scholarly journals Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Julia Isenring ◽  
Annelies Geirnaert ◽  
Christophe Lacroix ◽  
Marc J. A. Stevens
Keyword(s):  
Gut Microbiota ◽  
Fold Change ◽  
Strain History ◽  
Sequencing Analysis ◽  
Nutritive Medium ◽  
Gut Colonization ◽  
In The Wild ◽  
Probiotic Strains ◽  
Adhesin Protein

Abstract Background Auto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota. Results L. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1–4.4 log2-fold-change and LP_RS05230 (marR) at 4.5–5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype. Conclusions L. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

2012 ◽  
Vol 57 (1) ◽  
pp. 436-444 ◽  
Author(s):  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Izuru Ando ◽  
Kunihiro Hirahara ◽  
Tsutomu Shibata ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Resistance ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
Resistance Mutations ◽  
In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

In vitro fermentation of Cucumis sativus fructus extract by canine gut microbiota in combination with two probiotic strains

2019 ◽  
Vol 63 ◽  
pp. 103585
Author(s):  
Benedetta Belà ◽  
Maria Magdalena Coman ◽  
Maria Cristina Verdenelli ◽  
Cinzia Bianchi ◽  
Giulia Pignataro ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Cucumis Sativus ◽  
In Vitro Fermentation ◽  
Probiotic Strains

scholarly journals Efficient Protection of Probiotics for Delivery to the Intestinal tract by Cellulose Sulphate Encapsulation

2020 ◽  
Author(s):  
Walter Henry Gunzburg ◽  
Myo Myint Aung ◽  
Pauline Toa ◽  
Shirelle Ng ◽  
Eliot Read ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Cell Encapsulation ◽  
Stomach Acid ◽  
Intestinal Delivery ◽  
Subsequent Exposure ◽  
Probiotic Strains ◽  
A Cell ◽  
Cellulose Sulphate ◽  
Micro Organisms

Abstract Background: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic micro-organisms can improve health. However, orally delivered probiotic micro-organisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic micro-organisms are still not effective enough.Results : We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS) that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for up to 4 hours without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by levels of cellulase found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract.Conclusion: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeast from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

scholarly journals Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Walter H. Gunzburg ◽  
Myo Myint Aung ◽  
Pauline Toa ◽  
Shirelle Ng ◽  
Eliot Read ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Intestinal Tract ◽  
Cell Encapsulation ◽  
Stomach Acid ◽  
Intestinal Delivery ◽  
Subsequent Exposure ◽  
Probiotic Strains ◽  
A Cell ◽  
Cellulose Sulphate

Abstract Background Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. Results We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. Conclusion Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

scholarly journals Efficient Protection of Microorganisms for Delivery to the Intestinal tract by Cellulose Sulphate Encapsulation

2020 ◽  
Author(s):  
Walter Henry Gunzburg ◽  
Myo Myint Aung ◽  
Pauline Toa ◽  
Shirelle Ng ◽  
Eliot Read ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Intestinal Tract ◽  
Cell Encapsulation ◽  
Stomach Acid ◽  
Intestinal Delivery ◽  
Subsequent Exposure ◽  
Probiotic Strains ◽  
A Cell ◽  
Cellulose Sulphate

Abstract Background Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough.Results We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS) that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for up to 4 hours without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase in levels found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract.Conclusion Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

scholarly journals In Vitro Gut Modeling as a Tool for Adaptive Evolutionary Engineering of Lactiplantibacillus plantarum

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Julia Isenring ◽  
Annelies Geirnaert ◽  
Alex R. Hall ◽  
Christoph Jans ◽  
Christophe Lacroix ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Model Organism ◽  
Strain Improvement ◽  
Evolutionary Engineering ◽  
Bacterial Strains ◽  
Content Type ◽  
Colonic Microbiota ◽  
Gut Colonization ◽  
Fermentation Model

