U.S. patent application number 16/181590 was filed with the patent office on 2019-05-09 for microbial ecology shift assay.
This patent application is currently assigned to Second Genome, Inc.. The applicant listed for this patent is Second Genome, Inc.. Invention is credited to Peter DiStefano, Nadir Mahmood, Rachel Steger.
Application Number | 20190136287 16/181590 |
Document ID | / |
Family ID | 51625140 |
Filed Date | 2019-05-09 |
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United States Patent
Application |
20190136287 |
Kind Code |
A1 |
Steger; Rachel ; et
al. |
May 9, 2019 |
Microbial Ecology Shift Assay
Abstract
The disclosure provides assay methods for characterizing the
effects of an agent on a microbiome of a subject. Moreover, the
disclosure provides methods for practical applications of assay
results. The biological sample is extracted and the microbial
population is enumerated by using signals or markers specific to
the microbial species. The enumerated population is subjected to
the action of one or many therapeutic agents and the efficiency is
assessed by deriving a score based on the effects in the individual
samples and in the population of samples.
Inventors: |
Steger; Rachel; (Sunnyvale,
CA) ; DiStefano; Peter; (Southborough, MA) ;
Mahmood; Nadir; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Second Genome, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Second Genome, Inc.
South San Francisco
CA
|
Family ID: |
51625140 |
Appl. No.: |
16/181590 |
Filed: |
November 6, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14775722 |
Sep 14, 2015 |
10150982 |
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PCT/US2014/022844 |
Mar 10, 2014 |
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16181590 |
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61784629 |
Mar 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/06 20130101; G01N
33/569 20130101; G01N 2500/10 20130101 |
International
Class: |
C12Q 1/06 20060101
C12Q001/06; G01N 33/569 20060101 G01N033/569 |
Claims
1. A method for determining the effects of an agent on the
microbiota of a subject, the method comprising: a) preparing a
first reaction mixture that comprises a first portion of a sample
from a first subject in contact with an agent; b) preparing a
second reaction mixture that comprises a second portion of the
sample, wherein the second reaction mixture does not comprise the
agent; c) obtaining an enumeration of the abundance of one or more
microbial taxa in said first and second reaction mixtures; d)
determining the effects of the agent on the microbiota of the first
subject, based on a comparison of the enumeration of the abundance
of the one or more microbial taxa in the first and second reaction
mixtures; e) performing a functionality assay on the first and
second reaction mixtures, wherein the functionality assay comprises
a barrier function assay that measures activity of the microbiota
supernatants on barrier permeability; f) determining the effects of
the agent on the microbiota of the first subject, based on a
comparison of the results of the functionality assays on the first
and second reaction mixtures; and g) selecting the first subject
and/or one or more additional subjects for use of the agent in a
method of treatment for a condition selected from inflammatory
bowel disease (IBD), Crohn's Disease (CD), colitis, and
pouchitis.
2.-4. (canceled)
5. The method of claim 1, wherein said agent is used to treat said
condition in said first subject and/or said additional
subjects.
6. The method of claim 1, wherein said agent is a drug.
7. The method of claim 6, wherein said drug is an antibiotic.
8.-10. (canceled)
11. The method of claim 1, wherein said first subject is of a
different type of species than said additional subjects.
12. (canceled)
13. The method of claim 1, wherein the volume volumes of said first
and second reaction mixtures are at most 1 mL.
14.-18. (canceled)
19. The method of claim 1, wherein obtaining an enumeration of the
abundance of one or more microbial taxa comprises enumerating the
abundance of at least 60 microbial taxa in said first and second
reaction mixtures.
20.-39. (canceled)
40. A method for screening test agents for use in treatment of IBD,
CD, colitis, or pouchitis, comprising: a) preparing at least ten
individual first reaction mixtures that each comprises an aliquot
of a first portion of a sample from a first subject who has a
condition selected from IBD, CD, colitis, or pouchitis, wherein the
first portion in each individual reaction mixture is in contact
with a chemically distinct test agent, and preparing a second
reaction mixture that comprise a second portion of the sample and
does not comprise the test agents; b) obtaining an enumeration of
the abundance of one or more microbial taxa in each said first and
second reaction mixtures c) determining the effects of each of the
test agents on the microbiota of the first subject, based on a
comparison of the enumeration of the abundance of the one or more
microbial taxa in the first and second reaction mixtures; d)
performing a functionality assay on each of the first and second
reaction mixtures, wherein the functionality assay comprises a
barrier function assay that measures activity of the microbiota
supernatants on barrier permeability; e) determining the effects of
the agent on the microbiota of the first subject, based on a
comparison of the results of the functionality assays on the first
and second reaction mixtures; and f) making a decision regarding
the utility of each of said at least ten agents to be used as a
drug for treatment of a condition selected from IBD, CD, colitis,
and pouchitis based upon a comparison of the results of the
enumeration of the abundance of the one or more microbial taxa and
the functionality assays on the microbiota in the first and second
reaction mixtures.
41.-47. (canceled)
48. A method for determining the suitability of an agent for use as
a treatment for IBD, CD, colitis, or pouchitis, comprising: a)
preparing a first reaction mixture that comprises a first sample
from a first subject, wherein said first subject has a condition
selected from IBD, CD, colitis, and pouchitis, wherein the first
sample is in contact with an agent; b) preparing a second reaction
mixture that comprises a second sample from a second subject who
does not have said condition, wherein the second sample is in
contact with the agent; c) obtaining an enumeration of the
abundance of one or more microbial taxa in said first and second
reaction mixtures; d) performing a functionality assay on the first
and second reaction mixtures, wherein the functionality assay
comprises a barrier function assay that measures activity of the
microbiota supernatants on barrier permeability; in said second
reaction mixture; e) making a decision regarding the suitability of
said agent to be used as a therapeutic drug for treatment of a
condition selected from IBD, CD, colitis, and pouchitis, based upon
a comparison of the results of the enumeration of the abundance of
the one or more microbial taxa and the functionality assays on the
microbiota in the first and second reaction mixtures.
49.-50. (canceled)
51. The method of claim 1, wherein said sample is obtained from the
gut and/or comprises fecal or cecal material from the first
subject.
52. The method of claim 1, wherein said microbial taxa are
operational taxonomic units (OTUs).
53. The method of claim claim 1, wherein said enumeration of the
abundance of one or more microbial taxa or taxon-identifying
chemical species is completed by detecting an entity selected from
the group consisting of a nucleic acid, a lipid, a carbohydrate, a
protein, a peptide, a small molecule, and combinations thereof.
54. The method as in claim 1, wherein said sample is processed into
a slurry.
55. The method as in claim 1, wherein said reaction mixture is
incubated in an anaerobic atmosphere.
56. The method of claim 1, wherein obtaining the enumeration of the
abundance of one or more microbial taxa in the first and second
reaction mixtures comprises amplifying and sequencing DNA
comprising the variable region of the 16S rRNA gene from the first
and second reaction mixtures.
57. The method of claim 1, wherein the barrier function assay
comprises contacting and incubating the first and second reaction
mixtures with HT29, HT29 MTX, or CaCo2 cells, or mouse colon
organotypic colon cells.
58. The method of claim 1, wherein the barrier function assay
comprises incubating the first and second reaction mixtures with a
labeled Dextran.
59. The method of claim 1, wherein the first and/or additional
subjects are selected from a mammal, a rodent, a mouse, a rat, a
dog, a cat, a hamster, a monkey, a pig, a guinea pig, a gerbil, a
rabbit, a cow, a lamb, a horse, and a human.
60. The method of claim 1, wherein the first subject and/or
additional subjects are human.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/784,629, filed Mar. 14, 2013, said
application is incorporated herein by reference in its entirety for
all purposes.
BACKGROUND
[0002] Various microbiota found in a living organism provide many
crucial contributions to its host, including, for example, aiding
digestion, aiding in the development of immune systems, and/or
imparting resistance to pathogenic colonization. Even a slight
fluctuation in the symbiotic balance between microbiota and its
host may be deleterious to the host, possibly leading to a
pathological condition. For example, perturbations in the human gut
may lead to conditions such as Clostridium difficile infection or
inflammatory bowel disease (IBD). The composition of a microbial
community can undergo changes as a result of interactions between
the microbiota and a host's immune and metabolic systems, and/or
interactions between the microbiota and exogenous agents. In one
example, human exposure to antibiotics is known to have both
short-term and long-term effects on the composition of various host
microbiota, including those of the gut. The ability to monitor
various degrees of change in the microbiome is of utility in
diagnosing and treating disease.
SUMMARY
[0003] This disclosure provides in vitro assay methods for
determining the effects of an agent on the microbiota of a given
subject. Moreover, this disclosure provides methods for
interpreting and/or utilizing the results of an assay described
herein.
[0004] An aspect of the disclosure is a method for providing
counseling to a subject comprising obtaining a sample from a first
subject; contacting the sample with an agent in a reaction mixture;
obtaining an enumeration of the abundance of one or more microbial
taxa or taxon-identifying chemical species in the reaction mixture
after contacting the sample with the agent; and providing
counseling regarding the exposure of the agent to the first subject
and/or one or more additional subjects using the enumeration or a
manipulation of the enumeration.
[0005] The method may include the selection of the first subject
and/or the additional subjects for use of the agent. In some cases,
the first subject and/or the additional subjects have a condition.
The condition may be selected from the group consisting of:
Clostridium difficile infection, inflammatory bowel disease (IBD),
a condition of the gut, Crohn's Disease (CD), irritable bowel
syndrome (IBS), stomach ulcers, colitis, neonatal necrotizing
enterocolitis, or gastroesophageal reflux disease (GERD), cystic
fibrosis, chronic obstructive pulmonary disease, rhinitis, atopy,
asthma, acne, a food allergy, obesity, periodontal disease,
diarrhea, constipation, functional bloating, gastritis, lactose
intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof. In some cases, the agent may be used to treat the
condition in the first subject and/or the additional subjects.
[0006] Moreover, the agent may be a drug. In some cases, the drug
is selected from the group consisting of: an approved drug, a drug
available on the market, a withdrawn, a drug in pre-clinical
development, a drug in clinical development, a drug under
regulatory review, an unapproved drug, and combinations thereof.
Additionally, the drug may be an antibiotic.
[0007] Counseling can include a variety of types of information. In
some cases, counseling includes information selected from the group
consisting of: the enumeration, information regarding the efficacy
of the agent, information regarding the safety of the agent,
information regarding the safety of the agent when administered
with one or more different agents, information regarding the
efficacy of the agent when administered with one or more different
agents, a recommendation to use the agent, a recommendation to
continue to use of the agent, a recommendation to not use the
agent, a recommendation to discontinue use of the agent, providing
a ranked list of possible agents or combination of agents for use
or continued use, a recommendation for the addition of one or more
different agents to a regimen comprising the agent, a
recommendation for monitoring use of the agent over time, a
recommendation for a dose of the agent, a recommendation regarding
the propensity of an agent to cause a condition, and combinations
thereof.
[0008] The first subject may be a pet or subject under the care or
ownership of another subject.
[0009] Counseling may include the generation of a report.
[0010] In some cases, counseling may be provided by any of the
following: a person, a company, a representative of a health-care
organization, a health-care organization, a government official, a
government office, a public health organization, a consultant, via
a subscription service, via an online vendor, via a printed
publication, via live audio, via an audio recording, via postal
mail, via email, via telephone, via the internet, and combinations
thereof.
[0011] It may be the case that first subject is of a different type
of species than the additional subjects.
[0012] The first subject may be selected from the group consisting
of a species of a mammal, a species of a rodent, a species of a
mouse, a species of a rat, a species of a dog, a species of a cat,
a species of a hamster, a species of a monkey, a species of a pig,
a species of a squirrel, a species a guinea pig, a species of a
gerbil, a species of a bird, a species of a hydra, a species of a
rabbit, a species of a fish, a species of a frog, a species of a
cow, a species of a lamb, a species of a chicken, a species of
Drosphilia, a species of Xenopus, a species of horse, and a
human.
[0013] The additional subjects may be selected from the group
consisting of a species of a mammal, a species of a rodent, a
species of a mouse, a species of a rat, a species of a dog, a
species of a cat, a species of a hamster, a species of a monkey, a
species of a pig, a species of a squirrel, a species a guinea pig,
a species of a gerbil, a species of a bird, a species of a hydra, a
species of a rabbit, a species of a fish, a species of a frog, a
species of a cow, a species of a lamb, a species of a chicken, a
species of Drosphilia, a species of Xenopus, a species of horse,
and a human.
[0014] In some examples, the agent is a food, such, as a component
of a diet.
[0015] In some examples, the agent is selected from the group
consisting of a microbe, a virus, a fecal transplant, a drug, an
antibiotic, a food, a beverage, a beauty care product, makeup,
hairspray, lotion, a cosmetic, lip balm, sunscreen, a fragrance, a
personal hygiene product, shampoo, soap, shower gel, conditioner,
chemically treated wipes, hand sanitizer, an allergen, a household
chemical, bleach, ammonia, a caustic, fertilizer, a gardening
chemical, paint, paint thinner, a stain repellant, a water
repellant, a wound dressing, a bandage, a liquid bandage, a wound
antiseptic, hydrogen peroxide, an industrial chemical, a solvent,
an acid, a hazardous chemical, water, an environmental sample, a
soil sample, an aerosol that may be inhaled via the nose or throat,
a topical pain reliever, an Epsom salt, a material used to make
clothing, a polynucleotide, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), RNA capable of RNA-interference (RNAi), a
polypeptide, a protein, a recombinant protein, a lipid, cells
conditioned to secrete a chemical substance, microbial metabolites,
metabolites, detergents, surfactants, liposomes, drug delivery
vehicles, and combinations thereof.
[0016] Additionally, the volume of the reaction mixture can vary.
In some cases the volume of the reaction mixture may be at most 1
mL or at most 0.5 mL.
[0017] Counseling may include the use of a reference enumeration.
The reference enumeration may be generated from samples obtained
from a subject of a different species than the first subject.
[0018] Furthermore, the additional subjects may be a population.
For example, the first subject may be a human and the population
may be the human population.
[0019] In some cases, counseling may be completed with the aid of a
computer.
[0020] Another aspect of the disclosure provides a method
comprising: obtaining a sample from a first subject; contacting the
sample with an agent in a reaction mixture, wherein the agent is
selected from the group consisting of: a microbe, a virus, a fecal
transplant, a drug, an antibiotic, a food, a beverage, a beauty
care product, makeup, hairspray, lotion, a cosmetic, lip balm,
sunscreen, a fragrance, a personal hygiene product, shampoo, soap,
shower gel, conditioner, chemically treated wipes, hand sanitizer,
an allergen, a household chemical, bleach, ammonia, a caustic,
fertilizer, a gardening chemical, paint, paint thinner, a stain
repellant, a water repellant, a wound dressing, a bandage, a liquid
bandages, a wound antiseptic, hydrogen peroxide, an industrial
chemical, a solvent, an acid, a hazardous chemical, water, an
environmental sample, a soil sample, an aerosol that may be inhaled
via the nose or throat, a topical pain reliever, an Epsom salt, a
material used to make clothing, a polynucleotide, deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), RNA capable of RNA-interference
(RNAi), a polypeptide, a protein, a recombinant protein, a lipid,
cells conditioned to secrete a chemical substance, microbial
metabolites, metabolites, detergents, surfactants, liposomes, drug
delivery vehicles, and combinations thereof; and obtaining an
enumeration of the abundance of one or more microbial taxa or
taxon-identifying chemical species in the reaction mixture after
contacting the sample with the agent.
[0021] The enumeration or a numerical manipulation of the
enumeration is used for the selection of the first subject and/or
additional subjects for use of the agent. The first subject may be
of a different type of species than the additional subjects.
[0022] The first subject may be selected from the group consisting
of a species of a mammal, a species of a rodent, a species of a
mouse, a species of a rat, a species of a dog, a species of a cat,
a species of a hamster, a species of a monkey, a species of a pig,
a species of a squirrel, a species a guinea pig, a species of a
gerbil, a species of a bird, a species of a hydra, a species of a
rabbit, a species of a fish, a species of a frog, a species of a
cow, a species of a lamb, a species of a chicken, a species of
Drosphilia, a species of Xenopus, a species of horse, and a
human.
[0023] The additional subjects may be selected from the group
consisting of a species of a mammal, a species of a rodent, a
species of a mouse, a species of a rat, a species of a dog, a
species of a cat, a species of a hamster, a species of a monkey, a
species of a pig, a species of a squirrel, a species a guinea pig,
a species of a gerbil, a species of a bird, a species of a hydra, a
species of a rabbit, a species of a fish, a species of a frog, a
species of a cow, a species of a lamb, a species of a chicken, a
species of Drosphilia, a species of Xenopus, a species of horse,
and a human.
[0024] The first subject and/or the additional subjects may have a
condition, such as, for example any of the following: Clostridium
difficile infection, inflammatory bowel disease (IBD), a condition
of the gut, Crohn's Disease (CD), irritable bowel syndrome (IBS),
stomach ulcers, colitis, neonatal necrotizing enterocolitis, or
gastroesophageal reflux disease (GERD), cystic fibrosis, chronic
obstructive pulmonary disease, rhinitis, atopy, asthma, acne, a
food allergy, obesity, periodontal disease, diarrhea, constipation,
functional bloating, gastritis, lactose intolerance, visceral
hyperalgesia, colic, pouchitis, diverticulitis, allergies, asthma,
sinusitis, chronic obstructive pulmonary disorder (COPD),
depression, attention deficit hyperactivity disorder (ADHD),
autism, Alzheimers, migraines, multiple sclerosis (MS), Lupus,
arthritis, Type 2 diabetes, obesity, non alcoholic steato hepatitis
(NASH), non alcoholic fatty liver disease (NAFLD), risk of
infarction/cardiovascular risk, heart failure, cancer, dental
caries, gingivitis, oral cancer, oral mucositis, bacterial
vaginosis, fertility, sinusitis, allergies, cystic fibrosis, lung
cancer, psoriasis, atopic dermatis, methicillin-resistant
staphylococcus aureus (MRSA), colorectal cancer, vancomycin
resistant enterococcus, and combinations thereof.
[0025] Moreover, the agent may be used to treat the condition in
the first subject and/or the additional subjects. In some cases,
the agent is a drug selected, for example, from the group
consisting of an approved drug, a drug available on the market, a
withdrawn, a drug in pre-clinical development, a drug in clinical
development, a drug under regulatory review, an unapproved drug,
and combinations thereof. The drug may be an antibiotic.
[0026] Furthermore, the method may be used to make a decision
regarding the utility of the agent. The utility may include the
efficacy and/or safety or the agent. The method may also include
determining the propensity of the agent to cause a condition, such
as, for example, any of the following: Clostridium difficile
infection, inflammatory bowel disease (IBD), a condition of the
gut, Crohn's Disease (CD), irritable bowel syndrome (IBS), stomach
ulcers, colitis, neonatal necrotizing enterocolitis, or
gastroesophageal reflux disease (GERD), cystic fibrosis, chronic
obstructive pulmonary disease, rhinitis, atopy, asthma, acne, a
food allergy, obesity, periodontal disease, diarrhea, constipation,
functional bloating, gastritis, lactose intolerance, visceral
hyperalgesia, colic, pouchitis, diverticulitis, allergies, asthma,
sinusitis, chronic obstructive pulmonary disorder (COPD),
depression, attention deficit hyperactivity disorder (ADHD),
autism, Alzheimers, migraines, multiple sclerosis (MS), Lupus,
arthritis, Type 2 diabetes, obesity, non alcoholic steato hepatitis
(NASH), non alcoholic fatty liver disease (NAFLD), risk of
infarction/cardiovascular risk, heart failure, cancer, dental
caries, gingivitis, oral cancer, oral mucositis, bacterial
vaginosis, fertility, sinusitis, allergies, cystic fibrosis, lung
cancer, psoriasis, atopic dermatis, methicillin-resistant
staphylococcus aureus (MRSA), colorectal cancer, vancomycin
resistant enterococcus, and combinations thereof.
[0027] In other cases, the method may be used to make a decision
regarding the utility of a drug or a food, including food that is a
component of a diet.
[0028] The first subject may be a pet or subject under the care or
ownership of another subject.
[0029] Moreover, the method may be used to generate a list of
preferred agents.
[0030] Additionally, the method may include the use of a reference
enumeration that may be, for example, generated from samples
obtained from a subject of a different species than the first
subject.
[0031] It may be the case that the additional subjects are a
population. For example, the first subject may be a human and the
population may be the human population.
[0032] A method may be completed with the aid of a computer and the
internet.
[0033] A further aspect of the disclosure provides a method
comprising: obtaining a sample from a first subject; contacting the
sample with an agent in a reaction mixture; obtaining an
enumeration of the abundance of at least 60 microbial taxa or
taxon-identifying chemical species in the reaction mixture after
contacting the sample with the agent. The enumeration or a
numerical manipulation of the enumeration may be used for the
selection of the first subject and/or additional subjects for use
of the agent. In some cases, the first subject may be of a
different type of species than the additional subjects.
