Methods and compositions for reducing clostridium difficile infection

Abstract

The present invention relates to methods and compositions for reducing the risk and severity of C. difficile infection. It is based, at least in part, on the discovery that a restricted fraction of the gut microbiota, including the bacterium Clostridium scindens, contributes substantially to resistance against C. difficile infection. Without being bound by any particular theory, it is believed that this is achieved through the biosynthesis of secondary bile acids.

1 - 48 . (canceled) 
     
     
         49 . A recombinant cell expressing a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the recombinant cell comprises one or more exogenous nucleic acids encoding a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the one or more exogenous nucleic acids are operably linked to a promoter. 
     
     
         50 . The recombinant cell of  claim 49 , wherein the one or more exogenous nucleic acids comprises a baiCD gene encoding a 7α-hydroxysteroid dehydrogenase enzyme. 
     
     
         51 . The recombinant cell of  claim 50 , wherein the 7α-hydroxysteroid dehydrogenase is a bacterial 7α-hydroxysteroid dehydrogenase, wherein the bacteria is selected from the group consisting of  Clostridium scindens, Clostridium hiranonis, Clostridium hylemonae, Clostridium perfringens, Clostridium sordellii, Proteocatella sphenisci,  Lachnospiraceae 5_1_57FAA, Clostridiales VE202-05, and Clostridiales VE202-26. 
     
     
         52 . The recombinant cell of  claim 49 , wherein the cell is a  bacterium,  or spore thereof. 
     
     
         53 . The recombinant cell of  claim 52 , wherein the  bacterium  is selected from the group consisting of  Clostridium scindens, Lactobacillus, Lactococcus, Bacillus, Bifidobacterium,  and attenuated and non-monocytogenes  Listeria  strains. 
     
     
         54 . The recombinant cell of  claim 49 , wherein the bile acid-inducible (bai) 7α/β-dehydroxylation operon is expressed in an amount sufficient to transform a primary bile acid to a secondary bile acid by 7α/β-dehydroxylation. 
     
     
         55 . A composition comprising two or more isolated bacteria or spores thereof, selected from the group consisting of  Clostridium scindens, Barnesiella intestihominis, Blautia hansenii,  and  Pseudoflavonifractor capillosus,  wherein the two or more isolated bacteria or spores thereof are in a formulation for administration to a subject. 
     
     
         56 . The composition of  claim 55 , wherein the composition comprises a combination of  Clostridium scindens, Barnesiella intestihominis, Blautia hansenii,  and  Pseudoflavonifractor capillosus.    
     
     
         57 . The composition of  claim 55 , wherein the composition is formulated for oral or rectal administration. 
     
     
         58 . The composition of  claim 57 , wherein the composition formulated for oral or rectal administration further comprises a probiotic  bacterium,  probiotic yeast, or a combination thereof. 
     
     
         59 . The composition of  claim 57 , wherein the composition for oral or rectal administration is a liquid, suspension, dried powder, tablet, capsule or food product. 
     
     
         60 . A method for reducing the risk of  Clostridium difficile  infection in a subject, reducing the severity of  Clostridium difficile  infection in a subject, decreasing the amount of  Clostridium difficile  toxin in a subject, increasing resistance to  Clostridium difficile  infection in a subject, reducing risk of developing  Clostridium difficile -associated disease in a subject, treating  Clostridium difficile -associated disease in a subject, preventing a  Clostridium difficile -associated disease in a subject, or decreasing the severity of one or more symptoms of an intestinal disorder in a subject, comprising administering, to a subject in need of such treatment,
 (i) an effective amount of a composition comprising two or more isolated bacteria or spores thereof, selected from the group consisting of  Clostridium scindens, Barnesiella intestihominis, Blautia hansenii,  and  Pseudoflavonifractor capillosus,  wherein the two or more isolated bacteria or spores thereof are in a formulation for administration to a subject;   (ii) an effective amount of a recombinant cell expressing a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the recombinant cell comprises one or more exogenous nucleic acids encoding a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the one or more exogenous nucleic acids are operably linked to a promoter; or   (iii) an effective amount of an agent selected from the group consisting of an enzyme that converts a bile acid to a secondary bile acid, a secondary bile acid, deoxycholic acid, lithocholic acid, purified bacteria or spores thereof expressing an enzyme that converts a bile acid to a secondary bile acid, and combinations thereof.   
     
