Effect of the consumption of brazzein and monellin, two recombinant sweet-tasting proteins, on rat gut microbiota.

Sweet-tasting proteins (SPs) are proteins of plant origin initially isolated from tropical fruits. They are thousands of times sweeter than sucrose and most artificial sweeteners. SPs are a class of proteins capable of causing a sweet taste sensation in humans when interacting with the T1R2/T1R3 receptor. SP thaumatin has already been introduced in the food industry in some countries. Other SPs, such as monellin and brazzein, are promising products. An important stage in researching SPs, in addition to confirming the absence of toxicity, mutagenicity, oncogenicity, and allergenic effects, is studying their influence on gut microbiota. In this paper we describe changes in the composition of rat gut microbiota after six months of consuming one of two recombinant SPs-brazzein or monellin. A full length 16S gene sequencing method was used for DNA library barcoding. The MaAsLin2 analysis results showed noticeable fluctuations in the relative abundances of Anaerocella delicata in brazzein-fed rat microbiota, and of Anaerutruncus rubiinfantis in monellin-fed rat microbiota, which, however, did not exceed the standard deviation. The sucrose-fed group was associated with an increase in the relative abundance of Faecalibaculum rodentium, which may contribute to obesity. Overall, prolonged consumption of the sweet proteins brazzein and monellin did not significantly change rat microbiota and did not result in the appearance of opportunistic microbiota. This provides additional evidence for the safety of these potential sweeteners.

Systemic metabolic depletion of gut microbiome undermines responsiveness to melanoma immunotherapy.

Immunotherapy has proven to be a boon for patients battling metastatic melanoma, significantly improving their clinical condition and overall quality of life. A compelling link between the composition of the gut microbiome and the efficacy of immunotherapy has been established in both animal models and human patients. However, the precise biological mechanisms by which gut microbes influence treatment outcomes remain poorly understood. Using a robust dataset of 680 fecal metagenomes from melanoma patients, a detailed catalog of metagenome-assembled genomes (MAGs) was constructed to explore the compositional and functional properties of the gut microbiome. Our study uncovered significant findings that deepen the understanding of the intricate relationship between gut microbes and the efficacy of melanoma immunotherapy. In particular, we discovered the specific metagenomic profile of patients with favorable treatment outcomes, characterized by a prevalence of MAGs with increased overall metabolic potential and proficiency in polysaccharide utilization, along with those responsible for cobalamin and amino acid production. Furthermore, our investigation of the biosynthetic pathways of short-chain fatty acids, known for their immunomodulatory role, revealed a differential abundance of these pathways among the specific MAGs. Among others, the cobalamin-dependent Wood-Ljungdahl pathway of acetate synthesis was directly associated with responsiveness to melanoma immunotherapy.

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson's Disease.

About 15% of patients with parkinsonism have a hereditary form of Parkinson's disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

Consistent Stool Metagenomic Biomarkers Associated with the Response To Melanoma Immunotherapy.

The human gut microbiome plays an important role in both health and disease. Recent studies have demonstrated a strong influence of the gut microbiome composition on the efficacy of cancer immunotherapy. However, available studies have not yet succeeded in finding reliable and consistent metagenomic markers that are associated with the response to immunotherapy. Therefore, the reanalysis of the published data may improve our understanding of the association between the composition of the gut microbiome and the treatment response. In this study, we focused on melanoma-related metagenomic data, which are more abundant than are data from other tumor types. We analyzed the metagenomes of 680 stool samples from 7 studies that were published earlier. The taxonomic and functional biomarkers were selected after comparing the metagenomes of patients showing different treatment responses. The list of selected biomarkers was also validated on additional metagenomic data sets that were dedicated to the influence of fecal microbiota transplantation on the response to melanoma immunotherapy. According to our analysis, the resulting cross-study taxonomic biomarkers included three bacterial species: Faecalibacterium prausnitzii, Bifidobacterium adolescentis, and Eubacterium rectale. 101 groups of genes were identified to be functional biomarkers, including those potentially involved in the production of immune-stimulating molecules and metabolites. Moreover, we ranked the microbial species by the number of genes encoding functionally relevant biomarkers that they contained. Thus, we put together a list of potentially the most beneficial bacteria for immunotherapy success. F. prausnitzii, E. rectale, and three species of bifidobacteria stood out as the most beneficial species, even though some useful functions were also present in other bacterial species. IMPORTANCE In this study, we put together a list of potentially the most beneficial bacteria that were associated with a responsiveness to melanoma immunotherapy. Another important result of this study is the list of functional biomarkers of responsiveness to immunotherapy, which are dispersed among different bacterial species. This result possibly explains the existing irregularities between studies regarding the bacterial species that are beneficial to melanoma immunotherapy. Overall, these findings can be utilized to issue recommendations for gut microbiome correction in cancer immunotherapy, and the resulting list of biomarkers might serve as a good stepping stone for the development of a diagnostic test that is aimed at predicting patients' responses to melanoma immunotherapy.

