VARIATIONS OF BACTERIAL-POPULATIONS IN HUMAN FECES MEASURED BY FLUORESCENT IN-SITU HYBRIDIZATION WITH GROUP-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in huma n fecal samples. A set of two probes was specific for species of the B acteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rect ale group. The temperature of dissociation of each of the probes was d etermined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluo rescent in situ hybridization (FISH). The new probes were used in init ial FISH experiments to enumerate human fecal bacteria. The combinatio n of the two Bacteroides-specific probes detected a mean of 5.4 x 10(1 0) cells per g (dry weight) of feces; the Clostridium coccoides-Eubact erium rectale group-specific probe detected a mean of 7.2 x 10(10) cel ls per g (dry weight) of feces. The Clostridium histolyicum, Clostridi um lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of th e fecal flora. The normal biological variations within the fecal popul ations of the volunteers were determined and indicated that these vari ations should be considered when evaluating the effects of agents modu lating the flora.