Laboratory: MOLECULAR BIOMARKERS OF THE MICROBIOTA OF THE GASTROINTESTINAL TRACT

PERIMED

Head: Prof. Ilia ILIEV, PhD, Director of Centre of Technologies at Plovdiv University

The applying an interdisciplinary approach to scientific researches including the both areas of modern biological science and medicine, will contribute to obtaining of new knowledge in developing a strategy for individualization (personalization of prevention and treatment) in restoring and maintaining the balance of intestinal microbiota in human. All this will contribute to the prophylaxis and concomitant treatment of socially significant diseases such as metabolic syndrome, diabetes and colon cancer.

The results which are expected from the planned scientific researches in the work package will represent significant added value in three areas of modern biotechnology – for medicine and human health; food biotechnology to produce new raw-materials and healthy food and for the industry in the manufacture of new bio-active components.

The key fields in the study of the human microbiota are:

  • The microflora in the gastro-intestinal tract perform vital functions, including the processing of foods, digestion of a complex of indigestible polysaccharides and synthesis of vitamins;
  • The development of molecular techniques now allows the complete identification of individual microorganisms in sophisticated communities of the human microbiota in the context of the human health status;
  • Changes in the balance of the intestinal microflora have been identified in connection with a number of diseases of the liver and involved in the pathophysiology of non-alcoholic fatty liver, alcoholic liver disease. More specifically – bacterial overgrowth in the small intestine associated with cirrhosis, bacteremia, and hepatic encephalopathy;
  • The modulation of the microflora by exogenous factors such as antibiotics, is an established strategy in the management and prevention of a number of complications of chronic liver diseases;
  • The clinical evidences about the effects of probiotics, prebiotics and other bioactive substances on the balance of intestinal microbiota and the correlation with the prevention and treatment of liver diseases are scarce;
  • The relationship between the composition and quantity of the intestinal microbiota and the occurrence of the colon cancer is intensively investigated by checking of two hypotheses: 1 – the presence of raised number of specific bacteria species that are responsible for the initiation of colon cancer and 2 – differences in the balance between all the microorganisms of the intestinal microbiota, which are prerequisite for the start of the disease.

On many questions related to the importance of human microbiome for the health status remains to be answered:

  1. It is necessary to do a full analysis of microbiota using metagenomic sequencing of all the bacterial genes to identify microorganisms, defining human health or a disease condition.
  2. Is it possible by influencing the composition and quantity of microbiota to be able directly affect the disease?;
  3. It is essential by handling human microflora to be performed also a control over its metabolic capacity, as an innovative approach to the treatment and prevention of various diseases.

The scientific researches within the work package are directed to find answers to these questions. The main aim of the planned researches is a study of the correlation between the balance of the human intestinal microbiota and some social diseases such as diabetes, liver diseases, cancer of the colon and creation of a new concept for the prevention and treatment of these diseases by the application of a multidisciplinary approach to the researches and the principles of personalized medicine

Tasks:

  1. To examine the correlation between the composition and quantity of microorganisms in the gut microbiota on the one hand and diets in patients with the metabolic syndrome and diabetes;
  2. A study of fecal microbiota potential for non-invasive detection of colon cancer in patients in Bulgaria;
  3. To study the influence of probiotics and prebiotics on the balance of the microbiota and their enzyme activities responsible for the utilization of various carbohydrates;

To study the influence of probiotics and prebiotics on the balance of microbiota and the activity of enzymes responsible for the production of bioactive carcinogenic components (β-glucuronidase, β-glucosidase, azoreductase, nitroreductase) in patients with metabolic syndrome, diabetes and cancer of the large intestine.

The planned scientific researches are aimed to:

  • Creation of innovative science-based algorithm for exploration and evaluation of the composition and quantity of microorganisms in the intestinal microbiota in patients with metabolic syndrome and diabetes with the possibility of its application in other university centers in the country;
  • Developing of a model for a multidisciplinary approach in the scientific researches in the area of early diagnostics and prevention of colorectal carcinoma and the possibilities for prevention of undesired toxicity during the individualized therapy of patients;
  • Developing of a model for a multidisciplinary approach in the scientific researches and demonstration the possibilities for correlation of intestinal dysbiosis in patients with metabolic syndrome, diabetes and colon cancer by carrying out of concomitant application of probiotics and prebiotics;
  • Developing of a model for the individual approach to recovery of the microbial dysbiosis in patients with metabolic syndrome, diabetes, and colon cancer;
  • Developing of new biomarkers for early diagnostics of colorectal carcinoma with non-invasive methods for the analysis of fecal microbiota;
  • Developing of a series of symbiotic functional additives based on the metabolic features of the probiotic strains and functional characteristics of the prebiotics with specific structures which can be applied for the recovery of the intestinal microbiota in patients with metabolic syndrome, diabetes and colorectal carcinoma.