     
         61 . The method of  claim 60 , wherein the symptoms of an intestinal disorder are selected from the group consisting of frequency and/or volume of diarrhea; fever; abdominal cramping, pain, and/or tenderness; elevated level of white blood cells in the blood; loss of serum albumin; weight loss; appearance of pseudomembrane in the intestinal and/or rectal mucosa; and combinations thereof. 
     
     
         62 . The method of  claim 60 , wherein the enzyme that converts a bile acid to a secondary bile acid is a 7α-hydroxysteroid dehydrogenase enzyme. 
     
     
         63 . The method of  claim 60 , wherein the recombinant cell, composition or agent is administered to the subject in an amount effective to inhibit proliferation of  Clostridium difficile  in the subject. 
     
     
         64 . The method of  claim 60 , wherein the agent is a purified  bacterium  or spore thereof, selected from the group consisting of  Clostridium scindens, Clostridium hiranonis, Clostridium hylemonae, Clostridium perfringens, Clostridium sordellii, Proteocatella sphenisci,  Lachnospiraceae 5_1_57FAA, Clostridiales VE202-05, Clostridiales VE202-26, and combinations thereof. 
     
     
         65 . The method of  claim 64 , wherein the agent further comprises a second  bacterium  or spore thereof selected from the group consisting of  Barnesiella intestihominis, Blautia hansenii, Pseudoflavonifractor capillosus  and combinations thereof. 
     
     
         66 . The method of  claim 60 , further comprising administering to the subject, an antibiotic, an immunotherapeutic agent, an herbal remedy, a probiotic, or combinations thereof. 
     
     
         67 . The method of  claim 60 , further comprising identifying a subject with a  Clostridium difficile  infection, or at risk for  Clostridium difficile  infection, comprising
 (i) obtaining an intestinal microbiota sample from a subject and determining the level of one or more  bacterium  present in the intestinal microbiota sample that can convert a primary bile acid to a secondary bile acid;   comparing the level of the one or more  bacterium  in the sample with a reference  bacterium  level; and   administering the recombinant cell, composition or agent to the subject when the level of one or more  bacterium  in the sample is lower than the  bacterium  reference level;   (ii) obtaining an intestinal microbiota sample from a subject and determining the activity or level of 7α-hydroxysteroid dehydrogenase enzyme present in the intestinal microbiota sample;   comparing the activity or level of 7α-hydroxysteroid dehydrogenase enzyme in the sample with a reference 7α-hydroxysteroid dehydrogenase enzyme activity or level; and   administering the recombinant cell, composition or agent to the subject when the activity or level of 7α-hydroxysteroid dehydrogenase enzyme in the sample is lower than the reference 7α-hydroxysteroid dehydrogenase enzyme activity or level; or   (iii) obtaining an intestinal microbiota sample from a subject and quantifying the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid present in the intestinal microbiota sample;   comparing the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid in the intestinal microbiota sample with a reference bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid level; and   administering the recombinant cell, composition or agent to the subject when the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid in the intestinal microbiota sample is lower than the reference level.   
     
