Fig. 3: Methionine supplementation potentiates tigecycline activity against tet(X)-carrying bacteria.

a Percent survival of E. coli DH5α (pUC19) in the presence of Tig, Met, or both. Tig, tigecycline, 1-fold MIC; Met, L-methionine, 20 mM. b Percent survival of E. coli DH5α (pUC19-tet(X4)) in the presence of Tig (1-fold MIC) or Tig plus Met (5, 10, 20 mM) for 6 h. c The impact of adding Met (20 mM) before or after the addition of antibiotic (Tig, 1-fold MIC) on bacterial survival at 6 h. d Percent survival of clinic-sourced E. coli IF28 (tet(X4)) in the presence of Tig (1-fold MIC), Met (20 mM), or both. e Percent survival of clinic-sourced E. coli B3-1 (tet(X4)) in the presence of Tig (1-fold MIC), or Tig plus Met (20 mM) for 6 h. f Growth curves of E. coli B3-1 (tet(X4)) within 12 h in the presence of Tig (32 μg/mL), Met (5, 10, 20 mM) or both. The mean of three biological replicates is shown and error bars represent the SEM. g Time-dependent killing curves of E. coli B3-1 (tet(X4)) treated by Tig (16 or 32 μg/mL) and Met (20 or 40 mM). h Bacterial LIVE/DEAD BacLight viability in the presence of Tig (16 or 32 μg/mL) or Met (20 or 40 mM) or both via flow cytometry analysis. Viable cells were demonstrated using green fluorescence (SYTO 9 staining, 1.67 mM) with an excitation/emission wavelength of 485 nm/498 nm, while dead cells were shown using red fluorescence (Propidium Iodide staining, 10 mM) with an excitation/emission wavelength of 535 nm/615 nm. i Confocal scanning microscope of the living and dead states of E. coli B3-1 (tet(X4)) when Tig was added alone or in combination with Met. Red was used to label dead bacteria and green was used to dye living bacteria. j Percent survival of E. coli B3-1 (tet(X4)) in the addition of other upstream or downstream metabolites of Met, covering cystathionine (CYSTAT), L-homoserine (Homoser), L-homocysteine (L-Hcy), 5’-Methylthioadenosine (MTA), S-Adenosyl-L-homocysteine (SAH), and S-Adenosyl-L-methionine (SAM). An overview of the changes of Met-related pathways were presented, including methionine salvage, cysteine and methionine metabolism, and a part of glycine, serine, and threonine metabolism. Red, up-regulated genes in E. coli DH5α (pUC19-tet(X4)) compared with E. coli DH5α (pUC19); Green, down-regulated genes in E. coli DH5α (pUC19-tet(X4)) compared with E. coli DH5α (pUC19). Data in a––g and j were displayed as mean ± SEM. Three biological replicates were carried out. Statistical significance in a–e & j were assessed by an unpaired two-tailed Student’s t-test. ns, not significant.