U.S. patent application number 14/344967 was filed with the patent office on 2014-11-20 for media supplements and methods to culture human gastrointestinal anaerobic microorganisms.
This patent application is currently assigned to UNIVERSITY OF GUELPH. The applicant listed for this patent is Emma Allen-Vercoe, Julie McDonald. Invention is credited to Emma Allen-Vercoe, Julie McDonald.
Application Number | 20140342438 14/344967 |
Document ID | / |
Family ID | 47882498 |
Filed Date | 2014-11-20 |
United States Patent
Application |
20140342438 |
Kind Code |
A1 |
Allen-Vercoe; Emma ; et
al. |
November 20, 2014 |
MEDIA SUPPLEMENTS AND METHODS TO CULTURE HUMAN GASTROINTESTINAL
ANAEROBIC MICROORGANISMS
Abstract
A media supplement for culturing anaerobic bacteria is provided
which comprises a filtrate of effluent from a chemostat vessel in
which a target bacterial ecosystem has been cultured. Methods of
using the supplement for culturing or isolating anaerobic microbial
strains or communities, particularly anaerobic bacteria from the
human gut, are also provided.
Inventors: |
Allen-Vercoe; Emma; (Guelph,
CA) ; McDonald; Julie; (Guelph, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allen-Vercoe; Emma
McDonald; Julie |
Guelph
Guelph |
|
CA
CA |
|
|
Assignee: |
UNIVERSITY OF GUELPH
Guelph
ON
|
Family ID: |
47882498 |
Appl. No.: |
14/344967 |
Filed: |
September 14, 2012 |
PCT Filed: |
September 14, 2012 |
PCT NO: |
PCT/CA2012/050641 |
371 Date: |
May 5, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61534456 |
Sep 14, 2011 |
|
|
|
Current U.S.
Class: |
435/252.4 ;
435/252.1; 435/253.6 |
Current CPC
Class: |
C12R 1/145 20130101;
A61K 35/747 20130101; C12N 1/20 20130101; C12R 1/01 20130101; A61P
31/04 20180101; A61P 1/12 20180101; A61P 29/00 20180101; A61K 35/74
20130101; A61K 35/745 20130101 |
Class at
Publication: |
435/252.4 ;
435/252.1; 435/253.6 |
International
Class: |
C12N 1/20 20060101
C12N001/20 |
Claims
1-56. (canceled)
57. A media supplement for culturing anaerobic bacteria, the media
supplement comprising a filtrate of effluent from a chemostat
vessel in which a target bacterial ecosystem has been cultured,
wherein the target bacterial ecosystem has been cultured in culture
media which is Media 1, Media 1 comprising: 0.4% w/v Peptone; 0.4%
w/v Yeast extract; 0.4% w/v NaHCO.sub.3; 0.4% w/v Pectin; 0.4% w/v
Xylan; 0.4% w/v Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified
wheat starch; 0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v
L-cysteine HCl; 0.0002% w/v CaCl.sub.2; 0.0002% w/v NaCl; 0.0008%
w/v K.sub.2HPO.sub.4; 0.0008% w/v KH.sub.2PO.sub.4; 0.0002% w/v
MgSO.sub.4; 0.0001% w/v Hemin; and 0.00002% w/v menadione.
58. The media supplement of claim 57, wherein the target bacterial
ecosystem has been cultured in culture media which comprises
mucin.
59. The media supplement of claim 57, wherein the target bacterial
ecosystem is Defined Experimental Community 1 (DEC-1), Defined
Experimental Community 2 (DEC-2) or Defined Experimental Community
3 (DEC-3), wherein: (a) DEC-1 comprises the following bacterial
strains: Bacteroides ovatus, Bifidobacterium adolescentis,
Bifidobacterium longum, Collinsella aerofaciens, Eubacterium
rectale, Faecalibacterium prausnitzii, and Parabacteroides
distasonis; (b) DEC-2 comprises the following bacterial strains:
Bacteroides ovatus, Bacteroides vulgatus, Bifidobacterium
adolescentis, Bifidobacterium longum, Collinsella aerofaciens,
Eubacterium rectale, Faecalibacterium prausnitzii, Parabacteroides
distasonis, Roseburia inulinivorans, and Ruminococcus obeum; and
(c) DEC-3 comprises the following bacterial strains: Akkermemsia
muciniphila, Alistipes putredinis, Alistipes shahfi, Bacteroides
ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis,
Bacteroides vulgatus, Bifidobacterium longum, Collinsella
aerofaciens, Coprococcus comes, Dorea formicigenerans, Eubacterium
rectale, Faecalibacterium prausnitzii, Odoribacter splanchnicus,
Oscillibacter valericigenes, Ruminococcus bromii 1, Ruminococcus
bromii 2, Ruminococcus obeum, and Ruminococcus sp. 1.
60. The media supplement of claim 57, wherein the target ecosystem
which has been cultured in the chemostat comprises a community of
bacterial strains representing an enterotype of human gut, wherein
the enterotype is the Bacteroides enterotype, the Prevotella
enterotype or the Ruminococcus enterotype.
61. A method for preparing a media supplement for culturing
anaerobic bacteria, said method comprising the steps of: a)
culturing a target bacterial ecosystem in culture media in a
single-stage chemostat under conditions replicating normal human
colonic gastrointestinal tract, in equilibrium; b) collecting
effluent from the chemostat; and c) filtering the effluent through
a 0.2 .mu.m filter to remove bacterial cells, in order to produce
the media supplement; wherein the culture media is Media 1, which
comprises: 0.4% w/v Peptone; 0.4% w/v Yeast extract; 0.4% w/v
NaHCO.sub.3; 0.4% w/v Pectin; 0.4% w/v Xylan; 0.4% w/v
Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified wheat starch;
0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v L-cysteine HCl;
0.0002% w/v CaCl.sub.2; 0.0002% w/v NaCl; 0.0008% w/v
K.sub.2HPO.sub.4; 0.0008% w/v KH.sub.2PO.sub.4; 0.0002% w/v
MgSO.sub.4; 0.0001% w/v Hemin; and 0.00002% w/v menadione.
62. The method of claim 61, further comprising a step of
centrifuging the effluent at 14,000 rpm for 10 minutes and
collecting the supernatant before step c), and wherein the effluent
supernatant is then filtered in step c).
63. The method of claim 61, further comprising filtering the
effluent or effluent supernatant sequentially through a 1.0 .mu.m
filter, a 0.8 .mu.m filter, and a 0.45 .mu.m filter, before
filtering through the 0.2 .mu.m filter.
64. The method of claim 61, wherein the target bacterial ecosystem
comprises Defined Experimental Community 1 (DEC-1), Defined
Experimental Community 2 (DEC-2), or Defined Experimental Community
3 (DEC-3), wherein DEC-1, DEC-2 and DEC-3 are defined as follows:
DEC-1 comprises the following bacterial strains: Bacteroides
ovatus, Bifidobacterium adolescentis, Bifidobacterium longum,
Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium
prausnitzii, and Parabacteroides distasonis; DEC-2 comprises the
following bacterial strains: Bacteroides ovatus, Bacteroides
vulgatus, Bifidobacterium adolescentis, Bifidobacterium longum,
Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium
prausnitzii, Parabacteroides distasonis, Roseburia inulinivorans,
and Ruminococcus obeum; and DEC-3 comprises the following bacterial
strains: Akkermansia munciniphila, Alistipes putredinis, Alistipes
shahii, Bacteroides ovatus, Bacteroides thetaiotaomicron,
Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium
longum, Collinsella aerofaciens, Coprococcus comes, Dorea
formicigenerans, Eubacterium rectale, Faecalibacterium prausnitzii,
Odoribacter splanchnicus, Oscillibacter valericigenes, Ruminococcus
bromii 1, Ruminococcus bromii 2, Ruminococcus obeum, and
Ruminococcus sp. 1.
65. The method of claim 61, wherein the target ecosystem which has
been cultured in the chemostat comprises a community of bacterial
strains representing an enterotype of human gut, wherein the
enterotype is the Bacteroides enterotype, the Prevotella enterotype
or the Ruminococcus enterotype.
66. The method of claim 61, wherein the culture media comprises
mucin.
67. The method of claim 61, wherein the chemostat has a system
retention time of 24 hours.
68. The method of claim 61, wherein the conditions replicating
normal human colonic gastrointestinal tract comprise: a temperature
of about 37.degree. C.; a pH of about 6.9 to 7; a system retention
time of 24 hours; and maintenance of anaerobic conditions in the
chemostat.
69. A media supplement obtainable by the method of claim 61.
70. A method of isolating anaerobic bacteria from human gut,
comprising: a) culturing a target bacterial ecosystem in culture
media in a single-stage chemostat under conditions replicating
normal human colonic gastrointestinal tract, until equilibrium is
reached; b) diluting the culture and plating onto Fastidious
anaerobe agar (FAA) supplemented with the media supplement of claim
57, and optionally supplemented with defibrinated sheep blood; c)
incubating plates in an anaerobe chamber; d) purifying individual
anaerobic bacterial colonies grown in step (c); and e) optionally,
culturing the purified individual anaerobic bacterial colonies from
step (d) in liquid culture in a single-stage chemostat under
conditions replicating normal human colonic gastrointestinal tract,
optionally wherein the media supplement of claim 57 is used to
supplement culture media at about 1% v/v to about 10% v/v; such
that isolates of anaerobic bacteria are obtained.
71. The method of claim 70, wherein the anaerobe chamber contains
an atmosphere of N.sub.2, CO.sub.2 or H.sub.2, or a mixture
thereof.
72. The method of claim 70, wherein the target bacterial ecosystem
cultured in step (a) is a human fecal sample.
73. The method of claim 72, wherein the human fecal sample which is
cultured is a 10% w/v fecal slurry supernatant or a 20% w/v fecal
slurry supernatant.
74. The method of claim 70, wherein the culture media of step a) is
Media 1 comprising: 0.4% w/v Peptone; 0.4% w/v Yeast extract; 0.4%
w/v NaHCO.sub.3; 0.4% w/v Pectin; 0.4% w/v Xylan; 0.4% w/v
Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified wheat starch;
0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v L-cysteine HCl;
0.0002% w/v CaCl.sub.2; 0.0002% w/v NaCl; 0.0008% w/v
K.sub.2HPO.sub.4; 0.0008% w/v KH.sub.2PO.sub.4; 0.0002% w/v
MgSO.sub.4; 0.0001% w/v Hemin; and 0.00002% w/v menadione.
75. The method of claim 70, wherein the anaerobic bacteria obtained
is Faecalibacterium prausnitzii or Ruminococcus callidus
(ATCC27760).
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 61/534,456, filed on Sep. 14, 2011, the entire
contents of which are hereby incorporated by reference. This
application is related to copending international PCT application
titled "Method for Treatment of Disorders of the Gastrointestinal
System," filed Sep. 14, 2012, the entire contents of which are
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods for culturing microbial
communities from the human distal colon and methods for isolating
anaerobic microbes from such communities, as well as media
supplements for use in such methods.
BACKGROUND OF THE INVENTION
[0003] The human gut is the most densely inhabited ecosystem on
Earth (Marchesi and Shanahan, 2007). Like other complex microbial
ecosystems, the human microbiota has not been sampled to completion
(Eckburg et al., 2005). This is because the individual species of
the gut microbiota are difficult to culture axenically in vitro
(Hart et al., 2002). In fact, of the 500+ bacterial species which
colonize the human intestinal tract, about 75% have not been
cultured using conventional techniques (Duncan et al., 2007;
Eckburg et al., 2005; Hayashi et al., 2002). It is recognized that
novel culture techniques are required to grow these "unculturable"
microorganisms.
[0004] Studies of gut microbiota have been hampered by a lack of
model systems. While in vivo models can provide researchers with
physiologically relevant experimental models, they have several
drawbacks. For example, different study participants can each have
unique, host-specific community profiles representing their gut
microbiota, making comparison of the gut microbiota between
subjects difficult, especially when attempting to correlate the
effects of a treatment to changes in the gut microbiota. In vivo
models also often limit the dynamic monitoring of the gut
microbiota by deriving their data from end-point measurements.
Experiments involving humans or animals require research ethics
approval which can limit the experiments conducted on an
individual's gut microbiota in vivo.
[0005] In an attempt to improve upon the drawbacks of in vivo
models, several in vitro models have been developed. These in vitro
systems range from simple batch culture vessels to complex
continuous culture or themostat systems (Macfarlane, G. T. and
Macfarlane, S., Curr. Opin. Biotechnol., 18(2): 156-62, 2007).
Using chemostats, communities seeded from fresh feces can reach a
steady-state that closely resembles in vivo distal gut communities.
Being a host-free system, chemostats supporting gut microbiota make
ideal vessels in which to study microbial perturbations that
directly result from the addition of exogenous stimuli in isolation
from the effects of these stimuli on host physiology, making them
useful for mechanistic studies (Macfarlane, G. T. and Macfarlane,
S., Curr. Opin. Biotechnol., 18(2): 156-62, 2007).
[0006] In vitro models also provide several other advantages over
in vivo models in studies of the human gut microbiota. In vitro
studies are generally inexpensive and easy to set-up. They also
allow for the strict control of factors that influence the
environment while still facilitating frequent and simple sampling
of the simulated gut communities. However, while chemostats provide
a useful tool to investigate the microbial ecology of the gut,
operational parameters vary widely between different models in
different laboratories, often without experimental validation.
Preparation of the inocula, composition of the media, and retention
time of the vessel are parameters which can vary between different
studies.
[0007] To represent a valid model of the human distal gut,
communities which develop within chemostat vessels should share
some similarity to the fecal inoculum from which the gut community
was derived. The microbial ecosystem of the gut is a highly diverse
community, and it is therefore important that communities grown in
artificial systems also retain a high level of diversity (including
species richness and evenness). Finally, the reproducibility and
stability of these communities must be established and
characterized before experimentation can begin. This means that
microbial communities developed within these models must be
thoroughly analyzed and compared to in vivo communities before the
validity of a system can be confirmed.
[0008] Chemostat and fecal communities can be monitored using
molecular methods such as Denaturing Gradient Gel Electrophoresis
(DGGE). Currently, there is a lack of standardization between DGGE
analysis methods used in different research laboratories. Methods
of DGGE analysis vary from visual inspection to methods utilizing
statistical analysis software (such as GelCompar.TM., BioNumerics,
GeneTools, Quantity One.TM., etc.). Monitoring of communities using
computer software allows for more reliable and detailed analysis of
DGGE gels and provides more data on the composition and structure
of microbial communities, the stability of the community, and the
similarity between profiles. However, laboratories utilizing these
analysis programs do not use consistent methods when analyzing
their DGGE gels and report varying data on their communities. If
the analysis of DGGE gels can be standardized then this will
facilitate the comparison between the chemostat communities from
different laboratories.
[0009] It would be desirable therefore to be provided with
chemostat models of the human distal gut that are stable,
reproducible and biologically significant, as well as more complete
methods for the assessment and verification of such models.
SUMMARY OF THE INVENTION
[0010] There are provided herein methods for culturing
microorganisms that normally live in the human large intestine.
Methods for developing and characterizing microbial communities
from the human distal colon and for assessing and/or verifying such
communities are provided herein.
[0011] In an aspect, there are provided methods for culturing
microorganisms from the human distal colon using a media supplement
termed "Liquid Gold". Liquid Gold refers to a 0.2 .mu.m filtrate of
spent culture media or effluent from a chemostat vessel in which a
target ecosystem is cultured. Liquid Gold is used to supplement
culture media, e.g., standard laboratory media. Supplementation
with Liquid Gold allows culturing of "unculturable" microorganisms,
i.e., microorganisms which are refractory to culture axenically
using traditional methods. Without wishing to be limited by theory,
it is believed that Liquid Gold provides `growth signals` to
previously uncultured microbes to enhance their axenic growth in
vitro and hence allow them to be cultured and grown in vitro. It is
known in the art that certain microbes may grow well within a
microbial ecosystem, but are refractory to growth in isolation;
presumably the larger bacterial community in the ecosystem in some
way "supports" the growth of the microbes. We report herein that
this support can be provided by Liquid Gold to allow isolation of
certain microbes and to establish their growth as a pure isolate in
vitro, separate from the rest of the ecosystem.
