Multilocus genotyping for classification and genetic structuring of Lactobacillus casei: insights from source and geographical origin.

The aim of the study in this article is to systematise the newly introduced strains of Lactobacillus based on determining the nucleotide sequence of a particular set of their genes (loci). The primary approach employed to address this issue involves conducting a laboratory experiment. During this experiment, a thorough examination was carried out on a set of organic compounds consisting of small DNA elements from the Lactobacillus genus. The Multilocus genotyping method served as the central technique, complemented by additional molecular-biological and population methods. These additional methods were utilized to determine the extent of phylogenetic similarity among pure cultures of Lactobacillus and to classify them accordingly. The article presents the gene isolates that were used for Multilocus typing; the number of L. casei isolates suitable for Multilocus genotyping was revealed; the gene alleles that allowed classifying L. casei isolates into five sequencing types were revealed; the effectiveness of genetic typing method for Multilocus sequencing was substantiated. The article is of practical value for microbiologists and geneticists in the field of molecular biology, as well as for technologists in the food industry. With the development of applied methods in genetic systematics, it has become possible to study pure culture of Lactobacillus species. The application of modern methods of genotypic classification of Lactobacillus species will make it possible to increase the efficiency of using better and safer products in the food industry and medicine.

Metagenomic Analysis of Koumiss in Kazakhstan.

INTRODUCTION: Koumiss is a low-alcohol product made from fermented mare's milk, which is popular in Kazakhstan, Russia, and other countries of Central Asia, China, and Mongolia. Natural mare's milk is fermented in symbiosis of two types of microorganisms (lactobacteria and yeast). Koumiss's microbial composition varies depending on the geographical, climatic, and cultural conditions. Based on a phenotypic characteristic from samples, Wu, R. and colleagues identified the following bacteria isolated in inner Mongolia, an autonomous region of China: L.casei, L.helveticus, L.plantarum, L.coryniformis subsp. coryniformis, L.paracasei, L.kefiranofaciens, L.curvatus, L.fermentum, and W.kandleri. Studies of the yeast composition in koumiss also showed significant variations. Thus, there were Saccharomyces unisporus related 48.3% of isolates, to Kluyveromyces marxianus (27.6%), Pichia membranaefaciens (15.0%), and Saccharomyces cerevisiae (9.2%) from 87 isolated yeast cultures. The purpose of this study was to examine the bacterial composition in koumiss. METHODS: To extract DNA, 1.8 ml of fermented milk was centrifuged to generate a pellet, which was suspended in 450 µl of lysis buffer P1 from the Powerfood Microbial DNA Isolation kit (MoBio Laboratories Inc, USA). Amplification of the microflora was used to determine the composition of a fragment of the gene 16S rRNA and ITS1. Plasmid library with target insertion was obtained on the basis of height copy plasmid vectors producing high pGem-T. The definition of direct nucleotide sequencing was performed by the method of Sanger using a set of "BigDye Terminanor v 3.1 Cycle sequencing Kit with automatic genetic analyzer ABI 3730xl (Applied Biosystems, USA). Informax Vector NTI Suite 9, Sequence Scanner v 1.0 software package used for the analysis. RESULTS: Our studies showed that in the most samples of koumiss isolated from Akmola region (Central Kazakhstan) prevailed the following bacteria species: Lactobacillus diolivorans, Lactobacillus acidophilus, L. casei, L. curvatus yeast genus Torula (62.4%) and Saccharomyces cerevisiae (37.6%). CONCLUSION: Thus, the first metagenomic research of koumiss, which was conducted in Kazakhstan, showed significant variations in microbial composition.

Lactobacillus for Vaginal Microflora Correction.

