Blood microbiome: controlling contamination for reliable data

Circulating microbiome advances rely on strong contamination-aware metagenomics using negative controls

Schierwagen and colleagues (2019) described the circulating microbiome from different compartments accessible during the implantation of a transjugular intrahepatic portosystemic shunt (TIPS). The portal vein (first venous outflow in gut–liver axis), liver outflow, central venous blood and peripheral venous blood were analyzed using Vaiomer’s approach for low bacterial biomass samples. Taxonomic profiles showed blood compartment-specific bacterial signatures. Inflammatory cytokines were also correlated to bacterial genera showing again the link between blood microbiome and systemic inflammation. Aerobic and anaerobic cultivations were performed and isolates belonged to Staphylococcus and Acinetobacter. Both bacterial genera were abundantly represented in the sequencing results demonstrating that at least some members of the blood microbiota are viable.

Although exciting, these results raised concerns. Hornung and colleagues published a response to Schierwagen’s study in 2020. They compared the blood microbiome profile to the previously published description of common kit contaminants and established that they were extremely similar. Therefore this shed doubt on the validity of the findings from Schierwagen’s paper, especially as Schierwagen and colleagues (2019) did not report negative controls.

Although not initially reported, Schierwagen and colleagues (2020) did perform such negative controls as they were encompassed within Vaiomer’s targeted metagenomic approach and included in the DNA extraction and amplification steps. Negative controls showed over a 1000-fold difference in 16S rRNA gene copies with the biological samples and distinct taxonomic profiles. This analysis of negative controls is more important than using a fixed list of bacterial contaminants. Indeed, each experiment has its own contaminants and two different experiments done under different conditions cannot be compared. Also, Vaiomer’s approach is dedicated to low bacterial biomass samples and so it is unlikely to find the same contaminants as studies that did not perform the same extensive optimizations and developments.

These three papers highlight two important concepts when dealing with low bacterial biomass samples: 1) although useful to understand the burden of contamination a fixed list of common bacterial contaminants cannot be blindly applied to every study and 2) negative controls are essential to prove reliable and trustworthy data.

CITATIONS

Circulating microbiome in blood of different circulatory compartments

In collaboration with Vaiomer

Schierwagen R, Alvarez-Silva C, Madsen MSA, Kolbe CC, Meyer C, Thomas D, Uschner FE, Magdaleno F, Jansen C, Pohlmann A, Praktiknjo M, Hischebeth GT, Molitor E, Latz E, Lelouvier B, Trebicka J, Arumugam M. Gut. 2019 Mar;68(3):578-580. doi: 10.1136/gutjnl-2018-316227.

No abstract available.

Response to: ‘Circulating microbiome in blood of different circulatory compartments’ by Schierwagen et al

Hornung BVH, Zwittink RD, Ducarmon QR, Kuijper EJ. Gut. 2020 Apr;69(4):789-790. doi: 10.1136/gutjnl-2019-318601. Epub 2019 Apr 6. PMID: 30954950.

No abstract available.

Trust is good, control is better: technical considerations in blood microbiome analysis

In collaboration with Vaiomer

Schierwagen R, Alvarez-Silva C, Servant F, Trebicka J, Lelouvier B, Arumugam M. Gut. 2020 Jul;69(7):1362-1363. doi: 10.1136/gutjnl-2019-319123. Epub 2019 Jun 15. PMID: 31203205; PMCID: PMC7306979.

No abstract available.

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