ΦCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis

peer-reviewedCrAssphages are an extensive and ubiquitous family of tailed bacteriophages, predicted to infect bacteria of the order Bacteroidales. Despite being found in ~50% of individuals and representing up to 90% of human gut viromes, members of this viral family have never been isolated in culture and remain understudied. Here, we report the isolation of a CrAssphage (ΦCrAss001) from human faecal material. This bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming previous in silico predictions of the likely host. DNA sequencing demonstrates that the bacteriophage genome is circular, 102 kb in size, and has unusual structural traits. In addition, electron microscopy confirms that ΦcrAss001 has a podovirus-like morphology. Despite the absence of obvious lysogeny genes, ΦcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium, and is able to maintain itself in continuous host culture during several weeks

RNA phage biology in a metagenomic era

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies

Бактериальная транслокация из кишечника: микробиологические, иммунологические и патофизиологические аспекты

Bacterial translocation (BT) is both pathology and physiology phenomenon. In healthy newborns it accompanies the process of establishing the autochthonous intestinal microbiota and the host microbiome. In immunodeficiency it can be an aethio-pathogenetic link and a manifestation of infection or septic complications. The host colonization resistance to exogenous microbic colonizers is provided by gastrointestinal microbiota in concert with complex constitutional and adaptive defense mechanisms. BT may be result of barrier dysfunction and self-purification mechanisms involving the host myeloid cell phagocytic system and opsonins. Dynamic cell humoral response to microbial molecular patterns that occurs on the mucous membranes initiates receptor signaling pathways and cascade of reactions. Their vector and results are largely determined by cross-reactivity between microbiome and the host genome. Enterocyte barriers interacting with microbiota play leading role in providing adaptive, homeostatic and stress host reactivity. Microcirculatory ischemic tissue alterations and inflammatory reactions increase the intestinal barrier permeability and BT. These processes a well as mechanisms for apoptotic cells and bacteria clearance are justified to be of prospective research interest. The inflammatory and related diseases caused by alteration and dysfunction of the intestinal barrier are reasonably considered as diseases of single origin. Maternal microbiota affects the formation of the innate immune system and the microbiota of the newborn, including intestinal commensal translocation during lactation. Deeper understanding of intestinal barrier mechanisms needs complex microbiological, immunological, pathophysiological, etc. investigations using adequate biomodels, including gnotobiotic animals.Бактериальная транслокация (БТ) из кишечника представляет патологическое и физиологическое явление. БТ наблюдается в процессе установления аутохтонной кишечной микробиоты и микробиома хозяина при взаимодействии с комменсалами, сопровождая естественный иммуногенез, и при патологии (например, иммунодефицитах), представляя этиопатогенетическое звено инфекционного процесса и септических осложнений. Эволюционно выработанные механизмы взаимодействия хозяина и микробиоты, взаимно противоположной направленности, обусловлены инвазионными свойствами микроорганизмов и противостоящими им защитно-барьерными механизмами хозяина. Колонизационная резистентность хозяина, контактирующего с экзогенной микробиотой, обеспечивается комплексом конституциональных и адаптивных механизмов с ведущим участием комменсальной микробиоты. БT в результате дисфунции барьеров контролируется механизмами самоочищения с вовлечением миелоидной клеточной фагоцитарной системы и опсонинов. Возникающая на слизистых оболочках динамично развивающаяся клеточно-гуморальная реакция на молекулярные микробные паттерны инициирует рецепторно-сигнальные и каскадные реакции, вектор и результат которых определяются перекрестным взаимодействием генома и микробиома хозяина. Рассмотрена ультраструктура энтероцитарных барьеров, взаимодействующих с микробиотой (симбионты и патобионты), в обеспечении адаптационно-гомеостатической реактивности хозяина. Обоснованы исследования микроциркуляторного звена в патогенезе ишемических тканевых повреждений и воспаления, повышающих проницаемость кишечного барьера и БТ, а также механизмов очищения от бактерий и апоптотических клеток. Воспалительные и другие заболевания, напрямую связанные с барьерными нарушениями кишечника, обоснованно считаются болезнями кишечного барьера. Материнская микробиота влияет на формирование врожденного иммунитета и микробиоты новорожденного, в том числе путем БТ кишечных комменсалов с грудным молоком. Перспективны комплексные микробиологические, иммунологические, патофизиологические и другие исследования с использованием адекватных биомоделей, включая гнотобиотов