INTRODUCTION: Despite the significant progress made in prevention, diagnosis, and treatment, there is still a high rate of vaginal dysbiosis in Kazakh women. The use of antibiotics in the treatment of vaginal dysbiosis contributes to the elimination of pathogens as well as microflora, which can lead to a decrease in local immunity and more favorable conditions for infection spread. The most physiologically safe and promising method for the restoration of vaginal biocenosis is the use of probiotics administered by a vaginal route. METHODS: We have allocated 64 of cultures of Lactobacillus from the vaginal epithelium of healthy women of reproductive age and women with diagnosed bacterial vaginosis (BV). Identification of cultures was performed by PCR analysis of 16S ribosomal RNA. Evaluation of biological significance was determined by the following criteria: high antagonistic activity against Candida albicans, Escherichia coli, Serratia marcescens, Proteus mirabilis, Klebsiella ozaenae, and Staphylococcus aureus; and production of hydrogen peroxide, resistance to antibiotics, adhesive activity. We studied the symbiotic relationship of selected biologically active of cultures to each other and received options for consortiums with properties of probiotics through co-cultivation. RESULTS: Results of genotyping showed that the isolated lactobacilli belong to the seven species: L. fermentum, L. salivarius, L. gasseri, L. crispatus, L. jensenii, L. plantarum, and L. delbrueskii. L. fermentum, L. salivarius, L. gasseri, and L. jensenii occur in women with suspected BV. The highest percentage of occurrence in the vagina of healthy women was L. fermentum (28%). Most strains of lactobacilli possess high inhibitory activity for all test-strains, except Candida albicans (37.5%). 56% of studied cultures revealed high adhesion to human erythrocytes. All lactobacillus strains were resistant to metronidazole, 80% to kanamycin, 57% to vancomycin, and sensitivity to roxithromycin, amoxiclav, ampicillin was diagnosed in all strains. 50% of cultures showed a moderate sensitivity to gentamicin and cefazolin. In a study of peroxide-producing activity, 80% of the cultures exhibited peroxide-producing activity. As a result of screening, the 7 most active strains of lactobacilli were selected for development of 10 variants of probiotic consortia. Also, there was increase of adhesive activity in the consortia compared to other components. These consortia can be used for the treatment of BV in addition to metronidazole. CONCLUSION: The probiotic consortia identified in this study had high antagonistic, adhesive properties, and resistance to metronidazole. These probiotics can potentially be used for the development of biological products for the treatment and prevention of bacterial vaginosis.

Effect of Probiotic Consortium on the Local Inflammatory Process in Chronic Periodontitis.

INTRODUCTION: Inflammatory periodontal disease is one of the major concerns of researchers and clinicians, because it can lead to tooth loss and an increased risk of systemic pathologies, even at the age of 35. The purpose of this study was to determine the effects of gelatin-based probiotic consortium on the local and general factors of inflammation in rats with chronic periodontitis. METHODS: The study object was a complex of probiotic bacteria based in an odourless 6% gelatin plate with neutral flavour. A cellular biomass of the consortium consists of following lactobacilli: Lactobacillus casei subsp. pseudoplantarum, Lactobacillus caseisubsp.casei, L.fermentum, and L. helveticus. The viable cell number was 2.5 × 109 CFU/ml. The model of chronic periodontitis was reproduced in the white random-bred rats that weighed 160-220g, by keeping them on a low-protein diet. After three months, symptoms associated with medium and severe chronic periodontitis were observed in the rats. Application was carried out on the oral mucosa of rats 1 time per day for 14 days. The stickers lacking consortium of microorganisms were used as the placebo. The "Solcoseril" gel was chosen as a comparator. The hematologic, biochemical, and morphological characteristics were investigated. RESULTS: A complete clearance of periodontal pockets was observed during an objective examination of the experimental group rats on the 14th day of the experiment. Moreover, a gingival mucous turned pink, and there were no cyanosis tissues. The local changes were accompanied by improvement in hematological parameters, such as a reduction of blood eosinophilia and neutrophilia, and a recovery of the white blood cells number to the normal degree within the group that received the probiotic complex. A decrease of the acute plethora of microvasculature was observed morphologically as a result of the treatment. There were signs of basal layer activation of the stratified squamous epithelium with a merger of the acanthosis outgrowths and a formation of the fibrotic nodules. Biochemical investigations did not show significant changes in the indicators. CONCLUSIONS: In the settings of the chronic periodontitis model, the use of gelatin-based probiotic consortium consisting of Lactobacillus casei subsp. pseudoplantarum, Lactobacillus caseisubsp.casei, L.fermentum, L. helveticus. at 2.5 × 109 CFU/ml viable cell numbers lead to the reduction of the local inflammatory manifestations of the periodontitis in 14 days of treatment.

