Screening of novel secondary bile acid-producing intestinal bacteria and clarification of the mechanism of formation of colon-cancer promoter

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Isolation of novel intestinal bacteria converting primary bile acids such as cholic acid (CA) or chenodeoxycholic acid (CDCA) into colon cancer-promoting secondary bile acids such as deoxycholic acid (DCA) or lithocholic acid (LCA), respectively, was conducted from fecal samples of humans.Total 619 strains were isolated from fecal samples in anaerobic chamber. Their activities for the formation of secondary bile acids including DCA and LCA were examined by culturing them in the medium containing CA or CDCA followed by the detection of the reaction products by TLC, HPLC and GC-MS analyses. As the results, eight strains were obtained as the secondary bile-acid producers. Determination of 16SrRNA gene sequence revealed that two of them producing DCA or LCA were Clostridium scindens and C.leptum, both of which are known producers for DCA or LCA. The other six strains were found to produce 7-oxo-lithocholic acid (3α-hydroxy-7-oxo-5β-cholanoic acid, 7-keto-LCA) from CDCA. These strains consisted of already know producers for 7-keto-LCA including Escherichia coli(three strains) and Bacteroides fragilis (two strains) and one previously not described strain, Bacteroides intestinalis. Therefore, this strain was selected as a novel producer and designated B. intestinalis AM-1. This strain produced 7-keto-DCA from CA. The same activities were also detected in the type strain of B. intestinalis JCM13265^TConversion reaction of strain AM-1 was compared with those of E. coliHB101 and B. fragilis JCM11019^T as the reference strains. It was revealed that strain AM-1 showed conversion yield of more than 90% with lower growth level than the other two strains, resulting in higher activity of conversion per cell than the others. However, we did not observe significant differences in the activities of 7α-hydroxysteroid dehydrogenase, a responsible enzyme producing 7-keto-LCA, in these three strains.