual habitat products can include abiotic materials
(including undigested food) or it can include unwanted
microorganisms. Substantially free of residual habitat products may
also mean that the bacterial composition contains no detectable
cells from a human or animal and that only microbial cells are
detectable.
[0054] In one embodiment, substantially free of residual habitat
products may also mean that the bacterial composition contains no
detectable viral (including bacterial viruses (i.e., phage)),
fungal, mycoplasmal contaminants. In another embodiment, it means
that fewer than 1.times.10.sup.-2%, 1.times.10.sup.-3%,
1.times.10.sup.-4%, 1.times.10.sup.-5%, 1.times.10.sup.-6%,
1.times.10.sup.-7%, 1.times.10.sup.-8 of the viable cells in the
bacterial composition are human or animal, as compared to microbial
cells. There are multiple ways to accomplish this degree of purity,
none of which are limiting. Thus, contamination may be reduced by
isolating desired constituents through multiple steps of streaking
to single colonies on solid media until replicate (such as, but not
limited to, two) streaks from serial single colonies have shown
only a single colony morphology. Alternatively, reduction of
contamination can be accomplished by multiple rounds of serial
dilutions to single desired cells (e.g., a dilution of 10.sup.-8 or
10.sup.-9), such as through multiple 10-fold serial dilutions. This
can further be confirmed by showing that multiple isolated colonies
have similar cell shapes and Gram staining behavior. Other methods
for confirming adequate purity include genetic analysis (e.g. PCR,
DNA sequencing), serology and antigen analysis, enzymatic and
metabolic analysis, and methods using instrumentation such as flow
cytometry with reagents that distinguish desired constituents from
contaminants.
[0055] "Phylogenetic tree" refers to a graphical representation of
the evolutionary relationships of one genetic sequence to another
that is generated using a defined set of phylogenetic
reconstruction algorithms (e.g. parsimony, maximum likelihood, or
Bayesian). Nodes in the tree represent distinct ancestral sequences
and the confidence of any node is provided by a bootstrap or
Bayesian posterior probability, which measures branch
uncertainty.
[0056] "Operational taxonomic unit (OTU, plural OTUs)" refers to a
terminal leaf in a phylogenetic tree and is defined by a specific
genetic sequence and all sequences that share sequence identity to
this sequence at the level of species. A "type" or a plurality of
"types" of bacteria includes an OTU or a plurality of different
OTUs, and also encompasses a strain, species, genus, family or
order of bacteria. The specific genetic sequence may be the 16S
sequence or a portion of the 16S sequence or it may be a
functionally conserved housekeeping gene found broadly across the
eubacterial kingdom. OTUs share at least 95%, 96%, 97%, 98%, or 99%
sequence identity. OTUs are frequently defined by comparing
sequences between organisms. Sequences with less than 95% sequence
identity are not considered to form part of the same OTU.
[0057] "Clade" refers to the set of OTUs or members of a
phylogenetic tree downstream of a statistically valid node in a
phylogenetic tree. The clade comprises a set of terminal leaves in
the phylogenetic tree that is a distinct monophyletic evolutionary
unit.
[0058] In microbiology, "16S sequencing" or "16S rRNA" or
"165-rRNA" or "16S" refers to sequence derived by characterizing
the nucleotides that comprise the 16S ribosomal RNA gene(s). The
bacterial 16S rDNA is approximately 1500 nucleotides in length and
is used in reconstructing the evolutionary relationships and
sequence similarity of one bacterial isolate to another using
phylogenetic approaches. 16S sequences are used for phylogenetic
reconstruction as they are in general highly conserved, but contain
specific hypervariable regions that harbor sufficient nucleotide
diversity to differentiate genera and species of most bacteria, as
well as fungi.
[0059] The "V1-V9 regions" of the 16S rRNA refers to the first
through ninth hypervariable regions of the 16S rRNA gene that are
used for genetic typing of bacterial samples. These regions in
bacteria are defined by nucleotides 69-99, 137-242, 433-497,
576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465
respectively using numbering based on the E. coli system of
nomenclature. Brosius et al., Complete nucleotide sequence of a 16S
ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805
(1978). In some embodiments, at least one of the V1, V2, V3, V4,
V5, V6, V7, V8, and V9 regions are used to characterize an OTU. In
one embodiment, the V1, V2, and V3 regions are used to characterize
an OTU. In another embodiment, the V3, V4, and V5 regions are used
to characterize an OTU. In another embodiment, the V4 region is
used to characterize an OTU. A person of ordinary skill in the art
can identify the specific hypervariable regions of a candidate 16S
rRNA (in SEQ ID NOs 1-1,864) by comparing the candidate sequence in
question to the reference sequence and identifying the
hypervariable regions based on similarity to the reference
hypervariable regions.
[0060] The terms "subject" or "patient" refers to any animal
subject including humans, laboratory animals (e.g., primates, rats,
mice), livestock (e.g., cows, sheep, goats, pigs, turkeys,
chickens), and household pets (e.g., dogs, cats, rodents, etc.).
The subject or patient may be healthy, or may be suffering from an
infection due to a gastrointestinal pathogen or may be at risk of
developing or transmitting to others an infection due to a
gastrointestinal pathogen.
[0061] The term "pathobiont" refer to specific bacterial species
found in healthy hosts that may trigger immune-mediated pathology
and/or disease in response to certain genetic or environmental
factors. Chow et al., (2011) Curr Op Immunol. Pathobionts of the
intestinal microbiota and inflammatory disease. 23: 473-80. Thus, a
pathobiont is a pathogen that is mechanistically distinct from an
acquired infectious organism. Thus, the term "pathogen" includes
both acquired infectious organisms and pathobionts.
DETAILED DESCRIPTION
Bacterial Compositions
[0062] Provided are bacteria and combinations of bacteria of the
human gut microbiota with the capacity to meaningfully provide
functions of a healthy microbiota or catalyze an augmentation to
the resident microbiome when administered to mammalian hosts. In
particular, provided are synergistic combinations that treat,
prevent, delay or reduce the symptoms of diseases, disorders and
conditions associated with a dysbiosis. Representative diseases,
disorders and conditions potentially associated with a dysbiosis,
which are suitable for treatment with the compositions and methods
as described herein, are provided in Tables 8 and 9. Without being
limited to a specific mechanism, it is thought that such
compositions inhibit the growth, proliferation, and/or colonization
of one or a plurality of pathogenic bacteria in the dysbiotic
microbiotal niche, so that a healthy, diverse and protective
microbiota colonizes and populates the intestinal lumen to
establish or reestablish ecological control over pathogens or
potential pathogens (e.g., some bacteria are pathogenic bacteria
only when present in a dysbiotic environment). Inhibition of
pathogens includes those pathogens such as C. difficile, Salmonella
spp., enteropathogenic E coli, multi-drug resistant bacteria such
as Klebsiella, and E. coli, Carbapenem-resistent Enterobacteriaceae
(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL),
and vancomycin-resistant Enterococci (VRE).
[0063] The bacterial compositions provided herein are produced and
the efficacy thereof in inhibiting pathogenic bacteria is
demonstrated as provided in further detail herein.
[0064] In particular, in order to characterize those antagonistic
relationships between gut commensals that are relevant to the
dynamics of the mammalian gut habitat, provided is an in vitro
microplate-based screening system that demonstrates the efficacy of
those bacterial compositions, including the ability to inhibit (or
antagonize) the growth of a bacterial pathogen or pathobiont,
typically a gastrointestinal microorganism. These methods provide
novel combinations of gut microbiota species and OTUs that are able
to restore or enhance ecological control over important gut
pathogens or pathobionts in vivo.
[0065] Bacterial compositions may comprise two types of bacteria
(termed "binary combinations" or "binary pairs") or greater than
two types of bacteria. Bacterial compositions that comprise three
types of bacteria are termed "ternary combinations". For instance,
a bacterial composition may comprise at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, or at least 40, at least 50 or
greater than 50 types of bacteria, as defined by species or
operational taxonomic unit (OTU), or otherwise as provided herein.
In one embodiment, the composition comprises at least two types of
bacteria chosen from Table 1.
[0066] In another embodiment, the number of types of bacteria
present in a bacterial composition is at or below a known value.
For example, in such embodiments the bacterial composition
comprises 50 or fewer types of bacteria, such as 49, 48, 47, 46,
45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29,
28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, or 10 or fewer, or 9 or fewer types of bacteria, 8 or fewer
types of bacteria, 7 or fewer types of bacteria, 6 or fewer types
of bacteria, 5 or fewer types of bacteria, 4 or fewer types of
bacteria, or 3 or fewer types of bacteria. In another embodiment, a
bacterial composition comprises from 2 to no more than 40, from 2
to no more than 30, from 2 to no more than 20, from 2 to no more
than 15, from 2 to no more than 10, or from 2 to no more than 5
types of bacteria.
[0067] In some embodiments, bacterial compositions are provided
with the ability to exclude pathogenic bacteria. Exemplary
bacterial compositions are demonstrated to reduce the growth rate
of one pathogen, C. difficile, as provided in the Examples, wherein
the ability of the bacterial compositions is demonstrated by
assessing the antagonism activity of a combination of OTUs or
strains towards a given pathogen using in vitro assays.
[0068] In some embodiments, bacterial compositions with the
capacity to durably exclude C. difficile, are developed using a
methodology for estimating an Ecological Control Factor (ECF) for
constituents within the human microbiota. The ECF is determined by
assessing the antagonistic activity of a given commensal strain or
combination of strains towards a given pathogen using an in vitro
assay, resulting in observed levels of ecological control at
various concentrations of the added commensal strains. The ECF for
a commensal strain or combination of strains is somewhat analogous
to the longstanding minimal inhibitory concentration (MIC)
assessment that is employed in the assessment of antibiotics. The
ECF allows for the assessment and ranking of relative potencies of
commensal strains and combinations of strains for their ability to
antagonize gastrointestinal pathogens. The ECF of a commensal
strain or combination of strains may be calculated by assessing the
concentration of that composition that is able to mediate a given
percentage of inhibition (e.g., at least 10%, 20%, 50%, 70%, 75%,
80%, 85%, 90%, 95%, or 100%) of a target pathogen in the in vitro
assay. Provided herein are combinations of strains or OTUs within
the human microbiota that are able to significantly reduce the rate
of gastrointestinal pathogen replication within the in vitro assay.
These compositions are capable of providing a safe and effective
means by which to affect the growth, replication, and disease
severity of such bacterial pathogens.
[0069] Bacterial compositions may be prepared comprising at least
two types of isolated bacteria, wherein a first type and a second
type are independently chosen from the species or OTUs listed in
Table 1 and SEQ ID NOs. 1-1,864. Certain embodiments of bacterial
compositions with at least two types of isolated bacteria
containing binary pairs are reflected in Table 2. Additionally, a
bacterial composition may be prepared comprising at least two types
of isolated bacteria, wherein a first OTU and a second OTU are
independently characterized by, i.e., at least 95%, 96%, 97%, 98%,
99% or including 100% sequence identity to, sequences listed in SEQ
ID NOs. 1-1,864. Generally, the first bacteria and the second
bacteria are not the same OTU. The sequences provided in SEQ ID
NOs. 1-1,864 are full 16S sequences. Therefore, in one embodiment,
the first and/or second OTUs may be characterized by the full 16S
sequences listed in SEQ ID NOs. 1-1,864. In another embodiment, the
first and/or second OTUs may be characterized by one or more of the
variable regions of the 16S sequence (V1-V9). These regions in
bacteria are defined by nucleotides 69-99, 137-242, 433-497,
576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465
respectively using numbering based on the E. coli system of
nomenclature. (See, e.g., Brosius et al., Complete nucleotide
sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS
75(10):4801-4805 (1978)). In some embodiments, at least one of the
V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to
characterize an OTU. In one embodiment, the V1, V2, and V3 regions
are used to characterize an OTU. In another embodiment, the V3, V4,
and V5 regions are used to characterize an OTU. In another
embodiment, the V4 region is used to characterize an OTU.