[0012] Thus, in an aspect there is provided a method for growing
anaerobic bacteria comprising culturing the bacteria in a chemostat
under conditions replicating normal human colonic gastrointestinal
tract in equilibrium and then purifying individual anaerobic
bacteria into pure isolates.
[0013] In another aspect, there are provided microbial communities
from the human distal colon. In an embodiment, there is provided a
single-stage chemostat model of the human distal gut. In an
embodiment, microbial communities are stable, reproducible, and/or
biologically significant.
[0014] In another aspect, there is provided herein a media
supplement for culturing microbes termed "Liquid Gold." "Liquid
Gold" refers to filtered effluent from the chemostat, i.e., the
effluent forced out of the chemostat through pressure
differentials; it drips into sterile bottles, housed behind the
chemostat, via tubing. When the bottle is full, it is sealed and
can be stored at +4.degree. C. until needed. The effluent is passed
through a 0.2 .mu.m, e.g, a 0.22 .mu.m filter (Durapore, Millipore,
USA), to remove bacterial cells to produce cell-free Liquid Gold,
which is used to supplement culture media (usually added to 1% v/v,
3% v/v, 5% v/v, 7% v/v or 10% v/v). Liquid Gold is essentially
supernatant from a culture of microbes, containing a plethora of
signaling molecules, growth factors and so on. In an embodiment,
Liquid Gold is used to supplement culture media at 3% v/v. It
should be understood that Liquid Gold will differ depending on the
microbial community from which it is produced.
[0015] In an embodiment, there is provided a media supplement for
culturing anaerobic bacteria, the media supplement comprising a
filtrate of effluent from a chemostat vessel in which a target
bacterial ecosystem has been cultured. The filtrate may be, e.g., a
0.2 .mu.m filtrate. In one embodiment, the culture media in which
the target bacterial ecosystem is cultured is standard culture
media. In another embodiment, the culture media is Media 1. In an
embodiment, the culture media comprises mucin. Mucin may be present
in the culture media at a concentration of about 1-10%. In an
embodiment, mucin is present in the culture media at a
concentration of 4 g/L. In an embodiment, a human fecal sample has
been cultured in the chemostat. The human fecal sample may be, for
example, a 10% w/v fecal slurry supernatant or a 20% w/v fecal
slurry supernatant. In another embodiment, Defined Experimental
Community 1 (DEC-1), Defined Experimental Community 2 (DEC-2) or
Defined Experimental Community 3 (DEC-3) has been cultured in the
chemostat. In yet another embodiment, the target bacterial
ecosystem comprises at least one, at least three, at least five, at
least 8, at least 10, at least 15, or at least 25 of the bacterial
strains listed in Table 1, Table 2 or Table 3. In a further
embodiment, the target ecosystem which has been cultured in the
chemostat comprises a community of bacterial strains representing
an enterotype of human gut, e.g., the Bacteroides, the Prevotella
or the Ruminococcus enterotype. In another embodiment, the
anaerobic bacteria are bacteria found in the human gut
microbiome.
[0016] In an embodiment, the chemostat vessel used in the methods
and preparations of the invention is a single-stage chemostat.
[0017] In an embodiment, there is provided a media supplement for
culturing anaerobic bacteria, wherein the media supplement is
prepared by: a) culturing a target bacterial ecosystem in culture
media in a single-stage chemostat under conditions replicating
normal human colonic gastrointestinal tract, in equilibrium; b)
collecting effluent from the chemostat; and c) filtering the
effluent through a 0.2 .mu.m filter to remove bacterial cells, in
order to produce the media supplement. In an embodiment, the method
for preparing the media supplement further comprises a step of
centrifuging the effluent at 14,000 rpm for 10 minutes and
collecting the supernatant before step c), wherein the effluent
supernatant is then filtered in step c). In another embodiment, the
method for preparing the media supplement further comprises
filtering the effluent or effluent supernatant sequentially through
a 1.0 .mu.m filter, a 0.8 .mu.m filter, and a 0.45 .mu.m filter,
before filtering through the 0.2 .mu.m filter. In an embodiment,
the 0.2 .mu.m filter is a 0.22 .mu.m filter. The culture media may
be, e.g., standard culture media or Media 1.
[0018] In an embodiment, the target bacterial ecosystem is obtained
by culturing a human fecal sample, e.g., a 10% w/v fecal slurry
supernatant or a 20% w/v fecal slurry supernatant. In another
embodiment, the target bacterial ecosystem comprises Defined
Experimental Community 1 (DEC-1), Defined Experimental Community 2
(DEC-2), or Defined Experimental Community 3 (DEC-3). In another
embodiment, the target bacterial ecosystem comprises a community of
bacterial strains representing an enterotype of human gut, e.g.,
the Bacteroides, the Prevotella or the Ruminococcus enterotype. In
yet another embodiment, the target bacterial ecosystem comprises at
least one, at least three, at least five, at least 8, at least 10,
at least 15, or at least 25 of the bacterial strains listed in
Table 1, Table 2 or Table 3.
[0019] In an embodiment, the culture media is Media 1. In another
embodiment, the culture media comprises mucin, e.g., at a
concentration of 1-10%, e.g., at a concentration of 4 g/L. In an
embodiment, the chemostat is a single-stage chemostat. In an
embodiment, the chemostat has a system retention time of 24 hours.
In another embodiment, the conditions replicating normal human
colonic gastrointestinal tract comprise: a temperature of about
37.degree. C.; a pH of about 6.9 to 7; a system retention time of
24 hours; and maintenance of anaerobic conditions in the chemostat.
In another embodiment, the conditions replicating normal human
colonic gastrointestinal tract further comprise culturing the
target bacterial ecosystem in culture media containing mucin, e.g.,
mucin at a concentration of 1-10%, e.g., 4 g/L.
[0020] In an embodiment, there is provided herein a use of the
media supplement of the invention for growing anaerobic bacteria,
wherein the media supplement is used to supplement culture media in
a liquid culture at about 1% v/v to about 10% v/v. In an
embodiment, the media supplement is used to supplement culture
media at about 3% v/v. In an embodiment, the liquid culture is
grown in a chemostat. The media supplement may be added to the
culture media before culturing begins, or during culturing of the
anaerobic bacteria. There is also provided herein a use of the
media supplement of the invention for growing anaerobic bacteria,
wherein the media supplement is used to supplement solid culture
media, e.g., solid culture media in a Petri dish. In an embodiment,
the media supplement is added to FAA plates at a final
concentration of 3%.
[0021] In some embodiments, for the uses provided herein, the
anaerobic bacteria are bacteria found in human gut of a healthy
subject. In an embodiment, the anaerobic bacteria are
Faecalibacterium prausnitzii or Ruminococcus callidus
(ATCC27760).
[0022] In an embodiment, there is provided herein a method of
isolating anaerobic bacteria from human gut, comprising: a)
culturing a target bacterial ecosystem in culture media (e.g.,
standard culture media, Media 1) in a single-stage chemostat under
conditions replicating normal human colonic gastrointestinal tract,
until equilibrium is reached; b) diluting the culture and plating
onto Fastidious anaerobe agar (FAA) supplemented with the media
supplement of the invention, and optionally supplemented with
defibrinated sheep blood; c) incubating plates in an anaerobe
chamber; d) purifying individual anaerobic bacterial colonies grown
in step (c); and e) optionally, culturing the purified individual
anaerobic bacterial colonies from step (d) in liquid culture in a
single-stage chemostat under conditions replicating normal human
colonic gastrointestinal tract, optionally wherein the media
supplement of the invention is used to supplement culture media at
about 1% v/v to about 10% v/v; such that isolates of anaerobic
bacteria are obtained. In an embodiment, the media supplement is
used to supplement the culture media in step (e) at about 3% v/v.
In another embodiment, the media supplement is added at a final
concentration of 3% in step (b). In yet another embodiment, the
defibrinated sheep blood is added at a final concentration of 5%.
In a still further embodiment, the conditions replicating normal
human colonic gastrointestinal tract comprise: a temperature of
about 37.degree. C.; a pH of about 6.9 to 7; a system retention
time of 24 hours; and maintenance of anaerobic conditions in the
chemostat. In an embodiment, the conditions replication normal
human colonic gastrointestinal tract further comprise culturing in
culture media to which mucin has been added. In an embodiment,
bacteria are cultured in the chemostat in steps (a) and (e) under
reduced atmosphere with controlled levels of partial pressure of
N.sub.2:CO.sub.2:H.sub.2. For example, the preparation may be under
N.sub.2, CO.sub.2 or H.sub.2, or a mixture thereof. In an
embodiment, the mixture thereof is N.sub.2:CO.sub.2:H.sub.2. In
another embodiment, anaerobic conditions are maintained by bubbling
filtered nitrogen gas through the cultures in steps (a) and
(e).
[0023] In some embodiments, the target bacterial ecosystem cultured
in step (a) is a human fecal sample, e.g., a 10% w/v fecal slurry
supernatant or a 20% w/v fecal slurry supernatant. In an
embodiment, standard culture media is used in the chemostat. In an
embodiment, Media 1 is used in the chemostat.
[0024] In an embodiment, Faecalibacterium prausnitzii or
Ruminococcus callidus (ATCC27760) is isolated. In another
embodiment, a pure isolate of Faecalibacterium prausnitzii,
Clostridium aldenense 1, Clostridium aldenense 2, Clostridium
hathewayi 1, Clostridium hathewayi 2, Clostridium hathewayi 3,
Clostridium thermocellum, Ruminococcus bromii 2, Ruminococcus
torques 4, Ruminococcus torques 5, Clostridium cocleatum (e.g.,
Clostridium cocleatum 21FAA1), Eubacterium desmolans (e.g.,
Eubacterium desmolans 48FAA1), Eubacterium limosum 13LG,
Lachnospira pectinoshiza, Ruminococcus productus (e.g.,
Ruminococcus productus 27FM), Ruminococcus obeum (e.g.,
Ruminococcus obeum 11FM1), Blautia producta, or Clostridium
thermocellum is obtained.
[0025] In an embodiment, there is provided herein a pure isolate of
Faecalibacterium prausnitzii, Clostridium aldenense 1, Clostridium
aldenense 2, Clostridium hathewayi 1, Clostridium hathewayi 2,
Clostridium hathewayi 3, Clostridium thermocellum, Ruminococcus
bromii 2, Ruminococcus torques 4, Ruminococcus torques 5,
Clostridium cocleatum (e.g., Clostridium cocleatum 21FAA1),
Eubacterium desmolans (e.g., Eubacterium desmolans 48FAA1),
Eubacterium limosum 13LG, Lachnospira pectinoshiza, Ruminococcus
productus (e.g., Ruminococcus productus 27FM), Ruminococcus obeum
(e.g., Ruminococcus obeum 11FM1), Blautia producta, or Clostridium
thermocellum. A pure isolate may be obtained, e.g., using methods
provided herein.
[0026] In an embodiment, there is provided a method of culturing a
microbial community from human gut, comprising: a) obtaining a
fecal sample from a healthy human subject; b) inoculating a culture
with the fecal sample; and c) culturing the culture in culture
media (e.g., standard culture media, Media 1, etc.) in a
single-stage chemostat under conditions replicating normal human
colonic gastrointestinal tract, until equilibrium is reached; such
that a microbial community comprising bacterial strains found in
human gut is obtained. In an embodiment, the microbial community
represents a human gut enterotype. In another embodiment, the fecal
sample obtained in step (a) is prepared as a 10% w/v fecal slurry
supernatant or a 20% w/v fecal slurry supernatant before
inoculating the culture in step (b).
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0028] For a better understanding of the invention and to show more
clearly how it may be carried into effect, reference will now be
made by way of example to the accompanying drawings, which
illustrate aspects and features according to preferred embodiments
of the present invention, and in which:
[0029] FIG. 1 shows a single-stage chemostat vessel developed by
modifying a Multifors fermentation system which was used for
growing the isolated bacterial strains as described herein.
[0030] FIG. 2 shows a clustering Tree based on Dice similarity
coefficient and Unweighted Pair Group Method with Arithmetic Mean
(UPGMA) correlation of the DGGE profiles showing the 10% fecal
inocula prepared from Donor 2 feces on several different donations
over an 8 month period. The predominant bacterial species from this
healthy donor remained stable over time.
[0031] FIG. 3 shows a clustering Tree based on Dice similarity
coefficient and UPGMA correlation of the DGGE profiles showing the
10% fecal inocula prepared from four different donors (donors 1-4).
Each donor had a unique profile, with the profiles from some donors
more similar to each other than others (e.g., Donors 2 and 3).
[0032] FIG. 4 shows a clustering Tree based on Dice similarity
coefficient and UPGMA correlation of the DGGE profiles showing the
10% vs. 20% fecal inocula prepared from Donor 2 feces on two
different donations. The 10% and 20% inocula were very similar to
each other, therefore justifying the use of the 10% inocula (which
requires less fecal donation and is easier to administer to the
chemostat vessel upon inoculation).
[0033] FIG. 5 shows the reproducibility of two chemostat vessels
(V1 and V2) seeded with identical fecal inoculum from Donor 2. A)
DGGE profiles showing communities on days 0, 10, 26 and 28; B)
Correlation coefficients (expressed as percentages) comparing the
profiles of each vessel at the same time point, plotted over the
course of the experiment; C) Community dynamics as shown using
moving window correlation analysis. Similarity of the community
within each vessel was calculated by comparing the profile of day
(x) and day (x-2); D) Shannon Diversity Index (H') plot
representing the community diversity of each vessel over the course
of the experiment; E) Range weighted richness (Rr) plot
representing the richness in each vessel over the course of the
experiment; F) Shannon equitability index (E.sub.H) plot
representing the community evenness values from each vessel over
the course of the experiment. Without mucin, two vessels could be
run in parallel and maintain identical communities, reaching steady
state at about 26-28 days post-inoculation.
[0034] FIG. 6 shows a comparison of the chemostat media used in our
laboratory ("Medial"; used to feed V1) to a previously published
medium (V6; Walker et al., Appl. Environ. Microbiol.,
71(7):3692-700, 2005). The same fecal inoculum (from Donor 2, 10%)
was used to seed each vessel. A) DGGE profiles showing communities
on days 0, 10, 26 and 36; B) Correlation coefficients (expressed as
percentages) comparing the profiles of each vessel at the same time
point, plotted over the course of the experiment; C) Community
dynamics as shown using moving window correlation analysis.
Similarity of the community within each vessel was calculated by
comparing the profile of day (x) and day (x-2); D) Shannon
Diversity Index (H') plot representing the community diversity of
each vessel over the course of the experiment; E) Range weighted
richness (Rr) plot representing the richness in each vessel over
the course of the experiment; F) Shannon equitability index
(E.sub.H) plot representing the community evenness values from each
vessel over the course of the experiment. Comparison shows that the
media recipe we developed (Medial) provides a suitable medium to
grow a stable and diverse chemostat community when compared to the
previously published medium.
[0035] FIG. 7 shows a comparison of a 65 hour retention time (V1)
to a 24 hour retention time (V2). The same fecal inoculum (from
Donor 2, 10%) was used to seed each vessel. A) DGGE profiles
showing communities on days 0, 6, 10 and 14; B) Correlation
coefficients (expressed as percentages) comparing the profiles of
each vessel at the same time point, plotted over the course of the
experiment; C) Community dynamics as shown using moving window
correlation analysis. Similarity of the community within each
vessel was calculated by comparing the profile of day (x) and day
(x-2); D) Shannon Diversity Index (H') plot representing the
community diversity of each vessel over the course of the
experiment; E) Range weighted richness (Rr) plot representing the
richness in each vessel over the course of the experiment; F)
Shannon equitability index (E.sub.H) plot representing the
community evenness values from each vessel over the course of the
experiment. Increasing the retention time from the biologically
significant value of 24 hours to 65 hours resulted in a community
which was less similar to its inoculum and did not maintain a
higher level of diversity.
