Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

Abstract

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the speciesBacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and theClostridium lituseburense groups. Another probe was designed for the genera Streptococcus andLactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectalegroup. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectalegroup-specific probe detected a mean of 7.2 × 1010cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, andStreptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 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 the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

Quantitative, longitudinal profiling of the primate fecal microbiota reveals idiosyncratic, dynamic communities

“…This suggests that these allelic probes were reporting on distinct subpopulations of the bacterial group, and thus neither is reporting on the entire group present at any one time. Regarding absolute coverage, as noted above, the two direct quantification methods report maximum values within one (slot blots) or one-half (qPCR) order of magnitude of the 10 11 bacteria per gram of feces reported by microscopy and fluorescence in situ hybridization (Langendijk et al, 1995;Franks et al, 1998;Suau et al, 1999;Harmsen et al, 2002). We consider this a rather good agreement, given the previously documented diversity of targets in fecal specimens.…”

Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes

“…Molecular analyses have mainly been targeted at ribosomal RNA and, more speci¢cally, at 16S rRNA [7]. Applied to faecal microbiota, molecular approaches based on the direct study of 16S rRNA genes [5] or using 16S rRNA probe hybridisation [8,9] have revealed the predominance of four phylogenetic groups gathering the six dominant cultivable genera. The Bacteroides group comprises the genus Bacteroides and also encloses the genera Prevotella and Porphyromonas.…”

“…Although molecular methods were consistent in terms of dominant groups [12], a more thorough analysis of the data acquired independently revealed di¡erences in the proportions of each phylogenetic group, depending on the approach used. For instance, the Bacteroides group is by far the main group in terms of rRNA index when using dot-blot hybridisation [9,13], while the C. coccoides group is the main one in terms of the number of cells when using £uorescent in situ hybridisation (FISH) [8]. These inconsistencies between studies could be explained by di¡erent probe spec-i¢cities or sampling strategies.…”

Identification and real-time qPCR quantification of a nitrite-N degrading bacterial strain in aquatic water

“…It is important to develop an effective monitoring tool to directly detect and quantify selected probiotic bacteria (Souza et al 2012;Pandiyan, Balaraman, Thirunavukkarasu, George, Subaramaniyan, Manikkam & Sadayappan 2013;Skjermo, Bakke, Dahle & Vadstein 2015). Fluorescent in situ hybridization (FISH) has been widely applied in medical research, but its application to environmental research has been slower for its labourand cost-intensive with low throughput (Langendijk, Schut, Jansen, Raangs, Kamphuis, Wilkinson & Welling 1995;Franks, Harmsen, Raangs, Jansen, Schut & Welling 1998;Harmsen, Raangs, He, Degener & Welling 2002). An indirect enzyme-linked immunosorbent assay (indirect ELISA) was developed to detect and quantify bacteria cells by specific antigen-antibody interactions (Charneux, Lorin, Vitry, Antonicelli, Reguiai, Barbe & Bernard 2011;Su, Li, Pan & Xue 2016).…”