Randomized Clinical Trial: Efficacy of a New Synbiotic in Adults with Metabolic Syndrome.

INTRODUCTION: Metabolic syndrome is a lifestyle disease and is a frequent problem among the adult population. Human gut microbiota plays a key role in the development of metabolic syndrome. Recently, the gut microbiota has emerged as an important contributor to the development of obesity and metabolic disorders through its interactions with environmental (e.g. diet) and genetic factors. The aim of this study was to research the effects of synbiotic on the gut microbiota and host metabolism. METHODS: We conducted a double-blind, randomized, placebo-controlled trial. Our sample included 180 adults (ages 30-89) with symptoms of metabolic syndrome, who were allocated to either placebo or synbiotic group. The main inclusion criteria were: blood pressure of around 130/90 mmHg; raised fasting plasma glucose (FPG) >100 mg/dL (5.6 mmol/L), previous diagnosis of type 2 diabetes, dyslipidemia triglycerides (TG) of 1.70 mmol/L, a high-density lipoprotein cholesterol (HDL-C) of 0.90 mmol/L in males and 1.0 mmol/L in females, and central obesity with a waist/hip ratio > 0.90 in males or > 0.85 in females or a body mass index > 30 kg/m2. RESULTS: We enrolled 90 adults in the placebo group and 90 in the synbiotic group. The two groups had similar demographic and clinical characteristics. Consent was signed by all patients. All patients underwent clinical and laboratory evaluation, including complete blood tests, glucose test, glycosylated hemoglobin, total cholesterol and triglycerides, cholesterol, LDL, HDL plasma, immunogram, and coprogram. All patients were interviewed with a questionnaire that included 200 questions related to diet, lifestyle, and health. Synbiotic were used by patients in a dose of 200 grams twice a day. The duration of applying of the synbiotic was 90 days.To study the composition of the intestinal microbiota, stool samples were collected before and after applying the synbiotic. The microbial composition will be determined by analyzing the locus of 16S rDNA. CONCLUSION: This ongoing study is currently undergoing microbial composition analysis in order to establish the efficacy of the new synbiotic in adults with metabolic syndrome.

Complete Genome Sequence of the Probiotic Lactic Acid Bacterium Lactobacillus Rhamnosus.

INTRODUCTION: Lactobacilli are a bacteria commonly found in the gastrointestinal tract. Some species of this genus have probiotic properties. The most common of these is Lactobacillus rhamnosus, a microoganism, generally regarded as safe (GRAS). It is also a homofermentative L-(+)-lactic acid producer. The genus Lactobacillus is characterized by an extraordinary degree of the phenotypic and genotypic diversity. However, the studies of the genus were conducted mostly with the unequally distributed, non-random choice of species for sequencing; thus, there is only one representative genome from the Lactobacillus rhamnosus clade available to date. The aim of this study was to characterize the genome sequencing of selected strains of Lactobacilli. METHODS: 109 samples were isolated from national domestic dairy products in the laboratory of Center for life sciences. After screaning isolates for probiotic properties, a highly active Lactobacillus spp strain was chosen.Genomic DNA was extracted according to the manufacturing protocol (Wizard® Genomic DNA Purification Kit). The Lactobacillus rhamnosus strain was identified as the highly active Lactobacillus strain accoridng to its morphological, cultural, physiological, and biochemical properties, and a genotypic analysis. RESULTS: The genome of Lactobacillus rhamnosus was sequenced using the Roche 454 GS FLX (454 GS FLX) platforms. The initial draft assembly was prepared from 14 large contigs (20 all contigs) by the Newbler gsAssembler 2.3 (454 Life Sciences, Branford, CT). CONCLUSION: A full genome-sequencing of selected strains of lactic acid bacteria was made during the study.

Health benefits of new symbiotic "NAR".

