Development of methods for the detection and quantification of 7alpha-dehydroxylating clostridia, Desulfovibrio vulgaris, Methanobrevibacter smithii, and Lactobacillus plantarum in human feces.
BACKGROUND
Mounting evidence suggests a relationship between bacterial metabolism of certain dietary and endogenous factors and the development of colorectal cancer. Deoxycholic acid (DCA) is a well studied co-carcinogen and bio-transformation product of 7alpha-dehydroxylating Clostridia. H2S is a cytotoxic metabolite produced primarily by sulfate-reducing bacteria (SRB). The production of methane indicates low levels of active SRB. Lactic acid bacteria (LAB) have received attention recently due to their putative anti-tumor properties.


METHODS
Human stool was spiked with pure cultures of bacteria and diluted in several enriched media. Each dilution titer was analyzed for the presence of the organism by PCR and biochemical assays. Duplicate stool aliquots were stored under various conditions for a 1 month period at -20 degrees C to test viability and detection.


RESULTS
Growth and enumeration of each spiked organism was confirmed by PCR and biochemical assays. The combination of bead beating and chemical lysis steps produced the greatest DNA yields. PCR assays detected as low as 75 fg target DNA. The ability to detect Methanobrevibacter smithii, and Desulfovibrio vulgaris by either PCR or biochemical assay declined significantly after storage at -20 degrees C for 1 month.


CONCLUSIONS
Accurate detection and quantification of each bacterium using the described methods resulted when stool was processed immediately after collection. Storage of some members of the gut flora results in decrease in or loss of viability.
Ridlon
Jason M
JM
Department of Microbiology/Immunology, Medical College of Virginia Campus, Virginia, Commonwealth University, PO Box 980678, Richmond, VA, USA.
McGarr
Sean E
SE
Hylemon
Phillip B
PB
D015373:Bacterial Typing Techniques; D001681:Biological Assay; D003013:Clostridium / Q000302:isolation & purification*; D019175:DNA Methylation; D016969:Desulfovibrio vulgaris / Q000302:isolation & purification* / Q000502:physiology; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006900:Hydroxylation; D048191:Lactiplantibacillus plantarum / Q000302:isolation & purification* / Q000502:physiology; D041581:Methanobrevibacter / Q000302:isolation & purification* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity