Temperature and denaturating gradient gel electrophoresis analysis of 16S rRNA from human faecal samples

The gastrointestinal tract (GIT) of mammals harbours a complex community of obligate and facultative anaerobic bacteria. The composition of the GIT microbiota is dependent on the physiological condition, age, genetics, and diet of the host. During the past 5 years a number of molecular fingerprinting methods have been developed to characterise complex communities based on 16S rRNA sequence diversity. This paper describes the use of temperature and denaturing gradient gel electrophoresis (T/DGGE) of bacterial 16S rRNA/DNA in faecal samples from humans, in which special attention was given to the quantification of the sequence diversity. After birth the GIT community develops into a relatively simple community consisting of 1-8 major types within three months. Adults show more complex, but remarkably constant patterns. These patterns are hardly affected by changes in diet. Significant differences were observed between different individuals, particularly between genetically unrelated persons. In general, bacterial communities of faecal samples from genetically related adults (i.e. twins, brothers, sisters) show higher similarity of DGGE banding patterns than those from genetically unrelated individuals, although occasionally all persons in one family show highly similar profiles. The DGGE banding patterns of humans are significantly different from those of other mammals, such as pigs, gorillas and cats, indicating that genetic factors of the host affect the composition of the GI-tract flora. The major bacterial groups were identified by cloning and sequencing of the dominant 16S rDNA molecules. At least three sequences with highest homology to Ruminococcus obeum and Eubacterium halii and Fusobacterium prausnitzii were present in all human subjects and are likely to play a universal role in the GI-tract. Other sequences were found in variable ratios in different individuals