INTRODUCTION: The immune-modulatory effects of synbiotics and their ability to reduce free radical levels may be useful for functional food that is able to be active throughout whole period of colonization of the gastrointestinal tract.The aim of the present study was to investigate the immune-modulatory and antioxidant effects of the synbiotic product "NАR," a probiotic beverage. METHODS: The presence of IL-2, IL-4, IL-6, IL-8, IL-10, αTNF, γIFN, Ig A, Ig M, and Ig E was studied in vitro using a solid immunosorbent analysis. The total antioxidant activities of superoxide dismutase and glutathione reductase were determined by a spectrophotometry using the Sigma-Aldrich sets. RESULTS: Studies of the immune-modulatory properties of the synbiotic product NAR showed 1.7 fold increase of γINF levels (p<0.01) in blood after consumption of the synbiotic product "NAR" in comparison to control values, whereas the concentrations of IL-4 and Ig E decreased 2.0 times (treatment: 9.3; control: 18.7; p<0.01) and 1.3 times (p<0.1), respectively. The consumption of the synbiotic product "NAR" caused an increase in the proportion of γINF/IL 4 (treatment: 15.4; control: 4.4; p<0.01), which indicates a reduction in functional activity of Th2-type lymphocytes in comparison with the function of Th1 cells.Our study showed a high level of the total antioxidant activity of the synbiotic product (67.4 mmol/ml). The antioxidant activity of the intact cells of consortium (15.3 mM/ml), which was the basis for the preparation of the symbiotic product, is several times lower than the activity observed in the symbiotic samples.Expression of SOD is one of the mechanisms of antioxidant stress radicals inactivation by bacteria. The analysis identified a superoxide dismutase activity of synbiotic product (1.42 U/mg protein). A glutathione reductase activity of the synbiotic product was elevated (0.06 U/ml). CONCLUSION: The majority of the inflammatory mediators found in the blood after the consumption of symbiotic product NAR were inflammatory mediators that activate a cellular component of the resistance. Moreover, the symbiotic product has a high antioxidant activity.

Antioxidant activity of the probiotic consortium in vitro.

INTRODUCTION: Available evidence suggests that probiotics have different biological functions that depend on several mechanisms, such as antioxidant and DNA-protective activities. The probiotic consortium includes bacterial cultures such as Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, and other bacterial cultures isolated from traditional Kazakh dairy products (ayran, kumys, shubat, and healthy clinical material). The aim of this study was to investigate the total antioxidant activity of the consortium of probiotic bacteria and to determine the activity of superoxide dismutase, glutathione reductase, and DNA-protective action. MATERIAL AND METHODS: In vitro comet assay was used to determine the antigenotoxicity of the probiotic consortium. Total antioxidant activity was determined using a method of analysis with Trolox as the equivalent. The analysis method of superoxide dismutase activity assesses the inhibition rate of the nitroblue tetrazolium reduction to formazan by superoxide dismutase. Determination of glutathione reductase activity is based on the measurement of the NADPH oxidation speed. RESULTS: A significantly high level of the total antioxidant activity of the probiotic consortium intact cells (15.3 mM/ml) was observed whereas the activity index of lysate was 11.1 mM/ml.The superoxide dismutase activity of probiotic consortium lysate was evaluated, with values that peaked at 0.24 U/mg protein. The superoxide dismutase activity of the consortium was lower in comparison to L.fernentum E-3 and L.fernentum E-18 cultures with values of 0.85 U/mg and 0.76 U/mg protein, respectively. SOD activity of probiotic consortium whole cells was not observed, which is typical for lactic acid bacteria.Glutathione reductase plays an important role in the optimal protection from oxidative stress. Glutathione reductase activity of the studied probiotic consortium was low; moreover, the activity of the lysate was two times higher than the activity of the cells reaching 0.01 units/ml. Investigations by Dr. Li have shown that the intracellular glutathione may give a significant protection of Lactococcus from the damaging action of H2O2, even at very low concentrations.The data from our study suggests that the co-incubation of the epithelial cells with probiotic bacteria reduces the percentage of damaged cells (damage index-0.60). CONCLUSION: The studied probiotic consortium has antigenotoxic and antioxidant activities. Preparations and products of this probiotic consortium may serve as a protective component in the intestinal microbial ecosystem.

