Novel metal peroxide nanoboxes restrain Clostridioides difficile infection beyond the bactericidal and sporicidal activity

Abstract Clostridioides difficile spores are considered as the major source responsible for the development of C. difficile infection (CDI), which is associated with an increased risk of death in patients and has become an important issue in infection control of nosocomial infections. Current treatment against CDI still relies on antibiotics, which also damage normal flora and increase the risk of CDI recurrence. Therefore, alternative therapies that are more effective against C. difficile bacteria and spores are urgently needed. Here, we designed an oxidation process using H2O2 containing PBS solution to generate Cl− and peroxide molecules that further process Ag and Au ions to form nanoboxes with Ag–Au peroxide coat covering Au shell and AgCl core (AgAu‐based nanoboxes). The AgAu‐based nanoboxes efficiently disrupted the membrane structure of bacteria/spores of C. difficile after 30–45 min exposure to the highly reactive Ag/Au peroxide surface of the nano structures. The Au‐enclosed AgCl provided sustained suppression of the growth of 2 × 107 pathogenic Escherichia coli for up to 19 days. In a fecal bench ex vivo test and in vivo CDI murine model, biocompatibility and therapeutic efficacy of the AuAg nanoboxes to attenuate CDI was demonstrated by restoring the gut microbiota and colon mucosal structure. The treatment successfully rescued the CDI mice from death and prevented their recurrence mediated by vancomycin treatment. The significant outcomes indicated that the new peroxide‐derived AgAu‐based nanoboxes possess great potential for future translation into clinical application as a new alternative therapeutic strategy against CDI.

and therapeutic efficacy of the AuAg nanoboxes to attenuate CDI was demonstrated by restoring the gut microbiota and colon mucosal structure.The treatment successfully rescued the CDI mice from death and prevented their recurrence mediated by vancomycin treatment.The significant outcomes indicated that the new peroxidederived AgAu-based nanoboxes possess great potential for future translation into clinical application as a new alternative therapeutic strategy against CDI.

Translational Impact Statement
Compared to vancomycin treatment, the peroxide-AgAu nanoboxes exhibit higher biocompatibility, attenuate Clostridioides difficile infection (CDI), restore the gut microbiota and colon mucosal structure, and prevent CDI recurrence.This new nanodrug possesses great potential for future translation into clinical application as a new alternative therapeutic strategy against CDI.

| INTRODUCTION
Clostridioides difficile is a major causative pathogen of nosocomial infections worldwide, with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon.
Immunocompromised conditions and the application of broadspectrum antibiotics, which can disrupt the normal intestinal microbiota, are some of the major risk factors for the development of C. difficile infection (CDI). 1 The incidence of CDI has progressively increased in the United States over the past decade, 2,3 which places substantial clinical and economic burdens on hospitals. 4,5CDI was associated with an increased risk of death, new long-term care facility transfer, and new short-term skilled nursing facility transfer. 6C. difficile is frequently transmitted in health care settings via health care workers, so CDI is an important issue in infection control. 7ostridioides difficile spores are considered a significant transmissible infection through the fecal-oral route and are most frequently attributed to the healthcare settings.][10][11] Antibiotics that alter the gut microbiota are the driving force behind the depletion of secondary bile acid production in the gut.3][14] Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of CDI, clinical recurrence rates remain high.C. difficile recurrence is the most frequent complication of infection affected by the amount or viability of C. difficile spores in the gut lumen or reinfection with spores from the environment.The confirmation of CDI recurrence is recognized as gut microbiota disruption coincident with viable C. difficile spores.Therefore, developing novel and better therapeutic interventions targeted to C. difficile spores or vegetative cells (bacteria) is imperative and needs to be further investigated.
Previous studies have demonstrated good inhibition of C. difficile spores by chemical disinfectants such as glutaraldehyde (20 mg/mL), sodium hypochlorite (0.25 mg/mL), H 2 O 2 (15 mg/mL), and formaldehyde (5 mg/mL). 15However, these chemical products are too toxic, causing dermal or tissue irritation, and are acceptable for environmental usage but are not suitable for treatment in patients.Moreover, currently available antibiotics target bacteria rather than spores.In addition, resistance to multiple antibiotics is becoming a common feature of C. difficile strains. 16Therefore, alternative nonantibiotic agents that target both C. difficile bacteria and spores are needed, and dual-function materials will shed light on preventing recurrence and avoiding the development of drug resistance in CDI.
Recently, resistance to multiple antibiotics has become a common feature of newly emergent strains, and an increasing number of nanoparticle types are regarded as potential new-generation broad-spectrum antimicrobial agents. 174][35] However, these current antibacterial nanoparticles mainly target to vegetative cells, and the sporicidal activity of these nanoparticles has rarely been explored.Yamaguchi and his coworkers reported that WO 3 at high concentrations (up to 25 mg/50 mL) 36 with long-term (24 h) visible light irradiation could only result in a 2.5-log reduction in Bacillus subtilis (gram-positive bacteria) spores, probably due to the high rigidity of the spore coats.
Herein, we designed an oxidation process with H 2 O 2 in PBS solution and the regeneration of Cl À and peroxide molecules as well as  (XRD; Bruker D8 Discover, Karlsruhe, Germany) was used to record the crystal structure change of AgAu-based nanoboxes.

| Preparation of Ag nanocube
Ag nanocubes were prepared from the single crystalline seed using a NaHS-mediated polyol reduction process at a high temperature from our previous report. 39Briefly, 10 mL of ethylene glycol (EG) solution was first heated to 160 C for 1 h, followed by 0.24 mL of NaHS (3 mM) addition for 2 min under magnetic stirring.Next, 0.5 mL of HCl (3 mM, dispersed in EG) and 2.5 mL of a PVP (20 mg/mL, dispersed in EG) solution were added to the previous solution and reacted for an additional 2 min.Finally, 0.8 mL of CF 3 COOAg (282 mM in EG) was injected into the refluxed mixture and reacted for 1 h.The final solution with greenish-gray color was quenched in an ice bath, washed with acetone and water three times, and finally dispersed into DI water for further use.