[0036] FIG. 8 shows the effect of mucin on the diversity of distal
gut communities grown in a single-stage chemostat. The same fecal
inoculum (from Donor 2, 10%) was used to seed each vessel. A) DGGE
profiles showing communities on days 0 and 24; B) Correlation
coefficients (expressed as percentages) comparing the profiles of
V1 (no mucin) to V5 and V6 (with mucin) on days 0 and 24; C)
Correlation coefficients (expressed as percentages) comparing the
profiles of V1 (no mucin) to the average values from V5 and V6
(with mucin) on days 0 and 24; D) Shannon Diversity Index (H')
representing the community diversity of each vessel on days 0 and
24; E) Range weighted richness (Rr) representing the richness in
each vessel on days 0 and 24; F) Shannon equitability index (J)
representing the community evenness values from each vessel on days
0 and 24. Addition of mucin to the chemostat resulted in increases
in community diversity, richness, and evenness.
[0037] FIG. 9 shows a schematic description of measures used to
characterize microbial ecological communities (dynamics, diversity,
evenness and richness). The schematic diagram explains basic
ecological concepts (including community dynamics, diversity,
evenness, and richness). A) Community dynamics represents the
changes within the community over a fixed time frame using moving
window correlation analysis (Marzorati, M. et al., Environ.
Microbiol., 10:1571-1581, 2008; Possemiers, S. et al., FEMS
Microbiol. Ecol., 49: 495-507, 2004); B) Shannon diversity index is
a measure of community diversity which takes both species richness
(number of species present) and evenness (relative species
abundance) into account (Gafan, G. P. et al., J. Clin. Microbiol.,
43: 3971-3978, 2005); c) Shannon equitability index describing
community evenness, or the degree to which the numbers of
individuals are evenly divided between the different species of the
community (Pielou, E. C. 1975. Ecological diversity. Wiley, New
York); D) Community richness refers to the number of species
present in the ecosystem; this measure does not take relative
species abundance into account.
[0038] FIG. 10 depicts DGGE profiles comparing fecal communities to
the communities present in the chemostat vessels immediately
following inoculation. Two different chemostat runs were compared
for each healthy donor (Donors 5 and 6). The fecal inocula used to
seed the chemostat vessels was very similar to the starting fecal
donation and not altered significantly by the process of preparing
the inoculum.
[0039] FIG. 11 depicts DGGE profiles comparing fecal communities
from two different healthy donors (Donors 5 and 6). Each donor
provided a sample on two different occasions. Donors 5 and 6 had
different DGGE profiles. The DGGE profiles from both donors were
consistent between donations.
[0040] FIG. 12 depicts DGGE profiles comparing fecal communities
present in the chemostat vessels immediately following inoculation
to the steady state communities (samples obtained 36 days
post-inoculation) for two different healthy donors (donors 5 and
6). Two different vessels were seeded with identical fecal inoculum
for each chemostat run and two different chemostat runs were
compared for each healthy donor. By DGGE, the fecal inocula from
the same donor on two different occasions were more similar to each
other than to fecal inocula from the other donor. Also, the steady
state communities seeded with feces from the same donor were more
similar to each other between chemostat runs than to the
communities seeded with feces from another donor.
[0041] FIG. 13 shows community analysis of two identical chemostat
vessels modeling the human distal gut. Each vessel was seeded with
identical fecal inocula prepared from the feces of a healthy donor
(donor 5). Parameters were calculated by analyzing DGGE patterns of
general Bacteria (V3 region of the 16S gene) using GeneTools
statistical analysis software. Samples were analyzed every two days
throughout the duration of the experiment (days 0-48). The vertical
dashed line represents the beginning of steady state conditions. A:
Correlation coefficients (expressed as percentages) comparing the
profiles of each vessel at the same time point, plotted over the
course of the experiment. The horizontal dotted line represents the
cut-off threshold calculated by comparing the similarity of
identical marker lanes run on a single DGGE gel. The horizontal
dashed line represents the cut-off threshold -5% and allows for a
5% difference in similarity between the profiles of each vessel. Up
until day 48, both vessels were able to maintain very similar DGGE
profiles. B: Correlation coefficients comparing the profiles of
samples taken from each vessel over the course of the experiment to
its starting inocula. The horizontal dotted line represents the
cut-off threshold and the horizontal dashed line represents the
cut-off threshold -5%. While the steady state community was
different from the starting inoculum, the similarity was relatively
consistent over time. C: Community dynamics as shown using moving
window correlation analysis. Variability of the community within
each vessel was calculated by comparing the profile of day (x) and
day (x-2). The horizontal dotted line represents 100-(cut-off
threshold) and the horizontal dashed line represents 100-(cut-off
threshold -5%). By day 36 the communities within both vessels had
reached steady state (when confirmed by visual inspection of the
DGGE profiles between vessels). D: Shannon Diversity Index (H) plot
representing the corrected community diversity of each vessel over
the course of the experiment. The horizontal dotted line represents
the average Shannon diversity index value of the starting inocula.
The diversity in both vessels was similar to each other, stable
over time, and similar to that of the starting inocula. E:
Community richness (S) plot represented by plotting the number of
corrected observed bands in each DGGE gel against time. The
horizontal dotted line represents the average richness value of the
starting inocula. The richness in both vessels was similar to each
other, stable over time, and similar to that of the starting
inocula. F: Shannon equitability index (E.sub.H) plot representing
the corrected community evenness values from each vessel over the
course of the experiment. The horizontal dotted line represents the
average Shannon equitability index value of the starting inocula.
The evenness in both vessels was similar to each other, stable over
time, and similar to that of the starting inocula.
[0042] FIG. 14 shows representative plates demonstrating growth of
the Faecalibacterium prausnitzii strain, which showed differential
growth in response to Liquid Gold media supplement included in the
agar media preparation at 3%. Plates were inoculated with identical
inocula and incubated at 37.degree. C. for 3 days under total
anaerobic conditions. Plate A: Fastidious anaerobe agar (FAA)
supplemented with 5% defibrinated sheep blood alone. Plate B: FAA
supplemented with 5% defibrinated sheep blood and 3% filtered
(cell-free) Liquid Gold media supplement (from Donor 6). Growth was
clearly enhanced by addition of Liquid Gold media supplement to the
media.
DETAILED DESCRIPTION OF THE INVENTION
[0043] According to a broad aspect of the invention there are
provided herein novel methods of culturing anaerobic microorganisms
or microbes using a single-stage chemostat system. There is also
provided a novel media supplement, termed "Liquid Gold," for use in
culturing such microorganisms or microbes, in particular those
which are traditionally difficult to grow. Methods provided herein
can be used, inter alia, to culture enterotypes of the human gut
and to provide models of bacterial communities of the human distal
colon. Methods provided herein are particularly suited to culturing
anerobic bacteria, such as those found in the human gut.
[0044] The human gastrointestinal tract contains vast numbers of
bacteria, collectively called the intestinal microbiota. The
commensal gut flora contribute to host defense by priming the
dendritic cells of the immune system, producing bactericidal
products that kill pathogenic bacteria, inhibiting the colonization
of pathogenic bacteria and competing with pathogens for food and
for binding sites along the intestinal epithelial cell surface, a
phenomenon collectively known as "colonization resistance" (Stecher
B. and Hardt W. D., Trends Microbiol. (2008), 16:107-14; Rolfe, R.
D., Infect. Immun. (1984), 45:185-91).
[0045] Recent studies have suggested that intestinal or gut
enterotypes may not be specific to an individual but, rather, are
representative of different states of equilibrium that exist in the
gut microbiota in response to dietary stimuli. It has been reported
that the human gut microbiome, that is, the community of organisms
that live symbiotically within humans, occurs in certain set
varieties or "enterotypes." Three main enterotypes of the human
gut, which vary in species and functional composition, have been
identified to date, and are termed Bacteroides, Prevotella and
Ruminococcus (Arumugam, M. et al., Nature 12; 473(7346):174-80,
Epub Apr. 20, 2011).
[0046] In an aspect, there are provided herein culture methods for
culturing enterotypes of the human gut. As reported herein, we
harvested fecal samples from donors of each enterotype and used a
single-stage chemostat system to culture a bacterial community
modelling the community of the donor's distal intestine or gut.
Thus, in an embodiment there are provided herein methods for
culturing bacterial communities which model enterotypes of the
human gut. Methods provided herein can be used to form microbial
communities which are stable, reproducible, diverse, and/or
biologically significant, in terms of modeling the human distal
colon. As reported herein, steady-state communities are generally
produces at about one-month, e.g., at approximately 26 days,
post-inoculation with a fecal sample, using methods described
herein.
[0047] In addition, there are provided herein three Defined
Experimental Communities (DECs) of microorganisms from human fecal
samples. As described, using methods provided herein we subcultured
fecal samples from donors representing different enterotypes. Fecal
samples were harvested and used to generate DECs of microorganisms
modeling enterotypes of the human gut. In an embodiment, three DECs
(referred to herein as DEC-1, DEC-2 and DEC-3) are provided.
[0048] DEC-1 comprises intestinal bacterial strains that were
isolated and purified from donor stool from a donor who had not
received antibiotics in the last 5 years (this donor is also
referred to herein as "Donor 6"). DEC-1 includes 33-strains
(representing a total of 26 species), as shown in Table 1; this DEC
has been used successfully to treat two patients (Kingston General
Hospital) with recurrent Clostridium difficile infection,
demonstrating that the DEC successfully models a microbial
community of the human distal colon. Strains were speciated using
the 16S rRNA full-length sequence and the GreenGenes database
(http://greengenes.lbl.gov/cgi-bin/nph-blast_interface.cgi).
[0049] DEC-2 (from the same donor as DEC-1) includes all the
strains in DEC-1 as well as additional bacterial species. Bacterial
strains included in DEC-2 are shown in Table 3.
[0050] DEC-3 includes isolates of bacterial species shown in Table
2. DEC-3 was isolated from a male donor, 43 yrs old, with no
history of antibiotic use in the 6 years prior to stool donation
(referred to herein as "Donor 5"). Notably, DEC-3 contains a number
of microbes which are either known or speculated to be highly
beneficial and enriched in healthy individuals, such as Akkermansia
muciniphila, Faecalibacterium prausnitzii, Bifidobacterium spp.,
and Adlercreutzia equolifasciens.
[0051] In an embodiment, methods are provided herein for culturing
bacterial communities which model enterotypes of the human gut use
a single-stage chemostat system. This system has been optimized for
growing gastrointestinal microbes. In an embodiment, there is
provided a chemostat system using the following culture media,
referred to herein as "Media 1": Peptone (0.4% w/v); Yeast extract
(0.4% w/v); NaHCO3 (0.4% w/v); Pectin (from citrus, 0.4% w/v);
Xylan (from beechwood, 0.4% w/v); Arabinogalactan (0.4% w/v);
Casein (0.6% w/v); unmodified wheat starch (1% w/v); inulin (0.2%
w/v); bile salts (0.1% w/v); L-cysteine HCl (0.1% w/v); CaCl2
(0.0002% w/v); NaCl (0.0002% w/v); K2HPO4 (0.0008% w/v); KH2PO4
(0.0008% w/v); MgSO4 (0.0002% w/v); Hemin (0.0001% w/v); menadione
(0.00002% w/v); mucin (porcine, 0.004% w/v)
[0052] In an embodiment, the culture media used in methods of the
invention, e.g., standard culture media, Media 1, etc., further
comprises mucin. It will be understood by the skilled artisan that
mucin from any source which is available and affordable can be
used. For example, mucin from mammalian sources, such as bovine
mucin, porcine mucin, etc., may be used. In an embodiment, porcine
mucin is used.
[0053] In another embodiment, there is provided herein a media
supplement termed "Liquid Gold" and its use to supplement standard
laboratory culture media to enhance growth capabilities for
microbes that were otherwise considered "unculturable," such as,
e.g., certain gastrointestinal, anaerobic microbes. "Liquid Gold"
refers to the effluent from a chemostat in which a bacterial
community is grown, i.e., the effluent forced out of the chemostat
through pressure differentials. Effluent drips into sterile
bottles, housed behind the chemostat, via tubing. When the bottle
is full, it can be sealed and stored at +4.degree. C. until needed.
The effluent is passed through a filter, e.g., a 0.22 .mu.m filter,
to remove bacterial cells to produce cell-free Liquid Gold, which
is used to supplement culture media.
[0054] The optimal amount of Liquid Gold to be added to a culture
will vary depending on experimental conditions and the microbes to
be grown, and will be determined by the skilled artisan using
standard techniques. In an embodiment, Liquid Gold is added to 1%
v/v, 3% v/v, 5% v/v, 7% v/v or 10% v/v. In a particular embodiment,
Liquid Gold is used to supplement growth media at 3% v/v.
[0055] Liquid Gold is essentially a supernatant from a microbial
culture and includes a plethora of signaling molecules, growth
factors, and so on. It should be understood that the composition of
a Liquid Gold preparation will depend on the microbial community
from which it is produced. Different types of Liquid Gold can thus
be made by growing different bacterial communities in a chemostat.
For example, "Native Liquid Gold" is produced from chemostat
effluent from culturing complete native feces in a chemostat, e.g.,
a single-stage chemostat system, as described below. "DEC Liquid
Gold" is produced from chemostat effluent from culturing a Defined
Experimental Community (such as, e.g., DEC-1) in a chemostat, e.g.,
a single-stage chemostat system, as described below. As used
herein, "DEC-1 Liquid Gold" refers to Liquid Gold produced from
chemostat effluent from culturing the DEC-1 community; "DEC-2
Liquid Gold" refers to Liquid Gold produced from chemostat effluent
from culturing the DEC-2 community; and "DEC-3 Liquid Gold" refers
to Liquid Gold produced from chemostat effluent from culturing the
DEC-3 community. As reported herein, fecal samples were harvested
from donors of different enterotypes and cultured, and we were
therefore able to produce several different types of Liquid Gold
media supplement, including, e.g., DEC-1 Liquid Gold, DEC-2 Liquid
Gold, DEC-3 Liquid Gold, and Native Liquid Gold.
TABLE-US-00001 TABLE 1 Intestinal bacterial strains isolated and
purified from donor stool in DEC-1. Closest species match, inferred
by alignment of 16SrRNA Relative abundance sequence to GreenGenes %
identity to (by biomass) database* closest match in DEC-1
Acidaminococcus intestinalis 100 +++ Bacteroides ovatus 99.52 +
Bifidobacterium adolescentis 99.79 ++ (2 different strains) 99.79
++ Bifidobacterium longum 99.86 +++ (2 different strains) 99.16 +++
Blautia producta** 96.43 + Clostridium cocleatum 91.92 +
Collinsella aerofaciens 98.73 + Dorea longicatena 99.62 + (2
different strains) 99.60 + Escherichia coli 99.80 + Eubacterium
desmolans 94.90 + Eubacterium eligens 98.15 +++++ Eubacterium
limosum 97.05 + Eubacterium rectale 99.59 +++++ (4 different
strains) 99.60 +++++ 99.19 +++++ 99.53 +++++ Eubacterium ventriosum
100 ++ Faecalibacterium prausnitzii 99.17 +++++ Lachnospira
pectinoshiza 95.22 + Lactobacillus casei/paracasei 99.47 +
Lactobacillus casei 99.74 + Parabacteroides distasonis 99.45 ++
Raoultella sp. 99.40 + Roseburia faecalis 99.65 ++ Roseburia
intestinalis 100 ++ Ruminococcus torques 99.15 +++ (2 different
strains) 99.29 +++ Ruminococcus obeum 94.89 + (2 different strains)
94.69 + Streptococcus mitis.sup..PSI. 99.79 + *Closest species
match was inferred by alignment of 16SrRNA sequence to GreenGenes
database; note that in some cases 16SrRNA gene sequences could not
resolve identity beyond genus, and that closest match does not
infer definitive speciation. Shaded boxes indicate strains that are
likely novel species (and in some cases, genera). Note that some
representative strains identify with the same species by 16SrRNA
gene sequence alignment, but we believe them to be different
strains based on differences in colony morphology, antibiotic
resistance patterns and growth rates. **Also referred to as
Ruminococcus productus. .sup..PSI.Identifies with Strep. mitis but
is not .alpha.-hemolytic.
