Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models

<p>Abstract</p> <p>Background</p> <p>Accurate assessment of probiotics with targeted anti-<it>Salmonella </it>activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original <it>in vitro </it>intestinal models closely mimicking the complex <it>in vivo </it>conditions of the large intestine. Effluents from continuous <it>in vitro </it>three-stage fermentation colonic models of <it>Salmonella </it>Typhimurium infection inoculated with immobilized child microbiota and <it>Salmonella </it>were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of <it>Salmonella</it>, addition of two bacteriocinogenic strains, <it>Bifidobacterium thermophilum </it>RBL67 (thermophilicin B67) and <it>Escherichia coli </it>L1000 (microcin B17), and inulin were tested on <it>Salmonella </it>growth and interactions with epithelial cell layers. <it>Salmonella </it>adhesion and invasion were investigated and epithelial integrity assessed by transepithelial electrical resistance (TER) measurements and confocal microscopy observation. Data from complex effluents were compared with pure <it>Salmonella </it>cultures.</p> <p>Results</p> <p><it>Salmonella </it>in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log₁₀cfu/ml effluent. Invasion of cell-associated <it>Salmonella </it>was up to 50-fold lower in complex reactor samples compared to pure <it>Salmonella </it>cultures. It further depended on environmental factors, with 0.2 ± 0.1% being measured with proximal, 0.6 ± 0.2% with transverse and 1.3 ± 0.7% with distal reactor effluents, accompanied by a similar high decrease of TER across cell monolayers (minus 45%) and disruption of tight junctions. Subsequent addition of <it>E. coli </it>L1000 stimulated <it>Salmonella </it>growth (6.4 ± 0.6 log₁₀cfu/ml effluent of all 3 reactors) and further decreased TER, but led to 10-fold decreased invasion efficiency when tested with distal reactor samples. In contrast, presence of <it>B. thermophilum </it>RBL67 revealed a protective effect on epithelial integrity compared to previous <it>E. coli </it>L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced <it>Salmonella </it>growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors.</p> <p>Conclusions</p> <p>Our results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during <it>Salmonella </it>infection, providing an efficient system biology tool for preclinical development of new antimicrobials.</p

Set up of a new in vitro model to study dietary fructans fermentation in formula-fed babies

A new in vitro fermentation model with immobilised infant faecal microbiota simulating the proximal colon of a formula-fed baby was developed and used to test the effects of known prebiotic fructans. Intestinal fermentation, based on a previously developed colonic fermentation model, using a new feeding medium simulating a formula-fed infant ileal chyme, was carried out for seventy-one consecutive days divided into four stabilisation periods intercalated with four prebiotic treatment periods. At the end of the first stabilisation period, total bacterial concentration in colonised beads and in faecal sample was similar, metabolite concentrations returned to stabilisation values after each treatment period. As expected, the four prebiotic treatments significantly increased the bifidobacterial populations, whereas they decreased bacteroides and clostridia. No difference was observed in the prebiotic effect of these substrates selected. The treatments significantly increased total production of SCFA and decreased ammonia compared to stabilisation periods. Long-term stability of the system together with the reproducibility of the known prebiotic effects highlights the potential of the present model to quantify and compare the effects of different substrates in a formula-fed infant microbiota within the same fermentation experimen

The composition and metabolic activity of child gut microbiota demonstrate differential adaptation to varied nutrient loads in an in vitro model of colonic fermentation

The extent to which the dietary loads of simple sugars, carbohydrates, protein, and fiber impact colonic fermentation in children is unknown. This study assessed the impact of dietary energy on gut microbial communities and metabolism using a three-stage in vitro continuous fermentation model. Two separate models, replicating the proximal, transverse, and distal colon regions, were inoculated with immobilized fecal microbiota from one of two female children. Three different fermentation media were designed to examine the effects of prevalent Western dietary trends on gut microbiota. Media compositions reflected obese (high energy), normal weight (normal energy), and anorectic (low energy) child dietary intakes and were alternately supplied to each microbiota during separate fermentation periods. Gut microbiota demonstrated differential metabolic and compositional adaptation to varied substrate availability. High energy medium was strongly butyrogenic, resulting in significant stimulation of butyrate-producing members of clostridia cluster XIVa, whereas members of cluster IV demonstrated greater adaptive variability. Normal and low energy nutrient loads induced significantly less metabolic activity in both microbiota, with low energy medium inducing a broad reorganization of the commensal community structure. These results suggest a concerted metabolic adaptation in response to nutrient load, exercised by different microbial populations, indicating substantial redundancy in gastrointestinal metabolic pathway

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

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

A proof of concept infant-microbiota associated rat model for studying the role of gut microbiota and alleviation potential of Cutibacterium avidum in infant colic

