|Wells, James - Jim|
Submitted to: Clinica Chimica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2003
Publication Date: 5/1/2003
Citation: WELLS, J., WILLIAMS, K.B., WHITEHEAD, T.R., HEUMAN, D.M., HYLEMON, P.B. 2003. DEVELOPMENT AND APPLICATION OF A POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION AND ENUMERATION OF BILE ACID 7 ALPHA-DEHYDROXYLATING BACTERIA IN HUMAN FECES. CLINICA CHIMICA ACTA. 331:127-134. Interpretive Summary: Primary bile acids produced in the gall bladder are converted in the human intestinal tract to secondary bile acids by members of the intestinal bacteria population. These secondary bile acids have been correlated with an increased risk of colon cancer and cholesterol gallstone disease. The number of bacteria that can convert primary bile acids vary within the human population. We have developed a polymerase chain reaction (PCR)-based assay to detect bacteria in the feces capable of producing secondary bile acids. The PCR assay targets a particular gene that encodes for an enzyme important in the production of the secondary bile acids. This new method should prove useful for monitoring levels of bacteria in human fecal samples that produce secondary bile acids and may identify people predisposed to gallstones or colon cancer.
Technical Abstract: Secondary bile acids are synthesized in the human colon from the bacterial 7 alpha-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 the bile acid 7 alpha-dehydroxylation all belong to the genus Clostridium. We developed a polymerase chain reaction (PCR)-based assay that can specifically amplify the baiCD gene, specific to bile acid 7 alpha-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. The PCR assay was effective in detecting the baiCD gene in several strains known to exhibit bile acid 7 alpha-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 7 alpha-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. This new method should be useful for the monitoring of levels of bile acid 7 alpha-dehydroxylating bacteria in human fecal samples.