[0070] Methods for Determining 16S Sequence. OTUs may be defined
either by full 16S sequencing of the rRNA gene, by sequencing of a
specific hypervariable region of this gene (i.e. V1, V2, V3, V4,
V5, V6, V7, V8, or V9), or by sequencing of any combination of
hypervariable regions from this gene (e.g. V1-3 or V3-5). The
bacterial 16S rDNA is approximately 1500 nucleotides in length and
is used in reconstructing the evolutionary relationships and
sequence similarity of one bacterial isolate to another using
phylogenetic approaches. 16S sequences are used for phylogenetic
reconstruction as they are in general highly conserved, but contain
specific hypervariable regions that harbor sufficient nucleotide
diversity to differentiate genera and species of most microbes.
Using well known techniques, in order to determine the full 16S
sequence or the sequence of any hypervariable region of the 16S
sequence, genomic DNA is extracted from a bacterial sample, the 16S
rDNA (full region or specific hypervariable regions) amplified
using polymerase chain reaction (PCR), the PCR products cleaned,
and nucleotide sequences delineated to determine the genetic
composition of 16S gene or subdomain of the gene. If full 16S
sequencing is performed, the sequencing method used may be, but is
not limited to, Sanger sequencing. If one or more hypervariable
regions are used, such as the V4 region, the sequencing may be, but
is not limited to being, performed using the Sanger method or using
a next-generation sequencing method, such as an Illumina
(sequencing by synthesis) method using barcoded primers allowing
for multiplex reactions.
[0071] Bacterial Compositions exclusive of certain bacterial
species or strains. In one embodiment, the bacterial composition
does not comprise at least one of Enterococcus faecalis (previously
known as Streptococcus faecalis), Clostridium innocuum, Clostridium
ramosum, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides
thetaoiotaomicron, Escherichia coli (1109 and 1108-1), Clostridum
bifermentans, and Blautia producta (previously known as
Peptostreptococcus productus).
[0072] In another embodiment, the bacterial composition does not
comprise at least one of Acidaminococcus intestinalis, Bacteroides
ovatus, two species of Bifidobacterium adolescentis, two species of
Bifidobacterium longum, Collinsella aerofaciens, two species of
Dorea longicatena, Escherichia coli, Eubacterium eligens,
Eubacterium limosum, four species of Eubacterium rectale,
Eubacterium ventriosumi, Faecalibacterium prausnitzii,
Lactobacillus casei, Lactobacillus paracasei, Paracateroides
distasonis, Raoultella sp., one species of Roseburia (chosen from
Roseburia faecalis or Roseburia faecis), Roseburia intestinalis,
two species of Ruminococcus torques, and Streptococcus mitis.
[0073] In another embodiment, the bacterial composition does not
comprise at least one of Bamesiella intestinihominis; Lactobacillus
reuteri; a species characterized as one of Enterococcus hirae,
Enterococus faecium, or Enterococcus durans; a species
characterized as one of Anaerostipes caccae or Clostridium indolis;
a species characterized as one of Staphylococcus warneri or
Staphylococcus pasteuri; and Adlercreutzia equolifaciens.
[0074] In another embodiment, the bacterial composition does not
comprise at least one of Clostridium absonum, Clostridium
argentinense, Clostridium baratii, Clostridium bifermentans,
Clostridium botulinum, Clostridium butyricum, Clostridium
cadaveris, Clostridium camis, Clostridium celatum, Clostridium
chauvoei, Clostridium clostridioforme, Clostridium cochlearium,
Clostridium difficile, Clostridium fallax, Clostridium felsineum,
Clostridium ghonii, Clostridium glycolicum, Clostridium
haemolyticum, Clostridium hastiforme, Clostridium histolyticum,
Clostridium indolis, Clostridium innocuum, Clostridium irregulare,
Clostridium limosum, Clostridium malenominatum, Clostridium novyi,
Clostridium oroticum, Clostridium paraputrificum, Clostridium
perfringens, Clostridium piliforme, Clostridium putrefaciens,
Clostridium putrificum, Clostridium ramosum, Clostridium
sardiniense, Clostridium sartagoforme, Clostridium scindens,
Clostridium septicum, Clostridium sordellii, Clostridium
sphenoides, Clostridium spiroforme, Clostridium sporogenes,
Clostridium subterminale, Clostridium symbiosum, Clostridium
tedium, Clostridium tetani, Clostridium welchii, and Clostridium
villosum.
[0075] In another embodiment, the bacterial composition does not
comprise at least one of Clostridium innocuum, Clostridum
bifermentans, Clostridium butyricum, Bacteroides fragilis,
Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains
of Escherichia coli, and Lactobacillus sp.
[0076] In another embodiment, the bacterial composition does not
comprise at least one of Clostridium bifermentans, Clostridium
innocuum, Clostridium butyricum, three strains of Escherichia coli,
three strains of Bacteroides, and Blautia producta (previously
known as Peptostreptococcus productus).
[0077] In another embodiment, the bacterial composition does not
comprise at least one of Bacteroides sp., Escherichia coli, and non
pathogenic Clostridia, including Clostridium innocuum, Clostridium
bifermentans and Clostridium ramosum.
[0078] In another embodiment, the bacterial composition does not
comprise at least one of more than one Bacteroides species,
Escherichia coli and non-pathogenic Clostridia, such as Clostridium
butyricum, Clostridium bifermentans and Clostridium innocuum.
[0079] In another embodiment, the bacterial composition does not
comprise at least one of Bacteroides caccae, Bacteroides
capillosus, Bacteroides coagulans, Bacteroides distasonis,
Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis,
Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii,
Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus,
Bacteroides pneumosintes, Bacteroides putredinis, Bacteroides
pyogenes, Bacteroides splanchnicus, Bacteroides stercoris,
Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides
uniformis, Bacteroides ureolyticus, and Bacteroides vulgatus.
[0080] In another embodiment, the bacterial composition does not
comprise at least one of Bacteroides, Eubacteria, Fusobacteria,
Propionibacteria, Lactobacilli, anaerobic cocci, Ruminococcus,
Escherichia coli, Gemmiger, Desulfomonas, and
Peptostreptococcus.
[0081] In another embodiment, the bacterial composition does not
comprise at least one of Bacteroides fragilis ss. Vulgatus,
Eubacterium aerofaciens, Bacteroides fragilis ss. Thetaiotaomicron,
Blautia producta (previously known as Peptostreptococcus productus
II), Bacteroides fragilis ss. Distasonis, Fusobacterium
prausnitzii, Coprococcus eutactus, Eubacterium aerofaciens III,
Blautia producta (previously known as Peptostreptococcus productus
I), Ruminococcus bromii, Bifidobacterium adolescentis, Gemmiger
formicilis, Bifidobacterium longum, Eubacterium siraeum,
Ruminococcus torques, Eubacterium rectale III-H, Eubacterium
rectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridium
leptum, Bacteroides fragilis ss. A, Eubacterium biforme,
Bifidobacterium infantis, Eubacterium rectale III-F, Coprococcus
comes, Bacteroides capillosus, Ruminococcus albus, Eubacterium
formicigenerans, Eubacterium hallii, Eubacterium ventriosum I,
Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II,
Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus
cailidus, Butyrivibrio crossotus, Acidaminococcus fermentans,
Eubacterium ventriosum, Bacteroides fragilis ss. fragilis,
Bacteroides AR, Coprococcus catus, Eubacterium hadrum, Eubacterium
cylindroides, Eubacterium ruminantium, Eubacterium CH-1,
Staphylococcus epidermidis, Peptostreptococcus BL,Eubacterium
limosum, Bacteroides praeacutus, Bacteroides L, Fusobacterium
mortiferum I, Fusobacterium naviforme, Clostridium innocuum,
Clostridium ramosum, Propionibacterium acnes, Ruminococcus
flavefaciens, Ruminococcus AT, Peptococcus AU-1, Eubacterium AG,
-AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus, -ss. d, -ss.
f, Bacteroides L-1, L-5; Fusobacterium nucleatum, Fusobacterium
mortiferum, Escherichia coli, Streptococcus morbiliorum,
Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius,
Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH,
-CC; Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H,
-AG-M, -AJ, -BN-1; Bacteroides clostridiiformis ss.
clostridiiformis, Bacteroides coagulans, Bacteroides orails,
Bacteroides ruminicola ss. brevis, -ss. ruminicola, Bacteroides
splanchnicus, Desuifomonas pigra, Bacteroides L-4, -N-i;
Fusobacterium H, Lactobacillus G, and Succinivibrio A.
[0082] Inhibition of Bacterial Pathogens. The bacterial
compositions offer a protective or therapeutic effect against
infection by one or more GI pathogens of interest.
[0083] A list of exemplary bacterial pathogens is provided in Table
5.
[0084] In some embodiments, the pathogenic bacterium is selected
from the group consisting of Yersinia, Vibrio, Treponema,
Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia,
Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium,
Listeria, Leptospira, Legionella, Klebsiella, Helicobacter,
Haemophilus, Francisella, Escherichia, Ehrlichia, Enterococcus,
Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila,
Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifido
bacterium, Bacillus, multi-drug resistant bacteria, extended
spectrum beta-lactam resistant Enterococci (ESBL),
Carbapenem-resistent Enterobacteriaceae (CRE), and
vancomycin-resistant Enterococci (VRE).
[0085] In some embodiments, these pathogens include, but are not
limited to, Aeromonas hydrophila, Campylobacter fetus, Plesiomonas
shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium
botulinum, Clostridium difficile, Clostridium perfringens,
enteroaggregative Escherichia coli, enterohemorrhagic Escherichia
coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia
coli (such as, but not limited to, LT and/or ST), Escherichia coli
0157:H7, Helicobacter pylori, Klebsiellia pneumonia, Lysteria
monocytogenes, Plesiomonas shigelloides, Salmonella spp.,
Salmonella typhi, Salmonella paratyphi, Shigella spp.,
Staphylococcus spp., Staphylococcus aureus, vancomycin-resistant
enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio
parahaemolyticus, Vibrio vulnificus, and Yersinia
enterocolitica.
[0086] In one embodiment, the pathogen of interest is at least one
pathogen chosen from Clostridium difficile, Salmonella spp.,
pathogenic Escherichia coli, vancomycin-resistant Enterococcus
spp., and extended spectrum beta-lactam resistant Enterococci
(ESBL).
[0087] In Vitro Assays Substantiating Protective Effect of
Bacterial Compositions.
[0088] In one embodiment, provided is an In Vitro Assay utilizing
competition between the bacterial compositions or subsets thereof
and C. difficile. Exemplary embodiments of this Assay are provided
herein and in the Examples.
[0089] In another embodiment, provided is an In Vitro Assay
utilizing 10% (wt/vol) Sterile-Filtered Feces. Provided is an in
vitro assay to test for the protective effect of the bacterial
compositions and to screen in vitro for combinations of microbes
that inhibit the growth of a pathogen. The assay can operate in
automated high-throughput or manual modes. Under either system,
human or animal feces may be re-suspended in an anaerobic buffer
solution, such as pre-reduced PBS or other suitable buffer, the
particulate removed by centrifugation, and filter sterilized. This
10% sterile-filtered feces material serves as the base media for
the in vitro assay. To test a bacterial composition, an
investigator may add it to the sterile-filtered feces material for
a first incubation period and then may inoculate the incubated
microbial solution with the pathogen of interest for a second
incubation period. The resulting titer of the pathogen may be
quantified by any number of methods such as those described below,
and the change in the amount of pathogen is compared to standard
controls including the pathogen cultivated in the absence of the
bacterial composition. The assay is conducted using at least one
control. Feces from a healthy subject may be used as a positive
control. As a negative control, antibiotic-treated feces or
heat-treated feces may be used. Various bacterial compositions may
be tested in this material and the bacterial compositions
optionally compared to the positive and/or negative controls. The
ability to inhibit the growth of the pathogen may be measured by
plating the incubated material on C. difficile selective media and
counting colonies. After competition between the bacterial
composition and C. difficile, each well of the in vitro assay plate
is serially diluted ten-fold six times, and plated on selective
media, such as but not limited to cycloserine cefoxitin mannitol
agar (COMA) or cycloserine cefoxitin fructose agar (CCFA), and
incubated. Colonies of C. difficile are then counted to calculate
the concentration of viable cells in each well at the end of the
competition. Colonies of C. difficile are confirmed by their
characteristic diffuse colony edge morphology as well as
fluorescence under UV light.