     
         68 . A method of diagnosing a subject with a  Clostridium difficile  infection, or at risk for  Clostridium difficile  infection, comprising
 (i) obtaining an intestinal microbiota sample from a subject and determining the level of one or more  bacterium  present in the intestinal microbiota sample that can convert a primary bile acid to a secondary bile acid;   comparing the level of the one or more  bacterium  in the sample with a reference  bacterium  level; and   diagnosing the subject as having a  Clostridium difficile  infection, or at risk for  Clostridium difficile  infection, when the level of the one or more  bacterium  in the sample is lower than the  bacterium  reference level;   (ii) obtaining an intestinal microbiota sample from a subject and determining the activity or level of 7α-hydroxysteroid dehydrogenase enzyme present in the intestinal microbiota sample;   comparing the activity or level of 7α-hydroxysteroid dehydrogenase enzyme in the sample with a reference 7α-hydroxysteroid dehydrogenase enzyme activity or level; and   diagnosing the subject as having a  Clostridium difficile  infection, or at risk for  Clostridium difficile  infection, when the activity or level of 7α-hydroxysteroid dehydrogenase enzyme in the sample is lower than the reference 7α-hydroxysteroid dehydrogenase enzyme activity or level; or   (iii) obtaining an intestinal microbiota sample from a subject and quantifying the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid present in the intestinal microbiota sample;   comparing the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid present in the intestinal microbiota sample with a reference bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid level; and   diagnosing the subject as having a  Clostridium difficile  infection, or at risk for  Clostridium difficile  infection, when the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid present in the intestinal microbiota sample is lower than the reference level.   
     
     
         69 . A kit comprising (i) a recombinant cell expressing a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the recombinant cell comprises one or more exogenous nucleic acids encoding a bile acid-inducible (bai) 7α/β-dehydroxylation operon, wherein the one or more exogenous nucleic acids are operably linked to a promoter, or (ii) a composition comprising two or more isolated bacteria or spores thereof, selected from the group consisting of  Clostridium scindens, Barnesiella intestihominis, Blautia hansenii,  and  Pseudoflavonifractor capillosus,  wherein the two or more isolated bacteria or spores thereof are in a formulation for administration to a subject. 
     
     
         70 . The method of  claim 66 , wherein, the antibiotic administered to the subject is not an antibiotic selected from the group consisting of a β-lactam antibiotic, clindamycin, a cephalosporin, a quinolone antibiotic, levofloxacin, fluoroquinolone, a macrolide antibiotic, trimethoprim, and a sulfonamide antibiotic. 
     
     
         71 . The method of  claim 68 , further comprising administering an antibiotic to the subject, wherein
 when the level of one or more  bacterium  in the sample is equal to or greater than the  bacterium  reference level,   when the activity or level of 7α-hydroxysteroid dehydrogenase enzyme in the sample is greater than the reference 7α-hydroxysteroid dehydrogenase enzyme activity or level, or   when the level of bile acid-inducible (bai) 7α/β-dehydroxylation operon nucleic acid present in the sample is greater than the reference level,   the antibiotic administered to the subject is not an antibiotic selected from the group consisting of a β-lactam antibiotic, clindamycin, a cephalosporin, a quinolone antibiotic, levofloxacin, fluoroquinolone, a macrolide antibiotic, trimethoprim, and a sulfonamide antibiotic.   
     
     
         72 . The recombinant cell of  claim 49 , wherein the recombinant cell further comprises one or more exogenous nucleic acids encoding a bile salt hydrolase. 
     
     
         73 . The recombinant cell of  claim 49 , wherein the recombinant cell further comprises one or more exogenous nucleic acids encoding a protein that confers antibiotic resistance or sensitivity to the cell; or
 wherein the recombinant cell is formulated in a composition comprising a second recombinant cell, wherein the second recombinant cell expresses one or more exogenous nucleic acids selected from the group consisting of a nucleic acid encoding a bile salt hydrolase, a nucleic acid encoding an antibiotic resistance gene, a nucleic acid encoding an antibiotic susceptibility gene, and combinations thereof.   
     
     
         74 . The method of  claim 60 , wherein the subject is receiving antibiotic therapy. 
     
     
         75 . The method of  claim 60 , wherein the  Clostridium difficile -associated disease is  Clostridium difficile  colitis or pseudomembranous colitis.