[0034] The first subject may be selected from the group consisting
of a species of a mammal, a species of a rodent, a species of a
mouse, a species of a rat, a species of a dog, a species of a cat,
a species of a hamster, a species of a monkey, a species of a pig,
a species of a squirrel, a species a guinea pig, a species of a
gerbil, a species of a bird, a species of a hydra, a species of a
rabbit, a species of a fish, a species of a frog, a species of a
cow, a species of a lamb, a species of a chicken, a species of
Drosphilia, a species of Xenopus, a species of horse, and a
human.
[0035] The additional subjects may be selected from the group
consisting of a species of a mammal, a species of a rodent, a
species of a mouse, a species of a rat, a species of a dog, a
species of a cat, a species of a hamster, a species of a monkey, a
species of a pig, a species of a squirrel, a species a guinea pig,
a species of a gerbil, a species of a bird, a species of a hydra, a
species of a rabbit, a species of a fish, a species of a frog, a
species of a cow, a species of a lamb, a species of a chicken, a
species of Drosphilia, a species of Xenopus, a species of horse,
and a human.
[0036] In some cases, the first subject and/or the additional
subjects have a condition. The condition may be, for example,
Clostridium difficile infection, inflammatory bowel disease (IBD),
a condition of the gut, Crohn's Disease (CD), irritable bowel
syndrome (IBS), stomach ulcers, colitis, neonatal necrotizing
enterocolitis, or gastroesophageal reflux disease (GERD), cystic
fibrosis, chronic obstructive pulmonary disease, rhinitis, atopy,
asthma, acne, a food allergy, obesity, periodontal disease,
diarrhea, constipation, functional bloating, gastritis, lactose
intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof.
[0037] In some cases, the agent may be used to treat the condition
in the first subject and/or the additional subjects.
[0038] In some cases, the agent is a drug, selected, for example,
from the group consisting of: an approved drug, a drug available on
the market, a withdrawn, a drug in pre-clinical development, a drug
in clinical development, a drug under regulatory review, an
unapproved drug, and combinations thereof. It may be that the drug
is an antibiotic.
[0039] The method may be used to make a decision regarding the
utility of the agent, wherein the utility may include the efficacy
and/or safety or the agent.
[0040] The method may also include determining the propensity of
the agent to cause a condition, such as, for example, any of the
following: Clostridium difficile infection, inflammatory bowel
disease (IBD), a condition of the gut, Crohn's Disease (CD),
irritable bowel syndrome (IBS), stomach ulcers, colitis, neonatal
necrotizing enterocolitis, or gastroesophageal reflux disease
(GERD), cystic fibrosis, chronic obstructive pulmonary disease,
rhinitis, atopy, asthma, acne, a food allergy, obesity, periodontal
disease, diarrhea, constipation, functional bloating, gastritis,
lactose intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof.
[0041] Additionally, the method may be used to make a decision
regarding the utility of a drug or food, including a food that is a
component of a diet.
[0042] Moreover, the first subject may be a pet or subject under
the care or ownership of another subject.
[0043] In some cases, the method is used to generate a list of
preferred agents and/or may include the use of a reference
enumeration. The reference enumeration may be generated from
samples obtained from a subject of a different species than the
first subject.
[0044] In one example, the additional subjects are a population. In
another example, the first subject is a human and the population is
the human population.
[0045] A method may be completed with the aid of a computer and/or
the internet.
[0046] It may be that at least 10000, 1000, 100, or 10 microbial
taxa or taxon-identifying chemical species are enumerated.
[0047] Another aspect of the disclosure is a method comprising:
obtaining a sample from a first subject; contacting the sample with
an agent in a reaction mixture; obtaining an enumeration of the
abundance of one or more microbial taxa or taxon-identifying
chemical species in the reaction mixture after contacting the
sample with the agent, wherein the volume of the reaction mixture
is at most 1 milliliter (mL). In some cases, the enumeration or a
numerical manipulation of the enumeration is used for the selection
of the first subject and/or additional subjects for use of the
agent. The first subject may be of a different type of species than
the additional subjects.
[0048] The first subject may be selected from the group consisting
of a species of a mammal, a species of a rodent, a species of a
mouse, a species of a rat, a species of a dog, a species of a cat,
a species of a hamster, a species of a monkey, a species of a pig,
a species of a squirrel, a species a guinea pig, a species of a
gerbil, a species of a bird, a species of a hydra, a species of a
rabbit, a species of a fish, a species of a frog, a species of a
cow, a species of a lamb, a species of a chicken, a species of
Drosphilia, a species of Xenopus, a species of horse, and a
human.
[0049] The additional subjects is selected from the group
consisting of a species of a mammal, a species of a rodent, a
species of a mouse, a species of a rat, a species of a dog, a
species of a cat, a species of a hamster, a species of a monkey, a
species of a pig, a species of a squirrel, a species a guinea pig,
a species of a gerbil, a species of a bird, a species of a hydra, a
species of a rabbit, a species of a fish, a species of a frog, a
species of a cow, a species of a lamb, a species of a chicken, a
species of Drosphilia, a species of Xenopus, a species of horse,
and a human.
[0050] Moreover, the first subject and/or the additional subjects
may have a condition, such as, for example, Clostridium difficile
infection, inflammatory bowel disease (IBD), a condition of the
gut, Crohn's Disease (CD), irritable bowel syndrome (IBS), stomach
ulcers, colitis, neonatal necrotizing enterocolitis, or
gastroesophageal reflux disease (GERD), cystic fibrosis, chronic
obstructive pulmonary disease, rhinitis, atopy, asthma, acne, a
food allergy, obesity, periodontal disease, diarrhea, constipation,
functional bloating, gastritis, lactose intolerance, visceral
hyperalgesia, colic, pouchitis, diverticulitis, allergies, asthma,
sinusitis, chronic obstructive pulmonary disorder (COPD),
depression, attention deficit hyperactivity disorder (ADHD),
autism, Alzheimers, migraines, multiple sclerosis (MS), Lupus,
arthritis, Type 2 diabetes, obesity, non alcoholic steato hepatitis
(NASH), non alcoholic fatty liver disease (NAFLD), risk of
infarction/cardiovascular risk, heart failure, cancer, dental
caries, gingivitis, oral cancer, oral mucositis, bacterial
vaginosis, fertility, sinusitis, allergies, cystic fibrosis, lung
cancer, psoriasis, atopic dermatis, methicillin-resistant
staphylococcus aureus (MRSA), colorectal cancer, vancomycin
resistant enterococcus, and combinations thereof.
[0051] In some cases, the agent may be used to treat the condition
in the first subject and/or the additional subjects. For example,
the agent may be drug that is, for example, selected from the group
consisting of: an approved drug, a drug available on the market, a
withdrawn, a drug in pre-clinical development, a drug in clinical
development, a drug under regulatory review, an unapproved drug,
and combinations thereof. It may be that the drug is an
antibiotic.
[0052] The method may be used to make a decision regarding the
utility of the agent, including the efficacy and/or safety or the
agent. Also, the method may include determining the propensity of
the agent to cause a condition, such, as for example, any of the
following: Clostridium difficile infection, inflammatory bowel
disease (IBD), a condition of the gut, Crohn's Disease (CD),
irritable bowel syndrome (IBS), stomach ulcers, colitis, neonatal
necrotizing enterocolitis, or gastroesophageal reflux disease
(GERD), cystic fibrosis, chronic obstructive pulmonary disease,
rhinitis, atopy, asthma, acne, a food allergy, obesity, periodontal
disease, diarrhea, constipation, functional bloating, gastritis,
lactose intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof.
[0053] The method may be used to make a decision regarding the
utility of a drug or food, including a food that is a component of
a diet.
[0054] The first subject may be a pet or subject under the care or
ownership of another subject.
[0055] Moreover, the method may be used to generate a list of
preferred agents and/or may include the use of a reference
enumeration. The reference enumeration may be generated from
samples obtained from a subject of a different species than the
first subject.
[0056] In some cases, the additional subjects are a population. In
one instance, the first subject is a human and the population is
the human population.
[0057] The method may be completed with the aid of a computer and
the internet. In some cases, the volume of the reaction mixture is
at most 1 mL, at most 0.75 mL, or at most 0.5 mL.
[0058] Another aspect of the disclosure provides a method
comprising: obtaining a set of agents comprising at least 10
chemically distinct agents; obtaining a sample from a first
subject; contacting an aliquot of the sample with each of the at
least ten agents in an individual reaction mixture; obtaining an
enumeration of the abundance of one or more microbial taxa or
taxon-identifying chemical species in each the individual reaction
mixture after the separately contacting an aliquot of the sample
with each of the at least ten agents; and making a decision
regarding the utility of each of the at least ten agents to be used
as a drug based upon the enumerating.
[0059] The set may comprise at least 100, at least 1000, at least
10000, or at least 50000 chemically distinct agents.
[0060] The decision may include deciding to initiate or continue
development of the agents of the set into drugs, deciding to begin
a clinical-trial using one or more of the agents of the set,
determining the safety of the agents of the set to be used as
drugs, determining the efficacy of the agents of the set to be used
as drugs to treat a condition. The condition, for example, may be
Clostridium difficile infection, inflammatory bowel disease (IBD),
a condition of the gut, Crohn's Disease (CD), irritable bowel
syndrome (IBS), stomach ulcers, colitis, neonatal necrotizing
enterocolitis, or gastroesophageal reflux disease (GERD), cystic
fibrosis, chronic obstructive pulmonary disease, rhinitis, atopy,
asthma, acne, a food allergy, obesity, periodontal disease,
diarrhea, constipation, functional bloating, gastritis, lactose
intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof.
[0061] The decision may be made, for example, by a drug-research
organization or regulatory agency.
[0062] Another aspect of the disclosure provides a method
comprising: obtaining a first sample from a first subject;
contacting the first sample with an agent in a first reaction
mixture; obtaining a first enumeration of the abundance of one or
more microbial taxa or taxon-identifying chemical species in the
first reaction mixture after the contacting the first sample with
the one or more agents, wherein the volume of the first reaction
mixture is less than about 1 mL; obtaining a second sample from a
second subject; contacting the second sample with the agent to form
a second reaction mixture; obtaining a second enumeration of the
abundance of the microbial taxa or taxon-identifying chemical
species in the second reaction mixture after the contacting the
second sample with the one or more agents, wherein the volume of
the second reaction mixture is less than about 1 mL; and comparing
the second enumeration of the microbial taxa or taxon-identifying
chemical species in the second reaction mixture with the first
enumeration of the microbial taxa or taxon-identifying chemical
species in the first reaction mixture.
[0063] Another aspect of the disclosure provides a method
comprising: obtaining a first fecal sample from a first subject,
wherein the first subject has a condition; contacting the first
fecal sample with an agent in a first reaction mixture; obtaining a
first enumeration of the abundance of one or more microbial taxa or
taxon-identifying chemical species in the first reaction mixture
after the contacting the first fecal sample with the one or more
agents, wherein the volume of the first reaction mixture is less
than about 1 mL; obtaining a second sample from a second subject;
contacting the second sample with the agent to form a second
reaction mixture; obtaining a second enumeration of the abundance
of the microbial taxa or taxon-identifying chemical species in the
second reaction mixture after the contacting the second sample with
the one or more agents, wherein the volume of the second reaction
mixture is less than about 1 mL; and comparing the second
enumeration of the microbial taxa or taxon-identifying chemical
species in the second reaction mixture with the first enumeration
of the microbial taxa or taxon-identifying chemical species in the
first reaction mixture; and making a decision regarding the
suitability of the agent to be used as a therapeutic drug based
upon the comparing.
[0064] The condition may be, for example, any of the following:
Clostridium difficile infection, inflammatory bowel disease (IBD),
a condition of the gut, Crohn's Disease (CD), irritable bowel
syndrome (IBS), stomach ulcers, colitis, neonatal necrotizing
enterocolitis, or gastroesophageal reflux disease (GERD), cystic
fibrosis, chronic obstructive pulmonary disease, rhinitis, atopy,
asthma, acne, a food allergy, obesity, periodontal disease,
diarrhea, constipation, functional bloating, gastritis, lactose
intolerance, visceral hyperalgesia, colic, pouchitis,
diverticulitis, allergies, asthma, sinusitis, chronic obstructive
pulmonary disorder (COPD), depression, attention deficit
hyperactivity disorder (ADHD), autism, Alzheimers, migraines,
multiple sclerosis (MS), Lupus, arthritis, Type 2 diabetes,
obesity, non alcoholic steato hepatitis (NASH), non alcoholic fatty
liver disease (NAFLD), risk of infarction/cardiovascular risk,
heart failure, cancer, dental caries, gingivitis, oral cancer, oral
mucositis, bacterial vaginosis, fertility, sinusitis, allergies,
cystic fibrosis, lung cancer, psoriasis, atopic dermatis,
methicillin-resistant staphylococcus aureus (MRSA), colorectal
cancer, vancomycin resistant enterococcus, and combinations
thereof.
[0065] A sample used in a method described herein may be, for
example, earwax, sweat, breast milk, hair, blood, bile,
cerebrospinal fluid, lymphatic fluid, semen, vaginal discharge,
menstrual fluid, feces, sputum, urine, saliva, secretions from open
wounds, secretions from the eye, skin tissue (e.g., a skin biopsy),
subcutaneous tissue, muscle tissue, adipose tissue, and a
combination thereof.
[0066] Furthermore, a sample may be obtained from, for example, the
gut, the vagina, the penis, a testicle, the cervix, the respiratory
system, the ear, the skin, the rectum, the kidney, the liver, the
spleen, the lung, the pancreas, the small intestine, the
gallbladder, the lymph nodes, the colon, a nasal passage, the
central nervous system, an oral cavity, a sinus, a nare, the
urogenital tract, an udder, an auditory canal, a breast, an open
wound, the eye, fat, muscle, and combinations thereof.
[0067] In some instances, microbial taxa evaluated in a method may
be operational taxonomic units (OTUs). OTUs may be formed by
clustering nucleic acid sequences of microbial organisms based on
gene sequence homology. In some cases, the gene is a 16S ribosomal
ribonucleic acid (rRNA). OTUs may be characterized by microbes
having at least 80% 16S RNA sequence homology.
[0068] In other instances, microbial taxa evaluated in a method may
be domains, kingdoms, phyla, classes, orders, families, genera, and
single species. Microbial taxa may or may not be derived from
parsimonious phylogenetic trees.
[0069] Additionally, an enumeration of the abundance of one or more
microbial taxa or taxon-identifying chemical species in a method
may be completed by detecting, for example, a nucleic acid, a
lipid, a carbohydrate, a protein, a peptide, a small molecule, and
combinations thereof. The nucleic acid may be, for example,
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a
combination of RNA and DNA. In some examples, the nucleic acid is
all or a portion of a 16S ribosomal RNA (rRNA) gene or the 16S rRNA
product of the gene.
[0070] The detecting may be completed using nucleic acid sequencing
methods such as, for example, shotgun sequencing, polymerase chain
reaction, real-time polymerase chain reaction, ligase chain
reaction, single-molecule real-time sequencing, ion torrent
sequencing, pyrosequencing, sequencing by synthesis, sequencing by
ligation, chain termination sequencing, massively parallel
signature sequencing, polony sequencing, SOLiD sequencing, DNA
nanoball sequencing, heliscope single molecule sequencing, single
molecule real time sequencing, nanopore sequencing, mass
spectrometry sequencing, microfluidic sequencing, high-throughput
sequencing, Illumina sequencing, HiSeq sequencing, MiSeq
sequencing, and combinations thereof.
[0071] In some examples, detecting is completed by using one or
more sequence-specific oligonucleotide probes that may be arranged,
for example, in a nucleic acid array or arranged on microbeads.
[0072] In some examples, detecting may be completed using nucleic
acid barcoding.
[0073] Moreover, it may be that a nucleic acid is the V1, V2, V3,
V4, V5, V6, V7, V8, and/or V9 region of a 16S rRNA gene.
[0074] In some cases, the enumeration of the abundance of at least
one microbial taxa or taxon-identifying chemical species in a
method is completed by analyzing a proteome, a transcriptome, a
metabolome, a metagenome, and combinations thereof.
[0075] Furthermore, a method may include a sample that is processed
into a slurry.
[0076] A reaction mixture of a method may be incubated at a
temperature of about 34.degree. C. to about 42.degree. C. In some
cases, the reaction mixture is incubated at a temperature of about
37.degree. C.
[0077] A reaction mixture may be incubated from about 1 day to
about 5 days. In some examples, the reaction mixture is incubated
for about 1 day.
[0078] A reaction mixture may be incubated in an anaerobic
incubation atmosphere.
INCORPORATION BY REFERENCE
[0079] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0081] FIG. 1 is a schematic depicting an example system for
executing methods of the disclosure.
[0082] FIG. 2 is a plot summarizing a principal component analysis
(PCoA) for microplate experiments with respect to Donor 1 as
described in Example 1.
[0083] FIG. 3 is a plot summarizing a principal component analysis
(PCoA) for microplate experiments with respect to Donor 2 as
described in Example 1.
[0084] FIG. 4 is a plot summarizing a principal component analysis
(PCoA) for microplate experiments with respect to Donor 3 as
described in Example 1.
[0085] FIG. 5 is a plot summarizing a principal component analysis
(PCoA) for tube experiments with respect to differing incubation
times as described in Example 1.
[0086] FIG. 6 is a plot summarizing a principal component analysis
(PCoA) for microplate and tube and experiments comparing micronized
assays to those of larger volume as described in Example 1.
[0087] FIG. 7 is a bar chart summarizing the proportions of OTUs
classified at the phylum level for experiments described in Example
1.
[0088] FIG. 8 is a bar chart summarizing the proportions of OTUs
classified at the species level for experiments described in
Example 1.
[0089] FIG. 9 is a bar chart summarizing the bacterial and archael
richness for OTUs classified at the phylum level for experiments
described in Example 1.
[0090] FIG. 10 is a bar chart summarizing the bacterial and archael
richness for OTUs classified at the species level for experiments
described in Example 1.
[0091] FIG. 11 is a bar chart summarizing the results of
experiments described in Example 4.
[0092] FIG. 12 is a bar chart summarizing the results of
experiments described in Example 4.
[0093] FIG. 13 is a bar chart summarizing the results of
experiments described in Example 4.
[0094] FIG. 14 is a bar chart summarizing the results of
experiments described in Example 4.
[0095] FIG. 15 is a bar chart summarizing the results of
experiments described in Example 4.
[0096] FIG. 16 is a bar chart summarizing the results of
experiments described in Example 4.
DETAILED DESCRIPTION
[0097] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0098] As used herein, the singular forms "a," "an," and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Furthermore, to the extent that the
terms "including", "includes", "having", "has", "with", "such as",
or variants thereof, are used in either the specification and/or
the claims, such terms are not limiting and are intended to be
inclusive in a manner similar to the term "comprising".
[0099] The term "about," as used herein, generally refers to a
range that is 15% greater than or less than a stated numerical
value within the context of the particular usage. For example,
"about 10" would include a range from 8.5 to 11.5.
[0100] The term "agent," as used herein, generally refers to any
material evaluated for its effect on a microbiome.
[0101] The term "condition," as used herein, generally refers to
any abnormal physical and/or mental state of a living organism.
[0102] The term "microbiome," as used herein, generally refers to
the totality, or a subset of the totality, of microbes, their
genetic elements (genomes), and interactions with a particular
environment. Such an environment, for example, may be a region of a
living organism.
[0103] The term "microbiota," as used herein, generally refers to
the microflora and/or microfauna in an ecosystem. Such an
ecosystem, for example, may be in a host living organism, or a
particular region within a host living organism.
[0104] The term "taxon-identifying chemical species," as used
herein, generally refers to any chemical species whose detection
may be used to identify an associated microbial taxa. In some
cases, the detection of a taxon-identifying chemical species may
indicate the abundance of an associated microbial taxon in a
microbial community. In other cases, or in parallel, a
taxon-identifying chemical species may indicate the functionality
of an associated microbial taxon within a microbial community.
Non-limiting examples of a taxon-identifying chemical species
include a product of microbe gene expression, a product of microbe
metabolism (e.g., a metabolite), a chemical component of a
microbe's structure, a chemical species secreted by a microbe, and
a product of microbe respiration.
[0105] The term "sample," as used herein, generally refers to a
specimen, or a portion of a specimen, obtained from a subject.
[0106] The terms "taxa," or "taxon," as used herein, generally
refers to a group of microbes adjudged to be a unit. Microbes may
be classified into taxa by a host of different types of
characteristics. Several example classification schemes are
described below.
[0107] The microbiota of a living organism may be altered when
exposed to exogenous agents without consideration to how such
agents may affect the microbiota. The impact of an agent on the
composition and/or functionality of a subject's microbiota may not
be thoroughly considered during the development of an agent,
including the case of a therapeutic drug, such as an antibiotic.