[0090] In another embodiment, the in vitro assay utilizes
Antibiotic-Treated Feces. In an alternative embodiment, and instead
of using 10% sterile-filtered feces, human or animal feces may be
resuspended in an anaerobic buffer solution, such as pre-reduced
PBS or other suitable buffer. The resuspended feces is treated with
an antibiotic, such as clindamycin, or a cocktail of several
antibiotics in order to reduce the ability of feces from a healthy
subject to inhibit the growth of C. difficile; this material is
termed the antibiotic-treated matrix. While not being bound by any
mechanism, it is believed that beneficial bacteria in healthy
subjects protects them from infection by competing out C.
difficile. Treating feces with antibiotics kills or reduces the
population of those beneficial bacteria, allowing C. difficile to
grow in this assay matrix. Antibiotics in addition to clindamycin
that inhibit the normal flora include ceftriaxone and
piperacillin-tazobactam and may be substituted for the clindamycin.
The antibiotic-treated matrix is centrifuged, the supernatant
removed, and the pelleted material resuspended in
filter-sterilized, diluted feces in order to remove any residual
antibiotic. This washed antibiotic-treated matrix may be used in
the in vitro assay described above in lieu of the 10%
sterile-filtered feces.
[0091] Alternatively, the ability to inhibit the growth of the
pathogen may be measured by quantitative PCR (qPCR). Standard
techniques may be followed to generate a standard curve for the
pathogen of interest. Genomic DNA may be extracted from samples
using commercially-available kits, such as the Mo Bio
Powersoil.RTM.-htp 96 Well Soil DNA Isolation Kit (Mo Bio
Laboratories, Carlsbad, Calif.), the Mo Bio Powersoil.RTM. DNA
Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the
QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to
the manufacturer's instructions. The qPCR may be conducted using
HotMasterMix (5PRIME, Gaithersburg, Md.) and primers specific for
the pathogen of interest, and may be conducted on a MicroAmp.RTM.
Fast Optical 96-well Reaction Plate with Barcode (0.1mL) (Life
Technologies, Grand Island, N.Y.) and performed on a BioRad
C1000.TM. Thermal Cycler equipped with a CFX96.TM. Real-Time System
(BioRad, Hercules, Calif.), with fluorescent readings of the FAM
and ROX channels. The Cq value for each well on the FAM channel is
determined by the CFX Manager.TM. software version 2.1. The
log.sub.10(cfu/ml) of each experimental sample is calculated by
inputting a given sample's Cq value into linear regression model
generated from the standard curve comparing the Cq values of the
standard curve wells to the known log.sub.10(cfu/m1) of those
samples. The skilled artisan may employ alternative qPCR modes.
[0092] Also provided are In Vivo Assay Establishing Protective
Effect of Bacterial Compositions. Provided is an in vivo mouse
model to test for the protective effect of the bacterial
compositions against C. difficile. In this model (based on Chen, et
al., A mouse model of Clostridium difficile associated disease,
Gastroenterology 135(6):1984-1992 (2008)), mice are made
susceptible to C. difficile by a 7 day treatment (days -12 to -5 of
experiment) with 5 to 7 antibiotics (including kanamycin, colistin,
gentamycin, metronidazole and vancomycin and optionally including
ampicillin and ciprofloxacin) delivered via their drinking water,
followed by a single dose with Clindamycin on day -3, then
challenged three days later on day 0 with 10.sup.4 spores of C.
difficile via oral gavage (i.e., oro-gastric lavage). Bacterial
compositions may be given either before (prophylactic treatment) or
after (therapeutic treatment) C. difficile gavage. Further,
bacterial compositions may be given after (optional) vancomycin
treatment (see below) to assess their ability to prevent recurrence
and thus suppress the pathogen in vivo. The outcomes assessed each
day from day -1 to day 6 (or beyond, for prevention of recurrence)
are weight, clinical signs, mortality and shedding of C. difficile
in the feces. Weight loss, clinical signs of disease, and C.
difficile shedding are typically observed without treatment.
Vancomycin provided by oral gavage on days -1 to 4 protects against
these outcomes and serves as a positive control. Clinical signs are
subjective, and scored each day by the same experienced observer.
Animals that lose greater than or equal to 25% of their body weight
are euthanized and counted as infection-related mortalities. Feces
are gathered from mouse cages (5 mice per cage) each day, and the
shedding of C. difficile spores is detected in the feces using a
selective plating assay as described for the in vitro assay above,
or via qPCR for the toxin gene as described herein. The effects of
test materials including 10% suspension of human feces (as a
positive control), bacterial compositions, or PBS (as a negative
vehicle control), are determined by introducing the test article in
a 0.2 mL volume into the mice via oral gavage on day -1, one day
prior to C. difficile challenge, on day 1, 2 and 3 as treatment or
post-vancomycin treatment on days 5, 6, 7 and 8. Vancomycin, as
discussed above, is given on days 1 to 4 as another positive
control. Alternative dosing schedules and routes of administration
(e.g. rectal) may be employed, including multiple doses of test
article, and 10.sup.3 to 10.sup.10 of a given organism or
composition may be delivered.
[0093] Methods for Preparing a Bacterial Composition for
Administration to a Subject.
[0094] Methods for producing bacterial compositions may include
three main processing steps, combined with one or more mixing
steps. The steps are: organism banking, organism production, and
preservation.
[0095] For banking, the strains included in the bacterial
composition may be (1) isolated directly from a specimen or taken
from a banked stock, (2) optionally cultured on a nutrient agar or
broth that supports growth to generate viable biomass, and (3) the
biomass optionally preserved in multiple aliquots in long-term
storage.
[0096] In embodiments using a culturing step, the agar or broth may
contain nutrients that provide essential elements and specific
factors that enable growth. An example would be a medium composed
of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L
citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric
ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin
chloride, 2 mg/L calcium chloride, 1 mg/L menadione. A variety of
microbiological media and variations are well known in the art
(e.g. R. M. Atlas, Handbook of Microbiological Media (2010) CRC
Press). Medium can be added to the culture at the start, may be
added during the culture, or may be intermittently/continuously
flowed through the culture. The strains in the bacterial
composition may be cultivated alone, as a subset of the bacterial
composition, or as an entire collection comprising the bacterial
composition. As an example, a first strain may be cultivated
together with a second strain in a mixed continuous culture, at a
dilution rate lower than the maximum growth rate of either cell to
prevent the culture from washing out of the cultivation.
[0097] The inoculated culture is incubated under favorable
conditions for a time sufficient to build biomass. For bacterial
compositions for human use this is often at 37.degree. C.
temperature, pH, and other parameter with values similar to the
normal human niche. The environment may be actively controlled,
passively controlled (e.g., via buffers), or allowed to drift. For
example, for anaerobic bacterial compositions (e.g., gut
microbiota), an anoxic/reducing environment may be employed. This
can be accomplished by addition of reducing agents such as cysteine
to the broth, and/or stripping it of oxygen. As an example, a
culture of a bacterial composition may be grown at 37.degree. C.,
pH 7, in the medium above, pre-reduced with 1 g/L cysteineHCl.
[0098] When the culture has generated sufficient biomass, it may be
preserved for banking. The organisms may be placed into a chemical
milieu that protects from freezing (adding `cryoprotectants`),
drying (`lyoprotectants`), and/or osmotic shock
(`osmoprotectants`), dispensing into multiple (optionally
identical) containers to create a uniform bank, and then treating
the culture for preservation.
[0099] Containers are generally impermeable and have closures that
assure isolation from the environment. Cryopreservation treatment
is accomplished by freezing a liquid at ultra-low temperatures
(e.g., at or below -80.degree. C.). Dried preservation removes
water from the culture by evaporation (in the case of spray drying
or `cool drying`) or by sublimation (e.g., for freeze drying, spray
freeze drying). Removal of water improves long-term bacterial
composition storage stability at temperatures elevated above
cryogenic. If the bacterial composition comprises spore forming
species and results in the production of spores, the final
composition may be purified by additional means such as density
gradient centrifugation preserved using the techniques described
above. Bacterial composition banking may be done by culturing and
preserving the strains individually, or by mixing the strains
together to create a combined bank. As an example of
cryopreservation, a bacterial composition culture may be harvested
by centrifugation to pellet the cells from the culture medium, the
supernate decanted and replaced with fresh culture broth containing
15% glycerol. The culture can then be aliquoted into 1 mL
cryotubes, sealed, and placed at -80.degree. C. for long-term
viability retention. This procedure achieves acceptable viability
upon recovery from frozen storage.
[0100] Organism production may be conducted using similar culture
steps to banking, including medium composition and culture
conditions. It may be conducted at larger scales of operation,
especially for clinical development or commercial production. At
larger scales, there may be several subcultivations of the
bacterial composition prior to the final cultivation. At the end of
cultivation, the culture is harvested to enable further formulation
into a dosage form for administration. This can involve
concentration, removal of undesirable medium components, and/or
introduction into a chemical milieu that preserves the bacterial
composition and renders it acceptable for administration via the
chosen route. For example, a bacterial composition may be
cultivated to a concentration of 10.sup.10 CFU/mL, then
concentrated 20-fold by tangential flow microfiltration; the spent
medium may be exchanged by diafiltering with a preservative medium
consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium
phosphate buffer. The suspension can then be freeze-dried to a
powder and titrated.
[0101] After drying, the powder may be blended to an appropriate
potency, and mixed with other cultures and/or a filler such as
microcrystalline cellulose for consistency and ease of handling,
and the bacterial composition formulated as provided herein.
[0102] Formulations. Provided are formulations for administration
to humans and other subjects in need thereof. Generally the
bacterial compositions are combined with additional active and/or
inactive materials in order to produce a final product, which may
be in single dosage unit or in a multi-dose format.
[0103] In some embodiments the composition comprises at least one
carbohydrate. A "carbohydrate" refers to a sugar or polymer of
sugars. The terms "saccharide," "polysaccharide," "carbohydrate,"
and "oligosaccharide" may be used interchangeably. Most
carbohydrates are aldehydes or ketones with many hydroxyl groups,
usually one on each carbon atom of the molecule. Carbohydrates
generally have the molecular formula C.sub.nH.sub.2nO.sub.n. A
carbohydrate may be a monosaccharide, a disaccharide,
trisaccharide, oligosaccharide, or polysaccharide. The most basic
carbohydrate is a monosaccharide, such as glucose, sucrose,
galactose, mannose, ribose, arabinose, xylose, and fructose.
Disaccharides are two joined monosaccharides. Exemplary
disaccharides include sucrose, maltose, cellobiose, and lactose.
Typically, an oligosaccharide includes between three and six
monosaccharide units (e.g., raffinose, stachyose), and
polysaccharides include six or more monosaccharide units. Exemplary
polysaccharides include starch, glycogen, and cellulose.
Carbohydrates may contain modified saccharide units such as
2'-deoxyribose wherein a hydroxyl group is removed, 2'-fluororibose
wherein a hydroxyl group is replace with a fluorine, or
N-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,
2'-fluororibose, deoxyribose, and hexose). Carbohydrates may exist
in many different forms, for example, conformers, cyclic forms,
acyclic forms, stereoisomers, tautomers, anomers, and isomers.
[0104] In some embodiments the composition comprises at least one
lipid. As used herein a "lipid" includes fats, oils, triglycerides,
cholesterol, phospholipids, fatty acids in any form including free
fatty acids. Fats, oils and fatty acids can be saturated,
unsaturated (cis or trans) or partially unsaturated (cis or trans).
In some embodiments the lipid comprises at least one fatty acid
selected from lauric acid (12:0), myristic acid (14:0), palmitic
acid (16:0), palmitoleic acid (16:1), margaric acid (17:0),
heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1),
linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic
acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1),
eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),
eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0),
docosenoic acid (22:1), docosapentaenoic acid (22:5),
docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0).
In some embodiments the composition comprises at least one modified
lipid, for example a lipid that has been modified by cooking.