| Preparation of AgAu@PSMA nanocubes and
AgAu-based nanoboxes PSMA (2.5 mL of an 8.9 μM solution) and 250 μL of 12 mM Ag nanocube 39 were added to a two-neck round-bottomed flask with rapid magnetic stirring.Four milliliters of HAuCl 4 solution (0.125, 0.25, 0.5, or 1 mM) was added into the flask by using a dropwise addition process to get AgAu@PSMA nanocubes.Then, 200 μL of HNO 3 solution (0.22 M) was added immediately, followed by stirring for another 5 min until the color became stable.Then, 2.5 mL of 1Â phosphate-buffered saline (PBS) and 2.5 mL of 1.4 M H 2 O 2 were added into the flask and heated in an 80 C oil bath under reflux for 4 min, then quenched in an iced-water bath.The solution was centrifuged at 7000 rpm for 10 min and followed three times wash to collect AgAu-based nanoboxes stored in deionized water.

| Preparation of Au@MB
Twenty-five microliters of MB (5 mM) was added into 4.5 mL of HAuCl 4 (1.1 mM) solution mixed with 0.5 mL of TNA (2.5 mM) to prepare Au@MB. 40(2 M) were added to the Fe mixture.The Fe 3 O 4 @Chl was dispersed in deionized water after purification. 42

| SERS measurement
The 10 μL of aliquot samples with 0.5 mM [Ag] was dropped on Si substrate for SERS measurement.SERS spectra were acquired from a Jobin-Yvon LabRAM high-resolution micro-Raman spectrometer (Horiba iHR 320) with 785 nm laser excitation (DPSSL Driver II), 7 mW of power, 1200 grating number, 10 s of acquisition time and integrated into an Olympus BX53 microscope with Â20 objective lens.Each raw spectrum (200-1800 cm À1 ) was baseline corrected to remove the fluorescence background.

| LDI-MS detection of AgAu-based nanoboxes
An AutoflexIII LDI time-of-flight (TOF/TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive ion reflectron mode was used, with a SmartBeam laser (Nd:YAG, 355 nm, pulse width 6 ns, pulse duration 200 ns at 100 Hz) serving as the laser source, for the mass spectrometry experiments.The ions produced during laser ablation were stabilized by a delayed extraction period of 10 ns and then accelerated by a voltage ranging from +20 kV to À20 kV in the reflectron mode.Two hundred target positions were chosen for each sample using the random walk function for a total of 2000 laser shots with a power density of 18.3 kW/cm 2 .

| Bacterial culture
Clostridioides difficile CCUG 37780, a nontoxigenic strain, was used in nanoparticle-spore interaction.C. difficile VPI 10463, a toxigenic strain, was used in CDI animal model.C. difficile was cultured from stock bacteria stored at À80 C to CDC anaerobe 5% sheep blood agar (BD) in 37 C incubator under anaerobic conditions.After 48 h, colonies were transferred into brain-heart infusion supplemented medium (BD) with 5 mg/mL yeast extract (MO BIO) and 0.1% L-cysteine (AMRESCO ® ).Bacteria grow in 37 C anaerobic incubator for 2 days.

| Purification of spores
The C. difficile spores were prepared with mixture of 70% SMC medium and 30% BHIS medium (63 g Bacto peptone, 3.5 g protease peptone, 11.1 g BHIS medium, 1.5 g yeast extract, 1.06 g Tris base, 0.7 g NH 4 SO 4 , and 15 g agar/L) in 6-well dish, and then were incubated at 37 C under anaerobic condition (Thermo Fisher, Oxoid Ltd., Basingstoke, England) for 6-7 days.The spores were harvested from the 6-well dish with 10-15 mL of ice-cold sterile MQ water, vortexed for 5 min and then placed at 4 C overnight.The spore pellet was used ice-cold sterile MQ to wash five times and centrifuged at 8000 rpm for 20 min.Then, pellet was suspended with 200 μL of ice-cold sterile MQ.The suspension was spread on top of a 1 mL 50% (wt/vol) Nycodenz solution (Sigma-Aldrich, St. Louis, MO) and centrifuged at 10,800 rpm for 60 min.The purified spores were washed five times with ice-cold sterile MQ to remove extra Nycodenz solution, and stored in dark tube at 4 C.

| Spore staining viability assay
Dropped 1 μL of purified spore solution on the glass slide and dried on air.Then, used malachite green solution (Sigma-Aldrich, 50 g/L in water) to cover dried samples while heating the dye on heat plate for 5 min.Then, washed slide under running water and stained by Safarin O (Sigma, 5 g/L in water) for 15 s.Then, the slide was washed under the running water again.The spores showed green and the vegetative cells showed red under the light microscopy.

| Spore viability test
Purified spores were heated at 60 C for 30 min before test.C. difficile spores were diluted in fresh sterile MQ to an optical density (OD = 600 nm) of 0.2.Then treated with 100 ppm concentration of nanoparticles for 30 min.Next, spores were germinated by adding 10 mM taurocholate acid for 12 min.Finally, samples were diluted with BHIS and spread on CDC plates.The colony forming units (CFUs) at each dilution were examined after 48 h 37 C anaerobic culture.The survival rates were calculated using the following formula: (control CFU À treatment CFU)/control CFU Â 100%.

| Spore germination assay
Purified spores were heated at 60 C for 30 min before test.C. difficile spores were diluted in fresh sterile MQ to an optical density (OD = 600 nm) of 0.2.Then treated with different concentration of nanoparticles for 30 min and centrifuged at 8000 rpm for 20 min to get the spore pellets.Finally, 90 μL BHIS and 10 μL 10 mM taurocholate acid were added for spore germination induction.

| DPA release assay
Dipicolinic acid (DPA) release assay was measured by terbium fluorescence. 43Purified spores were heated at 60 C for 30 min before test and re-suspended in germination buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 100 mM glycine).Seventy-five microliters of spore were adjusted to an optical density (OD = 600 nm) of 0.

| Vegetative bacterial cells viability test
Minimum bactericidal concentration (MBC): C. difficile spores (10 4 CFUs) were treated with 100 ppm of silver nanoparticle for 15 min.Then, samples were diluted with fresh BHIS and spread on CDC plates.The CFUs at each dilution were examined after 48 h 37 C anaerobic culture.The survival rates were calculated using the following formula: Minimum inhibition concentration (MIC): E. coli (10 6 CFUs) were treated with 0-10 ppm concentration of AgAu-based nanoboxes, and the OD600 of the bacterial suspension was measured at multiple time points using microplate reader for bacterial survival rate calculation using the following formula: (OD600 treatment )/OD600 control ) Â 100%.
Ten microliters of C. difficile (10 7 CFUs) vegetative cells were added to 96-well plates.Then, treated with 190 μL fresh BHIS containing different concentrations of the category of nanoparticles under 37 C anaerobic conditions for 48 h.The results were observed after 2 days of culture.

