Clostridium difficile colonization and antibiotics response in PolyFermS continuous model mimicking elderly intestinal fermentation

Abstract Background Clostridium difficile (CD), a spore-forming and toxin-producing bacterium, is the main cause for antibiotic-associated diarrhea in the elderly. Here we investigated CD colonization in novel in vitro fermentation models inoculated with immobilized elderly fecal microbiota and the effects of antibiotic treatments. Methods Two continuous intestinal PolyFermS models inoculated with different immobilized elder microbiota were used to investigate selected factors of colonization of CD in proximal (PC, model 1) and transverse-distal (TDC, model 1 and 2) colon conditions. Colonization of two CD strains of different PCR ribotypes, inoculated as vegetative cells (ribotype 001, model 1) or spores (ribotypes 001 and 012, model 2), was tested. Treatments with two antibiotics, ceftriaxone (daily 150 mg L−1) known to induce CD infection in vivo or metronidazole (twice daily 333 mg L−1) commonly used to treat CD, were investigated in TDC conditions (model 2) for their effects on gut microbiota composition (qPCR, 16S pyrosequencing) and activity (HPLC), CD spore germination and colonization, and cytotoxin titer (Vero cell assay). Results CD remained undetected after inoculating vegetative cells in PC reactors of model 1, but was shown to colonize TDC reactors of both models, reaching copy numbers of up to log10 8 mL−1 effluent with stable production of toxin correlating with CD cell numbers. Ceftriaxone treatment in TDC reactors showed only small effects on microbiota composition and activity and did not promote CD colonization compared to antibiotic-free control reactor. In contrast, treatment with metronidazole after colonization of CD induced large modifications in the microbiota and decreased CD numbers below the detection limit of the specific qPCR. However, a fast CD recurrence was measured only 2 days after cessation of metronidazole treatment. Conclusions Using our in vitro fermentation models, we demonstrated that stable CD colonization in TDC reactors can be induced by inoculating CD vegetative cells or spores without the application of ceftriaxone. Treatment with metronidazole temporarily reduced the counts of CD, in agreement with CD infection recurrence in vivo. Our data demonstrate that CD colonized an undisturbed microbiota in vitro, in contrast to in vivo observations, thus suggesting an important contribution of host-related factors in the protection against CD infection

Low iron availability in continuous in vitro colonic fermentations induces strong dysbiosis of the child gut microbial consortium and a decrease in main metabolites

Iron (Fe) deficiency affects an estimated 2 billion people worldwide, and Fe supplements are a common corrective strategy. The impact of Fe deficiency and Fe supplementation on the complex microbial community of the child gut was studied using in vitro colonic fermentation models inoculated with immobilized fecal microbiota. Chyme media (all Fe chelated by 2,2′-dipyridyl to 26.5 mg Fe L−1) mimicking Fe deficiency and supplementation were continuously fermented. Fermentation effluent samples were analyzed daily on the microbial composition and metabolites by quantitative PCR, 16S rRNA gene 454-pyrosequencing, and HPLC. Low Fe conditions (1.56 mg Fe L−1) significantly decreased acetate concentrations, and subsequent Fe supplementation (26.5 mg Fe L−1) restored acetate production. High Fe following normal Fe conditions had no impact on the gut microbiota composition and metabolic activity. During very low Fe conditions (0.9 mg Fe L−1 or Fe chelated by 2,2′-dipyridyl), a decrease in Roseburia spp./Eubacterium rectale, Clostridium Cluster IV members and Bacteroides spp. was observed, while Lactobacillus spp. and Enterobacteriaceae increased consistent with a decrease in butyrate (−84%) and propionate (−55%). The strong dysbiosis of the gut microbiota together with decrease in main gut microbiota metabolites observed with very low iron conditions could weaken the barrier effect of the microbiota and negatively impact gut healt

In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota

Recently, the concept of prebiotics has been revisited to expand beyond non-digestible oligosaccharides, and the requirements for selective stimulation were extended to include microbial groups other than, and additional to, bifidobacteria and lactobacilli. Here, the gut microbiota-modulating effects of well-known and novel prebiotics were studied. An in vitro fermentation screening platform (i-screen) was inoculated with adult fecal microbiota, exposed to different dietary fibers that had a range of concentrations (inulin, alpha-linked galacto-oligosaccharides (alpha-GOS), beta-linked GOS, xylo-oligosaccharides (XOS) from corn cobs and high-fiber sugar cane, and beta-glucan from oats), and compared to a positive fructo-oligosaccharide (FOS) control and a negative control (no fiber addition). All dietary fibers displayed prebiotic activity, with beta-glucan showing more distinct effects on the microbial composition and metabolism compared to the other fibers. Beta-glucan induced the growth of Prevotella and Roseburia with a concomitant increase in propionate production. Inulin and both forms of GOS and XOS had a strong bifidogenic effect on the microbial composition. A dose-response effect was observed for butyrate when exposed to beta-glucan and inulin. The findings of this study support the potential for alpha-GOS, XOS, and oat beta-glucan to serve as novel prebiotics, due to their association with the positive shifts in microbiome composition and short-chain fatty acid production that point to potential health benefits

