Depletion of multidrug‐resistant uropathogenic Escherichia coli BC1 by ebselen and silver ion

Abstract Ebselen, an organo‐selenium compound with well‐characterized toxicology and pharmacology, recently exhibited potent antibacterial activity against glutathione (GSH)‐negative bacteria by disrupting redox homeostasis. In this paper, we show that ebselen and silver ion in combination exert strong bactericidal activity against multidrug‐resistant (MDR) uropathogenic Escherichia coli (UPEC) BC1, a model MDR GSH‐positive bacterium. The mechanisms were found to involve consumption of total intracellular GSH and inhibition of thioredoxin reductase activity, which was highly related to reactive oxygen species up‐regulation. Furthermore, the therapeutic efficacy of ebselen and silver ion against UPEC‐induced cystitis was assessed in a mouse model. Treatment with ebselen and silver ion significantly reduced bacterial loads, down‐regulated the expression levels of tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) on‐site and decreased white/red blood cell counts in mild cystitis model mice, which demonstrated the anti‐inflammatory property of these agents. In addition, ebselen and silver ion also exhibited significantly high protective ability (100%) against acute cystitis infections. These results together may lay the foundation for further analysis and development of ebselen and silver ion as antibacterial agents for treatment of MDR UPEC infections.

There are two major cellular thiol-dependent redox systems, the thioredoxin (Trx) and glutathione (GSH) systems, which maintain cellular redox homeostasis and protect cells from oxidative stress. 10,11 The Trx system is composed of Trx, thioredoxin reductase (TrxR) and NADPH, while glutaredoxin (Grx), glutathione reductase (GR), GSH and NADPH constitute the GSH/Grx system. 12,13 Both systems are important in DNA synthesis and repair, cell proliferation and antioxidant defences. 14,15 The Trx system is widespread in all living organisms, and the GSH/Grx system is absent in the majority of Gram-positive bacteria (GSH-negative bacteria). 14,16 Thus, the maintenance of intracellular redox balance in GSH-negative bacteria is principally dependent on the Trx system. It is worth noting that in the pathological process of UTI, UPEC infection reduces the levels of antioxidant enzymes including GR and glutathione peroxidase (GPx) in urinary tissue. In the late stage of UPEC infection, the levels of GSH decrease, while lipid peroxidation increases significantly in the urinary tissue. 17 This decline in antioxidant enzymes provides a more suitable microenvironment for elevating the ROS level in the urinary system, leading to cell and tissue damage and increasing the severity of bacterial infection.
As an organo-selenium compound, 2-phenyl-1,2 benzisoselenazol-3(2H)-one (ebselen) is known to be a classic GPx mimic and a substrate of mammalian TrxR. 16 It is also a clinical trial drug with well-characterized toxicology and pharmacology for stroke, neurodegenerative disease, bipolar disorder, COVID-19, tobramycin-induced ototoxicity, chemotherapy-induced hearing loss. 18,19 Previous researches have shown that ebselen can protect against oxidative injuries in various tissues, [20][21][22] and recent studies have demonstrated that ebselen possesses antibacterial activity against GSH-negative bacteria. 16,18,23,24 Our group further confirmed that ebselen could work synergistically with silver ion to kill types of GSH-positive bacteria, 18 including multidrug-resistant (MDR) E coli, and the effective concentration of silver ion was significantly reduced. Despite the gradual increase of reports concerning the antibacterial activity of ebselen and silver ion, few studies have focused on its involvement in bacteria-caused host inflammation. In this paper, the bactericidal activity of ebselen and silver ion against the MDR UPEC BC1 strain has been detected in vitro by visible spectrophotometry, DTNB, flow cytometry, qPCR and Western blot assays. Furthermore, the mild and acute mouse cystitis models were constructed and the therapeutic efficacy, the regulation of UPEC-induced inflammatory properties and the potential clinical applications of ebselen and silver ion against MDR UPEC were also assessed. BALB

| Reagents
Bacterial cells were cultured in Luria Bertani (LB) medium (EMD Millipore Rabbit anti-sheep IgG-HRP, goat anti-mouse H&L, anti-TNF-α, anti-IFN-γ and anti-DnaK antibodies were from Santa Cruz. IgG2a mouse monoclonal antibody was from VIROGEN, Lowry protein assay was from Bio-Rad DC™, and all the other reagents were from Sigma-Aldrich.