The Gene Expression Profile Differs in Growth Phases of the Bifidobacterium Longum Culture.

To date, transcriptomics have been widely and successfully employed to study gene expression in different cell growth phases of bacteria. Since bifidobacteria represent a major component of the gut microbiota of a healthy human that is associated with numerous health benefits for the host, it is important to study them using transcriptomics. In this study, we applied the RNA-Seq technique to study global gene expression of B. longum at different growth phases in order to better understand the response of bifidobacterial cells to the specific conditions of the human gut. We have shown that in the lag phase, ABC transporters, whose function may be linked to active substrate utilization, are increasingly expressed due to preparation for cell division. In the exponential phase, the functions of activated genes include synthesis of amino acids (alanine and arginine), energy metabolism (glycolysis/gluconeogenesis and nitrogen metabolism), and translation, all of which promote active cell division, leading to exponential growth of the culture. In the stationary phase, we observed a decrease in the expression of genes involved in the control of the rate of cell division and an increase in the expression of genes involved in defense-related metabolic pathways. We surmise that the latter ensures cell survival in the nutrient-deprived conditions of the stationary growth phase.

The effects of Levilactobacillus brevis on the physiological parameters and gut microbiota composition of rats subjected to desynchronosis.

BACKGROUND: All living organisms have developed during evolution complex time-keeping biological clocks that allowed them to stay attuned to their environments. Circadian rhythms cycle on a near 24 h clock. These encompass a variety of changes in the body ranging from blood hormone levels to metabolism, to the gut microbiota composition and others. The gut microbiota, in return, influences the host stress response and the physiological changes associated with it, which makes it an important determinant of health. Lactobacilli are traditionally consumed for their prophylactic and therapeutic benefits against various diseases, namely, the inflammatory bowel syndrome, and even emerged recently as promising psychobiotics. However, the potential role of lactobacilli in the normalization of circadian rhythms has not been addressed. RESULTS: Two-month-old male rats were randomly divided into three groups and housed under three different light/dark cycles for three months: natural light, constant light and constant darkness. The strain Levilactobacillus brevis 47f was administered to rats at a dose of 0.5 ml per rat for one month and The rats were observed for the following two months. As a result, we identified the biomarkers associated with intake of L. brevis 47f. Changing the light regime for three months depleted the reserves of the main buffer in the cell-reduced glutathione. Intake of L. brevis 47f for 30 days restored cellular reserves of reduced glutathione and promoted redox balance. Our results indicate that the levels of urinary catecholamines correlated with light/dark cycles and were influenced by intake of L. brevis 47f. The gut microbiota of rats was also influenced by these factors. L. brevis 47f intake was associated with an increase in the relative abundance of Faecalibacterium and Roseburia and a decrease in the relative abundance of Prevotella and Bacteroides. CONCLUSIONS: The results of this study show that oral administration of L. brevis 47f, for one month, to rats housed under abnormal lightning conditions (constant light or constant darkness) normalized their physiological parameters and promoted the gut microbiome's balance.

Separation of Donor and Recipient Microbial Diversity Allows Determination of Taxonomic and Functional Features of Gut Microbiota Restructuring following Fecal Transplantation.

Fecal microbiota transplantation (FMT) is currently used in medicine to treat recurrent clostridial colitis and other intestinal diseases. However, neither the therapeutic mechanism of FMT nor the mechanism that allows the donor bacteria to colonize the intestine of the recipient has yet been clearly described. From a biological point of view, FMT can be considered a useful model for studying the ecology of host-associated microbial communities. FMT experiments can shed light on the relationship features between the host and its gut microbiota. This creates the need for experimentation with approaches to metagenomic data analysis which may be useful for the interpretation of observed biological phenomena. Here, the recipient intestine colonization analysis tool (RECAST) novel computational approach is presented, which is based on the metagenomic read sorting process per their origin in the recipient's post-FMT stool metagenome. Using the RECAST algorithm, taxonomic/functional annotation, and machine learning approaches, the metagenomes from three FMT studies, including healthy volunteers, patients with clostridial colitis, and patients with metabolic syndrome, were analyzed. Using our computational pipeline, the donor-derived and recipient-derived microbes which formed the recipient post-FMT stool metagenomes (successful microbes) were identified. Their presence is well explained by a higher relative abundance in donor/pre-FMT recipient metagenomes or other metagenomes from the human population. In addition, successful microbes are enriched with gene groups potentially related to antibiotic resistance, including antimicrobial peptides. Interestingly, the observed reorganization features are universal and independent of the disease. IMPORTANCE We assumed that the enrichment of successful gut microbes by lantibiotic/antibiotic resistance genes can be related to gut microbiota colonization resistance by third-party microbe phenomena and resistance to bacterium-derived or host-derived antimicrobial substances. According to this assumption, competition between the donor-derived and recipient-derived microbes as well as host immunity may play a key role in the FMT-related colonization and redistribution of recipient gut microbiota structure.

