Background
Intestinal microbes have been postulated to play an important role in the development of colorectal cancer. Recently developed methods for preserving and delivering fecal samples at ambient temperature to the laboratory for molecular analysis of bacterial constituents were used to test associations of bacterial populations with epidemiologic risk factors for colorectal cancer.
Material and Method
Real-time PCR targeting 16S rRNA gene sequences was used to quantify three intestinal bacterial groups relative to total DNA in stool samples preserved with RNAlater from 62 subjects. Subjects' medical and family history, race, diet, weight, height, and personal habits including smoking were obtained through structured questionnaires.
Results
Bacteroides DNA proportions were relatively stable among individuals and relatively independent of dietary intake or other personal factors. Clostridium (coccoides group) DNA was positively associated with total fat and vitamin C intake. Desulfovibrio DNA amount tended to be higher in African Americans than in other races. Furthermore, Desulfovibrio DNA increased progressively with pack-years of cigarette smoking. The relative DNA quantity (%) was more than 17 times higher in the subjects who smoked at least 15 pack-years compared with never-smokers (P-value for a linear trend =0.001). In addition, Desulfovibrio DNA (%) decreased with increased calcium, vitamin E, and dietary fiber intake. However, only smoking remained significant in multivariable analysis.
Conclusions
Although the study was limited by its sample size, these results suggest that smoking (or possibly unmeasured dietary confounders) may exert modulatory effects on the bacterial populations of the gastrointestinal tract. The study also demonstrates collection, preservation, and sample delivery procedures suitable for large epidemiological studies.

Keywords: Questionnaires, RNA, Ribosomal, 16S - genetics, Polymerase Chain Reaction, Multivariate Analysis, Michigan, Diet, Feces - microbiology, Desulfovibrio - genetics, Colorectal Neoplasms - microbiology, Clostridium - genetics, Body Mass Index, Bacteroides - genetics, African Americans, Smoking - adverse effects