TABLE-US-00002 TABLE 2 Intestinal bacterial strains isolated and
purified from donor stool in DEC-3. No. Strain Closest species
.sup.c % ID.sup.a 1 11 TSAB Adlercreutzia equolifaciens 99.76% 2 18
FAA SS Akkermansia muciniphila 100% 3 9 FAA NB Alistipes finegoldii
99.27% 4 19 D5 FAA Alistipes putredinis 97.15% 5 15 D5 FAA
Alistipes shahii 99.85% 6 5 D5 FAA SS Alistipes sp. 100% 7 5 D5 FAA
Bacteroides capillosus 96.98% 8 12 FAA Bacteroides cellulosilyticus
99.46% 9 9 D5 FAA Bacteroides eggerthii 100% 10 1 D6 FAA SS
Bacteroides ovatus 100% 11 23 FAA Bacteroides thetaiotaomicron 100%
12 1 TSAB Bacteroides uniformis 100% 13 17 BHI Bacteroides vulgatus
99.85% 14 3 FAA SS AER. Bacillus circulans 100% 15 1 D5 FAA SS AER.
Bacillus simplex 98.70% 16 1 D6 FAA Bifidobacterium longum 100% 17
18 D6 FAA SS Blautia hydrogenotrophica 100% 18 8 FAA Blautia sp.
99.15% 19 4 TSA SS Blautia/Clostridium coccoides 99.85% 20 1 D6 FAA
SS AER. Brevibacillus parabrevis 97.60% 21 3 MRS SS Catabacter
hongkongensis 98.65% 22 16 TSA SS Catabacter sp. 99.05% 23 10 TSAB
Catenibacterium mitsuokai 99.40% 24 13 D6 FAA SS Clostridium
aldenense 1 92.04% 25 21 D6 FAA SS Clostridium aldenense 2 92.24%
26 13 D5 FAA SS Clostridium asparagiforme 94.37% 27 3 D6 FAA SS
Clostridium bolteae 99.84% 28 6 D5 FAA Clostridium celerecrescens
94.48% 29 13 D6 FAA Clostridium hathewayi 1 92.19% 30 21 FAA NB SS
Clostridium hathewayi 2 91.28% 31 10 FAA Clostridium hathewayi 3
92.99% 32 11 FAA Clostridium hathewayi 4 98.64% 33 6 D6 FAA SS
Clostridium hylemonae 1 99.85% 34 8 D5 FAA SS Clostridium hylemonae
2 97.85% 35 5 FAA SS Clostridium inocuum 99.12% 36 11B D5 FAA SS
Clostridium lavalense 99.08% 37 16 D5 FAA SS Clostridium leptum
93.92% 38 4 TSA Clostridium orbiscindens 96.21% 39 14 TSA
Clostridium ramosum 96.14% 40 5 D6 FAA SS Clostridium scindens
99.82% 41 16 BHI SS Clostridium staminisolvens 95.40% 42 17 D5 FAA
SS Clostridium sulfatireducens 96.63% 43 2 FAA SS Clostridium
symbiosum 99.83% 44 16 BHI Clostridium thermocellum 90.83% 45 18 D5
FAA Clostridium sp. 1 99.16% 46 2 BHI SS Clostridium sp. 2 97.16%
47 20 D5 FAA Clostridium sp. 3 95.51% 48 16 D6 FAA SS Clostridium
sp. 4 98% 49 9 D5 FAA SS Clostridium sp. 5 97.88% 50 5 TSA
Clostridium sp. 6 96.95% 51 6 FAA Collinsella aerofaciens 100% 52
17 D5 FAA Coprococcus catus 99.19% 53 1 BHI Coprococcus comes
99.70% 54 13 FAA Coprococcus eutactus 96.49% 55 5 NA Dorea
formicigenerans 99.49% 56 1 D5 FAA Dorea longicatena 100% 57 1 FAA
SS AER. Escherichia coli 100% 58 5 TSAB Eubacterium biforme 98.76%
59 11 NA SS Eubacterium callanderi 98.08% 60 19 FAA NB SS
Eubacterium dolichum 93.23% 61 20 FAA Eubacterium eligens 96.78% 62
9 TSAB SS Eubacterium fissicatena 97.67% 63 1 BHI SS Eubacterium
limosum 99.25% 64 5 D6 FAA Eubacterium rectale 100% 65 13 BHI
Eubacterium siraeum 93.57% 66 8 MRS Eubacterium ventriosum 97.37%
67 22 D6 FAA Eubacterium xylanophilum 1 97.39% 68 15 FAA SS
Eubacterium xylanophilum 2 96.53% 69 23 D6 FAA SS Eubacterium sp.
94.31% 70 5 FAA NB Faecalibacterium prausnitzii .sup.b 100% 71 24
FAA Gemmiger/Subdoligranulum 98.79% formicilis/variabile 1 72 19 D5
FAA SS Gemmiger/Subdoligranulum 95.18% formicilis/variabile 2 73 17
D6 FAA SS Holdemania filiformis 97.51% 74 1 FAA NB SS AER.
Microbacterium schleiferi 99.34% 75 7 FAA NB SS AER. Micrococcus
luteus 97.04% 76 21 D6 FAA Odoribacter splanchnicus 100% 77 24 D6
FAA SS Oscillibacter valericigenes 95.16% 78 6 FAA NB Oscillibacter
sp. 98.74% 79 16 FAA Parabacteroides gordonii 99.81% 80 6 D6 FAA
Parabacteroides merdae 100% 81 10 D5 FAA SS Parasutterella
excrementihominis 100% 82 22 FAA Phascolarctobacterium sp. 99.85%
83 10 D5 FAA Roseburia faecalis 1 99.84% 84 9 D6 FAA Roseburia
faecalis 2 96.76% 85 9A BHI Roseburia hominis 99.04% 86 17 TSA
Roseburia intestinalis 100% 87 11 TSA Roseburia sp. 95.07% 88 23 D5
FAA Ruminococcus albus 96.96% 89 6 FAA NB SS Ruminococcus bromii 1
100% 90 17 FAA SS Ruminococcus bromii 2 92.83% 91 17 TSAB
Ruminococcus lactaris 94.46% 92 2 FAA NB Ruminococcus luti 98.91%
93 15 TSA Ruminococcus obeum 99.06% 94 4 FAA Ruminococcus torques 1
99.27% 95 11 FAA Ruminococcus torques 2 100% 96 8 D6 FAA SS
Ruminococcus torques 3 96.47% 97 9B D6 FAA SS Ruminococcus torques
4 91.94% 98 13 FAA NB Ruminococcus torques 5 91.47% 99 5 BHI
Ruminococcus sp. 1 94.32% 100 11 FAA NB Ruminococcus sp. 2 98.04%
101 4 D6 FAA SS Ruminococcus sp. 3 97.05% 102 4 FAA SS AER.
Staphylococcus epidermidis 99.82% 103 1 FAA NB SS Streptococcus
mitis 100% 104 11 FAA NB SS Streptococcus thermophilus 100% 105 12
D6 FAA SS Synergistes sp. 95.83% 106 16 D5 FAA Turicibacter
sanguinis 100% .sup.a% ID for each species was determined using the
16SrRNA gene database, Green Genes. Average length of sequences
used to obtain identification was 550 nucleotides. (Green Genes
BLAST interface to 16S data URL:
http://greengenes.lbl.gov./cgi-bin/nph-blast interface.cgi) .sup.b
The strain Faecalibacterium prausnitzii 5 FAA NB requires Liquid
Gold for growth. A 3% final volume of Liquid Gold produced from the
chemostat where the donor fecal sample was cultured was used to
supplement FAA plates. Growth was observed after 48 hours. .sup.c
Multiple strains of the same species are denoted by a number
following the species name. For example, Clostridium aldenense 1
and 2 are two different strains of the same species. Shaded boxes
indicate strains that are likely novel species (and in some cases,
genera)
TABLE-US-00003 TABLE 3 Intestinal bacterial strains isolated and
purified from donor stool in DEC-2. Closest species match, inferred
by alignment of 16SrRNA sequence to GreenGenes database*
Acetobacterium sp. Acidaminococcus intestinalis Anaerostipes hadrus
Atopobium minutum Bacteroides fragilis Bacteroides ovatus
Bacteroides vulgatus Bifidobacterium adolescentis (2 different
strains) Bifidobacterium longum (2 different strains) Blautia
coccoides Blautia producta Clostridium aldenense Clostridium
citroniae Clostridium cocleatum Clostridium hathewayi Clostridium
lactatifermentans Clostridium orbiscindens Collinsella aerofaciens
Dorea longicatena (2 different strains) Escherichia coli
Eubacterium desmolans Eubacterium eligens Eubacterium fissicatena
Eubacterium limosum Eubacterium rectale (4 different strains)
Eubacterium sp. (unclassified) (3 different strains) Eubacterium
ventriosum Faecalibacterium prausnitzii Lachnospira pectinoshiza
Lactobacillus casei Lactobacillus paracasei Parabacteroides
distasonis Raoultella sp. Roseburia faecalis Roseburia hominis
Roseburia intestinalis Roseburia inulinivorans Ruminococcus torques
(2 different strains) Ruminococcus obeum (2 different strains)
Streptococcus mitis *Closest species match was inferred by
alignment of 16SrRNA sequence to GreenGenes database; note that in
some cases 16SrRNA gene sequences could not resolve identity beyond
genus, and that closest match does not infer definitive speciation.
Shaded boxes indicate strains in DEC-2 that are NOT in DEC-1.
[0056] Liquid Gold can be stored at 4.degree. C. for weeks at a
time without losing its capacity to support microbial growth,
indicating that Liquid Gold's ability to support or promote growth
is stable and can be preserved for a prolonged period of time.
Liquid Gold can also be frozen and preserved for future use.
[0057] In an embodiment, DEC-1 Liquid Gold is provided herein. In
another embodiment, DEC-2 Liquid Gold is provided herein. In yet
another embodiment, DEC-3 Liquid Gold is provided herein. In
another embodiment, Native Liquid Gold is provided herein. It
should be understood that Liquid Gold can be produced from
culturing many different fecal samples and/or combinations of the
human intestinal strains provided herein, and that such types of
Liquid Gold are encompassed by the present invention. For example,
Liquid Gold may be produced by culturing at least one, at least
three, at least five, at least 8, at least 10, at least 15, or at
least 25 of the bacterial strains listed in Table 1, Table 2 or
Table 3. In an embodiment, Liquid Gold is produced by culturing all
of the strains listed in Table 1, Table 2 or Table 3. In another
embodiment, Liquid Gold is produced by culturing some of the
strains listed in Table 1, Table 2 or Table 3.
[0058] In a further embodiment, methods of using Liquid Gold to
support and/or enhance growth of microbes, e.g., human intestinal
anaerobic microbes, are provided herein. In an embodiment, Liquid
Gold is used to supplement standard laboratory culture media in a
liquid culture, e.g., in a chemostat. For example, Liquid Gold may
be added to culture media before culturing begins, or during
culturing of microbes. Alternatively, Liquid Gold may be added to
plates, e.g., solid media in a dish such as a petri dish, to
support or enhance growth of microbes on the solid media. It will
be understood that many variations are possible and are encompassed
by the present invention.
[0059] In an embodiment, Liquid Gold is made by growing bacterial
communities using the culture media referred to herein as "Media
1", prepared as follows:
[0060] Media 1 is prepared in the following steps (for 2 L):
[0061] Mixture 1:
[0062] The following reagents are dissolved in 1800 mL of distilled
water (ddH.sub.2O): peptone water, 4 g (Oxoid Limited); Yeast
extract, 4 g (Oxoid Limited); NaHCO.sub.3, 4 g (Sigma); CaCl.sub.2,
0.02 g (Sigma); Pectin (from citrus), 4 g (Sigma); Xylan (from
beechwood), 4 g (Sigma); Arabinogalactan, 4 g (Sigma); Starch (from
wheat, unmodified), 10 g (Sigma); Casein, 6 g (Sigma); inulin (from
Dahlia tubers), 2 g (Sigma); NaCl, 0.2 g (Sigma). Water
(ddH.sub.2O) is added to 1900 mL, as the volume is reduced to 1800
mL after autoclaving. The mixture is sterilized by autoclaving at
121.degree. C. for 60 min and allowed to cool overnight.
[0063] Mixture 2:
[0064] The following reagents are dissolved in 100 mL of distilled
water (Mixture 2A): K.sub.2HPO.sub.4, 0.08 g; KH.sub.2PO.sub.4,
0.08 g; MgSO.sub.4, 0.02 g; Hemin, 0.01 g; Menadione, 0.002 g. Bile
salts (1 g) is dissolved in 50 mL of distilled water (Mixture 2B).
L-cysteine HCl (1 g) is also dissolved in 50 mL of distilled water
(Mixture 2C). After Mixtures 2B and 2C dissolve they are added to
Mixture 2A resulting in the formation of a fine white precipitate.
This precipitate is then dissolved by the drop-wise addition of 6M
KOH until a clear, brown solution is formed (Mixture 2). This
mixture (200 mL total volume) is sterilized by filtering through a
0.22 .mu.m filter.
[0065] Culture media ("Media 1"): Mixture 2 (0.2 L) is aseptically
added to mixture 1 (1.8 L), in order to reach the final volume of 2
L. To prevent future foaming, 5 mL of antifoam B silicone emulsion
(J. T. Baker) is aseptically added to each 2 L bottle of media.
[0066] In an embodiment, mucin is added to Media 1 (to make "Media
1+mucin"). In this embodiment, mixture 1 is prepared by adding 1600
mL of ddH.sub.2O before autoclaving. The mucin addition is prepared
by dissolving 8 g mucin (e.g., from porcine stomach, type II) in
200 mL of ddH.sub.2O, and autoclaved for 20 minutes. Mixture 2 is
prepared as described above. After autoclaving, mixture 2 (0.2 L)
and the mucin solution (0.2 L) are aseptically added to mixture 1
(1.6 L), in order to reach the final volume of 2 L. Again, 5 mL of
antifoam B silicone emulsion is aseptically added to each 2 L
bottle of media.
[0067] In another embodiment, Liquid Gold is made by growing
bacterial communities using standard culture media, many of which
are known in the art. In an embodiment, mucin is added to the
culture media. For example, mucin may be added at a concentration
of 1-10%, e.g., 4 g/L. The amount of mucin to be used will vary
depending on culture conditions and is determined by the skilled
artisan based on common general knowledge and routine methods.
[0068] In an embodiment, in order to produce Liquid Gold, a 10% w/v
fecal slurry supernatant (referred to herein as a "10% inoculum) is
cultured in a chemostat. In another embodiment, in order to produce
Liquid Gold, a 20% w/v fecal slurry supernatant (referred to herein
as a "20% inoculum) is cultured in a chemostat. In other
embodiments, 5-25%, 5%, 10%, 15%, 20% or 25% inoculums are cultured
in a chemostat, e.g., a single-stage chemostat system, to produce
Liquid Gold. It should be understood that any % inoculum can be
used to inoculate a chemostat vessel to produce Liquid Gold, as
long as a sufficient amount of the fecal sample is present to seed
the vessel, and the resulting fecal slurry is not too thick or
viscous to work with.
