The effect of primer choice and short read sequences on the outcome of 16S rRNA gene based diversity studies

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

A comparison of the in vitro effects of 2'fucosyllactose and lactose on the composition and activity of gut microbiota from infants and toddlers

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(R)]) 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(R) study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2'-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME(R). 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

Synbiotic effect of Bifidobacterium lactis CNCM I-3446 and bovine milk-derived oligosaccharides on infant gut microbiota

Background: This study evaluated the impact ofBifidobacterium animalisssp.lactisCNCM I-3446, Bovine Milk-derived OligoSaccharides (BMOS) and their combination on infant gut microbiota in vitro. In addition, a novel strategy consisting of preculturingB. lactiswith BMOS to further enhance their potential synbiotic effects was assessed. Method: Short-term fecal batch fermentations (48 h) were used to assess the microbial composition and activity modulated by BMOS alone,B. lactisgrown on BMOS or dextrose alone, or their combinations on different three-month-old infant microbiota. Results: 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 provedB. lactisability 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, preculturingB. lactiswith BMOS strengthened further the early modulation of microbiota activity observed after 6 h. Conclusion: This study demonstrated the strong synbiotic effect of BMOS andB. lactison the infant gut microbiota, and suggests a strategy to improve its effectiveness in an otherwise low-Bifidobacteriummicrobiota

A novel non-digestible, carrot-derived polysaccharide (cRG-I) selectively modulates the human gut microbiota while promoting gut barrier integrity : an integrated in vitro approach

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 alpha-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 asBifidobacterium longum,Bifidobacterium adolescentis,Bacteroides dorei,Bacteroides ovatus,Roseburia hominis,Faecalibacterium prausnitzii, andEubacterium hallii. 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

Fructans with varying degree of polymerization enhance the selective growth of Bifidobacterium animalis subsp. lactis BB-12 in the human gut microbiome in vitro

Synbiotics aim to improve gastrointestinal health by combining pre- and probiotics. This study evaluated combinations of Bifidobacterium animalis subsp. lactis BB-12 with seven fructans: oligofructoses (OF1-OF2; low degree of polymerization (DP)), inulins (IN1-IN2-IN3; high DP) and OF/IN mixtures (OF/IN1-OF/IN2). During monoculture incubations, all fructans were fermented by BB-12 as followed from increased BB-12 numbers and increased acetate and lactate concentrations, with most pronounced fermentation for low DP fructans (OF1-OF2). Further, short-term colonic incubations for three human donors revealed that also in presence of a complex microbiota, all fructans (particularly OF1) consistently selectively enhanced the growth of BB-12. While each fructan as such already increased Bifidobacteriaceae numbers with 0.94-1.26 log(cells/mL), BB-12 co-supplementation additionally increased Bifidobacteriaceae with 0.17-0.46 log(cells/mL). Further, when co-supplemented with fructans, BB-12 decreased Enterobacteriaceae numbers (significant except for IN1-IN3). At metabolic level, all fructans decreased pH due to increased acetate and lactate production, while OF/IN2-IN1-IN2-IN3 also stimulated propionate and butyrate production. BB-12 co-supplementation further increased propionate and butyrate for OF/IN2-IN3 and IN1-IN2, respectively. Overall, combinations of BB-12 with fructans are promising synbiotic concepts, likely due to intracellular consumption of low DP-fructans by BB-12 (either present in starting product or released upon fermentation by indigenous microbes), thereby enhancing effects of the co-administered fructan

Influence of probiotic bacteria on gut microbiota composition and gut wall function in an in-vitro model in patients with Parkinson's disease

We report here the potential role of a 4-strain probiotic suspension for use with patients with Parkinson's disease (PD). Stool samples from a group of three patients with diagnosed PD were used to create microbiotas in an in-vitro gut model. The effects of dosing with an oral probiotic suspension (Symprove) on bacterial composition and metabolic activity in the microbiotas was evaluated over 48 h and compared with healthy controls. Additionally, the effect of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. In general, the relative proportions of the main bacterial phyla in the microbiotas of PD patients differed from those of healthy subjects, with levels of Firmicutes raised and levels of Bacteroidetes reduced. Dosing with probiotic resulted in a change in bacterial composition in the microbiotas over a 48 h period. Several other indicators of gut health changed upon dosing with the probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased and levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased. Tight junction integrity was seen to improve with probiotic dosing and wound healing was seen to occur faster than a control. The data suggest that if development and/or progression of PD is influenced by gut microbiota dysbiosis then supplementation of the diet with a properly formulated probiotic may be a useful adjunct to standard treatment in clinic