| Sustained antibacterial experiment
Escherichia coli (106 CFUs) was daily added into 10 ppm of AgAu-based nanoboxes and measured OD600 by microplate reader at 24 h after bacteria addition.The experiment was continuously recorded until the bacteria grew in AgAucontaining broth.The same concentration of AgNO 3 , Ag@PVP, and Ag nanocube was also performed to monitor the sustained antibacterial time.

| ROS determination
To

| TEM analysis of spores and vegetative cells
The interaction between the nanoparticles and C. difficile spores and vegetative cells was observed by TEM.The spores were treated with 25 ppm nanoparticles for 30 min.The samples were washed three times by sterile MQ and dropped 10 μL onto the copper grid.The samples were observed at least 4 h vacuum dried.

| The effect of the nanoparticles on animal guts microbiota
The genetic distribution ratio was estimated by real-time PCR analysis.After normal mice had been sacrificed, cecum content was collected and treated with different concentrations of nanoparticles or MQ for 24 h.The DNA in the cecum content was extracted via the High Pure PCR Template Preparation Kit (Roche).Primers used in this study can refer to Table S1.

| Statistical analysis
GraphPad Prism 6 was used for all statistical analyses.Values were reported as mean ± SEM.All tests in this study were done in triplicate.
One-way analysis of variance (ANOVA) and then Tukey's multiple comparison test, Student's t-test were used for comparisons between groups, and differences were considered to be statistically significant with p-value < 0.05.
XRD patterns (Figure 1c) demonstrated that the fractions of the AgCl crystal structure over metallic Ag-/Au-related composites increased with increasing HAuCl 4 concentration.We used HADDF-EDS line scan to prove the formation of an AgCl@Au-rich shell, as discussed later.
As verified by the AAS measurement in Figure 1d, the Au/Ag ratio increased in the AgAu-based nanoboxes with increasing HAuCl 4 concentrations from 0.125 to 0.5 mM.Interestingly, the mixture of Ag@PSMA nanocubes with 0.125-0.5 mM HAuCl 4 in the presence of H 2 O 2 /PBS resulted in the disappearance of the original SPR bands (Figure 1e) compared to their precursors (nonoxidation products; Figure S2).Notably, a new peak appeared at 256 nm, which indicated AgCl crystal formation. 46The far IR absorption over 1000 nm for these AgAu 0.125 nanoboxes (Figure 1e) could be attributed to the existence of the thin-layer AgAu nanowalls decorated with dielectric AgCl nanocrystals. 47AAS measurements showed that the HAuCl 4 concentration at 1 mM did not dramatically decrease the Au count by oxidizing Ag atoms (Figure 1d), and the excess Au ions remained in the Obviously, the production of the AgCl nanocrystals was assisted by reacting Ag@PSMA nanocubes in PBS/H 2 O 2 solution with a high HAuCl 4 concentration.It is possible that oxidizing Ag 0 with a large amount of HAuCl 4 led to the release of more Ag + ions according to the galvanic replacement reaction, 39,51,52 and then were bound to the carboxylate groups in the PSMA layer.These released Ag ions further combined with Cl À ions and recrystallized in PBS solution, producing massive AgCl crystals in the XRD analysis in Figure 1c.These structures also attach to the AgAu@PSMA nanowalls (Figure 1a).Upon exposure to H 2 O 2 -containing PBS solution, the Ag ions at the PSMA polymer also generated peroxide materials. 49,53Note that Au(0) at 87.2 eV (Au 4f 7/2 ) and at 83.6 eV (Au 4f 5/2 ) was the primary derived from the AuAg alloy on the surface of the AgAu 0.5 and AgAu 1.0 nanoboxes.It is possible that the PSMA-bundled and Au-related thick shell aided in binding the metal ions 45,48 released from the dissolved nanocube templates, resulting in the difficult outward diffusion of Ag ions from the particle interior.These Ag ion species could rapidly bind to Cl ions in the PBS solution to form AgCl crystals that further attached to the inner walls of the AgAu 0.5 nanoboxes (Figure 2g) and the AgAu 1.0 nanoboxes.Because the Au(III) species are significantly presented on particles surface (Figure 2c) under excess HAuCl 4 at 1.0 mM (Figure S3), the formation of Au-based peroxide materials (Figure 2d) should originate from the homogeneous reaction between metal ions and H 2 O 2 54 at the carboxylate complex on the particle surface of the AgAu 1.0 nanoboxes, as illustrated in Figure 1a.The peak deconvolution analyses showed a tendency of the formation of Au-rich nanoshell structures (Figure 2c).Accordingly, the occupancy of Au-rich structures masked the AgCl and AgOO materials at the surface structures (Figure 2b).The surface measurement in FT-IR spectra was used to determine the vibration peaks of the O O at 831 cm À1 for AgAu 1.0 nanoboxes (Figure S5), which is located in similar positions to CaOO 55 and the Au peroxide complex. 54wever, due to the insufficient TEM resolution and limitation of the EDS mapping sensitivity, we were unable to identify the O elements to distinguish them on the interface of AgAu 1.0 nanoboxes at this time, and further investigation is needed in the future.
Next, we utilized LDI-MS to investigate the fine structures of AgAu-based nanoboxes (Figure S6).The existence of silver and chloride species, such as Ag 2 Cl and Ag 3 Cl 2 , for all samples, would be originated from the AgCl crystals in consistence with the XRD and XPS results (Figures 1c and 2a).Consistently, the population of Ag  S6b-d).
Figure S7 shows the dissolution experiment of the four AgAubased nanoboxes in water.The results confirmed that Ag-ion dissolution slowed down for the AgAu 1.0 nanoboxes, and the total released amount (2.7%) was less than that 5.1% of release rate from the AgCl crystals at 24 h (Figure S7a), indicating the formation of thick Au-rich nanowalls that protect the AgCl nanocrystals and delay Ag + release.A slow Ag + ion release feature demonstrated a promising long-term antibacterial efficacy.Notably, the concentrations of Ag + in the solution at 10 min were higher for the AgAu 0.125 nanoboxes (3.1%) than those for 2.4% by AgAu 0.5 and the 1.8% by AgAu 1.0 nanoboxes, which was attributed to the direct dissolution of AgCl nanocrystals from the AgAu 0.125 nanoboxes.Compared to 2.7% of Ag ions at 24 h, the dissolution of Au was relatively low at 1.4% in AgAu 1.0 nanoboxes (Figure S7b) due to its stable and inert structure of alloy-based nanocomposite.