Indeed, complications with the consumption of or exposure to a
particular agent may be due to unfavorable disruption of
microbiota.
[0108] Additionally, an agent may be useful for treating a
condition related to an abnormal state of the microbiota of a
living organism. An agent may be used as a therapeutic, for
example, to shift the composition and/or function of the microbiota
of a condition-afflicted subject toward the composition and/or
function of a normal (i.e., healthy) subject. Indeed, both in vivo
and in vitro assays may be useful in assessing the suitability of
an agent for use as a therapeutic.
[0109] Shortcomings to assessing the impact of an agent on
microbiota may be due to the lack of available, reproducible, and
standardized methods for assessing the differential impact of an
agent on the composition and functionality of various microbial
communities within a living organism. In one aspect, the successful
development of such methods requires that various challenges be
overcome including the fact that many microbial communities are
often characterized by intrinsic variations, including across host
species, subjects of a species, and across time. In another aspect,
safety regulations regarding agent use in subjects may make it
difficult to assess the differential impact of unapproved and/or
potentially harmful agents. As a result, reliance upon in vitro or
ex vivo assays may be desirable and/or necessary. Nevertheless, the
successful development of reliable methods that enable accurate,
reproducible assessment of the potential of an agent to alter
microbial communities of a living organism could offer an important
tool for assessing the impact and/or therapeutic utility of an
agent.
[0110] Recognized herein is a need for methods for reproducible
assessment of the differential impact of an agent on the
composition and functionality of microbial communities in a host
living organism. "Composition" as used herein may generally refer
to the makeup of a microbial community and may include either or
both of the number of microbes and types of microbes of the
particular microbial community. "Functionality" as used herein may
generally refer to the capability of a microbial community to
exercise its regular homeostatic activities with non-limiting
examples that include metabolism, respiration, and gene
expression.
[0111] This disclosure provides methods and systems for
characterizing the effects of one or more agents on at least one
microbial organism of a subject host. In one aspect, the disclosure
provides methods for in vitro assays that may be used to
characterize the effects of an agent on the microbiota of a given
subject. Methods generally rely on the enumeration of microbial
taxa found in samples obtained from subjects, when the samples are
contacted with one or more agents of interest.
[0112] Enumerations may be made directly such that microbial taxa
are enumerated or may be made indirectly such that an enumeration
of a taxon-identifying chemical species associated with a taxon of
interest is completed. In some cases, both types of enumerations
are made. Enumerating taxon-identifying chemical species may be
useful in assessing changes to either or both of abundance and
functionality with respect to a microbial taxon. Enumerations may
be completed at one or more time points prior to, during, and/or
after contacting samples with the agent of interest.
[0113] In another aspect, this disclosure provides methods for
estimating the effects of an agent on the microbiota of a first
subject, using samples taken from a second subject. The first
subject may be of the same species as the second subject or may be
of a different species. More specifically, such methods generally
include contacting a sample obtained from the second subject with
an agent of interest and enumerating microbial taxa of interest in
the sample in order to estimate the effects of the agent on the
microbial taxa in the first subject.
[0114] In yet another aspect, this disclosure provides methods for
both interpreting the results of an in vitro assay described herein
and enabling practical use of such results in a variety of
applications.
[0115] This disclosure also provides systems that may be useful in
executing and/or interpreting any method described herein.
Microbial Ecology Shift Assay (MESA)
[0116] This disclosure provides in vitro assay methods for
determining the effects of an agent on the microbiota of a given
subject. Generally speaking, a method comprises the steps of: (a)
obtaining a sample from a first subject; (b) contacting the sample
with an agent in a reaction mixture; and (c) enumerating the
abundance of one or more microbial taxa or taxon-identifying
chemical species in the reaction mixture, after contacting the
sample with the agent. Enumerations of microbial taxa may be direct
such that microbial taxa of interest are directly enumerated, may
be indirect such that taxon-identifying chemical species are
enumerated, or may be a combination of both direct and indirect
methods. Detection of taxon-identifying chemical species associated
with microbial taxa of interest may also quantitatively measure the
functionality of the microbial taxa of interest.
Subjects and Samples
[0117] Assays may be used to determine the effects of an agent on
microbiota in a variety of different types of subjects. A subject
may be any entity capable of hosting a microbiome and for which a
microbiome may be identified. Non-limiting examples of different
types of subjects include a species of a mammal, a species of a
rodent, a species of a mouse, a species of a rat, a species of a
dog, a species of a cat, a species of a hamster, a species of a
monkey, a species of a pig, a species of a squirrel, a species a
guinea pig, a species of a gerbil, a species of a bird, a species
of a hydra, a species of a rabbit, a species of a fish, a species
of a frog, a species of a cow, a species of a lamb, a species of a
chicken, a species of Drosphilia, a species of Xenopus, a species
of horse and a human. In some examples, a subject may be a species
of a common laboratory animal, such as a species of mouse or rat.
Moreover, a subject may be a wild-type species or may be a
genetically-modified species. Furthermore, a subject may be
gnotobiotic. A gnotobiotic subject may be, for example, a subject
of a murine species lacking microbiota that is transplanted with
human microbiota.
[0118] The type of sample used in an assay may vary depending on,
for example, the subject of interest, the ease of obtaining the
sample from the subject of interest, the amount of sample that may
be obtained from a subject, the relationship between the microbial
taxa of interest and a condition of interest, the site from which a
sample is obtained, and combinations thereof. A sample may be any
form of matter that comprises microbiota of the subject from which
the sample is obtained, including, for example, a biological fluid
or solid matter of biological origin. Non-limiting examples of
types of samples include earwax, sweat, breast milk, hair, blood,
bile, cerebrospinal fluid, lymphatic fluid, semen, vaginal
discharge, menstrual fluid, feces, sputum, urine, saliva,
secretions from open wounds, secretions from the eye, skin tissue
(e.g., a skin biopsy), subcutaneous tissue, muscle tissue, adipose
tissue, cells obtained from tissues, and combinations thereof.
[0119] Moreover, samples may be obtained from a variety of sources,
including both internal environments, external surfaces, and body
cavities. Non-limiting examples of sample sources include the gut,
the vagina, the penis, a testicle, the cervix, the respiratory
system, the ear, the skin, the rectum, the kidney, the liver, the
spleen, the lung, the pancreas, the small intestine, the
gallbladder, the lymph nodes, the colon, the cecum, a nasal
passage, the central nervous system, an oral cavity, a sinus, a
nare, the urogenital tract, an udder, an auditory canal, a breast,
an open wound, the eye, muscle, fat, and combinations thereof. In
some examples, samples may be obtained to indirectly represent
microbial communities in other parts of a subject from which they
were obtained. For example, fecal samples may be studied to assess
the effect of an agent on microbial communities of the gut.
Moreover, surgical means may be used to obtain samples from
internal tissues, such, as, for example, the gut.
[0120] A solid sample obtained from a subject may be suspended in a
liquid or a mixture of liquids to form a slurry. An aliquot of the
slurry may then be used in an assay. A slurry may be useful, for
example, in minimizing the abundance variability of appropriate
microbial taxa between replicate aliquots of a solid sample, aiding
in the preparation of a solid sample for a reaction mixture,
preserving a solid sample, and combinations thereof. Additionally,
a slurry may be in the form of a sample homogenate, wherein the
size of the sample has been substantially reduced by mechanical
(e.g., milling, sonication) and/or chemical means (e.g., treatment
in concentrated acid, caustic) in the liquid comprising the
slurry.
[0121] The concentration (referring to the percentage weight of
sample per unit volume (w:v) slurry) of a slurry formed from a
sample may vary depending upon, for example, the availability of
sample, the availability of liquid(s) in which solid material is
suspended, the size of a vessel in which a sample slurry is
incubated with an agent, the particle size of the sample in the
slurry, and combinations thereof. The concentration of a slurry
used in an assay may be, for example, from about 0.01% to about
30%. In other examples, the concentration of a slurry used in an
assay may be from about 0.1% to about 10%. In other examples, the
concentration of a slurry used in an assay may be from about 3% to
about 10%. In other examples, the concentration of a slurry used in
an assay may be from about 1% to about 3%. In other examples, the
concentration of a slurry used in an assay may be from about 0.1%
to about 1%. In still other examples, the concentration of a slurry
used in an assay may be about 0.01%, 0.01%, 1%, 2%, 3%, 4%, 5%, 6%,
7%, 8%, 9%, 10%, 20%, 30% or more.
[0122] In some examples, the concentration of a slurry used in an
assay may be at least 0.01%. In other examples, the concentration
of a slurry used in an assay may be at least 0.1%. In other
examples, the concentration of a slurry used in an assay may be at
least 1%. In other examples, the concentration of a slurry used in
an assay may be at least 3%. In other examples, the concentration
of a slurry used in an assay may be at least 10%. In still other
examples, the concentration of a slurry used in an assay may be at
least 0.01%, 0.01%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%,
or 30%.
[0123] The type of liquid that may be used to form a sample slurry
may vary, depending on, for example, the compatibility (i.e.,
degree to which liquid does not interfere with the sample) of the
liquid with the sample, the necessary ionic strength or pH of the
slurry, the compatibility of the liquid with other components
(e.g., an agent of interest), temperature, level of oxygen in the
liquid, level of nitrogen in the liquid, level of carbon dioxide in
the liquid, and combinations thereof. Non-limiting examples of
liquids that may be used to form a sample slurry include water, a
buffer (e.g., phosphate buffered saline (PBS)), cell culture media,
minimal media, conditioned media, serum, broth, blood broth, liquid
agar, and combinations thereof.
[0124] Liquid samples obtained from a subject may be diluted with
another liquid or a mixture of other liquids. An aliquot of the
dilution may then be used in an assay. Dilution may be necessary,
for example, to aid in sufficiently detecting microbial taxa,
ensuring an appropriate proper sample-to-agent (or other reaction
mixture component) ratio in reaction mixtures, preserving a sample
after it is obtained from a subject, and combinations thereof.
Non-limiting examples of liquids used for dilution include water, a
buffer (e.g., phosphate buffered saline (PBS)), cell culture media,
minimal media, conditioned media, serum, broth, blood broth, liquid
agar, and combinations thereof.
[0125] Samples may be used immediately after acquisition from a
subject, immediately after pre-processing (e.g., forming a sample
slurry, diluting a liquid sample), or may be stored, either after
receipt from a subject or after pre-processing, in a storage medium
composition (e.g., storage medium comprising 1-20% ethanol, 1-10%
glycerol, a buffer (e.g., phosphate buffered saline (PBS), and/or
1-10% dimethylsulfoxide (DMSO)), at reduced temperatures (for
example, at or above -80.degree. C.), and/or under anaerobic
conditions.
[0126] A sample may be obtained from a single subject or samples
may be obtained from multiple subjects of a group (e.g., a group of
subjects all afflicted with a particular condition, a group of
normal (i.e., healthy subjects), or multiple subjects of different
groups. In the cases where samples are obtained from multiple
subjects of a group, a pooled sample, to be used in an assay, may
be generated from samples obtained from one or more subjects in the
group. Alternatively, samples obtained from each subject in a group
may be used in an assay separately.
Agents
[0127] The type of agent used in an assay may vary. An agent may be
virtually any chemical species or combination of chemical species
capable of being incubated with a given sample in a given reaction
mixture. Non-limiting examples of agents that may be evaluated in
an assay include a microbe (e.g., including bacteria), a virus, a
prebiotic, a probiotic, a synbiotic, feces, cecal contents, a fecal
transplant, a fecal slurry, a supernatant obtained from a fecal
slurry, a cecal contents slurry, a supernatant obtained from a
cecal contents slurry, a drug, an antibiotic, a food, a beverage, a
nutraceutical, a supplement, a beauty care product (e.g., makeup,
hairspray, lotion, cosmetics, lip balm, sunscreen, fragrances),
personal hygiene product (e.g., shampoo, soap, shower gel,
conditioner, chemically treated wipes, hand sanitizer), an
allergen, a household chemical (e.g., bleach, ammonia, caustic
household cleaning mixtures, fertilizer, gardening chemicals,
paint, paint thinner, stain/water repellant (e.g.,
Scotchguard.TM.)), wound dressings (e.g., bandages, liquid
bandages), wound antiseptics (e.g., hydrogen peroxide), an
industrial chemical (e.g., solvents, caustics, acids), a hazardous
chemical, water from a municipal water source, an environmental
sample (e.g., soil samples, water samples from natural sources),
aerosols that may be inhaled via the nose or throat, topical pain
relievers (e.g., Epsom salts), materials used to make clothing, a
polynucleotide (e.g., deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), and combinations thereof), RNA capable of RNA-interference
(RNAi), a polypeptide, a protein, a recombinant protein, a lipid, a
carbohydrate, a sugar, a cell conditioned to secrete a chemical
species, a microbial metabolite, a non-microbial metabolite, a
detergent, a surfactant, a liposome, a drug delivery vehicle, a
nanoparticle, and combinations thereof.
[0128] An agent may be a drug. The drug may be an approved drug
already available in the marketplace, a drug previously available
in the marketplace but subsequently withdrawn, a drug in
development, an unapproved drug, or a chemical entity not already
indicated as a drug. Comprehensive listings of drugs that may be
agents in an assay can be found in reference materials such as the
U.S. Food and Drug Administration Orange Book or the Merck Index,
which are both incorporated herein in entirety by reference.
Moreover, an agent may be formulated in a pharmaceutical
composition that comprises the agent and, optionally, other desired
species such as, for example, an excipient, stabilizer, carrier, or
other agent included for sufficient agent delivery to its site of
action and/or agent efficacy. Such a pharmaceutical composition may
be used in an assay.
[0129] An agent may be evaluated in the absence of other agents or
may be evaluated in combination with one or more other agents. In
cases where a combination of agents is evaluated in an assay, the
number of agents evaluated in combination may vary. In some
examples, the number of agents evaluated in combination is from 2
agents to 100 agents. In other examples, the number of agents
evaluated in combination is from 2 agents to 10 agents. In other
examples, the number of agents evaluated in combination is from 2
agents to 5 agents. In still other examples, the number of agents
evaluated in combination is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or
more agents.
[0130] In some examples, the number of agents evaluated in
combination is at least 2 agents. In other examples, the number of
agents evaluated in combination is at least 10 agents. In other
examples, the number of agents evaluated in combination is at least
50 agents. In still other examples, the number of agents evaluated
in combination is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or
more agents
[0131] A set of related, chemically distinct agents may be
evaluated in an assay, including a portion of a set. All agents in
a set may be evaluated at once, a subset of agents in the set may
be evaluated at once, or each agent in the set may be evaluated
separately from the others. Non-limiting examples of a set of
agents may be various diets of a food, a set of related drugs
(e.g., a set of HMG-CoA reductase inhibitors, a set of drugs in
development, a set of beta-blockers, a set of carbamazepines, etc.,
a set of drugs used in combination therapy, a set of compounds in a
drug development pipeline, a set of approved drugs), a set of
common household cleaners, a set of probiotics, a set of
prebiotics, a set of medical foods, a set of natural products, a
set of compounds generally recognized as safe (GRAS), a set of any
of the agent types described herein, and combinations thereof.
[0132] The number of agents in a set may vary, depending upon, for
example, the relationship between the agents of a set. For example,
the number of agents in a set may be at least 2 agents. In other
examples, the number of agents in a set may be at least 10 agents.
In other examples, the number of agents in a set may be at least
1,000 agents. In other examples, the number of agents in a set may
be at least 10,000 agents. In other examples, the number of agents
in a set may be at least 100,000 agents. In other examples, the
number of agents in a set may be at least 1,000,000 agents. In
still other examples, the number of agents in a set may be at least
2, 10, 100, 1,000, 10,000, 100,000, or 1,000,000 agents.
[0133] In some examples, the number of agents in a set may be from
1 agent to 1,000,000 agents. In other examples, the number of
agents in a set may be from 1 agent to 100,000 agents. In other
examples, the number of agents in a set may be from 1 agent to
10,000 agents. In other examples, the number of agents in a set may
be from 1 agent to 1,000 agents. In other examples, the number of
agents in a set may be from 1 agent to 100 agents. In other
examples, the number of agents in a set may be from 1 agent to
1000. In still other examples, the number of agents in a set may be
2, 10, 100, 1,000, 10,000, 100,000, or 1,000,000 agents.
[0134] An agent may be in any physical phase, including solid,
liquid, or gas. Additionally, an agent may be dissolved in a liquid
or mixture of liquids such that a solution of the agent is formed.
Alternatively, an agent may not be readily soluble in a liquid or
mixture of liquids, and instead forms a suspension when introduced
into a liquid or mixture of liquids.
Reaction Mixtures
[0135] In general, a sample may be combined with one or more agents
of interest and any additional reagents to form a reaction mixture
that is then subjected to the appropriate incubation conditions. In
one example, control reaction mixtures do not comprise the agent of
interest. Additional reagents may be necessary, for example, to
preserve the sample; to dilute a sample; to maintain the
appropriate pH of the reaction mixture; to slurry a sample; to
solubilize a sample; to maintain the appropriate ionic strength of
a reaction mixture; to improve the dispersion of a sample that has
been slurried; to maintain appropriate conditions for resident
microbiota; to solubilize an agent, to bring a reaction mixture up
to appropriate volume, to improve detection of signals, to improve
the differentiation of different signals during detection, to
detect one or more specific signals, to query a particular readout,
and combinations thereof. Non-limiting examples of additional
reagents that are comprised in a reaction mixture include culture
media, water, buffer (e.g., phosphate buffered saline (PBS)),
glycerol, DMSO, Triton-X-100, adenosine triphosphate (ATP),
vitamins, stearic acid, lithocholic acid, bile salts, cysteine,
hemin, Tween-80, and combinations thereof.
[0136] A reaction mixture may be a liquid reaction mixture (e.g.,
liquid sample+liquid additional reagents+agent of interest
dissolved in a liquid), may be a solid reaction mixture (e.g.,
solid sample+solid reagents+solid agent of interest), or may be a
combination thereof (e.g. slurried sample+liquid additional
reagents+agent of interest in solution).
[0137] The volume of a reaction mixture may vary depending on, for
example, the availability of sample, the size of available vessels
in which to contain a reaction mixture, the availability of
additional reagents, the requirements for sufficient detection of
microbial taxa or taxon-identifying chemical species, conditions
(e.g., temperature, oxygen-content, humidity, pH, incubation time,
etc.) required for incubation, the particular microbial taxa or
taxon-identifying chemical species of interest, the need for
high-throughput methods, and combinations thereof. In some
examples, the volume of a reaction mixture may be from about 0.01
milliliters ("mL") to about 10 mL. In other examples, the volume of
a reaction mixture may be from about 0.1 mL to about 1 mL. In other
examples, the volume of a reaction mixture may be from about 0.1 mL
to about 0.5 mL. In still other examples, the volume of a reaction
mixture may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,
0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50,
0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 2.0,
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mL, or more.
[0138] In some examples, the volume of a reaction mixture may be at
least 0.01 mL. In other examples, the volume of a reaction mixture
may be at least 0.1 mL. In other examples, the volume of a reaction
mixture may be at least 1 mL. In still other examples, the volume
of a reaction mixture may be at least 0.01, 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40,
0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95,
1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mL, or more.
[0139] In some examples, the volume of a reaction mixture may be at
most 10 mL. In other examples, the volume of a reaction mixture may
be at most 1 mL. In other examples, the volume of a reaction
mixture may be at most 0.5 mL. In other examples, the volume of a
reaction mixture may be at most 0.1 mL. In still other examples,
the volume of a reaction mixture may be at most 0.01, 0.02, 0.03,
0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30,
0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85,
0.90, 0.95, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0
mL.
[0140] The amount of a solid sample in a reaction mixture may vary
depending on, for example, the type of sample, the availability of
sample, the abundance of microbial taxa or taxon-identifying
chemical species of interest in the sample, the concentration at
which a sample slurry comprising the solid sample was generated,
the availability of an agent of interest, the necessary conditions
for sufficient detection of microbial taxa or taxon-identifying
chemical species of interest, conditions (e.g., temperature,
oxygen-content, humidity, pH, incubation time, etc.) required for
incubation, the type of additional reagents in a reaction mixture,
the concentration of additional reagents in a reaction mixture, the
particular microbial taxa or taxon-identifying chemical species of
interest, and combinations thereof. The amount of a solid sample in
a reaction mixture may be, for example, from about 1 nanogram
("ng") to about 100 milligrams ("mg"). In other examples, the
amount of a solid sample in a reaction mixture may be from about 10
ng to about 100 mg. In other examples, the amount of a solid sample
in a reaction mixture may be from about 10 ng to about 1 mg. In
still other examples, the amount of a solid sample in a reaction
mixture may be about 1, 10, 100, or 500 ng, 1, 10, 100, or 500
micrograms (".mu.g"), 1, 10, or 100 mg.
