The degradation of cholic acid and deoxycholic acid by Bacteroides species under strict anaerobic conditions.

Microbial transformation of bile acids at the 3a-, 7aand 12a-hydroxyl positions, under anaerobic conditions, was first reported in Alcaligenes fueculis (Hughes & Schmidt, 1942). Other gut bacteria have also been shown to contain hydroxy steroid dehydrogenases (Aries & Hill, 1970). The most common of the three reactions, 7a-hydroxy steroid dehydrogenation, has been reported in many strains of Escherichiu coli (Haslewood et ul., 1973) and Bacteroidesfragilis (Macdonald et ul., 1975). Furthermore 3a-, 7aand 12a-hydroxy steroid dehydrogenase activity has been shown with cell-free preparations of Clostridium perfringens (Macdonald et a / . , 1976). Strains of Clostridium pnraprrtrific~im isolated from human faeces desaturate the bile acid nucleus to form 4-en-3-one (pH optimum 7.5) and 1,4-dien-3-one (pH optimum 8.5) products (Aries et a/ . , 197 1 ), and strains of Bucteroidesfragilis and Bacteroides thetaiotaomicron exhibit both 3aand 7a-dehydroxylase activity (Edenharder et ul., 1976). To date no evidence has been presented for side-chain cleavage by any bacterium indigenous to the human intestinal tract or for nuclear dehydrogenation in Bacteroides spp. This communication reports, in addition to side-chain cleavage of cholic acid and deoxycholic acid, both dehydroxylase and dehydrogenase activity in two Bacteroides spp. The nuclear dehydrogenation reactions carried out by the human intestinal flora have been implicated in the aetiology of colon cancer (Hill, 1974), but have been little studied because of the complex nutritional requirements of these bacteria. Bucteroides spp. capable of metabolizing sodium cholate and deoxycholate have been isolated from human faecal material. The isolates designated Bacteroides frugilis XF23 and Bucteroides thetaiotaomicron E59 have the ability to dehydroxylate cholic acid and oxidize both bile acids during anaerobic growth to yield metabolites with the I ,4-dien-3one structure. Anaerobic metabolism of bile acids by Bacteroides spp. was carried out in modified Todd-Hewitt broth ( I 5g/l) as basal medium containing (g/l): L-cysteine hydrochloride, 0.01 ; 1 M-NaOH, 0.01 ml; Na2S,9H,0, 1 .O; Na2C03, 8.0; MgS04,7H20, 0.1 ; bile salt, 1 .O; the final pH was 7.0 and it was dispensed to fill screw-cap bottles. The medium was autoclaved and on cooling was transferred to an anaerobic glove bag under an atmosphere of CO2/N2 (1 :9). The medium was gassed for lOmin with the above gas phase, and during this procedure the medium was inoculated aseptically with Bucteroides spp. The cultures were incubated at 37°C for 6 weeks. After acidification to pH4.0, the steroids were extracted into redistilled ethyl acetate, the solvent being removed by evaporation. Steroid products were separated by preparative t.1.c. on silica-gel GF254 plates and those with the 1,4-dien-3-one structure were detected under U.V. light; other products were detected by their colour with anisaldehyde reagent (Kritchevsky et a/., 1963). Products were identified with reference to U.V. and i.r. spectra and t.1.c. and g.1.c. mobilities of known standards (Barnes et ul., 1976). Retention times for g.1.c. were measured relative to 5a-cholestane and t.1.c. analysis was performed on Kieselgel GFZS4 in the solvent systems methanol/methylene chloride ( I : 19, v/v) and trimethylpentane/ethyl acetate/acetic acid (5 : 5 : I , by vol.). Mobilities on t.1.c. were measured relative to androsta-l,4-diene-3,17-dione. The results show (Fig. 1) that both Bucteroides spp. have the ability to dehydroxylate, dehydrogenate and carry out side-chain cleavage of cholic acid (I), yielding three acidic and three neutral products, namely: 3a,l2a-dihydroxy-5~-choIanic acid (11); 12ahydroxy-3-oxo-5~-cho1-24-oic acid (111); 12a-hydroxy-3-oxopregna-1,4-dien-20-carboxylic acid (IV); 12~-hydroxyandrosta-4-ene-3,17-dione (V) ; 128-hydroxyandrosta1,4-diene-3,17-dione (VT) and 12a-hydroxyandrosta-1,4-diene-3,17-dione (Vll). Both species also are able to dehydrogenate and carry out side-chain cleavage of deoxy-