Clinical specimens
Whole blood samples were collected from patients with suspected sepsis at Toyama University Hospital and Nagaresugi Geriatric Hospital. All procedures were performed under a protocol approved by the Ethics Committee at the University of Toyama and Nagaresugi Geriatric Hospital, and written informed consent was obtained from all patients. The methods were carried out in accordance with the approved guidelines.
Steps in the novel pretreatment procedure
For the following steps, all steps were performed at room temperature unless the temperature was specifically described.
- Pelletizing platelets and bacteria from whole blood: A total of 5 mL of venous blood was collected in plasma separation EDTA tubes (Vacutainer® PPTTM Plasma Preparation Tube; BD Biosciences, CA, USA). The blood samples were then centrifuged at 1100×g for 10 minutes to spin down the blood cells, and the resulting supernatant fractions (2 mL), which were plasma and bacteria, were used. The supernatants were equally divided into two parts (1 ml each) and were centrifuged again at 2,000×g for 10 minutes, and then 900 μL of the supernatant fraction was removed, with particular care taken to avoid disturbing the pellets.
- Proteolysis of the platelet plasma membrane: Next, 1 mL (10% w/v) of Aroase NP-10 (Yakult Pharmaceutical Industry, Tokyo, Japan) dissolved in molecular-grade distilled water (UltraPureTM DNase/RNase-Free Distilled Water; Thermo Fisher Scientific, MA, USA) was added to the pellets, and the mixture was pipetted 10 times and subsequently incubated at 37 °C for 10 minutes. After incubation, the mixture was centrifuged again at 2,000×g for 5 minutes, and then 1 mL of the supernatant fractions was removed, with particular care taken to avoid disturbing the pellets.
- Osmotic swelling of platelets: Next, 800 μL of hypotonic solution at pH 7.9, which consists of 10 mM HEPES at pH 7.9 (DOJINDO Molecular Technologies, Tokyo, Japan), 1.5 mM KCl (Wako Pure Chemical Industries, Tokyo, Japan) and 10 mM MgCl2·6H2O (Wako Pure Chemical Industries) dissolved in molecular-grade distilled water (UltraPureTM DNase/RNase-Free Distilled Water; Thermo Fisher Scientific) and then autoclaved, was added to the pellets, and the mixture was pipetted 10 times and subsequently incubated at room temperature for 5 minutes.
- Destruction of the platelet plasma membrane: After incubation, 200 μL of Detergent A, which consists of 2% saponin (SERVA Electrophoresis GmbH, Heidelberg, Germany) and 0.05% SDS (Wako Pure Chemical Industries) dissolved in 1× phosphate-buffered saline (PBS) at pH 7.4 (Thermo Fisher Scientific), was added to the pellets. The mixture was pipetted 10 times and subsequently centrifuged at 2,000×g for 5 minutes, and then 1 mL of the supernatant fractions was removed, with particular care taken to avoid disturbing the pellets. Next, 1 mL of Detergent A was added again to the pellets, and the mixture was pipetted 20 times and subsequently incubated at room temperature for 15 minutes.
- Pelletizing bacteria with minimal ATP background of human origin: After incubation, the mixture was centrifuged at 2,000×g for 5 minutes, and 1 mL of the supernatant fractions was removed, with particular care taken to avoid disturbing the pellets. Next, 1 mL of Muller-Hinton medium (Muller-Hinton II broth, cation-adjusted; BD Biosciences) was added to the pellets, and the mixture was gently turned upside down several times and subsequently centrifuged again at 2,000×g for 5 minutes. After centrifugation, 1 mL of the supernatant fraction was removed, with particular care taken to avoid disturbing the pellets. This pellet-washing process was then repeated again, with 1 mL of Muller-Hinton medium added to the pellets and the mixture gently turned upside down several times before being centrifuged again at 2,000×g for 5 minutes. After centrifugation, 1 mL of the supernatant fraction was removed, with particular care taken to avoid disturbing the pellets. The final product obtained was the bacterial pellet from whole blood with minimal ATP background of human origin.
Shaking culture in the presence or absence of antibacterial drugs
After the pretreatment procedure, 1 mL of Muller-Hinton medium was added to the pellets. These mixtures were then pooled and equally divided into two parts again. Shaking culture was then performed in the presence or absence of LVFX (Sigma-Aldrich, St. Louis, Missouri, USA) 2.0 μg/ml at 37 °C for 0, 2, 4, 6, 12 and 24 h.
