Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends

Abstract

The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. -36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species-of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)-conventional molecular ecological approaches, such as PCR-DGGE and qPCR, still remain a very useful complementary tool.

Linking Microbial Community Structure to Function in Representative Simulated Systems

“…Specifically, rare OTUs and singletons were removed, three biological replicates under each condition were tested, and all samples were collected and analyzed in the same manner to ensure more-reproducible data. The relatively large amount of bacteriodetes in the microbial community in the absence of the pathogen agrees with the findings for previously studied in vitro models (17,23,51,52) and human gut microbiomes (7,53). The majority of the real colon microbiomes have a higher concentration of firmicutes than bacteriodetes (7,53).…”

Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women

“…We used previously described methods to determine microbial ecology parameters per sample: richness, defined as the median number of genera per sample (Marzorati et al, 2008); the Shannon diversity index (Shannon, 1948); and evenness, expressed as a Co-value with 0 representing complete evenness and 100 as complete unevenness (Marzorati et al, 2013). We focused the evenness calculations on the five most abundant bacteria in each cluster to reduce the influence of the long tail of minority species (Marzorati et al, 2013).…”

“…We focused the evenness calculations on the five most abundant bacteria in each cluster to reduce the influence of the long tail of minority species (Marzorati et al, 2013). To compare cumulative Co-values per cluster, an average sample per cluster was generated by calculating median S/B ratios per genus across the samples in that cluster.…”

High Throughput Sequencing: An Overview of Sequencing Chemistry

“…In Pyrosequencing, pyrophosphate (PPi) released during the DNA polymerization reaction is detected and used as an indicator of specific base incorporation. It involves detection of the base incorporated in the daughter DNA by DNA polymerase, with feeding and removing, all the four bases sequentially and detecting the pyrophosphate released by cascade of enzymes that emit light [7,19].…”

“…In contrast to the short read length of SGS technologies already available, third generation sequencing (TGS) results in longer read length at low cost. TGS is the ultimate dream of biologists, as it will bring freedom from amplification artifacts and bias [7]. In the sequencing reactions, the nucleotide base assignment is referred to as ''Base calling'' which is scanned for error probability by Phred software.…”