Genotype frequencies of polymorphic MDR1 variants in the Kazakhstani population.

INTRODUCTION: Statins appear to be handled by an ATP-dependent membrane transporter and three SNPs (C1236T (rs1128503), G2677T (rs2032582), and C3435T (rs1045642), which capture the common genetic variation at this locus. Individuals, who carry the T allele at each SNP (i.e., the T-T-T haplotype), have higher systemic exposure to simvastatin.A triallelic thymine (T) - guanine (G) - adenine (A), which is a point mutation at nucleotide 2677 in exon 22, leads to ABCB1 in a non-synonymous codons (GCT alanine, TCT serine, threonine ACT) at position 893 in a cytoplasmic loop of ATP-dependent membrane transporters. METHODS: Blood samples from healthy individuals were collected in the Republican Diagnostic Center, Astana, Kazakhstan. The research samples included 461 healthy people. Genomic DNA was extracted from peripheral blood using the 'salting out' procedure. For the MDR1 exon 21, 2677G>T/A (Ala893Ser/Thr) polymorphism was genotyped by PCR sequencing by the use of dye-terminator (ABI 3730xl sequencer). RESULTS: The GG allele appeared in 23% of samples, the GA in 6.7%, the GT in 44%, the non-G heterozygote in 4.5%, and the non-G homozygote in 18%. These results are consistent with previously published data. Importantly, the frequency of 2677T alleles in our group was 15.4%. This represents the lowest frequency of this allele compared to published data in different populations. The frequency of the 2677T allele in Asians and Caucasians varies from 38 to 62%, and is 15% for African Americans. On the other hand, the 2677A allele frequency in the Japanese varies from 15 to 22%, and in Caucasians from 2% and 4%. The 2677A allele frequency has been found in 4.6% of samples. CONCLUSIONS: Our study further emphasizes differences between various Asian populations and the importance of repeating this genetic study in different ethnic groups.

Influence of Probiotic Consortium on TH1 and TH2 Immune Response.

INTRODUCTION: The main role of probiotics is to maintain homeostasis in the intestines and improve bowel protective function. The aim of the present study is to investigate immuno-modulatory effects of a probiotic consortium. METHODS: Observations were carried out in vitro. The presence of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, IgA, IgM, and IgE was studied using a solid-phase enzyme immunosorbent assay on the VECTOR-BEST sets (Russia). RESULTS: Immunomodulatory properties of the probiotic consortium were studied, which consisted of the following strains: Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, and Bifidobacterium bifidum. Elevated concentrations of INFγ in control samples decreased 3.9 times (p < 0.05) after a saturation of blood with the probiotic consortium. Significant reduction of cytokine levels after the probiotic effects of the consortium was observed in IL-10 by 2.1 times (p < 0.05) and IgA by 1.87 times (p < 0.0005). There was a significant increase in the levels of IL-4, IgE, IL-6, and IL-8 by 1.3 (p < 0.005), 1.1 (p < 0.5), 18.0 (p < 0.005), and 6 (p < 0.05) times, respectively, in comparison with the control samples. IL-4 and INFγ have different effects on the synthesis of IgE. Soluble low affinity receptors FcɛRII (CD23) in association with IL-4 facilitate a differentiation of the B-lymphocytes in IgE-synthesizing cells, while γ-INF inhibits this process. It is known that the intracellular expression of γ-INF and IL-4 is the most reliable marker for Th1 and Th2 immune responses, respectively. The conducted studies determined that the ratio of INF-γ/IL-4 was 0.9 (control 4.8, P < 0.005) after the saturation of the blood cells with probiotic consortium. NF-γ/IL4 ratio decreased by 5.3 times compared with a control value, which indicates a reduction in the functional activity of Th1 type lymphocytes in comparison with the function of Th2 cells. CONCLUSION: The application of the probiotic consortium results in the maintenance of homeostasis by the stimulation of immune function through the activation of humoral immunity. Moreover, the probiotic application changes the orientation of the immunological memory causing the cancellation of the recruitment of Th1 cells in the response.