Genome Features of Probiotic Bifidobacteria Determining Their Strain-Specific Properties.

The aim of the study was to analyze the genome features of the probiotic strains Bifidobacterium longum 379, Bifidobacterium bifidum 1, and Bifidobacterium bifidum 791 and study their antiviral activity. Materials and Methods: Whole genome sequencing of three strains of bifidobacteria was performed on the MiSeq platform (Illumina Inc., USA). The genomes were annotated using the Prokka v. 1.11 utility and RAST genomic server. The individual genetic determinants were searched using the ResFinder 3.2, PathogenFinder, PlasmidFinder, RAST, and Bagel 4 software. The antiviral activity of the strains against influenza A viruses was studied using MDCK cells (Madin-Darby canine kidney cells), the epidemic strain of influenza A/Lipetsk/1V/2018 (H1N1 pdm09) (EPI_ISL_332798), the highly pathogenic avian influenza virus A/common gull/Saratov/1676/2018 (H5N6) strain (EPI_ISL_336925), and neutral red vital dye. Results: The genomes of all studied strains contained determinants responsible for utilization of carbohydrates of plant origin; the genes of key enzymes for the synthesis of tryptophan and folic acid are present in the genomes of B. longum 379 and B. bifidum 791. A feature of the B. bifidum 791 genome is the presence of determinants responsible for the synthesis of thermostable type I bacteriocins - flavucin and lasso peptide. The B. bifidum 791 strain was found to show pronounced antiviral activity against both the strains of influenza A, the supernatant of which suppressed viral replication in vitro up to a dilution of 1:8, and the cells inhibited viral reproduction up to a concentration of 6·106 CFU/ml. Conclusion: The analysis of complete genomes of B. longum 379, B. bifidum 1, and B. bifidum 791 showed features that determine their strain-specific properties, the findings on which were previously made empirically based on indirect signs. In the genomes of B. longum 379 and B. bifidum 791 strains, in contrast to B. bifidum 1 strain, key enzymes for the synthesis of tryptophan and folic acid were found. These substances have an impact on the human body in many ways, including having a thymoleptic effect (reducing emotional stress, irritability, anxiety, eliminating lethargy, apathy, melancholy, anxiety) and regulating cognitive activity. The presence of determinants responsible for the synthesis of thermostable type I bacteriocins in the genome of B. bifidum 791 strain determines its pronounced antiviral activity.

Colon Microbiocenosis and Its Correction in Patients Receiving Programmed Hemodialysis.

The aim of the investigation was to study the species composition of colon microbiocenosis in patients with chronic kidney disease receiving programmed hemodialysis treatment and to evaluate the efficacy of its correction using a new immobilized synbiotic. MATERIALS AND METHODS: Samples of colon microbiota from 62 patients undergoing programmed hemodialysis were studied before and after a course of diet therapy that included probiotic components, in particular, the immobilized synbiotic LB-complex L. Isolation of microorganisms was carried out according to our original method; for bacteria identification, a MALDI-TOF Autoflex speed mass spectrometer (Bruker Daltonik, Germany) was used in the Biotyper program mode. The results were assessed using the criteria proposed by the authors and based on the OST 91500.11.0004-2003. The efficacy of the immobilized synbiotic was determined based on the clinical data, questionnaires, and bacteriological tests. RESULTS: In patients receiving programmed hemodialysis (before the start of the diet therapy), chronic moderate inflammation and azotemia were found. Dysbiotic changes in microbiocenosis were revealed in all the examined patients; in the absence or suppression of lacto- and bifidoflora, the number and diversity of Bacteroides spp., Clostridium spp., Collinsella spp., Eggerthella spp. and other bacteria increased, which was consistent with the theory of functional redundancy of gut microbiota. From the answers to the questionnaires, a decrease in the quality of life was found (up to 70 points out of 100) according to six of the eight scales used. After the combined therapy using the synbiotic LB-complex L in the study group, 56% of the examined patients showed their microbiocenosis restored to normal; no grade III dysbiosis was detected in any patient. There was a significant decrease in CRP and ESR in these patients and an improvement in the quality of life by criteria reflecting physical health. CONCLUSION: In patients receiving programmed hemodialysis, the addition of a probiotic component in the diet therapy restores the evolutionarily determined structure of the microbiocenosis, normalizes its functions, and leads to an overall improvement in health and quality of life.

[CHARACTERISTICS OF BIOLOGICAL AND MOLECULAR-GENETIC PROPERTIES OF LACTOBACILLUS FERMENTUM 90 TC-4 PROBIOTIC STRAIN].

AIM: Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods. MATERIALS AND METHODS: Object of the study--Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), "DNA-sorb B" kitwas used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly of genome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), "CLC Bio Assembly" and genome server RAST (rast.nmpdr.org). RESULTS: L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fer- mentum species has been proven. CONCLUSION: Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.

[Lactobacillus fermentum 90 TC-4 taxonomic status confirmation using whole genome sequencing and MALDI TOF mass spectrum].

With the use of whole genome sequencing, the taxonomic status of Lactobacillus fermentum 90 TC-4 strain from Russian collections were studied. Complex analysis of phenotypical and genetic properties was conducted using phenotypic and molecular genetic methods. The main characteristics of the genome and biochemical activity profile of the strain were determined. A comparative analysis of the mass spectrum of ribosomal proteins of the strain, its biochemical properties, a fragment of 16S rRNA gene sequencing, and the entire genome revealed that the present strain belongs to the species L. fermentum, confirming its taxonomic status in accordance with modern taxonomy.

[Indication and identification of Lactobacillus spp. with the use of polymerase chain reaction].

AIM: The aim of the work is the development of laboratory test for indication and identification of Lactobacillus spp. by the polymerase chain reaction. MATERIALS AND METHODS: The work is developed on the base of the GenBank/EMBL data about genetic sequences of the Lactobacillus spp. The sequences of DNA were studied with the help of the ClustalW program. The strains of the Lactobacillus spp., which are the object of the research, have been registered in Russian collection of industrial microorganisms. RESULTS: The laboratory test of nested-PCR for indication and identification L. plantarum, L. fermentum, L. acidophilus, L. delbrueckii, L. casei, L. rhamnosus was performed. The specificity of the nested-PCR was correlated with the control analyses of monoculture Lactobacillus spp. and commercial products. CONCLUSION: The new developed laboratory nested-PCR test may be use in control system of milk foods enriched by probiotic microorganisms.