Development and application of a polymerase chain reaction assay for the detection and enumeration of bile acid 7alpha-dehydroxylating bacteria in human feces.
BACKGROUND
Secondary bile acids are synthesized in the human colon from the bacterial 7alpha-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 7alpha-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 7alpha-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 7alpha-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 7alpha-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 7alpha-dehydroxylating bacteria in human fecal samples.
Wells
James E
JE
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, PO Box 980678, Richmond, VA 23298-0678, USA.
Williams
Kristopher B
KB
Whitehead
Terence R
TR
Heuman
Douglas M
DM
Hylemon
Phillip B
PB
D002793:Cholic Acids / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006900:Hydroxylation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D002793:Cholic Acids; D017931:DNA Primers; D012336:RNA, Ribosomal, 16S