BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is chara... more BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is characterized by thrombocytopenia and severe thrombosis. Platelet function during patient recovery in the medium-/long-term has not been investigated fully. Here, we undertook a 3-month study, assessing the recovery of a VITT patient and assessing platelet morphology, granule content and dense-granule release at two distinct time points during recovery.Case PresentationA 61 year-old female was admitted to hospital 15 days post ChAdOx1 nCov-19 vaccination. Hematological parameters and peripheral blood smears were monitored over 3 months. Platelet morphology and granule populations were assessed using transmission electron microscopy (TEM) at two distinct time points during recovery, as was agonist-induced platelet dense-granule release. Upon admission, the patient had reduced platelet counts, increased D-dimer and high anti-PF4 antibodies with multiple sites of cerebral venous sinus thrombosis...
Additional file 7: Figure S4. Multiple site-specific recombinase-encoding genes and evidence of d... more Additional file 7: Figure S4. Multiple site-specific recombinase-encoding genes and evidence of dynamic recombinations in the genome of B. xylanisolvens APCS1/XY. a Circular map of genome (the innermost circle [green and purple], GC skew; circle two [black], relative G+C content; circles three and four [red and dark blue], open reading frames identified on the positive and negative DNA strands respectively; circle five [orange], tRNA and rRNA genes. circle six, genes annotated as Sus-like surface-associated glycan utilisation proteins [green] and TonB-dependent nutrient receptor [light blue]; circle seven [black], genes annotated as invertases, integrases and recombinases). To the right of the genome map, circular maps of the two associated circular plasmids are shown; pBXS1-1 and pBXS1-2. Annotated features are coloured and labelled; b Distribution of length of Oxford Nanopore sequencing reads used for dynamic genome recombination analysis; c Distribution of percentage identity in ...
Additional file 12: Table S6. Primer sequences specific to each crAss-like phage strains detected... more Additional file 12: Table S6. Primer sequences specific to each crAss-like phage strains detected following sequencing of subject ID: 924 faeces post fermentation.
Additional file 2:. Supplementary Dataset. Spreadsheets of numerical data and raw unedited photog... more Additional file 2:. Supplementary Dataset. Spreadsheets of numerical data and raw unedited photographs required for reproduction of Figs.1, 2, 3 and 4
Additional file 2: Figure S2. Diversity index of fermentates generated with and without selective... more Additional file 2: Figure S2. Diversity index of fermentates generated with and without selective conditions. a 16S diversity index. In the presence of antibiotics alpha-diversity, evenness and richness are decreased. b Virome diversity index. Under selective enrichment there is a reduction for each index in parallel with bacteriome reduction. Error bars indicate standard deviation between triplicate fermentations (n = 3).
Additional file 6: Figure S3. Phylogenetic tree of large terminase subunits encoded by published ... more Additional file 6: Figure S3. Phylogenetic tree of large terminase subunits encoded by published complete genomes of crAss-like phages. Protein sequences were aligned using MUSCLE, approximately-maximum-likelihood phylogenetic trees were generated using FastTree. Branch support values calculated using SH-test. Tree tip colours correspond to candidate genera as proposed in [19]. ΦcrAss002 label is highlighted in red. Previously well-characterised uncultured phage genomes and cultured isolates are highlighted in boldface font. Accession numbers of genomes in NCBI GenBank/RefSeq/WGS databases are provided.
Additional file 13: Table S7. Primers specific to the SIHUMI bacterial community, ΦcrAss001 host ... more Additional file 13: Table S7. Primers specific to the SIHUMI bacterial community, ΦcrAss001 host B. intestinalis 919/174, and ΦcrAss002 host B. xylanisolvens APCS1/XY.
Additional file 5: Table S3. Metadata associated with ΦcrAss002 and related phages of candidate g... more Additional file 5: Table S3. Metadata associated with ΦcrAss002 and related phages of candidate genus IV identified by in silico analyses.
Additional file 3: Table S1. Bacterial isolates identified following Sanger sequencing. The isola... more Additional file 3: Table S1. Bacterial isolates identified following Sanger sequencing. The isolates were enriched from subject ID: 924 faeces with the aid of antibiotic selective enrichment to promote Bacteroidales growth. FAA; Fastidious anaerobic agar, YCFA; yeast extract, casitone, fatty acids agar, CBA; Columbia blood agar.
Additional file 8: Figure S5. Transmission electron micrographs showing vesicle-like structures o... more Additional file 8: Figure S5. Transmission electron micrographs showing vesicle-like structures on the surface of B. xylanisolvens APCS1/XY cells. Micrographs were prepared from cross-sections of soft agar collected lawns of B. xylanisolvens APCS1/XY with and without spotting of ΦcrAss002 lysates.
Additional file 1: Figure S1. Graphical representation of the key experimental steps taken in the... more Additional file 1: Figure S1. Graphical representation of the key experimental steps taken in the isolation of ΦcrAss002.
Background The crAss-like phages are ubiquitous and highly abundant members of the human gut viro... more Background The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. Results Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other “benign” forms of phage infection when the host is stably present a...
BackgroundThe gut phageome comprises a complex phage community of thousands of individual strains... more BackgroundThe gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual’s core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible.ResultsFaeces from a healthy donor with high levels of crAss-like phage was used to in...
BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is chara... more BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) post SARS-CoV-2 vaccination is characterized by thrombocytopenia and severe thrombosis. Platelet function during patient recovery in the medium-/long-term has not been investigated fully. Here, we undertook a 3-month study, assessing the recovery of a VITT patient and assessing platelet morphology, granule content and dense-granule release at two distinct time points during recovery.Case PresentationA 61 year-old female was admitted to hospital 15 days post ChAdOx1 nCov-19 vaccination. Hematological parameters and peripheral blood smears were monitored over 3 months. Platelet morphology and granule populations were assessed using transmission electron microscopy (TEM) at two distinct time points during recovery, as was agonist-induced platelet dense-granule release. Upon admission, the patient had reduced platelet counts, increased D-dimer and high anti-PF4 antibodies with multiple sites of cerebral venous sinus thrombosis...
Additional file 7: Figure S4. Multiple site-specific recombinase-encoding genes and evidence of d... more Additional file 7: Figure S4. Multiple site-specific recombinase-encoding genes and evidence of dynamic recombinations in the genome of B. xylanisolvens APCS1/XY. a Circular map of genome (the innermost circle [green and purple], GC skew; circle two [black], relative G+C content; circles three and four [red and dark blue], open reading frames identified on the positive and negative DNA strands respectively; circle five [orange], tRNA and rRNA genes. circle six, genes annotated as Sus-like surface-associated glycan utilisation proteins [green] and TonB-dependent nutrient receptor [light blue]; circle seven [black], genes annotated as invertases, integrases and recombinases). To the right of the genome map, circular maps of the two associated circular plasmids are shown; pBXS1-1 and pBXS1-2. Annotated features are coloured and labelled; b Distribution of length of Oxford Nanopore sequencing reads used for dynamic genome recombination analysis; c Distribution of percentage identity in ...
Additional file 12: Table S6. Primer sequences specific to each crAss-like phage strains detected... more Additional file 12: Table S6. Primer sequences specific to each crAss-like phage strains detected following sequencing of subject ID: 924 faeces post fermentation.
Additional file 2:. Supplementary Dataset. Spreadsheets of numerical data and raw unedited photog... more Additional file 2:. Supplementary Dataset. Spreadsheets of numerical data and raw unedited photographs required for reproduction of Figs.1, 2, 3 and 4
Additional file 2: Figure S2. Diversity index of fermentates generated with and without selective... more Additional file 2: Figure S2. Diversity index of fermentates generated with and without selective conditions. a 16S diversity index. In the presence of antibiotics alpha-diversity, evenness and richness are decreased. b Virome diversity index. Under selective enrichment there is a reduction for each index in parallel with bacteriome reduction. Error bars indicate standard deviation between triplicate fermentations (n = 3).
Additional file 6: Figure S3. Phylogenetic tree of large terminase subunits encoded by published ... more Additional file 6: Figure S3. Phylogenetic tree of large terminase subunits encoded by published complete genomes of crAss-like phages. Protein sequences were aligned using MUSCLE, approximately-maximum-likelihood phylogenetic trees were generated using FastTree. Branch support values calculated using SH-test. Tree tip colours correspond to candidate genera as proposed in [19]. ΦcrAss002 label is highlighted in red. Previously well-characterised uncultured phage genomes and cultured isolates are highlighted in boldface font. Accession numbers of genomes in NCBI GenBank/RefSeq/WGS databases are provided.
Additional file 13: Table S7. Primers specific to the SIHUMI bacterial community, ΦcrAss001 host ... more Additional file 13: Table S7. Primers specific to the SIHUMI bacterial community, ΦcrAss001 host B. intestinalis 919/174, and ΦcrAss002 host B. xylanisolvens APCS1/XY.
Additional file 5: Table S3. Metadata associated with ΦcrAss002 and related phages of candidate g... more Additional file 5: Table S3. Metadata associated with ΦcrAss002 and related phages of candidate genus IV identified by in silico analyses.
Additional file 3: Table S1. Bacterial isolates identified following Sanger sequencing. The isola... more Additional file 3: Table S1. Bacterial isolates identified following Sanger sequencing. The isolates were enriched from subject ID: 924 faeces with the aid of antibiotic selective enrichment to promote Bacteroidales growth. FAA; Fastidious anaerobic agar, YCFA; yeast extract, casitone, fatty acids agar, CBA; Columbia blood agar.
Additional file 8: Figure S5. Transmission electron micrographs showing vesicle-like structures o... more Additional file 8: Figure S5. Transmission electron micrographs showing vesicle-like structures on the surface of B. xylanisolvens APCS1/XY cells. Micrographs were prepared from cross-sections of soft agar collected lawns of B. xylanisolvens APCS1/XY with and without spotting of ΦcrAss002 lysates.
Additional file 1: Figure S1. Graphical representation of the key experimental steps taken in the... more Additional file 1: Figure S1. Graphical representation of the key experimental steps taken in the isolation of ΦcrAss002.
Background The crAss-like phages are ubiquitous and highly abundant members of the human gut viro... more Background The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. Results Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other “benign” forms of phage infection when the host is stably present a...
BackgroundThe gut phageome comprises a complex phage community of thousands of individual strains... more BackgroundThe gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual’s core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible.ResultsFaeces from a healthy donor with high levels of crAss-like phage was used to in...
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