Bacteriological agar

The yeast strain analyzed in the current study was C. tropicalis ATCC MYA3404 (Broad Institute, USA). It is one of the main soil contaminants in sugarcane plantations, and had been isolated from a fermenter tank in Brazil. Yeast cultures were maintained in a yeast extract-peptone-dextrose medium [YEPD; 2% dextrose w/v (Synth—Diadema, Brazil), 1% yeast extract w/v (Kasvi—Curitiba, Brazil), 2% peptone w/v (Acumedia—Indaiatuba, Brazil), and 2% bacteriological agar w/v (Himedia—West Chester, Pennsylvania, USA)] at 30 °C.

S. aureus was procured from microbial type culture collection and gene bank (MTCC), Chandigarh. The freeze-dried culture was suspended in 3% tryptone soya broth (TSB) and plated onto tryptone soya broth (HiMedia, Mumbai) supplemented with 1.5% bacteriological agar (HiMedia, Mumbai). The plates were incubated at 37°C overnight. The following day, colonies were picked up from the plates and cultured in TSB. The cultures were further confirmed by Gram's staining [6 (link)].

The cultures of B. pseudomallei were grown on Ashdown’s agar medium containing 4% Glycerol (Fisher Scientific, #CAS 56-81-5), 1% Tryptone Soya Broth (HiMedia, #M011), 0.5 mg/L Crystal Violet (HiMedia, #GRM961), 5 mg/L Neutral Red (HiMedia, #RM122), and 1.5% Bacteriological Agar (HiMedia, #GRM026) supplemented with 5 mg/L of Gentamicin (HiMedia, #RM461) at 37 °C for 48–72 h [16 (link)]. The other cultures used in the study were grown on Brain Heart Infusion (BHI) agar medium (HiMedia, #M211) at 37 °C for 16 to 48 h. Obtained single colonies were inoculated into BHI broth (HiMedia, #M210) for DNA extraction. The genomic DNA from bacterial cells was extracted using DNeasy Blood and Tissue kit (Qiagen, #69504) in accordance with the manufacturer’s protocol. The purity and quantity of DNA were measured using NanoDrop (Thermo). Isolated purified bacterial genomic DNA was aliquoted and stored at -20 °C till further use.

All reagents were obtained from standard lab suppliers. Bacteriological agar and Brain Heart Infusion (BHI) broth for the media preparation were the products of HiMedia Laboratories (Mumbai, India; lot number: BCBH4004V), with BHI consisting of (g/L) calf brain infusion from 200 g (12.5), beef heart infusion from 250 g (5), peptone (10), sodium chloride (5), disodium chloride D (+) glucose (2), disodium hydrogen phosphate (2.5) at pH 7.4. Vitamin B12 or cyanocobalamin was from ThermoFisher, (
Massachusetts, USA; number A14894). In addition, we also tested three clinical isolates using a trace metal solution (details in Supplementary Table 1,
Extended data [
Ahmad 2023 (link)]).

The microbial strains (bacterial and fungal) used in the study were obtained from the Institute of Microbial Technology (MTCC-IMTECH), Chandigarh and National Collection of Pathogenic Fungi (NCPF), Post-Graduate Institute of Medical Education and Research (PGIMER), Chandigarh. The bacterial strains Escherichia coli MTCC 2961, Staphylococcus aureus MTCC 3160, Enterococcus faecalis MTCC 439, Vibrio cholera MTCC 3906, Streptococcus pyogenes MTCC 442 were cultured in Mullar Hinton broth (MHB, HiMedia, India). The fungal strains Candida albicans NCPF 400034, Candida glabrata MTCC3019, Candida krusei NCPF 44002, Candida parapsilosis NCPF 450002, Candida keyfer, NPCPF 410004 and Candida tropicalis NPCPF420007 were cultured in yeast extract-peptone-dextrose (YEPD broth, HiMedia, India) and RPMI 1640 media (HiMedia, India). For agar plates, 2.5% (w/v) bacteriological agar (HiMedia, India) was added to the medium. The strains were stored with 15% glycerol at -80°C as frozen stocks. The cells were freshly revived on respective agar plates from the stock before each experiment [43 (link),44 (link)].