| The inhibition of UPEC BC1 growth by ebselen and silver ion
The inhibition of UPEC BC1 growth by ebselen and silver ion was measured by visible spectrophotometer at 600 nm. UPEC BC1 cells were grown (37°C, 220 rpm) 8 hours and diluted 100 times that to be treated with serial concentrations of ebselen and silver ion for 16,20 and 24 hours at 37°C in a 96-well plate, and the absorbance values at 600 nm were measured. ceftazidime was used as negative control, and amikacin was used as positive control.

| The disruption of UPEC BC1 cell membrane by ebselen and silver ion
The inhibition efficiency of ebselen and silver ion against UPEC BC1 was detected by Nuclear staining reagent propidium iodide (PI). BC1 cells were grown (37°C, 220 rpm) until an OD 600 nm of 0.4, which were treated with 80 μmol/L ebselen and 5 μmol/L silver ion (1.09 mmol/L amikacin was used as positive control; 1.17 mmol/L ceftazidime was used as negative control) for 20 minutes at 37°C. Cells were stained with 5 μg/mL PI for 30 min at 37°C , and the fluorescence was measured by flow cytometry (BECKMAN COULTER, AW15093).

| Effect of ebselen and silver ion on UPEC BC1 bacterial morphology
UPEC BC1 was grown until an OD 600 nm of 0.4 and separately treated for 20 minutes with 80 μmol/L ebselen and 5 μmol/L silver ion, 80 μmol/L ebselen, 5 μmol/L silver ion, 1.09 mmol/L amikacin, 1.17 mmol/L ceftazidime and PBS. Cells were obtained by centrifugation (4°C, 13 000 rpm, 15 minutes) and fixed with 2.5% glutaraldehyde. The morphology and structure of E coli cells were observed by transmission electron microscopy (Hitachi H-7500).  Table 2, and the cycling protocol included an initial denaturation at 95°C for 30 seconds followed by 40

| Protein S-glutathionylation in UPEC BC1 treated with ebselen and silver ion
Total protein S-glutathionylation (S-PSSG) of 80 μmol/L ebselen and 5 μmol/L silver ion-treated E coli cells were detected by Western blotting. Cells were cultured, washed and resuspended in lysis buffer (as described above) containing 30 mmol/L Iodoacetamide (IAM).
Western blotting assay was performed with IgG2a mouse monoclonal antibody for glutathione-protein complexes.

| Determination of ROS production in UPEC BC1 treated with ebselen and silver ion
UPEC BC1 was grown until an OD 600 nm of 0.4 in LB medium and incubated with 80 μmol/L ebselen and 5 μmol/L silver ion for 20 minutes. The BC1 cells were stained with 10 μmol/L H 2 DCFH-DA for 30 minutes at 37°C. After incubation, the ROS production was quantified by flow cytometry (BECKMAN COULTER, AW15093).

| Mild mouse cystitis model assay
Sixty healthy male BALB/c mice were divided into six groups randomly (n = 10). The bladders of female mice were infected via transurethral catheterization with 50 µL of MDR UPEC BC1 (×10 6 ).
Group A was left infected as the control; groups B-F were inoculated, and 20 mg/kg ceftazidime, 25 mg/kg ebselen, 6 mg/kg silver ion, 25 mg/kg ebselen plus 6 mg/kg silver ion, 10 mg/Kg amikacin and DMSO were administered i.p. on the 1st, 3rd and 5th days postinfection, respectively. The bacterial loads were calculated by counting the colony formation unity (CFU) derived from homogenates of urinary tissue.

| Immunohistochemical analysis
Mice bladders were bisected, fixed in formalin (10%), and embedded in paraffin. Paraffin sections (4 µm thick) were used to detect cytokine levels by immunohistochemical (IHC) analysis. Anti-TNF-α and anti-IFN-γ antibodies were used for the quantification of cytokines in tissue.

| Routine urine and blood tests
Urine of mice was collected 2, 4 and 6 days post-infection and diluted with normal saline to 1 mL, and the white blood cells (WBCs) and red blood cells (RBCs) in urine were determined by urine sediment. For investigation of liver and kidney function, blood was collected and centrifuged at 3000 rpm for 10 minutes. Serum alanine transaminase (ALT), aspartate aminotransferase (AST), urea and creatinine were determined.

