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.

Droplet digital PCR improves absolute quantification of viable lactic acid bacteria in faecal samples.

Analysing correlations between the observed health effects of ingested probiotics and their survival in digestive tract allows adapting their preparations for food. Tracking ingested probiotic in faecal samples requires accurate and specific tools to quantify live vs dead cells at strain level. Traditional culture-based methods are simpler to use but they do not allow quantifying viable but non-cultivable (VBNC) cells and they are poorly discriminant below the species level. We have set up a viable PCR (vPCR) assay combining propidium monoazide (PMA) treatment and either real time quantitative PCR (qPCR) or droplet digital PCR (ddPCR) to quantify a Lactobacillus rhamnosus and two Lactobacillus paracasei subsp. paracasei strains in piglet faeces. Adjustments of the PMA treatment conditions and reduction of the faecal sample size were necessary to obtain accurate discrimination between dead and live cells. The study also revealed differences of PMA efficiency among the two L. paracasei strains. Both PCR methods were able to specifically quantify each strain and provided comparable total bacterial counts. However, quantification of lower numbers of viable cells was best achieved with ddPCR, which was characterized by a reduced lower limit of quantification (improvement of up to 1.76 log10 compared to qPCR). All three strains were able to survive in the piglets' gut with viability losses between 0.78 and 1.59 log10/g faeces. This study shows the applicability of PMA-ddPCR to specific quantification of small numbers of viable bacterial cells in the presence of an important background of unwanted microorganisms, and without the need to set up standard curves. It also illustrates the need to adapt PMA protocols according to the final matrix and target strain, even for closely related strains. The PMA-ddPCR approach provides a new tool to quantify bacterial survival in faecal samples from a preclinical and clinical trial.

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

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.

Novel method based on chromogenic media for discrimination and selective enumeration of lactic acid bacteria in fermented milk products.

Microbial analyses of fermented milk products require selective methods to discriminate between close species simultaneously present in high amounts. A culture-based method combining novel chromogenic agar media and appropriate incubation conditions was developed to enumerate lactic acid bacteria (LAB) strains in fermented milk. M1 agar, containing two chromogenic substrates, allowed selective enumeration of Lactobacillus rhamnosus, two strains of Lactobacillus paracasei subsp. paracasei and Streptococcus salivarius subsp. thermophilus based on differential ß-galactosidase and ß-glucosidase activities. Depending on the presence of some or all of the above strains, M1 agar was supplemented with L-rhamnose or vancomycin and incubations were carried out at 37 °C or 44 °C to increase selectivity. A second agar medium, M2, containing one chromogenic substrates was used to selectively enumerate ß-galactosidase producing Lactobacillus delbrueckii subsp. bulgaricus at 47 °C. By contrast with the usual culture media, the chromogenic method allowed unambiguous enumeration of each species, including discrimination between the two L. paracasei, up to 10(9) CFU/g of fermented milk. In addition, the relevance of the method was approved by enumerating reference ATCC strains in pure cultures and fermented milk product. The method could also be used for enumerations on non-Danone commercial fermented milk products containing strains different from those used in this study, showing versatility of the method. To our knowledge, this is the first description of a chromogenic culture method applied to selective enumeration of LAB.

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.