[0141] In some examples, the amount of a solid sample in a reaction
mixture may be at least 1 ng. In other examples, the amount of a
solid sample in a reaction mixture may be at least 1 .mu.g. In
other examples, the amount of a solid sample in a reaction mixture
may be at least 1 mg. In other examples, the amount of a solid
sample in a reaction mixture may be at least 10 mg. In still other
examples, the amount of a solid sample in a reaction mixture may be
at least 1, 10, 100, or 500 ng, 1, 10, 100, or 500 .mu.g, 1, 10, or
100 mg.
[0142] In some examples, the amount of a solid sample in a reaction
mixture may be at most 100 mg. In other examples, the amount of a
solid sample in a reaction mixture may be at most 10 mg. In other
examples, the amount of a solid sample in a reaction mixture may be
at most 1 mg. In other examples, the amount of a solid sample in a
reaction mixture may be at most 1 .mu.g. In still other examples,
the amount of a solid sample in a reaction mixture may be at most
1, 10, 100, or 500 ng, 1, 10, 100, or 500 .mu.g, 1, 10, or 100
mg.
[0143] In cases where a slurry is added to a reaction volume, the
volume percentage of a slurry in a reaction mixture may vary
depending on, for example, the concentration of the prepared
slurry. For example, the volume percentage of slurry in a reaction
mixture may be from about 0.1% to about 100%. In other examples,
the volume percentage of slurry in a reaction mixture may be from
about 0.1% to 10%. In other examples, the volume percentage of
slurry in a reaction mixture may be from about 0.1% to 2%. In still
other examples, the volume percentage of slurry in a reaction
mixture may be at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,
0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.5%, 3.0%,
3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,
9.0%, 9.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
99%.
[0144] In some examples, the volume percentage of slurry in a
reaction mixture may be at least about 0.1%. In other examples, the
volume percentage of slurry in a reaction mixture may be at least
about 1%. In other examples, the volume percentage of slurry in a
reaction mixture may be at least about 10%. In other examples, the
volume percentage of slurry in a reaction mixture may be at least
about 50%. In still other examples, the volume percentage of slurry
in a reaction mixture may be at least about 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%,
2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%,
8.0%, 8.5%, 9.0%, 9.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 99%.
[0145] In some examples, the volume percentage of slurry in a
reaction mixture may be at most about 50%. In other examples, the
volume percentage of slurry in a reaction mixture may be at most
about 10%. In other examples, the volume percentage of slurry in a
reaction mixture may be at most about 1%. In other examples, the
volume percentage of slurry in a reaction mixture may be at most
about 0.1%. In still other examples, the volume percentage of
slurry in a reaction mixture may be at most about 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%,
2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%,
7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 99%.
[0146] The concentration (referring to the percentage volume of
liquid sample per unit volume of reaction mixture) of a liquid
sample in a reaction mixture may vary depending on, for example,
the type of sample, the availability of sample, the abundance of
microbial taxa or taxon-identifying chemical species of interest in
the sample, the availability of an agent of interest, the necessary
conditions for sufficient detection of microbial taxa or
taxon-identifying chemical species of interest, conditions (e.g.,
temperature, oxygen-content, humidity, pH, incubation time, etc.)
required for incubation, the particular microbial taxa or
taxon-identifying chemical species of interest, and combinations
thereof. The concentration of a liquid sample in a reaction mixture
may be, for example, from about 0.01% to about 50%. In other
examples, the concentration of a liquid sample in a reaction
mixture may be from about 0.05% to about 20%. In other examples,
the concentration of a liquid sample in a reaction mixture may be
from about 0.05% to 10%. In still other examples, the concentration
of a liquid sample in a reaction mixture may be about 0.01%, 0.1%,
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%,
or 50%.
[0147] In some examples, the concentration of a liquid sample in a
reaction mixture may be at least 0.01%. In other examples, the
concentration of a liquid sample in a reaction mixture may be at
least 0.1%. In other examples, the concentration of a liquid sample
in a reaction mixture may be at least 1%. In other examples, the
concentration of a liquid sample in a reaction mixture may be at
least 5%. In still other examples, the concentration of a liquid
sample in a reaction mixture may be at least 0.01%, 0.1%, 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or
50%.
[0148] In some examples, the concentration of a liquid sample in a
reaction mixture may be at most 5%. In other examples, the
concentration of a liquid sample in a reaction mixture may be at
most 1%. In other examples, the concentration of a liquid sample in
a reaction mixture may be at most 0.1%. In other examples, the
concentration of a liquid sample in a reaction mixture may be at
most 0.01%. In still other examples, the concentration of a liquid
sample in a reaction mixture may be at most 0.01%, 0.1%, 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or
50%.
[0149] The concentration (referring to the percentage weight of
agent per unit volume of reaction mixture) of an agent of interest
in a reaction mixture may vary depending on, for example, the type
of agent, the availability of the agent of interest, the
availability of sample, the concentration of an agent in a solution
added to a reaction mixture, the abundance of microbial taxa or
taxon-identifying chemical species of interest in a sample, the
necessary conditions for sufficient detection of microbial taxa or
taxon-identifying chemical species of interest, conditions (e.g.,
temperature, oxygen-content, humidity, pH, incubation time, etc.)
required for incubation, the particular microbial taxa or
taxon-identifying chemical species of interest, and combinations
thereof. The concentration of an agent in a reaction mixture may be
from about from about 0.01% to about 50%. In other examples, the
concentration of an agent in a reaction mixture may be from about
0.05% to about 20%. In other examples, the concentration of an
agent in a reaction mixture may be from about 0.05% to 10%. In
still other examples, the concentration of an agent in a reaction
mixture may be about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%.
[0150] In some examples, the concentration of an agent in a
reaction mixture may be at least 0.01%. In other examples, the
concentration of an agent in a reaction mixture may be at least
0.1%. In other examples, the concentration of an agent in a
reaction mixture may be at least 1%. In other examples, the
concentration of an agent in a reaction mixture may be at least 5%.
In still other examples, the concentration of an agent in a
reaction mixture may be at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%.
[0151] In some examples, the concentration of an agent in a
reaction mixture may be at most 5%. In other examples, the
concentration of an agent in a reaction mixture may be at most 1%.
In other examples, the concentration of an agent in a reaction
mixture may be at most 0.1%. In other examples, the concentration
of an agent in a reaction mixture may be at most 0.01%. In still
other examples, the concentration of an agent in a reaction mixture
may be at most 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 15%, 20%, 25%, 30%, 40%, or 50%.
[0152] A reaction mixture may be contained in a variety of
different types of vessels. The vessel chosen to contain a reaction
mixture may depend, for example, on the volume of a reaction
mixture, available equipment for incubating the reaction mixture,
the number of reaction mixtures desired for evaluation, and
combinations thereof. Non-limiting examples of vessels that may be
used to contain a reaction mixture include centrifuge tubes,
beakers, flasks, graduated cylinders, bottles, micro-centrifuge
tubes, test tubes, cuvettes, microwell plates (referred to herein
as a "microplate"--e.g., comprising at least about 2 wells, 4
wells, 6, wells, 8 wells, 12 wells, 24 wells, 36 wells, 48 wells,
54 wells, 60 wells, 66 wells, 72 wells, 78 wells, 84 wells, 90
wells or 96 wells, 144 wells, 192 wells, 384 wells, 768, 1536
wells, 3072 wells, 6144 wells, 12228 wells, or more), capillary
tubes, and tissue culture plates (e.g., at least 10 mm diameter, at
least 35 mm diameter, at least 100 mm diameter, at least 150 mm
diameter).
[0153] A single reaction mixture may be generated for a sample or
multiple reaction mixtures may be generated for a given sample.
Identical replicate reaction mixtures may be used for replicate
incubations or differing incubations. For example, a sample slurry
may be generated from a solid sample and aliquots of the sample
slurry used to generate replicate reaction mixtures each comprising
identical components in identical amounts. The replicate reaction
mixtures may also be incubated at the same conditions.
Alternatively, replicate aliquots of the sample slurry may be used
to evaluate different conditions, such as, for example, differing
incubation conditions, differing levels of an agent of interest in
the reaction mixture, or entirely different types of agents in the
reaction mixture. Moreover, in analogous fashion, a liquid sample
may be obtained and aliquots of the sample may be used to generate
replicate reaction mixtures.
[0154] In the case where replicate reaction mixtures are prepared
for a sample, each reaction mixture may be contained, for example,
in a well of a microplate or the like. Micronization of multiple
reaction mixtures into a vessel designed to contain multiple
reaction mixtures, such as a microplate, may be useful for
achieving higher assay throughput, as a larger number of reaction
mixtures may be more easily incubated and/or evaluated at once when
compared to other means, such as, for example, single reaction
mixture tubes.
Incubation Conditions
[0155] After a reaction mixture is prepared, the reaction mixture
may then be incubated at a given set of conditions. In some cases,
control reaction mixtures may not be subject to incubation.
Conditions necessary for a given incubation may vary depending on,
for example, the type of sample, the type of agent, the amount or
concentration of sample in a reaction mixture, in interactions of
an agent with microbial taxa of interest, the amount or
concentration of an agent of interest in a reaction mixture, the
abundance of microbial taxa or taxon-identifying chemical species
of interest in a reaction mixture, the type of microbial taxa or
taxon-identifying chemical species of interest, the size of a
reaction mixture, the amount of additional reagents in a reaction
mixture, the type of additional reagents in a reaction mixture, the
availability of necessary equipment for incubation, the number of
reaction mixtures, and combinations thereof. Moreover, non-limiting
examples of conditions that may be included in executing an assay
include time of incubation, temperature, relative humidity, levels
of oxygen (O.sub.2) in an incubation atmosphere, levels of carbon
dioxide (CO.sub.2) in an incubation atmosphere, and levels of
nitrogen (N.sub.2) in an incubation atmosphere.
[0156] The time for which a reaction mixture is incubated (herein
referred to as "incubation time") may vary. For example, a reaction
mixture may be subject to an incubation time from about 1 minute
("min") to about 5 days. In other examples, a reaction mixture may
be subject to an incubation time from about 2 hours ("hr") to about
2 days. In other examples, a reaction mixture may be subject to an
incubation time from about 12 hours to 2 days. In still other
examples, a reaction mixture may be subject to an incubation time
from about 1 min, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hr, 1, 2, 3, 4, or 5
days.
[0157] In some examples, a reaction mixture may be subject to an
incubation time of at least 1 min. In other examples, a reaction
mixture may be subject to an incubation time of at least 1 hr. In
other examples, a reaction mixture may be subject to an incubation
time of at least 1 day. In still other examples, a reaction mixture
may be subject to an incubation time of at least 1 min, or 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, or 24 hr, 1, 2, 3, 4, or 5 days.
[0158] In some examples, a reaction mixture may be subject to an
incubation time of at most 2 days. In other examples, a reaction
mixture may be subject to an incubation time of at most 12 hrs. In
other examples, a reaction mixture may be subject to an incubation
time of at most 6 hrs. In still other examples, a reaction mixture
may be subject to an incubation time of at most 1 min, or 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, or 24 hr, 1, 2, 3, 4, or 5 days.
[0159] The temperature at which a reaction mixture is incubated
(herein referred to as "incubation temperature") may vary. The
incubation temperature may be held constant throughout the course
of incubation or may be made to vary. For example, the incubation
temperature may be from about 2 degrees Celsius (".degree. C.") to
about 60.degree. C. In other examples, the incubation temperature
may be from about 4.degree. C. to about 50.degree. C. In other
examples, the incubation temperature may be from about 4.degree. C.
to about 42.degree. C. In still other examples, the incubation
temperature may be about 1.degree. C., 2.degree. C., 3.degree. C.,
4.degree. C., 5.degree. C., 6.degree. C., 7.degree. C., 8.degree.
C., 9.degree. C., 10.degree. C., 11.degree. C., 12.degree. C.,
13.degree. C., 14.degree. C., 15.degree. C., 16.degree. C.,
17.degree. C., 18.degree. C., 19.degree. C., 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., 35.degree. C., 36.degree. C.,
37.degree. C., 38.degree. C., 39.degree. C., 40.degree. C.,
41.degree. C., 42.degree. C., 43.degree. C., 44.degree. C.,
45.degree. C., 46.degree. C., 47.degree. C., 48.degree. C.,
49.degree. C., 50.degree. C. or 60.degree. C.
[0160] In some examples, the incubation temperature is at least
1.degree. C. In other examples, the incubation temperature is at
least 10.degree. C. In other examples, the incubation temperature
is at least 20.degree. C. In other examples, the incubation
temperature is at least 30.degree. C. In other examples, the
incubation temperature is at least 40.degree. C. In still other
examples, the incubation temperature is at least 1.degree. C.,
2.degree. C., 3.degree. C., 4.degree. C., 5.degree. C., 6.degree.
C., 7.degree. C., 8.degree. C., 9.degree. C., 10.degree. C.,
11.degree. C., 12.degree. C., 13.degree. C., 14.degree. C.,
15.degree. C., 16.degree. C., 17.degree. C., 18.degree. C.,
19.degree. C., 20.degree. C., 21.degree. C., 22.degree. C.,
23.degree. C., 24.degree. C., 25.degree. C., 26.degree. C.,
27.degree. C., 28.degree. C., 29.degree. C., 30.degree. C.,
31.degree. C., 32.degree. C., 33.degree. C., 34.degree. C.,
35.degree. C., 36.degree. C., 37.degree. C., 38.degree. C.,
39.degree. C., 40.degree. C., 41.degree. C., 42.degree. C.,
43.degree. C., 44.degree. C., 45.degree. C., 46.degree. C.,
47.degree. C., 48.degree. C., 49.degree. C., 50.degree. C. or
60.degree. C.
[0161] In some examples, the incubation temperature is at most
60.degree. C. In other examples, the incubation temperature is at
most 50.degree. C. In other examples, the incubation temperature is
at most 30.degree. C. In other examples, the incubation temperature
is at most 30.degree. C. In other examples, the incubation
temperature is at most 20.degree. C. In still other examples, the
incubation temperature is at most 1.degree. C., 2.degree. C.,
3.degree. C., 4.degree. C., 5.degree. C., 6.degree. C., 7.degree.
C., 8.degree. C., 9.degree. C., 10.degree. C., 11.degree. C.,
12.degree. C., 13.degree. C., 14.degree. C., 15.degree. C.,
16.degree. C., 17.degree. C., 18.degree. C., 19.degree. C.,
20.degree. C., 21.degree. C., 22.degree. C., 23.degree. C.,
24.degree. C., 25.degree. C., 26.degree. C., 27.degree. C.,
28.degree. C., 29.degree. C., 30.degree. C., 31.degree. C.,
32.degree. C., 33.degree. C., 34.degree. C., 35.degree. C.,
36.degree. C., 37.degree. C., 38.degree. C., 39.degree. C.,
40.degree. C., 41.degree. C., 42.degree. C., 43.degree. C.,
44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C. or 60.degree. C.
[0162] The relative humidity at which a reaction mixture is
incubated (herein referred to as "incubation humidity") may vary.
The incubation humidity may be held constant throughout the course
of incubation or may be made to vary. For example, the incubation
humidity may be from about 0% to about 100%. In some examples, the
incubation humidity may be from about 20% to 100%. In some
examples, the incubation humidity may be from about 30% to about
100%. In still other examples, the incubation humidity may be about
0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
[0163] In some examples, the incubation humidity may be at least
0.1%. In some examples, the incubation humidity may be at least
10%. In some examples, the incubation humidity may be at least 30%.
In still other examples, the incubation humidity may be at least
0.1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.
[0164] In some examples, the incubation humidity may be at most
100%. In some examples, the incubation humidity may be at most 75%.
In some examples, the incubation humidity may be at most 25%. In
still other examples, the incubation humidity may be at most 0.1%,
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%.
[0165] The levels of oxygen (O.sub.2) in an incubation atmosphere
may vary. An "incubation atmosphere" as used herein generally
refers to the local environment in which the incubation of a
reaction mixture takes place. In some cases, an incubation
atmosphere is anaerobic in that the incubation atmosphere comprises
no oxygen. Alternatively, an incubation atmosphere is aerobic. In
some examples, an incubation atmosphere comprises a volume
percentage of oxygen from about 0.5% oxygen to about 100% oxygen.
In other examples, an incubation atmosphere comprises a volume
percentage of oxygen from about 0.5% oxygen to about 50% oxygen. In
other examples, an incubation atmosphere comprises a volume
percentage of oxygen from about 0.5% oxygen to about 10% oxygen. In
other examples, an incubation atmosphere comprises a volume
percentage of oxygen from about 0.5% oxygen to about 5% oxygen. In
still other examples, an incubation atmosphere comprises a volume
percentage of oxygen of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% oxygen.
[0166] In some examples, an incubation atmosphere comprises a
volume percentage of oxygen at least about 0.5% oxygen. In other
examples, an incubation atmosphere comprises a volume percentage of
oxygen at least about 5% oxygen. In other examples, an incubation
atmosphere comprises a volume percentage of oxygen at least about
10% oxygen. In other examples, an incubation atmosphere comprises a
volume percentage of oxygen at least about 50% oxygen. In still
other examples, an incubation atmosphere comprises a volume
percentage of oxygen at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 99% oxygen.
[0167] In some examples, an incubation atmosphere comprises a
volume percentage of oxygen at most about 50% oxygen. In other
examples, an incubation atmosphere comprises a volume percentage of
oxygen at most about 10% oxygen. In other examples, an incubation
atmosphere comprises a volume percentage of oxygen at most about 5%
oxygen. In other examples, an incubation atmosphere comprises a
volume percentage of oxygen at most about 0.5% oxygen. In still
other examples, an incubation atmosphere comprises a volume
percentage of oxygen at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 99% oxygen.
[0168] The levels of carbon dioxide (CO.sub.2) in an incubation
atmosphere may vary. In some cases, an incubation atmosphere
comprises no carbon dioxide. In some examples, an incubation
atmosphere comprises a volume percentage of carbon dioxide from
about 0.5% carbon dioxide to about 100% carbon dioxide. In other
examples, an incubation atmosphere comprises a volume percentage of
carbon dioxide from about 0.5% carbon dioxide to about 50% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide from about 0.5% carbon dioxide
to about 10% carbon dioxide. In other examples, an incubation
atmosphere comprises a volume percentage of carbon dioxide from
about 0.5% carbon dioxide to about 5% carbon dioxide. In still
other examples, an incubation atmosphere comprises a volume
percentage of carbon dioxide of about 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 99% carbon dioxide.
[0169] In some examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at least about 0.5% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at least about 5% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at least about 10% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at least about 50% carbon
dioxide. In still other examples, an incubation atmosphere
comprises a volume percentage of carbon dioxide at least about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
carbon dioxide.
[0170] In some examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at most about 50% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at most about 10% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at most about 5% carbon
dioxide. In other examples, an incubation atmosphere comprises a
volume percentage of carbon dioxide at most about 0.5% carbon
dioxide. In still other examples, an incubation atmosphere
comprises a volume percentage of carbon dioxide at most about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
carbon dioxide.
[0171] The levels of nitrogen (N.sub.2) in an incubation atmosphere
may vary. In some cases, an incubation atmosphere comprises no
nitrogen. In some examples, an incubation atmosphere comprises a
volume percentage of nitrogen from about 0.5% nitrogen to about
100% nitrogen. In other examples, an incubation atmosphere
comprises a volume percentage of nitrogen from about 0.5% nitrogen
to about 50% nitrogen. In other examples, an incubation atmosphere
comprises a volume percentage of nitrogen from about 0.5% nitrogen
to about 10% nitrogen. In other examples, an incubation atmosphere
comprises a volume percentage of nitrogen from about 0.5% nitrogen
to about 5% nitrogen. In still other examples, an incubation
atmosphere comprises a volume percentage of nitrogen of about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
nitrogen.
[0172] In some examples, an incubation atmosphere comprises a
volume percentage of nitrogen at least about 0.5% nitrogen. In
other examples, an incubation atmosphere comprises a volume
percentage of nitrogen at least about 5% nitrogen. In other
examples, an incubation atmosphere comprises a volume percentage of
nitrogen at least about 10% nitrogen. In other examples, an
incubation atmosphere comprises a volume percentage of nitrogen at
least about 50% nitrogen. In still other examples, an incubation
atmosphere comprises a volume percentage of nitrogen at least about
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
nitrogen.
[0173] In some examples, an incubation atmosphere comprises a
volume percentage of nitrogen at most about 50% nitrogen. In other
examples, an incubation atmosphere comprises a volume percentage of
nitrogen at most about 10% nitrogen. In other examples, an
incubation atmosphere comprises a volume percentage of nitrogen at
most about 5% nitrogen. In other examples, an incubation atmosphere
comprises a volume percentage of nitrogen at most about 0.5%
nitrogen. In still other examples, an incubation atmosphere
comprises a volume percentage of nitrogen at most about 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
nitrogen.