| The rapid, efficient, and prolonged bactericidal ability of AgAu-based nanoboxes against pathogenic E. coli O157
To determine the antibacterial activity of the AgAu-based nanoboxes, we first used the clinical Shiga toxin-producing E. coli O157:H7 strain, which is an easier cultured enterohemorrhagic bacterial strain that growth in a facultative anaerobic condition that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) in humans, as the target microorganism.A total of 1 Â 10 6 E. coli were incubated with 0-5 ppm AgAu-based nanoboxes for 1-24 h (Figure 3a-c).The 1 h treatment (Figure 3a) already demonstrated dramatic antibacterial activity (10%-45% inhibition) at 0.25 ppm and a significant depletion of the bacteria at 2.5 ppm by the AgAu 1.0 nanoboxes.As shown in Figure 3b, the killing efficiency at 1.25 ppm to E. coli upon 6 h incubation follows the order: AgAu 1.0 > AgAu 0.5 > AgAu 0.25 > AgAu 0.125 .
The minimum antibacterial concentrations (MICs) of the AgAu-based nanoboxes at 6 and 24 h of incubation were 1.25 ppm (Figure 3b,c).
In addition to the solution-based antibacterial experiments, a similar tendency of highly depletive bacteria by Au-rich nanoboxes (i.e., AgAu 0.5 and AgAu 1.0 ) could also be observed in the agar plating bactericidal assay (Figure 3d).The TEM images showed that the AgAu 1.0 nanoboxes, as a model group, were closely attached to the surface of E. coli O157:H7 (Figure 3e) after 45 min of reaction time.The bacterial membrane structure became disintegrated, suggesting that the bacterial cells were seriously damaged by AgAu 1.0 nanoboxes.Therefore, the extremely low inhibition concentration at 2.5 ppm with AgAu 1.0 nanoboxes at 1 h (Figure 3a) could injure the bacteria, which might be due to the exposure of highly active spots at the sharp edges of the nanobox structure compared to the 1 h treatment of 10 ppm AgAu nanosphere structure in our previous work. 48We next performed a redox experiment with different AgAu-based nanoboxes as catalysts for the interfacial transfer of electrons from NaBH 4 (oxidation) to 4-nitrophenol (reduction).As shown in Figure S8, the peroxide surface composite of AgAu 1.0 -based nanoboxes provided a faster reaction below 3 min and complete conversion from 4-nitrophenol to 4-aminophenol at 30 min when compared with the static state generation of 4-AP with the metallic AgAu@PSMA 1.0 nanocube (without reaction in H 2 O 2 and PBS).AgAu 1.0 nanoboxes also exhibited efficient and superior oxidation of aminophenol to form quinone 56,57 (Figure S9).These results provided clear evidence that the delicate peroxide surface structure easily triggered redox reaction of the biomolecules in the natural state bacteria.Perhaps the outermost surface membrane of bacterial cells was oxidized when these AgAu 1.0 nanoboxes contacted the cell surface and drew their electrons because of their strong oxidation powers of the Au(III), Au(I), and Ag(I) complex at the nanowalls, [58][59][60] and specific peroxide surface structures 49 (Figure 2a-d).The oxidized bacterial membrane of the normal microorganism would make dysfunction of the electron transport chain. 61 verify bacterial injury, we used DCFHDA to measure the intracellular ROS changes in E. coli O157:H7 after coincubation with AgAubased nanoboxes.Figure 3f shows that all the bacteria presented high intracellular ROS levels with the four nanobox treatments in 30 min.
Such high ROS production in the cell body would affect the subsequent duplication of bacteria, leading to not only reduced bacterial proliferation but also cell death.
We also demonstrated that the AgAu-based nanoboxes have no nanozyme properties 62 due to no superoxide, singlet oxygen, or hydroxyl radical generation when reacted with H 2 O 2 and under 660 nm irradiation (Figure S10).These results confirm the elimination of additional injury caused by these ROS to microorganisms.
In addition, AgAu 1.0 nanoboxes exhibited long-term antibacterial activity compared with AgAu 0.125 nanoboxes (Figure 3g).This activity yielded a dramatic depletion of bacteria of up to 5 Â 10 6 by using AgAu 1.0 nanoboxes (Figure 3h).Compared with AgNO 3 at 10 ppm with 1 Â 10 6 bacterial cells (Figure S11), our AgAu-based nanoboxes showed a superior extended sustainable antibacterial period from 6 days (AgAu 0.125 ) to 19 days (AgAu 1.0 ), as shown in Figure 3g.Therefore, it was realized that AgCl crystals (Figure 1c) surrounded by the Au-rich shell protection layer of the AgAu 1.0 nanoboxes could prolong the amount of Ag ions by its slow and continuous in situ release (Figure S7a) from AgCl and thus possessed long-term antibacterial activity.At concentrations of $0.035 ppm of Au ions and 0.0675 ppm of Ag ions, based on the release rates of 1.4% Au and 2.7% Ag (Figure S7) from the 2.5 ppm AgAu 1.0 nanoboxes, the 24 h-MIC data showed no significant reduction of bacteria growth (Figure S12).We further removed the AgCl and peroxides on the AgAu-based nanoboxes by washing with saturated NaCl to form dissolved metal chloride complexes.Figure S13 shows a significant decrease in antibacterial activity against E. coli O157:H7.
To mimic the effect of high concentrations of aminothiols in biological fluids as well as intracellular environments on the AgAu-based nanoboxes, we pre-incubated the nanoboxes with cellular-abundant GSH (10 mM) for 24 h.The S 2p signals 63,64 from the Ag S and/or Au S bonds were detectable in Figure S14a, indicating the successful immobilization of GSH on the surface of AgAu 1.0 nanoboxes.
Figure S14b shows that the fractions of peroxide (O O) reduced from 38.3% to 17.3% when compared to as-prepared AgAu 1.0 nanobox's surface structure.They lacked significant change in the fraction of Ag O between as-prepared AgAu 1.0 nanoboxes (33.4%) and GSHpassivated AgAu 1.0 nanoboxes (35.5%).As shown in Figure S14c, we found that the GSH-passivated AgAu 1.0 nanoboxes weakened the antibacterial activity, which agrees with other compromised antibacterial ability of thiol-stabilized AgNPs, 65,66 being possibly due to the hinder the bacterial membrane destruction upon the particle attachment (Figure S14d).Figure S14c shows that the AgAu 1.0 nanoboxes perform similar tendency in killing bacteria if the GSH concentration in the incubation medium decreased to 1 mM.