Drift of the Subgingival Periodontal Microbiome during Chronic Periodontitis in Type 2 Diabetes Mellitus Patients.

Since periodontitis and type 2 diabetes mellitus are complex diseases, a thorough understanding of their pathogenesis requires knowing the relationship of these pathologies with other disorders and environmental factors. In this study, the representability of the subgingival periodontal microbiome of 46 subjects was studied by 16S rRNA gene sequencing and shotgun sequencing of pooled samples. We examined 15 patients with chronic periodontitis (CP), 15 patients with chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM), and 16 healthy subjects (Control). The severity of generalized chronic periodontitis in both periodontitis groups of patients (CP and CPT2DM) was moderate (stage II). The male to female ratios were approximately equal in each group (22 males and 24 females); the average age of the subjects was 53.9 ± 7.3 and 54.3 ± 7.2 years, respectively. The presence of overweight patients (Body Mass Index (BMI) 30-34.9 kg/m2) and patients with class 1-2 obesity (BMI 35-45.9 kg/m2) was significantly higher in the CPT2DM group than in patients having only chronic periodontitis or in the Control group. However, there was no statistically significant difference in all clinical indices between the CP and CPT2DM groups. An analysis of the metagenomic data revealed that the alpha diversity in the CPT2DM group was increased compared to that in the CP and Control groups. The microbiome biomarkers associated with experimental groups were evaluated. In both groups of patients with periodontitis, the relative abundance of Porphyromonadaceae was increased compared to that in the Control group. The CPT2DM group was characterized by a lower relative abundance of Streptococcaceae/Pasteurellaceae and a higher abundance of Leptotrichiaceae compared to those in the CP and Control groups. Furthermore, the CP and CPT2DM groups differed in terms of the relative abundance of Veillonellaceae (which was decreased in the CPT2DM group compared to CP) and Neisseriaceae (which was increased in the CPT2DM group compared to CP). In addition, differences in bacterial content were identified by a combination of shotgun sequencing of pooled samples and genome-resolved metagenomics. The results indicate that there are subgingival microbiome-specific features in patients with chronic periodontitis associated with type 2 diabetes mellitus.

Gene Networks Underlying the Resistance of Bifidobacterium longum to Inflammatory Factors.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host's immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter's growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro. By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.

Shifts in the Human Gut Microbiota Structure Caused by Quadruple Helicobacter pylori Eradication Therapy.

The human gut microbiome plays an important role both in health and disease. Use of antibiotics can alter gut microbiota composition, which can lead to various deleterious events. Here we report a whole genome sequencing metagenomic/genomic study of the intestinal microbiota changes caused by Helicobacter pylori (HP) eradication therapy. Using approaches for metagenomic data analysis we revealed a statistically significant decrease in alpha-diversity and relative abundance of Bifidobacterium adolescentis due to HP eradication therapy, while the relative abundance of Enterococcus faecium increased. We have detected changes in general metagenome resistome profiles as well: after HP eradication therapy, the ermB, CFX group, and tetQ genes were overrepresented, while tetO and tetW genes were underrepresented. We have confirmed these results with genome-resolved metagenomic approaches. MAG (metagenome-assembled genomes) abundance profiles have changed dramatically after HP eradication therapy. Focusing on ermB gene conferring resistance to macrolides, which were included in the HP eradication therapy scheme, we have shown a connection between antibiotic resistance genes (ARGs) and some overrepresented MAGs. Moreover, some E. faecium strains isolated from stool samples obtained after HP eradication have manifested greater antibiotic resistance in vitro in comparison to other isolates, as well as the higher number of ARGs conferring resistance to macrolides and tetracyclines.

VERA: agent-based modeling transmission of antibiotic resistance between human pathogens and gut microbiota.

MOTIVATION: The resistance of bacterial pathogens to antibiotics is one of the most important issues of modern health care. The human microbiota can accumulate resistance determinants and transfer them to pathogenic microbiota by means of horizontal gene transfer. Thus, it is important to develop methods of prediction and monitoring of antibiotics resistance in human populations. RESULTS: We present the agent-based VERA model, which allows simulation of the spread of pathogens, including the possible horizontal transfer of resistance determinants from a commensal microbiota community. The model considers the opportunity of residents to stay in the town or in a medical institution, have incorrect self-treatment, treatment with several antibiotics types and transfer and accumulation of resistance determinants from commensal microorganism to a pathogen. In this model, we have also created an assessment of optimum observation frequency of infection spread among the population. Investigating model behavior, we show a number of non-linear dependencies, including the exponential nature of the dependence of the total number of those infected on the average resistance of a pathogen. As the model infection, we chose infection with Shigella spp., though it could be applied to a wide range of other pathogens. AVAILABILITY AND IMPLEMENTATION: Source code and binaries VERA and VERA.viewer are freely available for download at github.com/lpenguin/microbiota-resistome. The code is written in Java, JavaScript and R for Linux platform. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Long-term impact of fecal transplantation in healthy volunteers.