Enumerating Microbial Taxa and/or Taxon-Identifying Chemical
Species and Data Analysis
[0174] Microbial taxa may be enumerated by a variety of means
depending upon the desired route and/or available instrumentation.
In some cases, microbial taxa may be enumerated by quantitatively
detecting one or more taxon-identifying chemical species associated
with a given microbial taxon in a sample. Moreover, enumerations of
taxon-identifying chemical species may be used to indicate function
of a given microbial taxon. Non-limiting examples of such chemical
species include small-molecules (including metabolites), proteins,
lipids, nucleic acids, and/or carbohydrates.
[0175] In some cases, a detected molecule may be a common
structural component of a group of organisms comprised in a
microbial taxon. For example, a protein type or lipid associated
with the plasma membrane of a microbe may be detected. In addition,
a molecule secreted may be detected. For example, some bacteria are
known to produce short-chain fatty acids such as butyrate,
propionate, valerate, and acetate. Secretion of a species such as
butyrate, for example, may be the common characteristic used to
group organisms into a given microbial taxon. The detection of
butyrate may then be used to enumerate the abundance of the
respective microbial taxon in a sample. Moreover, a molecule, for
example, may be a common metabolite produced by organisms within a
given microbial taxon. Detection of that metabolite may then be
used to enumerate the abundance of that microbial taxon in a sample
and/or the functionality of that taxon. Furthermore, detection of
one or more molecules in combination may be used to enumerate a
microbial taxon.
[0176] Detection of a molecule may be achieved with a variety of
methods that include spectroscopic methods. Non-limiting examples
of spectroscopic methods that may be used in enumerating microbial
taxa include optical methods (e.g., UV-Vis absorbance,
fluorescence, bioluminescence, Fourier-transform infrared (FT-IR)
spectroscopy), nuclear magnetic resonance (NMR) spectroscopy,
dynamic light scattering, and mass spectrometry.
[0177] Nucleic acids may be detected and quantified in order to
enumerate microbial taxa. Such methods may be especially useful in
cases where microbial taxa are operational taxonomic units (OTUs)
distinguished by one or more gene sequence homologies. Detected
nucleic acids may be deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), or combinations thereof. Nucleic acids may be detected
generically, without respect to sequence, or may be detected in a
sequence specific manner. In cases where sequence specific
detection is desired, detection of a nucleic acid may be completed
by the detection of a full-length gene sequence or may be completed
by the detection of a partial-length gene sequence.
[0178] Moreover, nucleic acids may be downstream molecules
synthesized as the result of gene transcription and/or metagenomic
molecules present in a living organism. In general, a metagenomic
molecule may be a genetic molecule that may be recovered from an
environmental sample, such as a living organism. For example, in
the case of the 16S ribosomal RNA (rRNA) gene, genomic DNA
corresponding, in whole or part, to regions of the 16S rRNA gene,
messenger RNA (mRNA) transcripts, in whole or part, of the 16S rRNA
gene, and/or functional 16S rRNA may be detected and used to
enumerate the abundance of a microbial taxon characterized by
sequence homology of a particular 16S rRNA gene sequence.
[0179] Nucleic acid sequencing methods may be used to detect and
quantify sequence specific nucleic acids such that they are used to
enumerate the abundance of a microbial taxon characterized by
homology of the detected sequence amongst organisms clustered into
the microbial taxon. Non-limiting examples of sequencing methods
that may be used include shotgun sequencing, polymerase chain
reaction, real-time polymerase chain reaction, ligase chain
reaction, single-molecule real-time sequencing, ion torrent
sequencing, pyrosequencing, sequencing by synthesis, sequencing by
ligation, chain termination sequencing, massively parallel
signature sequencing, polony sequencing, SOLiD sequencing, DNA
nanoball sequencing, heliscope single molecule sequencing, single
molecule real time sequencing, nanopore sequencing, mass
spectrometry sequencing, microfluidic sequencing, high-throughput
sequencing, Illumina sequencing, HiSeq sequencing, MiSeq
sequencing, or combinations thereof. Sequencing may be completed
such that full-length genes are sequenced or partial-length genes
are sequenced.
[0180] Sequence-specific detection of nucleic acids may also be
completed with oligonucleotide probes. An oligonucleotide probe may
be capable of hybridizing with a full-length or partial-length gene
sequence of interest. Moreover, an oligonucleotide probe may be
labeled with a detectable tag, such as a fluorescent dye, that may
be detected. Alternatively, nucleic acid to be probed may be
labeled such that its binding with the oligonucleotide probe is
detected (via an attached label). An oligonucleotide probe may be a
primer or a longer, different type of oligonucleotide. The
oligonucleotide probe may the same type of nucleic acid as the
target (e.g., DNA target and DNA oligonucleotide) or the
oligonucleotide probe may be a different type of nucleic acid than
the target (e.g., DNA target and RNA probe). Non-limiting examples
of a label linked to an oligonucleotide probe may be a fluorescent
dye, absorbent chemical species, radiolabel, quantum dot, or
nanoparticle. Moreover, an oligonucleotide probe may also include a
quencher (a molecule used, for example, to inhibit fluorescence).
Probes useful in real-time polymerase chain reactions may be useful
in sequence specific detection. Non-limiting examples of such
probes include TaqMan probes, TaqMan Tamara probes, TaqMan MGB
probes, or Lion probes.
[0181] Prior to sequencing, nucleic acids to be sequenced may be
amplified.
[0182] Oligonucleotide probes may be immobilized to an array such
that the binding of a target nucleic acid sequence is detected. In
some examples, such oligonucleotide probes may be immobilized in
one or more arrays. Each oligonucleotide probe is assigned a
specific position in the array such that the position corresponds
to the oligonucleotide probe. Nucleic acids to-be-detected may be
labeled with an agent capable of being detected. Hybridization of a
labeled nucleic acids to a complementary, immobilized sequence
results in accumulation of detectable label at the signal which can
then be identified indirectly as presence of the a given sequence.
Nucleic acids to-be-analyzed may be exposed to an oligonucleotide
probe array without size reduction or may be fragmented in order to
ensure that the size of the to-be-analyzed nucleic acid is more
similar to the oligonucleotide probes arranged on the array. Size
similarity may result in better nucleic acid binding to
oligonucleotide probes of the array. Oligonucleotide probe arrays
have been generated for taxonomic analyses based on the
sequence-specific detection of nucleic acids. Non-limiting examples
of such arrays include the G2 PhyloChip.TM. and G3 PhyloChip.TM..
The selection of oligonucleotide probes, the construction of each
array, methods for obtaining data, and methods for analysis of data
obtained from each array are described in detail in U.S. Patent
Application Publication No. 2009/0291858 and U.S. Patent
Application Publication No. 2012/0165215 which are both
incorporated in entirety herein by reference.
[0183] Oligonucleotide probes may be immobilized on microbeads.
Binding of nucleic acids to oligonucleotide probes arranged on
microbeads and detection of such nucleic acids is completed in an
analogous fashion to that mentioned above for oligonucleotides,
such that nucleic acids to-be-analyzed are labeled and their
hybridization with an oligonucleotide probe results in the
accumulation of detectable signal that can be indirectly
interpreted as the presence of a sequence specific region of
nucleic acid. Again, nucleic acids to-be-analyzed may be exposed to
oligonucleotide probes on microbeads without size reduction or may
be fragmented in order to ensure that the size of the
to-be-analyzed nucleic acid is more similar to the oligonucleotide
probes arranged on the microbeads.
[0184] DNA barcording may aid in enumerating microbial taxa. DNA
samples from multiple subjects and time points may be PCR amplified
using primers that incorporate a unique DNA barcode in addition to
the 16S rRNA priming sites. Produced amplicons may then be pooled
together and sequenced in a single batch.
[0185] Enumeration of microbial taxa may also be achieved by other
means such as analyzing proteomes, transcriptomes, metabolomes, or
combinations thereof. For example, microbial RNA transcripts,
proteins, non-16S genes, etc. may be profiled and their abundance
used to determine the impact of the agent on the microbial
communities.
[0186] An enumeration of microbial taxa and/or taxon-identifying
chemical species in a reaction mixture (herein referred to as
"enumeration") may be completed both prior to and at one or more
time points after adding an agent to the reaction mixture. An
aliquot of the reaction mixture may be used for enumerations at
each desired time point or the entire reaction mixture may be used
for a single time point. In one example, enumerations are completed
at a time point immediately following the addition of an agent to a
reaction mixture and at subsequent time points after addition of
the agent. Moreover, enumerations determined for a reaction mixture
may be back-calculated (using the appropriate assay protocol,
reagent ratios, etc.) such that an enumeration is determined for
the original sample.
[0187] The number of time points for which microbial taxa and/or
taxon-identifying chemical species are enumerated may vary
depending upon, for example, the number of microbial taxa to be
enumerated, the duration of agent action (if any) on the microbial
taxa and/or taxon-identifying chemical species of interest,
incubation conditions, and combinations thereof. For example, the
number of time points for which microbial taxa and/or
taxon-identifying chemical species are enumerated is at least 1
time point. In other examples the number of time points for which
microbial taxa and/or taxon-identifying chemical species are
enumerated is at least 10 time points. In other examples, the
number of time points for which microbial taxa and/or
taxon-identifying chemical species are enumerated is at least 30
time points. In still other examples, the number of time points for
which microbial taxa and/or taxon-identifying chemical species are
enumerated is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
or more time points.
[0188] The time interval between time points for which microbial
taxa and/or taxon-identifying chemical species are enumerated may
vary. Intervals may be equally spaced between time points (e.g.,
for example, a time point is taken every 5 minutes) or intervals
may be spaced such that intervals are different between different
time points (e.g., a time point is taken at 5 minutes, 10 minutes,
20 minutes, 50 minutes, 100 minutes, etc.). Moreover, the duration
of a time interval between time points may vary. In some examples,
the duration of the time interval between time points may be at
least about 1 second. In some examples, the duration of the time
interval between time points may be at least about 1 minute. In
other examples, the duration of the time interval between time
points may be at least about 1 hour. In other examples, the
duration of the time interval may be at least 1 day. In other
examples, the duration of the time interval may be at least 5 days.
In still other examples, the duration of the time interval may be
about 0.01, 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, or 60 minutes
or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 hours, or 1,
1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6,
3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 12, 14,
16, 18, or 20 days.
[0189] An enumeration may be determined once for a reaction mixture
at a given time point or may be determined using replicate
enumerations obtained from aliquots of the reaction mixture. In
cases, where replicate enumerations are used, an average
enumeration may be calculated for a time point. Moreover,
enumerations may be completed for identical, replicate reaction
mixtures and subsequently reported collectively as an average.
[0190] The number of microbial taxa or taxon-identifying chemical
species that are enumerated may vary. Number variance may vary, for
example, due to the number of species or taxon- identifying
chemical species that are present in a microbial community of
interest. Some microbial communities of interest may possess
greater numbers of relevant taxa or taxon- identifying chemical
species that are present. In some examples, the number of microbial
taxa or taxon-identifying chemical species that are enumerated is
from 1 microbial taxa or taxon- identifying chemical species to
1,000,000 microbial taxa or taxon-identifying chemical species. In
other examples, the number of microbial taxa or taxon-identifying
chemical species that are enumerated is from 1 microbial taxa or
taxon-identifying chemical species to 100,000 microbial taxa or
taxon-identifying chemical species. In other examples, the number
of microbial taxa or taxon-identifying chemical species that are
enumerated is from 1 microbial taxa or taxon-identifying chemical
species to 10,000 microbial taxa or taxon-identifying chemical
species. In other examples, the number of microbial taxa or
taxon-identifying chemical species that are enumerated is from 1
microbial taxa or taxon-identifying chemical species to 100
microbial taxa or taxon-identifying chemical species. In other
examples, a single microbial taxon or taxon-identifying chemical
species is enumerated.
[0191] In some examples, the number of microbial taxa or
taxon-identifying chemical species that are enumerated is at least
1 microbial taxon or taxon-identifying chemical species. In other
examples, the number of microbial taxa or taxon-identifying
chemical species that are enumerated at least 100 microbial taxa or
taxon-identifying chemical species. In other examples, the number
of microbial taxa or taxon-identifying chemical species that are
enumerated is at least 1,000 microbial taxa or taxon-identifying
chemical species. In other examples, the number of microbial taxa
or taxon-identifying chemical species that are enumerated is at
least 10,000 microbial taxa or taxon-identifying chemical species.
In other examples, the number of microbial taxa or
taxon-identifying chemical species that are enumerated is at least
100,000 microbial taxa or taxon-identifying chemical species. In
still other examples, the number of microbial taxa or
taxon-identifying chemical species enumerated is at least 1, 10,
100, 1000, 10,000, 100,000, or 1,000,000 microbial taxa or
taxon-identifying chemical species.
[0192] Moreover, all possible microbial taxa within a given
taxonomic classification scheme or all known taxon-identifying
chemical species may be enumerated. Alternatively, one or more
particular subsets of all possible microbial taxa within a given
taxonomic classification scheme or taxon-identifying chemical
species may be enumerated.
[0193] The particular microbial taxa and/or taxon-identifying
chemical species that are enumerated may be derived from microbial
taxa determined from experiments. Such experiments may include
experiments that have identified certain microbial taxa that are
important to a specific disease condition or general health of a
subject.
[0194] Completed enumerations may be manipulated in order to better
interpret the results of an assay and/or compare the results of an
assay with that of another. Numerical manipulations may be made,
for example, using an algorithm that may be useful in interpreting
the results of an assay. Such numerical manipulations may be made
for all enumerated microbial taxa and/or taxon-identifying chemical
species, a subset of all enumerated microbial taxa and/or
taxon-identifying chemical species, or a single enumerated
microbial taxon and/or taxon-identifying chemical species. An
example algorithm is a difference algorithm wherein an enumeration
completed after adding an agent to a reaction mixture is subtracted
from an enumeration completed prior to adding the agent to the
reaction mixture. The magnitude of the calculated difference may
quantitatively indicate the degree to which the agent affects the
respective microbial taxon of interest in the subject of
interest.
[0195] In another example, an algorithm may be used to compare an
enumeration to one or more reference enumerations. For example, a
first sample may be obtained from a subject afflicted with a
condition and contacted with an agent of interest in a first
reaction mixture. Microbial taxa or taxon-identifying chemical
species of interest in the first reaction mixture are enumerated
after the agent is added to the first reaction mixture. A second
sample may be obtained from a normal (i.e., healthy) subject,
prepared in a second reaction mixture, with no agent added to the
second reaction mixture. The appropriate microbial taxa and/or
taxon-identifying chemical species of interest enumerated in the
first reaction mixture may then also be enumerated in the second
reaction mixture. An example algorithm whereby the two reaction
mixtures are compared includes taking the difference between the
enumerations for the two reaction mixtures. The magnitude of the
calculated difference may quantitatively indicate the degree to
which an agent drives the levels and/or function of the microbial
taxon of interest in the afflicted subject toward the levels and/or
function of the microbial taxon observed in the normal subject.
[0196] Enumerations of each microbial taxa and/or taxon-identifying
chemical species may be collected and used for completing a
principal component analysis (PCoA). A PCoA analysis may be used to
generate UniFrac distances between different treatment groups
(e.g., samples obtained from an afflicted subject and contacted
with an agent vs. samples obtained from a normal subject and not
contacted with the agent). A UniFrac distance generally refers to a
metric that is a distance measure between organismal communities
using phylogenetic information. Such a metric may be useful in
quantitatively describing the degree of similarity/dissimilarity
between different treatment groups. Such measures of
similarity/dissimilarity may be used to identify shifts in
microbiomes.
[0197] Dissimilarity measures are generally with respect to two
samples with a result that is a matrix of all pair-wise sample
dissimilarities. This matrix may be more manageable for creating an
overview because statistical techniques such as hierarchical
clustering or ordinations can be employed to visualize the
important patterns of variation across samples. The particular
dissimilarity measures used may depend on assessment of changes in
the abundances of microbes, the incidence (presence/absence) of
microbes, whether to consider information on phylogenetic
relationships between microbial taxa or to treat the microbial taxa
as unrelated bins, and combinations thereof.
[0198] In considering a pair of samples, the Sorensen index may be
sensitive to overlap. Specifically, the Sorensen index is the ratio
of the number of microbial taxa or metabolites simultaneously
detected in both samples compared to the sum of the microbial taxa
or metabolites in both samples. Moreover, the Sorensen index
provides insights but stops short of using the phylogenetic
relationships that exist among all bacteria, something that is
offered by unweighted Unifrac calculations. Since bacteria are
taxonomically classified into phyla, classes, orders, families,
genera and species, differences between samples can incorporate the
magnitude of the genetic difference between the microbes present in
each sample. Using Unifrac, if two species are in the same family
the pair-wise dissimilarity may be less than if those two species
were in distinct families. In cases where phylogenetic
relationships between taxa are known, unweighed Unifrac
dissimilarity may be preferable over Sorensen dissimilarity.
[0199] The basic sample-to-sample dissimilarity measurement using
the abundance of each microbial taxa is carried out by the
Bray-Curtis index. The Bray-Curtis function performs a pair-wise
normalization by dividing the sum of differences by the sum of all
abundances which is helpful when abundance metrics across samples
are imperfectly scaled. The index is sensitive to the difference in
abundance observed between the same taxa across pairs of samples.
To integrate both the relationships between taxa and the abundance
fluctuations of those taxa across samples, the weighted Unifrac
measure may be used. Unifrac can aid in resolving subtle variations
in composition between microbial communities.
Combination with Additional Assays
[0200] Assays described herein may be used in combination with
additional assays depending on need. In some instances, results
from an assay described herein may be used execute an additional
assay or results from an additional assay may be used to execute an
assay described herein. Non-limiting examples of additional assays
that may used with an assay described herein include a blood assay,
a urine assay, a fecal assay, a cerebrospinal fluid assay, a saliva
assay, a sputum assay, an assay performed on a biopsy, an assay
performed on part of the reproductive system, a cardiovascular
assay, a respiratory assay, a cognitive assay, a reproductive
assay, a liver function assay, a kidney function assay, a thyroid
assay, a locomotor assay, an ocular assay, and combinations
thereof.
Functionality Assays
[0201] In addition to enumerating the abundance of one or more
microbial taxa or taxon-identifying chemical species, functionality
assays may be used to determine the effects of an agent on a
sample, including the effects of microbial agents (e.g., bacterial
agents) on tissue or cellular samples obtained from a subject. The
tissue or cellular samples may be representative of a particular
anatomical region of the subject (e.g., fecal samples may be
representative of the gut). In such cases, a method may comprise
the steps of: (a) obtaining a sample (e.g., a tissue sample or cell
sample) from a first subject; (b) contacting the sample with an
agent in a reaction mixture; and (c) evaluating the functionality
of the sample upon contact with the agent. When contacting an agent
with a sample, any suitable incubation condition(s) may be used,
including incubation conditions (e.g., incubation temperature,
incubation humidity, atmospheric oxygen content, atmospheric carbon
dioxide content, atmospheric nitrogen content, incubation time,
etc.) described elsewhere herein.
[0202] For example, an agent in a functionality assay may comprise
one or more microbes, such as, for example bacteria. The microbes
may be obtained from any sample type or sample source described
herein, such as, for example, feces, cecal contents, or other
matter (including others described herein) produced by a living
organism. In some cases, the microbes may be microbes obtained from
samples already contacted with another agent.
[0203] In cases where microbes are obtained from solid matter
(e.g., feces or cecal contents) produced by a living organism, the
solid matter may be obtained from the living organism, homogenized
if necessary (e.g., in the case of tissue samples), and used as in
agent. In some cases, solid matter may be optionally homogenized
and slurried. The slurry may be incubated at appropriate conditions
(e.g., incubation temperature, incubation time, incubation
atmosphere, etc. as described elsewhere herein) in order to
propagate microbes in the slurry. The solid content of the slurry
may then be separated (e.g., via centrifugation) from the liquid
component and the supernatant comprising microbes from the matter
obtained from the living organism used as an agent. In some cases,
the solid content of the slurry may be used as an agent.
[0204] The agent may then be contacted with a sample of interest
(e.g., cell sample (e.g., HT29, HT29 MTX or CaCo2 cells), cells
grown from tissue samples, tissue samples, organotypic cultures
derived from a tissue (e.g., colon), etc.) and incubated with the
agent at desired conditions (e.g., e.g., incubation temperature,
incubation time, incubation atmosphere, etc. as described elsewhere
herein). In some cases, antibiotics may be added to reaction
mixtures in order to determine the effect of diminished microbial
functionality in the assay.
[0205] After contact with the sample of interest, the functionality
of the sample may be assessed to determine the effects of the agent
on the sample. Functionality may be assessed, for example, via a
barrier function assay (including assays where the permeation of
high molecular weight, fluorescent labels, such as FITC-Dextran,
are measured), a cell viability assay (e.g., via Live/Dead.TM.
staining or DAPI staining), a cytokine production assay (including
where cytokines are measured via an ELISA assay), and/or an immune
response assay.
