Chocolate agar

The serogroup B strain MC58 and the unencapsulated MC58 cap- knock-out mutant were used for binding experiments [47 (link)]. H44/76 strain was used to measure serum bactericidal antibody titers. Bacteria were routinely grown on GC agar (BD Biosciences, Milan, Italy) or Chocolate Agar plates at 37°C and 5% CO₂ overnight. The serogroup B strain 2996 was used for in vivo experiments. Bacteria were washed twice in non-pyrogenic PBS and resuspended to the desired concentrations before injection.

AN11251 was provided by Calibr at Scripps Research (San Diego, CA, USA). All the other chemicals were purchased from Sigma (St Louis, MO, USA) without further purification. Rats were purchased from Charles River (Wilmington, NC, USA) and fasted overnight before dosing. Yeast Extract (OXOID, from Thermo Fisher (Waltham, MA, USA)), Sheep Blood (Yuanye Bio-Technology in Shanghai, China), Haemophilus Test Medium Base (HTM, HalingBio in Shanghai, China), Tryptic Soy Broth (TSA, BD (Franklin Lakes, NJ, USA)), Cation Adjusted Muller-Hinton broth (CAMHB, BD), 7H9 broth (BD), OADC (BD), glycerol (Sigma), Tyloxapol (Sigma), and Chocolate Agar (BD) were from commercial sources as indicated. Rifampicin (RIF, Sigma), Ciprofloxacin (CIP, MedChemExpress (South Brunswick, NJ, USA)), Alamar Blue^TM Cell Viability Reagent from Thermo Fisher, and other chemical reagents were purchased.

CSF samples from patients with suspected tuberculous meningitis (clinical suspicion, HIV-positivity, or no improvement on conventional antibiotic treatment) were tested for Mycobacterium tuberculosis with the GeneXpert MTB/RIF assay (Dx System Version 4.0c, Cepheid Inc., CA, US) [19 (link)]. CSF was processed as recommended by the manufacturer: a 2:1 volume of the reagent supplied with the assay was added directly to the sample. The mixture was vortexed and incubated at room temperature for 15 min. Two mL of the reagent-sample mix was then transferred to the Xpert cartridge and analysed.
Blood culture bottles were incubated in an automated blood culture incubator BACTEC FX40 system (BD) for a maximum of five days and flagged as either “negative” or “positive” for bacterial growth. Positive blood cultures were analysed using the FilmArray blood culture identification (BCID) panel (BioMerieux, France) [20 (link)], and cultured onto blood agar (BD), MacConkey agar (BD) and chocolate agar (BD), with the same routine bacterial identification as for CSF cultures.
Other investigations, such as malaria testing using peripheral blood smear and May-Grünwald-Giemsa staining, rapid testing for HIV, haematological and biochemical analyses were performed according to routine practice at JUSH.

Native lung tissues were transferred into mixing vessels (ProbeAX; AxonLab) containing 5 mL of physiological saline and were homogenized using a dispersion instrument (ULTRA-TURRAX® Tube Drive; AxonLab). The homogenates were inoculated (0.1 mL aliquots) onto aerobic blood agar, MacConkey agar, chocolate agar, and anaerobic blood agar plates (BD Diagnostics) and into thioglycollate broth (Oxoid). Plates were incubated at 35°C and 37 °C aerobically, in an atmosphere containing 5% carbon dioxide and anaerobically (Genbox anaer, bioMérieux) for up to 14 days, respectively. Cloudy thioglycollate broths were sub-cultured onto plates. Colonies were identified using matrix-assisted-laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF MS) using the Vitek® MS (bioMérieux) or MALDI Biotyper^TM (Bruker) instruments or by 16S rRNA gene sequencing (Gorkiewicz et al., 2003 (link)).

CSF samples were processed according to standard procedures at JUSH which include bacteriological culture, macroscopic assessment, protein and glucose measurements, manual WBC count with differential count and Gram staining. India ink staining and rapid cryptococcal antigen testing (CrAg, IMMY, OK, USA) were performed upon request. Gram stain was performed on the primary sample, usually without centrifugation due to lack of equipment and/or consumables. CSF glucose and protein measurements, were performed when reagents were available. CSF samples were cultured onto blood agar (BD) and/or chocolate agar (BD) for 72 h at 35 °C in a CO₂-enriched incubator (candle jar). Routine bacterial identification was based on colony morphology, Gram staining and standard biochemical reactions [17 ].

Positive BCs were Gram stained and sub-cultured overnight on Columbia blood (bioMérieux) and chocolate agar (BD, Heidelberg, Germany) at 37°C and 5% CO₂. The next day, a single bacterial colony was transferred from the agar plate to a single well of a Vitek^® MS-DS target slide (bioMérieux). One microliter of matrix (Vitek^® MS-CHCA, [3.1g/100μl alpha-Cyano-4-hydroxycinnamic acid]) (bioMérieux) was added and the mixture was air dried. The slide was analyzed in the Vitek^® MS mass spectrometer (bioMérieux, [337 nm nitrogen laser, 19.9 kV, maximum pulse rate 50 Hz, mass range 2000–20000 Da]) and only results with confidence level > 99% were used for identification. If no identification could be obtained, biochemical identification using the Vitek^® 2 (bioMérieux) and/or the 16S rDNA sequencing [20 (link)] were performed.