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ABSTRACT Inflammatory bowel diseases are characterized by chronic intestinal inflammation and alterations in the gut microbiota composition. Bacteroides fragilis, which secretes outer membrane vesicles (OMVs) with polysaccharide A (PSA), can moderate the inflammatory response and possibly alter the microbiota composition. In this study, we created a murine model of chronic sodium dextran sulfate (DSS)-induced intestinal colitis and treated it with B. fragilis OMVs. We monitored the efficiency of OMV therapy by determining the disease activity index (DAI) and performing histological examination (HE) of the intestine before and after vesicle exposure. We also analyzed the microbiota composition using 16S rRNA gene sequencing. Finally, we evaluated the volatile compound composition in the animals’ stools by HS-GC/MS to assess the functional activity of the microbiota. We observed more effective intestinal repair after OMV treatment according to the DAI and HE. A metabolomic study also revealed changes in the functional activity of the microbiota, with a predominance of phenol and pentanoic acid in the control group compared to the group treated with DSS and the group treated with OMVs (DSS OMVs). We also observed a positive correlation of these metabolites with Saccharibacteria and Acetivibrio in the control group, whereas in the DSS group, there was a negative correlation of phenol and pentanoic acid with Lactococcus and Romboutsia. According to the metabolome and sequencing data, the microbiota composition of the DSS-treated OMV group was intermediate between that of the control and DSS groups. OMVs not only have an anti-inflammatory effect but also contribute to the recovery of the microbiota composition.IMPORTANCEBacteroides fragilis vesicles contain superficially localized polysaccharide A (PSA), which has unique immune-modulating properties. Isolated PSA can prevent chemically induced colitis in a murine model. Outer membrane vesicles (OMVs) also contain digestive enzymes and volatile metabolites that can complement the anti-inflammatory properties of PSA. OMVs showed high therapeutic activity against sodium dextran sulfate-induced colitis, as confirmed by histological assays. 16S rRNA sequencing of fecal samples from different inflammatory stages, supplemented with comprehensive metabolome analysis of volatile compounds conducted by HS-GC/MS, revealed structural and functional alterations in the microbiota composition under the influence of OMVs. Correlation analysis of the OMV-treated and untreated experimental animal groups revealed associations of phenol and pentanoic acid with Lactococcus, Romboutsia, Saccharibacteria, and Acetivibrio.

Inflammatory bowel diseases are extremely common throughout the world. However, in most cases, it is asymptomatic at the initial stage. Therefore, it is important to develop non-invasive diagnostic methods that allow identification of the IBD risks in a timely manner. It is well known that gastrointestinal microbiota secrete volatile compounds (VOCs) and their composition may change in IBD. We propose a non-invasive method to identify the dynamics of IBD development in the acute and remission stage at the level of VOCs in model of dextran sulfate sodium (DSS) with chemically induced colitis measured by headspace GC/MS (HS GC/MS). Methods: VOCs profile was identified using a headspace GC/MS (HS GC/MS). GC/MS data were processed using MetaboAnalyst 5.0 and GraphPad Prism 8.0.1 software. The disease activity index (DAI) and histological method were used to assess intestinal inflammation. The peak of intestinal inflammation activity was reached on day 7, according to the disease activity index. Histological examination data showed changes in the intestine due to different stages of inflammation. As the acute inflammation stage was reached, the metabolomic profile also underwent changes, especially at the short-fatty acids level. A higher relative amounts of acetic acid (p value < 0.025) and lower relative amounts of propanoic acid (p value < 0.0005), butanoic acid (p value < 0.005) and phenol 4-methyl- (p value = 0.053) were observed in DSS7 group on day 7 compared to the control group. In remission stage, disease activity indexes decreased, and the histological picture also improved. But metabolome changes continued despite the withdrawal of the DSS examination. A lower relative amounts of propanoic acid (p value < 0.025), butanoic acid (p value < 0.0005), pentanoic acid (p value < 0.0005), and a significant de-crease of hexanoic acid (p value < 0.0005) relative amounts were observed in the DSS14 group compared to the control group on day 14. A model of DSS-induced colitis in rats was successfully implemented for metabolomic assessment of different stages of inflammation. We demonstrated that the ratios of volatile compounds change in response to DSS before the appearance of standard signs of inflammation, determined by DAI and histological examination. Changes in the volatile metabolome persisted even after visual intestine repair and it confirms the high sensitivity of the microbiota to the damaging effects of DSS. The use of HS GC/MS may be an important addition to existing methods for assessing inflammation at early stages.