Establishing the relationship between gut microbiota and host health has become a main target of research in the last decade. Human gut microbiota-associated animal models represent one alternative to human research, allowing for intervention studies to investigate causality. Recent cohort and in vitro studies proposed an altered gut microbiota and lactate metabolism with excessive H$_{2}$ production as the main causes of infant colic. To evaluate H$_{2}$ production by infant gut microbiota and to test modulation of gut colonizer lactose- and lactate-utilizer non-H$_{2}$-producer, Cutibacterium avidum P279, we established and validated a gnotobiotic model using young germ-free rats inoculated with fecal slurries from infants younger than 3 months. Here, we show that infant microbiota-associated (IMA) rats inoculated with fresh feces from healthy (n = 2) and colic infants (n = 2) and fed infant formula acquired and maintained similar quantitative and qualitative fecal microbiota composition compared to the individual donor's profile. We observed that IMA rats excreted high levels of H$_{2}$, which were linked to a high abundance of lactate-utilizer H$_{2}$-producer Veillonella. Supplementation of C. avidum P279 to colic IMA rats reduced H$_{2}$ levels compared to animals receiving a placebo. Taken together, we report high H$_{2}$ production by infant gut microbiota, which might be a contributing factor for infant colic, and suggest the potential of C. avidum P279 in reducing the abdominal H$_{2}$ production, bloating, and pain associated with excessive crying in colic infants

EASIROC, an Easy & Versatile ReadOut Device for SiPM

AbstractEASIROC, standing for Extended Analogue Si-pm Integrated ReadOut Chip is a 32 channels fully analogue front end ASIC dedicated to readout SiPM detectors. This low power and highly versatile ASIC was developed from the chip SPIROC[1] which has been designed for the Analogue Hadronic Calorimeter foreseen at the International Linear Collider. EASIROC integrates a 4.5V range 8-bit DAC per channel for individual SIPM gain adjustment. A multiplexed charge measurement from 160 fC up to 320 pC is available thanks to 2 analogue outputs. These charge paths are made of 2 variable gain preamplifiers followed by 2 tuneable shapers and a track and hold. A trigger path integrates a fast shaper followed by a discriminator the threshold of which is set by an integrated 10-bit DAC. These 32 trigger outputs can be used for timing measurements. The power consumption is lower than 5 mW/channel and unused features can be powered OFF to decrease the power. The chip has been designed in AMS 0.35μm SiGe technology and 4000 dies have been produced in 2010. Its versatility allows its use in many photo detector experiments and is already used for PEBS, MuRAY, J-PARC and medical imaging

Stability of the Maternal Gut Microbiota During Late Pregnancy and Early Lactation

Scarce research has been performed to assess whether the human maternal gut microbiota undergoes changes during the perinatal period. Therefore, in the present study, gut microbiota composition of seven healthy mothers(to-be) was assessed at different time points during the perinatal period (i.e. weeks 3-7 prepartum and days 3-6, 9-14, and 25-30 postpartum) using quantitative polymerase chain reaction (qPCR) and pyrosequencing, and was complemented by short-chain fatty acids (SCFA) and calprotectin quantification using high-performance liquid chromatography and enzyme-linked immunosorbent assay, respectively. qPCR revealed the predominance of members of the Firmicutes, Bacteroides, and Bifidobacterium without detectable changes over the perinatal period. Pyrosequencing supported these data in terms of microbiota stability for any population at any taxonomic level, although ratios of members of the Actinobacteria and Bacteroidetes differed between the two methods. However, the number of operational taxonomic units observed by pyrosequencing was subjected to fluctuations and the relative abundance of Streptococcus decreased numerically postpartum (P=0.11), which may indicate that aberrancies in subdominant populations occur perinatally. Furthermore, total fecal SCFA concentrations, particularly the branched-chain fatty acids isobutyrate and isovalerate, were higher than for non-pregnant subjects throughout the perinatal period. This suggests metabolic changes and increased energy extraction via proteolytic, in addition to saccharolytic fermentation, accompanied by low-grade inflammation based on fecal calprotectin levels. Our data show that the maternal gut microbiota remained stable over the perinatal period despite altered metabolic activity and low-grade inflammation; however, it remains to be confirmed whether changes preceded earlier during pregnancy and succeeded later postpartum

Milk sialyllactose influences colitis in mice through selective intestinal bacterial colonization

The presence of particular oligosaccharides in mother’s milk influences bacterial colonization of the newborn mouse intestine and susceptibility to dextran sodium sulfate-driven colitis

Selective proliferation of intestinal Barnesiella under fucosyllactose supplementation in mice

The oligosaccharides 2-fucosyllactose and 3-fucosyllactose are major constituents of human breast milk but are not found in mouse milk. Milk oligosaccharides have a prebiotic action, thus affecting the colonisation of the infant intestine by microbiota. To determine the specific effect of fucosyllactose exposure on intestinal microbiota in mice, in the present study, we orally supplemented newborn mice with pure 2-fucosyllactose and 3-fucosyllactose. Exposure to 2-fucosyllactose and 3-fucosyllactose increased the levels of bacteria of the Porphyromonadaceae family in the intestinal gut, more precisely members of the genus Barnesiella as analysed by 16S pyrosequencing. The ability of Barnesiella to utilise fucosyllactose as energy source was confirmed in bacterial cultures. Whereas B. intestinihominis and B. viscericola did not grow on fucose alone, they proliferated in the presence of 2-fucosyllactose and 3-fucosyllactose following the secretion of linkage-specific fucosidase enzymes that liberated lactose. The change in the composition of intestinal microbiota mediated by fucosyllactose supplementation affected the susceptibility of mice to dextran sulphate sodium-induced colitis, as indicated by increased resistance of mice subjected to 2-fucosyllactose supplementation for 6 weeks. The present study underlines the ability of specific milk oligosaccharides to change the composition of intestinal microbiota and thereby to shape an intestinal milieu resilient to inflammatory disease