Brain heart infusion agar

Strains used in this study were E. coli (DH10B) One Shot® TOP 10 (Invitrogen) and various Francisella strains (see Table 1). The iglC mutant strain of Fth strain LVS was kindly provided by Anders Sjöstedt (Golovliov et al., 2003 (link)). For genes and abbriviations used, see Table 1.
E. coli was cultivated in Luria-Bertani (LB) medium or on LB agar. The antibiotic concentrations used for E. coli were chloramphenicol (Cm) 40 μg ml⁻¹ and kanamycin (Km) 40 μg ml⁻¹. Francisella strains were cultivated in medium T (Pavlovich and Mishan'kin, 1987 (link); Becker et al., 2016 (link)), on medium T-based agar plates (MT-KH agar: medium T containing 2.4 g l⁻¹ of activated charcoal, 14.3 g l⁻¹ of agar and 9.5 g l⁻¹ of hemoglobin), or on HCA agar (Brain Heart Infusion Agar [Liofilchem, Roseto degli Abruzzi, Italy] with 10% sheep blood). The antibiotic concentrations used for Francisella were 10 μg ml⁻¹ for chloramphenicol and 12 μg ml⁻¹ for kanamycin.
The human macrophage-like cell line U937 (ATCC CRL-1593.2) (growth medium RPMI 1640 + 10% FCS [purchased from PAA, Pasching, Austria]) was used to investigate the intracellular multiplication of Francisella strains. U937 cells were cultivated at 37°C and 5% CO₂.

We investigated the immunogenicity of 3 clinical strains of Escherichia coli (randomly named S1, S2, and S3) isolated from the urine of HbSS subjects. These bacterial strains were obtained after 24-hour urine culture on UriSelect 4 agar and identified. Thereafter, bacterial strains were stored on microbeads (Microorganism Preservation System – Protect, Technical Service Consultants Ltd) at –20 °C for 3 months. Bacteria were then cultured on the UriSelect 4 medium for 24 hours at 37 °C. One colony was selected and cultured in Oxoid Mueller-Hinton Broth (ThermoFisher Scientific). Bacteria were inactivated at 56 ± 1 °C for 6 hours in a water bath,^{20 (link)} followed by triple washing with Dulbecco's phosphate buffered saline (DPBS) (ThermoFisher Scientific). To assess the complete inactivation, a bacterial culture on brain heart infusion agar (Liofilchem, Inc) was performed.
After complete inactivation of bacteria, tubes containing the culture broth were centrifuged, and the pellet was collected and added to 1 mL of DPBS. The protein concentration was measured by Bradford assay. Bacterial antigens obtained were frozen at –20 °C for further use.