Media supplements and methods to culture human gastrointestinal anaerobic microorganisms

Abstract

A media supplement for culturing anaerobic bacteria is provided which comprises a filtrate of effluent from a chemostat vessel in which a target bacterial ecosystem has been cultured. Methods of using the supplement for culturing or isolating anaerobic microbial strains or communities, particularly anaerobic bacteria from the human gut, are also provided.

1 - 56 . (canceled) 
     
     
         57 . A media supplement for culturing anaerobic bacteria, the media supplement comprising a filtrate of effluent from a chemostat vessel in which a target bacterial ecosystem has been cultured, wherein the target bacterial ecosystem has been cultured in culture media which is Media 1, Media 1 comprising: 0.4% w/v Peptone; 0.4% w/v Yeast extract; 0.4% w/v NaHCO 3 ; 0.4% w/v Pectin; 0.4% w/v Xylan; 0.4% w/v Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified wheat starch; 0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v L-cysteine HCl; 0.0002% w/v CaCl 2 ; 0.0002% w/v NaCl; 0.0008% w/v K 2 HPO 4 ; 0.0008% w/v KH 2 PO 4 ; 0.0002% w/v MgSO 4 ; 0.0001% w/v Hemin; and 0.00002% w/v menadione. 
     
     
         58 . The media supplement of  claim 57 , wherein the target bacterial ecosystem has been cultured in culture media which comprises mucin. 
     
     
         59 . The media supplement of  claim 57 , wherein the target bacterial ecosystem is Defined Experimental Community 1 (DEC-1), Defined Experimental Community 2 (DEC-2) or Defined Experimental Community 3 (DEC-3), wherein:
 (a) DEC-1 comprises the following bacterial strains:  Bacteroides ovatus, Bifidobacterium adolescentis, Bifidobacterium longum, Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium prausnitzii , and  Parabacteroides distasonis;      (b) DEC-2 comprises the following bacterial strains:  Bacteroides ovatus, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium longum, Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium prausnitzii, Parabacteroides distasonis, Roseburia inulinivorans , and  Ruminococcus obeum ; and   (c) DEC-3 comprises the following bacterial strains:  Akkermemsia muciniphila, Alistipes putredinis, Alistipes shahfi, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea formicigenerans, Eubacterium rectale, Faecalibacterium prausnitzii, Odoribacter splanchnicus, Oscillibacter valericigenes, Ruminococcus bromii  1 , Ruminococcus bromii  2 , Ruminococcus obeum , and  Ruminococcus  sp. 1.   
     
     
         60 . The media supplement of  claim 57 , wherein the target ecosystem which has been cultured in the chemostat comprises a community of bacterial strains representing an enterotype of human gut, wherein the enterotype is the  Bacteroides  enterotype, the  Prevotella  enterotype or the  Ruminococcus  enterotype. 
     
     
         61 . A method for preparing a media supplement for culturing anaerobic bacteria, said method comprising the steps of:
 a) culturing a target bacterial ecosystem in culture media in a single-stage chemostat under conditions replicating normal human colonic gastrointestinal tract, in equilibrium;   b) collecting effluent from the chemostat; and   c) filtering the effluent through a 0.2 μm filter to remove bacterial cells, in order to produce the media supplement;   wherein the culture media is Media 1, which comprises: 0.4% w/v Peptone; 0.4% w/v Yeast extract; 0.4% w/v NaHCO 3 ; 0.4% w/v Pectin; 0.4% w/v Xylan; 0.4% w/v Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified wheat starch; 0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v L-cysteine HCl; 0.0002% w/v CaCl 2 ; 0.0002% w/v NaCl; 0.0008% w/v K 2 HPO 4 ; 0.0008% w/v KH 2 PO 4 ; 0.0002% w/v MgSO 4 ; 0.0001% w/v Hemin; and 0.00002% w/v menadione.   
     
     
         62 . The method of  claim 61 , further comprising a step of centrifuging the effluent at 14,000 rpm for 10 minutes and collecting the supernatant before step c), and wherein the effluent supernatant is then filtered in step c). 
     
     
         63 . The method of  claim 61 , further comprising filtering the effluent or effluent supernatant sequentially through a 1.0 μm filter, a 0.8 μm filter, and a 0.45 μm filter, before filtering through the 0.2 μm filter. 
     
