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

从人类粪便样本中分离出一种新型肠道细菌，分别将胆酸（CA）或鹅去氧胆酸（CDCA）等初级胆汁酸转化为促进结肠癌的次级胆汁酸，如去氧胆酸（DCA）或石胆酸（LCA）。厌氧室共分离出619株细菌。通过在含有CA或CDCA的培养基中培养，并通过TLC、HPLC和GC-MS分析检测反应产物，研究了它们形成DCA和LCA二级胆汁酸的活性。结果得到8株产二级胆汁酸的菌株。16SrRNA基因序列的测定表明，产生DCA或LCA的梭状芽胞杆菌分别为scindens梭状芽胞杆菌和leptum梭状芽胞杆菌，它们都是已知的DCA或LCA的制造者。另外6株菌株由CDCA产生7-氧基胆酸（3α-羟基-7-氧基-5β-胆酸，7-酮- lca）。这些菌株由已知的7-酮- lca的生产者组成，包括大肠杆菌（3株）和脆弱拟杆菌（2株），以及一种以前未被描述的菌株肠拟杆菌。因此，该菌株被选为新的生产者，命名为B. testinalis AM-1。菌株AM-1的转化反应与大肠杆菌hb101和脆弱杆菌JCM11019^T的转化反应进行了比较。结果表明，菌株AM-1的转化率在90%以上，但其生长水平低于其他两种菌株，导致其单位细胞转化活性高于其他两种菌株。然而，我们没有观察到这三个菌株的7α-羟基类固醇脱氢酶（一种产生7-酮- lca的酶）的活性有显著差异。

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.