The antibacterial activities of COL against the tested strains after single and triple combinations

Time-kill curves of Salmonella strains JS, S16, and S30 with COL alone and in combinations.

a-f Samples were treated with different concentration of COL (0.1 or 2 mg/L), alone or in drug combinations, for 12 h. When used in combination, 1/8 MIC of AS or EDTA was added to a final concentration of 156.3 or 15.6 mg/L, respectively. Counts of CFU/mL were performed on all cultures at each time point, and data are mean ± SD from representative of three independent experiments. CON indicates the negative control group.

AS, EDTA, and COL affected membrane integrity, PMF, ROS, and H2O2 levels in S16 and S30 strains.

Different concentration of COL (0.1 or 2 mg/L) were used alone or in combination with AS and EDTA. When used in combination, 1/8 MIC of AS or EDTA was added to a final concentration of 156.3 or 15.6 mg/L, respectively.

a-d AS, EDTA, and COL affected membrane integrity as measured by fluorescence probes NPN and PI. Error bars indicated standard deviations for 3 replicas (* p < 0.001, ns not significant). CON indicates the negative control group, and SOV indicates the solvent-exposed group.

e-h Disruption of PMF is shown by measuring the dissipation of electric potential (Δψ) (a, b) and osmoticcomponent (ΔpH) (c, d). Error bars indicate standard deviations for 3 replicas (* p < 0.001, ns not significant). CON indicates the negative control group, and SOV indicates the solvent-exposed group.

i-l Intracellular accumulation of ROS (i, j) and H2O2 (k, l) in S16 and S30 strains after 1 h treatment. Data were shown in the mean of triplicates ± SD (* p < 0.001, ns not significant). CON indicates the negative control group, SOV indicates the solvent-exposed group, and POS indicates the positive control group.

Morphological changes of S16 (mcr-1−) strain.

The images were obtained after the treatment with COL (2 mg/L) alone or in combination with 1/8 MIC of AS (156.3 mg/L) or EDTA (15.6 mg/L). CON indicates the negative control group, and SOV indicates the solvent-exposed group. Red arrows indicate the cell damages characterized by folds, crevices, or depressions.

KEGG pathway analysis of SDEGs in S16 (a, b, c) and S30 (d, e, f) strains within the AEC .vs. C, AEC .vs. AC, and AEC .vs. EC groups.

Samples were harvested after the trestment of COL (2 mg/L) alone or in combination with 1/8 MIC of AS (156.3 mg/L) or EDTA (15.6 mg/L) for 6 h. Pathway name and number of down-regulated (blue), up-regulated (red) genes in each pathway are indicated in parentheses on the left (down/up). Highlighted with red circles are the pathways that SDEGs mainly enriched and appeared simultaneously in different comparison groups.

The SDEGs detected in two component system (a, b), flagellar assembly (c, d) and ABC transporters (e, f) pathways among different comparison groups, within S16 and S30 strains.

Samples were harvested after the treatment of COL (2 mg/L) alone or in combination with 1/8 MIC of AS (156.3 mg/L) or EDTA (15.6 mg/L) for 6 h. Labels in each square indicate the log2 (fold change) of corresponding genes. Squares without label and gray background indicate the data are not credible (p > 0.05, |log2Fold Change| < 1.0). Background colors indicate the expression levels of the respective genes, red = up-regulated, blue = down-regulated. log2FC: log2Fold Change.

The SDMs detected in archidonic acid metabolism, degradation of aromatic compounds, taurine and hypotaurine metabolism pathways among different comparison groups, within S16 (a) and S30 (b) strains.

Samples were harvested after the treatment of COL (2 mg/L) alone or in combination with 1/8 MIC of AS (156.3 mg/L) or EDTA (15.6 mg/L) for 6 h. Labels in each square indicate the fold changes of corresponding metabolites. Squares without label and gray background indicate the data are not credible (VIP < 1.0, 0.833 > Fold Change < 1.2 or Fold Change ≤ 0.833, p ≥ 0.05). Background colors indicate the fold changes of the respective metabolites, red = increased, blue = decreased.

AS and EDTA potentiate colistin activity against Salmonella S30 (mcr-1+) in vivo.

Kunming mice (n=6 per group) were intraperitoneally given a non-lethal dose of Salmonella S30 (1.31 × 105 CFU), then treated with PBS, COL (10 mg/kg), AS (15 mg/kg), AS (15 mg/kg) + COL (10 mg/kg), EDTA (50 mg/kg) + COL (10 mg/kg), and AS (15 mg/kg) + EDTA (50 mg/kg)+ COL (10 mg/kg) by intraperitoneal injection. Bacterial loads were determined in spleen and liver and bacterial counts were computed and presented as the mean ± SD log10 CFU/mL. The p values were determined by one-way ANOVA (* p < 0.001, ns not significant).

Scheme summarizing the proposed mechanisms that AS and EDTA enhancing the antibacterial effect of COL.

①COL and EDTA cause the membrane damages by targeting LPS and chelating cations, which punch holes in both the outer membrane (OM) and inner membrane (IM). ②AEC combination collapses the Δψ component of Proton Motive Force (PMF) in Salmonella. ③The down-regulated lipid compounds prostaglandins (PGs) lead to perturbtion of membrane phospholipids in cell membranes. ④Accumulation of toxic compounds (eg. styrene, ROS et al) could accelerate the destruction of cell membrane. ⑤The down-regulation of chemotaxis, flagellar assembly, Salmonella infection related genes indicate impaired virulence of Salmonella. ⑥The down-regulation of genes in ABC transporters indicate impaired stress tolerance of Salmonella. ⑦AEC treatment results in the down-rugulation of flagellar assembly related genes and defective of flagellum. ⑧AS was capable of significantly enhancing the antibacterial activities of antibiotics against E. coli, via inhibition of the efflux pumps. ⑨AS could be developed as the a MCR-1 inhibitor, which may work synergistically with the EDTA chelation to inhibit MCR-1 and contribute to reverse the COL resistance of mcr-1-harboring Salmonella strains. FC: Fold Change.

Intracellular accumulation of ROS in S16 and S30 strains after 6 h treatment.

The number of DEGs are identified in S16 and S30 strains among different comparison groups.

KEGG pathway analysis of SDEGs in S16 (a, b) and S30 (c, d) strains within the AC .vs. C, and EC .vs. C groups.

(a) The SDEGs detected in Salmonella infection and ribosome pathways; (b) Expression level of mcr-1; (c, d) efflux pump activity in S16 or S30 strain; (e) Putative pattern of interaction between AS and MCR-1 protein.