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

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

Shannon diversity index of fecal samples and reactors of PolyFermS models.

<p>The Shannon index was assessed in fecal donors 2 and 3, all reactors of model 2 and reactors IR, CR, TR3 and TR4 of model 3 of three last days at the end of stabilization phase. A higher Shannon index reflects a more diverse community (in abundance and evenness).</p

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)

Daily mean SCFA concentrations in fermentation effluents of IR of PolyFermS models measured by HPLC.

<p>Initial stabilization: stabilization period in continuous mode to reach pseudo steady-state.; stabilized operation mode: continuous operation mode during pseudo steady-state conditions. <b>(A)</b> Model 2 and <b>(B)</b> model 3; (♦) acetate, (●) butyrate, and (■) propionate.</p

Correlations between genus-level phylogenetic groups and metabolites (SCFA, BCFA) of three last days at the end of stabilization period of model 2.

<p>The correlations, assessed by Spearman are indicated by either red (positive) and blue (negative), the significant correlations (q < 0.05) are indicated by ‘+’. Only genus related phylotypes > 0.1% and with at least one significant correlation with metabolites are depicted. Parentheses indicate an unknown genus belonging to a family or order.</p

Metabolites concentration (mM) and ratios (%) measured by HPLC in effluent samples of models’ reactors at the end of the stabilization period.

<p>PC, proximal colon reactor; DC, distal colon reactor; IR, inoculum reactor; CR, control reactor; TR, test reactor</p><p>Data are means ± SD of three last days at the end of the stabilization period; samples were analyzed in duplicate. ND, not detected</p><p>Values with different letters are significantly different within one model (P < 0.05)</p><p>Metabolites concentration (mM) and ratios (%) measured by HPLC in effluent samples of models’ reactors at the end of the stabilization period.</p

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

qPCR enumeration of bacterial groups in fecal inocula and effluent samples of models’ reactors at the end of the stabilization period.

<p>PC, proximal colon reactor; DC, distal colon reactor; IR, inoculum reactor; CR, control reactor; TR, test reactor; ND, not detected</p><p>¹Data are mean log₁₀ copies 16S rRNA gene g^-1 feces; samples were analyzed in duplicate.</p><p>²Data are mean log₁₀ copies 16S rRNA gene g^-1 fermentation effluent ± SD of three last days at the end of the stabilization period; samples were analyzed in duplicate. Values with different letters are significantly different within one model (P < 0.05)</p><p>qPCR enumeration of bacterial groups in fecal inocula and effluent samples of models’ reactors at the end of the stabilization period.</p