[0105] In some embodiments the composition comprises at least one
supplemental mineral or mineral source. Examples of minerals
include, without limitation: chloride, sodium, calcium, iron,
chromium, copper, iodine, zinc, magnesium, manganese, molybdenum,
phosphorus, potassium, and selenium. Suitable forms of any of the
foregoing minerals include soluble mineral salts, slightly soluble
mineral salts, insoluble mineral salts, chelated minerals, mineral
complexes, non-reactive minerals such as carbonyl minerals, and
reduced minerals, and combinations thereof.
[0106] In some embodiments the composition comprises at least one
supplemental vitamin. The at least one vitamin can be fat-soluble
or water soluble vitamins. Suitable vitamins include but are not
limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K,
riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine,
thiamine, pantothenic acid, and biotin. Suitable forms of any of
the foregoing are salts of the vitamin, derivatives of the vitamin,
compounds having the same or similar activity of the vitamin, and
metabolites of the vitamin.
[0107] In some embodiments the composition comprises an excipient.
Non-limiting examples of suitable excipients include a buffering
agent, a preservative, a stabilizer, a binder, a compaction agent,
a lubricant, a dispersion enhancer, a disintegration agent, a
flavoring agent, a sweetener, and a coloring agent.
[0108] In some embodiments the excipient is a buffering agent.
Non-limiting examples of suitable buffering agents include sodium
citrate, magnesium carbonate, magnesium bicarbonate, calcium
carbonate, and calcium bicarbonate.
[0109] In some embodiments the excipient comprises a preservative.
Non-limiting examples of suitable preservatives include
antioxidants, such as alpha-tocopherol and ascorbate, and
antimicrobials, such as parabens, chlorobutanol, and phenol.
[0110] In some embodiments the composition comprises a binder as an
excipient. Non-limiting examples of suitable binders include
starches, pregelatinized starches, gelatin, polyvinylpyrolidone,
cellulose, methylcellulose, sodium carboxymethylcellulose,
ethylcellulose, polyacrylamides, polyvinyloxoazolidone,
polyvinylalcohols, C.sub.12-C.sub.18 fatty acid alcohol,
polyethylene glycol, polyols, saccharides, oligosaccharides, and
combinations thereof.
[0111] In some embodiments the composition comprises a lubricant as
an excipient. Non-limiting examples of suitable lubricants include
magnesium stearate, calcium stearate, zinc stearate, hydrogenated
vegetable oils, sterotex, polyoxyethylene monostearate, talc,
polyethyleneglycol, sodium benzoate, sodium lauryl sulfate,
magnesium lauryl sulfate, and light mineral oil.
[0112] In some embodiments the composition comprises a dispersion
enhancer as an excipient. Non-limiting examples of suitable
dispersants include starch, alginic acid, polyvinylpyrrolidones,
guar gum, kaolin, bentonite, purified wood cellulose, sodium starch
glycolate, isoamorphous silicate, and microcrystalline cellulose as
high HLB emulsifier surfactants.
[0113] In some embodiments the composition comprises a disintegrant
as an excipient. In some embodiments the disintegrant is a
non-effervescent disintegrant. Non-limiting examples of suitable
non-effervescent disintegrants include starches such as corn
starch, potato starch, pregelatinized and modified starches
thereof, sweeteners, clays, such as bentonite, micro-crystalline
cellulose, alginates, sodium starch glycolate, gums such as agar,
guar, locust bean, karaya, pecitin, and tragacanth. In some
embodiments the disintegrant is an effervescent disintegrant.
Non-limiting examples of suitable effervescent disintegrants
include sodium bicarbonate in combination with citric acid, and
sodium bicarbonate in combination with tartaric acid.
[0114] In some embodiments the excipient comprises a flavoring
agent. Flavoring agents can be chosen from synthetic flavor oils
and flavoring aromatics; natural oils; extracts from plants,
leaves, flowers, and fruits; and combinations thereof. In some
embodiments the flavoring agent is selected from cinnamon oils; oil
of wintergreen; peppermint oils; clover oil; hay oil; anise oil;
eucalyptus; vanilla; citrus oil such as lemon oil, orange oil,
grape and grapefruit oil; and fruit essences including apple,
peach, pear, strawberry, raspberry, cherry, plum, pineapple, and
apricot.
[0115] In some embodiments the excipient comprises a sweetener.
Non-limiting examples of suitable sweeteners include glucose (corn
syrup), dextrose, invert sugar, fructose, and mixtures thereof
(when not used as a carrier); saccharin and its various salts such
as the sodium salt; dipeptide sweeteners such as aspartame;
dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana
(Stevioside); chloro derivatives of sucrose such as sucralose; and
sugar alcohols such as sorbitol, mannitol, sylitol, and the like.
Also contemplated are hydrogenated starch hydrolysates and the
synthetic sweetener
3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide,
particularly the potassium salt (acesulfame-K), and sodium and
calcium salts thereof.
[0116] In some embodiments the composition comprises a coloring
agent. Non-limiting examples of suitable color agents include food,
drug and cosmetic colors (FD&C), drug and cosmetic colors
(D&C), and external drug and cosmetic colors (Ext. D&C).
The coloring agents can be used as dyes or their corresponding
lakes.
[0117] The weight fraction of the excipient or combination of
excipients in the formulation is usually about 99% or less, such as
about 95% or less, about 90% or less, about 85% or less, about 80%
or less, about 75% or less, about 70% or less, about 65% or less,
about 60% or less, about 55% or less, 50% or less, about 45% or
less, about 40% or less, about 35% or less, about 30% or less,
about 25% or less, about 20% or less, about 15% or less, about 10%
or less, about 5% or less, about 2% or less, or about 1% or less of
the total weight of the composition.
[0118] The bacterial compositions disclosed herein can be
formulated into a variety of forms and administered by a number of
different means. The compositions can be administered orally,
rectally, or parenterally, in formulations containing
conventionally acceptable carriers, adjuvants, and vehicles as
desired. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, or intrasternal injection
and infusion techniques. In an exemplary embodiment, the bacterial
composition is administered orally.
[0119] Solid dosage forms for oral administration include capsules,
tablets, caplets, pills, troches, lozenges, powders, and granules.
A capsule typically comprises a core material comprising a
bacterial composition and a shell wall that encapsulates the core
material. In some embodiments the core material comprises at least
one of a solid, a liquid, and an emulsion. In some embodiments the
shell wall material comprises at least one of a soft gelatin, a
hard gelatin, and a polymer. Suitable polymers include, but are not
limited to: cellulosic polymers such as hydroxypropyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC),
methyl cellulose, ethyl cellulose, cellulose acetate, cellulose
acetate phthalate, cellulose acetate trimellitate,
hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl
cellulose succinate and carboxymethylcellulose sodium; acrylic acid
polymers and copolymers, such as those formed from acrylic acid,
methacrylic acid, methyl acrylate, ammonio methylacrylate, ethyl
acrylate, methyl methacrylate and/or ethyl methacrylate (e.g.,
those copolymers sold under the trade name "Eudragit"); vinyl
polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl
acetate, polyvinylacetate phthalate, vinylacetate crotonic acid
copolymer, and ethylene-vinyl acetate copolymers; and shellac
(purified lac). In some embodiments at least one polymer functions
as taste-masking agents.
[0120] Tablets, pills, and the like can be compressed, multiply
compressed, multiply layered, and/or coated. The coating can be
single or multiple. In one embodiment, the coating material
comprises at least one of a saccharide, a polysaccharide, and
glycoproteins extracted from at least one of a plant, a fungus, and
a microbe. Non-limiting examples include corn starch, wheat starch,
potato starch, tapioca starch, cellulose, hemicellulose, dextrans,
maltodextrin, cyclodextrins, inulins, pectin, mannans, gum arabic,
locust bean gum, mesquite gum, guar gum, gum karaya, gum ghatti,
tragacanth gum, funori, carrageenans, agar, alginates, chitosans,
or gellan gum. In some embodiments the coating material comprises a
protein. In some embodiments the coating material comprises at
least one of a fat and an oil. In some embodiments the at least one
of a fat and an oil is high temperature melting. In some
embodiments the at least one of a fat and an oil is hydrogenated or
partially hydrogenated. In some embodiments the at least one of a
fat and an oil is derived from a plant. In some embodiments the at
least one of a fat and an oil comprises at least one of glycerides,
free fatty acids, and fatty acid esters. In some embodiments the
coating material comprises at least one edible wax. The edible wax
can be derived from animals, insects, or plants. Non-limiting
examples include beeswax, lanolin, bayberry wax, carnauba wax, and
rice bran wax. Tablets and pills can additionally be prepared with
enteric coatings.
[0121] Alternatively, powders or granules embodying the bacterial
compositions disclosed herein can be incorporated into a food
product. In some embodiments the food product is a drink for oral
administration. Non-limiting examples of a suitable drink include
fruit juice, a fruit drink, an artificially flavored drink, an
artificially sweetened drink, a carbonated beverage, a sports
drink, a liquid diary product, a shake, an alcoholic beverage, a
caffeinated beverage, infant formula and so forth. Other suitable
means for oral administration include aqueous and nonaqueous
solutions, emulsions, suspensions and solutions and/or suspensions
reconstituted from non-effervescent granules, containing at least
one of suitable solvents, preservatives, emulsifying agents,
suspending agents, diluents, sweeteners, coloring agents, and
flavoring agents.
[0122] In some embodiments the food product is a solid foodstuff.
Suitable examples of a solid foodstuff include without limitation a
food bar, a snack bar, a cookie, a brownie, a muffin, a cracker, an
ice cream bar, a frozen yogurt bar, and the like.
[0123] In some embodiments, the compositions disclosed herein are
incorporated into a therapeutic food. In some embodiments, the
therapeutic food is a ready-to-use food that optionally contains
some or all essential macronutrients and micronutrients. In some
embodiments, the compositions disclosed herein are incorporated
into a supplementary food that is designed to be blended into an
existing meal. In some embodiments, the supplemental food contains
some or all essential macronutrients and micronutrients. In some
embodiments, the bacterial compositions disclosed herein are
blended with or added to an existing food to fortify the food's
protein nutrition. Examples include food staples (grain, salt,
sugar, cooking oil, margarine), beverages (coffee, tea, soda, beer,
liquor, sports drinks), snacks, sweets and other foods.
[0124] In one embodiment, the formulations are filled into gelatin
capsules for oral administration. An example of an appropriate
capsule is a 250 mg gelatin capsule containing from 10 (up to 100
mg) of lyophilized powder (10.sup.8 to 10.sup.11 bacteria), 160 mg
microcrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesium
stearate. In an alternative embodiment, from 10.sup.5 to 10.sup.12
bacteria may be used, 10.sup.5 to 10.sup.7, 10.sup.6 to 10.sup.7,
or 10.sup.8 to 10.sup.10, with attendant adjustments of the
excipients if necessary. In an alternative embodiment an
enteric-coated capsule or tablet or with a buffering or protective
composition may be used.
[0125] In one embodiment, the number of bacteria of each type may
be present in the same amount or in different amounts. For example,
in a bacterial composition with two types of bacteria, the bacteria
may be present in from a 1:10,000 ratio to a 1:1 ratio, from a
1:10,000 ratio to a 1:1,000 ratio, from a 1:1,000 ratio to a 1:100
ratio, from a 1:100 ratio to a 1:50 ratio, from a 1:50 ratio to a
1:20 ratio, from a 1:20 ratio to a 1:10 ratio, from a 1:10 ratio to
a 1:1 ratio. For bacterial compositions comprising at least three
types of bacteria, the ratio of type of bacteria may be chosen
pairwise from ratios for bacterial compositions with two types of
bacteria. For example, in a bacterial composition comprising
bacteria A, B, and C, at least one of the ratio between bacteria A
and B, the ratio between bacteria B and C, and the ratio between
bacteria A and C may be chosen, independently, from the pairwise
combinations above.
[0126] Methods of Treating a Subject.