The team that will work on the proposed work package has over 15 years experience in the study of transferase enzymes production of oligosaccharides with prebiotic potential and is recognizable among the international scientific community. In the framework of several national and international projects within the department of “Biochemistry and Microbiology” at the Faculty of Biology of Plovdiv University is built and operated Center for the Study of prebiotics.

The summarized examples illustrate how the accumulated genomic data for microorganisms with proven practical significance can be exploited for the discovery of new enzymes with potential use for the production of new bio-materials with a wide range of applications, such as polysaccharides and oligosaccharide with specific linkages. The research in this direction – from fully sequenced genomes of lactic acid bacteria to identification of new genes and characterization of their products – are tasks whose solution would help build innovative knowledge and application possibilities.
The project results will provide the industry with innovative solutions for the development of biopharmaceutical biotechnology, including biotransformation and food biotechnology.

The attached achievements of the project can be used as fundamental in the development of technologies to create new biopolymer systems.

1. Molecular biological methods:

  • Isolation of total DNA from fecal samples and preparation for sequencing. The identification of microorganisms in the microbiota take place on the basis of two universal primers for bacteria – fluorescently labeled primer 27F [5′- (6FAM) AGAGTTTGATCCTGGCTCAG-3′] and unlabelled reverse primer 355R'(5′-GCTGCCTCCCGTAGGAGT-3′). Sequencing, bioinformatics analysis of data and their statistical processing is supported by the associated partner IATA-CSIC;
  • Bioinformatics analysis of genes in microorganisms of the intestinal microbiota encoding enzymes for utilization of carbohydrates, their transport through the cell membrane and carbohydrate metabolism;
  • Isolation, sequencing and cloning of the most important genes encoding enzymes with the highest activity against glycosides and oligosaccharides with different glycosidic linkages;
  • 16S rRNA sequencing of DNA samples from fecal samples using the primer of the V4 region na16S rRNA gene (F515 50-GTGCCAGCMGCCGCGGTAA-30, R806 50-GGACTACH VGGGTWT-CTAAT-30) for colorectal cancer.

2. Biochemical analysis:

  • Analysis of the activity of enzymes responsible for carbohydrate metabolism of gut microbiota and its individual strains.
  • Analysis of key enzymes of the main metabolic pathways of carbohydrates (glycosyltransferases, β-glucosidases, etc.) by spectrophotometric, luminometric methods of analysis.
  • Analysis of enzymes responsible for the production of bioactive carcinogenic components (β-glucuronidase, β-glucosidase azoreductases and nitroreductase).

3. Electrophoretic methods:

  • Standard methods for detection of PCR products and the protein profile of cellular proteins of the isolated microorganisms, and in situ assays and detection of specific enzymes of carbohydrate metabolism.

4. Chromatographic methods:

  • Separation and quantification of oligosaccharides and glycosides by HPLC on a C18 column, with mobile phase acetonitrile / water;
  • Analysis of metabolites, biologically active substances (BAS) during the cultivation of strains of bifidobacteria and lactobacilli on the media in the presence of glycosides and oligosaccharides.
  • Determining the concentrations of lactate, acetate, ethanol, residual amounts of oligosaccharides using the HPLC system and a gas chromatograph.

5. Microbiological methods:

  • Isolation of bifidobacterial strains from faecal samples of patients and initial screening by positive test for fructose 6 phosphate fructokinase;
  • An in vitro test for antagonistic activity of bifidobacteria strains against test microorganisms Acinetobacter calcoaceticus, Enterococcus faecalis, Salmonella thyphimurium, Sphingomonas paucimobilis, Staphylococcus lentus;
  • Phenotypic characteristics and kinetics of growth and optimization of the parameters of the cultivation in the presence of glycosides and oligosaccharides by using “Biolog” system;
  • Isolation and purification of β-glucosidase and phospho glucosidase by cells of lactobacilli – the destruction of the cell mass isolation of enzymes;
  • Study of the antimicrobial activity of the isolated and purified biologically active substances synthesized from lactobacilli, in the presence of glycosides and oligosaccharides.
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