Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We ...wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment.
Modulation of the gut microbiome as a means to improve human health has recently gained increasing interest. In this study, it was investigated whether cRG-I, a carrot-derived pectic polysaccharide, ...enriched in rhamnogalacturonan-I (RG-I) classifies as a potential prebiotic ingredient using novel in vitro models. First, digestion methods involving α-amylase/brush border enzymes demonstrated the non-digestibility of cRG-I by host-derived enzymes versus digestible (starch/maltose) and non-digestible controls (inulin). Then, a recently developed short-term (48 h) colonic incubation strategy was applied and revealed that cRG-I fermentation increased levels of health-promoting short-chain fatty acids (SCFA; mainly acetate and propionate) and lactate comparable but not identical to the reference prebiotic inulin. Upon upgrading this fermentation model by inclusion of a simulated mucosal environment while applying quantitative 16S-targeted Illumina sequencing, cRG-I was additionally shown to specifically stimulate operational taxonomic units (OTUs) related to health-associated species such as
,
,
,
,
,
, and
. Finally, in a novel model to assess host-microbe interactions (Caco-2/peripheral blood mononuclear cells (PBMC) co-culture) fermented cRG-I increased barrier integrity while decreasing markers for inflammation. In conclusion, by using novel in vitro models, cRG-I was identified as a promising prebiotic candidate to proceed to clinical studies.
NUTRIOSE® (Roquette, Lestrem, France) is a resistant dextrin with well-established prebiotic effects. This study evaluated the indirect effects of pre-digested NUTRIOSE® on host immune response and ...gut barrier integrity. Fecal samples from eight healthy donors were inoculated in a Colon-on-a-plate® system (ProDigest, Ghent, Belgium) with or without NUTRIOSE® supplementation. Following 48 h fermentation, colonic suspensions were tested in a Caco-2/THP1-Blue™ co-culture system to determine their effects on gut barrier activity (transepithelial electrical resistance) and immune response following lipopolysaccharide stimulation. Additionally, changes in short-chain fatty acid levels (SCFA) and microbial community composition following a 48 h fermentation in the Colon-on-a-plate® system were measured. Across all donors, immune-mediated intestinal barrier damage was significantly reduced with NUTRIOSE®-supplemented colonic suspensions versus blank. Additionally, IL-6 and IL-10 levels were significantly increased, and the level of the neutrophil chemoattractant IL-8 was significantly decreased with NUTRIOSE®-supplemented colonic suspensions versus blank in the co-culture models following lipopolysaccharide stimulation. These beneficial effects of NUTRIOSE® supplementation were likely due to increased acetate and propionate levels and the enrichment of SCFA-producing bacteria. NUTRIOSE® was well fermented by the colonic bacteria of all eight donors and had protective effects on inflammation-induced disruption of the intestinal epithelial barrier and strong anti-inflammatory effects.
Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk ...oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2'fucosyllactose (2'-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem M-SHIME
) with fecal samples from 3-month-old breastfed (BF) infants as well as 2-3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME
study. A key finding was the strong and immediate increase in the relative abundance of
following 2'-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME
. At the metabolic level, while decreasing branched-chain fatty acids, 2'-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2'-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.
The present study examined the suitability of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid grouping of bacterial isolates, i.e. ...dereplication. Dereplication is important in large-scale isolation campaigns and screening programs since it can significantly reduce labor intensity, time and costs in further downstream analyses. Still, current dereplication techniques are time consuming and costly. MALDI-TOF MS is an attractive tool since it performs fast and cheap analyses with the potential of automation. However, its taxonomic resolution for a broad diversity of bacteria remains largely unknown. To verify the suitability of MALDI-TOF MS for dereplication, a total of 249 unidentified bacterial isolates retrieved from the rhizosphere of potato plants, were analyzed with both MALDI-TOF MS and repetitive element sequence based polymerase chain reaction (rep-PCR). The latter technique was used as a benchmark. Cluster analysis and inspection of the profiles showed that for 204 isolates (82%) the taxonomic resolution of both techniques was comparable, while for 45 isolates (18%) one of both techniques had a higher taxonomic resolution. Additionally, 16S rRNA gene sequence analysis was performed on all members of each delineated cluster to gain insight in the identity and sequence similarity between members in each cluster. MALDI-TOF MS proved to have higher reproducibility than rep-PCR and seemed to be more promising with respect to high-throughput analyses, automation, and time and cost efficiency. Its taxonomic resolution was situated at the species to strain level. The present study demonstrated that MALDI-TOF MS is a powerful tool for dereplication.
► MALDI-TOF MS fit for dereplication at the species-to-strain level. ► MALDI-TOF MS is a high-throughput dereplication tool. ► Dereplication tool for broad range of bacterial species. ► MALDI-TOF has high reproducibility, low operational costs and labor intensity.
This study evaluated the impact of
ssp.
CNCM I-3446, Bovine Milk-derived OligoSaccharides (BMOS) and their combination on infant gut microbiota in vitro. In addition, a novel strategy consisting of ...preculturing
with BMOS to further enhance their potential synbiotic effects was assessed.
Short-term fecal batch fermentations (48 h) were used to assess the microbial composition and activity modulated by BMOS alone,
grown on BMOS or dextrose alone, or their combinations on different three-month-old infant microbiota.
