Human serum

Determination of survival in 50% human serum was performed as previously described [23 (link)]. Briefly, overnight cultures were diluted 1:100 in 5 mL of fresh LB and incubated with shaking at 37 °C until OD₆₀₀ reached 0.5 McFarland standard turbidity. Then, 1000 µL of the bacterial suspension was pelleted (7500× g for 5 min at rt) and resuspended in 1 mL of phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA). One hundred microliters of the sample were seeded in a 96-well microtiter plate containing 100 µL of human serum (United States origin; Sigma-Aldrich, St. Louis, MO, USA) per well (resulting in a final concentration of 50% human serum and approximately 10⁸ CFU mL⁻¹). Following this, 20 µL of each sample was withdrawn and serial dilutions were plated on LB agar plates and incubated overnight at 37 °C to determine the size of the inoculum. The inoculated microtiter plates were incubated for 4 h at 37 °C without agitation. Thereafter, the number of surviving CFU mL⁻¹ was determined by plating out serial dilutions and incubating at 37 °C overnight. The positive control in each experiment was the serum-resistant PBIO1289 (initially designated as IMT10740 [78 (link),79 (link)]). The serum-sensitive W3110 served as the negative control. Serum resistance was expressed as log₂ fold change in CFU mL⁻¹ after treatment with respect to inoculum size.

Growth curves in THB were performed in biological duplicate. Wild-type and ΔnrdM strains were grown overnight as described and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 in approximately 12 mL THB. The OD₆₀₀ was monitored every hour using a Thermo Scientific Genesys 30 spectrophotometer. For growth curves in the presence of human serum, biological triplicate overnight cultures were grown in streptococcal defined medium (45 (link), 46 (link), 72 (link)) and diluted to an OD₆₀₀ of 0.1 in a defined medium supplemented with 5% vol/vol human serum (Sigma-Aldrich). The OD₆₀₀ was monitored at 3, 6, and 24 h as described above.

Streptavidin-coated paramagnetic beads (100 μl, 1 μm diameter; Life Technologies) were saturated with 20 μg of enzymatically monobiotinylated DBLMSP or a Cd4 tag-alone control, isolated with a magnet, and washed three times with PBS before incubating with 1 ml of filtered human serum (Sigma) for one h at 4 °C. Beads were washed four times with 1 ml of PBS and eluted with 200 μl of 1% SDS. 20 μl were resolved by SDS-PAGE under reducing conditions and stained with SYPRO Orange (Sigma), and the gel image captured on a Typhoon 9400 phosphorimaging device (GE Healthcare).
Anti-human IgM agarose beads (Sigma) were incubated with long term parasite culture supernatants from the IT4 var1 and var13 strains grown in the presence of human serum or control culture medium without parasite for 1 week at 37 °C. After five washes in PBS, the beads were resuspended in loading buffer in the presence or absence of DTT, and eluates were blotted onto nitrocellulose membranes (Amersham Biosciences Protran) followed by blocking in PBS, 0.1% Tween 20, 5% nonfat milk powder) and incubated for 1 h with a rabbit anti-full-length DBLMSP antibody at a 1:100 dilution. After further washes, the membrane was incubated with an anti-rabbit HRP-conjugated IgG secondary antibody (1:1000; Sigma) and developed using 3′,3′-diaminobenzidine (DAKO) according to the manufacturer's instructions.

Peripheral blood from healthy donors was acquired from the National Health Service blood service under ethics license REC 05/Q0401/108 (University of Manchester). Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque Plus; Amersham Pharmacia Biotech). Human monocyte-derived macrophages were derived as described previously (Davies and Gordon, 2005 (link)). In brief, CD14⁺ cells were isolated by positive selection from peripheral blood mononuclear cells using magnetic beads (CD14 MicroBeads; Miltenyi Biotec) and cultured at 10⁶ cells/ml in serum-free media (X-Vivo 10; Lonza) supplemented with 1% human serum (Sigma-Aldrich). After 24 h, monocytes were washed with PBS (Sigma-Aldrich) to remove nonadherent cells and cultured in X-Vivo media with 1% human serum. After 3 d of incubation, adherent cells were washed with PBS and cultured in standard DMEM-based media (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen), 1% penicillin and streptomycin (Gibco), 1% l-glutamine (Gibco), and 1% Hepes (Sigma-Aldrich) for 6 days to generate monocyte-derived macrophages, phenotyped to be CD14⁺, CD11a⁺, CD3⁻, CD56⁻, and CD19⁻. Cells were washed with PBS, and media was replaced every 3 d.

Human serum was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), and the Human serum (99 μL) was spiked with heparin solution (1 μL). The spiked samples contained varying heparin concentration (36 μg/mL–1800 μg/mL). Spiked samples (1 μL) were added directly into 2 mL of 10 mM HEPES (pH 8.0) solution containing CTX3-MB or CTX3-aptamer sensors. Then, the fluorescence spectra were measured using excitation and emission wavelengths at 480 and 520 nm, respectively.

All assays were performed in M9 minimal medium (10.5g/L M9 broth [Amresco], 0.2% casamino acids, 0.1M CaCl₂, 0.4% glucose, 1M MgSO₄, 0.25% nicotinic acid, 0.33% thiamine in H₂O) unless otherwise stated. To determine MICs, an M9 culture of MG1655 was growth overnight at 37°C with shaking, then diluted to OD₆₀₀ = 0.002. We then inoculated each well with approximately 1,000 cells (2 μl of culture into 200 μl of medium per well). Plates were incubated at 37°C unless otherwise stated. Small-molecule gradients were diluted in 2-fold dilution series unless otherwise stated. MIC values (Table 1) are calculated following 24 hours incubation at 37°C. MIC values are calculated as >90% growth inhibition unless otherwise stated. MIC values for MDR strains, which grow more rapidly than lab strains, were calculated following 24 hours incubation at 37°C. When calculating MICs in the presence of human serum, we used standard techniques but substituted up to 20% of the media volume with human serum (Sigma).

For each condition, wild-type B. pseudomallei K96243; B. thailandensis E264; B. pseudomallei ΔpglL and B. thailandensis ΔpglL were cultured overnight from a single colony in 5 ml sterile Luria–Bertani (LB) broth at 37°C with shaking. All strains employed in this study exhibited comparable growth rates when cultured in the media used. Bacteria concentrations were standardized by adjusting to an OD₅₉₀ of 1 and then further diluted 1:100 in fresh LB. A total of 10 μl of bacterial solutions were added in triplicate to flat bottom 96-well plates containing 100 μl of titrated polymixin B or human serum (both, Sigma-Aldrich, Gillingham, UK) at the concentrations described. For heat inactivation, human serum was incubated at 65°C for 1 h. After 24 h incubation at 37°C, colony-forming unit (CFU) assay was performed by serial dilution of each sample well in sterile phosphate-buffered saline (PBS; Sigma, UK) before plating on fresh LB-agar and incubation for a further 24 h at 37°C for colony enumeration.