[0206] Results of a functionality assay may be used for any
suitable purpose, including the various uses for the results of
assays described elsewhere herein. In some cases, a sample in a
functionality assay can serve as `reporter` host to determine the
effects of microbial agents (e.g., bacteria) on cellular functions.
Where microbial agents (e.g., bacteria) are obtained from the
products of another assay, the effects of the microbial agents in a
functionality assay may be used to assess the functionality of the
microbial agents. For example, in cases where a first agent is
contacted with a first sample in a first assay in a reaction
mixture, the microbes (e.g., bacteria) in the reaction mixture can
then be used as second agents in a functionality assay. The
observed effects of the microbes in the functionality assay may be
used to assess the functionality of the microbes obtained after
contacting the first agent with the first sample in the first
assay.
Methods for Utilizing the Results of a MESA Assay
[0207] This disclosure provides methods for interpreting and/or
utilizing the results of an assay described herein. The in vitro
nature of assays may be especially beneficial to probing the
effects of an agent on microbial taxa of interest, without the
potential for agent toxicity when investigated in vivo. In one
aspect, the results of assays may be used to make inferences about
the effects of an agent for a population of subjects. In another
aspect, assays may aid in decision-making regarding the utility of
an agent in a variety of applications that include, for example,
health care and health safety decision-making. This disclosure also
provides methods for counseling such that healthcare and health
safety decision-making may be disseminated to subjects in want or
need.
Reference Enumerations
[0208] In general, an enumeration of an assay (referred to herein
as a "test enumeration") or any numerical manipulation of the
enumeration, may be considered in isolation or may be considered
with respect to one or more reference enumerations or numerical
manipulations of a reference enumeration. In general, a reference
enumeration may be any enumeration used as a control for a test
enumeration. A reference enumeration, for example, may be an
enumeration generated for a sample(s) (or reaction mixture
comprising a sample) obtained from a subject(s) of different type
than that of the test enumeration; may be an enumeration generated
for samples obtained from a subject not contacted with an agent of
interest; may be an enumeration generated for a sample contacted
with an agent different than that of interest; and combinations
thereof.
[0209] In one example, a test enumeration is completed for a sample
obtained from a subject afflicted with a particular condition and
contacted with an agent of interest. The same enumeration is also
completed for a sample obtained from a normal (i.e., healthy)
subject and not contacted with the agent, and the enumerations
serves as a reference enumeration. The reference enumeration may be
used, with or without further data analysis, for example, to
determine whether or not the agent can drive the level and/or
function of the enumerated microbial taxa in the afflicted subject
toward the level and/or function of the enumerated microbial taxa
in the normal subject.
[0210] In another example, a first and second sample is obtained
from a subject afflicted with a condition. A test enumeration is
completed for the first sample, which is contacted with an agent of
interest. The same enumeration is completed for the second sample,
which is not contacted with the agent of interest, and the
enumeration serves as a reference enumeration. The reference
enumeration may be used, with or without further data analysis, for
example, to determine whether or not the agent affects the level
and/or function of the enumerated microbial taxa in the afflicted
subject.
[0211] In yet another example, two samples are obtained from a
subject afflicted with a condition and two samples are obtained
from a normal (i.e., healthy) subject. One sample from each subject
is contacted with an agent of interest and two respective test
enumerations are generated. The remaining two samples are not
contacted with the agent of interest and each serve as a reference
enumeration for each test enumeration for the respective subject.
The reference enumeration for each subject may be used, with or
without further data analysis, for example, to determine whether or
not the agent affects the level and/or function of the enumerated
microbial taxa in each subject. Moreover, the comparisons of each
reference enumeration to its respective test enumeration may be
used to determine whether or not the agent affects the level and/or
function of the enumerated microbial taxa in the subject afflicted
with the condition differently than it does in the normal
subject.
[0212] A reference enumeration may represent a threshold in a given
application. A threshold value generally should be considered with
respect to the particular microbial taxa evaluated and/or any other
available information (e.g., additional assays). In some examples,
higher-than-threshold values for a test enumeration may be desired.
In other examples, lower-than-threshold values for a test
enumeration may be desired.
Estimating Effects of an Agent on Subjects of a Subject
Population
[0213] An assay described herein may be used to estimate the
effects of an agent on subjects of a population. For example,
assays may be completed with respect to an agent of interest for a
collection of samples obtained from various subjects of a group
selected to represent a population. The collection of results from
the assays may then be used, with or without further analysis, to
generalize the effects of the agent on any subject of the
population. For example, assays with respect to an agent of
interest may be completed for samples obtained from 100 subjects
all afflicted with a condition. The set of assay results may used,
with or without further analysis, to generalize the effect of the
agent on the appropriate microbial taxa in any subjects afflicted
with the condition.
[0214] A population may be, for example, subjects all afflicted
with a condition, subjects not afflicted with a condition, subjects
all of the same race, subjects all of the same ethnicity, subjects
all of the same gender, subjects all of a particular age or age
group, subjects all of a particular phenotype (e.g., subjects that
all have red hair, subjects that all have blue eyes, subjects all
of a certain height, etc.), subjects all of a particular level in a
phylogenic classification scheme, and combinations thereof. Indeed,
a population may be the totality of subjects in a group identified
by a unifying feature. Moreover, a population may be divided into a
plurality of sub-populations based on an additional unifying
feature (e.g., subjects that all have red hair and blue eyes).
[0215] The number of subjects in a group selected to represent a
population may vary. In some examples, the number of test subjects
in a group selected to represent a population is 1 subject. In some
examples, the number of test subjects in a group selected to
represent a population is at least 2 subjects. In other examples,
the number of test subjects in a group selected to represent a
population is at least 100 subjects. In other examples, the number
of test subjects in group selected to represent a population is at
least 1000 subjects. In other examples, the number of test subjects
in a group selected to represent a population is at least 10000
subjects. In still other examples, the number of test subjects in a
group selected to represent a population is at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 2000, 3000, 4000, 5000, 10000 or more subjects.
[0216] In some examples, the number of test subjects in a group
selected to represent a population is from 2 subjects to 10000
subjects. In other examples, the number of test subjects in a group
selected to represent a population is from 2 subjects to 1000
subjects. In other examples, the number of test subjects in group
selected to represent a population is from 2 subjects to 100
subjects. In other examples, the number of test subjects in a group
selected to represent a population is from 2 subjects to 10
subjects.
[0217] In some cases, an assay completed for one type of subject or
population may be used to estimate the effects of an agent on one
or more different types of subjects or population. For example, an
assay for a subject of one species of a living organism (i.e., one
type of subject) may be used to estimate the effects of the agent
in a subject of another species of a living organism (i.e., another
type of subject). Analogously, assays completed to estimate the
effects of an agent in the population of subjects of one species of
living organism may be used to estimate the effects of the agent in
the population of subjects of another species of living
organism.
[0218] Indeed, assays completed using a subject(s) of any of the
species described herein may be used to estimate, with or without
further analysis, the effects of an agent on a subject(s) of any
other species described herein. For example, an assay completed for
a species of a common laboratory animal such as a mouse or rat may
be used to estimate the effects of an agent on a human.
Non-limiting examples of additional species pairs include: a human
and a species of mouse; a human and a species of rat; a human and a
species of dog; a human and a species of monkey; a human and a
species of rabbit; a human and a species of pig; a dog and a
species of mouse; a dog and a species of rat; a cat and a species
of mouse; a cat and a species of rat.
Healthcare and Health-Safety Decision-Making
[0219] An assay described herein may aid in healthcare and/or
health-safety related decision- making with respect to an agent or
a combination of agents. Samples may be obtained from a subject or
a group of subjects, assayed with an agent or a combination of
agents desired for healthcare-related use, and the results of an
assay(s) used in making decisions regarding the acceptability of
the agent or combination of agents and/or the use of such entities
in the subject or group of subjects studied. Decisions made for a
group of subjects may be extended to a population for which the
subjects of the group are selected to represent. Moreover,
decisions made for a subject, group of subjects, or population may
be extended to one or more different types of subject, group of
subjects, or population.
[0220] Healthcare decisions may include, for example, deciding upon
the utility (e.g., efficacy, safety, etc.) of an agent or
combination of agents in a subject or population; and selection of
subjects for particular use(s) of an agent or combination of
agents. More specific examples of healthcare related decisions
include: determining the utility of an agent or combination of
agents to be used as a drug(s); selecting subjects to use a drug or
combination of drugs; determining the utility of an agent or
combination of agents to be used as a supplement(s); selecting
subjects to use a supplement or combination of supplements;
determining the utility of a food or combination of foods (e.g., a
diet); selecting subjects for a particular food or diet;
determining the utility of a beauty product or a combination of
beauty products; selecting subjects to use a beauty care product or
combination of beauty care products; determining the utility of a
personal hygiene product or combination of personal hygiene
products; selecting subjects to use a personal hygiene product or
combination of personal hygiene products; determining the
propensity of an agent to cause a condition in a subject or
population where the condition is associated with an undesirable
shift in local microbial populations caused by the agent;
determining the safety of a new chemical; determining the safety of
a hazardous material, and combinations thereof.
Decision-making with Respect to a Drug or Other Consumer Healthcare
Product
[0221] An assay described herein may aid in determining the utility
of an agent to be used as a drug or a plurality of agents to be
used as a combination of drugs. Determining the utility of an agent
to be used as a drug or a combination of agents to be used as a
combination of drugs may include evaluating the potential
therapeutic efficacy and/or safety of the agent or combination of
agents. Such determinations may be made, for example, with respect
to the capability of an agent or combination of agents to cause a
desirable change in the levels and/or function of one or more
microbial taxa in a subject(s) from which a sample(s) is obtained.
In cases where a condition is thought to be linked to a microbiome,
a desirable change with respect to a potential drug(s) generally
refers to a shift in the levels and/or function of microbial taxa
associated with a condition toward levels and/or function
associated with a healthy state all while minimizing any unwanted
and/or harmful changes (i.e., side-effects) to other microbial
taxa. In cases where a condition is not thought to be linked to a
microbiome, a desirable change with respect to a potential drug(s)
generally refers to minimized disruption of a microbiome. In some
cases, two or more agents may be considered in combination therapy
such that one or more of the agents aids in ameliorating the
deleterious effects of any others.
[0222] In some cases, it may be determined, based upon one or more
assays described herein, that an agent or combination of agents may
not alter or may unfavorably alter the levels and/or function of
one or more microbial taxa to the extent that the agent or
combination of agents is potentially inefficacious and/or unsafe
(i.e. toxic). In other cases, it may be determined, based upon one
or more assays described herein, that an agent or combination of
agents favorably alters the levels and/or function of one or more
microbial taxa to the extent that the agent or combination of
agents is potentially efficacious and/or safe. In still other
cases, it may be determined that assays or investigations in
addition to one or more assays described herein may be required to
determine the utility of an agent or combination of agents as a
drug(s).
[0223] An agent studied for use as a drug may be any chemical
species, including any example agent described herein. Moreover, an
assay may be completed post-approval of a drug such that an already
approved drug (prescription or over-the-counter) is further
evaluated for utility, including efficacy with respect to an
indication not yet approved for treatment with the drug and/or
safety with respect to a microbiome. Analogously, previously
approved drugs that have been withdrawn from the market may be
further evaluated for utility using an assay described herein.
Moreover, an agent that is currently in pre-clinical or clinical
development, or an agent that is currently available but has not
yet been considered for use as a drug may also be evaluated for
utility as a drug.
[0224] In particular, antibiotic therapies may be of especially
important interest as they are generally designed to stunt growth
the growth of (e.g., bacteriostatic agents) and/or kill (e.g.,
bactericidal agents) bacteria. Unfortunately, an antibiotic
designed to reduce the levels and/or function of harmful bacteria
may also reduce the levels and/or function of bacterial populations
considered to be beneficial. Thus, assays may be especially useful
in decision-making with respect to the utility of an antibiotic or
of an antibiotic used in combination with other drugs or
agents.
[0225] An assay described herein may aid in utility determinations
for agents implicated as potential or already-available other
consumer healthcare products such as supplements, beauty products,
and/or personal hygiene products in analogous fashion to that
described above for drugs. Again, any chemical species may be
considered for use as any of these types of agents. Assays may aid
in making utility determinations when an agent or agents of any of
these types is considered with respect to the use of a drug. For
example, an assay described herein may be used to determine the
utility of one or more supplements and/or drugs when the
supplement(s) and drug(s) are used in combination.
[0226] An assay described herein may aid in determining the utility
of an agent or a combination of agents during development and/or
regulatory agency (e.g., the U.S. Food and Drug Administration
(FDA)) evaluations of an agent. For example, during pre-clinical
development and/or clinical trials of a drug, an assay described
herein may be used by a drug research and development organization
to make decisions regarding initiating or continuing development of
a particular agent. In one example, it may be determined that a set
of agents (novel, non-novel, or a combination thereof) may be
useful as drugs to treat one or more conditions. At any point
during development, an assay employing one or more agents of the
set may be used to, for example, select agents from the set for
initiating or continuing pre-clinical development; select agents
from the set for initiating or continuing clinical trials; and/or
determining the mechanism of action of one or more agents of the
set. The use of high-throughput assays described herein may be
especially useful in screening large groups of potential drugs.
[0227] Moreover, at any point during the development process of an
agent, an assay described herein may be used (by either or both of
a drug development organization or regulatory agency) to assess the
acceptability of an agent to receive approval (e.g., for
prescription use or for over-the-counter use in the case of a drug)
for use in a population of subjects (e.g., for use in humans)
and/or the dosage at which an agent or combination of agents can be
safely and/or effectively administered. Additionally, an assay
described herein may be used post-approval of an agent such that a
regulatory agency makes a decision as to whether or not the
approved agent should remain on the market and/or whether changes
to an approved/recommended dosage should be made.
[0228] An assay described herein may be used to aid in selecting a
subject for use of a drug, combination of drugs, other consumer
healthcare product, combination of other consumer healthcare
products, and combinations thereof. For example, a subject
afflicted with a condition may be in want or need of a drug
approved and available for the treatment of the condition. A sample
may be obtained from the subject and assayed with respect to the
drug using methods described herein. The results of the assay, with
respect to drug efficacy and/or safety, may then be used to
determine whether or not the subject may benefit from treatment
with the drug and, thus, whether or not the subject should commence
using the drug.
[0229] An assay described herein may be used to aid in selecting a
population for use of a drug, combination of drugs, other consumer
healthcare product, combination of other consumer healthcare
products, and combinations thereof. For example, assays may be
completed for samples obtained from a group of subjects selected to
represent a population with respect to a drug or combination of
drugs. The results of the assays may be used to aid in making
decisions with respect to use of the drug or combination of drugs
in the population. In one example, it may be observed, after
completing an assay described herein, that key microbial taxa
respond favorably in a group of human subjects selected for a
clinical trial of an agent. Such an observation may be used in
deciding whether humans in general. In another example, it may be
observed that, after completing an assay described herein, key
microbial taxa respond unfavorably to a drug in a group of women
selected to represent the population of women, to the extent that
the drug may not be useful in women. The results of the assay may
then be used in deciding whether or not women in general should use
the drug.
Decision-Making with Respect to Foods and Diets
[0230] An assay described herein may aid in determining a
preference for a food (which may be a beverage) or a combination of
foods, including a diet. The preference for a food or diet may be
based upon a number of factors that include, for example, the
safety of a food or diet and/or the propensity of a food or diet to
cause a change in one or more microbial populations of a host
subject that ingests the food or diet. Non-limiting examples of
diets include a South Beach Diet, a Dukin diet, a Stillman diet, an
Atkins Diet, a gluten-free diet, a ketogenic diet, a low-residue
diet, a liquid diet, a vegetarian diet, a low-calorie diet (e.g.,
Weight Watchers.TM., Jenny Craig.TM., Nutrisystems.TM.), a low-fat
diet, a low-carbohydrate diet, a low-protein diet, a low-monosodium
glutamate (MSG) diet, a detox diet, an elimination diet, a specific
carbohydrate diet, a diabetic diet, a dietary approaches to stop
hypertension diet (DASH) diet, a best bet diet, an organic diet,
and combinations thereof.
[0231] Assays may be completed for virtually any type of food
and/or diet. In some cases, determinations are made between the
same type of food that is obtained from a plurality of sources
(e.g., beef obtained from grass-fed livestock vs. beef obtained
from livestock fed on a concentrated diet of grain, soy, corn and
other supplements such as steroids and antibiotics). Foods or diets
may include, for example, already available foods or diets that are
available on the market, foods or diets that have been withdrawn
for the market, foods or diets that are currently in development,
foods or diets that have yet-to-be-developed; and agents that are
currently available but have not yet been considered for use as
foods or as foods in a diet. Moreover, an assay described herein
may be used to make decisions regarding a food or combination of
foods in a subject or group of subjects also treated with a drug or
other agent.
[0232] The results of an assay evaluating a food or combination of
foods may be used in analogous fashion to that described above for
drugs and other consumer healthcare products, such that the results
are used to decide whether a food or combination of foods should be
consumed. Moreover, selection of a subject and/or population for a
particular food or diet may also be completed analogous to
subject/population selection described above for drugs and other
consumer healthcare products.
Decision-Making with Respect to the Propensity to Cause a
Condition
[0233] An assay described herein may aid in determining the
propensity of an agent to cause one or more conditions. Indeed, an
in vitro assay described herein may be of particular value in
assessing any deleterious effects of an agent in a subject prior to
actually administering the agent to the subject in vivo. A number
of conditions are known to be associated with the presence and
composition of particular microbial communities. For example, the
intestinal gut microbiota provides many crucial functions to its
host, including contribution to digestion, the development of the
immune system, and resistance to pathogenic colonization. Even a
slight fluctuation in the symbiotic balance may be deleterious to
the host, leading to pathological conditions such as, for example,
Clostridium difficile infection or inflammatory bowel disease
(IBD). As a result, it is important to monitor the effects of
agents on microbiota as they may cause conditions to arise in an
administered host. Other non-limiting examples of conditions that
may be caused by an agent include a condition of the gut, Crohn's
Disease (CD), irritable bowel syndrome (IBS), stomach ulcers,
colitis, neonatal necrotizing enterocolitis, or gastroesophageal
reflux disease (GERD), cystic fibrosis, chronic obstructive
pulmonary disease, rhinitis, atopy, asthma, acne, a food allergy,
obesity, periodontal disease, diarrhea, constipation, functional
bloating, gastritis, lactose intolerance, visceral hyperalgesia,
colic, pouchitis, diverticulitis, allergies, asthma, sinusitis,
chronic obstructive pulmonary disorder (COPD), depression,
attention deficit hyperactivity disorder (ADHD), autism,
Alzheimers, migraines, multiple sclerosis (MS), Lupus, arthritis,
Type 2 diabetes, obesity, non alcoholic steato hepatitis (NASH),
non alcoholic fatty liver disease (NAFLD), risk of
infarction/cardiovascular risk, heart failure, cancer, dental
caries, gingivitis, oral cancer, oral mucositis, bacterial
vaginosis, fertility, sinusitis, allergies, cystic fibrosis, lung
cancer, psoriasis, atopic dermatis, methicillin-resistant
staphylococcus aureus (MRSA), colorectal cancer, acne, vancomycin
resistant enterococcus, and combinations thereof.
[0234] The capability of an agent or combination of agents to cause
a condition can be evaluated using an assay described herein for
virtually any agent, including any of the example agents described
herein. In cases where disease states are already present, the
propensity of an agent to further exacerbate the symptoms and/or
progression of a condition and/or cause a form of toxicity may also
be evaluated.
Lists of Recommended Agents
[0235] An assay described herein may be used to aid in generating a
list of preferred or recommended agents. A list may include, for
example, the specific agents recommended and appropriate use
regimens (e.g., dose, dosing frequency, exposure limits, etc.) and
may also include agents not-recommended with or without a rationale
for withholding recommendation. Lists may rank agents, categorize
agents (e.g., by condition, drug-class, etc.), or may rank and
categorize agents. Lists may be compiled such that recommended (or
non-recommended) agents or combinations of agents are those that
have assay results at or above (or below) a given threshold. Such a
threshold may be determined, for example, from one or more
reference enumerations.
Counseling
[0236] The results of an assay described herein may be used to
provide counseling services to those in want or need. In general,
counseling generally comprises the steps of: (a) obtaining a sample
from a first subject; (b) contacting said sample with an agent in a
reaction mixture; (c) obtaining an enumeration of the abundance of
one or more microbial taxa or taxon-identifying chemical species in
said reaction mixture after contacting the sample with the agent;
and (d) providing counseling regarding the exposure of said agent
to the first subject and/or one or more additional subjects using
the enumeration or a numerical manipulation of the enumeration.