[0069] In an embodiment, in order to produce Liquid Gold, a
chemostat system is used where the growth medium is continuously
fed into the chemostat vessel at a rate of 400 mL/day (16.7
mL/hour) to give a retention time of 24 hours, a value set to mimic
the retention time of the distal gut (Cummings, J. H. et al., Gut
(1976), 17:210-18). In another embodiment, the growth medium is
continuously fed into the chemostat at a rate of about 148 mL/day
(6.2 mL/hour) to give a retention time of 65 hours. In an
embodiment, Liquid Gold is produced from a chemostat having a
system retention time of about 20 to about 70 hours, about 20
hours, about 22 hours, about 24 hours, about 26 hours, about 28
hours, or about 30 hours, about 40 hours, about 50 hours, about 60
hours, about 65 hours, or about 70 hours.
[0070] We report herein that several novel bacterial species from
the human gut have been cultured by supplementation of culture
media with Liquid Gold (e.g., 3% v/v Liquid Gold). Several
bacterial species have been isolated for the first time using the
methods provided herein, including using Liquid Gold. In an aspect,
there is provided herein a method of culturing Faecalibacterium
prausnitzii, wherein the growth media is supplemented with Liquid
Gold. In some embodiments, there is provided herein a method of
culturing strains which have not been previously isolated, e.g.,
Clostridium aldenense 1, Clostridium aldenense 2, Clostridium
hathewayi 1, Clostridium hathewayi 2, Clostridium hathewayi 3,
Clostridium thermocellum, Ruminococcus bromii 2, Ruminococcus
torques 4, Ruminococcus torques 5, Clostridium cocleatum (e.g.,
Clostridium cocleatum 21 FAA1), Eubacterium desmolans (e.g.,
Eubacterium desmolans 48FAA1), Eubacterium limosum 13LG,
Lachnospira pectinoshiza, Ruminococcus productus (e.g.,
Ruminococcus productus 27FM), Ruminococcus obeum (e.g.,
Ruminococcus obeum 11FM1), Blautia producta, and/or Clostridium
thermocellum, wherein the growth media is supplemented with Liquid
Gold.
[0071] In an aspect, there are provided herein certain isolated
bacterial strains, which have not been previously isolated. In an
embodiment, there is provided herein an isolate of Faecalibacterium
prausnitzii. In another embodiment, there is provided herein an
isolate of Clostridium aldenense 1, Clostridium aldenense 2,
Clostridium hathewayi 1, Clostridium hathewayi 2, Clostridium
hathewayi 3, Clostridium thermocellum, Ruminococcus bromii 2,
Ruminococcus torques 4, Ruminococcus torques 5, Clostridium
cocleatum (e.g., Clostridium cocleatum 21FAA1), Eubacterium
desmolans (e.g., Eubacterium desmolans 48FAA1), Eubacterium limosum
13LG, Lachnospira pectinoshiza, Ruminococcus productus (e.g.,
Ruminococcus productus 27FM), Ruminococcus obeum (e.g.,
Ruminococcus obeum 11FM1), Blautia producta, or Clostridium
thermocellum.
[0072] Culture conditions for exemplary bacterial strains isolated
using methods provided herein are given in Table 4.
TABLE-US-00004 TABLE 4 Culture conditions for anaerobic strains
isolated from a human fecal sample. Growth media used Relative for
synthetic stool growth Strain Closest species Colony morphology
preparations.sup.1 rate.sup.2 18 Eubacterium rectale Small, FAA +
5% DSB +++ FAA white/translucent 10 Dorea longicatena Small/medium,
FAA + 5% DSB +++ FAA opaque, somewhat mucoid 42 Dorea longicatena
Medium, opaque, FAA + 5% DSB +++ FAA 1 pitting 31 Roseburia Medium,
opaque FAA + 5% DSB + 3% LG +++ FAA 1 intestinalis 6 MRS
Lactobacillus Medium, white, sticky FAA + 5% DSB +++
casei/paracasei 1 FAA Eubacterium rectale Pinpoint, FAA + 5% DSB
+++ opaque/white 27 FM Ruminococcus Small, white, dry FAA + 5% DSB
+ productus 30 Ruminococcus Small, white, dry FAA + 5% DSB +++ FAA
torques 2 MRS Ruminococcus Medium, FAA + 5% DSB + obeum
white/opaque, sticky 6 FM 1 Eubacterium rectale Medium, FAA + 5%
DSB + 3% LG +++ white/opaque, sticky 2 FAA Bifidobacterium Small,
brown, FAA + 5% DSB +++ longum translucent, metallic sheen, sticky
39 Roseburia faecalis Medium, FAA + 5% DSB + 3% LG +++ FAA 1
white/opaque, pitting 14 LG Acidaminococcus Large, white FAA + 5%
DSB +++ 2 intestinalis 5 FM Parabacteroides Small, white, FAA + 5%
DSB +++ distasonis translucent 21 Clostridium Medium, FAA + 5% DSB
+++ FAA 1 cocleatum white/opaque, very pitting/difficult to scrape,
sticky 20 Bifidobacterium Pin, brown/opaque, FAA + 5% DSB +++ MRS 1
adolescentis slight metallic sheen, sticky 48 Eubacterium Pinpoint,
FAA + 5% DSB + FAA 1 desmolans white/opaque, sticky 5 MM
Bacteroides ovatus Small, FAA + 5% DSB +++ 1 white/translucent 4 FM
1 Bifidobacterium Pinpoint, translucent, FAA + 5% DSB +++ longum
yellow, dry, pitting, metallic sheen, sticky 11 FM Ruminococcus
Small, white/opaque, FAA + 5% DSB ++ 1 obeum translucent F1
Eubacterium Pinpoint, FAA + 5% DSB + 3% LG + FAA 1 eligens
pink/purple/opaque 25 Lactobacillus casei Small, white/opaque, FAA
+ 5% DSB +++ MRS 1 sticky 13 LG Eubacterium Small, off- FAA + 5%
DSB + 3% LG +++ limosum white/opaque 9 FAA Ruminococcus Small,
white/opaque, FAA + 5% DSB +++ torques translucent 47 Eubacterium
Sticky, small FAA + 5% DSB +++ FAA ventriosum 3 FM 2 Collinsella
Pinpoint, FAA + 5% DSB +++ aerofaciens white/opaque, translucent,
dry 11 Bifidobacterium Small, yellow/opaque, FAA + 5% DSB +++ FAA 1
adolescentis mucoid 34 Lachnospira Pinpoint, yellow FAA + 5% DSB +
3% LG ++ FAA 1 pectinoshiza 40 Faecalibacterium Pinpoint,
transparent FAA+3%LG +++ FAA prausnitzii 29 Eubacterium rectale
small, white/opaque, FAA + 5% DSB +++ FAA 1 translucent .sup.1FAA:
Fastidious anaerobe agar, commercially available as Lab90; DSB:
Defibrinated sheep blood, commercially available; LG: Liquid Gold,
a clarified, filtered effluent supernatant from chemostat
communities seeded from healthy fecal communities, required by a
number of synthetic stool strains for optimal growth.
.sup.2Relative growth rate; on average plates were incubated for 3
days at 37.degree. C. under anaerobic conditions.
[0073] As used herein, "anaerobic" bacteria refers to bacteria
which are facultatively anaerobic as well as bacteria which are
strictly anaerobic.
[0074] As used herein, "standard culture media" refers to common
and/or commercially available growth media for microorganisms, such
as nutrient broths and agar plates, of which many variations are
known in the art. Standard culture media generally contains at
least a carbon source for bacterial growth, e.g., a sugar such as
glucose; various salts which are required for bacterial growth,
e.g., magnesium, nitrogen, phosphorus, and/or sulfur; and water.
Non-limiting examples of standard culture media include Lysogeny
broth (LB), A1 broth, and culture media described herein. Standard
culture media for use in methods provided herein will be selected
by a skilled artisan based on common general knowledge. The terms
"standard culture media" and "standard laboratory culture media"
are used interchangeably herein.
[0075] As used herein, the terms "pure isolate," "single isolate"
and "isolate" are used interchangeably to refer to a culture
comprising a single bacterial species or strain, e.g., grown
axenically, in isolation from other bacterial species or
strains.
[0076] For strains listed in the tables herein, the closest
bacterial species was determined using the 16S rRNA full-length
sequences, which were aligned with the NAST server (DeSantis, T. Z.
Jr. et al., Nucleic Acids Res., 34:W394-W399 (2006)) and were then
classified using the GreenGenes classification server (DeSantis, T.
Z. Jr. et al., Appl. Environ. Microbiol., 72:5069-5072 (2006)), as
described below. The most specific name in the GreenGenes
classification was used and we report the DNA maximum likelihood
score for each classification.
EXAMPLES
[0077] The present invention will be more readily understood by
referring to the following examples, which are provided to
illustrate the invention and are not to be construed as limiting
the scope thereof in any manner.
[0078] Unless defined otherwise or the context clearly dictates
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. It should be understood that
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention.
Materials and Methods
Single-Stage Chemostats and Inoculation
[0079] We developed a single-stage chemostat vessel to model the
human distal gut microbiota by modifying a Multifors fermentation
system (Infors, Switzerland; shown in FIG. 1). Conversion from a
fermentation system into a chemostat was accomplished by blocking
off the condenser and bubbling nitrogen gas through the culture.
The pressure build up forced the waste out of a metal tube
(formerly a sampling tube) at a set height and allowed for the
maintenance of a 400 mL working volume.
[0080] Throughout the duration of the experiment, the vessels were
kept anaerobic by bubbling filtered nitrogen gas (Praxair) through
the culture. Temperature (37.degree. C.) and pH (set to 7.0;
usually fluctuated around 6.9 to 7 in the culture) were
automatically controlled and maintained by the computer-operated
system. The system maintained the culture pH using 5% (v/v) HCl
(Sigma) and 5% (w/v) NaOH (Sigma). The growth medium was
continuously fed into the vessel at a rate of 400 mL/day (16.7
mL/hour) to give a retention time of 24 hours, a value set to mimic
the retention time of the distal gut (Cummings, J. H. et al., Gut
(1976), 17:210-18). Another retention time of 65 hours (.about.148
mL/day, 6.2 mL/hour) was also tested to determine the effect of
retention time on the composition of the chemostat community.
[0081] Since the growth medium contained components which cannot
survive sterilization by autoclaving (see below), the vessels were
autoclaved with 400 mL of ddH.sub.2O. During autoclaving, the waste
pipes were adjusted so the metal tube reached the bottom of the
vessel. Once the vessel cooled it was fitted to the rest of the
computer operated unit, filtered nitrogen gas was bubbled through
the water to pressurize and drain the vessel. The waste pipe was
then raised to the working volume (400 mL) and 300 mL of sterile
media was pumped into the vessel. The vessel was then left
stirring, heating, and degassing overnight. To check for
contamination within the vessel, each vessel was aseptically
sampled and plated out (both aerobically and anaerobically) on
fastidious anaerobe agar (FAA) supplemented with 5% defibrinated
sheep blood. This procedure was repeated one day before inoculation
and immediately prior to inoculation to ensure contamination was
avoided.
Collection and Preparation of Fecal Inocula
[0082] Fresh fecal samples were provided by a healthy female donor
(42 years old, with no history of antibiotic use in the 10 years
prior to stool donation; "Donor 6") or by a healthy male donor (43
years old, with no history of antibiotic use in the 6 years prior
to stool donation; "Donor 5"). Other donors also provided fecal
samples (e.g., Donor 1, Donor 2, etc.). All donors were healthy
subjects from 38 to 43 years of age with no recent history of
antibiotic treatment. Research Ethics Board (REB) approval was
obtained for fecal collection and use in these experiments.
[0083] To prepare the inoculum, freshly voided stool samples were
collected and immediately placed in an anaerobic chamber (in an
atmosphere of 90% N.sub.2, 5% CO.sub.2 and 5% H.sub.2). A 10% (w/v)
fecal slurry was immediately prepared by macerating 5 g of fresh
feces in 50 mL of anaerobic phosphate buffered saline (PBS) for 1
minute using a stomacher (Tekmar Stomacher Lab Blender, made by
Seward). The resulting fecal slurry was centrifuged for 10 minutes
at 1500 rpm to remove large food residues. The resulting
supernatant was used as the inoculum for this study. The 10%
original w/v fecal slurry supernatant is referred to herein as the
"10% inoculum". We also compared a 10% inoculum to a 20% (w/v)
inoculum to determine whether any differences existed between these
two concentrations. The 20% (w/v) inoculum was prepared in the same
manner as the 10% inoculum except that 10 g of feces was added to
50 mL of anaerobic PBS prior to homogenization. Again, the inoculum
derived from the 20% original w/v fecal slurry supernatant is
referred to herein as the "20% inoculum".
Inoculation Process
[0084] To give a final working volume of 400 mL, 100 mL of 10%
inocula was added to the 300 mL of sterile media in each vessel.
Since the thickness of the fecal inoculum made it difficult to seed
the vessel through the septum using a needle, the inoculum was
added to the vessel through the waste pipe using a syringe.
Immediately following inoculation the pH controls were turned on so
the vessel pH was adjusted to and maintained at a pH of about 6.9
to 7.0. During the first 24 hours post-inoculation the communities
were grown in batch culture to allow the community to adjust from
in vivo to in vitro conditions and avoid culture washout. During
this period the vessels were heated, degassed and stirred with
continuous pH adjustment. After this 24 hour period the feed pumps
were turned on and the vessels were run as chemostats. Fresh
culture medium was added continuously and waste was continuously
removed.
[0085] In the chemostat, culture conditions and media supply were
maintained constant. The chemostat system was generally set with a
retention time of 24 hours to mimic distal gut transit time.
Preparation of the Growth Medium
[0086] A culture growth medium for the chemostat was developed
based on media used in previous studies attempting to mimic the
human gut (Gibson, G. R. et al., Appl. Environ. Microbiol.,
54(11):2750-5, 1988; Lesmes, U. et al., J. Agric. Food Chem., 56:
5415-5421, 2008). Due to the large amount of medium used by each
vessel, medium was prepared in 2 L volumes. The culture medium was
prepared in the following steps (for 2 L):
[0087] Mixture 1:
[0088] The following reagents were dissolved in 1800 mL of
distilled water (ddH.sub.2O): peptone water, 4 g (Oxoid Limited);
Yeast extract, 4 g (Oxoid Limited); NaHCO.sub.3, 4 g (Sigma);
CaCl.sub.2, 0.02 g (Sigma); Pectin (from citrus), 4 g (Sigma);
Xylan (from beechwood), 4 g (Sigma); Arabinogalactan, 4 g (Sigma);
Starch (from wheat, unmodified), 10 g (Sigma); Casein, 6 g (Sigma);
inulin (from Dahlia tubers), 2 g (Sigma); NaCl, 0.2 g (Sigma).
Water (ddH.sub.2O) was added to 1900 mL, as the volume is reduced
to 1800 mL after autoclaving. The mixture was sterilized by
autoclaving at 121.degree. C. for 60 min and allowed to cool
overnight.
[0089] Mixture 2:
[0090] The following reagents (all purchased from Sigma) were
dissolved in 100 mL of distilled water (Mixture 2A):
K.sub.2HPO.sub.4, 0.08 g; KH.sub.2PO.sub.4, 0.08 g; MgSO.sub.4,
0.02 g; Hemin, 0.01 g; Menadione, 0.002 g. Bile salts (1 g) was
dissolved in 50 mL of distilled water (Mixture 2B). L-cysteine HCl
(1 g) was also dissolved in 50 mL of distilled water (Mixture 2C).
