Culturable and Non-Culturable Blood Microbiota of Healthy Individuals

Abstract

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.

Human Blood Bacteriome: Eubiotic and Dysbiotic States in Health and Diseases

“…Whole blood may be the most representative specimen for the term blood bacteriome because it consists of all elements of the blood. The most abundant phylum of whole blood bacteriome in several studies was Proteobacteria, followed by Firmicutes and Actinobacteria [13,14,21,[23][24][25]28]. Unexpectedly, Blood cell components may favor Proteobacteria as they occupied the highest proportion of bacteriome among types of the blood specimens [22,24,25,[28][29][30][31][32].…”

“…Thereby, the blood microbiome may be an appropriate term at the present time instead of blood microbiota, which is still controversial. Most studies into the blood microbiome focused on the bacteriome, and the archeome, virome, and mycobiome were scantly explored [13][14][15].…”

New Paradigms for Familiar Diseases: Lessons Learned on Circulatory Bacterial Signatures in Cardiometabolic Diseases

“…Routinely, microbiome studies will usually consist of the following procedures before any analytical pipeline can be implemented: sample collection, DNA extraction, and sequencing library preparation (possibly with or without bacterial enrichment techniques such as culture [39,126,127] or bacterial DNA enrichment), highthroughput sequencing technologies [128] such as amplicon-based sequencing (e. g., 16 S rRNA gene), shotgun metagenomic sequencing [129], as well as RNA sequencing-based approaches [38]. Because procedural controls, which take contamination of samples during collection and cross-contamination during processing and sequencing into account, are rarely implemented, studies in low microbial biomass samples relying on sensitive metagenomic techniques highly confound and inflate the diagnostic implications for microbial contribution to disease [130].…”

“…In the meantime, a commendable body of work has combined classic molecular and microbiological techniques to evidence the presence of bacterial cells and sequences in the blood of presumably healthy humans, either as a study cohort or as healthy controls for specific disease states (recently reviewed in 26 ). These methods include quantitative polymerase chain reaction (qPCR) 27 28 29 30 31 32 33 34 35 of 16 S rRNA or targeted bacterial genes 29 35 , fluorescence in-situ hybridization (FISH) and other employment of fluorescent probes 35 , transmission electron microscopy 36 , dark field microscopy 36 , PCR followed by electrospray ionization-mass spectrometry 37 as well as classical bacterial culture 32 38 39 . Several of these results have been met with criticism, considering the long-standing dogma of the blood being an innocuous environment and controversy regarding visual confirmation of bacteria deemed by some authors as L-form bacteria and by others as merely membrane vesicles or aggregated proteins 40 .…”

Another Look at the Contribution of Oral Microbiota to the Pathogenesis of Rheumatoid Arthritis: A Narrative Review

“…Those bacteria cannot usually be cultivated, although some may grow slowly in aerobic or anaerobic cultures even in healthy subjects [68]. In this respect, some authors advocated the use of testing a medium supplemented with a high concentration of vitamin K (1 mg/mL), and culturing at 43 • C for 24 h [70,71], to demonstrate that the bacterial DNA found in blood is not just cell-free DNA, but belongs to still alive bacteria and fungi, some being able to multiply in the erythrocytes [70,71].…”