Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella

Abstract

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.

Keywords: Salmonella; colistin; combination therapy; mcr-1; resistance.

Figure 1

Resistance reversal rate (%) of colistin when baicalin and EDTA are used alone or in combination. BAI, baicalin. The reversal rate of colistin resistance was calculated as follows: the number of strains whose MICs of colistin reversed to sensitive after the combination was divided to the number of all strains that were colistin-resistant in this study.

Figure 2

Baicalin and EDTA potentiates colistin activity against Salmonella in vitro. BAI, baicalin. COL, colistin. (A) Time-kill curves of the Salmonella JS in the presence of baicalin (1,250 mg/L), EDTA (62.5 mg/L) or colistin (sub-MIC) alone or in combination for 24 h. (B) (C) (D) Time-kill curves of the clinical strain SR03, SR04 and SR14 (mcr-1) in the presence of baicalin (1,250 mg/L), EDTA (125 mg/L or 62.5 mg/L) or colistin (sub-MIC) alone or in combination for 24 h. Data are representative of 3 independent experiments and shown as mean ± SD.

Figure 3

mcr-1 gene expression in E.coli DH5α (pUC18-mcr-1) in the absence of antibiotic and in the presence of baicalin (625 mg/L), EDTA (31.25 mg/L) or colistin (2 mg/L) alone or in combination. BAI, baicalin. COL, colistin. Significant differences were evaluated by one-way ANOVA analysis and shown with ***P < 0.001.

Figure 4

Putative pattern of interaction among baicalin, EDTA and MCR-1 protein. (A) The interactions formed between the amino acid residues (stick) and the docked baicalin and EDTA molecule (ball and stick) in the MCR-1 binding site. (B) Interaction of planar amino acids among baicalin, EDTA and MCR-1 molecule.

Figure 5

Transcriptomic analysis of SR14 treated by baicalin, EDTA, and baicalin plus EDTA. KEGG enrichment analysis (A) of the differential expression genes (DEGs) in SR14 after exposure to baicalin plus EDTA. The x and y axis in A represent the expression changes and corresponding statistically significant degree, respectively. (B) Selected DEGs involved in TCA cycle, antioxidant response, LPS modification, ABC transporters, multidrug efflux pump and Salmonella infection and virulence. B, baicalin alone; E, EDTA alone; B+E, baicalin in combination with EDTA.

Figure 6

Relative expression of LPS modification and multidrug efflux pumps pathways related genes. Data were presented as mean ± SD from three biological replicates. Significant differences were evaluated by one-way ANOVA analysis and shown with **P < 0.01, ***P < 0.001.

Figure 7

Baicalin and EDTA rescues colistin activity in three animal infection models. Decreased bacterial load in the mouse abdominal cavity infection model by combination therapy. Female KM mice (n=6 per group) were intraperitoneally infected given a non-lethal dose of Salmonella SR04 and SR14 (mcr-1) (1.0 × 10⁵ cfus), and treated with a single dose of colistin(20 mg/kg), colistin + baicalin (20 + 50 mg/kg), colistin + EDTA (20 + 10 mg/kg) or a combination of the three drugs (20 + 50 + 10 mg/kg), or PBS by intraperitoneal injection. P values were determined by Mann-Whitney U test. P values (*P < 0.05, **P < 0.01 and ***P < 0.001) are reported. Data are presented as mean ± SD.

Figure 8

Scheme summarizing the mechanisms of rescuing colistin susceptibility of baicalin in combination with EDTA. BAI, baicalin. (A) Promoting intracellular accumulation of colistin in drug-resistant bacteria by inhibiting the multidrug efflux pumps and ABC transporters. (B) Inhibit the LPS modification by reducing the expression of TCSs PmrAB and PhoPQ systems. (C) Enhances bacterial oxidative damage by accelerating the TCA and inhibiting the oxidation-reduction system. (D) attenuates Salmonella virulence by inhibiting the expression of T3S system and flagellin in Salmonella.