Microorganisms and their hosts communicate with each other by secreting numerous components. This cross-kingdom cell-to-cell signaling involves proteins and small molecules, such as metabolites. These compounds can be secreted across the membrane via numerous transporters and may also be packaged in outer membrane vesicles (OMVs). Among the secreted components, volatile compounds (VOCs) are of particular interest, including butyrate and propionate, which have proven effects on intestinal, immune, and stem cells. Besides short fatty acids, other groups of volatile compounds can be either freely secreted or contained in OMVs. As vesicles might extend their activity far beyond the gastrointestinal tract, study of their cargo, including VOCs, is even more pertinent. This paper is devoted to the VOCs secretome of the Bacteroides genus. Although these bacteria are highly presented in the intestinal microbiota and are known to influence human physiology, their volatile secretome has been studied relatively poorly. The 16 most well-represented Bacteroides species were cultivated; their OMVs were isolated and characterized by NTA and TEM to determine particle morphology and their concentration. In order to analyze the VOCs secretome, we propose a headspace extraction with GC–MS analysis as a new tool for sample preparation and analysis of volatile compounds in culture media and isolated bacterial OMVs. A wide range of released VOCs, both previously characterized and newly described, have been revealed in media after cultivation. We identified more than 60 components of the volatile metabolome in bacterial media, including fatty acids, amino acids, and phenol derivatives, aldehydes and other components. We found active butyrate and indol producers among the analyzed Bacteroides species. For a number of Bacteroides species, OMVs have been isolated and characterized here for the first time as well as volatile compounds analysis in OMVs. We observed a completely different distribution of VOC in vesicles compared to the bacterial media for all analyzed Bacteroides species, including almost complete absence of fatty acids in vesicles. This article provides a comprehensive analysis of the VOCs secreted by Bacteroides species and explores new perspectives in the study of bacterial secretomes in relation the intercellular communication.

14 августа 2023 г. на 76-м году жизни после не продолжительной болезни скоропостижно скончался выдающийся ученый, отличный организатор, прекрасный клиницист, эрудированный педагог, общественный деятель, доктор медицинских наук, профессор, академик РАН Сергей Павлович Миронов.

Primary cardiac channelopathies are a group of diseases wherein the role of DNA testing in aiding diagnosis and treatment-based decision-making is gaining increasing attention. However, in some cases, evaluating the pathogenicity of new variants is still challenging. We report an accurate multistage assessment of a rare genetic variant in the SCN5A gene using next-generation sequencing (NGS) techniques and Sanger sequencing. Female sportsman (14 years old) underwent genetic counseling and DNA testing due to QT interval prolongation registered during ECG Holter monitoring. Genetic testing of the proband was performed in two independent laboratories. Primary DNA testing was performed by WES using the Ion ProtonTM System. Target panel sequencing of 11 genes was performed using PGM Ion Torrent. Search for variants in non-canonical and canonical exons 6 was performed by Sanger sequencing. The cascade familial screening and control re-sequencing were provided for proband with identified genetic variant p.S216L (g.38655290G>A, NM_198056.2:c.647C>T, and rs41276525) in the canonical exon 6 of the SCN5A gene after receiving data from another laboratory. Control Sanger and NGS sequencing revealed the absence p.S216L in the canonical exon 6 and confirmed the presence of p.S216L (g.38655522G>A, c.647C>T, and rs201002736) in the non-canonical exon 6 of the SCN5A gene. The identified variant was re-interpreted. The non-canonical transcripts of the exon 6 of the SCN5A gene is poorly represented in cardiac tissue (gnomAD). The detected variant was found in proband’s healthy mother. The correct interpretation of genetic data requires close cooperation between clinicians and researchers. It can help to avoid financial costs and stress for proband’s and families.