After Mixtures 2B and 2C dissolved they were added to Mixture 2A
resulting in the formation of a fine white precipitate. This
precipitate was then dissolved by the drop-wise addition of 6M KOH
until a clear, brown solution was formed (Mixture 2). This mixture
(200 mL total volume) was sterilized by filtering through a 0.22
.mu.m filter.
[0091] Culture media ("Media 1"): Mixture 2 (0.2 L) was aseptically
added to mixture 1 (1.8 L), in order to reach the final volume of 2
L. To prevent future foaming, 5 mL of antifoam B silicone emulsion
(J. T. Baker) was aseptically added to each 2 L bottle of media.
The media was stored at 4.degree. C. until use for a maximum of two
weeks. A bit of media was plated out on FAA (aerobically and
anaerobically) the day before adding to chemostat and immediately
after taking off the chemostat, to check for contamination.
[0092] The media was pumped into each vessel using a peristaltic
pump whose speed is controlled by the computer-operated system. To
pump media from the bottles into the vessel, standard GL-45 glass
bottle lids (VWR) had holes drilled into them to fit two stainless
steel metal tubes. When Mixture 1 was prepared, the media bottle
had all the required silicone tubing and 0.22 .mu.m filters
attached (see FIG. 1).
[0093] Each vessel was fed from one media bottle with a 2 L volume
of media. Since the tubing which supplied the media to the vessel
was also changed as each media bottle was changed, this helped to
prevent back-growth of bacteria from the vessel into the sterile
media reservoir. Each media bottle was plated out on supplemented
FAA and grown both aerobically and anaerobically before each bottle
was added to the chemostat and after each bottle was removed from
the chemostat.
[0094] We compared our culture media (Media 1) to a media
previously described in the literature (Walker, A. W. et al., Appl.
Environ. Microbiol., 71(7): 3692-700, 2005), since pH and peptide
supply can alter bacterial populations and short-chain fatty acid
ratios within microbial communities from human colon. This media
was prepared using a similar method as was used to prepare our
media, only the composition of the two mixtures changed. The
chemostat media described in Walker et al. was prepared in the
following steps (for 2 L):
[0095] Mixture 1:
[0096] The following reagents were dissolved in 1800 mL of
distilled water: peptone water, 12 g; NaHCO.sub.3, 6.4 g; pectin
(from citrus), 1.2 g; xylan (from beechwood), 1.2 g;
arabinogalactan, 1.2 g; starch (wheat, unmodified), 10 g; casein
hydrolysate, 12 g; amylopectin, 1.2 g. This mixture was sterilized
in an autoclave at 121.degree. C. for 60 min.
[0097] Mixture 2:
[0098] L-cysteine HCl (1 g) was dissolved in 100 mL of distilled
water (Mixture 2A). Bile salts (1 g) were dissolved in 100 ml of
distilled water (Mixture 2B). Mixture 2B was added to Mixture 2A
resulting in the formation of a fine white precipitate. The pH of
the solution was then adjusted by the drop-wise addition of 6M KOH
until a clear, colourless solution was formed. This mixture (200 mL
total volume) was sterilized by filtering through a 0.22 .mu.m
filter.
[0099] Chemostat Media:
[0100] Mixture 2 (0.2 L) was aseptically added to mixture 1 (1.8
L), in order to reach the final volume of 2 L. To prevent future
foaming, 5 mL of antifoam B silicone emulsion was aseptically added
to each 2 L bottle of media. The media was stored at 4.quadrature.C
until use for a maximum of two weeks.
[0101] To determine whether the addition of mucin to our culture
media (Media 1) had an effect on the composition and structure of
healthy distal gut communities, we compared one vessel fed with our
culture media (without mucin) to two vessels fed with our culture
media (containing mucin). The chemostat media with mucin was
prepared in a similar manner as our culture media without mucin,
with a couple of changes. Firstly, mixture 1 was prepared by adding
1600 mL of ddH.sub.2O before autoclaving. The mucin addition was
prepared by dissolving 8 g mucin (from porcine stomach, type II) in
200 mL of ddH.sub.2O, and autoclaved for 20 minutes. Mixture 2 was
prepared as described above. After autoclaving, mixture 2 (0.2 L)
and the mucin solution (0.2 L) were aseptically added to mixture 1
(1.6 L), in order to reach the final volume of 2 L. Again, 5 mL of
antifoam B silicone emulsion was aseptically added to each 2 L
bottle of media. The media was also stored at 4.degree. C. until
use for a maximum of two weeks.
Sampling
[0102] Each chemostat vessel was sampled daily by removing 4 mL of
culture through the septum using a sterile needle and syringe.
Samples were transferred into two screw-capped tubes which were
stored at -80.degree. C. to archive. During weekdays, 10 drops of
antifoam B silicone emulsion was added through the septum by a
syringe and needle at 9 am and 5 .mu.m (20 drops per day total). On
weekends, 20 drops of antifoam was added to each vessel around 12
.mu.m. This amount of antifoam added to each vessel daily (in
conjunction with the amount of antifoam present in the media) was
sufficient to prevent foaming in our system using a 24 hour
retention time.
DNA Extraction
[0103] The DNA used for DGGE analysis was extracted using a
protocol involving a combination of bead beating, the Omega Bio-Tek
E.Z.N.A..RTM. Stool DNA Kit, and the Promega Maxwell.RTM.16 DNA
Purification Kit. The first half of the protocol follows the June
2009 revision of the E.Z.N.A..RTM. Stool DNA Kit protocol with a
few alterations. Briefly, we added 200 .mu.L of liquid chemostat or
fecal sample, 300 .mu.L of SLX buffer from the E.Z.N.A. kit, 10
.mu.L of 20 mg/mL proteinase K (in 0.1 mM CaCl.sub.2) and 200 mg of
glass beads to a screw-capped tube and bead beat for 4.times.45
seconds (3 minutes total). The samples were then incubated at
70.degree. C. for 10 minutes, 95 .quadrature.C for 5 minutes and on
ice for 2 minutes. Next we added 100 .mu.L of Buffer P2 from the
E.Z.N.A. kit to each tube and vortexed them for 30 seconds. This
was followed by incubation on ice for 5 minutes and centrifugation
at 14500.times.g for 5 minutes. The supernatant was then
transferred into a new 1.5 mL tube and 200 .mu.L of HTR reagent
from the E.Z.N.A. kit was added to each tube using wide bore tips.
The samples were then vortexed for 10 seconds and incubated at room
temperature for 2 minutes. The tubes were then centrifuged at
14500.times.g for 2 minutes and the supernatant was transferred
into Maxwell cartridges. The remainder of the DNA extraction
protocol was carried out as described in the Maxwell.RTM.16 DNA
Purification Kit protocol (Promega).
PCR and DGGE
[0104] The V3 region (339-539 bp, Escherichia coli numbering) of
the 16S rRNA gene was amplified using primers HDA1 and HDA2-GC
(Walter, J. et al., Appl. Environ. Microbiol., 66(1): 297-303,
2000). The PCR master mix consisted of Tsg DNA polymerase (Bio
Basic) and 1.times. Thermopol buffer (with 2 mM MgSO.sub.4, NEB),
using DNA (extracted as described above) as a template. The cycling
conditions were as follows: 92 .quadrature.C for 2 min (92.degree.
C. for 1 min, 55.degree. C. for 30 sec, 72.degree. C. for 1
min).times.35; 72.degree. C. for 10 min. Three identical 50 .mu.L
PCR reactions were set up for each sample using 2 .mu.L of DNA
template. Each sample was concentrated using the EZ-10 Spin Column
PCR Products Purification Kit (Bio Basic) by loading all three PCR
reactions from each sample onto the same column and eluting in 45
.mu.L of warm HPLC grade water. Once the PCR reactions were
concentrated 10 .mu.L of DGGE loading dye (0.05 g bromophenol blue
in 10 ml 1.times.TAE) was added to each sample.
[0105] A DGGE ladder created from five laboratory strains was used
to normalize the gel. This ladder consisted of V3 DGGE PCR
reactions from laboratory strains 1/2/53 (Coprobacillus), 30/1 A
(Enterococcaceae), 5/2/43 FAA (Veillonella), 1/1/41 A1 FAA CT2
(Peptostreptococcaceae), and 7/6/55B FAA (Propionibacterium). DNA
from these strains was extracted using the method described by
Strauss et al. (Strauss, J. et al., Anaerobe, 14(6): 301-9, 2008).
The PCR reactions used to generate the amplicons to construct the
ladder were prepared as described above, except 1 PCR reaction was
prepared per stain. The five different PCR reactions were pooled
and 62.5 .mu.L of DGGE loading dye was added to the mixture.
[0106] The protocol used for DGGE analysis was developed based on a
protocol using the DCode System (Bio-Rad Laboratories, Hercules,
Calif., USA) described by Muyzer et al. (Muyzer, G. et al., Antonie
Van Leeuwenhoek, 73(1): 127-41, 1998). 40 .mu.L of the PCR/dye
mixture was loaded onto each lane of the gel. The polyacrylamide
gels consisted of 8% (v/v) polyacrylamide (37.5:1
acrylamide/bisacrylamide) in 0.5.times.TAE (TAE is Tris base,
acetic acid and EDTA buffer, made using the following recipe: Tris
base [tris(hydroxymethyl)aminomethane] (0.048% w/v); glacial acetic
acid (17.4M) (0.011% v/v); EDTA disodium salt (0.0037% w/v)). The
amplicons were separated using a denaturating gradient of 30-55%.
Electrophoresis was performed in 0.5.times.TAE buffer at a constant
temperature of 60.degree. C. for 5 h at 120 V. Gels were stained
for 10 minutes in ethidium bromide solution (in 1.times.TAE, Sigma
Aldrich) and destained for 10 minutes in ddH.sub.2O. Images were
captured using the SynGene G-Box gel documentation system and
GeneSnap software (version 6.08.04). The gels were normalized for
saturation while the images were captured.
DGGE Statistical Analyses
[0107] DGGE gel images were analyzed using the Syngene GeneTools
software (version 4.01.03, Perkin Elmer). The image background was
subtracted using rolling disc subtraction with a radius of 30
pixels. Lanes were manually detected and set on each gel image.
Analysed bands were both automatically and manually detected for
each profile.
[0108] The profiles were matched using the "profile" type under the
matching parameters menu with a set tolerance of 1%. Dendrograms
were drawn using the Unweighted Pair Group Method with Arithmetic
Mean (UPGMA). Profile comparison resulted in an automatically
generated similarity matrix and dendrogram. Similarity index values
range from 0 to 1, with values of 0 indicating two profiles have no
bands in common, while values of 1 indicate the two profiles have
identical banding patterns. Percent similarity values were
calculated by multiplying the similarity index value by 100.
[0109] Comparing Two Vessels on Day (x):
[0110] The similarity of two vessels was determined plotting the %
similarity of V(x) vs. V(y) against the day the sample was taken.
This analysis was carried out for samples taken every two days
beginning at Day 0.
[0111] Community Dynamics:
[0112] Community dynamics represents the changes within a community
over a fixed time frame. Moving window analysis was performed by
plotting the similarity between consecutive sampling points. In
this case we chose to plot Day (x-2) vs. Day (x). We found that
this time interval was adequate and did not cause us to miss large
fluctuations in the community dynamics and was in agreement with
previous literature (Possemiers, S. et al., FEMS Microbiol. Ecol.,
49(3): 495-507, 2004). This analysis resulted in the generation of
a graph which was used to assess the stability of the community as
well as to determine how long it took the vessel to reach steady
state. An example of a moving window correlation plot illustrating
community dynamics is shown in FIG. 13c.
[0113] The rate of change (.DELTA.t) can then be calculated as
100-the average of the respective moving window curve data points
(Marzorati, M. et al., Environ. Microbiol., 10(6): 1571-81, 2008).
The larger the change between the profiles of the consecutive
sampling points the higher the .DELTA.t value. However, since an
initial stabilization period is noted as the community transitions
from an in vivo to an in vitro environment, values may vary
depending on the period chosen (Marzorati, M. et al., Environ.
Microbiol., 10(6): 1571-81, 2008). According to two papers by
Wittebolle et al. (Marzorati, M. et al., Environ. Microbiol.,
10(6): 1571-81, 2008; Wittebolle, L. et al., J. Appl. Microbiol.,
107(2):385-94, 2009), a low .DELTA.t value ranges from 0-5%, a
medium value ranges from 5-15%, and a high value is 15+%. Steady
state is reached once the curve of the graph remains above the set
threshold. We considered our chemostat communities to be stable (at
steady state) once a low .DELTA.t value (0-5%) was maintained by
the community.
Shannon Diversity Index
[0114] The Shannon index is a commonly used mathematical measure of
community diversity which takes into account both species richness
(number of species present) and evenness (relative species
abundance). The Shannon diversity index (H') is calculated as shown
below (Marzorati, M. et al., Environ. Microbiol., 10(6):1571-81,
2008):
H ' = - i = 1 S ( p i ln p i ) ##EQU00001##
where: [0115] H'=the value of the Shannon diversity index [0116]
p.sub.i=the proportion of the ith species [0117] ln=the natural
logarithm of p.sub.i [0118] S=total number of species in the
community (richness) [0119] .SIGMA.=sum from species 1 to species
S. The minimum value of the Shannon index is zero, which is equal
to the value of H' for a community with a single species (i.e., a
monoculture with no diversity). The H' value increases as community
richness and evenness increase. Because of this, an increase in H'
may be the result of an increase in species richness, an increase
in species evenness, or an increase in both. This is a flaw in the
index and the reason that care should be taken when using this
measure of diversity. H' values have been found to range from 1.5
(low species richness and evenness) to 3.5 (high species evenness
and richness) in natural systems (MacDonald, G. M., 2003,
Biogeography: Space, Time and Life, John Wiley & Sons, Inc.,
U.S.A., pg 409). However, we find it more important to use the
Shannon index to measure and track changes in the diversity of
samples as compared to the original fecal inoculum (Gafan, G. P. et
al., J. Clin. Microbiol., 43(8): 3971-8, 2005).
Range-Weighted Richness
[0120] Range-weighted richness (Rr) is a measure of community
richness that takes the percentage of denaturant needed to describe
the diversity of the community into account when analyzing DGGE
gels (Marzorati, M. et al., Environ. Microbiol., 10(6):1571-81,
2008). Rr is calculated as shown below:
Rr=N.sup.2.times.D.sub.g
where: [0121] Rr=Range-weighted richness [0122] N=total number of
bands in the pattern [0123] D.sub.g=denaturing gradient comprised
between the first and last band of the pattern. Low Rr values are
less than 10, medium Rr values range from 10 to 30, and high Rr
values are greater than 30 (Marzorati, M. et al., Environ.
Microbiol., 10(6):1571-81, 2008).
Shannon's Equitability
[0124] Community evenness is the degree to which the numbers of
individuals are evenly divided between the different species of the
community. Community evenness can be assessed by calculating
Shannon's equitability (E.sub.H; Marzorati, M. et al., Environ.
Microbiol., 10(6):1571-81, 2008). E.sub.H is calculated by dividing
Shannon index (H') by H.sub.max (where H.sub.max is ln S). This is
shown below as follows:
E.sub.H=H'/H.sub.max=H'/ln S
E.sub.H values range from 0-1, with a value of 0 representing
complete community unevenness and a value of 1 representing
complete community evenness. Increases in the evenness result in an
increase in community diversity (Pielou, E. C. 1975. Ecological
diversity. Wiley, New York).
Example 1
Threshold for DGGE Analysis
[0125] A study by Possemiers et al. (Possemiers, S. et al., FEMS
Microbiol. Ecol., 49(3):495-507, 2004) established a threshold
stability criterion of 80% similarity (or 20% variability) for DGGE
studies based on within-gel variability seen between identical
marker lanes. According to this study, the threshold can be used in
conjunction with moving window correlation analysis to monitor the
dynamics of the community. This approach allows us to examine the
similarity of a vessel to itself over time. The 80% similarity
threshold can then be used to determine how long it takes a vessel
to rise above this cut-off and reach steady state.
