The synergistic activity of SBC3 in combination with Ebselen against Escherichia coli infection

Abstract

Escherichia coli ranks as the number one clinical isolate in the past years in China according to The China Antimicrobial Surveillance Network (CHINET), and its multidrug-resistant (MDR) pathogenic strains account for over 160 million cases of dysentery and one million deaths per year. Here, our work demonstrates that E. coli is highly sensitive to the synergistic combination of SBC3 [1,3-Dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver (I) acetate] and Ebselen, which shows no synergistic toxicity on mammalian cells. The proposed mechanism for the synergistic antibacterial effect of SBC3 in combination with Ebselen is based on directly inhibiting E. coli thioredoxin reductase and rapidly depleting glutathione, resulting in the increase of reactive oxygen species that cause bacterial cell death. Furthermore, the bactericidal efficacy of SBC3 in combination with Ebselen has been confirmed in mild and acute peritonitis mice. In addition, the five most difficult to treat Gram-negative bacteria (including E. coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa) are also highly sensitive to a synergistic combination of SBC3 and Ebselen. Thus, SBC3 in combination with Ebselen has potential as a treatment for clinically important Gram-negative bacterial infections.

Keywords: Escherichia coli; SBC3; ebselen (PubChem CID: 3194); peritonitis; redox homeostasis; thiol-dependent redox system.

FIGURE 1

Effects of SBC3 in combination with Ebselen on the growth of bacteria and mammalian cells. (A–J) Bacteria overnight cultures were diluted 1:1,000 into 100 μL of LB medium in 96 micro-well plates and treated with 100 μL serial dilutions of SBC3 and Ebselen. (A) E. coli DHB4. (B) E. coli BC1. (C) A. baumannii ATCC 19606. (D) E. cloacae ATCC 700323. (E) P. aeruginosa ATCC 27853. (F) K. pneumoniae ATCC 700603. (G–J) The toxicity of SBC3 and Ebselen on the growth of MEFs and S-KN-SH. Cells were treated with serial concentrations of SBC3 and Ebselen for 24 h and the cell toxicity was detected by ATP assay. Data are presented by Student’s t test, p < 0.05 constitutes statistical significance.

FIGURE 2

Antibacterial effect of Ebselen and SBC3 on E. coli DHB4. E. coli DHB4 was cultured until an A₆₀₀ = 0.4 and treated for 30 min with SBC3 and Ebselen. (A) Cell viability was represented by measuring A₆₀₀. The growth curves showed a synergistic antibacterial effect of SBC3 and Ebselen on E. coli DHB4. Data are presented by Student’s t test, p < 0.05 constitutes statistical significance. (B) Transmission electron microscopy showed the morphology change of DHB4 after the treatment with SBC3 and Ebselen at 300,000× (upper row) or 150,000× (lower row) magnification.

FIGURE 3

Antibacterial effect of Ebselen and SBC3 on DHB4 targeting TDRS system and producing of ROS. E. coli DHB4 was cultured until an A₆₀₀ = 0.4 and treated for 30 min with SBC3 and Ebselen. (A) TrxR activity endpoint was detected using DTNB reduction assay in the presence of Trx in DHB4 extracts. (B) Trx activity endpoint was detected using DTNB reduction assay in the presence of TrxR in DHB4 extracts. (C) GSH activity endpoint was detected using DTNB reduction assay in the presence of GR in DHB4 extracts. (D,E) Mean fluorescent intensity (MFI) of H₂DCF-DA-stained DHB4 was used to detect ROS level by flow cytometry. (F) UV-vis at A₆₀₀ has been used to present DTT could save E. coli DHB4 from SBC3 and Ebselen treatment. (G) Flow cytometry has been used to detect DTT could statistically down-regulate the intracellular ROS production level of E. coli DHB4. Data are presented by ONE-WAY ANOVA, p < 0.05 constitutes statistical significance.

FIGURE 4

SBC3 with Ebselen in combination directly disrupted bacterial TDRS systems. DHB4 was cultured until an A₆₀₀ = 0.4 and treated for 30 min with serial concentrations of SBC3 and Ebselen. (A,B) Trx1 protein level was measured by Western blot. (C) trxa (Trx1) mRNA expression level was tested by qPCR. (D,E) S-PSSG protein level was measured by Western blot. (F) grxa (Grx1) mRNA expression level was tested by qPCR. For Western blot, DnaK was used as a reference; for qPCR, rrsA was used as a reference. Data are presented by ONE-WAY ANOVA, p < 0.05 constitutes statistical significance.

FIGURE 5

Therapeutic efficacy of SBC3 and Ebselen in treating E. coli BC1-induced peritonitis. (A) MDR BC1 was cultured overnight, and mice (n = 15) were injected i.p. with DMSO, 2 mg/kg SBC3, 25 mg/kg Ebselen, 2 mg/kg SBC3 and 25 mg/kg Ebselen in combination on the day 1- and 3- before infection. On day 0, mice infected with 100 μL 2 × 10⁸ CFU/ml to construct an acute peritonitis model. Overall survival was observed. Data are presented by log-rank (Mantel-Cox) test, p < 0.05 constitutes statistical significance. (B) MDR BC1 was cultured overnight, and mice (n = 10) were injected i.p. with DMSO, 2 mg/kg SBC3, 25 mg/kg Ebselen, 2 mg/kg SBC3 and 25 mg/kg Ebselen in combination on the day 1- and 3- before infection. On day 0, mice infected with 100 μL 2 × 10⁷ CFU/ml to construct a mild peritonitis model. The bacterial load was calculated by counting the colonies. Data are presented by chi-square test, p < 0.05 constitutes statistical significance. To evaluate the effects of SBC3 in combination with Ebselen on the kidney and liver function of mice, the sera of mice were collected 14 days post-infection, and contents of CRE (C), ALT (D), AST (E), and UA (F) were measured. Data are presented by Student’s t test, p < 0.05 constitutes statistical significance.