The Origin of Plasma-Derived Bacterial Extracellular Vesicles in Healthy Individuals and Patients with Inflammatory Bowel Disease: A Pilot Study

The gastrointestinal tract harbors the gut microbiota, structural alterations of which (dysbiosis) are linked with an increase in gut permeability (“leaky gut”), enabling luminal antigens and bacterial products such as nanosized bacterial extracellular vesicles (BEVs) to access the circulatory system. Blood-derived BEVs contain various cargoes and may be useful biomarkers for diagnosis and monitoring of disease status and relapse in conditions such as inflammatory bowel disease (IBD). To progress this concept, we developed a rapid, cost-effective protocol to isolate BEV-associated DNA and used 16S rRNA gene sequencing to identify bacterial origins of the blood microbiome of healthy individuals and patients with Crohn’s disease and ulcerative colitis. The 16S rRNA gene sequencing successfully identified the origin of plasma-derived BEV DNA. The analysis showed that the blood microbiota richness, diversity, or composition in IBD, healthy control, and protocol control groups were not significantly distinct, highlighting the issue of ‘kit-ome’ contamination in low-biomass studies. Our pilot study provides the basis for undertaking larger studies to determine the potential use of blood microbiota profiling as a diagnostic aid in IBD.

Culturable and Non-Culturable Blood Microbiota of Healthy Individuals

Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals.

Blood microbiota composition in Iranian pre-diabetic and type 2 diabetic patients

BACKGROUND: Type 2 diabetes as the most prevalent metabolic disorder, is one of the major causes of morbidity and mortality worldwide. Recent studies suggest that body microbiota may play a role in developing metabolic disorders including type 2 diabetes. The objective of the present study was to investigate the blood microbiota composition in Iranian pre-diabetic and type 2 diabetic patients compared to healthy individuals. METHODS: Blood samples were taken after 12-h fasting from 90 participants, 30 healthy individuals, 30 type 2 diabetes patients and 30 pre-diabetic participants. The buffy coat layer separated by centrifugation at 800 and DNA was extracted using a column-based method. Composition and load of blood microbiota was evaluated by real-time PCR method using genus specific 16S rRNA primers. RESULTS: The load of Akkermansia, and Faecalibacterium was higher in normal volunteers compared to pre-diabetic and type 2 diabetes group (p< 0.05). The load of Bifidobacterium was higher in normal volunteers compared to type 2 diabetes patients (p= 0.02). In contrast, the load of Lactobacillus and Escherichia coli was higher in pre-diabetics and type 2 diabetes patients compared to normal volunteers (p< 0.05). The load of Bacteroides fragilis was not statistically different between studied groups but it was higher in males compared to female group (p= 0.04). the load of other bacteria was not significantly different between male and female participants. CONCLUSION: There is difference between microbiota composition in white blood cells of pre-diabetic and type 2 diabetes patients compared to healthy people. Determination of blood microbiota pattern may have a role in diagnosis and preventive of type 2 diabetes in a certain population. For more clarification about correlation between blood microbiota and type 2 diabetes, larger studies with more participants in different ethnical populations is suggested.

Identification of Lung and Blood Microbiota Implicated in COVID-19 Prognosis

The implications of the microbiome on Coronavirus disease 2019 (COVID-19) prognosis has not been thoroughly studied. In this study we aimed to characterize the lung and blood microbiome and their implication on COVID-19 prognosis through analysis of peripheral blood mononuclear cell (PBMC) samples, lung biopsy samples, and bronchoalveolar lavage fluid (BALF) samples. In all three tissue types, we found panels of microbes differentially abundant between COVID-19 and normal samples correlated to immune dysregulation and upregulation of inflammatory pathways, including key cytokine pathways such as interleukin (IL)-2, 3, 5-10 and 23 signaling pathways and downregulation of anti-inflammatory pathways including IL-4 signaling. In the PBMC samples, six microbes were correlated with worse COVID-19 severity, and one microbe was correlated with improved COVID-19 severity. Collectively, our findings contribute to the understanding of the human microbiome and suggest interplay between our identified microbes and key inflammatory pathways which may be leveraged in the development of immune therapies for treating COVID-19 patients.