| Statistical analyses
Statistical analyses were performed by GraphPad Prism 6.0 (GraphPad Software). Means of data between two groups were contrasted using Student's t test. Sample rates between two groups were tested with chi-square analysis. Overall survival was analysed by the Gehan-Breslow-Wilcoxon test. P values of <0.05 were considered to be significant.

| Antibacterial activity of ebselen and silver ion against uropathogenic E coli
The antibacterial effects of ebselen and silver ion on the growth of BC1 were detected by visible spectrophotometer. As shown in Figure 1A-C, silver ion alone inhibited BC1 growth with a 90% minimal inhibition concentration (MIC 90 ) of 32 μmol/L after incubation, while the addition of 4 μmol/L ebselen effectively reduced the MIC 90 of silver ion to 0.5 μmol/L. Meanwhile, 4 μmol/L ebselen and 0.5 μmol/L silver ion showed no synergistic toxicity on human cells. 18 These results demonstrate that ebselen and silver ion exerted significant selective synergistic toxicity against UPEC over mammalian cells. Furthermore, 64 μg/mL (109 μmol/L) amikacin was used as positive control, and 64 μg/mL (117 μmol/L) ceftazidime was used as negative control. The result in Figure 1D showed that 4 μmol/L ebselen and 0.5 μmol/L silver ion could completely inhibit bacterial growth and were more effective than amikacin (P < 0.00000000000001).
Propidium iodide (PI) nuclear staining, which reflects bacte-  (Figure 2A; Figure S1). Thus, most cells were dead after ebselen and silver ion treatment, which showed significantly greater effect than did ebselen (P = 0.0003), silver ion (P = 0.0005) or the control (P = 0.0004). The results demonstrate that UPEC BC1 was highly sensitive to ebselen and silver ion in combination.
The effect of ebselen and silver ion on the morphology of UPEC BC1 was assessed by transmission electron microscopy ( Figure 2B).

| Antibacterial activity of ebselen and silver ion targeting uropathogenic E coli TRX and GSH systems
The effects of ebselen and silver ion on bacterial TrxR activity, Trx expressions and total GSH level in UPEC BC1 were measured.
The results showed that the combination of 80 μmol/L ebselen Taken together, the above results suggest that ebselen and silver ion inhibited Trx activity and depleted GSH in UPEC BC1.

| The up-regulation of the intracellular expression level of ROS
The mean fluorescent intensity (MFI) of ROS in BC1 cells was detected by flow cytometry, and the result showed that the ROS production level in BC1 cells treated with 80 μmol/L ebselen plus 5 μmol/L silver ion was clearly up-regulated when compared with the control (P = 0.007), ebselen (P = 0.006) and silver ion (P = 0.0003) (Figure 4; Figure S2).
These results show that ebselen and silver ion constitute an effective antibacterial combination by inhibiting UPEC BC1 TrxR and depletion of GSH in vitro, and ROS production is one of the key mechanisms of its bactericidal activity.

| The depletion of UPEC BC1 in the mouse cystitis model by ebselen and silver ion
Cystitis is among the most common bacterial infections of human. The mouse provides an excellent and tractable model system for cystitis caused by UPEC. 28 As ebselen and silver ion have been demonstrated to be safe in our previous studies, 18

| The effects of ebselen and silver on inflammatory properties of mice with cystitis
An IHC assay was used to measure the presence of pro-inflammatory cytokines, including TNF-α and FN-γ. As shown in Figure 6, ebselen and silver ion significantly reduced the expression of both tested proinflammatory cytokines compared to mice treated with DMSO. For TNF-α, the group treated with ebselen plus silver ion exhibited the greatest reduction in expression, followed by amikacin and the control ( Figure 6). For IFN-γ, the group treated with ebselen plus silver ion also showed the greatest reduction in expression, followed by amikacin and DMSO ( Figure 6).
The urine and peripheral blood from different groups of mice were collected. The WBCs and RBCs counts in urine were determined, and the results showed that ebselen and silver ion statistically reduced the numbers of WBCs (P = 0.03, Figure 7A) and RBCs (P = 0.0009, Figure 7B) compared with the control. Furthermore, ALT, AST, urea and creatinine in mouse blood serum were also detected, and the results showed that ebselen and silver ion treatment had no influence on the ALT (P > 0.05, Figure 7C) or AST (P > 0.05, Figure 7D) levels; meanwhile, the combined treatment up-regulated the urea (P = 0.0003, Figure 7E) and down-regulated the creatinine (P = 0.06, Figure 7F).
Overall, ebselen and silver ion treatment groups exhibited the greatest effects in reducing the UPEC BC1-caused inflammation, and the activity of these drugs was considerably higher than that of amikacin. These results demonstrate that ebselen and silver ion may assist the recovery of infected urinary tracts, and their abilities to do so are considerably higher than that of amikacin.