BACKGROUND: Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes. RESULTS: We have performed a combined analysis of three healthy volunteers before and after capsule FMT by evaluating their general condition, adverse clinical effects, changes of basic laboratory parameters, and several immune markers. Intestinal microbiota samples were evaluated by 16S rRNA gene and shotgun sequencing. The data analysis demonstrated profound shift towards the donor microbiota taxonomic composition in all volunteers. Following FMT, all the volunteers exhibited gut colonization with donor gut bacteria and persistence of this effect for almost ∼1 year of observation. Transient changes of immune parameters were consistent with suppression of T-cell cytotoxicity. FMT was well tolerated with mild gastrointestinal adverse events, however, one volunteer developed a systemic inflammatory response syndrome. CONCLUSIONS: The FMT leads to significant long-term changes of the gut microbiota in healthy volunteers with the shift towards donor microbiota composition and represents a relatively safe procedure to the recipients without long-term adverse events.

MetaCherchant: analyzing genomic context of antibiotic resistance genes in gut microbiota.

Motivation: Antibiotic resistance is an important global public health problem. Human gut microbiota is an accumulator of resistance genes potentially providing them to pathogens. It is important to develop tools for identifying the mechanisms of how resistance is transmitted between gut microbial species and pathogens. Results: We developed MetaCherchant-an algorithm for extracting the genomic environment of antibiotic resistance genes from metagenomic data in the form of a graph. The algorithm was validated on a number of simulated and published datasets, as well as applied to new 'shotgun' metagenomes of gut microbiota from patients with Helicobacter pylori who underwent antibiotic therapy. Genomic context was reconstructed for several major resistance genes. Taxonomic annotation of the context suggests that within a single metagenome, the resistance genes can be contained in genomes of multiple species. MetaCherchant allows reconstruction of mobile elements with resistance genes within the genomes of bacteria using metagenomic data. Application of MetaCherchant in differential mode produced specific graph structures suggesting the evidence of possible resistance gene transmission within a mobile element that occurred as a result of the antibiotic therapy. MetaCherchant is a promising tool giving researchers an opportunity to get an insight into dynamics of resistance transmission in vivo basing on metagenomic data. Availability and implementation: Source code and binaries are freely available for download at https://github.com/ctlab/metacherchant. The code is written in Java and is platform-independent. Cotanct: ulyantsev@rain.ifmo.ru. Supplementary information: Supplementary data are available at Bioinformatics online.

Draft genomes of Enterococcus faecium strains isolated from human feces before and after eradication therapy against Helicobacter pylori.

The abundance of Enterococci in the human intestinal microbiota environment is usually < 0.1% of the total bacterial fraction. The multiple resistance to antibiotics of the opportunistic Enterococcus spp. is alarming for the world medical community because of their high prevalence among clinically significant strains of microorganisms. Enterococci are able to collect different mobile genetic elements and transmit resistance to antibiotics to wide range of Gram-positive and Gram-negative species of microorganisms, including the transmission of vancomycin resistance to methicillin-resistant strains of Staphylococcus aureus. The number of infections caused by antibiotics resistant strains of Enterococcus spp. is increasing. Here we present a draft genomes of Enterococcus faecium strains. These strains were isolated from human feces before and after (1 month) Helicobacter pylori eradication therapy. The samples were subject to whole-genome sequencing using Illumina HiSeq. 2500 platform. The data is available at NCBI https://www.ncbi.nlm.nih.gov/bioproject/PRJNA412824.

Data on gut metagenomes of the patients with Helicobacter pylori infection before and after the antibiotic therapy.

Antibiotic therapy can lead to the disruption of gut microbiota community with possible negative outcomes for human health. One of the diseases for which the treatment scheme commonly included antibiotic intake is Helicobacter pylori infection. The changes in taxonomic and functional composition of microbiota in patients can be assessed using "shotgun" metagenomic sequencing. Ten stool samples were collected from 4 patients with Helicobacter pylori infection before and directly after the H. pylori eradication course. Additionally, for two of the subjects, the samples were collected 1 month after the end of the treatment. The samples were subject to "shotgun" (whole-genome) metagenomic sequencing using Illumina HiSeq platform. The reads are deposited in the ENA (project ID: PRJEB18265).