Abolishment of morphology‑based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021−March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratifed by vote in 2022. Signifcant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved

Ruthenibacterium lactatiformans gen. nov., sp.nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces

Two novel strains of Gram-stain-negative, rod-shaped, obligately anaerobic, non-spore-forming, non-motile bacteria were isolated from the faeces of healthy human subjects. The strains, designated as 585-1T and 668, were characterized by mesophilic fermentative metabolism, production of d-lactic acid, succinic acid and acetic acid as end products of d-glucose fermentation, prevalence of C18 : 1 ω9, C18 : 1 ω9 aldehyde, C16 : 0 and C16 : 1 ω7c fatty acids, presence of glycine, glutamic acid, lysine, alanine and aspartic acid in the petidoglycan peptide moiety and lack of respiratory quinones. Whole genome sequencing revealed the DNA G+C content was 56.4–56.6 mol%. The complete 16S rRNA gene sequences of the two strains shared 91.7/91.6 % similarity with Anaerofilum pentosovorans FaeT, 91.3/91.2 % with Gemmiger formicilis ATCC 27749T and 88.9/88.8 % with Faecalibacterium prausnitzii ATCC 27768T. On the basis of chemotaxonomic and genomic properties it was concluded that the strains represent a novel species in a new genus within the family Ruminococcaceae , for which the name Ruthenibacterium lactatiformans gen. nov., sp. nov. is proposed. The type strain of Ruthenibacterium lactatiformans is 585-1T (=DSM 100348T=VKM B-2901T)

Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

peer-reviewedBackground Faecalibacterium prausnitzii is a ubiquitous member of the human gut microbiome, constituting up to 15% of the total bacteria in the human gut. Substantial evidence connects decreased levels of F. prausnitzii with the onset and progression of certain forms of inflammatory bowel disease, which has been attributed to its anti-inflammatory potential. Two phylogroups of F. prausnitzii have been identified, with a decrease in phylogroup I being a more sensitive marker of intestinal inflammation. Much of the genomic and physiological data available to date was collected using phylogroup II strains. Little analysis of F. prausnitzii genomes has been performed so far and genetic differences between phylogroups I and II are poorly understood. Results In this study we sequenced 11 additional F. prausnitzii genomes and performed comparative genomics to investigate intraspecies diversity, functional gene complement and the mobilome of 31 high-quality draft and complete genomes. We reveal a very low level of average nucleotide identity among F. prausnitzii genomes and a high level of genome plasticity. Two genomogroups can be separated based on differences in functional gene complement, albeit that this division does not fully agree with separation based on conserved gene phylogeny, highlighting the importance of horizontal gene transfer in shaping F. prausnitzii genomes. The difference between the two genomogroups is mainly in the complement of genes associated with catabolism of carbohydrates (such as a predicted sialidase gene in genomogroup I) and amino acids, as well as defense mechanisms. Conclusions Based on the combination of ANI of genomic sequences, phylogenetic analysis of core proteomes and functional differences we propose to separate the species F. prausnitzii into two new species level taxa: F. prausnitzii sensu stricto (neotype strain A2–165T = DSM 17677T = JCM 31915T) and F. moorei sp. nov. (type strain ATCC 27768T = NCIMB 13872T).This research was conducted with the financial support of Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273, a Science Foundation Ireland’s Spokes Programme which is co-funded under the European Regional Development Fund under Grant Number SFI/14/SP APC/B3032, and a research grant from Janssen Biotech, Inc