| D ISCUSS I ON
Until now, UPEC has been the most common pathogenic bacteria, 29 accounting for 95% of community-acquired infections and 50% of hospital-acquired infections. 30 The failure of UTI treatment and F I G U R E 3 Antibacterial effect of ebselen and silver ion on UPEC BC1 targeting Trx and GSH systems. UPEC BC1 was cultured to OD 600 nm = 0.4 and treated with for 20 min with DMSO, 640 μg/mL ceftazidime, 80 μmol/L ebselen, 5 μmol/L silver ion, 80 μmol/L ebselen plus 5 μmol/L silver ion or 640 μg/mL amikacin. TrxR activity in slope (A) or end-point (B) was detected using DTNB reduction assay in the presence of Trx in BC1 extracts; (C) trxa (Trx1) mRNA expression level was tested by qPCR (normalized levels with respect to the reference rrs); D and E, Trx1 protein level was measured by Western blot (normalized levels with respect to the reference DnaK); GSH amount in slope (F) or end-point (G) was detected using DTNB reduction in the presence of GR in BC1 extracts; H and I, The P-GSSG level of total proteins was detected by Western blot, and the expression level of protein in 14 kDa was qualified. In recent studies, we found out that ebselen and silver ion have potential antimicrobial activity against GSH-positive bacteria, including MDR E coli and A baumannii. 18,26,27 In this study, the bactericidal activity against MDR UPEC BC1 of ebselen and silver ion was confirmed by visible spectrophotometry, transmission electron microscopy, DTNB assay and flow cytometry in vitro. These results showed that ebselen and silver ion exerted potent bactericidal activity against the MDR UPEC BC1 strain, which was closely related to the bacterial TrxR inhibition and GSH depletion.
Since bacterial Trx and GSH systems are important for reducing various critical cellular antioxidants to maintain the intracellular redox environment balance, inhibition of TrxR and depletion of GSH are highly related to the excessive production of ROS. 18 To detect the intracellular ROS expression, cells treated with ebselen and silver ion were stained by H 2 DCFH-DA, and the FACS results confirmed that ebselen and silver ion inhibited electron transfer, which enormously influences the ROS removal. This result demonstrates that ROS elevation is a major player in determining the bacterial fates, which is consistent with many reports from other groups that ROS produced by some clinically used antibiotics contribute to their bactericidal efficacy. 24,33,34 Using  To evaluate the effects of drugs on the liver and kidney function of mice, the sera of mice were collected, and contents of ALT (C), AST (D), urea (E) and creatinine (F) were measured communities (IBCs). 43 Their ability to attach and enter into cells in the bladder is a limiting step for their pathogenicity. 44 52,53 In this experiment, we found that the pro-inflammatory cytokines, WBCs and RBCs were significantly reduced following treatment with ebselen and silver ion. Declines in TNF-α, IFN-γ and WBCs in bladder may be highly related to the anti-inflammatory effect of ebselen. 54 Meanwhile, the decrease in RBCs in urine can be explained as follows: ebselen can inhibit endothelium-mediated diastole by scavenging peroxides 55 and inhibit oxidative stress of endothelial cells 56 to alleviate the inflammation-induced vasodilation-related hematuresis in cystitis. Meanwhile, it can down-regulate the levels of TNF-α and IFN-γ to reduce inflammatory injury. In addition, when ebselen and silver ion were used together, the ALT level showed no difference from that of the control group. This is because ebselen was able to alleviate the liver damage from different causes 57,58 and reduce the toxicity of heavy metals caused by methylmercury and manganese chloride in vivo, including silver ion. 22,59 The increase in blood urea has many causes, and it is not as useful a biomarker as serum creatinine for reflecting renal function.
Thus, based on our results, ebselen and silver ion have no effect on liver and renal function. These results suggest that ebselen and silver ion may be able to serve as alternative agents to amikacin to help treat MDR UPEC BC1-induced cystitis, and the sensitivity of bacteria to ebselen and silver ion is dependent on the antioxidant systems present in the bacteria.

ACK N OWLED G EM ENTS
We are grateful for the support of the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.