[0127] In some embodiments the proteins and compositions disclosed
herein are administered to a patient or a user (sometimes
collectively referred to as a "subject"). As used herein
"administer" and "administration" encompasses embodiments in which
one person directs another to consume a bacterial composition in a
certain manner and/or for a certain purpose, and also situations in
which a user uses a bacteria composition in a certain manner and/or
for a certain purpose independently of or in variance to any
instructions received from a second person. Non-limiting examples
of embodiments in which one person directs another to consume a
bacterial composition in a certain manner and/or for a certain
purpose include when a physician prescribes a course of conduct
and/or treatment to a patient, when a parent commands a minor user
(such as a child) to consume a bacterial composition, when a
trainer advises a user (such as an athlete) to follow a particular
course of conduct and/or treatment, and when a manufacturer,
distributer, or marketer recommends conditions of use to an end
user, for example through advertisements or labeling on packaging
or on other materials provided in association with the sale or
marketing of a product.
[0128] The bacterial compositions offer a protective and/or
therapeutic effect against infection by one or more GI pathogens of
interest and thus may be administered after an acute case of
infection has been resolved in order to prevent relapse, during an
acute case of infection as a complement to antibiotic therapy if
the bacterial composition is not sensitive to the same antibiotics
as the GI pathogen, or to prevent infection or reduce transmission
from disease carriers. These pathogens include, but are not limited
to, Aeromonas hydrophila, Campylobacter fetus, Plesiomonas
shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium
botulinum, Clostridium difficile, Clostridium perfringens,
enteroaggregative Escherichia coli, enterohemorrhagic Escherichia
coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia
coli (LT and/or ST), Escherichia coli 0157:H7, Helicobacter pylori,
Klebsiella pneumonia, Lysteria monocytogenes, Plesiomonas
shigelloides, Salmonella spp., Salmonella typhi, Shigella spp.,
Staphylococcus, Staphylococcus aureus, vancomycin-resistant
Enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio
parahaemolyticus, Vibrio vulnificus, and Yersinia
enterocolitica.
[0129] In one embodiment, the pathogen may be Clostridium
difficile, Salmonella spp., pathogenic Escherichia coli,
Carbapenem-resistent Enterobacteriaceae (CRE), extended spectrum
beta-lactam resistant Enterococci (ESBL) and vancomycin-resistant
Enterococci (VRE). In yet another embodiment, the pathogen may be
Clostridium difficile.
[0130] The present bacterial compositions may be useful in a
variety of clinical situations. For example, the bacterial
compositions may be administered as a complementary treatment to
antibiotics when a patient is suffering from an acute infection, to
reduce the risk of recurrence after an acute infection has
subsided, or when a patient will be in close proximity to others
with or at risk of serious gastrointestinal infections (physicians,
nurses, hospital workers, family members of those who are ill or
hospitalized).
[0131] The present bacterial compositions may be administered to
animals, including humans, laboratory animals (e.g., primates,
rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys,
chickens), and household pets (e.g., dogs, cats, rodents).
[0132] In the present method, the bacterial composition is
administered enterically, in other words by a route of access to
the gastrointestinal tract. This includes oral administration,
rectal administration (including enema, suppository, or
colonoscopy), by an oral or nasal tube (nasogastric, nasojejunal,
oral gastric, or oral jejunal), as detailed more fully herein.
[0133] A. Pretreatment Protocols
[0134] Prior to administration of the bacterial composition, the
patient may optionally have a pretreatment protocol to prepare the
gastrointestinal tract to receive the bacterial composition. In
certain embodiments, the pretreatment protocol is advisable, such
as when a patient has an acute infection with a highly resilient
pathogen. In other embodiments, the pretreatment protocol is
entirely optional, such as when the pathogen causing the infection
is not resilient, or the patient has had an acute infection that
has been successfully treated but where the physician is concerned
that the infection may recur. In these instances, the pretreatment
protocol may enhance the ability of the bacterial composition to
affect the patient's microbiome.
[0135] As one way of preparing the patient for administration of
the microbial ecosystem, at least one antibiotic may be
administered to alter the bacteria in the patient. As another way
of preparing the patient for administration of the microbial
ecosystem, a standard colon-cleansing preparation may be
administered to the patient to substantially empty the contents of
the colon, such as used to prepare a patient for a colonscopy. By
"substantially emptying the contents of the colon," this
application means removing at least 75%, at least 80%, at least
90%, at least 95%, or about 100% of the contents of the ordinary
volume of colon contents. Antibiotic treatment may precede the
colon-cleansing protocol.
[0136] If a patient has received an antibiotic for treatment of an
infection, or if a patient has received an antibiotic as part of a
specific pretreatment protocol, in one embodiment the antibiotic
should be stopped in sufficient time to allow the antibiotic to be
substantially reduced in concentration in the gut before the
bacterial composition is administered. In one embodiment, the
antibiotic may be discontinued 1, 2, or 3 days before the
administration of the bacterial composition. In one embodiment, the
antibiotic may be discontinued 3, 4, 5, 6, or 7 antibiotic
half-lives before administration of the bacterial composition. In
another embodiment, the antibiotic may be chosen so the
constituents in the bacterial composition have an MIC50 that is
higher than the concentration of the antibiotic in the gut.
[0137] MIC50 of a bacterial composition or the elements in the
composition may be determined by methods well known in the art.
Reller et al., Antimicrobial Susceptibility Testing: A Review of
General Principles and Contemporary Practices, Clinical Infectious
Diseases 49(11):1749-1755 (2009). In such an embodiment, the
additional time between antibiotic administration and
administration of the bacterial composition is not necessary. If
the pretreatment protocol is part of treatment of an acute
infection, the antibiotic may be chosen so that the infection is
sensitive to the antibiotic, but the constituents in the bacterial
composition are not sensitive to the antibiotic.
[0138] Routes of Administration
[0139] The bacterial compositions of the invention are suitable for
administration to mammals and non-mammalian animals in need
thereof. In certain embodiments, the mammalian subject is a human
subject who has one or more symptoms of a dysbiosis.
[0140] When the mammalian subject is suffering from a disease,
disorder or condition characterized by an aberrant microbiota, the
bacterial compositions described herein are suitable for treatment
thereof. In some embodiments, the mammalian subject has not
received antibiotics in advance of treatment with the bacterial
compositions. For example, the mammalian subject has not been
administered at least two doses of vancomycin, metronidazole and/or
or similar antibiotic compound within one week prior to
administration of the therapeutic composition. In other
embodiments, the mammalian subject has not previously received an
antibiotic compound in the one month prior to administration of the
therapeutic composition. In other embodiments, the mammalian
subject has received one or more treatments with one or more
different antibiotic compounds and such treatment(s) resulted in no
improvement or a worsening of symptoms.
[0141] In some embodiments, the gastrointestinal disease, disorder
or condition is diarrhea caused by C. difficile including recurrent
C. difficile infection, ulcerative colitis, colitis, Crohn's
disease, or irritable bowel disease. Beneficially, the therapeutic
composition is administered only once prior to improvement of the
disease, disorder or condition. In some embodiments the therapeutic
composition is administered at intervals greater than two days,
such as once every three, four, five or six days, or every week or
less frequently than every week. Or the preparation may be
administered intermittently according to a set schedule, e.g., once
a day, once weekly, or once monthly, or when the subject relapses
from the primary illness. In another embodiment, the preparation
may be administered on a long-term basis to subjects who are at
risk for infection with or who may be carriers of these pathogens,
including subjects who will have an invasive medical procedure
(such as surgery), who will be hospitalized, who live in a
long-term care or rehabilitation facility, who are exposed to
pathogens by virtue of their profession (livestock and animal
processing workers), or who could be carriers of pathogens
(including hospital workers such as physicians, nurses, and other
health care professionals).
[0142] In embodiments, the bacterial composition is administered
enterically. This preferentially includes oral administration, or
by an oral or nasal tube (including nasogastric, nasojejunal, oral
gastric, or oral jejunal). In other embodiments, administration
includes rectal administration (including enema, suppository, or
colonoscopy). The bacterial composition may be administered to at
least one region of the gastrointestinal tract, including the
mouth, esophagus, stomach, small intestine, large intestine, and
rectum. In some embodiments it is administered to all regions of
the gastrointestinal tract. The bacterial compositions may be
administered orally in the form of medicaments such as powders,
capsules, tablets, gels or liquids. The bacterial compositions may
also be administered in gel or liquid form by the oral route or
through a nasogastric tube, or by the rectal route in a gel or
liquid form, by enema or instillation through a colonoscope or by a
suppository.
[0143] If the composition is administered colonoscopically and,
optionally, if the bacterial composition is administered by other
rectal routes (such as an enema or suppository) or even if the
subject has an oral administration, the subject may have a
colon-cleansing preparation. The colon-cleansing preparation can
facilitate proper use of the colonoscope or other administration
devices, but even when it does not serve a mechanical purpose it
can also maximize the proportion of the bacterial composition
relative to the other organisms previously residing in the
gastrointestinal tract of the subject. Any ordinarily acceptable
colon-cleansing preparation may be used such as those typically
provided when a subject undergoes a colonoscopy.
[0144] Dosages and Schedule for Administration
[0145] In some embodiments the bacteria and bacterial compositions
are provided in a dosage form. In some embodiments the dosage form
is designed for administration of at least one OTU or combination
thereof disclosed herein, wherein the total amount of bacterial
composition administered is selected from 0.1 ng to 10 g, 10 ng to
1 g, 100 ng to 0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg, or from
10-15 mg. In some embodiments the bacterial composition is consumed
at a rate of from 0.1 ng to 10 g a day, 10 ng to 1 g a day, 100 ng
to 0.1 g a day, 0.1 mg to 500 mg a day, 1 mg to 100 mg a day, or
from 10-15 mg a day, or more.
[0146] In some embodiments the treatment period is at least 1 day,
at least 2 days, at least 3 days, at least 4 days, at least 5 days,
at least 6 days, at least 1 week, at least 2 weeks, at least 3
weeks, at least 4 weeks, at least 1 month, at least 2 months, at
least 3 months, at least 4 months, at least 5 months, at least 6
months, or at least 1 year. In some embodiments the treatment
period is from 1 day to 1 week, from 1 week to 4 weeks, from 1
month, to 3 months, from 3 months to 6 months, from 6 months to 1
year, or for over a year.
[0147] In one embodiment, from 10.sup.5 and 10.sup.12
microorganisms total may be administered to the patient in a given
dosage form. In one mode, an effective amount may be provided in
from 1 to 500 ml or from 1 to 500 grams of the bacterial
composition having from 10.sup.7 to 10.sup.11 bacteria per ml or
per gram, or a capsule, tablet or suppository having from 1 mg to
1000 mg lyophilized powder having from 10.sup.7 to 10.sup.11
bacteria.
[0148] Those receiving acute treatment may receive higher doses
than those who are receiving chronic administration (such as
hospital workers or those admitted into long-term care
facilities).
[0149] Any of the preparations described herein may be administered
once on a single occasion or on multiple occasions, such as once a
day for several days or more than once a day on the day of
administration (including twice daily, three times daily, or up to
five times daily). Or the preparation may be administered
intermittently according to a set schedule, e.g., once weekly, once
monthly, or when the patient relapses from the primary illness. In
another embodiment, the preparation may be administered on a
long-term basis to individuals who are at risk for infection with
or who may be carriers of these pathogens, including individuals
who will have an invasive medical procedure (such as surgery), who
will be hospitalized, who live in a long-term care or
rehabilitation facility, who are exposed to pathogens by virtue of
their profession (livestock and animal processing workers), or who
could be carriers of pathogens (including hospital workers such as
physicians, nurses, and other health care professionals).
[0150] Patient Selection
[0151] Particular bacterial compositions may be selected for
individual patients or for patients with particular profiles. For
example, 16S sequencing may be performed for a given patient to
identify the bacteria present in his or her microbiota. The
sequencing may either profile the patient's entire microbiome using
16S sequencing (to the family, genera, or species level), a portion
of the patient's microbiome using 16S sequencing, or it may be used
to detect the presence or absence of specific candidate bacteria
that are biomarkers for health or a particular disease state, such
as markers of multi-drug resistant organisms or specific genera of
concern such as Escherichia. Based on the biomarker data, a
particular composition may be selected for administration to a
patient to supplement or complement a patient's microbiota in order
to restore health or treat or prevent disease. In another
embodiment, patients may be screened to determine the composition
of their microbiota to determine the likelihood of successful
treatment.