[0126] In order to apply this threshold to our studies, a similar
analysis was carried out on the marker lanes from our DGGE gels. We
found an average of 80.4.+-.8.9% similarity, or 19.6.+-.8.9%
variability, between these marker lanes. Since these values
correspond well with the values used in the Possemiers et al.
study, a threshold of 80% similarity was also used for our
analyses. In some cases individual within gel variation was used as
a cut-off.
[0127] In the Examples below, we analyzed the colonization process
in two identical vessels to determine whether these vessels can be
run in parallel and still maintain identical communities. We also
compared different concentrations of fecal inocula, different
compositions of media, and different system retention times to
optimize the operation of our chemostat system.
Example 2
Comparison of Same Donor Over Time
[0128] The DGGE pattern of 10% inocula from Donor 2 (a 38-year old
healthy female) over an 8 month period is shown in FIG. 2. As seen
by visual inspection, the variation in the profiles seems to be due
to differences in band brightness, not the appearance or
disappearance of bands. The inocula isolated from this donor had an
average correlation coefficient of 76.9.+-.8.7%. Slight differences
between profiles were shown in FIG. 2 where samples showed slightly
higher similarity depending on the time of sample collection.
Overall, the gut microbiota of this healthy donor remained stable
over time.
[0129] The gut microbiota of this donor maintained a high diversity
over time, with an average Shannon-Weaver index value of
3.39.+-.0.08. The donor's microbiota also maintained a very high
average range-weighted richness at 776.5.+-.27.7. Finally, the
community evenness was stable over time, with an average Shannon
equitability value of 0.82.+-.0.02.
Example 3
Comparison of Different Donors
[0130] We used DGGE to compare fecal inocula isolated from four
different healthy donors (from 38 to 43 years of age with no recent
history of antibiotic treatment) (FIG. 3). We found that the fecal
community of each donor was different from the communities of other
individuals (as expected, Tannock, G. W., Eur. J. Clin. Nutr., 56
Suppl. 4:S44-9, 2002). The average correlation coefficient between
the inocula from different donors was 74.4.+-.8.4%.
[0131] The gut microbiota of all four donors had high diversity,
with an average Shannon-Weaver index value of 3.42.+-.0.04. The
donor microbiota also maintained a very high average range-weighted
richness at 585.3.+-.26.2. The community evenness between the
different donors was quite similar, with an average Shannon
equitability value of 0.86.+-.0.01.
[0132] We also used DGGE to compare two different communities,
seeded by fecal samples from two different healthy donors, each of
whom donated on at least two separate occasions. Donor 5 made two
donations, about 5 months apart: "Run 20" was inoculated on Oct.
28, 2011, and "Run 22" was inoculated on Mar. 23, 2012. Donor 6
made two donations about 6 months apart: "Run 16" was inoculated on
Feb. 10, 2011, and "Run 19" was inoculated on Aug. 3, 2011. We
asked how similar are the inocula from the two different donors to
each other; whether we would see the same loss of diversity from
each donor; and how different are the two donors from each
other.
[0133] Results are shown in FIGS. 10-13. FIG. 10 shows that fecal
inocula used to seed the chemostat vessels was very similar to the
starting fecal donation, and not altered significantly by the
process of preparing the inoculum. FIG. 11 shows that DGGE profiles
from Donors 5 and 6 were consistent between donations. FIG. 12
shows that, by DGGE, the fecal inocula from the same donor on two
different occasions were more similar to each other than to fecal
inocula from another donor. Also, the steady state communities
seeded with feces from the same donor were more similar to each
other between chemostat runs than to communities seeded with feces
from another donor. FIG. 13 shows that evenness in two vessels with
the same inocula was similar to each other, stable over time, and
similar to that of the starting inocula.
Example 4
Comparison of 10% Vs. 20% Inocula
[0134] DGGE was used to assess whether a 10% or 20% inoculum was
better suited to seed a chemostat vessel by maintaining a higher
diversity (FIG. 4). Within-group comparisons of the 10% inocula
gave a correlation coefficient of 98.1%, while the 20% inocula gave
a correlation coefficient of 98.4%. Between-group comparisons of
the 10% and 20% inocula gave an average correlation coefficient of
97.9.+-.0.7%. With little differences between the within- and
between-group values, little differences in the concentrations of
inocula were observed.
[0135] There were no differences between the diversity of the 10%
and 20% inocula. The Shannon-Weaver index values for both the 10%
and 20% inocula were high, with values of 3.38 and 3.37,
respectively. Also, the range-weighted richness values for both the
10% and 20% inocula were very high, with values of 802.9 and 810.0,
respectively. There were also no differences between the evenness
of the 10% and 20% inocula. The 10% and 20% inocula both had
Shannon equitability values of 0.82.
[0136] Both inocula were similar to the fecal samples, with
correlation coefficients of 86.4% for the 10% inocula and 80.9% for
the 20% inocula. These inocula were also similar to the respective
pellets formed during inocula preparation, with correlation
coefficients of 73.7% for the 10% inoculum and 75.5% for the 20%
inoculum (Table 5).
TABLE-US-00005 TABLE 5 Correlation coefficients for the 10% inocula
and the 20% inocula. 10% inoculum 20% inoculum Feces vs. inoculum
86.4 80.9 Inoculum vs. pellet 73.7 75.5 Feces vs. pellet 86.4
85.7
Example 5
Comparison of Two Vessels Run in Parallel
[0137] DGGE was used to monitor the composition, diversity, and
dynamics of two identical chemostat vessels (V1 and V2). Each
vessel was seeded with fecal inocula from the same healthy donor to
determine whether these two vessels could maintain identical
communities.
[0138] The inocula used to seed each vessel were very similar to
each other and immediately after inoculation the correlation
coefficients of samples taken from each vessel was 91.3%. The
composition of each vessel varied from each other between days 2-8,
however the communities within each vessel became more similar to
each other between days 10-28, with an average correlation
coefficient of 94.7.+-.2.0%.
[0139] GeneTools (statistical analysis software; Syngene) only
takes species richness into account when calculating its similarity
indices. Both vessels were 95.6% similar to each other on day 10
based on the GeneTools analysis and therefore shared most of their
bands between the profiles. However, the vessels differed from each
other in terms of the brightness of these bands. Upon visual
inspection supported by measures of evenness, both vessels showed
identical communities in terms of banding patterns and band
brightness by day 26.
[0140] During the initial 10 days of the experiment the communities
in both vessels were unstable and had high .DELTA.t values as
determined using moving window analysis (FIG. 5). Between days
0-10, both vessels had similar high .DELTA.t values, with averages
of 25.1.+-.13% for V1 and 20.8.+-.9.0% for V2 (p>0.10). Between
days 24 and 28, V1 and V2 had similar low dynamics, with .DELTA.t
values of 1.6.+-.0.2% for V1 and 3.9.+-.1.6% for V2
(p>0.10).
[0141] The community diversity was very high throughout the
duration of the experiment, however an initial drop was observed
between days 0 and 10, with Shannon-Weaver index values dropping
from 3.38 to 2.93 for V1 and from 3.38 to 3.00 for V2. This drop
evened out by day 14, giving an average Shannon-Weaver index value
of 3.03.+-.0.06 for V1 and 2.98.+-.0.10 for V2 between days 14 and
28 (p>0.10). The community range-weighted richness also saw a
drop between days 0 to 10, with values dropping from 578.6 to 341.7
for V1 and from 582.4 to 325.7 for V2. Like the diversity, the drop
in range-weighted richness values evened out by day 14, giving
average values of 303.0.+-.43.1 for V1 and 299.0.+-.31.6 for V2
between days 14 and 28 (p>0.10). We also observed a drop in
community evenness during the initial 10 days of the experiment.
The Shannon equitability values dropped slightly from 0.86 to 0.80
for V1 and 0.86 to 0.82 for V2. This drop evened out by day 14,
giving average values of 0.83.+-.0.01 for V1 and 0.82.+-.0.02 for
V2 between days 14 and 28 (p>0.10) (Table 6).
[0142] To determine the biological significance of our steady-state
communities we compared the profiles of our vessels immediately
after inoculation to our samples from steady-state (day 26), as
shown in FIG. 5. We found that V1 and V2 were 96.3% similar to each
other immediately following inoculation, and 96.0% similar to each
other 26 days post-inoculation. However, V1 day 0 and V1 day 26
were 40.2% similar to each other, while V2 day 0 and V2 day 26 were
39.3% similar to each other.
TABLE-US-00006 TABLE 6 Comparison of two communities cultured
separately with fecal inocula from the same healthy donor, in
Vessel #1 (V1) and Vessel #2 (V2). Time Result of period paired
Parameter V1 V2 (Days x-y) t-test Dynamics (Dy) 25.1 .+-. 13% 20.8
.+-. 9.0% 0-10 p > 0.10 1.6 .+-. 0.2% 3.9 .+-. 1.6% 24-28 p >
0.10 Shannon index (H) 3.03 .+-. 0.06 2.98 .+-. 0.10 14-28 p >
0.10 Range-weighted 303.0 .+-. 43.1 299.0 .+-. 31.6 14-28 p >
0.10 richness (Rr) Shannon 0.83 .+-. 0.01 0.82 .+-. 0.02 14-28 p
> 0.10 equitability (E.sub.H)
Example 6
Comparison of Different Media
[0143] Our laboratory developed a culture media recipe based on two
other recipes found in the literature, as described above (Gibson,
G. R., et al., Appl. Environ. Microbiol., 54(11): 2750-5, 1988;
Lesmes, U. et al., J. Agric. Food Chem., 56: 5415-5421, 2008). To
compare the effectiveness of our culture media (Media 1) we seeded
two vessels with the same inoculum from a healthy donor, but used
our media to feed one vessel (V1), while using the media recipe by
Walker et al., Appl. Environ. Microbiol. 71(7):3692-700, 2005 to
feed the other vessel (V6).
[0144] The inocula used to seed each vessel were very similar to
each other and immediately after inoculation the correlation
coefficients of samples taken from each vessel was 95.5%.
Throughout the course of the experiment, the two vessels varied
from each other based on their DGGE profiles, with correlation
coefficient values fluctuating above and below the 80% similarity
threshold between days 2 and 26 (FIG. 6). These vessels were only
consistently similar to each other between days 28 and 36, with an
average correlation coefficient of 88.9.+-.3.1%.
[0145] Between days 28 and 36, both vessels had similar low
dynamics as determined using moving window correlation, with
.DELTA.t values of 4.4.+-.2.1% for V1 and 5.5.+-.3.7% for V6
(p>0.10). On day 28, when V1 had already reached steady state,
V1 and its inoculum shared a correlation coefficient of 69.4%,
while V6 and its inoculum shared a correlation coefficient of
53.9%. On day 36, when V6 reached steady state, V1 and its inoculum
shared a correlation coefficient of 67.3%, while V6 and its
inoculum shared a correlation coefficient of 60.2%.
[0146] The diversity of the communities in both vessels was high
throughout the duration of the experiment; however an initial drop
in diversity was seen between days 0 and 10. The Shannon-Weaver
index values dropped from 3.54 to 2.84 for V1 and from 3.52 to 2.63
for V6 during this period. This drop evened out by day 14, giving
average values of 3.02.+-.0.06 for V1 and 2.92.+-.0.10 for V6
between days 14 and 36 (p<0.05). The Shannon index values became
similar between days 32 and 36, with average values of 2.96.+-.0.01
for V1 and 3.04.+-.0.07 for V6 (p>0.10). Also during the initial
10 days of this run, the range-weighted richness values dropped in
both vessels. These values dropped from 625.2 to 365.5 for V1 and
from 626.9 to 366.0 for V6. This drop evened out by day 14, giving
average range-weighted richness values of 333.0.+-.52.7 for V1 and
357.8.+-.41.7 for V6 between days 14 and 36 (p>0.10). A drop in
community evenness was also observed between days 0 and 10 of the
experiment. The Shannon equitability values dropped from 0.88 to
0.81 for V1 and 0.88 to 0.75 for V6. This drop evened out by day
14, giving average values of 0.85.+-.0.01 for V1 and 0.83.+-.0.02
for V6 between days 14 and 36 (p<0.05) (Table 7).
TABLE-US-00007 TABLE 7 Comparison of communities in two vessels
using different media, Vessel #1 (V1; our media) and Vessel #6 (V6;
Walker et al., Appl. Environ. Microbiol. 71(7): 3692-700, 2005
media). Time Result of period paired Parameter V1 V6 (Days x-y)
t-test Dynamics (Dy) 4.4 .+-. 2.1% 5.5 .+-. 3.7% 28-36 p > 0.10
Shannon index (H) 3.02 .+-. 0.06 2.92 .+-. 0.10 14-36 p < 0.05
Range-weighted 333.0 .+-. 52.7 357.8 .+-. 41.7 14-36 p > 0.10
richness (Rr) Shannon equitability 0.85 .+-. 0.01 0.83 .+-. 0.02
14-36 p < 0.05 (E.sub.H)
Example 7
Comparison of Two Different Retention Times
[0147] Two vessels modeling the distal gut were run in parallel in
an identical manner, except that the retention time for V1 was set
to 65 hours, while the retention time for V2 was set to 24 hours.
In this experiment we tested whether an increased retention time
would allow the more slow growing bacteria to establish themselves
within the vessel and therefore the community, increasing community
diversity.
[0148] The inocula used to seed each vessel were very similar to
each other and immediately after inoculation the correlation
coefficients of samples taken from each vessel was 96.7%. While V1
and V2 were reaching steady state they varied from each other and
by day 14 the correlation coefficient between V1 and V2 dropped to
78.0%.
[0149] FIG. 7 shows each vessel compared to their respective
inocula during days 0-10. Over the 10 day period shown V2 was more
similar to its inoculum than V1. V1 had an average correlation
coefficient of 48.3.+-.2.9% between days 4 and 10 while V2 had an
average correlation coefficient of 67.9.+-.5.4% (p<0.01).
Comparisons between the inocula and samples taken on day 10 showed
that V2 maintained a community that was more similar to its
inoculum, with a correlation coefficient of 75.7% for V2 and only
50.5% for V1.
[0150] Differences in community dynamics were observed during the
first 16 days of the experiment using the two different retention
times. Between days 10-16, V1 had a .DELTA.t value of 4.4.+-.1.9%
while V2 had a .DELTA.t of 9.9.+-.6.5% (p>0.10). If the
experiment had been allowed to run longer, we would have expected
to see a decrease in the dynamics of the community to a value more
similar to that of V1, as discussed previously.
[0151] An initial drop in community diversity was noted between
days 0 and 10 of the experiment. During this period the
Shannon-Weaver index values dropped from 3.44 to 3.06 for V1 and
from 3.44 to 3.05 for V2. These drops evened out by day 10 giving
average values of 3.11.+-.0.05 for V1 and 2.99.+-.0.05 for V2,
between days 10 and 16 (p>0.05). There was also an initial drop
in range-weighted richness values for each vessel, with values
dropping from 636.5 to 614.5 for V1 and from 633.9 to 486.1 for V2
between days 0 and 10. By day 10 V1 and V2 began to have similar
average range-weighted richness values, with 508.3.+-.78.5 for V1
and 433.6.+-.61.2 for V2 between days 10 and 16 (p>0.10).
Following the pattern observed in the other experiments, a drop in
evenness was observed between days 0 and 10. The Shannon
equitability values dropped from 0.86 to 0.77 for V1 and 0.85 to
0.79 for V2. This drop evened out by day 10, giving average values
of 0.80.+-.0.02 for V1 and 0.83.+-.0.01 for V2 between days 10 and
16 (p>0.05) (Table 8).
