Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis

Abstract

Prompt identification of causative pathogenic bacteria is imperative for the treatment of patients suffering from infectious diseases, including sepsis and pneumonia. However, current culture-based methodologies have several drawbacks including their limitation of use to culturable bacterial species. To circumvent these problems, we attempted to detect bacterial DNA in blood using next-generation DNA sequencing (NGS) technology. We conducted metagenomic and 16S ribosomal RNA (rRNA) gene amplicon sequencing of DNA extracted from bacteria-spiked blood using an Ion Personal Genome Machine. NGS data was analyzed using our in-house pipeline Genome Search Toolkit and database GenomeSync. The metagenomic sequencing analysis successfully detected three gram-positive and three gram-negative bacteria spiked in the blood, which was associated with a significant portion of non-bacterial reads, even though human blood cells were separated by low-speed centrifugation prior to DNA extraction. Sequencing analysis of seven variable regions of the 16S rRNA gene amplicon also successfully detected all six bacteria spiked in the blood. The methodology using 16S rRNA gene amplicon analysis was verified using DNA from the blood of six patients with sepsis and four healthy volunteers with potential pathogenic bacteria in the blood being identified at the species level. These findings suggest that our system will be a potential platform for practical diagnosis in the future.

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?

“…In the literature, there are no reports on the detection of bacteria directly in the blood using FISH, apart from the studies by our team, so we cannot compare the obtained results with other studies [13,20]. With the use of FISH, a high percentage of genetic material of Gram-negative bacteria in the blood samples of healthy individuals was detected, which would argue for the translocation of bacteria into the bloodstream and, generally, for the existence of bacterial DNAemia phenomenon, in completely healthy people [39][40][41]. The presence of bacterial ribosomal RNA signals the presence of bacterial cells, hence, also their DNA, since ribonucleic acid degrades very quickly, in contrast to deoxyribonucleic acid.…”

Rapid sequencing‐based diagnosis of infectious bacterial species from meningitis patients in Zambia

“…water) in our previous sequencing analyses. 24 In addition, as for the other bacterial species found in the Sample #12, Deinococcus proteolyticus, these are commonly found from materials, surfaces and dust contaminated by humans and animals as well as soil and sewage. 25 Therefore, these detected bacterial species in the Sample #12 likely reflect human contamination.…”

The heart microbiome of insectivorous bats from Central and South Eastern Europe

“…were found (Hornok et al 2019). Identification of infectious bacteria from the bloodstream, particularly in humans are usually performed using blood cultures, but different studies compared this method with the 16S metagenomics, showing that this later technique is more sensitive and specific (Decuypere et al 2016, Rutanga et al 2018, Watanabe et al 2018]. The blood tested for those studies was fresh however there are no studies that have tested the sensitivity in partly degraded heart or heart tissue.…”