Bood acqusition and bacterial spiking procedure
The project was approved by the Ethical committee of University Hospital Bratislava, Ružinov, decision No. EK/206/2019. All involved participants provided informed consent. The research was conducted in accordance with the Declaration of Helsinki. The blood collection was performed by medical personnel from 4 healthy young volunteers. In total, 72 ml of blood from each healthy individual were collected into K3EDTA blood collection tubes (Polaris method, Molzym method, Control sample) and serum collection tubes (Centrifugation method). The collected blood was aliquoted to obtain triplicates for each method. Non-spiked blood was used as a negative control. Escherichia coli CFT 073 and Staphylococcus aureus have been cultured overnight (approx. 20h) in LB broth (Sigma-Aldrich) prior to the spiking procedure. To determine the overall bacterial concentration, the optical density of bacterial suspension was measured at 600 nm (OD600). Afterward, the bacterial suspensions were diluted to concentrations from 104 to 101 colony forming units per milliliter (CFU /ml). Subsequently, these diluted samples were plated on the LB agar, and incubated overnight for retrospective validation. One ml of 1000 CFU /ml of each bacterial suspension was further spiked into the 9 ml serum blood collection tubes, and 0.375 ml of each bacterial suspension was spiked into the 3 ml K3EDTA blood collection tubes to achieve a final concentration of 100 CFU /ml of the whole sample. To simulate a real-life environment during which the sample needs to reach the laboratory, the samples were incubated for 30 minutes and administered for further processing (Figure 12). Spiked blood samples were plated on the LB agar and incubated overnight as a control for successful spiking process. All steps of the spiking process were performed under sterile conditions under the laminar flow hood.
Spiked blood in the serum collection tube was centrifuged for 2 000 g for 10 minutes. Afterward, the supernatant was collected without disturbing the middle layer, placed into sterile 5 ml Eppendorf tubes, and centrifuged at 20 000 g for 10 minutes. Then, the supernatant was carefully discarded, and the pellet was resuspended in 200 μl of sterile PBS. Subsequently, DNA isolation was performed.
For the Molzym DNA separation method, the whole treatment of the samples was performed according to the manufacturer’s instructions from the MolYsis ™ Complete5 kit (Molzym GmbH, Germany). Briefly, the host cells in a blood sample were lysed by adding a chaotropic buffer, while microbial cells remained unaffected. Then, the DNA released from host cells and dead cells were degraded by Molzym's MolDNase B. The suspension was further centrifuged. The pellet composed of microbial cells was then treated with BugLysis reagents to degrade cell walls of Gram-negative bacteria and Gram-positive bacteria and then digested by Proteinase K. The microbial DNA was extracted by using the spin column and then isolated by a specific buffer, 70% ethanol wash and final elution with deionized water. Finally, the isolation of DNA was carried out according to the manufacturer's instructions.
The Polaris method was performed according to published study . Briefly, spiked blood from the K3EDTA collection tube was divided into 1 ml triplicates. Subsequently, 2 ml of Polaris lysis solution composed of 500 mM sodium bicarbonate and 1% Triton-X (Sigma-Aldrich, USA) were added. The mixture was vigorously mixed for 5 seconds and incubated at room temperature for 5 min. After incubation, a Polaris neutralizing solution composed of 1 M Tris-HCl was added, and tubes were centrifuged at 2800 g for 10 minutes. The supernatant was carefully discarded, and 1 ml of sterile PBS was added to the pellet, and centrifugation at 20 000 g for 10 minutes followed. After the last centrifugation step, 200 μl of sterile PBS was added to the pellet, and the suspension was ready for DNA extraction.
Standard treatment (Qiagen method)
Two hundred μl of the whole blood was treated with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Bacterial DNA isolation
Extraction of total DNA was carried out using Qiagen DNA Mini Kit (Qiagen, Hilden, Germany). Additionally, for the repeated experiment with S. aureus, a lysozyme enzyme solution (Sigma-Aldrich, USA) was added to the suspension prior to Qiagen isolation according to manfucaturer instrucions for DNA from bacteria isolation.
Bacterial and Genomic DNA quantification
Bacterial 16s RT-qPCR
TaqMan primers (V7-V9 regions of 16S rRNA) and the probe (Microsynth, Switzerland) were used to detect bacterial DNA (Supplementary Table S1). A twenty μl reaction mixture was composed of 1 μl of each primer (0.5 μM), 0.5 μl of the probe (0.25 μM), 10 μl of TaqMan Fast Advance Master Mix (AppliedBiosystems, USA), 3.5 μl of distilled water, and 4 μl of the template sample. RT-qPCR program was set up to pre-incubation at 50 °C for 2 minutes, initial denaturation at 90 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds, annealing at 60 °C for 30 seconds and extension at 72 °C for 65 °C. Analysis was carried out on the Mastercycler Realplex 4 (Eppendorf, Germany).
For detection of eukaryotic DNA, Beta-2-microglobulin as a housekeeping gene was used (Supplementary Table S2). Ten μl reaction was set up, composed of 5 μl of SsoAdvanced universal SYBR® Green supermix (2x), 0.25 μl of forward and reverse primer (0.25 μM) (Bio-Rad, USA), 2 μl of PCR grade water, and 2.5 μl of template DNA. Cycling conditions were set up as follows: initial denaturation at 98 °C for 3 minutes followed by 40 cycles of denaturation at 98 °C for 15 seconds, annealing at 51 °C for 30 seconds, and extension at 60 °C for 30 seconds. After the end of the qPCR, all amplicons were melted to observe the melting curve of the present amplified products. The RT-qPCR analysis was performed on the Mastercycler Realplex 4 (Eppendorf, Germany).
All statistical analyses were performed using Graphpad prism 8.0.1 (La Jolla, California, USA). Non-parametric Kruskal-Wallis test with subsequent Dunn post-hoc correction tests was performed to analyze differences between the groups. P-value <0.05 was considered significant. Data are presented as mean +/- SD.