TABLE-US-00008 TABLE 8 Comparison of communities in two vessels run
with different retention times, Vessel #1 (V1; retention time of 65
hours) and Vessel #2 (V2; retention time of 24 hours). Time Result
of period paired Parameter V1 V6 (Days x-y) t-test Dynamics (Dy)
4.4 .+-. 1.9% 9.7 .+-. 5.3 10-16 p > 0.10 Shannon index (H) 3.11
.+-. 0.05 2.99 .+-. 0.05 10-16 p > 0.05 Range-weighted 508.3
.+-. 78.5 433.6 .+-. 61.2 10-16 p > 0.10 richness (Rr) Shannon
0.80 .+-. 0.02 0.83 .+-. 0.01 10-16 p > 0.05 equitability
(E.sub.H)
Example 8
Effect of Mucin on Gut Communities
[0152] Mucin is an important carbon source for the microbial
communities of the distal colon (Derrien, M. et al., Gut Microbes,
1(4):254-268, 2010). To determine whether mucin addition to our
culture media would allow us to develop communities which are more
diverse and more similar to the starting fecal material, we seeded
three vessels with the same inoculum from a healthy donor. One
vessel was fed using our culture media without mucin (V1), while
two other vessels were fed using our culture media supplemented
with 4 g/L mucin (V5 and V6).
[0153] The inocula used to seed each vessel were very similar to
each other and immediately after inoculation the similarity of
samples taken from each vessel ranged from 96.1% to 98.1% (FIG. 8).
On day 24, the communities in V5 and V6 shared a 92.4% similarity,
the communities in V1 and V5 shared a 61.0% similarity, and the
communities in V1 and V6 shared a 58.4% similarity (FIG. 8).
[0154] The communities in each vessel at day 24 were also compared
to the communities in each vessel at day 0. For V1, communities at
day 0 and day 24 were 56.5% similar; for V5, communities at day 0
and day 24 were 59.6% similar; and for V6, communities at day 0 and
day 24 were 48.9% similar.
[0155] The diversity in each vessel dropped between days 0 and 24,
however there was a larger decrease in diversity in the vessel
without mucin (V1) than there was in the vessels with mucin (V5,
V6), as shown in FIG. 8. The average Shannon-Weaver index value of
all three vessels on day 0 was 3.35.+-.0.02. At 24 days
post-inoculation, the Shannon-Weaver index values dropped to 2.96
for V1, 3.08 for V5, and 3.13 for V6.
[0156] The richness in each vessel also dropped between days 0 and
24, however there was a slightly larger decrease in richness in the
vessel without mucin (V1) than there was in the vessels with mucin
(V5, V6), as shown in FIG. 8. The average range-weighted richness
value of all three vessels on day 0 was 686.8.+-.2.8. At 24 days
post-inoculation, the range-weighted richness values dropped to
504.2 for V1, 526.8 for V5, and 526.2 for V6.
[0157] Finally, the evenness in each vessel dropped between days 0
and 24. There was a larger decrease in evenness in the vessel
without mucin (V1) than there was in the vessels with mucin (V5,
V6), as shown in FIG. 8. The average Shannon equitability index
value of all three vessels on day 0 was 0.84.+-.0.01. At 24 days
post-inoculation, the Shannon equitability index values dropped to
0.77 for V1, 0.80 for V5, and 0.81 for V6.
[0158] In sum, we have developed and characterized microbial
communities from the human distal colon that were stable,
reproducible, and biologically significant in a single-stage
chemostat model of the gut. We fully characterized the diversity,
stability, and similarity of these communities and also
characterized the fecal inoculations from the starting material to
use as a point of reference when analyzing our simulated
communities. We show that the microbial communities are
physiologically relevant, steady-state communities having
reproducible starting points. The in vitro communities closely
mimic the communities of the distal gut microbiota. We also
compared several fecal inocula from Donor 2 over an 8 month period,
and found that the predominant bacterial species from this healthy
donor remained stable over time (not shown). These values provide
us with a baseline to which we can compare our chemostat community
values. Our microbial chemostat communities maintained similar high
diversity, richness, and evenness values.
[0159] The results also show that one can develop reproducible
communities in our chemostat model as this donor fulfills our
criteria to donate (healthy, no recent history of antibiotics), as
one can collect a stool sample to use in future experiments that
will share similar profiles to a stool sample taken at an earlier
time. This means that similar steady-state communities can be
established that can be compared between chemostat runs using
complex microbial communities prepared from fresh fecal
samples.
[0160] Comparison of the fecal community from four different
healthy donors showed that each donor had a unique profile (as
expected, Tannock G. W., Eur. J. Clin. Nutr., 56 Suppl 4:S44-9,
2002). However, all four profiles showed similar diversity,
richness, and evenness values. This suggests that the fecal
microbiota from different healthy individuals share similar levels
of diversity (as assessed by DGGE).
[0161] We used DGGE to assess whether there was a significant
difference between 10% and 20% inocula used to seed a chemostat
vessel in terms of community structure and diversity. Based on the
% similarity, we found similar within- and between-group
differences for both concentrations of inocula. Both communities
also had similar community diversity, richness, and evenness
values. However, one obvious difference between the two
concentrations of inocula is the thickness of the inocula, as the
20% inoculum was much thicker than the 10% inocula. This made
inoculation with the 20% inocula much more difficult. This,
together with the fact that the 10% and 20% inocula were very
similar as assessed by DGGE, meant that a 10% inoculum was used for
all future studies.
[0162] We also compared the two concentrations of inocula to the
starting fecal material to assess whether the protocol used to
prepare the inocula might have altered the microbial community
structure. We found that both the 10% and 20% inocula were composed
of microbial communities which were representative of the starting
feces. This result shows that our protocol does not cause the
inoculum to vary significantly from the feces it was derived from,
making it a relevant seeding material to simulate the in vivo
community in our chemostat model. Differences between the fecal
inocula (the supernatant) and the pellet formed after
centrifugation of the fecal slurry may be due to bacteria adherent
to food residues that did not detach when homogenized. As these
populations probably represent more specialized niches, they are
not representative of the general luminal populations of interest
for our studies.
Example 9
Supplementing Microbial Growth Using Liquid Gold
[0163] Liquid Gold was obtained by filter-sterilization of a
donor-seeded chemostat sample, as described above. In brief, the
sample was centrifuged at 14,000 rpm for 10 minutes and the
supernatant was filtered sequentially through different sized
syringe filters in the following order: 1.0 .mu.m, 0.8 .mu.m, 0.45
.mu.m and finally 0.22 .mu.m. Sequential filtration was required to
allow removal of sediments, which readily clog the filters. Liquid
Gold was used to supplement FAA plates to a final concentration of
3%. Concentrations of 1%, 3%, 5% and 10% have been tested, and 3%
was found to be optimal (not shown).
[0164] Growth using Liquid Gold-supplemented FAA plates was
observed for Faecalibacterium prausnitzii and Ruminococcus callidus
(ATCC27760). For both of these species, no growth was observed
using unsupplemented FAA plates. Liquid Gold-supplemented FAA
plates yielded .about.30 colonies for Ruminococcus callidus and
.about.50 colonies for F. prausnitzii when streaked from frozen
stocks.
[0165] The F. prausnitzii strain used here was isolated from Donor
5. Liquid Gold plates supplemented with Donor 5 Liquid Gold was
used to grow the strain from frozen stock. R. callidus was grown on
Donor 5 Liquid Gold supplemented plates and Donor 6 Liquid Gold
supplemented plates. Growth was observed for both media types, but
plates supplemented with Donor 5 Liquid Gold yielded more growth
(30 colonies versus 5), suggesting that there are
growth-enhancement relevant differences between Liquid Gold from
different sources or donors.
[0166] FIG. 14 shows that growth of the F. prausnitzii strain
isolated from Donor 5 was enhanced by supplementation of culture
media with Liquid Gold media supplement from Donor 6. Plates were
inoculated with identical inocula and incubated at 37.degree. C.
for 3 days under total anaerobic conditions. Plate A: Fastidious
anaerobe agar supplemented with 5% defibrinated sheep blood alone;
Plate B: Fastidious anaerobe agar supplemented with 5% defibrinated
sheep blood and 3% filtered (cell-free) Liquid Gold (from donor 6).
FIG. 14 shows that growth was clearly enhanced by the addition of
Liquid Gold to the media at a concentration of 3%.
[0167] We have regularly stored Liquid Gold for several months at
4.degree. C., without a noticeable diminishment of effectiveness,
indicating that the growth-enhancing qualities of Liquid Gold are
stable.
[0168] In summary, the above examples show that the methods
described herein provide for preparation of stable and reproducible
communities, which can be used, e.g., to assess the effect of a
treatment on community composition and structure. For example, a
"test" vessel can be operated in parallel with a "control" vessel.
Running two identical vessels in parallel and ensuring they have
identical, steady-state communities at the time of treatment allows
one to determine that shifts in the community are due to the
treatment, and not to naturally occurring shifts in the
community.
[0169] As described above, we monitored the colonization of two
identical vessels set to mimic the distal colon for 28 days
post-inoculation. DGGE was used to monitor the composition,
diversity, and dynamics of two identical chemostat vessels (V1 and
V2) seeded with identical fecal inocula from a healthy donor. We
determined that two vessels could be run in parallel and maintain
identical communities.
[0170] We used moving window correlation to create stability
profiles by plotting the similarity values between day x and day
x-2 (FIGS. 5, 7). There was an increase in the rate of change
values as the communities transitioned from an in vivo to an in
vitro environment (days 0-10). During this period there was an
initial drop in community diversity in both vessels. When we looked
more closely at the communities by analyzing the richness and
evenness separately, we saw that the drop in diversity was more
influenced by the drop in richness than the drop in evenness. After
the transition period the communities stabilized and the Shannon
index, range-weighted richness, and Shannon's equitability values
were identical and reflected a stable community able to maintain
high diversity, richness, and evenness.
[0171] While the two vessels shared a similar community composition
by Day 10, they didn't develop identical bacterial communities that
were similar both in terms of species composition and abundance
until 26 days post-inoculation. Both communities reached steady
state as the rate of change values dropped below 5% by day 26
(achieved for both vessels between days 24-28).
[0172] Taken together, the results reported herein show that our
single-stage chemostat vessels can be seeded with the same fecal
community and produce communities that are stable, reproducible,
and diverse, reaching steady state after approximately 26 days
post-inoculation. Further, our single-stage chemostat was able to
develop two identical steady-state communities which were more
similar to each other than communities developed in multi-stage
chemostat systems (Van den Abbeele, P. et al., Appl. Environ.
Microbiol., 76(15): 5237-46, 2010). In our single-stage chemostat
model of the distal gut we found that the communities developed in
two identical vessels showed a correlation of 97.6% on day 26. At
this time the band brightness in these DGGE profiles was almost
identical. Overall, the single-stage chemostat model of the distal
gut produced more stable, reproducible communities than those grown
previously in multi-stage chemostats.
[0173] It is known that the composition of the gut microbiota
varies depending on the segment of the intestine being sampled
(Mai, V. and Morris, J. G. Jr., J. Nutr., 134(2):459-64, 2004;
Marteau, P. et al., Appl. Environ. Microbiol., 67(10):4939-42,
2001). Fresh fecal samples should be used to model the bacterial
communities of the distal gut lumen since the fecal bacteria are
more representative of the distal gut luminal microbiota than of
the microbiota from other segments of the intestine (Possemiers, S.
et al., FEMS Microbiol. Ecol., 49(3):495-507, 2004). Modelling the
distal gut in a single-stage system more accurately reflects the in
vivo environment to provide more biologically significant
results.
[0174] In addition, microbial diversity and community composition
within the gut is influenced by several physical, biochemical, and
physiological factors. One must assure that the simulated community
is as similar to the in vivo community as possible if the results
are to be extrapolated to the host, so it is important to control
and mimic these factors as closely as possible when designing in
vitro simulators. Computer-operated process controls of chemostat
models allow for experimental parameters such as pH, temperature,
feed rate, and oxygen levels to be continuously monitored and
automatically adjusted if deviations occur.
[0175] As described above, to determine the effectiveness of our
media recipe we set up two chemostat vessels: one vessel fed with
media prepared according to our recipe (V1), and another vessel fed
with media prepared according to a previously published recipe (V6,
Walker, et al., Appl. Environ. Microbiol., 71(7):3692-700, 2005).
The community in V1 was more similar to its inoculum than the
community in V6, meaning that the vessel fed using the Media 1
culture medium supported a community that was more representative
of the fecal microbiota than the community supported by the
previously published medium. Both vessels shared similar community
dynamics throughout the course of the experiment and similar rate
of change values between days 28 and 36. Community diversity and
evenness was higher in V1 between days 14 and 36, however, both
vessels shared similar range-weighted richness values during this
period. While both media can support diverse communities mimicking
those of the distal gut, our media recipe supports a community that
is more similar to the inoculum. Based on these observations, the
media recipe we developed provides a suitable medium to grow a
stable and diverse chemostat community.
[0176] As described above, we also investigated whether an
increased retention time would allow the more slow growing bacteria
to establish themselves within the vessel during the beginning of
the experiment (and therefore establish themselves within the
community), increasing community diversity. We did this to ensure
that the system retention time currently being used (24 hours) was
not high enough to prevent certain slow growing populations from
surviving within the system. We set up two chemostat vessels with
different retention times (V1 and V2). V1 had a longer retention
time (65 hrs), while V2 had a shorter, more biologically relevant
retention time (24 hrs). DGGE analysis showed that both vessels
developed different communities over time (see Table 8). Of these
two communities, V2 with a 24 hr retention time developed a
community that was more similar to its inoculum than the community
developed in V1. Both vessels had similar community dynamics;
however the dynamics in V2 was more variable. Both vessels were
similar to each other in terms of community diversity, richness,
and evenness. Overall, increasing the retention time from the
biologically significant value of 24 hours to 65 hours resulted in
a community which was less similar to its inoculum and did not
maintain a higher level of diversity.
[0177] We also compared the colonization process in three chemostat
vessels: one vessel fed with a medium without mucin (V1), and two
identical vessels fed with a medium supplemented with mucin (V5 and
V6). While no differences were observed between all three vessels
on day 0, by day 24 the vessels supplemented with mucin were
similar to each other, but different from the vessel that was not
supplemented with mucin. The addition of mucin to the chemostat
resulted in increases in community diversity, richness, and
evenness. The addition of mucin also allowed for the development of
communities which were more similar to the inoculum than
communities without mucin. These results show that it could be
advantageous, in certain embodiments, to include mucin as part of
the culture medium recipe.
[0178] Analysis of samples using the Shannon diversity index,
range-weighted richness, and Shannon equitability across several
gels resulted in variability in values for the same samples. To
correct for this, we ran the same samples at the end of one gel and
the beginning of the next gel. For example, if the first gel
contained the samples from days 0-10, the next gel would contain
the samples from days 10-20, and the next gel would contain the
samples from days 20-30, etc. We then added or subtracted the
calculated values for the overlapping days to correct for any
variation between gels. While the variability of the Shannon
diversity index and Shannon equitability values were relatively
small, larger variation was seen in the range-weighted richness
values. While between-gel variability is an inherent drawback of
using DGGE, it still provides us with estimates of community
composition and structure. More detailed analyses (such as
metagenomic analyses) can also be performed.
[0179] In conclusion, we have demonstrated that our single-stage
model of the human distal gut supported complex communities which
were stable, reproducible, and diverse. We also presented data to
support our optimized operational parameters including inoculum
concentration, media recipe, and system retention time.
[0180] Although this invention is described in detail with
reference to preferred embodiments thereof, these embodiments are
offered to illustrate but not to limit the invention. It is
possible to make other embodiments that employ the principles of
the invention and that fall within its spirit and scope as defined
by the claims appended hereto.
[0181] The contents of all documents and references cited herein
are hereby incorporated by reference in their entirety.
* * * * *
References