BMOS alone significantly induced acetate and lactate production (leading to pH decrease) and stimulated bifidobacterial growth in 10 donors. A further in-depth study on two different donors proved
ability to colonize the infant microbiota, regardless of the competitiveness of the environment. BMOS further enhanced this engraftment, suggesting a strong synbiotic effect. This was also observed at the microbiota activity level, especially in a donor containing low initial levels of bifidobacteria. In this donor, preculturing
with BMOS strengthened further the early modulation of microbiota activity observed after 6 h.
This study demonstrated the strong synbiotic effect of BMOS and
on the infant gut microbiota, and suggests a strategy to improve its effectiveness in an otherwise low-
microbiota.
Human gut microbiota has a fundamental role in human health, and diet is one of the most relevant factors modulating the gut microbial ecosystem. Fiber, fat, proteins, and micronutrients can shape ...microbial activity and structure. Much information is available on the role of defined prebiotic fibers on gut microbiota, but less known are the effects of intact dietary fiber sources on healthy gut ecosystems. This research investigated
in vitro
the short-term effect of 22 commercially available food sources of dietary fiber on gut microbiota activity pH, gas, short-chain fatty acids (SCFA), branched fatty acids (BCFA), lactate and specific composition of Firmicutes, Bacteroidetes, bifidobacteria, and lactobacilli populations. More than 80% (19 of 22) of the products were highly fermentable and induced SCFAs production, with specific product differences. In general, all the whole grain cereals had a similar effect on gut microbiota modulation, inducing acetate and butyrate production and increasing bifidobacteria levels. Incorporating and comparing a large variety of products, including “non-conventional” fiber sources, like konjac, bamboo fiber, or seeds fiber, about which there is little information, contributes to our knowledge on the modulatory activity of diverse food fiber sources on human gut microbiota, and therefore potential health promotion through dietary fiber diversification.
The human gut microbiota has been linked to the health status of the host. Modulation of human gut microbiota through pro- and prebiotic interventions has yielded promising results; however, the ...effect of novel prebiotics, such as chitin–glucan, on gut microbiota–host interplay is still not fully characterized. We assessed the effect of chitin–glucan (CG) and chitin–glucan plus Bifidobacterium breve (CGB) on human gut microbiota from the luminal and mucosal environments in vitro. Further, we tested the effect of filter-sterilized fecal supernatants from CG and CGB fermentation for protective effects on inflammation-induced barrier disruption and cytokine production using a co-culture of enterocytes and macrophage-like cells. Overall, CG and CGB promote health-beneficial short-chain fatty acid production and shift human gut microbiota composition, with a consistent effect increasing Roseburia spp. and butyrate producing-bacteria. In two of three donors, CG and CGB also stimulated Faecalibacterium prausniitzi. Specific colonization of B. breve was observed in the lumen and mucosal compartment; however, no synergy was detected for different endpoints when comparing CGB and CG. Both treatments included a significant improvement of inflammation-disrupted epithelial barrier and shifts on cytokine production, especially by consistent increase in the immunomodulatory cytokines IL10 and IL6.
Background: Prebiotics used as a dietary supplement, stimulate health-related gut microbiota (e.g., bifidobacteria, lactobacilli, etc.). This study evaluated potential prebiotic effects of an ...artichoke aqueous dry extract (AADE) using in vitro gut model based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME
).
Short-term colonic fermentations (48 h) of AADE, fructo-oligosaccharides (FOS), and a blank were performed. Microbial metabolites were assessed at 0, 6, 24, and 48 h of colonic incubation via measuring pH, gas pressure, lactate, ammonium, and short-chain fatty acids (SCFAs) levels. Community composition was assessed via targeted qPCRs.
After 24 and 48 h of incubation, bifidobacteria levels increased 25-fold with AADE (
< 0.05) and >100-fold with FOS (
< 0.05) compared to blank.
spp. levels only tended to increase with AADE, whereas they increased 10-fold with FOS. At 6 h, pH decreased with AADE and FOS and remained stable until 48 h; however, gas pressure increased significantly till the end of study. Acetate, propionate, and total SCFA production increased significantly with both at all time-points. Lactate levels initially increased but branched SCFA and ammonium levels remained low till 48 h.
AADE displayed prebiotic potential by exerting bifidogenic effects that stimulated production of health-related microbial metabolites, which is potentially due to inulin in AADE.
Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) cause intestinal discomfort in patients with irritable bowel syndrome (IBS). An enzyme mix (2500 SU invertase, 2400 ...GalU α-galactosidase, 10,000 ALU β-galactosidase) optimized for FODMAP digestion, and/or human milk oligosaccharides (HMO) (2'-FL, DFL, and LNnT), were evaluated for effects on microbial community activity and composition in short-term colonic incubations using the fecal microbiota of four patients with IBS-D symptoms under the following test conditions: (i) FODMAP, (ii) pre-digested (with enzyme mix) FODMAP, (iii) FODMAP + HMO, and (iv) pre-digested FODMAP + HMO. Pre-digested FODMAP reduced short-chain fatty acid (SCFA) production versus FODMAP; HMO restored this. A 10-day experiment with the simulator of the human intestinal microbial ecosystem (SHIME
), using fecal samples from two patients with IBS-D, further evaluated these findings. FODMAP resulted in decreased microbial diversity versus blank. Pre-digestion with the enzyme mix restored microbial diversity, improved FODMAP digestibility, and reduced gas pressure versus undigested FODMAP; however, SCFA production decreased. HMO restored SCFA production along with an increase in gas pressure and increased abundance of
. When used in combination, the FODMAP enzyme mix and HMO may resolve FODMAP-related IBS symptoms while maintaining a healthy gut microbiome via prebiotic activity.
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