Bactec plus aerobic f culture vials

Isolated Y. pestis colonies (grown on BHIA plates at 28 °C for 48 h) were suspended in BHI to an optical density (OD)_660nm value of 0.35 (~1–3 × 10⁸ CFU/mL), and dilution was performed as required. The bacterial suspension was then diluted 1:100 with whole human blood (10 mL) and injected into an aerobic blood culture bottle (BACTEC Plus Aerobic/F Culture Vials; BD, Cat. no. 442192). The bottle was incubated in a BACTEC™ FX40 instrument until it was indicated positive by this system. For the isolation of Y. pestis bacteria from blood components, we used the serum separation tube (SST) separation protocol as we described previously [35 (link)]. Briefly, 6 mL of blood culture was injected into the SST (Grainer Bio-One, Cat. no. 455071) and centrifuged at 1700× g for 15 min at room temperature (RT). The supernatant was discarded, and the bacterial lining on the gel matrix was resuspended to the original volume using MHB growth media. Resuspended bacteria were used for diagnostic tests, including identifying the bacteria by the specific phage-based bioluminescent assay and simultaneously determining their antibiotic susceptibility to various antibiotic agents by phage-based AST.

Yersinia strains were grown on BHIA (Brain heart infusion agar, BD Difco, Sparks, MD, USA, #241830) plates at 28 °C for 48 h. E. coli, P. auraginosa and S. typhimurium were grown at 37 °C for 24 h. Colonies were suspended in sterile phosphate buffered saline (PBS, Biological industries, Beth haemek, Israel, #02-023-1A) and added at a defined concentration into BACTEC plus aerobic/F culture vials (BD, MD, USA, #442192) supplemented with 10 mL of naïve human blood containing Citrate-phosphate-dextrose solution with adenine (CPDA) as an anticoagulant. The inoculated blood culture vials were then shaken at 150 rpm at 37 °C in a New Brunswick Scientific C76 water bath for different durations. Final colony forming units (CFU) counts for each vial were determined by plating 0.1 mL of serial 10-fold dilutions on BHIA plates and incubating for 48 h at 28 °C. F. tularensis subsp. holarctica vaccine strain LVS and B. anthracis Vollum strain were grown, as described in Reference [62 (link)]. The resulting blood cultures were processed, as described in the next section.

The ^△ strain DYY3 was isolated from a critically ill patient’s blood sample with a catheter-associated bloodstream infection. Briefly, the blood specimen was inoculated in a blood culture bottle (BD BACTEC Plus aerobic/F Culture Vials, Becton, Dickinson and Company, United States) at 35°C until it showed a positive result. For the isolation of ^△ strain DYY3, blood agar plates (bioMérieux, Marcy l’Etoile, France) were used, and the plates were incubated in a CO₂ incubator for 48 h. Later, dozens of single colonies were picked up from the blood agar plates. VITEK-MS automatic microbiological analyzer (bioMérieux, Marcy l’Etoile, France) was used to identify the taxonomic classification of the ^△ strain DYY3 according to the standard operation process using the VITEK MS IVD KB V3.2 database as the reference. The total length of the 16S rRNA sequence was amplified by PCR using the primers of 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and the amplified fragments were sequenced using a 3730XL sequencer (Applied Biosystems, United States).