Methods and compositions for increasing toxin production

Abstract

The invention provides methods and compositions (such as for example, culture media) for culturing Clostridium difficile and producing the C. difficile Toxins A and B.

1 - 85 . (canceled) 
     
     
         86 . A medium for culturing a  Clostridium difficile  bacterium comprising soy peptone, yeast extract, a buffering agent, a phosphate buffer, and at least one additive selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin, wherein the medium is at a pH of between 6.35 and 7.45. 
     
     
         87 . The medium of  claim 86 , wherein said the phosphate buffer comprises sodium phosphate, dibasic and potassium phosphate, monobasic. 
     
     
         88 . The medium of  claim 86 , wherein the at least one additive comprises:
 (a) adenosine at a concentration of between 0.8 and 1.2 mM, biotin at a concentration of between 40 and 60 nM, and azaserine at a concentration between 15 and 50 μM;   (b) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM;   (c) D-sorbitol;   (d) D-sorbitol at a concentration between 6 g/L and 20 g/L;   (e) D-sorbitol at a concentration of 12 g/L;   (f) chromium trioxide at a concentration of between 40 and 60 mg/L;   (g) chromium trioxide at a concentration of 50 mg/L;   (h) clindamycin at a concentration between 0.4 and 0.6 mg/L;   (i) clindamycin at a concentration of 0.5 mg/L;   (j) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;   (k) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;   (l) butyric acid at a concentration between 30 mM and 60 mM;   (m) butyric acid at a concentration selected from 30 mM and 60 mM;   (n) D(+)xylose at a concentration between 6 g/L and 10 g/L; or   (o) D(+)xylose at a concentration of 6 g/L.   
     
     
         89 . A bacterial culture comprising  Clostridium difficile  and culture medium, wherein said culture medium comprises at least one additive selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. 
     
     
         90 . The bacterial culture of  claim 89 , wherein said culture medium comprises:
 (a) D-sorbitol;   (b) D-sorbitol at a concentration between 6 g/L and 20 g/L;   (c) D-sorbitol at a concentration of 12 g/L;   (d) chromium trioxide at a concentration between 40 and 60 mg/L;   (e) chromium trioxide at a concentration of 50 mg/L;   (f) clindamycin at a concentration between 0.4 and 0.6 mg/L;   (g) clindamycin at a concentration of 0.5 mg/L;   (h) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;   (i) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;   (j) butyric acid at a concentration between 30 mM and 60 mM;   (k) butyric acid at a concentration selected from 30 mM and 60 mM;   (l) D(+)xylose at a concentration between 6 and 10 g/L.   (m) D(+)xylose at a concentration of 6 g/L; or   (n) soy peptone, yeast extract, KH 2 PO 4 , Na 2 HPO 4 , and NaHCO 3 , and wherein the culture is at a pH of between 6.35 and 7.45.   
     
     
         91 . The bacterial culture of  claim 89 , wherein said culture medium comprises at least two of said additives, optionally wherein:
 (a) said medium comprises adenosine at a concentration of between 0.8 and 1.2 mM, biotin at a concentration of between 40 and 60 nM, and azaserine at a concentration between 15 and 50 μM; or (b) said medium comprises adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM.   
     
     
         92 . A method of culturing  Clostridium difficile  comprising inoculating culture medium with  Clostridium difficile,  said medium comprising at least one additive selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, D-sorbitol, sucrose, and a combination of azaserine, adenosine, and biotin. 
     
     
         93 . The method of  claim 92 , wherein said culture medium comprises:
 (a) at least two of said additives;   (b) D-sorbitol;   (c) D-sorbitol at a concentration between 6 g/L and 20 g/L;   (d) D-sorbitol at a concentration of 12 g/L;   (e) chromium trioxide at a concentration between 40 and 60 mg/L;   (f) chromium trioxide at a concentration of 50 mg/L;   (g) clindamycin at a concentration between 0.4 and 0.6 mg/L;   (h) clindamycin at a concentration of 0.5 mg/L;   (i) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;   (j) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;   (k) butyric acid at a concentration between 30 mM and 60 mM;   (l) butyric acid at a concentration selected from 30 mM and 60 mM;   (m) D(+)xylose at a concentration between 6 and 10 g/L;   (n) D(+)xylose at a concentration of 6 g/L;   (o) adenosine at a concentration of between 0.8 and 1.2 mM, biotin at a concentration of between 40 and 60 nM, and azaserine at a concentration between 15 and 50 μM; or   (p) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and azaserine at a concentration of 50 μM.   
     
     
         94 . The method of  claim 93 , wherein said medium further comprises soy peptone, yeast extract, KH 2 PO 4 , Na 2 PO 4 , and NaHCO 3 , and wherein the culture is at a pH of between 6.35 and 7.45.