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Isolation and Characterization of Human Intestinal Bacteria Cytobacillus oceanisediminis NB2 for Probiotic Potential

ABSTRACT: Systemic characterization of the human gut microbiota highlighted its vast therapeutic potential. Despite having enormous potential, the non-availability of their culture representatives created a bottleneck to understand the concept of microbiome-based therapeutics. The present study is aimed to isolate and evaluate the probiotic potential of a human gut isolate. Physiochemical, morphological, and phylogenetic characterization of a human gut isolate identifies it as a rod-shaped gram-negative microbe taxonomically affiliated with the Cytobacillus genus, having an optimal growth at 37°C in a partially alkaline environment (pH 8.0). This human gut isolate showed continuous growth in the presence of salts (up to 7% NaCl and 10% KCl), antibiotics, metals and metalloids [silver nitrate (up to 2 mM); lead acetate (up to 2 mM); sodium arsenate (up to 10 mM); potassium dichromate (up to 2 mM)], gastric and intestinal conditions, diverse temperature (25–50°C), and pH (5–9) conditions making it fit to survive in the highly variable gut environment. Genomic characterization identified the presence of gene clusters for diverse bio-catalytic activity, stress response, and antimicrobial activity, as well as it indicated the absence of pathogenic gene islands. A combination of functional features like anti-amylase, anti-lipase, glutenase, prolyl endopeptidase, lactase, bile salt hydrolase, cholesterol oxidase, and anti-pathogenic activity is indicative of its probiotic potential in various disorders. This was further substantiated by the CaCo-2 cell line assay confirming its cellular adherence and biosafety. Conclusively, human gut isolate possessed significant probiotic potential that can be used to promote animal and human health.

SUBMITTER: Yadav M

PROVIDER: S-EPMC9326467 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: In the last decade, much attention has been drawn to probiotic bacteria in the context of inflammatory bowel disease (IBD), since the potential of certain strains to attenuate inflammation was demonstrated in several animal experiments and clinical studies. Data in humans elucidating the molecular mechanism of probiotic action are still scarce. To this end, we used an organ culture system of human colon mucosa and investigated the gene expression profiles after treatment with different probiotic bacteria in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)) stimulated samples using whole genome microarrays. Moreover, we analyzed changes occurring in the intestinal explants cultured for 8 hours when compared to fresh, directly frozen mucosa, in order to infer the suitability of the system to study an inflammatory stimulus and likely antiinflammatory responses. Results: Culturing intestinal colon fragments during 8 hours elicited differential gene expression in 283 genes, 229 upregulated and 54 downregulated. Upregulated genes were predominantly related to apoptosis, whereas downregulated genes encoded mitochondrial proteins. No specific enrichment of genes related to inflammation or immune response could be detected, confirming the suitability of the system to further study the inmunomodulatory/anti-inflammatory properties of Lactobacillus casei BL23 (BL23), L.plantarum 299v (LP299v) and L.plantarum 299v (A-) (LP299v (A-)), a mutant strain with reduced adhesive properties to enterocytes. Intestinal explants were stimulated with PMA/IO for 3 hours and subsequently incubated with probiotic bacteria for 4 h. ANOVA analysis (p ≤ 0,01) revealed 205 differentially expressed genes between Control, PMA/IO (Inflamed), and the 3 bacterial treatments. Most importantly, a number of PMA/IO induced genes related to immune response and immune system process such as IL-2, IFN-γ, IL17A and pro-inflammatory cytokines CXCL9 and CXCL11 were downregulated by BL23, LP299v and LP299v (A-). The behaviour of the three Lactobacillus strains was quite similar, although their presence induced differential expression of a small number of genes in a strain dependent manner. Conclusion: The human colon organ culture was found to be a suitable model for the study of inflammatory/anti-inflammatory stimuli, and therefore it constitutes a valuable tool to determine the inmunomodulatory effect of probiotic bacteria. The global transcriptional profile evoked by strains BL23, LP299v and LP299v (A-) in artificially inflamed tissue indicated a clear homeostasis restoring effect, including a decrease of the signals produced by activated T cells.

Project description:Background: In the last decade, much attention has been drawn to probiotic bacteria in the context of inflammatory bowel disease (IBD), since the potential of certain strains to attenuate inflammation was demonstrated in several animal experiments and clinical studies. Data in humans elucidating the molecular mechanism of probiotic action are still scarce. To this end, we used an organ culture system of human colon mucosa and investigated the gene expression profiles after treatment with different probiotic bacteria in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)) stimulated samples using whole genome microarrays. Moreover, we analyzed changes occurring in the intestinal explants cultured for 8 hours when compared to fresh, directly frozen mucosa, in order to infer the suitability of the system to study an inflammatory stimulus and likely antiinflammatory responses. Results: Culturing intestinal colon fragments during 8 hours elicited differential gene expression in 283 genes, 229 upregulated and 54 downregulated. Upregulated genes were predominantly related to apoptosis, whereas downregulated genes encoded mitochondrial proteins. No specific enrichment of genes related to inflammation or immune response could be detected, confirming the suitability of the system to further study the inmunomodulatory/anti-inflammatory properties of Lactobacillus casei BL23 (BL23), L.plantarum 299v (LP299v) and L.plantarum 299v (A-) (LP299v (A-)), a mutant strain with reduced adhesive properties to enterocytes. Intestinal explants were stimulated with PMA/IO for 3 hours and subsequently incubated with probiotic bacteria for 4 h. ANOVA analysis (p ? 0,01) revealed 205 differentially expressed genes between Control, PMA/IO (Inflamed), and the 3 bacterial treatments. Most importantly, a number of PMA/IO induced genes related to immune response and immune system process such as IL-2, IFN-?, IL17A and pro-inflammatory cytokines CXCL9 and CXCL11 were downregulated by BL23, LP299v and LP299v (A-). The behaviour of the three Lactobacillus strains was quite similar, although their presence induced differential expression of a small number of genes in a strain dependent manner. Conclusion: The human colon organ culture was found to be a suitable model for the study of inflammatory/anti-inflammatory stimuli, and therefore it constitutes a valuable tool to determine the inmunomodulatory effect of probiotic bacteria. The global transcriptional profile evoked by strains BL23, LP299v and LP299v (A-) in artificially inflamed tissue indicated a clear homeostasis restoring effect, including a decrease of the signals produced by activated T cells. Macroscopically healthy colonic intestinal tissue was obtained at surgery from 3 patients. Intestinal explants were treated with PMA and ionomycin for 3 h to induce pro-inflammatory conditions. Then, culture medium was changed and replaced with either medium or medium containing either Lactobacillus casei BL23, Lactobacillus plantarum 299v, or a nonadherent mutant of L. plantarum 299v (A-) and incubated for further 4 hours. In parallel, control intestinal explants were cultured without any treatment of PMA/ionomycin or probiotic bacteria and compared to directly frozen tissue in order to evaluate changes in gene expression which are due solely to the culture conditions.

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Isolation and Characterization of Human Intestinal Bacteria Cytobacillus oceanisediminis NB2 for Probiotic Potential

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