Roseoflavin

The effect of flavins or their analogs on parasites proliferation was evaluated through growth curves. Stationary phase parasites were inoculated in fresh SDM-79 or SDM-20 media (basal 20 nM riboflavin) supplemented to a defined riboflavin concentration. Initial density for T. cruzi Y-GFP, MJ Levin-GFP (referenced as wild-type) and MJ Levin-TcRibJ (referenced as TcRibJ) was 10⁷ parasite/mL, while initial density for T. brucei, L. (L.) mexicana, C. fasciculata and Phytomonas Jma was 10⁶ parasites/mL. Riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) were dissolved in water and the analogs lumiflavin, lumichrome and roseoflavin (all from Sigma-Adrich) dissolved in DMSO. Then, flavins and analogs were added to the culture media at the indicated concentrations. Parasites were daily counted using a hemocytometer chamber. Proliferation was calculated as the percentage of parasite counts relative to control condition values (flavin at 20 nM or without analogs) on the fifth day. Day 5 was chosen because it is when all parasite cultures tested reach stationary phase in control conditions (S1 Fig).

Pure culture of Lactobacillus plantarum-HDS27 (Gen Bank accession number–MK314098) Production medium—De Man, Rogosa and Sharpe agar (MRS) and Chemically Defined Medium (CDM) (2.5 g lactose, 2.5 g K₂HPO₄, 3.0 g KH₂PO₄, 0.6 g ammonium citrate, 1.0 g sodium acetate, 0.25 g cysteine-HC1, 5.0 g salt-free, vitamin-free casein hydrolysate, 10 mL vitamin solution, 10 mL metal solution, 10 mL nucleic acid bases solution) were procured from Hi-Media Laboratories (Mumbai, India). Roseoflavin from Sigma Aldrich (St. Louis, MO, United States).

S. epidermidis ATCC 12228 was cultured in TSB (Sigma) at 37°C. The bacterial pellet was harvested by centrifugation at 15 000 g for 5 min, suspended in PBS. Electricity produced by S. epidermidis was measured by the changes in voltage (mV) against time (min) using a digital multimeter (Lutron, DM‐9962SD, Sydney, Australia). Voltages were recorded every 5 s using a device equipped with a carbon felt (2.5 cm × 10 cm) and a carbon cloth (10 cm × 10 cm) (Homy Tech, Taoyuan, Taiwan) as anode and cathode respectively. The cathode was covered with a Nafion membrane N117 (6 cm × 6 cm) (Homy Tech) as a proton exchange membrane (PEM). Copper wires were used to link the anode and cathode to an external resistance (1000 Ω). S. epidermidis pretreated with or without 1 μM of roseoflavin (Sigma) for 24 h in the presence of LCC (TNJC corporation, Chiayi, Taiwan) was pipetted on the surface of the anode.