[0152] Combination Therapy
[0153] The bacterial compositions may be administered with other
agents in a combination therapy mode, including anti-microbial
agents and prebiotics. Administration may be sequential, over a
period of hours or days, or simultaneous.
[0154] In one embodiment, the bacterial compositions are included
in combination therapy with one or more anti-microbial agents,
which include anti-bacterial agents, anti-fungal agents, anti-viral
agents and anti-parasitic agents.
[0155] Anti-bacterial agents include cephalosporin antibiotics
(cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin,
cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole);
fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin,
avelox, and norflox); tetracycline antibiotics (tetracycline,
minocycline, oxytetracycline, and doxycycline); penicillin
antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin,
carbenicillin, vancomycin, and methicillin); and carbapenem
antibiotics (ertapenem, doripenem, imipenem/cilastatin, and
meropenem).
[0156] Anti-viral agents include Abacavir, Acyclovir, Adefovir,
Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine,
Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine,
Enfuvirtide, Etravirine, Famciclovir, Foscarnet, Fomivirsen,
Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir
Maraviroc, MK-2048, Nelfinavir, Nevirapine, Penciclovir,
Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,
Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,
Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,
Zalcitabine, Zanamivir and Zidovudine.
[0157] Examples of antifungal compounds include, but are not
limited to polyene antifungals such as natamycin, rimocidin,
filipin, nystatin, amphotericin B, candicin, and hamycin; imidazole
antifungals such as miconazole, ketoconazole, clotrimazole,
econazole, omoconazole, bifonazole, butoconazole, fenticonazole,
isoconazole, oxiconazole, sertaconazole, sulconazole, and
tioconazole; triazole antifungals such as fluconazole,
itraconazole, isavuconazole, ravuconazole, posaconazole,
voriconazole, terconazole, and albaconazole; thiazole antifungals
such as abafungin; allylamine antifungals such as terbinafine,
naftifine, and butenafine; and echinocandin antifungals such as
anidulafungin, caspofungin, and micafungin. Other compounds that
have antifungal properties include, but are not limited to
polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid,
flucytosine or 5-fluorocytosine, griseofulvin, and haloprogin.
[0158] In one embodiment, the bacterial compositions are included
in combination therapy with one or more corticosteroids,
mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives,
immunosuppressive drugs, cyclosporin A, mercaptopurine,
azathiopurine, prednisone, methotrexate, antihistamines,
glucocorticoids, epinephrine, theophylline, cromolyn sodium,
anti-leukotrienes, anti-cholinergic drugs for rhinitis,
anti-cholinergic decongestants, mast-cell stabilizers, monoclonal
anti-IgE antibodies, vaccines, and combinations thereof.
[0159] A prebiotic is a selectively fermented ingredient that
allows specific changes, both in the composition and/or activity in
the gastrointestinal microbiota that confers benefits upon host
well-being and health. Prebiotics may include complex
carbohydrates, amino acids, peptides, or other essential
nutritional components for the survival of the bacterial
composition. Prebiotics include, but are not limited to, amino
acids, biotin, fructooligosaccharide, galactooligosaccharides,
inulin, lactulose, mannan oligosaccharides, oligofructose-enriched
inulin, oligofructose, oligodextrose, tagatose,
trans-galactooligosaccharide, and xylooligosaccharides.
[0160] Methods for Characterization of Bacterial Compositions
[0161] In certain embodiments, provided are methods for testing
certain characteristics of bacterial compositions. For example, the
sensitivity of bacterial compositions to certain environmental
variables is determined, e.g., in order to select for particular
desirable characteristics in a given composition, formulation
and/or use. For example, the constituents in the bacterial
composition may be tested for pH resistance, bile acid resistance,
and/or antibiotic sensitivity, either individually on a
constituent-by-constituent basis or collectively as a bacterial
composition comprised of multiple bacterial constituents
(collectively referred to in this section as bacterial
composition).
[0162] pH Sensitivity Testing. If a bacterial composition will be
administered other than to the colon or rectum (i.e., through, for
example, but not limited to, an oral route), optionally testing for
pH resistance enhances the selection of bacterial compositions that
will survive at the highest yield possible through the varying pH
environments of the distinct regions of the GI tract. Understanding
how the bacterial compositions react to the pH of the GI tract also
assists in formulation, so that the number of bacteria in a dosage
form can be increased if beneficial and/or so that the composition
may be administered in an enteric-coated capsule or tablet or with
a buffering or protective composition. As the pH of the stomach can
drop to a pH of 1 to 2 after a high-protein meal for a short time
before physiological mechanisms adjust it to a pH of 3 to 4 and
often resides at a resting pH of 4 to 5, and as the pH of the small
intestine can range from a pH of 6 to 7.4, bacterial compositions
can be prepared that survive these varying pH ranges (specifically
wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, or as much as 100% of the bacteria can survive gut
transit times through various pH ranges). This may be tested by
exposing the bacterial composition to varying pH ranges for the
expected gut transit times through those pH ranges. Therefore, as a
nonlimiting example only, 18-hour cultures of bacterial
compositions may be grown in standard media, such as gut microbiota
medium ("GMM", see Goodman et al., Extensive personal human gut
microbiota culture collections characterized and manipulated in
gnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or another
animal-products-free medium, with the addition of pH adjusting
agents for a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1
hour, a pH of 4 to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5
to 3 hours. An alternative method for testing stability to acid is
described in U.S. Pat. No. 4,839,281. Survival of bacteria may be
determined by culturing the bacteria and counting colonies on
appropriate selective or non-selective media.
[0163] Bile Acid Sensitivity Testing. Additionally, in some
embodiments, testing for bile-acid resistance enhances the
selection of bacterial compositions that will survive exposures to
bile acid during transit through the GI tract. Bile acids are
secreted into the small intestine and can, like pH, affect the
survival of bacterial compositions. This may be tested by exposing
the bacterial compositions to bile acids for the expected gut
exposure time to bile acids. For example, bile acid solutions may
be prepared at desired concentrations using 0.05 mM Tris at pH 9 as
the solvent. After the bile acid is dissolved, the pH of the
solution may be adjusted to 7.2 with 10% HCl. Bacterial
compositions may be cultured in 2.2 ml of a bile acid composition
mimicking the concentration and type of bile acids in the patient,
1.0 ml of 10% sterile-filtered feces media and 0.1 ml of an 18-hour
culture of the given strain of bacteria. Incubations may be
conducted for from 2.5 to 3 hours or longer. An alternative method
for testing stability to bile acid is described in U.S. Pat. No.
4,839,281. Survival of bacteria may be determined by culturing the
bacteria and counting colonies on appropriate selective or
non-selective media.
[0164] Antibiotic Sensitivity Testing. As a further optional
sensitivity test, bacterial compositions may be tested for
sensitivity to antibiotics. In one embodiment, bacterial
compositions may be chosen so that the bacterial constituents are
sensitive to antibiotics such that if necessary they can be
eliminated or substantially reduced from the patient's
gastrointestinal tract by at least one antibiotic targeting the
bacterial composition.
[0165] Adherence to Gastrointestinal Cells. The bacterial
compositions may optionally be tested for the ability to adhere to
gastrointestinal cells. A method for testing adherence to
gastrointestinal cells is described in U.S. Pat. No. 4,839,281.
[0166] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification. The
embodiments within the specification provide an illustration of
embodiments and should not be construed to limit the scope. The
skilled artisan readily recognizes that many other embodiments are
encompassed. All publications and patents cited in this disclosure
are incorporated by reference in their entirety. To the extent the
material incorporated by reference contradicts or is inconsistent
with this specification, the specification will supersede any such
material. The citation of any references herein is not an admission
that such references are prior art.
[0167] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification, including claims, are to be understood as
being modified in all instances by the term "about." Accordingly,
unless otherwise indicated to the contrary, the numerical
parameters are approximations and may vary depending upon the
desired properties sought to be obtained. At the very least, and
not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims, each numerical parameter
should be construed in light of the number of significant digits
and ordinary rounding approaches.
[0168] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series.
EXAMPLES
[0169] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for. Examples of the techniques and protocols described
herein with regard to therapeutic compositions can be found in,
e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed), 1980.
Example 1.
Construction of Binary Pairs in a High-Throughput 96-Well
Format
[0170] To allow high-throughput screening of binary pairs, vials of
-80.degree. C. glycerol stock banks were thawed and diluted to 1 e8
CFU/mL. Each strain was then diluted 10.times. (to a final
concentration of 1e7 CFU/mL of each strain) into 200 uL of PBS+15%
glycerol in the wells of a 96-well plate. Plates were then frozen
at -80.degree. C. When needed, plates were removed from -80.degree.
C. and thawed at room temperature under anaerobic conditions when
testing in a CivSim with Clostridium difficile.
Example 2.
Construction of Ternary Combinations in a High-Throughput 96-Well
Format
[0171] To allow high-throughput screening of ternary combinations,
vials of -80.degree. C. glycerol stock banks were thawed and
diluted to 1e8 CFU/mL. Each strain was then diluted 10.times. (to a
final concentration of 1 e7 CFU/mL of each strain) into 200 uL of
PBS+15% glycerol in the wells of a 96-well plate. Plates were then
frozen at -80.degree. C. When needed for the assay, plates were
removed from -80.degree. C. and thawed at room temperature under
anaerobic conditions when testing in a CivSim with Clostridium
difficile.
Example 3.
Construction of a CivSim Assay to Screen for Ecobiotic.TM.
Compositions Inhibitory to the Growth of Clostridium difficile
[0172] An overnight culture of Clostridium difficile was grown
under anaerobic conditions in SweetB-Fosln or other suitable media
for the growth of C. difficile. SweetB-Fosln is a complex media
composed of brain heart infusion, yeast extract, cysteine,
cellobiose, maltose, soluble starch, and
fructooligosaccharides/inulin, and hemin, and is buffered with
MOPs. After 24 hr of growth the culture was diluted 100,000 fold
into a complex media such as SweetB-Fosln which is suitable for the
growth of a wide variety of anaerobic bacterial species. The
diluted C. difficile mixture was then aliquoted to wells of a
96-well plate (180 uL to each well). 20 uL of a unique binary pair
of potential inhibitory species was then added to each well at a
final concentration of 1 e6 CFU/mL of each species. Alternatively
the assay can be tested with binary pairs at different initial
concentrations (1e9 CFU/mL, 1e8 CFU/mL, 1e7 CFU/mL, 1e5 CFU/mL, 1e4
CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL). Control wells only inoculated with
C. difficile were included for a comparison to the growth of C.
difficile without inhibition. Additional wells were used for
controls that either inhibit or do not inhibit the growth of C.
difficile. One example of a positive control that inhibits growth
was a combination of Blautia producta, Clostridium bifermentans and
Escherichia coli. One example of a control that shows reduced
inhibition of C. difficile growth as a combination of Bacteroides
thetaiotaomicron, Bacteroides ovatus and Bacteroides vulgatus.
Plates were wrapped with parafilm and incubated for 24 hr at
37.degree. C. under anaerobic conditions. After 24 hr the wells
containing C. difficile alone were serially diluted and plated to
determine titer. The 96-well plate was then frozen at -80C before
quantifying C. difficile by qPCR assay.
Example 4.
Construction of a CivSim Assay to Screen for Bacterial Compositions
that Produce Diffusible Products Inhibitory to the Growth of
Clostridium difficile using a Filter Insert
[0173] The CivSim assay described above was modified by using a
0.22 uM filter insert (Millipore.TM. MultiScreen.TM. 96-Well Assay
Plates--Item MAGVS2210) in 96-well format to physically separate C.
difficile from the bacterial compositions. The C. difficile was
aliquoted into the 96-well plate while the bacterial compositions
were aliquoted into media on the filter overlay. The nutrient media
as in contact on both sides of the 0.22 uM filter, allowing
exchange of nutrients, small molecules and many macromolecules
(e.g., bacteriocins, cell-surface proteins, or polysaccharides) by
diffusion. In this embodiment, after 24 hr incubation, the filter
insert containing the bacterial compositions was removed. The plate
containing C. difficile was then transferred to a 96-well plate
reader suitable for measuring optical density (OD) at 600 nm. The
growth of C. difficile in the presence of different bacterial
compositions was compared based on the OD measurement.