Microbiota dysbiosis and functional outcome in acute ischemic stroke patients

Currently, few studies are reported on the composition of microbiota in stroke patients and the association with stroke prognosis. This study investigated the differing microbiota composition in stroke patients and confirmed the association of microbiota composition with poor functional outcome. Between January of 2018 and December of 2019, 198 patients with acute cerebral infarction were included in this study. For the case–control study, age and sex-matched normal healthy subjects (n = 200) were included when receiving their health screening examinations. We isolated bacterial extracellular membrane vesicles and extracted DNA from blood samples. Taxonomic assignments were performed by using the sequence reads of 16S rRNA genes following blood microbiota analysis. Statistical analysis was conducted appropriately by using Statistical Analysis System software. The mean age of the stroke patients were 63.7 ± 12.5 years, and the male sex was 58.5%. Of the total enrolled patients, poor functional outcome (modified Rankin Score ≥ 3) was noted in 19.7%. The principal component analysis of microbiota composition revealed significant differences between healthy control subjects and stroke patients. At the genus level, Aerococcaceae(f), ZB2(c), TM7-1(c), and Flavobacterium were significantly increased in stroke patients compared to the healthy controls, whereas Mucispirillum, rc4-4, Akkermansia, Clostridiales(o), Lactobacillus, and Stenotrophomonas were decreased considerably. For the functional outcome after ischemic stroke, Anaerococcus, Blautia, Dialister, Aerococcaceae(f), Propionibacterium, Microbacteriaceae(f), and Rothia were enriched in the group with good outcomes, whereas Ruminococcaceae(f) and Prevotella were enriched in the group with poor outcome. There was apparent dysbiosis of blood microbiota in patients with acute ischemic stroke compared to healthy people. Ruminococcaceae(f) and Prevotella were elevated in stroke patients with poor functional outcome.

Influence of periodontal treatment on blood microbiotas: a clinical trial

Objective To investigate the effects of periodontal treatment on the abundance and diversity of blood microbiota. Methods and Materials Twenty-seven periodontitis patients were randomly allocated to a control group (A) and two test groups (B1 and B2). Group A patients received full-mouth scaling and root planing (SRP), group B1 patients received subgingival glycine air polishing (GAP) right after SRP, and group B2 patients received subgingival glycine air polishing right before SRP. Peripheral blood samples were obtained at the baseline, the day after periodontal treatment, and 6 weeks after treatment and evaluated using nested polymerase chain reaction and 16SrRNA Gene Sequencing (Miseq platform). Results All participants exhibited significant improvements in the clinical parameters evaluated at the 6-week follow-up visit compared to the values at the baseline, but no significant differences were observed between the three groups. The total bacterial count was lowest in group B2. The bacterial species diversity (α-diversity) in group B1 was significantly higher (Chao-1 index, P = 0.03) and Porphyromonas and Pantoea were the dominant genera (linear discriminant analysis (LDA > 2)) in this group the day after treatment compared to the baseline. No significant difference was detected in the relative abundance and α-diversity of blood microbiota between the baseline and 6 weeks after treatment. Conclusion Local periodontal treatment merely disrupts the stability of blood microbiota in the short term. Periodontitis treatment using full-mouth SRP followed by adjunctive GAP is a promising approach to reduce the introduction of bacteria into the bloodstream during the procedure.

Blood Microbiome: A New Marker of Gut Microbial Population in Dogs?

The characterization of the microbial population in different compartments of the organism, such as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field have already investigated the fecal microbiome in healthy or sick subjects; however, the methodologies used in the different laboratories and the limited number of animals recruited in each experiment do not allow a straight comparison among published results. Previously, our research focused on the characterization of the microbial taxa variability in 340 fecal samples from 132 healthy dogs, collected serially from several in-house experiments. The results supported the responsiveness of microbiota to dietary and sex factors and allowed us to cluster dogs with high accuracy. For the present study, intestinal and blood microbiota of healthy dogs from different breeds, genders, ages and food habits were collected, with three principal aims: firstly, to confirm the results of our previous study regarding the fecal microbiome affected by the different type of diet; secondly, to investigate the existence of a blood microbial population, even in heathy subjects; and thirdly, to seek for a possible connection between the fecal and the blood microbiota. Limited researches have been published on blood microbiota in humans, and this is the first evidence of the presence of a bacterial population in the blood of dogs. Moreover, gut and blood microbiota can discriminate the animals by factors such as diet, suggesting some relationship between them. These preliminary results make us believe in the use of the blood microbiome for diagnostic purposes, such as researching and preventing gut inflammatory diseases.