| Sporicidal ability of nanoparticles against C. difficile spores
To examine the killing effect on the harmful and elusive spores, purified C. difficile spores ($10 7 ) were first prepared and then incubated with 0-100 ppm AgAu 1.0 nanoboxes for 30 min and were then treated with TA for germination and spreading on CDC plates.Germination was measured by tracking the loss of optical density at 600 nm over 12 min at room temperature (Figure 4a).As shown in Figure 4b, spore germination was dose-dependently inhibited by AgAu 1.0 nanoboxes at 25-100 ppm concentrations.Compared to the 96.4% and 74.1% of spore survival by 100 ppm AgAu@PSMA and Ag nanoplate, respectively, treatment with AgAu 1.0 nanoboxes at 100 ppm markedly suppressed the spore survival low to 8% (Figure 4c) after a 12-min germination process.Figure 4d,e shows a lack of germination inhibition by positively charged TNA/PEI-AgAu NPs and antibacterial TNAcoated Fe 3 O 4 (Fe 3 O 4 @TNA). 41PDT-functionalized Fe 3 O 4 @Chl 42 and PDT-functionalized Au@MB nanoparticles 40 were implemented for an additional comparison and showed insufficient or inferior suppression of C. difficile spore survival (Figure 4e).These nanoparticles were possibly not appropriate for attachment and/or caused remarkedly oxidation damage to C. difficile spores.
The limited sporicidal effect was further examined by measuring the release of dipicolinic acid (DPA), a dehydrated core of C. difficile spores that is biosynthesized during sporulation.As shown in Figure 4f, the fluorescence intensity at 545 nm of DPA was decreased by the AgAu 1.0 nanobox-treated spores in a dose-dependent manner.This release of DPA when incubated with 12.5-100 ppm AgAu 1.0 nanoboxes was superior, indicating the effective injurious/oxidation to the cell membrane (Figure 5a) by the particle's peroxide surface structures, when compared with the positive control group in a reaction at 100 C for 30 min (Figure 4f) and with metallic Ag nanoplates and AgAu@PSMA nanocubes at 100 ppm [Ag]   (Figure 4g).(Table 1).The metallic AgAu 0.125 @PSMA nanocubes need a 100 ppm sample dose to approach the bactericidal effect (Figure 5b), which might be due to less destruction of the cell membrane structure in the same incubation time (Figure 5a) and its low release amount of Ag ions in the solution (Figure S15).The peroxide surface structure combined with AgCl-based species stored in the core of the AgAu@AgCl nanoboxes was exceptionally injurious to the cell membrane and  S2).
To investigate the anti-spore and antimicrobial mechanism, TEM imaging (Figure 5c,d) was implemented to monitor the interaction between the AgAu 1.0 nanoboxes and C. difficile spores/vegetative cells.Compared to gram-negative bacteria, the cell wall composed of multilayer peptidoglycan of gram-positive bacteria was thick. 67However, the sturdy surface structures of the spores and vegetative cells were destroyed after treating with 100 ppm AgAu   F I G U R E 6 Legend on next page.
(Figure 6f).In addition, serum amyloid A (SAA), a major acute-phase protein that responds to infections, 68 was significantly decreased in the AgAu 1.0 -treated group compared to the CDI group (Figure 6g), while vancomycin-treated mice showed a higher infection index than that of the CDI group due to CDI recurrence.We also measured the changes in C. difficile growth, and the results showed that AgAu 1.0 could largely reduce the C. difficile numbers compared with that of the vancomycin-treated group (Figure 6h).The histopathological images of the colon also showed that AgAu 1.0 nanoboxes have therapeutic efficacy by attenuating CDI through reducing inflammation and showed protection of the murine colon by preserving mucosal integrity in treated mice compared to the damage to the colon mucosa caused by vancomycin (Figure 6i).These results indicated that AgAu 1.0 nanoboxes have better therapeutic outcomes than traditional antibiotic treatment.Although CDI recurrence was observed after Day 5 in the vancomycin-treated group, the therapeutic outcome at Day 5 was still effective, with an increased cecum weight of approximately double that in the CDI group (Figure S18a).The colon length in vancomycin-treated and AgAu 1.0 nanobox-treated CDI mice was significantly longer than that in the CDI control group (Figure S18b), and SAA was greatly attenuated in vancomycin-treated and AgAu 1.0 nanobox-treated CDI mice (Figure S18c).Combined with the histopathological images of the colon that showed a recovered mucosal structure in vancomycin-treated mice (Figure S18d), these data suggest that short-term treatment with antibiotics can successfully reverse the CDI in mice, but there is a risk of recurrence with longterm treatment that can be solved by alternative treatment with AgAu 1.0 nanoboxes.
A previous study 69 reported that DAPT-functional gold nanoparticles have excellent antibacterial ability without considering their impact on the normal flora, which plays critical role in preventing the invasion of other pathogens in the host's gut. 70However, clinical treatment of CDI relies on antibiotic usage, which damages the normal flora in the human gut tract and increases the risk of CDI recurrence.
Thus, an optimal antibacterial nanoparticle should be able to specifically target pathogens without disrupting the normal flora.In the long-term assessment of the posttreatment effect on the gut microbiota, we examined the effects of AgAu 1.0 nanoboxes on the mouse microbiome, and the genetic distribution was estimated by real-time PCR analysis. 71Compared to the impact of vancomycin on the gut microbiota, the AgAu 1.0 nanoboxes showed relatively better microbiome composition closed to normal flora while vancomycin caused worst microbiome alteration (Figure 6j).Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced by gut bacteria through saccharolytic fermentation of complex carbohydrates.SCFAs play a crucial role in energy supply, intestinal barrier integrity, mucus production, and inflammation protection. 72To further understand SCFA-producing bacterial genera, such as Bacteroides, Faecalibacterium, and Parabacteroides, real-time PCR for analyzing SCFA-producing enzyme genes was performed.Figure 6k shows that the administration of AgAu 1.0 nanoboxes restored the abundance of SCFA-producing enzyme genes.We found one of the butyrate-producing bacteria, Faecalibacterium prausnitzii, was increased after AgAu nanoboxes treatment (data not shown).These findings suggest the potential beneficial effects of AgAu 1.0 nanoboxes for treating CDI on gut health.These results revealed the potential of future application with AgAu 1.0 nanoboxes in infected animals and humans.