РАЗРАБОТКА ДИАГНОСТИЧЕСКОЙ ТЕСТ-СИСТЕМЫ ДЛЯ РАННЕЙ НЕИНВАЗИВНОЙ ДИАГНОСТИКИ РАКА ПРОСТАТЫ, ОСНОВАННОЙ НА КОЛИЧЕСТВЕННОЙ ДЕТЕКЦИИ МРНК ГЕНА PCA3 В ОСАДКЕ МОЧИ МЕТОДОМ ОТ-ПЦР В РЕЖИМЕ РЕАЛЬНОГО ВРЕМЕНИ

The wide introduction of prostatic specific antigen (PSA) determination into clinical practice has resulted in a larger number of prostate biopsies, while the lower age threshold for PSA has led to a larger number of unnecessary prostate biopsies. Hence, there is a need for new biomarkers that can detect prostate cancer. РCА3 is a noncoding messenger ribonucleic acid (mRNA) that is expressed exclusively in prostate cells.  The aim of the study  has been to develop a diagnostic test system for early non-invasive detection of prostate cancer based on PCA3 mRNA levels in urine sediment using quantitative reverse transcription polymerase chain reaction (qRT-PCR). As part of the study, a laboratory diagnostic test system prototype has been designed, an application methodology has been developed and specificity and sensitivity data of the method has been assessed. The diagnostic system has demonstrated its ability to detect significantly elevated levels of PCA 3/ KLK 3 in samples from prostate cancer (PCa) patients compared with those from healthy men. The findings have shown relatively high diagnostic sensitivity, specificity and negative-predictive values for an early non-invasive screening of prostate cancerШирокое внедрение в клиническую практику определения содержания простатического специфического антигена (ПСА) привело к увеличению числа выполняемых биопсий простаты, а снижение порога возрастных норм ПСА — к увеличению числа неоправданных биопсий. В связи с этим возникла необходимость в новых биомаркерах рака предстательной железы. РСА3 — некодирующая мРНК, которая экспрессируется исключительно клетками предстательной железы. Целью данной работы было разработать диагностическую тест-систему для ранней неинвазивной диагностики рака простаты, основанной на количественной детекции мРНК гена РСА3 в осадке мочи методом полимеразной цепной реакции (ПЦР) в режиме реального времени сопряженной с обратной транскрипцией (ОТ). В результате был создан лабораторный образец диагностической ПЦР-тест-системы и отработана методика его применения. Получены данные о специфичности и чувствительности разработанного метода. Показана способность диагностической системы выявлять значительное превышение параметра PCA3/KLK3 в образцах биоматериала, полученного от больных раком простаты, по сравнению с образцами от здоровых индивидуумов. В ходе проведенных исследований установлены достаточно высокие показатели диагностической чувствительности, специфичности и негативной предсказательной ценности для раннего неинвазивного скринингово обнаружения рака предстательной железы. 

Reproducible protocols for metagenomic analysis of human faecal phageomes

peer-reviewedAll sequence data used in the analyses were deposited in the Sequence read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under BioProject PRJNA407341. Sample IDs, meta data and corresponding accession numbers are summarised in Additional file 2: Table S2. All raw count tables, 16S taxonomic assignments, BLAST top hits for viral contigs and R code used for the analysis are available at (https://figshare.com/s/71163558b4f78e3e7ed6).Background Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing. Results Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 °C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples. Conclusions The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results

Изучение видового разнообразия бактерий рода Bifidobacterium кишечной микрофлоры с использованием метода MALDI-TOF масс-спектрометрии