Example 5.
Construction of a CivSim Assay to Screen for Bacterial Compositions
Inhibitory to the Growth of Clostridium difficile using Clostridium
difficile Selective Media for Quantification
[0174] The CivSim assay described above can be modified to
determine final C. difficile titer by serially diluting and plating
to C. difficile selective media (Bloedt et al 2009) such as CCFA
(cycloserine cefoxitin fructose agar, Anaerobe Systems), CDSA
(Clostridium difficile selective agar, which is cycloserine
cefoxitin mannitol agar, Becton Dickinson).
Example 6
Quantification of C. difficile using Quantitative PCR (qPCR)
Standard Curve Preparation
[0175] The standard curve was generated from a well on each assay
plate containing only pathogenic C. difficile grown in SweetB+Fosln
media as provided herein and quantified by selective spot plating.
Ser. dilutions of the culture were performed in sterile
phosphate-buffered saline. Genomic DNA was extracted from the
standard curve samples along with the other wells.
[0176] Genomic DNA Extraction
[0177] Genomic DNA was extracted from 5 .mu.l of each sample using
a dilution, freeze/thaw, and heat lysis protocol. 5 .mu.L of thawed
samples were added to 45 .mu.L of UltraPure water (Life
Technologies, Carlsbad, Calif.) and mixed by pipetting. The plates
with diluted samples were frozen at -20.degree. C. until use for
qPCR which includes a heated lysis step prior to amplification.
Alternatively the genomic DNA could be isolated using the Mo Bio
Powersoil.RTM.-htp 96 Well Soil DNA Isolation Kit (Mo Bio
Laboratories, Carlsbad, Calif.), Mo Bio Powersoil.RTM. DNA
Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.), or the
QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.) according to
the manufacturer's instructions.
[0178] qPCR Composition and Conditions
[0179] The qPCR reaction mixture contained 1.times. SsoAdvanced
Universal Probes Supermix, 900 nM of Wr-tcdB-F primer
(AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG (SEQ ID NO: 1916), IDT,
Coralville, Iowa), 900 nM of Wr-tcdB-R primer
(CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 1917), IDT, Coralville,
Iowa), 250 nM of Wr-tcdB-P probe
(6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 1918), Life
Technologies, Grand Island, N.Y.), and Molecular Biology Grade
Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 .mu.l (Primers
adapted from: Wroblewski, D. et al., Rapid Molecular
Characterization of Clostridium difficile and Assessment of
Populations of C. difficile in Stool Specimens, Journal of Clinical
Microbiology 47:2142-2148 (2009)). This reaction mixture was
aliquoted to wells of a Hard-shell Low-Profile Thin Wall 96-well
Skirted PCR Plate (BioRad, Hercules, Calif.). To this reaction
mixture, 2 .mu.l of diluted, frozen, and thawed samples were added
and the plate sealed with a Microseal `B` Adhesive Seal (BioRad,
Hercules, Calif.). The qPCR was performed on a BioRad C1000.TM.
Thermal Cycler equipped with a CFX96.TM. Real-Time System (BioRad,
Hercules, Calif.). The thermocycling conditions were 95.degree. C.
for 15 minutes followed by 45 cycles of 95.degree. C. for 5
seconds, 60.degree. C. for 30 seconds, and fluorescent readings of
the FAM channel. Alternatively, the qPCR could be performed with
other standard methods known to those skilled in the art.
[0180] Data Analysis
[0181] The Cq value for each well on the FAM channel was determined
by the CFX Manager.TM. 3.0 software. The log.sub.10(cfu/mL) of C.
difficile each experimental sample was calculated by inputting a
given sample's Cq value into a linear regression model generated
from the standard curve comparing the Cq values of the standard
curve wells to the known log.sub.10(cfu/mL) of those samples. The
log inhibition was calculated for each sample by subtracting the
log.sub.10(cfu/mL) of C. difficile in the sample from the
log.sub.10(cfu/mL) of C. difficile in the sample on each assay
plate used for the generation of the standard curve that has no
additional bacteria added. The mean log inhibition was calculated
for all replicates for each composition.
[0182] A histogram of the range and standard deviation of each
composition was plotted. Ranges or standard deviations of the log
inhibitions that were distinct from the overall distribution were
examined as possible outliers. If the removal of a single log
inhibition datum from one of the binary pairs that were identified
in the histograms would bring the range or standard deviation in
line with those from the majority of the samples, that datum was
removed as an outlier, and the mean log inhibition was
recalculated.
[0183] The pooled variance of all samples evaluated in the assay
was estimated as the average of the sample variances weighted by
the sample's degrees of freedom. The pooled standard error was then
calculated as the square root of the pooled variance divided by the
square root of the number of samples. Confidence intervals for the
null hypothesis were determined by multiplying the pooled standard
error to the z score corresponding to a given percentage threshold.
Mean log inhibitions outside the confidence interval were
considered to be inhibitory if positive or stimulatory if negative
with the percent confidence corresponding to the interval used.
Samples with mean log inhibition greater than the 99% confidence
interval (C.I) of the null hypothesis are reported as ++++, those
with a 95% <C.I. <99% as +++, those with a 90%
<C.I.<95% as ++, those with a 80% <C.I.<90% as + while
samples with mean log inhibition less than than the 99% confidence
interval (C.I) of the null hypothesis are reported as ----, those
with a 95% <C.I.<99% as ---, those with a 90% <C.I.<95%
as --, those with a 80% <C.I.<90% as -.
[0184] Many binary pairs inhibit C. difficile Table 3. 622 of 989
combinations show inhibition with a confidence interval >80%;
545 of 989 with a C.I.>90%; 507 of 989 with a C.I.>95%; 430
of 989 with a C.I. of >99%. Non-limiting but exemplary binary
pairs include those with mean log reduction greater than 0.366,
e.g. Allistipes shahii paired with Blautia producta, Clostridium
hathaweyi, or Colinsella aerofaciens, or Clostidium mayombei paired
with C. innocuum, C. tertium, Colinsella aerofaciens, or any of the
other 424 combinations shown in Table 5. Equally important, the
CivSim assay describes binary pairs that do not effectively inhibit
C. difficile. 188 of 989 combinations promote growth with >80%
confidence; 52 of 989 show a lack of inhibition with >90%
confidence; 22 of 989 show a lack of inhibition with >95%
confidence; 3 of 989, including B. producta combined with
Coprococcus catus, Alistipes shahii combined with Dorea
formicigenerans, and Eubacterium rectale combined with Roseburia
intestinalis, show a lack of inhibition with >99% confidence.
249 of 989 combinations are neutral in the assay, meaning they
neither promote nor inhibit C. difficile growth to the limit of
measurement.
[0185] Ternary combinations with mean log inhibition greater than
0.312 are reported as ++++ (.gtoreq.99% confidence interval (C.I.)
of the null hypothesis), those with mean log inhibition between
0.221 and 0. 312 as +++ (95% <C.I.<99%), those with mean log
inhibition between 0.171 and 0. 221as ++ (90% <C.I.<95%),
those with mean log inhibition between 0.113 and 0. 171 as + (80%
<C.I.<90%), those with mean log inhibition between -0.113 and
-0. 171 as - (80% <C.I.<90%), those with mean log inhibition
between -0. 171 and -0. 221 as -- (90% <C.I.<95%), those with
mean log inhibition between -0. 221and -0. 312 as --- (95%
<C.I.<99%), and those with mean log inhibition less than -0.
312 as ---- (99% <C.I.).
[0186] The CivSim shows that many ternary combinations inhibit C.
difficile. 39 of 56 combinations show inhibition with a confidence
interval >80%; 36 of 56 with a C.I.>90%; 36 of 56 with a
C.I.>95%; 29 of 56 with a C.I. of >99%. Non-limiting but
exemplary ternary combinations include those with mean log
reduction greater than 0.171, e.g. any combination shown in Table 4
with a score of ++++, such as Colinsella aerofaciens, Coprococcus
comes, and Blautia producta. Equally important, the CivSim assay
describes ternary combinations that do not effectively inhibit C.
difficile. 5 of 56 combinations promote growth with >80%
confidence; 2 of 56 promote growth with >90% confidence; 1 of
56, Coprococcus comes, Clostridium symbiosum and Eubacterium
rectale, promote growth with >95% confidence. 12 of 56
combinations are neutral in the assay, meaning they neither promote
nor inhibit C. difficile growth to the limit of measurement.
Example P1.
Full 16S Sequencing to Determine Operational Taxonomic Unit
(OTU)
[0187] Genomic DNA Extraction
[0188] Genomic DNA is extracted from pure microbial cultures using
a hot alkaline lysis method. 2 .mu.l of microbial culture is added
to 18 .mu.l of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the
mixture is incubated at 95.degree. C. for 30 minutes. Subsequently,
the samples are cooled to 4.degree. C. and neutralized by the
addition of 20 .mu.l of Neutralization Buffer (40 mM Tris-HCl) and
then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).
Alternatively, genomic DNA is extracted from pure microbial
cultures using commercially available kits such as the Mo Bio
Ultraclean.RTM. Microbial DNA Isolation Kit (Mo Bio Laboratories,
Carlsbad, Calif.) or by standard methods known to those skilled in
the art.
[0189] PCR
[0190] To amplify bacterial 16S rDNA, 2 .mu.l of extracted gDNA is
added to a 20 .mu.l final volume PCR reaction. The PCR reaction
also contains 1.times. HotMasterMix (5PRIME, Gaithersburg, Md.),
250 nM of 27f primer (AGRGTTTGATCMTGGCTCAG (SEQ ID NO: 1919), IDT,
Coralville, Iowa), and 250 nM of 1492r primer
(TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 1920), IDT, Coralville, Iowa),
with Molecular Biology Grade Water (Mo Bio Laboratories, Carlsbad,
Calif.) for the balance of the volume. Alternatively, other
universal bacterial primers or thermostable polymerases known to
those skilled in the art are used.
[0191] The PCR performed on commercially available thermocyclers
such as a BioRad MyCycler.TM. Thermal Cycler (BioRad, Hercules,
Calif.). The reactions are run at 94.degree. C. for 2 minutes
followed by 30 cycles of 94.degree. C. for 30 seconds, 51.degree.
C. for 30 seconds, and 68.degree. C. for 1 minute 30 seconds,
followed by a 7 minute extension at 72.degree. C. and an indefinite
hold at 4.degree. C. Following PCR, gel electrophoresis of a
portion of the reaction products is used to confirm successful
amplification of a .about.1.5 kb product.
[0192] PCR Cleanup
[0193] To remove nucleotides and oligonucleotides from the PCR
products, 1 .mu.l of HT ExoSap-IT (Affymetrix, Santa Clara, Calif.)
is added to 2.5 .mu.l of PCR product followed by a 15 minute
incubation at 37.degree. C. and then a 15 minute inactivation at
80.degree. C.
[0194] Sanger Sequencing
[0195] For each sample, two sequencing reactions are performed, one
using each primer: 27f and 1492r. 40 ng of ExoSap-IT-cleaned PCR
products are mixed with 25 pmol of sequencing primer and Molecular
Biology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 15
.mu.l total volume. This reaction is submitted to a commercial
sequencing organization such as Genewiz (South Plainfield, N.J.)
for Sanger sequencing.
Example P2.
V4 16S Sequencing to Determine Operational Taxonomic Unit (OTU)
[0196] Genomic DNA Extraction
[0197] Genomic DNA is extracted from pure microbial cultures using
a hot alkaline lysis method. 2 .mu.l of microbial culture is added
to 18 .mu.l of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the
mixture is incubated at 95.degree. C. for 30 minutes. Subsequently,
the samples are cooled to 4.degree. C. and neutralized by the
addition of 18 .mu.l of Neutralization Buffer (40 mM Tris-HCl) and
then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).