Low iron availability in continuous in vitro colonic fermentations induces strong dysbiosis of the child gut microbial consortium and a decrease in main metabolites

ISSN:0168-6496ISSN:1574-694

Design and Investigation of PolyFermS In Vitro Continuous Fermentation Models Inoculated with Immobilized Fecal Microbiota Mimicking the Elderly Colon

In vitro gut modeling is a useful approach to investigate some factors and mechanisms of the gut microbiota independent of the effects of the host. This study tested the use of immobilized fecal microbiota to develop different designs of continuous colonic fermentation models mimicking elderly gut fermentation. Model 1 was a three-stage fermentation mimicking the proximal, transverse and distal colon. Models 2 and 3 were based on the new PolyFermS platform composed of an inoculum reactor seeded with immobilized fecal microbiota and used to continuously inoculate with the same microbiota different second-stage reactors mounted in parallel. The main gut bacterial groups, microbial diversity and metabolite production were monitored in effluents of all reactors using quantitative PCR, 16S rRNA gene 454-pyrosequencing, and HPLC, respectively. In all models, a diverse microbiota resembling the one tested in donor’s fecal sample was established. Metabolic stability in inoculum reactors seeded with immobilized fecal microbiota was shown for operation times of up to 80 days. A high microbial and metabolic reproducibility was demonstrated for downstream control and experimental reactors of a PolyFermS model. The PolyFermS models tested here are particularly suited to investigate the effects of environmental factors, such as diet and drugs, in a controlled setting with the same microbiota source.ISSN:1932-620

In vitro Study of Lactobacillus paracasei CNCM I-1518 in Healthy and Clostridioides difficile Colonized Elderly Gut Microbiota

Consumption of probiotic bacteria can result in a transient colonization of the human gut and thereby in potential interactions with the commensal microbiota. In this study, we used novel PolyFermS continuous fermentation models to investigate interactions of the candidate probiotic strain Lactobacillus paracasei CNCM I-1518 (L. paracasei) with colonic microbiota from healthy elderly subjects using 16S rRNA gene amplicon sequencing and metatranscriptomics, or with microbiota in vitro-colonized with Clostridioides difficile (C. difficile NCTC 13307 and C. difficile DSM 1296)—an enteropathogen prevalent in the elderly population. Small changes in microbiota composition were detected upon daily addition of L. paracasei, including increased abundances of closely related genera Lactobacillus and Enterococcus, and of the butyrate producer Faecalibacterium. Microbiota gene expression was also modulated by L. paracasei with distinct response of the Faecalibacterium transcriptome and an increase in carbohydrate utilization. However, no inhibitory effect of L. paracasei was observed on C. difficile colonization in the intestinal models under the tested conditions. Our data suggest that, in the in vitro experimental conditions tested and independent of the host, L. paracasei has modulatory effects on both the composition and function of elderly gut microbiota without affecting C. difficile growth and toxin production

MOESM2 of Clostridium difficile colonization and antibiotics response in PolyFermS continuous model mimicking elderly intestinal fermentation

Additional file 2. Daily mean metabolites concentrations in ceftriaxone (=CRO) treated TR3 compared to CR during period A (CRO I) and B (CRO II)

Effects of colon-targeted vitamins on the composition and metabolic activity of the human gut microbiome– a pilot study

An increasing body of evidence has shown that gut microbiota imbalances are linked to diseases. Currently, the possibility of regulating gut microbiota to reverse these perturbations by developing novel therapeutic and preventive strategies is being extensively investigated. The modulatory effect of vitamins on the gut microbiome and related host health benefits remain largely unclear. We investigated the effects of colon-delivered vitamins A, B2, C, D, and E on the gut microbiota using a human clinical study and batch fermentation experiments, in combination with cell models for the assessment of barrier and immune functions. Vitamins C, B2, and D may modulate the human gut microbiome in terms of metabolic activity and bacterial composition. The most distinct effect was that of vitamin C, which significantly increased microbial alpha diversity and fecal short-chain fatty acids compared to the placebo. The remaining vitamins tested showed similar effects on microbial diversity, composition, and/or metabolic activity in vitro, but in varying degrees. Here, we showed that vitamins may modulate the human gut microbiome. Follow-up studies investigating targeted delivery of vitamins to the colon may help clarify the clinical significance of this novel concept for treating and preventing dysbiotic microbiota-related human diseases. Trial registration: ClinicalTrials.gov, NCT03668964. Registered 13 September 2018 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03668964

Microbial composition of fecal samples and reactors of PolyFermS models measured by 454 pyrosequencing.

<p>Relative abundance at <b>(A)</b> phylum level of fecal samples of donors 2 and 3, IR and DCI of model 2 and IR and CR of model 3 <b>(B)</b> family level and <b>(C)</b> genus level of fecal samples of donors 2 and 3, all reactors of model 2 and reactors IR, CR, TR3 and TR4 of model 3 identified by pyrosequencing of the V5-V6 hypervariable regions of the 16S rRNA gene. Effluent samples are average values of three last days at the end of the stabilization period. Parentheses indicate an unknown family belonging to an order or an unknown genus belonging to a family or order. Values < 1% are summarized in the group “others”.</p