ATP measurement of viable bacteria by ATP bioluminescence
In the following processes, the automated ATP bioluminometer provided an automated ATP measurement. The automated ATP bioluminometer is the prototype model developed by Hitachi, Ltd. (Tokyo, Japan) as an attomole-level ATP bioluminometer for detecting a single bacterium11.
Concerning reagents, the CheckLiteTM HS set (Kikkoman Biochemifa, Tokyo, Japan) and ATP standard sample (Kikkoman Biochemifa) were used. The CheckLiteTM HS set has an ATP eliminating reagent, ATP extracting reagent and bioluminescence reagent. Molecular-grade distilled water (UltraPureTM DNase/RNase-Free Distilled Water; Thermo Fisher Scientific) was used as sterile water for the washing solution and the dilution buffer to make dilution series of bacterial samples and ATP standard samples, respectively.
Regarding the ATP calibration curve, 2 dilution series of ATP standard sample (Kikkoman Biochemifa) were prepared: 100 to 2×109 amol/mL of ATP standard sample was prepared by dilution with sterile water, and 100 to 500 amol/mL of ATP standard sample was prepared by dilution with sterile water or ATP extracting reagent. The sterile water or ATP extracting reagent was used as blank samples. ATP bioluminescence was measured in the following steps: first, 10 μL of ATP standard sample diluted with sterile water or ATP extracting reagent in a reaction tube and 1 mL of bioluminescence reagent in a reaction tube were manually set in the automated ATP bioluminometer; 50 μL of bioluminescence reagent was then added to the sample by the ATP bioluminometer.
Finally, concerning the procedure for intracellular ATP measurement of bacteria, ATP eliminating reagent was diluted to 20% (v/v) with PBS at first. Next, 12.5 μL of 20% (v/v) ATP eliminating reagent was added to 12.5 μL of the bacterial sample after shaking culture. To remove extracellular ATP, the bacterial sample was incubated for 30 minutes, and then 25 μL of ATP extracting reagent was added to the bacterial sample or blank sample as a reference. To extract intracellular ATP, the samples were incubated for 1 minute. A total of 10 μL of each sample after the extraction of intracellular ATP was added to a reaction tube and set in the ATP bioluminometer. Bacterial intracellular ATP in the sample was mixed with bioluminescence reagent and measured for 30 seconds. Relative light intensities (RLUs) were calculated as the mean value for 5 seconds from the maximum intensity within 30 seconds after mixing the sample and bioluminescence reagent. Total bacterial intracellular ATP (amol) was determined by dividing the ATP bioluminescence (RLU) of the sample by the slope of ATP calibration curve (RLU/amol).
Measuring the recovery rates of viable bacteria after the pretreatment procedure
All 19 bacterial species had been detected in Toyama University Hospital within the past 6 months. The bacteria were obtained from clinical samples and identified by the MicroScan WalkAway system (Siemens Healthcare Diagnostics, Munich, Germany).
Prior to performing the measurement of recovery rates, bacteria were cultivated in Mueller-Hinton Broth at 37 °C for 12 h, and 2 bacterial suspensions (1 mL) were prepared. The pretreatment procedure was then performed for one bacterial suspension and not performed for the other one.
To measure the recovery rates of viable bacteria after the pretreatment procedure, ATP were measured in triplicate before and after the procedure using ATP bioluminescence as described above, and then recovery rates were calculated.
To measure the recovery rates of viable bacteria after the pretreatment procedure using the conventional colony-counting method, 50 µL of the diluted suspensions was inoculated onto Standard Methods BD Agar/Plate Count Agar (Fisher Scientific, Waltham, Massachusetts, USA) before and after the pretreatment procedure. After incubation at 35 °C for 12 h, the number of colony-forming units (CFU) was determined by counting the colonies grown on the agar plates in triplicate, and recovery rates were calculated.
Culture-based biochemical identification of bacteria
The whole blood samples (one aerobic and one anaerobic blood culture bottle) were collected simultaneously with the blood sample for Tm mapping identification and quantification from the same puncture site. The whole blood samples were then analyzed according to standard methods used by the Clinical Laboratory Center (certified ISO15189) at Toyama University Hospital. The blood culture procedures were performed using the BacT/ALERT 3D system (bioMerieux, Inc., Mercy-l’Etoile, France). Positive blood culture bottles were subcultured in the appropriate media and incubated aerobically or anaerobically until sufficient growth was present to proceed with testing (usually 18 to 24 h). The specific identification methods differed according to the organism, although they included the MicroScan WalkAway system (Siemens Healthcare Diagnostics), RapID ANA II (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and various latex agglutination and biochemical spot tests.