Experimental tests challenge the evidence of a healthy human blood microbiome.

The advent of next-generation sequencing (NGS) technologies has made it possible to investigate microbial communities in various environments, including different sites within the human body. Therefore, the previously established belief of the sterile nature of several body sites, including human blood, has now been challenged. However, metagenomics investigation of areas with an anticipated low microbial biomass may be susceptible to misinterpretation. Here, we critically evaluate the results of 16S targeted amplicon sequencing performed on total DNA collected from healthy donors' blood samples while incorporating specific negative controls aimed at addressing potential bias to supplement and strengthen the research in this area. We prepared negative controls by increasing the initial DNA quantity through sequences that can be recognized and subsequently discarded. We found that only three organisms were sporadically present among the samples, and this was mostly attributable to bacteria ubiquitously present in laboratory reagents. Despite not fully confirming or denying the existence of healthy blood microbiota, our results suggest that living bacteria, or at least their residual DNA sequences, are not a common feature of human blood in healthy people. Finally, our study poses relevant questions on the design of controls in this research area that must be considered in order to avoid misinterpreted results that appear to contaminate current high-throughput research.