ABSTRACT Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor’s gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization under colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence. IMPORTANCE Improvement of bacterial strains in regard to specific abiotic environmental factors is broadly used to enhance strain characteristics for processing and product quality. However, there is currently no multidimensional probiotic strain improvement approach for both abiotic and biotic factors of a colon microbiota. The continuous PolyFermS fermentation model allows stable and reproducible continuous cultivation of colonic microbiota and provides conditions akin to the host gut with high control and easy sampling. This study investigated the suitability of PolyFermS for adaptive evolutionary engineering of a probiotic model organism for lactobacilli, Lactiplantibacillus plantarum, to an adult human colonic microbiota. The application of PolyFermS controlled gut microbiota environment led to adaptive evolution of L. plantarum strains for enhanced gut colonization characteristics. This novel tool for strain improvement can be used to reveal relevant factors involved in gut microbiota colonization and develop adapted probiotic strains with improved functionality in the gut.

scholarly journals Multiple Selection Criteria for Probiotic Strains with High Potential for Obesity Management

Nutrients ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 713
Author(s):  
Jeanne Alard ◽  
Benoit Cudennec ◽  
Denise Boutillier ◽  
Véronique Peucelle ◽  
Amandine Descat ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Leptin Receptor ◽  
Body Weight Gain ◽  
Bifidobacterium Longum ◽  
Protective Effects ◽  
Metabolic Dysfunction ◽  
Diet Induced Obesity ◽  
Probiotic Strains

Since alterations of the gut microbiota have been shown to play a major role in obesity, probiotics have attracted attention. Our aim was to identify probiotic candidates for the management of obesity using a combination of in vitro and in vivo approaches. We evaluated in vitro the ability of 23 strains to limit lipid accumulation in adipocytes and to enhance the secretion of satiety-promoting gut peptide in enteroendocrine cells. Following the in vitro screening, selected strains were further investigated in vivo, single, or as mixtures, using a murine model of diet-induced obesity. Strain Bifidobacterium longum PI10 administrated alone and the mixture of B. animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 limited body weight gain and reduced obesity-associated metabolic dysfunction and inflammation. These protective effects were associated with changes in the hypothalamic gene expression of leptin and leptin receptor as well as with changes in the composition of gut microbiota and the profile of bile acids. This study provides crucial clues to identify new potential probiotics as effective therapeutic approaches in the management of obesity, while also providing some insights into their mechanisms of action.

scholarly journals Unexpected consequences of administering bacteriocinogenic probiotic strains for Salmonella populations, revealed by an in vitro colonic model of the child gut

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3342-3353 ◽  
Author(s):  
Annina Zihler ◽  
Mélanie Gagnon ◽  
Christophe Chassard ◽  
Anita Hegland ◽  
Marc J. A. Stevens ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Continuous Fermentation ◽  
Distal Colon ◽  
Proximal Colon ◽  
Second Phase ◽  
E Coli ◽  
Probiotic Strains ◽  
High Level ◽  
Microcin B17

New biological strategies for the treatment of Salmonella infection are needed in response to the increase in antibiotic-resistant strains. Escherichia coli L1000 and Bifidobacterium thermophilum RBL67 were previously shown to produce antimicrobial proteinaceous compounds (microcin B17 and thermophilicin B67, respectively) active in vitro against a panel of Salmonella strains recently isolated from clinical cases in Switzerland. In this study, two three-stage intestinal continuous fermentation models of Salmonella colonization inoculated with immobilized faeces of a two-year-old child were implemented to study the effects of the two bacteriocinogenic strains compared with a bacteriocin-negative mutant of strain L1000 on Salmonella growth, as well as gut microbiota composition and metabolic activity. Immobilized E. coli L1000 added to the proximal colon reactor showed a low colonization, and developed preferentially in the distal colon reactor independent of the presence of genetic determinants for microcin B17 production. Surprisingly, E. coli L1000 addition strongly stimulated Salmonella growth in all three reactors. In contrast, B. thermophilum RBL67 added in a second phase stabilized at high levels in all reactors, but could not inhibit Salmonella already present at a high level (>107 c.f.u. ml−1) when the probiotic was added. Inulin added at the end of fermentation induced a strong bifidogenic effect in all three colon reactors and a significant increase of Salmonella counts in the distal colon reactor. Our data show that under the simulated child colonic conditions, the microcin B17 production phenotype does not correlate with inhibition of Salmonella but leads to a better colonization of E. coli L1000 in the distal colon reactor. We conclude that in vitro models with complex and complete gut microbiota are required to accurately assess the potential and efficacy of probiotics with respect to Salmonella colonization in the gut.

