The active human gut microbiota differs from the total microbiota

Abstract

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around 75% of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis.Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.

Figure 1. Sequencing overview.

The image shows a general overview of the sequencing results. Abbreviations are defined as follow. Active fractions: PA, pyronin-Y activated; LC, low Cy5; HC, high Cy5. Total fractions: FS, Fecal Suspension; R, Ring fraction (see Methods section). Panel A reports the number of reads obtained from each sample/fraction. Panel B shows “violin plots” reporting on the X-axis the density of sequences at a given percentage of similarity (Y-axis) of the best matches from the database (see Methods section). Panel C reports the ordered percentage distribution of families for each sample/fraction; the gray dotted line labeled as “” defines the threshold used to define the two categories of URB and ORB.

Figure 2. Cluster analysis among samples.

Picture shows cluster analysis carried out for families table normalized to percentage. Clusters were obtained applying Bray-Curtis distance and complete agglomeration method.

Figure 3. Rarefaction analysis carried out at family taxonomic rank.

The X-axis shows the number of sequences in each sample/fraction, while the Y-axis shows the numbers of families encountered, respectively. Sample 4 lacks R and FS curves due to the very low number of sequences obtained from these two fractions (abbreviations as in Figure 1).

Figure 4. Phyla distribution.

The X-axis represents the proportion of phyla from total (FS and R fraction) and active (HC, LC and PA fractions) libraries respectively (abbreviations as in Figure 1).

Figure 5. Families histogram.

The histograms describe the distribution of URB and ORB families among total and active fractions. On the bottom, taxonomy ranks are reported. Bars describe percentage distribution of underrepresented (less than , left) and overrepresented (more than right) families. Blue and red bars describe median distribution of active (HC plus LC and PA fractions) and total (FS and R fraction) respectively. Gray bars indicate the maximum values for each family. Asterisks indicate statistically significant difference (p-value = 0.05) between active and total fractions (abbreviations as in Figure 1).

Figure 6. Cytometry dotplot.

The three panels show the distribution of events in FL8 PMT versus FL2 PMT. X and Y axis are defined in logarithmic arbitrary units. Gray histograms resume the distribution of events along FL2 PMT. Panel A shows unstained cells. Panel B shows cells stained with pyronin-Y with trigger in SS PMT; it is possible to appreciate the two populations of unstained and stained cells respectively (dotted black line separates the two populations). Panel C shows only pyronin-Y stained cells passing the threshold on FL2 PMT.