Background: The members of genus Bifidobacterium represent a significant part of intestinal microbiota in adults and predominate in infants. Species repertoire of the intestinal bifidobacteria is known to be subjected to major changes with age; however, many details of this process are still to be elucidated.Objective: Our aim was to study the diversity of intestinal bifidobacteria and changes of their qualitative and quantitative composition characteristics during the process of growing up using MALDI-TOF mass-spectrometric analysis of pure bacterial cultures.Methods: A cross-sectional study of bifidobacteria in the intestinal microbiota was performed in 93 healthy people of the ages from 1 month to 57 years. Strains were identified using Microflex LT MALDI-TOF MS, the confirmation was performed by 16S rRNA gene fragment sequencing.Results: 93% of isolated bifidobacterial strains were successfully identified using MALDI-TOF mass-spectrometry. At least two of the strains from each species were additionally identified by 16S rRNA gene fragment sequencing, in all of the cases the results were the same. It was shown that the total concentration of bifidobacteria decreases with age (p 0.001) as well as the frequency of isolation of Bifidobacterium bifidum (p =0.020) and Bifidobacterium breve (p 0.001), and the frequency of isolation of Bifidobacterium adolescentis, increases (p 0.001), representing the continuous process of transformation of microbiota.Conclusion: The method of MALDI-TOF mass spectrometry demonstrated the ability to perform rapid and reliable identification of bifidobacteria that allowed the study of changes in the quantitative and qualitative characteristics of human microbiota in the process of growing up.Представители рода Bifidobacterium представляют значительную часть микрофлоры кишечника взрослых людей и численно доминируют в микрофлоре младенцев. Известно, что видовой состав кишечных бифидобактерий подвергается сильным изменениям с возрастом, однако многие детали этого процесса остаются неясными.Цель исследования: изучить видовое разнообразие бифидобактерий кишечника и изменения их качественного и количественного состава в процессе взросления человека при помощи технологии MALDI-TOF масс-спектрометрического анализа белковых профилей чистых культур.Методы: кросс-секционное исследование разнообразия бифидобактерий в составе нормальной микрофлоры кишечника проведено у 93 человек в возрасте от 1 мес до 57 лет. Осуществляли выделе- ние чистых культур и их идентификацию на приборе Microflex LT MALDI-TOF MS (Bruker Daltonics, Германия), подтверждение реализовывали с использованием секвенирования фрагмента гена 16S рРНК.Результаты: с применением MALDI-TOF масс-спектрометрии было успешно идентифицировано 93% выделенных штаммов бифидобактерий. Минимум по 2 представителя от каждого из видов были дополнительно определены методом секвенирования фрагмента гена 16SрРНК; во всех случаях результаты методов совпали. Показано, что с возрастом происходит снижение общей концентрации бифидобактерий (p 0,001), уменьшается встречаемость видов Bifidobacterium bifidum (p =0,020) и Bifidobacterium breve (p 0,001), а встречаемость вида Bifidobacterium adolescentis увеличивается (p 0,001), отражая постепенные процессы перестройки микрофлоры.Заключение: метод MALDI-TOF масс-спектрометрии показал возможность быстрой и надежной идентификации бифидобактерий, позволившей провести исследование изменений количественных и качественных показателей микрофлоры человека в процессе взрослени

Stability of the human gut virome and effect of gluten-free diet

The human gut microbiome consists of bacteria, archaea, eukaryotes, and viruses. The gut viruses are relatively underexplored. Here, we longitudinally analyzed the gut virome composition in 11 healthy adults: its stability, variation, and the effect of a gluten-free diet. Using viral enrichment and a de novo assembly-based approach, we demonstrate the quantitative dynamics of the gut virome, including dsDNA, ssDNA, dsRNA, and ssRNA viruses. We observe highly divergent individual viral communities, carrying on an average 2,143 viral genomes, 13.1% of which were present at all 3 time points. In contrast to previous reports, the Siphoviridae family dominates over Microviridae in studied individual viromes. We also show individual viromes to be stable at the family level but to vary substantially at the genera and species levels. Finally, we demonstrate that lower initial diversity of the human gut virome leads to a more pronounced effect of the dietary intervention on its composition