[0237] An additional subject may be of the same subject type as the
first subject or may be of a different subject type. For example,
counseling to a human subject may be provided based off of
enumerations completed for a sample(s) obtained from another human
subject. In another example, counseling to a human subject may be
provided based off of enumerations completed for a sample(s)
obtained from a common laboratory animal, such as, for example, a
mouse or rat. In another example, counseling may be provided with
respect to one species of dog using enumerations for a sample(s)
obtained from subjects of another species of dog.
[0238] In some cases, enumerations of microbial taxa or
taxon-identifying chemical species or numerical manipulations of
such enumerations may be provided in a database and stored, for
example, in a computer. The database may be accessed in order to
provide counseling to subjects in want or need.
[0239] Counseling services may include deciding on a treatment
regimen for a subject with a condition. In some examples,
counseling may include deciding between two or more drugs available
for treatment of the condition and/or the dosage of the chosen
drug(s) that should be used for treatment.
[0240] In some examples, counseling may include advice for pursuing
fecal transplants. A sample may be obtained from a subject of want
or need of fecal transplant advice. The sample may be assayed with
an aliquot of donor fecal matter (which may be used in a
transplant) and the relevant microbial taxa or taxon-identifying
chemical species enumerated. Obtained enumerations or numerical
manipulations of such enumerations may then be used to counsel the
subject on the utility of pursuing a fecal transplant.
[0241] Counseling services may include the communication of a
variety of pieces of information with respect to use of an agent,
including recommendations. Non-limiting examples of such
information includes information regarding the results of an assay
for samples obtained from the subject seeking counseling, the
results of an assay for samples obtained from a subject different
than the subject seeking counseling, the results of an assay for
samples obtained from a group of subjects selected to represent a
population, information regarding the safety of an agent,
information regarding the efficacy of an agent, information
regarding the safety of an agent when administered with one or more
different agents, information regarding the efficacy of an agent
when administered with one or more different agents, a
recommendation to use or continue to use an agent or combination of
agents, a recommendation to not use or discontinue use of an agent
or combination of agents, providing a ranked list of possible
agents or combination of agents for use or continued use,
recommendations for the addition of one or more different agents to
a regimen comprising an agent or combination of agents,
recommendations for monitoring use of an agent over time,
recommendations for doses of an agent, recommendations regarding
the propensity of an agent to cause a condition, or combinations
thereof.
[0242] Counseling may be provided, for example, by a person, a
company, a representative of a health-care organization (e.g., a
hospital, hospital system, medical group, etc.), a health-care
organization, a government official, a government office, a
consultant, via a subscription service, via an online vendor, via a
printed publication, via live audio or an audio recording, via
postal mail, via email, via telephone, via the internet, and
combinations thereof. Counseling may be provided, for example, to a
person, a company, a representative of a health-care organization,
a consultant, a government official, a government office, and
combinations thereof.
[0243] Counseling may be provided on demand, in a single counseling
session, or in multiple counseling sessions. For example, on demand
counseling may be necessary where immediate intervention is
necessary to treat a condition.
[0244] Counseling may be provided for a pet or other subject under
to the ownership and/or care of another subject.
[0245] Counseling may be completed by or provided to a company or
organization. For example, an assay may be completed by an
organization (e.g., a business, a company, a special interest
group, an educational institution, etc.) and the results used to
counsel another subject, group of subjects, or organization.
[0246] Counseling may be provided by a public health organization.
For example, the results of assays completed for a group of
subjects selected to represent a population may be disseminated to
members of the population via government-sponsored
advertisements.
[0247] Counseling services may also include the generation of one
or more reports. Such reports may be given to a subject in want or
need in hard-copy form or may be transmitted electronically, such
as by email. Reports may include raw data obtained from detecting
microbial taxa or taxon-identifying chemical species, enumerating
microbial taxa or relating chemical species, any numerical
manipulations of an enumeration, changes of abundance of one or
more microbial taxa enumerated after contact with an agent, an
algorithm used to enumerate microbial taxa, an algorithm used to
numerically manipulate an enumeration, and combinations thereof. A
report may also include summaries of provided counseling, including
any of the various example pieces of information described
above.
Systems to Implement Methods
[0248] The disclosure provides specialized computer systems that
are configured to implement methods described herein, including the
execution of any part of an assay, enumerating microbial taxa
and/or taxon-identifying chemical species, further analysis of such
enumerations, and/or aiding in providing counseling services.
Specialized computer systems are generally capable of any of the
following: (a) controlling any equipment used to execute an assay;
(b) accepting raw data obtained from executing an assay; (c)
pre-processing raw data such that it is acceptable for entry into
algorithms used to enumerate microbial taxa and/or
taxon-identifying chemical species; (d) numerical manipulation of
an enumeration (e); interpreting, in whole or part, the results of
an assay; (f) storing any assay protocol, enumeration, numerical
manipulation of an enumeration, and/or interpretation of the
results of an assay; (g) generating and storing a report; and (h)
aiding in providing counseling. The system may include a computer
server ("server") that is programmed to implement the methods
described herein. FIG. 1 depicts a system 100 adapted to enable a
user to detect, analyze, and process raw data obtained an assay.
The system 100 includes a central computer server 101 that is
programmed to implement exemplary methods described herein. The
server 101 includes a central processing unit (CPU, also
"processor") 105 which can be a single core processor, a multi core
processor, or plurality of processors for parallel processing. The
server 101 also includes memory 110 (e.g. random access memory,
read-only memory, flash memory); electronic storage unit 115 (e.g.
hard disk); communications interface 120 (e.g. network adaptor) for
communicating with one or more other systems; and peripheral
devices 125 which may include cache, other memory, data storage,
and/or electronic display adaptors. The memory 110, storage unit
115, interface 120, and peripheral devices 125 are in communication
with the processor 105 through a communications bus (solid lines),
such as a motherboard. The storage unit 115 can be a data storage
unit for storing data. The server 101 may be operatively coupled to
a computer network ("network") 130 with the aid of the
communications interface 120. The network 130 can be the Internet,
an intranet and/or an extranet, an intranet and/or extranet that is
in communication with the Internet, a telecommunication or data
network. The network 130 in some cases, with the aid of the server
101, can implement a peer-to-peer network, which may enable devices
coupled to the server 101 to behave as a client or a server.
[0249] The storage unit 115 can store files, such as raw data files
from assays, assay protocols, databases of reference enumerations,
nucleic acid sequences used to enumerate microbial taxa, databases
of microbial taxa classification schemes, instructions to execute
numerical manipulations, enumerations, numerical manipulations of
enumerations, interpretations (e.g., reports, input notes, etc.) of
assay results, or any aspect of data associated with the executing
methods described herein.
[0250] The server can communicate with one or more remote computer
systems through the network 130. The one or more remote computer
systems may be, for example, personal computers, laptops, tablets,
telephones, Smart phones, or personal digital assistants. Moreover,
system 100 may be capable of accepting instructions over network
130 from one or more remote computer systems such that its data is
accessed (either by the remote computer systems or system 100).
Alternatively, system 100 is capable of accepting data stored,
analyzed, and/or interpreted on a remote system that is transmitted
to system 100 over network 130. Moreover, system 100 is also
capable of transmitting data stored, analyzed, and/or interpreted
by system 100 to one or more remote computers over network 130.
[0251] In some situations the system 100 includes a single server
101. In other situations, the system includes multiple servers in
communication with one another through an intranet, extranet and/or
the Internet.
[0252] The server 101 can be adapted to store raw data files from
assays, assay protocols, databases of reference enumerations,
nucleic acid sequences used to enumerate microbial taxa, databases
of microbial taxa classification schemes, instructions to execute
numerical manipulations, enumerations, numerical manipulations of
enumerations, interpretations (e.g., reports, input notes, etc.) of
assay results, or any aspect of data associated with the executing
methods described herein. Such information can be stored on the
storage unit 115 or the server 101 and such data can be transmitted
through a network, such as network 130.
[0253] Methods as described herein can be implemented by way of
machine (or computer processor) executable code (or software)
stored on an electronic storage location of the server 101, such
as, for example, on the memory 110, or electronic storage unit 115.
During use, the code can be executed by the processor 105. In some
cases, the code can be retrieved from the storage unit 115 and
stored on the memory 110 for ready access by the processor 105. In
some situations, the electronic storage unit 115 can be precluded,
and machine-executable instructions are stored on memory 110.
Alternatively, the code can be executed on a second computer system
140.
[0254] Aspects of the systems and methods provided herein, such as
the server 101, can be embodied in programming. Various aspects of
the technology may be thought of as "products" or "articles of
manufacture" typically in the form of machine (or processor)
executable code and/or associated data that is carried on or
embodied in a type of machine readable medium. Machine-executable
code can be stored on an electronic storage unit, such memory (e.g.
read-only memory, random-access memory, flash memory) or a hard
disk. "Storage" type media can include any or all of the tangible
memory of the computers, processors or the like, or associated
modules thereof, such as various semiconductor memories, tape
drives, disk drives and the like, which may provide non-transitory
storage at any time for the software programming. All or portions
of the software may at times be communicated through the Internet
or various other telecommunication networks. Such communications,
for example, may enable loading of the software from one computer
or processor into another, for example, from a management server or
host computer into the computer platform of an application server.
Thus, another type of media that may bear the software elements
includes optical, electrical, and electromagnetic waves, such as
used across physical interfaces between local devices, through
wired and optical landline networks and over various air-links. The
physical elements that carry such waves, such as wired or wireless
likes, optical links, or the like, also may be considered as media
bearing the software. As used herein, unless restricted to
non-transitory, tangible "storage" media, terms such as computer or
machine "readable medium" refer to any medium that participates in
providing instructions to a processor for execution.
[0255] Hence, a machine readable medium, such as
computer-executable code, may take many forms, including but not
limited to, tangible storage medium, a carrier wave medium, or
physical transmission medium. Non-volatile storage media can
include, for example, optical or magnetic disks, such as any of the
storage devices in any computer(s) or the like, such may be used to
implement the system. Tangible transmission media can include:
coaxial cables, copper wires, and fiber optics (including the wires
that comprise a bus within a computer system). Carrier-wave
transmission media may take the form of electric or electromagnetic
signals, or acoustic or light waves such as those generated during
radio frequency (RF) and infrared (IR) data communications. Common
forms of computer-readable media therefore include, for example: a
floppy disk, a flexible disk, hard disk, magnetic tape, any other
magnetic medium, a CD-ROM, DVD, DVD-ROM, any other optical medium,
punch cards, paper tame, any other physical storage medium with
patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM,
any other memory chip or cartridge, a carrier wave transporting
data or instructions, cables, or links transporting such carrier
wave, or any other medium from which a computer may read
programming code and/or data. Many of these forms of computer
readable media may be involved in carrying one or more sequences of
one or more instructions to a processor for execution.
[0256] Interpretation of the results of an assay, such as the
generation of a report, may be presented to a user with the aid of
a user interface, such as a graphical user interface (GUI).
Moreover, systems may be coupled to a printer device (not shown in
FIG. 1) that is capable of producing paper hard copies of any
information displayed to a user. Non-limiting examples of paper
hard copies that may be generated by the printer include reports
that summarize the experimental protocol for an assay, the results
of an assay, interpretations of the results of an assay,
recommendations based on the results of an assay, and/or producing
ranked lists of agents or groupings of agents based on the results
of an assay.
Microbial Taxa Classification Schemes
[0257] Microbial taxa may be classified according to a variety of
different schemes. Different classification schemes may result in
taxa of different microbial compositions. Moreover, a particular
taxon may comprise varied numbers of microbial species. In some
examples, a microbial taxon may comprise a single microbial
species. In other examples, a taxon may comprise from about 1
microbial species to about 1,000,000 microbial species. In other
examples, a taxon may comprise from about 1 microbial species to
about 10,000 microbial species. In other examples, a taxon may
comprise from about 1 microbial species to about 100 microbial
species. In other examples, a taxon may comprise from about 1
microbial species to about 10 microbial species. In still other
examples, a taxon may comprise about 1, 10, 100, 1,000, 10,000,
100,000, or 1,000,000 microbial species. Moreover, microbial taxa
may vary in the number of component microbial species comprised in
each microbial taxon.
[0258] Microbial taxa may be arranged according to parsimonious
trees such that nodes of the trees are species ordered in an
evolutionary hierarchy. Taxa may be grouped, for example, in clades
according to descendants of a node in the tree, such that all
descendants from a common ancestor (or node) are grouped within a
microbial taxon. Sub-taxa may also be derived for nodes at lower
levels of the tree in an analogous fashion. Alternatively, more
complicated schemes may be used to distinguish taxa within a
parsimonious tree.
[0259] Microbial taxa may be arranged according to classical
Linnaean taxonomy. Linnaean taxonomy generally relies on ordering
species at various ranks such that organisms at a given rank all
share one or more common characteristic. A common characteristic,
for example, may be a common anatomical or structural feature
shared by members of a given taxon. Non-limiting examples of
classical Linnaean taxonomy, in order of highest rank to lowest
rank, include: domains, kingdoms, phyla, classes, orders, families,
genera, or single species. In general, a genus name and species
name indicates a unique species using classical Linnaean
taxonomy.
[0260] Microbial taxa may be arranged as operational taxonomic
units (OTUs). For a thorough description of arrangement of
microbial taxa into OTUs, see U.S. Patent Application Publication
No. 2012/0165215 and U.S. Patent Application Publication No.
2009/0291858 which are both incorporated in their entirety herein
by reference. An operational taxon unit (OTU) refers to a group of
one or more organisms that can be represented as a node in a
clustering tree. The level of a cluster is determined by its
hierarchical order. In some examples, an OTU is a group tentatively
assumed to be a valid taxon for purposes of phylogenetic analysis.
In other examples, an OTU is any of the extant taxonomic units
under study. In other examples, an OTU is given a name and a rank.
For example, an OTU can represent a domain, a sub-domain, a
kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, a
sub-class, an order, a sub-order, a family, a subfamily, a genus, a
subgenus, or a species. In some cases, OTUs can represent one or
more organisms from the kingdoms eubacteria, protista, or fungi at
any level of a hierarchal order. In other cases, an OTU represents
a prokaryotic or fungal order. Moreover, OTUs may be derived for
example by a common physical attribute shared by its component
organisms or may be derived from evolutionary hierarchy.
[0261] Alternatively, OTUs may be derived by other means such as by
clustering organisms into OTUs by identifying of one or more
conserved genes and/or polynucleotide sequence homologies for
shared genes comprised in a plurality of organisms to-be-clustered.
Highly conserved polynucleotides usually show at least about 80%,
85%, 90%, 92%, 94%, 95%, or 97% homology across a domain, kingdom,
phylum, class, order, family or genus, respectively. The sequences
of these polynucleotides can be used for determining evolutionary
lineage or making a phylogenetic determination and are also known
as phylogenetic markers.
[0262] A database of nucleic acid sequences may be used to organize
organisms into particular OTUs based on one or more conserved genes
and/or highly homologous nucleic acid sequences shared by a group
of organisms. The choice of database that is used to assign
organisms to OTUs is dependent on a number of factors with
non-limiting examples that include the total number of sequences
within the database, the length of the overall sequences or the
length of highly conserved regions within the sequences listed in
the database, and the quality of the sequences therein. Typically,
databases with longer target regions of conserved sequence may
generally contain a larger total number of possible sequences that
can be compared. In some examples, the sequences in a database are
at least about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 4,000, 8,000, 16,000 or
24,000 nucleotides long. Moreover, databases with a larger number
of sequences may generally provide greater numbers of sequences
from which to choose. In some examples, a database contains at
least about 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,
80,000, 100,000, 200,000, 500,000, 1,000,000 or 2,000,000
sequences.
[0263] A database used for the selection of OTUs may comprise at
least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up
to 100% of the known sequences of the organisms to be clustered
into OTUs. The sequences for each individual organism in the
database can include more than about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 100% of the genome of the organism, or of the
non-redundant regions thereof.
[0264] A variety of existing databases may be used to assign
organisms to an OTU based on nucleic acid sequences. A listing of
almost 40,000 aligned 16S rRNA gene sequences greater than 1250
nucleotides in length can be found on the Greengenes web
application (greengenes.secondgenome.com), a publicly accessible
database run by the Greengenes Consortium. Other publicly
accessible databases include GenBank, Michigan State University's
ribosomal database project, the Max Planck Institute for Marine
Microbiology's Silva database, and the National Institute of
Health's NCBI. Proprietary sequence databases or combinations
created by amalgamating the contents of two or more private and/or
public databases can also be used to assign organisms to a given
OTU.
[0265] As noted above, OTUs may be arranged by sequence homology of
a conserved polynucleotide. The conserved polynucleotide may be
from a highly conserved gene or the conserved polynucleotide may be
from a highly conserved region of a gene with moderate or large
sequence variation. Moreover, the highly conserved polynucleotide
may be an intron, exon, or a linking section of nucleic acid that
separates two genes.
[0266] The highly conserved polynucleotide used to assign organisms
to OTUs may be a phylogenetic gene. Non-limiting examples of a
phylogenetic gene includes the 5.8S ribosomal ribonucleic acid
(rRNA) gene, 12S rRNA gene, 16S rRNA gene-prokaryotic, 16S rRNA
gene-mitochondrial, 18S rRNA gene, 23S rRNA gene, 28S rRNA gene,
gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, and the nifD
gene. For eukaryotic species, rRNA genes can be nuclear,
mitochondrial, or both. In some cases, the spacer region between
highly conserved segments of two genes can be used. For example,
the internal transcribed spacer (ITS) region between 16S and 23S
rRNA genes can be used to differentiate closely related taxa with
or without consideration of other rRNA genes, including conserved
sections of either the 16S or 23S rRNA gene.
[0267] Due to structural constraints necessary for proper
functioning of 16S rRNA when comprised in protein synthesis
machinery (e.g., ribosomes), specific regions throughout the gene
have a highly conserved polynucleotide sequence although
non-structural segments may have a high degree of variability.
Regions of the 16S rRNA gene that possess high levels of
variability include the V1, V2, V3, V4, V5, V6, V7, V8, and V9
regions of the gene. These and other regions of high variability
may be detected, for example, to distinguish/enumerate OTUs at a
single species level, while regions of less variability might be
used to distinguish OTUs that represent a subgenus, a genus, a
subfamily, a family, a sub-order, an order, a sub-class, a class, a
sub-phylum, a phylum, a sub-kingdom, or a kingdom. Such a
classification scheme may be useful for identifying closely related
microorganisms and OTUs from a background or pool of closely
related organisms.
[0268] Microbial taxa may be arranged by virtue of other
descriptors with non-limiting examples that include transcriptomes,
proteomes, metabolomes, and metagenomes. Such descriptors may be
both indicators of microbial compositions and functionality. In
some examples, microbial organisms may be arranged into taxa via
clusters of organisms with similar, full or partial transcriptomes.
Transcriptomes generally refer to a set of ribonucleic acid (RNA)
molecules of a living organism. RNA molecules may include mRNA,
rRNA, transfer RNA (tRNA), and other non-coding RNA. Transcriptomes
may be an entire set of all RNA molecules of a living organism or
may be a particular subset of RNA molecules. Moreover, taxa may be
arranged based on full organism transcriptomes or may be based on
partial transcriptomes.
[0269] In some examples, microbial organisms may be arranged into
taxa via clusters of organisms with similar proteomes. A proteome
generally refers to a set of proteins expressed by a living
organism. Proteomes may be an entire set of all proteins of a
living organism or may be a particular subset of proteins.
Moreover, taxa may be arranged based on full organism proteomes or
may be based on partial proteomes.
[0270] In some examples, microbial organisms may be arranged into
taxa via clusters of organisms with similar metabolomes. A
metabolome generally refers to a set of small-molecule metabolites
(such as metabolic intermediates, hormones and other signaling
molecules, and secondary metabolites) found within a living
organism. Metabolomes may be an entire set of all metabolites found
within a living organism or may be a particular subset of
metabolites.
[0271] Moreover, taxa may be arranged based on full organism
metabolomes or may be based on partial metabolomes.
[0272] In some examples, microbial organisms may be arranged into
taxa via clusters of organisms with similar metagenomes. A
metagenome generally refers to genetic material recovered directly
from environmental samples, such as for example a living organism.
Metagenomes may be an entire set of all genetic material found
within a living organism or may be a particular subset of genetic
material. Moreover, taxa may be arranged based on full metagenomes
or may be arranged based on partial metagenomes.
EXAMPLES
Example 1
MESA Assay
[0273] Experiments were carried out using three human donor fecal
samples. Samples were incubated in a micro-well plates as well as
larger volume tubes, in the presence of various carbon sources. 16S
rRNA sequencing was performed to determine shifts in microbiota
profiles across time and donor. Principle component analysis and
relative taxa frequency were determined to assess whether changes
occurred according to donor, carbon source addition, and time.
