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.

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.