| Biosafety and biocompatibility of the AgAubased nanoboxes
Notably, the biocompatibility of the AgAu 1.0 nanoboxes was additionally studied to demonstrate the potential medical applications, especially in the cells belonging to the digestive system that would directly contact the nanomaterials.Human normal oral keratinocyte (hNOK) commonly serves as normal cell control for the toxicology test of nanomaterials and has been used extensively in biological and cancer research. 73The result showed that AgAu 1.0 nanoboxes exhibited a higher cell survival rate than the AgAu 0.5 nanoboxes with close Au/Ag ratio (Figure 1d) and those Ag-rich AgAu 0.125 and AgAu 0.25 nanoboxes to hNOK cells (Figure S19), suggesting the improved biocompatibility by the exposure of Au-rich shell structure at AgAu 1.0 nanoboxes.In addition to in vitro test, we performed in vivo toxicology evaluation of AgAu 1.0 nanoboxes in healthy mice (Figure 7a).The body weight (Figure 7b) and colon length (Figure 7c,d) in AgAu 1.0 nanoboxestreated mice were not changed, similar to untreated animals.The kidney weight and histological examinations were the same as those of normal mice (Figure 7e), as was the liver (Figure 7f).It is worth mentioning that the mucosal integrity of the murine colon in the AgAu 1.0 nanobox-treated group remained normal in the in vivo study (Figures 6i and S18d).
Many previous studies of the cytotoxicity induced by Ag ions in silver nanoparticles. 74,75Silver has been used for at least six millennia to prevent microbial infections and was the most critical antimicrobial agent available before the introduction of antibiotics. 76,77However, its toxicity has also been a concern for modern medication.The toxicity of silver depends on its solubility and release of biologically active Ag + ions; for example, acute human mortality has been observed following an abortion procedure involving the intrauterine administration of 64 mg silver/kg silver nitrate, 78 while silver acetate has an LD 50 of 36.7 mg/kg in mice, 79 and silver chloride has an LD 50 higher than 10 g/kg in mice following oral administration.For Ag nanoparticles, orally administered nanoparticulate silver was not toxic to guinea pigs at acute doses of up to 5 g/kg/day, 80 suggesting that soluble compounds are always more cytotoxic than insoluble ones.The dosage of AgAu nanoboxes used in this study was only 2 mg/kg, which is far from the toxic threshold, and our results supported the nontoxicity to animals in vitro and in vivo.To approach proper protection, we speculated that the Au-rich capping nanolayer at the AgAu 1.0 nanoboxes could protect the AgCl from direct contact with the cell.These particles also exhibited a delayed release (Figure S7

2. 2 |
Characterizations Transmission electron microscopy (TEM, Hitachi H7500 TEM instrument at 80 kV) was utilized to determine the structures of the AgAubased nanoboxes.The absorption spectra of the AgAu-based nanoboxes were measured by a V-730 UV-Vis spectrophotometer from Jasco (USA).The Ag concentrations of AgAu-based nanoboxes were quantified by AAS (SensAA GBC, Australia).The FT-IR spectra were obtained by Fourier-transform infrared spectroscopy (JASCO FT/IR-4700) with a KBr plate.X-ray photoelectron spectra (XPS) (PHI 5000 VersaProbe, Japan) were utilized to measure AgAu-based nanoboxes by a Mg Kα source (12 kV and 10 mA).The binding energy scale was calibrated to the central C 1s peak at 284.5 eV.The thin film x-ray diffractometer F I G U R E 1 (a) Scheme of the synthesis reactions to fabricate the AgCl/AuAg nanoshells@Ag-based peroxide (AgAu 0.125 nanoboxes) and AgCl@Au-rich nanoshells@Au-based peroxide (AgAu 1.0 nanoboxes) using low and high Au ion concentrations in H 2 O 2 -containing PBS solution.(b) TEM images, (c) XRD pattern, (d) AAS measurements, and (e) UV-visible spectra of the AuAg-based nanoboxes.(f) Raman spectra of the mixed solution of 0.05 mM methylene blue and 0.5 mM [Ag] AuAg-based nanoboxes.