Assay Setup and DNA Extraction
[0274] A fecal sample was obtained from a human donor. 1 gram of
the fecal sample was added to 10 mL phosphate buffered saline (PBS)
to form a slurry of 10% (w/v) fecal sample. In triplicate, 40 .mu.L
of the above slurry was added to each of three wells in a 48 well
plate. 360 .mu.L of minimal bacterial broth medium (comprising 2
grams ("g")/liter ("L") of peptone water, 1 g/L of yeast extract,
0.1 g/L of NaCl, 0.04 g/L of K.sub.2HPO.sub.4, 0.01 g/liter of
MgSO.sub.4.7H.sub.2O, 0.01 g/L of CaCl.sub.2.2H.sub.2O, 2 g/L of
NaHCO.sub.3, 0.5 g/L of bile salts, 0.5 g/L of L-cysteine
hydrochloride, 50 mg/L of hemin, 10 .mu.L/L of vitamin K1, 2 mL/L
of Tween 80, and 0.05% (wt/vol) resazurin solution, pH 7) was then
added to each well to form a 400 .mu.L culture comprising 1% w/v
fecal slurry. 100 .mu.L culture is then collected for 16S rRNA gene
sequencing.
[0275] In triplicate, 200 .mu.L of the above slurry was added to
each of three tubes. 1.8 mL of minimal broth medium was then added
to each well to form a 2 mL culture comprising 1% w/v fecal slurry.
0.5 mL of the culture is then collected for 16S rRNA gene
sequencing.
[0276] DNA extraction of 100 .mu.L aliquots obtained from
microplate experiments revealed that 100 .mu.L of culture was not a
sufficient aliquot for obtaining sufficient amounts of DNA.
Instead, it was determined that all 400 .mu.L the total culture
volume was required.
[0277] DNA extraction of 0.5 mL aliquots obtained from tube
experiments revealed that 0.5 mL of culture was a sufficient
aliquot for obtaining sufficient amounts of DNA. However, it was
determined that a 1 mL aliquot was a better for DNA sequencing.
MESA Experiment
[0278] A fecal sample was obtained from each of three human donors,
Donor 1, Donor 2, and Donor 3. 1 gram of each fecal sample was
added to 10 mL phosphate buffered saline (PBS) to form three
separate slurries of 10% (w/v) fecal sample.
[0279] A total of 36 experiments was setup on a 48-well plate
(referred to herein as "microplate experiments"). For each slurry
obtained from Donor 1 Donor 2, or Donor 3, 40 .mu.L of the
respective slurry was added to each of twelve wells of the plate.
For each of these 12 wells, 360 .mu.L of water (control), water
comprising fructose, or water comprising fructose oligosaccharide
(FOS) were added. After addition of water, three of the twelve
wells comprised slurry+water; three of the twelve wells comprised
slurry+2% w/v fructose+water; three of the twelve wells comprised
slurry+0.5% w/v FOS+water; and three of the twelve wells comprised
slurry+2% w/v FOS+water. A summary of the experiments is shown in
Table 1. In Table 1, "Donor 1" represents slurry from Donor 1,
"Donor 2" represents slurry from Donor 2, and "Donor 3" represents
slurry from Donor 3. The microplate was then incubated for 24 hours
in an anaerobic chamber under 80% nitrogen, 10% carbon dioxide, and
10% hydrogen (H.sub.2). A total of 8 experiments were prepared each
in a vial (referred to herein as "tube experiments"). 200 .mu.L of
slurry from Donor 1 was added to each of the 8 vials. In 4 of the
vials, 1.8 mL of water comprising FOS was added for a final
concentration of 2% w/v FOS. For the other four vials, 1.8 mL of
water (control) was added. Two of the vials comprising FOS and the
two vials comprising water are incubated for 24 hours in an
anaerobic chamber (comprising 80% nitrogen, 10% carbon dioxide, and
10% hydrogen (H.sub.2)) at 37.degree. C. The other four vials are
incubated for 16 hours.
[0280] DNA was extracted from each sample (well or tube) and
quantified using a PicoGreen method. DNA is also extracted from an
aliquot of each slurry obtained from Donor 1, Donor 2, and Donor 3.
DNA comprising the variable region (V4) region of the 16S rRNA gene
was amplified and sequenced using the MiSeq platform from Ilumina.
Principle component analyses (PCoA) were performed to identify
Unifrac distances (degree of similarity/dissimilarity) of the
different treatment groups. Relative taxa representation for the
various treatment groups was determined at the phylum, class,
order, family, genus and species levels using operational taxonomic
units (OTUs) by comparing results to the GreenGenes database.
Results
[0281] In FIG. 2, PCoA and calculation of Unifrac distances shows
microbiota profiles of experiments related to Donor 1 in the
microplate experiments (n=3 for each experimental condition (e.g.,
no agent (control), 2% fructose, 0.5% FOS, 2% FOS)). As shown, a
difference in the microbiota profile of control experiments is
noticeable after the 24-hour incubation period ("nocarb"--dark gray
circles) when compared to non-incubated slurry ("inoc"--stars).
Hence, a "shift" in the profile can be observed. Also shown are
shifts in microbiota profiles for 2% fructose experiments
("fruc"--triangles), 0.5% FOS experiments ("FOSL"--black circles),
or 2% FOS experiments ("FOSH"--squares). As shown in FIG. 2, test
agents appear to shift microbiota differently from controls,
indicating that the tested agents may have an effect on the
evaluated microbial communities in Donor 1.
[0282] In FIG. 3, PCoA and calculation of Unifrac distances shows
microbiota profiles of experiments related to Donor 2 in the
microplate experiments (n=3 for each experimental condition). As
shown, a difference in the microbiota profile of control
experiments is noticeable after the 24-hour incubation period
("nocarb"--dark gray circles) when compared to non-incubated slurry
("inoc"--stars). Also shown are shifts in microbiota profiles for
2% fructose experiments ("fruc"--triangles), 0.5% FOS experiments
("FOSL"--black circles), or 2% FOS experiments ("FOSH"--squares).
As shown in FIG. 3, test agents appear to shift microbiota
differently from controls, indicating that the tested agents may
have an effect on the evaluated microbial communities in Donor
2.
[0283] In FIG. 4, PCoA and calculation of Unifrac distances shows
microbiota profiles of experiments related to Donor 2 in the
microplate experiments (n=3 for each experimental condition). As
shown, a difference in the microbiota profile of control
experiments is noticeable after the 24-hour incubation period
("nocarb"--dark gray circles) when compared to non-incubated slurry
("inoc"--stars). However, when compared to results from Donor 1
(FIG. 2) and/or Donor 2 (FIG. 3) shifts between control experiments
and non-incubated slurry occur to a lesser degree. Also shown are
shifts in microbiota profiles for 2% fructose experiments
("fruc"--triangles), 0.5% FOS experiments ("FOSL"--black circles),
or 2% FOS experiments ("FOSH"--squares). As shown in FIG. 4, test
agents appear to shift microbiota differently from controls,
indicating that the tested agents may have an effect on the
evaluated microbial communities in Donor 3. Also, test agents
appear to shift the microbiome from controls similarly in Donor 3.
Comparison of results between Donor 1 (FIG. 2), Donor 2 (FIG. 3),
and Donor 3 (FIG. 4), however, indicates that shifts of microbiota
when in contact with the set of test agents were noticeably
different between the three different donors.
[0284] In FIG. 5, PCoA and calculation of Unifrac distances shows
microbiota profiles of experiments in the tube experiments (n=2 for
each experimental condition (e.g., no agent (control)+16 hour
incubation ("nocarb_16 h"--squares); no agent (control)+24 hour
incubation ("nocarb_24 h"--triangles); 2% FOS+16 hour incubation
("FOSH_16h"--dark gray circles); 2% FOS+24 hour incubation
("FOSH_24 h"--black circles))). As shown, shifts in microbial
communities in control experiments between the two incubation times
are similar as are shifts in microbial communities in experiments
using FOS. Results suggest that a 16 hr or 24 hr incubation time
may both give similar results.
[0285] In FIG. 6, PCoA and calculation of Unifrac distances shows
microbiota profiles of 24 hour incubation time control
("nocarb_t"--black circles) and 2% FOS experiments
("FOSH_t"--squares) in the tube experiments and the control
("nocarb_m"--stars) and 2% FOS experiments ("FOSH_m"--triangles) in
microplate experiments (n=2 for tube experiments and n=3 for
microplate experiments). As shown, shifts in microbial communities
in control experiments between the two assay formats are similar as
are shifts in microbial communities in experiments using FOS.
Results suggest that a micronized version of an assay may give
similar results to an assay completed with larger volumes.
[0286] In FIG. 7, proportions of OTUs classified at the phylum
level are shown for all donors, experiment sets (e.g., tube or
microplate), and experimental conditions. FIG. 7 shows the top nine
richest taxa at the phylum rank and the relative proportion of OTUs
unclassified at either the phylum level or at all levels. Although
PCoA in FIG. 2, FIG. 3, and FIG. 4 show differences in microbiome
shifts between the three donors, the relative proportions of the
major phyla are similar between donors across experimental
conditions tested.
[0287] In FIG. 8, proportions of OTUs classified at the species
level are shown for all donors, experiment sets (e.g., tube or
microplate), and experimental conditions. FIG. 8 shows the top nine
richest taxa at the species rank and the relative proportion of
OTUs unclassified at either the species or at all levels. Although
PCoA in FIG. 2, FIG. 3, and FIG. 4 show differences in microbiome
shifts between the three donors, the relative proportions of the
major species are similar between donors across experimental
conditions tested.
[0288] Similar results (not shown) were obtained for analyses
conducted on OTUs classified at the class, order, family, and genus
levels.
[0289] In FIG. 9, the bacterial (gray) and archael (black) taxon
richness for OTUs classified at the phylum level is shown for all
donors, experiment sets, and experimental conditions. Taxon
richness refers to the number of unique phyla detected by V4 16S
rRNA gene sequencing. As shown in FIG. 9 and in cases where both
bacterial and archael taxon richness was evaluated for a sample,
the richness of bacterial and archael taxa at the phyla level are
similar.
[0290] In FIG. 10, the bacterial (gray) and archael (black) taxon
richness for OTUs classified at the species level is shown for all
donors, experiment sets, and experimental conditions. Taxon
richness refers to the number of unique species detected by V4 16S
rRNA gene sequencing. As shown in FIG. 10 and in cases where both
bacterial and archael taxon richness was evaluated for a sample,
the richness of bacterial and archael taxa at the species level are
similar.
TABLE-US-00001 TABLE 1 Setup of Microplate Experiments in Example 1
Donor1 + Donor1 + Donor1 + Donor1 + Donor3 + Donor3 + Donor3 +
Donor3 + Control 2% Fructose 0.5% FOS 2% FOS Control 2% Fructose
0.5% FOS 2% FOS Donor1 + Donor1 + Donor1 + Donor1 + Donor3 + Donor3
+ Donor3 + Donor3 + Control 2% Fructose 0.5% FOS 2% FOS Control 2%
Fructose 0.5% FOS 2% FOS Donor1 + Donor1 + Donor1 + Donor1 + Donor3
+ Donor3 + Donor3 + Donor3 + Control 2% Fructose 0.5% FOS 2% FOS
Control 2% Fructose 0.5% FOS 2% FOS Donor2 + Donor2 + Donor2 +
Donor2 + Control 2% Fructose 0.5% FOS 2% FOS Donor2 + Donor2 +
Donor2 + Donor2 + Control 2% Fructose 0.5% FOS 2% FOS Donor2 +
Donor2 + Donor2 + Donor2 + Control 2% Fructose 0.5% FOS 2% FOS
Example 2
Counseling and Subject Selection for Use of an Agent
[0291] A human subject is diagnosed with ulcerative colitis. A
fecal sample is obtained from the subject and pre-processed into a
slurry. The slurry is combined with an agent, approved for the
treatment of ulcerative colitis, and additional reagents to form a
reaction mixture. The reaction mixture is incubated at appropriate
conditions. After incubation, DNA is extracted from the reaction
mixture and the V4 region of the 16S rRNA gene is amplified
sequenced. Via sequencing, appropriate microbial taxa (e.g.,
microbial taxa determined to be associated with ulcerative colitis,
microbial known to be at risk for unwanted alteration, etc.) are
enumerated and numerically manipulated. A numerical manipulation
from an enumeration obtained from a group of subjects not afflicted
with ulcerative colitis (a reference enumeration) is obtained from
a stored database. A processor is used to compare the two numerical
manipulations and determines that contacting samples obtained from
the subject with the agent results in a shift in microbial taxa
abundance in the direction of that observed from the reference
enumeration. The results are thus used to select the subject for
use of the agent to treat the subject's ulcerative colitis. These
results are communicated to the subject in the form of counseling
administered at a healthcare facility by a health care
practitioner. During counseling the use of the agent as a therapy
for ulcerative colitis and a dosing regimen of the agent are
recommended. A summary of the results of the assay and the
recommendations provided during counseling are provided to the
subject on a printed report.
Example 3
Counseling and Subject Selection for Use of an Agent
[0292] A human subject is working with a new chemical process in a
chemical production facility. The process produces a new chemical
that may be potentially hazardous to microbiota of the human
subject. A fecal sample is obtained from the subject and
pre-processed into a slurry. An aliquot of the slurry is combined
with the chemical and additional reagents to form a reaction
mixture. A separate aliquot of the slurry is combined with just the
additional reagents. The reaction mixtures are incubated at
appropriate conditions. After incubation, DNA is extracted from
both reaction mixtures and the V4 region of the 16S rRNA gene is
amplified and sequenced. Via sequencing, appropriate microbial taxa
are enumerated for each reaction mixture and the obtained
enumerations numerically manipulated. A processor is used to
compare the two numerical manipulations and determines that
exposure of a sample obtained from the subject to the new chemical
results in no appreciable shift in microbial taxa abundance in the
direction of that observed for the sample not contacted with the
new chemical. The results are thus used to approve the subject's
use of and exposure to the new chemical. Results of the assay are
communicated to the subject in the form of counseling administered
at a healthcare facility by a health care practitioner. During
counseling, it is communicated to the subject that the use of and
exposure to the new chemical is cleared and an exposure limit is
recommended based on the results of the assay. A summary of the
results of the assay and the recommendations provided during
counseling are provided to the subject on a printed report.
Example 4
Functionality Assays
Methods
[0293] Cecal/fecal slurry preparation: Briefly, cecal or fecal
contents from human or mouse subjects were homogenized in anoxic
(e.g., oxygen-free) phosphate buffered saline (PBS) as a 10%
mixture, weight:volume. After centrifugation, slurries were diluted
to 1% in a minimal medium ("MESA medium") equilibrated in 10%
H.sub.2, 10% CO.sub.2, and 80% N.sub.2, consisting of 2 g/liter of
peptone water, 1 g/liter of yeast extract, 0.1 g/liter of NaCl,
0.04 g/liter of K.sub.2HPO.sub.4, 0.04 g/liter of KH.sub.2PO.sub.4,
0.01 g/liter of MgSO.sub.4-7H.sub.2O, 0.01 g/liter of
CaCl.sub.2-2H.sub.2O, 2 g/liter of NaHCO.sub.3, 0.5 g/liter of bile
salts, 0.5 g/liter of L-cysteine hydrochloride, 50 mg/liter of
hemin, 10 microliter of vitamin K1, 2 ml/liter of Tween 80, and
0.05%0 (wt/vol) resazurin solution, and at pH 7.0. Slurries were
cultured in an anaerobic cabinet for 4-48 hr under anaerobic
conditions. After incubation, slurries were pelleted and processed
for genomic DNA purification and amplicon library production,
followed by 16S rRNA gene sequencing using Ilumina MiSeq sequencing
technology.
[0294] A portion of supernatants obtained from pelleting were
placed on either HT29 MTX human epithelial cells or on mouse colon
organotypic cultures. In a parallel set of experiments,
supernatants were diluted 1:2 with phosphate buffered saline (PBS)
and added to cultures. MESA medium and diluted MESA medium (1:2
dilution) not containing homogenized material were also added to
either HT29 MTX human epithelial cells or mouse colon organotypic
cultures as a control.
[0295] HT29 MTX cells were grown as monolayer cultures in
trans-well plates. Organotypic cultures were obtained by splaying
mouse colon tissue and flushing with PBS containing Pen/Strep.
Three millimeter ("mm") sections were punched out using a skin
biopsy tool to obtain explants. Explants were placed in trans-well
plates with DMEM medium supplemented with calf serum. Cells or
organotypic cultures were incubated with supernatants or basal
medium for 24 hours. At the conclusion of incubation, barrier
function, cellular viability, and cytokine release were measured in
the cultures.
[0296] For HT29 MTX cells grown in monolayer, barrier function was
measured by quantifying the penetration of a 70 kDa FITC-Dextran
label through the monolayer. Increased penetration of fluorescence
through the monolayer was used as a measure of increased
permeability/barrier breakdown. Viability was measured by
quantifying DAPI label incorporation into the cultures and the
fluorescence signal quantified. Cytokines were measured in the
media of cultures by ELISA.
[0297] For organotypic cultures, barrier function was measured by
incorporation and quantification of 70 kDa FITC-Dextran into the
explant. Viability was measured by quantifying incorporation of the
Live/Dead.TM. fluorescence marker into the explant; increased
fluorescence served as an index of decreased viability of the
explant in response to supernatant additions. Cytokines were
measured in the media of cultures by ELISA. In parallel
experiments, antibiotics were added with supernatants to determine
the effect of diminished bacterial viability (e.g., due to
antibiotic treatment) on the various readouts.
[0298] In another set of experiments, live bacteria (e.g.,
cecum-derived bacteria) were placed on either the HT29 MTX cells or
the organotypic cultures for various times, and barrier function,
viability, and cytokines measured as described above for
supernatant additions. In parallel experiments, antibiotics were
added with the bacteria to determine the effect of diminished
bacterial viability on the various readouts.
Results
Barrier Function and Viability--HT29 MTX Cells
[0299] FIG. 11 shows the effect of various cecal-derived
supernatants or bacteria on barrier permeability in HT29 MTX cells.
FIG. 12 is a companion figure showing viability of HT29 MTX
cultures. Spent bacterial supernatants induced cell loss and
dextran leakage; while bacterial components induced barrier
breakdown in the absence of cell loss. Addition of antibiotics into
the cecal cultures mitigated the effect of both bacteria and their
spent supernatants on host function. "MESA Media" in FIGS. 11 and
12 refers to control basal medium used to generate slurries and
"Spent Cecum Media" refers to supernatants generated from cecal
slurries subject to anaerobic culture.
Cytokines--HT29 MTX Cells
[0300] FIG. 13 shows the effect of various cecal-derived
supernatants or bacteria on IL-8 cytokine production in HT29 MTX
cells. Spent bacterial supernatants (e.g., supernatants obtained
from cecal slurries subject to anaerobic conditions) and bacteria
induced IL-8 production from HT-29 MTX cells. Addition of
antibiotics into the cecal cultures mitigated the effect of both
bacteria and their spent supernatants on IL-8 responses. "MESA
Media" in FIG. 13 refers to control basal medium used to generate
slurries and "Spent Cecum Media" refers to supernatants generated
from cecal contents and subject to anaerobic culture.
Barrier Function and Viability--Organotypic Cultures
[0301] FIG. 14 shows the effect of various cecal-derived
supernatants or bacteria on barrier permeability in mouse
organotypic cultures. Spent bacterial supernatants (e.g.,
supernatants obtained from cecal slurries subject to anaerobic
conditions) induced dextran leakage; while bacterial components
induced barrier breakdown in the absence of cell loss. Addition of
antibiotics into the cecal cultures mitigated the affect of both
bacteria and their spent supernatants on host function.
[0302] FIG. 15 shows the effect of various cecal-derived
supernatants or bacteria on viability in mouse organotypic
cultures. Spent bacterial supernatants (e.g., supernatants obtained
from cecal slurries subject to anaerobic conditions) decreased
viability while bacterial components induced breakdown in the
absence of cell loss. Addition of antibiotics into the cecal
cultures mitigated the effect of both bacteria and their spent
supernatants on host function.
Cytokines--Organotypic Cultures
[0303] FIG. 16 shows the effect of various cecal-derived MESA media
or bacteria on KC (e.g., mouse IL-8) production in organotypic
cultures. Spent bacterial supernatants (e.g., supernatants obtained
from cecal slurries subject to anaerobic conditions) and bacteria
induced KC production in the explant cultures. Addition of
antibiotics into the cecal cultures mitigated the effect of both
bacteria and their spent supernatants on KC responses.
[0304] It should be understood from the foregoing that, while
particular implementations have been illustrated and described,
various modifications may be made thereto and are contemplated
herein. It is also not intended that the invention be limited by
the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned
specification, the descriptions and illustrations of the preferable
embodiments herein are not meant to be construed in a limiting
sense. Furthermore, it shall be understood that all aspects of the
invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. Various
modifications in form and detail of the embodiments of the
invention will be apparent to a person skilled in the art. It is
therefore contemplated that the invention shall also cover any such
modifications, variations and equivalents. It is intended that the
following claims define the scope of the invention and that methods
and structures within the scope of these claims and their
equivalents be covered thereby.
* * * * *