scholarly journals In vitro gut modeling as a tool for adaptive evolutionary engineering of Lactiplantibacillus plantarum

2020 ◽  
Author(s):  
Julia Isenring ◽  
Annelies Geirnaert ◽  
Alex Hall ◽  
Christoph Jans ◽  
Christophe Lacroix ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Model Organism ◽  
Strain Improvement ◽  
Probiotic Strain ◽  
Evolutionary Engineering ◽  
Bacterial Strains ◽  
Colonic Microbiota ◽  
Gut Colonization ◽  
Fermentation Model

AbstractResearch and marketing of probiotics demand for holistic strain improvement considering both, the biotic and abiotic gut environment. Here we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donors gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205 and intergenic region LP_RS05100<LP_RS05095 were found in recovered strains from different adaptation experiments. Combined addition of the reference strain NZ3400 and each of those mutants to the gut microbiota resulted in increased abundance of the corresponding mutant in PolyFermS microbiota after 10 days, showing the beneficial nature of these mutations. Our data show that the PolyFermS system is a suitable technology to generate adapted mutants for colonization in colonic conditions. Analysis thereof will provide knowledge about factors involved in gut microbiota colonization and persistence.ImportanceImprovement of bacterial strains to specific abiotic environmental factors is broadly used to enhance strain characteristics for processing and product quality. However, there is currently no multidimensional probiotic strain improvement approach for both, abiotic and biotic factors of a colon microbiota. The continuous PolyFermS fermentation model allows stable and reproducible continuous cultivation of colonic microbiota and provides conditions akin the host gut with high control and easy sampling. This study investigated the suitability of PolyFermS for adaptive evolutionary engineering of a probiotic model organism for lactobacilli, Lactiplantibacillus plantarum, to an adult human colonic microbiota. The application of PolyFermS controlled gut microbiota environment led to adaptive evolution of L. plantarum strains for enhanced gut colonization characteristics. This novel tool for strain improvement can be used to reveal relevant factors involved in gut microbiota colonization and develop adapted probiotic strains with enhanced functionality in the gut.

scholarly journals Escherichia coli Exopolysaccharides Induced by Ceftriaxone Regulated Human Gut Microbiota in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Baiyuan Li ◽  
Huahai Chen ◽  
Linyan Cao ◽  
Yunfei Hu ◽  
Dan Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gut Microbiota ◽  
Human Health ◽  
Molar Ratio ◽  
Composition Analysis ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Human Gut ◽  
Human Gut Microbiota

A stable intestinal microflora is an essential prerequisite for human health. This study investigated the interaction between Escherichia coli exopolysaccharides (named EPS-m2) and the human gut microbiota (HGM) in vitro. The EPS-m2 was produced by E. coli WM3064 when treated with ceftriaxone. The monosaccharide composition analysis revealed that EPS-m2 is composed of glucuronic acid, glucose, fucose, galactose/N-acetyl glucosamine, arabinose, xylose, and ribose with a molar ratio of approximately 77:44:29:28:2:1:1. The carbohydrates, protein, and uronic acids contents in EPS-m2 was 78.6 ± 0.1%, 4.38 ± 0.11%, and 3.86 ± 0.09%, respectively. In vitro batch fermentation experiments showed that 77% of EPS-m2 could be degraded by human fecal microbiota after 72 h of fermentation. In reverse, 16S rRNA gene sequencing analysis showed that EPS-m2 increased the abundance of Alistipes, Acinetobacter, Alloprevotella, Howardella, and Oxalobacter; GC detection illustrated that EPS-m2 enhanced the production of SCFAs. These findings indicated that EPS-m2 supplementation could regulate the HGM and might facilitate modulation of human health.

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