FeCl 3 •
6H 2 O (1 M) and Chl/Fe (0.14 M) were dissolved in 2 mL of HCl.Afterward, 0.5 mL of FeCl 2 •4H 2 O (2 M) and 20 mL of NH 4 OH 2 and then added to 96-well white plate.Next, spore suspension is mixed with equal volume of different concentrations of nanoparticle.Then, 50 μL germination buffer containing 40 mM taurocholate and 0.4 mM TbCl 3 was added to samples.The spores treated with TbCl 3 only were served as a negative control.The total DPA of the spores was extracted by boiling the samples for 30 min.The signal of DPAterbium was monitored by FlexStation 3 Multi-Mode Microplate Reader with excitation/emission at 270 and 545 nm, and cutoff at 520 nm.
determine intracellular ROS production in AgAu-based nanoboxtreated bacteria, 5 Â 10 6 bacteria were exposed to different concentrations (0-10 ppm [Ag] ) of AgAu-based nanoboxes at 37 C for 30 min.After exposure, the bacteria were centrifuged and washed three times with PBS.The bacterial pellets were suspended in 100 μL of Mueller-Hinton broth (MH broth) containing 20 μM 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) for 30 min, which can passively enter into the bacteria and react with ROS to form the highly fluorescent compound dichlorofluorescein for ROS quantification.After incubation with DCFH-DA, bacteria were washed three times and resuspended in PBS for fluorescence measurement by microplate reader with 495/520 nm excitation/emission spectra.The relative ROS level was expressed as the fold changes of fluorescence intensity compared to untreated bacteria.
Spore integrity was measured by LIVE/DEAD BacLight Bacterial Viability Kit.No treatment spores and treat-nanoparticle spores were mixed with SYTO9 and PI for 15 min.The samples were observed by fluorescence microscopy.
measurements for the (a) Cl, (b) Ag, (c) Au, and (d) O composites on the AgAu-based nanobox surface.The (e) Cl/Au and (f) Au/Ag ratio changes of AgAu-based nanoboxes were calculated from the XPS results.(g) HAADF and EDS elemental line scan/mapping images of AgAu 0.125 , AgAu 0.5 , and AgAu 1.0 nanoboxes.supernatant (Figure S3), suggesting that it has reached the maximum level of AgCl formation.We observed an SPR band at 711 nm for AgAu 1.0 nanoboxes, suggesting the generation of thick Au-based nanoshells.We further utilized a SERS experiment to demonstrate the gradual evolution of Raman signals from methylene blue for the solution mixture with the resulting nanoboxes from AgAu 0.125 to AgAu 1.0 (Figure 1f), proving the massive metallic nature at the surface of the AgAu 1.0 nanoboxes.The XPS spectra were analyzed to reveal the chemical states of AgAu 0.125-1.0nanoboxes (Figure 2a-d), and the element results in the surface analysis are presented in Figures 2e,f and S4.Figures 2e and S4a show the appearance of Cl/Au and Cl/Ag signals on the surface structure of the AgAu 0.125 nanoboxes and were markedly decreased in the AgAu 1.0 nanoboxes.The total Cl amount on the surface structure is 13.4%-17.4%for the AgAu 0.125-0.5 and 4.2% for the AgAu 1.0nanoboxes, respectively (FigureS4b).Figure2fshows an increase in the ratio of Au/Ag from 0.38 for the AgAu 0.125 nanoboxes to 1.08 for the AgAu 1.0 nanoboxes, indicating the formation of Au-rich surface structures (Figure1a).Consistently, the high-angle annular dark-field (HAADF) integrated energy-dispersive x-ray spectroscopy (EDS) elemental line scan and mapping (Figure2g) of a single AgAu 1.0 nanobox showed that the Ag and Cl atoms were primarily located in the interior area and were surrounded by the Au-based shell.When the HAuCl 4 concentration was decreased to 0.125 mM, the resultant AgAu 0.125 nanowalls (Figure2e) consisted of AgCl surface composites and AgAu nanocrystals,48 in agreement with the significant amount of Cl À and Ag(I) ions determined in the XPS surface analysis (FigureS4b).The layer structure of the AgAu 0.5 nanoboxes could be a possible hybrid structure of AgAu alloy and AgCl as shown in Figure1a.High-resolution XPS measurements of Cl 2p signals at 199.5 and 196.6 eV were clearly recorded in all nanobox samples (Figure2a).The Ag(I) ions of the AgCl crystal at 367.2 eV (Ag 3d5/2) and 373.2 eV (Ag 3d3/2) were determined in Figure2b, which depicts a decrease in the peak areas when increasing the Au amount in the AgAu-based nanoboxes.Intriguingly, the new Ag 3d5/2 at 367.8 eV and Ag 3d3/2 at 373.8 eV (Ag 3d3/2) are assigned to the AgOO structure.49The alloy-type Ag at 367.2 eV (Ag 3d5/2) and 373.2 eV (Ag 3d3/2)) became the predominant composition on the surface of the AgAu 1.0 nanoboxes compared with the perfected Ag(I)Cl and AgOO in other AgAu-based nanobox groups.It is known that the alloy type of the AgAu composition was more resistant to the oxidation and etching by H 2 O 2 compared with pure Ag materials.50However, the relative peaks of Au(III) ion species at 86.6 eV (Au 4f 7/2 ) and at 90.2 eV (Au 4f 5/2 ) and Au(I) ion species at 84.4 eV (Au 4f 7/2 ) and at 88.0 eV (Au 4f 5/2 ) could be observed and grew very well at the particle surface of the AgAu 1.0 nanoboxes (Figure2c).An O1s signal at 532.8 eV was assigned to the O O structure in the XPS spectra (Figure2d) in addition to Ag O (at 531.9 eV) of AgOO49 and C O (at 530.7 eV) of PSMA, as detected in Figure2d.

F I G U R E 5
The decrease in C. difficile vegetative cell viability by AgAu-based nanoboxes.(a) The schematic of AgAu 1.0 nanoboxes, but not other Ag-associated NPs or Ag ions, exhibiting bactericidal and sporicidal activity within 30 min.(b) C. difficile was treated with 100 ppm nanoparticles and then spread on CDC plates.Colony counts were determined for bacterial survival.TEM images of different silver nanoparticles interacting with (c) C. difficile spores and (d) vegetative cells by treatment with 100 ppm nanoparticles and incubation for 30 min.The AgAu 1.0 nanoboxes damaged spore (e and f) and vegetative cell integrity (g and h) determined by a LIVE/DEAD BacLight Bacterial Viability Kit.Scale bar = 10 μm [****p < 0.0001; **p < 0.01; one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test].sustained Ag ion donation to interact with biomolecules of C. difficile vegetative cells.We also compared the anti-C.difficile ability of AgAubased nanoboxes with metronidazole, which is the primary antibiotic used for CDI treatment.It appears that the MICs of AgAu 1.0 and metronidazole were 6.25 and 0.5 ppm, respectively (Table Figure S16b, the AgAu 1.0 nanoboxes exhibited dose-dependent bactericidal ability: 26.9% by 12.5 ppm, 38.4% by 25 ppm, and over 93% at 50-100 ppm.

3. 5 |
The therapeutic effects on CDI in vivoBased on the aforementioned successful therapeutic in vitro (Figures4 and 5) and ex vivo results (FigureS16) against CDI, we further established a recurrent CDI murine model and evaluated disease progression.The untreated CDI group and vancomycin-treated CDI group were added for comparisons of the enteric microflora (gut microbiota composition) in the murine infection model.At 48 h postoral infection of mice by toxigenic C. difficile BAA-1805 spores, the mice were gavaged with 100 μL of 500 ppm AgAu 1.0 nanoparticles (2 mg [Ag] /kg) and 50 mg/kg vancomycin every 24 h for 2 days (Figure6a).Compared to that of untreated CDI mice, the body weight of AgAu 1.0 nanobox-treated CDI mice started to increase and continuously recovered to that of untreated CDI mice and the corresponding mice before CDI infection (Figure6b).The trend of weight recovery with AgAu 1.0 nanoboxes was slower than that of the vancomycintreated CDI mice but continuously increased, while vancomycintreated CDI mice showed recurrent CDI with a drop in body weight after Day 5.This finding suggested that the sporicidal AgAu 1.0 nanoboxes could efficiently sustain the reversal of the CDI condition, while antibiotics can only temporally alleviate the CDI due to the insufficient sporicidal ability.The survival rate of mice was also rescued from 80% in the CDI group to 100% when treated with AuAg 1 (Figure6c).To examine the effect of nanoboxes on eliminating the C. difficile level in vivo, we reperformed a follow-up experiment by detecting C. difficile genes using PCR with stool samples collected on day of sacrifice, we demonstrated three of the higher tpi levels per mouse group, as shown in Figure6dand S17.The results showed a significant reduction in C. difficile residues presented in the stool samples after administering nanoboxes, especially in the AgAu 1.0 treatment.Regarding the clinical outcome, due to CDI recurrence, the cecum weight of the vancomycin-treated group was lighter than that of the AuAg-treated group on Day 11 (Figure 6e), and the colon length in the AuAg-treated group recovered nearly to that of the mock control T A B L E 1 C. difficile growth inhibition by silver nanoparticles.