     
         64 . The method of  claim 61 , wherein the target bacterial ecosystem comprises Defined Experimental Community 1 (DEC-1), Defined Experimental Community 2 (DEC-2), or Defined Experimental Community 3 (DEC-3), wherein DEC-1, DEC-2 and DEC-3 are defined as follows:
 DEC-1 comprises the following bacterial strains:  Bacteroides ovatus, Bifidobacterium  adolescentis,  Bifidobacterium longum, Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium prausnitzii , and  Parabacteroides distasonis;      DEC-2 comprises the following bacterial strains:  Bacteroides ovatus, Bacteroides vulgatus, Bifidobacterium  adolescentis,  Bifidobacterium longum, Collinsella aerofaciens, Eubacterium rectale, Faecalibacterium prausnitzii, Parabacteroides distasonis, Roseburia inulinivorans , and  Ruminococcus obeum ; and   DEC-3 comprises the following bacterial strains:  Akkermansia munciniphila, Alistipes putredinis, Alistipes shahii, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea formicigenerans, Eubacterium rectale, Faecalibacterium prausnitzii, Odoribacter splanchnicus, Oscillibacter valericigenes, Ruminococcus bromii  1 , Ruminococcus bromii  2 , Ruminococcus obeum , and  Ruminococcus  sp. 1.   
     
     
         65 . The method of  claim 61 , wherein the target ecosystem which has been cultured in the chemostat comprises a community of bacterial strains representing an enterotype of human gut, wherein the enterotype is the  Bacteroides  enterotype, the  Prevotella  enterotype or the  Ruminococcus  enterotype. 
     
     
         66 . The method of  claim 61 , wherein the culture media comprises mucin. 
     
     
         67 . The method of  claim 61 , wherein the chemostat has a system retention time of 24 hours. 
     
     
         68 . The method of  claim 61 , wherein the conditions replicating normal human colonic gastrointestinal tract comprise: a temperature of about 37° C.; a pH of about 6.9 to 7; a system retention time of 24 hours; and maintenance of anaerobic conditions in the chemostat. 
     
     
         69 . A media supplement obtainable by the method of  claim 61 . 
     
     
         70 . A method of isolating anaerobic bacteria from human gut, comprising:
 a) culturing a target bacterial ecosystem in culture media in a single-stage chemostat under conditions replicating normal human colonic gastrointestinal tract, until equilibrium is reached;   b) diluting the culture and plating onto Fastidious anaerobe agar (FAA) supplemented with the media supplement of  claim 57 , and optionally supplemented with defibrinated sheep blood;   c) incubating plates in an anaerobe chamber;   d) purifying individual anaerobic bacterial colonies grown in step (c); and   e) optionally, culturing the purified individual anaerobic bacterial colonies from step (d) in liquid culture in a single-stage chemostat under conditions replicating normal human colonic gastrointestinal tract, optionally wherein the media supplement of  claim 57  is used to supplement culture media at about 1% v/v to about 10% v/v;   
       such that isolates of anaerobic bacteria are obtained. 
     
     
         71 . The method of  claim 70 , wherein the anaerobe chamber contains an atmosphere of N 2 , CO 2  or H 2 , or a mixture thereof. 
     
     
         72 . The method of  claim 70 , wherein the target bacterial ecosystem cultured in step (a) is a human fecal sample. 
     
     
         73 . The method of  claim 72 , wherein the human fecal sample which is cultured is a 10% w/v fecal slurry supernatant or a 20% w/v fecal slurry supernatant. 
     
     
         74 . The method of  claim 70 , wherein the culture media of step a) is Media 1 comprising: 0.4% w/v Peptone; 0.4% w/v Yeast extract; 0.4% w/v NaHCO 3 ; 0.4% w/v Pectin; 0.4% w/v Xylan; 0.4% w/v Arabinogalactan; 0.6% w/v Casein; 1% w/v unmodified wheat starch; 0.2% w/v inulin; 0.1% w/v bile salts; 0.1% w/v L-cysteine HCl; 0.0002% w/v CaCl 2 ; 0.0002% w/v NaCl; 0.0008% w/v K 2 HPO 4 ; 0.0008% w/v KH 2 PO 4 ; 0.0002% w/v MgSO 4 ; 0.0001% w/v Hemin; and 0.00002% w/v menadione. 
     
     
         75 . The method of  claim 70 , wherein the anaerobic bacteria obtained is  Faecalibacterium prausnitzii  or  Ruminococcus callidus  (ATCC27760).