Development and application of a polymerase chain reaction assay for the detection and enumeration of bile acid 7α-dehydroxylating bacteria in human feces

Abstract

Background: Secondary bile acids are synthesized in the human colon from the bacterial 7α-dehydroxylation of primary bile acids. Increased levels of secondary bile acids have been correlated with an increased risk of colon cancer and cholesterol gallstone disease. Based on 16s rDNA sequence analysis, stock cultures of bacterial strains with bile acid 7α-dehydroxylation all belong to the genus Clostridium. Methods: We developed a polymerase chain reaction (PCR)-based assay that can specifically amplify the baiCD gene, specific to bile acid 7α-dehydroxylation pathway. The PCR primer pair was designed using known nucleotide sequences from two different baiCD genes described for Clostridium scindens VPI 12708 and Clostridium hiranonis TO931. Although the DNA sequences of these genes were <70% identical, several regions were conserved enough to design primers with little or no redundancy. Results: The PCR assay was effective in detecting the baiCD gene in several strains known to exhibit bile acid 7α-dehydroxylation activity. The PCR assay also detected the baiCD gene in DNA extracted from fecal dilution series and correlated with the levels of cholic acid 7α-dehydroxylating bacteria detected by activity assays. The PCR assay was sensitive enough to detect the baiCD gene in DNA samples extracted directly from as little as 0.5 mg feces. Conclusions: This new method should be useful for the monitoring of levels of bile acid 7α-dehydroxylating bacteria in human fecal samples.