Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia

Abstract

Blood is considered to be a sterile microenvironment, in which bacteria appear only periodically. Previously used methods allowed only for the detection of either viable bacteria with low sensitivity or selected species of bacteria. The Next-Generation Sequencing method (NGS) enables the identification of all bacteria in the sample with their taxonomic classification. We used NGS for the analysis of blood samples from healthy volunteers (n = 23) and patients with sepsis (n = 62) to check whether any bacterial DNA exists in the blood of healthy people and to identify bacterial taxonomic profile in the blood of septic patients. The presence of bacterial DNA was found both in septic and healthy subjects; however, bacterial diversity was significantly different (P = 0.002) between the studied groups. Among healthy volunteers, a significant predominance of anaerobic bacteria (76.2 %), of which most were bacteria of the order Bifidobacteriales (73.0 %), was observed. In sepsis, the majority of detected taxa belonged to aerobic or microaerophilic microorganisms (75.1 %). The most striking difference was seen in the case of Actinobacteria phyla, the abundance of which was decreased in sepsis (P < 0.001) and Proteobacteria phyla which was decreased in the healthy volunteers (P < 0.001). Our research shows that bacterial DNA can be detected in the blood of healthy people and that its taxonomic composition is different from the one seen in septic patients. Detection of bacterial DNA in the blood of healthy people may suggest that bacteria continuously translocate into the blood, but not always cause sepsis; this observation can be called DNAemia.Electronic supplementary materialThe online version of this article (doi:10.1007/s10096-016-2805-7) contains supplementary material, which is available to authorized users.

Applications of sequencing technology in clinical microbial infection

“…Among the healthy volunteers, anaerobic bacteria accounted for the main proportion (76.2%) of bacterial species, and the abundance of Actinobacteria phyla was significantly higher than that in patients with sepsis (76.3% in healthy volunteers and 31.0% in the sepsis patients), while among the sepsis patients, most species (75.1%) were aerobic or microaerobic microorganisms, and the abundance of phyla in the Proteobacteria was significantly higher in patients with sepsis than in the healthy volunteers (16.4% in healthy volunteers and 60.1% in sepsis patients). 51 Grumaz et al 52 The specific technical problems in the diagnosis of pathogens by mNGS include the following. First, mNGS is limited to roughly judging the species of pathogenic microorganisms and estimating the approximate proportion of those microorganisms, and the results of different tests or from different labs may vary.…”

Cultural Isolation and Characteristics of the Blood Microbiome of Healthy Individuals

“…On the bases of sampling 242 healthy adults at 18 different anatomical sites of the human body and sequencing the 16S rRNA genes the presence of 5177 microbial taxonomic profiles was proven, but only 800 of them could be cultured [1]. The observation of cell-free DNAemia is well described feature of the healthy blood [2] [3], but the presence of transient culturable blood microbiota in the blood of healthy individuals could also be supposed. We tested whether the presence of DNA in healthy blood is associated with DNAemia or it is due to existing blood microbiota.…”

“…Kalfin reported 100% positivity of the blood cultures. Subsequently several other authors questioned the existence of the blood microbiome or DNAemia in healthy individuals, Domingue (1977) [5], (1997) [7], Nikkari et al (2001) [8], Mc Laughlin et al (2002) [9], Moriyama et al (2008) [10], Markova (2015) [11], Damgaard (2015) [12] Dimova et al (2017) [13], Gosiewski et al (2017) [2] and Kowarsky et al (2017) [3]. The authors reported supporting electron microscopy, cultural and molecular data in favour of the existence of the blood microbiota in healthy individuals.…”

“…Most of the DNA extraction kits: Tissue and cells genomic Prep kit and Blood genomic Prep kit, (GE, USA); Genomic DNA purification kit, (Thermo Fisher Scientific, USA); QIAamp DNA isolation kit, (Qiagen, Germany) and the standard CTAB phenol/chlorophorm DNA extraction protocol were not successful for DNA extraction of the cultured blood microbiota. Successful DNA extrac- 2 O and incubated for 30 min at 37˚C in order to eliminate residual DNA contaminations associated with lysed nuclear blood cells, DNA from the culture medium or DNA traces in water. Centrifugation and washings were repeated three times.…”

Development of a Universal Fluorescent Probe for Gram‐Positive Bacteria

“…[1][2][3][4] Classification of bacteria is the fundamental first step for bacterial taxonomy determination and is mainly based on the differences in cellular composition, cellular metabolism, and cellular structure.G enetic analysis approaches,such as PCR (polymerase chain reaction), [5] FISH (fluorescence in situ hybridization), [6] and NGS (next generation sequencing) [7,8] are powerful techniques for the identification of bacteria, but these are often inappropriate in clinical diagnosis and wastewater treatment, which require ar apid diagnosis and in situ monitoring, due to the heavy preparative work and the huge amount of data required for in-depth analysis . Gram staining has been the gold standard technique for bacterial classification based on the different physical properties of the bacterial cell-wall structure. [1][2][3][4] Classification of bacteria is the fundamental first step for bacterial taxonomy determination and is mainly based on the differences in cellular composition, cellular metabolism, and cellular structure.G enetic analysis approaches,such as PCR (polymerase chain reaction), [5] FISH (fluorescence in situ hybridization), [6] and NGS (next generation sequencing) [7,8] are powerful techniques for the identification of bacteria, but these are often inappropriate in clinical diagnosis and wastewater treatment, which require ar apid diagnosis and in situ monitoring, due to the heavy preparative work and the huge amount of data required for in-depth analysis . Gram staining has been the gold standard technique for bacterial classification based on the different physical properties of the bacterial cell-wall structure.…”