Alternatively, genomic DNA is extracted from pure microbial
cultures using commercially available kits such as the Mo Bio
Ultraclean.RTM. Microbial DNA Isolation Kit (Mo Bio Laboratories,
Carlsbad, Calif.) or by standard methods known to those in skilled
in the art.
[0198] PCR
[0199] To amplify the V4 region of bacterial 16S rDNA, 2 .mu.l of
extracted gDNA is added to a 20 .mu.l final volume PCR reaction.
The PCR reaction also contains 1.times. HotMasterMix (5PRIME,
Gaithersburg, Md.), 200 nM of V4_515f_adapt
(AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGC GGTAA (SEQ
ID NO: 1921), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc
(CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO:
1922)_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO:
1923), IDT, Coralville, Iowa), with Molecular Biology Grade Water
(Mo Bio Laboratories, Carlsbad, Calif.) for the balance of the
volume. These primers incorporate adapters for Illumina sequencing
by synthesis. Optionally, identical replicate, triplicate, or
quadruplicate reactions may be performed. Alternatively other
universal bacterial primers or thermostable polymerases known to
those skilled in the art are used.
[0200] The PCR performed on commercially available thermocyclers
such as a BioRad MyCycler.TM. Thermal Cycler (BioRad, Hercules,
Calif.). The reactions are run at 94.degree. C. for 3 minutes
followed by 25 cycles of 94.degree. C. for 45 seconds, 50.degree.
C. for 1 minute, and 72.degree. C. for 1 minute 30 seconds,
followed by a 10 minute extension at 72.degree. C. and a indefinite
hold at 4.degree. C. Following PCR, gel electrophoresis of a
portion of the reaction products is used to confirm successful
amplification of a .about.0.4 kb product.
[0201] PCR Cleanup
[0202] To remove nucleotides and oligonucleotides from the PCR
products, the entire remaining volume of the PCR, or of the
multiple PCRs, is cleaned up using the Mo Bio Ultraclean.RTM.-htp
96 Well PCR Clean-up Kit (Mo Bio Laboratories, Carlsbad, Calif.)
according to the manufacturer's instructions or other commercially
available kits such as the QIAquick 96 PCR Purification Kit
(QIAGEN, Valencia, Calif.).
[0203] DNA Quantification & Pooling
[0204] The cleaned PCR products are quantified using the
Quant-iT.TM. PicoGreen.RTM. dsDNA Assay Kit (Life Technologies,
Grand Island, N.Y.) according to the manufacturer's instructions.
Following quantification, the barcoded cleaned PCR products are
combined such that each distinct PCR product is at an equimolar
ratio to create a prepared Illumina library.
[0205] Illumina Sequencing
[0206] The prepared library is sequenced on Illumina HiSeq or MiSeq
sequencers (Illumina, San Diego, Calif.) with cluster generation,
template hybridization, iso-thermal amplification, linearization,
blocking and denaturization and hybridization of the sequencing
primers performed according to the manufacturer's instructions.
16SV4SeqFw (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 1924)),
16SV4SeqRev (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 1923)),
and 16SV4Index (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 1925))
(IDT, Coralville, Iowa) are used for sequencing. This sequencing
can optionally be performed by a contract research organization
such as Metanome (Houston, Tex.), Ambry Genetics (Aliso Viejo,
Calif.), Edge Bio (Gaithersburg, Md.), or Covance (Princeton,
N.J.).
[0207] Taxonomic Assignment to Sequence Read Data
[0208] Nucleic acid sequences are analyzed and taxonomic and
phylogenetic assignments of specific OTUs are made using sequence
similarity and phylogenetic methods that are well known to those
skilled in the art, including but not limited to maximum likelihood
phylogenetic reconstruction (see e.g. Liu K, Linder C R, and Warnow
T. 2011. RAxML and FastTree: Comparing Two Methods for Large-Scale
Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731.
McGuire G, Denham M C, and Balding D J. 2001. Models of sequence
evolution for DNA sequences containing gaps. Mol. Biol. Evol 18:
481-490. Wrobel B. 2008. Statistical measures of uncertainty for
branches in phylogenetic trees inferred from molecular sequences by
using model-based methods. J. Appl. Genet. 49: 49-67.) From these
taxonomic assignments OTUs in the dataset are defined. The
certainty of the OTU call is defined based on the OTU's sequence
similarity to a reference nucleic acid sequence and the proximity
of the OTU sequence relative to one or more reference sequences in
the phylogeny. The specificity of an OTU's taxonomic and
phlylogenetic assignment determines whether the match is assigned
at the level of Family, Genus, Species, or Strain, and the
confidence of this assignment is determined based on the position
of bootstrap supported branches in the reference phylogenetic tree
relative to the placement of the OTU sequence being
interrogated.
Example P3.
Construction of an In Vitro Assay to Screen for Combinations of
Microbes Inhibitory to the Growth of Pathogenic E. coli
[0209] The in vitro assay is used to screen for combinations of
bacteria inhibitory to the growth of E. coli by modifying the media
used for growth of the pathogen inoculum. One of several choices of
media is used for growth of the pathogen such as Reinforced
Clostridial Media (RCM), Brain Heart Infusion Broth (BHI) or Luria
Bertani Broth (LB) (also known as Lysogeny Broth). E. coli is
quantified by using alternative selective media specific for E.
coli or using qPCR probes specific for the pathogen. For example,
aerobic growth on MacConkey lactose medium selects for enteric Gram
negatives, including E. coli. qPCR is conducted using probes
specific for the shiga toxin of pathogenic E. coli.
Example P4. Construction of an In Vitro Assay to Screen for
Combinations of Microbes Inhibitory to the Growth of
Vancomycin-Resistant Enterococcus (VRE)
[0210] The in vitro assay is used to screen for combinations of
bacteria inhibitory to the growth of Vancomycin-Resistant
Enterococcus spp. (VRE) by modifying the media used for growth of
the pathogen inoculum. Several choices of media are used for growth
of the pathogen such as Reinforced Clostridial Media (RCM), Brain
Heart Infusion Broth (BHI) or Luria Bertani Broth (LB). VRE is
quantified by using alternative selective media specific for VRE or
using qPCR probes specific for the pathogen. For example,
m-Enterococcus agar containing sodium azide is selective for
Enterococcus spp. and a small number of other species. Probes
specific to the van genes conferring vancomycin resistance are used
in the qPCR.
Example P5.
Testing of Bacterial Composition Against Salmonella
[0211] The in vitro assay is used to screen for combinations of
bacteria inhibitory to the growth of Salmonella spp. by modifying
the media used for growth of the pathogen inoculum. Several choices
of media are used for growth of the pathogen such as Reinforced
Clostridial Media (RCM), Brain Heart Infusion Broth (BHI) or Luria
Bertani Broth (LB). Salmonella spp. are quantified by using
alternative selective media specific for Salmonella spp. or using
qPCR probes specific for the pathogen. For example, MacConkey agar
is used to select for Salmonella spp. and the invA gene is targeted
with qPCR probes; this gene encodes an invasion protein carried by
many pathogenic Salmonella spp. and is used in invading eukaryotic
cells.
Example P6.
Method of Preparing the Bacterial Composition for Administration to
a Subject
[0212] Two strains for the bacterial composition are independently
cultured and mixed together before administration. Both strains are
independently be grown at 37.degree. C., pH 7, in a GMM or other
animal-products-free medium, pre-reduced with 1 g/L cysteineHCl.
After each strain reaches a sufficient biomass, it is preserved for
banking by adding 15% glycerol and then frozen at -80.degree. C. in
1 ml cryotubes.
[0213] Each strain is then be cultivated to a concentration of
10.sup.10 CFU/mL, then concentrated 20-fold by tangential flow
microfiltration; the spent medium is exchanged by diafiltering with
a preservative medium consisting of 2% gelatin, 100 mM trehalose,
and 10 mM sodium phosphate buffer, or other suitable preservative
medium. The suspension is freeze-dried to a powder and
titrated.
[0214] After drying, the powder is blended with microcrystalline
cellulose and magnesium stearate and formulated into a 250 mg
gelatin capsule containing 10 mg of lyophilized powder (10.sup.8 to
10.sup.11 bacteria), 160 mg microcrystalline cellulose, 77.5 mg
gelatin, and 2.5 mg magnesium stearate.
Example P7.
Method of Treating a Subject with a Bacterial Composition
[0215] A patient has suffered from recurrent bouts of C. difficile.
In the most recent acute phase of illness, the patient is treated
with an antibiotic sufficient to ameliorate the symptoms of the
illness. In order to prevent another relapse of C. difficile, the
patient is administered one of the present bacterial compositions.
Specifically, the patient is administered Bacillus circulans and
Roseburia inulinivorans at a dose of 10.sup.8 bacteria total in a
lyophilized form, specifically in a 250 mg gelatin capsule
containing 10 mg of lyophilized bacteria, 160 mg microcrystalline
cellulose, 77.5 mg gelatin, and 2.5 mg magnesium stearate. The
patient takes the capsule by mouth and resumes a normal diet after
4, 8, 12, or 24 hours. In another embodiment, the patient may take
the capsule by mouth before, during, or immediately after a
meal.
[0216] Feces is collected before and at 1 day, 3 days, 1 week, and
1 month after administration. The presence of C. difficile is found
in the feces before administration of the bacterial composition,
but feces collections after administration show reducing (such as
at least 50% less, 60%, 70%, 80%, 90%, or 95%) to no detectable
levels of C. difficile, as measured by qPCR, as described above.
ELISA for toxin protein or traditional microbiological
identification techniques may also be used.
[0217] As another measure of patient success, a positive response
may be defined as absence of diarrhea, which itself is defined as 3
or more loose or watery stools per day for at least 2 consecutive
days or 8 or more loose or watery stools in 48 hours, or persisting
diarrhea (due to other causes) with repeating (three times)
negative stool tests for toxins of C. difficile.
[0218] Treatment failure is defined as persisting diarrhea with a
positive C. difficile toxin stool test or no reduction in levels of
C. difficile, as measured by qPCR sequencing. ELISA or traditional
microbiological identification techniques may also be used.
Example P8.
Method of Treating a Subject with a Bacterial Composition
[0219] A patient has suffered from recurrent bouts of C. difficile.
In the most recent acute phase of illness, the patient is treated
with an antibiotic sufficient to ameliorate the symptoms of the
illness. In order to prevent another relapse of C. difficile, the
patient is administered one of the present bacterial compositions.
Specifically, the patient is administered a bacterial composition
containing two bacterial types from Table 1 or SEQ ID NOs. 1-1,864,
or a combination from Table 2, at a dose of 10.sup.8 bacteria total
in a lyophilized form formulated in an enteric coated capsule.
Example of the patient or samples derived from the patient is
expected to demonstrate at least one measure of success as
described herein (reducing levels of C. difficile as measured by
qPCR, ELISA, or traditional microbiological identification; absence
of diarrhea; persisting diarrhea with repeating (three times)
negative stool tests for toxins of C. difficile.
[0220] Other embodiments will be apparent to those skilled in the
art from consideration of the specification and practice of the
embodiments. Consider the specification and examples as exemplary
only, with a true scope and spirit being indicated by the following
claims.
TABLE-US-00001 Lengthy table referenced here
US20170165302A1-20170615-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00002 Lengthy table referenced here
US20170165302A1-20170615-T00002 Please refer to the end of the
specification for access instructions.
TABLE-US-00003 Lengthy table referenced here
US20170165302A1-20170615-T00003 Please refer to the end of the
specification for access instructions.
TABLE-US-00004 Lengthy table referenced here
US20170165302A1-20170615-T00004 Please refer to the end of the
specification for access instructions.
TABLE-US-00005 Lengthy table referenced here
US20170165302A1-20170615-T00005 Please refer to the end of the
specification for access instructions.
TABLE-US-00006 Lengthy table referenced here
US20170165302A1-20170615-T00006 Please refer to the end of the
specification for access instructions.
TABLE-US-00007 Lengthy table referenced here
US20170165302A1-20170615-T00007 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170165302A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170165302A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170165302A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References