6
The in vivo therapeutic effects of AgAu-based nanoboxes on CDI.(a) Scheme of protocol for CDI induction and antibiotic/ AgAu-based nanoboxes treatment timelines on murine model.The CDI disease progression is defined by (b) body weight loss and (c) survival rate; (d) C. difficile DNA determined by tpi PCR on mouse stool; (e) cecum weight; (f) colon length; (g) SAA concentrations; and (h) the C. difficile population determined by C. difficile Toxins A and B in the stool.(i) Microscopic examine of the colon tissues.(j) The genetic distribution ratio of colon microbiome with CDI/vancomycin/AgAu-based nanoboxes estimated by the real-time PCR analysis.(k) Short chain fatty acids (SCFAs) producing bacteria population quantitation [***p < 0.001; **p < 0.01; *p < 0.05; one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test].
) and thus resisted the increased Ag ion concentration in a concise time.The proper cell variability toleration, low injury to the colon, relatively less change in gut microbiota, and superb anti-C.difficile spores and bacteria demonstrate the practical utility of AgAu 1.0 nanoboxes to treat CDI.

HAuCl 4
concentrations in PBS/H 2 O 2 solution.The AgAu 1.0 nanoboxes exhibited improved bactericidal (E. coli and C. difficile) and sporicidal capabilities based on the high oxidation of the cell membrane structures in a short time.The subsequent release of Ag ions from the interior of the AgCl nanocrystals of AgAu 1.0 nanoboxes offered an increased killing efficiency of bacteria and spores with sustainable microorganism inhibition.From the results of the in vivo study, we demonstrated that the developed AgAu 1.0 nanoboxes could inhibit the survival of C. difficile spores and vegetative cells without causing significant colon mucosal damage and could prevent the recurrence of CDI.These AgAu 1.0 nanoboxes possessed greatly improved biocompatibility due to the Au-rich surface structures, which is promising for potential future translation into clinical application as a new alternative therapeutic strategy against CDI.AUTHOR CONTRIBUTIONS Li-Xing Yang: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (equal); software (equal); validation (equal); visualization (lead); writingoriginal draft (lead); writingreview and editing (lead).Yi-Hsin Lai: Conceptualization (equal); data curation (lead); formal analysis (lead); methodology (equal); software (lead); validation (equal); visualization (equal); writingoriginal draft (equal).Chun In Cheung: Data curation (lead); formal analysis (lead); methodology (equal); software (lead); visualization (equal); writingoriginal draft (lead).Zhi Ye: Data curation (lead); formal analysis (lead); methodology (equal); software (lead); visualization (equal); writingoriginal draft (lead).Tzu-Chi Huang: Data curation (lead); formal analysis (lead); methodology (lead); software (equal); visualization (equal); writingoriginal draft (equal).Yu-Chin Wang: Data curation (lead); formal analysis (lead); methodology (equal); software (equal); validation (equal); visualization (equal); writingoriginal draft (equal).Yu-Cheng Chin: Data curation (equal); formal analysis (equal); methodology (equal); validation (equal).Zi-Chun Chia: Formal analysis (supporting); validation (equal); writingoriginal draft F I G U R E 7 The in vivo toxicology evaluation of AgAu 1.0 nanoboxes in non-CDI murine model.(a) Scheme of protocol for AgAu 1.0 nanoboxes on murine model.The (b) body weight, (c) colon length, and the (d) colon images of mice with/without AgAu 1.0 nanoboxes.The (e) kidney and (f) liver weight and the corresponding IHC images of mice with/without AgAu 1.0 nanoboxes.All the results were collected after sacrifice of animals on Day 5.
Antibiotic-treated mice had been sacrificed; cecum content was collected and 200 μL for each tube.Then, added 50 μL C. difficile (10 7 CFU) and 50 μL different concentrations of AgAu nanoparticles 44is study used 8-weeks-old C57BL/6 mice for C. difficile infected murine model.Referred to our laboratory murine protocol,44mice were administrated with antibiotics mixture (kanamycin, 0.4 mg/mL; gentamicin, 0.035 mg/mL; colistin, 850 U/mL; vancomycin, 0.0045 mg/mL; metronidazole, 0.215 mg/mL; Sigma-Aldrich) in the drinking water for 5 days before CDI.However, vancomycin and metronidazole were stopped to feed to avoid killing C. difficile on the day before infection.Then, the mice were treated with proton pump inhibitor (PPI), Esomeprazole (40 mg/kg/day; Nexium ® ) by five times oral gavage per 12 h interval before CDI.On the day for CDI, mice were injected with clindamycin (4 mg/kg; Sigma-Aldrich) intraperitoneally and then treated with C. difficile spore suspension (1 Â 10 8 CFU) orogastrically.2.23 | Inhibition of C. difficile in a fecal bench ex vivo testThe CDI animal model was described above.After 48 h post-infection, the mice were gavaged with 100 μL 500 ppm nanoparticles every 24 h for 2 days.While the control CDI mice were gavaged with sterile MQ.Mice were weighed and scored daily to monitor symptoms of CDI until sacrificed after 120 h.CDI disease progression was defined by body weight loss, colon length, cecum weight, and PCR results of tpi gene (specific to C. difficile).
2 AuCl 2 and Ag 2 Au 2 Cl species increased when the Au-rich nanoboxes formed.The levels of Ag 2 and Ag 3 species decreased, accompanied by the generation of Au 3 and AgAu 2 molecules, as the Au concentrations increased in the AgAu nanobox structure at the same time.Besides, we found that the peroxide-based species such as Ag 2 AuCl 2 + H 5 O 2 , Ag 3 AuCl 2 + H 5 O 2 , and Ag 2 Au 2 Cl + H 8 O 4 were primarily detected in