Poly(D,l-lactide-co-glycolide) particles are metabolised by the gut microbiome and elevate short chain fatty acids

© 2024 The Authors. Published by Elsevier B.V. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/The production of short chain fatty acids (SCFAs) by the colonic microbiome has numerous benefits for human health, including maintenance of epithelial barrier function, suppression of colitis, and protection against carcinogenesis. Despite the therapeutic potential, there is currently no optimal approach for elevating the colonic microbiome's synthesis of SCFAs. In this study, poly(D,l-lactide-co-glycolide) (PLGA) was investigated for this application, as it was hypothesised that the colonic microbiota would metabolise PLGA to its lactate monomers, which would promote the resident microbiota's synthesis of SCFAs. Two grades of spray dried PLGA, alongside a lactate bolus control, were screened in an advanced model of the human colon, known as the M-SHIME® system. Whilst the high molecular weight (Mw) grade of PLGA was stable in the presence of the microbiota sourced from three healthy humans, the low Mw PLGA (PLGA 2) was found to be metabolised. This microbial degradation led to sustained release of lactate over 48 h and increased concentrations of the SCFAs propionate and butyrate. Further, microbial synthesis of harmful ammonium was significantly reduced compared to untreated controls. Interestingly, both types of PLGA were found to influence the composition of the luminal and mucosal microbiota in a donor-specific manner. An in vitro model of an inflamed colonic epithelium also showed the polymer to affect the expression of pro- and anti-inflammatory markers, such as interleukins 8 and 10. The findings of this study reveal PLGA's sensitivity to enzymatic metabolism in the gut, which could be harnessed for therapeutic elevation of colonic SCFAs.Peer reviewe

Ex vivo colonic fermentation of NUTRIOSE® exerts immuno-modulatory properties and strong anti-inflammatory effects

NUTRIOSE (R) (Roquette, Lestrem, France) is a resistant dextrin with well-established prebiotic effects. This study evaluated the indirect effects of pre-digested NUTRIOSE (R) on host immune response and gut barrier integrity. Fecal samples from eight healthy donors were inoculated in a Colon-on-a-plate (R) system (ProDigest, Ghent, Belgium) with or without NUTRIOSE (R) supplementation. Following 48 h fermentation, colonic suspensions were tested in a Caco-2/THP1-Blue (TM) 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 (R) system were measured. Across all donors, immune-mediated intestinal barrier damage was significantly reduced with NUTRIOSE (R)-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 (R)-supplemented colonic suspensions versus blank in the co-culture models following lipopolysaccharide stimulation. These beneficial effects of NUTRIOSE (R) supplementation were likely due to increased acetate and propionate levels and the enrichment of SCFA-producing bacteria. NUTRIOSE (R) 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

Efficacy of Rhizobacteria on plant growth promotion and disease suppression in vitro

This research examines the effect of selected rhizobacteria for pathogen antagonistic and plant growth promotion activities in potato. A total of 22 rhizobacterial isolates collected from potato fields were tested in vitro for antagonism against R. solani and also for their plant growth promotion, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase production, phosphate solubilization ability and pathogen suppression activities in potato. Of 22 isolates, 11 were identified as Pseudomonas spp., 10 were identified as Bacillus spp. and one as an Enterobacter sp. All 22 isolates showed antagonistic activity on plate assays against R. solani ranging from 26.9 to 53.4% while the positive controls namely, Bacillus subtilis FZB24® WG and Pseudomonas fluorescens CHAO suppressed the growth of R. solani by 21.3 and 19.8%. Four isolates (18%) positively produced the phytohormone IAA, 17 (77%) had ACC deaminase activity and 19 (86%) solubilized phosphorous. Bacteria were examined for their plant growth promotion and disease suppression effects in in vitro potato after one week bacterization followed by three weeks of pathogen challenge. Over half of the rhizobacterial strains showed a significant effect on plant growth in vitro with a total plant fresh weight ranging from 1.44 to 2.04 g while the control, Bacillus subtilis FZB24® WG and Pseudomonas fluorescens CHAO had a fresh weight of 1.34, 1.62 and 1.51 g, respectively. Disease suppression activity was measured in terms of plant weight which ranged from 1.10 to 1.76 g while the control, Bacillus subtilis FZB24® WG and Pseudomonas fluorescens CHAO had 1.15, 1.33 and 1.29 g, respectively. These preliminary results suggest that the rhizobacteria studied have potential as biocontrol agents