Octahedron Iron Oxide Nanocrystals Prohibited Clostridium difficile Spore Germination and Attenuated Local and Systemic Inflammation

Clinical management of Clostridium difficile infection is still far from satisfactory as bacterial spores are resistant to many chemical agents and physical treatments. Certain types of nanoparticles have been demonstrated to exhibit anti-microbial efficacy even in multi-drug resistance bacteria. However, most of these studies failed to show biocompatibility to the mammalian host cells and no study has revealed in vivo efficacy in C. difficile infection animal models. The spores treated with 500 µg/mL Fe3-δO4 nanoparticles for 20 minutes, 64% of the spores were inhibited from transforming into vegetative cells, which was close to the results of the sodium hypochlorite-treated positive control. By cryo-electron micro-tomography, we demonstrated that Fe3-δO4 nanoparticles bind on spore surfaces and reduce the dipicolinic acid (DPA) released by the spores. In a C. difficile infection animal model, the inflammatory level triple decreased in mice with colonic C. difficile spores treated with Fe3-δO4 nanoparticles. Histopathological analysis showed a decreased intense neutrophil accumulation in the colon tissue of the Fe3-δO4 nanoparticle-treated mice. Fe3-δO4 nanoparticles, which had no influence on gut microbiota and apparent side effects in vivo, were efficacious inhibitors of C. difficile spore germination by attacking its surface and might become clinically feasible for prophylaxis and therapy.

Phase-contrast microscopy showed that in the positive controls, to which taurocholate was added for 15 minutes, but not in the negative controls, the contrast of the spores transitioned from bright to dark (Fig. 2C). The contrasts of the spores treated with 500 μg/mL Fe 3-δ O 4 nanoparticles were not significantly different. The luminescence signal that accompanies DPA release was significantly higher during taurocholate-induced spore germination (Fig. 2D). However, the luminescence signal was less intense and less abundant in the Fe 3-δ O 4 -treated group than in the taurocholate-treated group. No luminescence signal was detected in the group of spores not treated with taurocholate because the spores did not release DPA.  The spores were first treated for 20 minutes with 500 μg/mL of 22-nm Fe 3-δ O 4 (■), 500 μg/mL of 14-nm Fe 3-δ O 4 (▲), 50 μg/mL of 22-nm Fe 3-δ O 4 (□), 50 μg/ mL of 14-nm Fe 3-δ O 4 (▵), or 3% sodium hypochlorite (•) and then were treated with taurocholate to induce germination. Both 14-nm and 22-nm Fe 3-δ O 4 nanoparticles had a similar dose-dependent effect on spore germination. (B) After C. difficile spores and 500 μg/mL of 22-nm Fe 3-δ O 4 nanoparticles or 3% sodium hypochlorite had been incubated for 20 minutes, the spores were plated on BHIS agar for a colony formation assay the next day. The level of colony-forming unit inhibition was similar for Fe 3-δ O 4 nanoparticle-and sodium hypochlorite-treated spores. (C) The spores were treated for 20 minutes with 500 μg/mL of 22-nm Fe 3-δ O 4 and then stimulated using 10 mM taurocholate. After 15 minutes, the phase contrast of the spores was recorded under a phase contrast microscope. (D) The DPA-release assay showed that Fe 3-δ O 4 nanoparticles-treated spores released less DPA than did the control group. Data are mean ± SEM. (***P < 0.001; one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison test).

Inhibitory effect of
between those treated with 500 μg/ml of Fe 3-δ O 4 nanoparticles and those treated with sodium hypochlorite, but CCUG 19126 was relevantly resistant to treatment with 50 μg/mL of Fe 3-δ O 4 nanoparticles (Fig. 3A).
To evaluate the bactericidal effect of Fe 3-δ O 4 nanoparticles on vegetative cells in different strains, three groups of vegetative cells-CCUG 37780, CCUG 19126, and BAA-1805-were first treated for 20 minutes with 500 μg/ mL of Fe 3-δ O 4 nanoparticles, spread on BHIS agar plates, and their CFUs were counted. The growth behavior of the Fe 3-δ O 4 -treated vegetative cells was not significantly different from that of the negative control groups; however, almost all the vegetative cells treated with 3% sodium hypochlorite died (Fig. 4). Treatment with Fe 3-δ O 4 nanoparticles did not damage C. difficile vegetative cells.  The mechanisms underlying Fe 3-δ O 4 -inhibited spore germination. To determine whether any additional underlying mechanisms allow Fe 3-δ O 4 nanoparticles to inhibit spore germination, we first excluded the sporicidal activity of the coating polymer poly-(styrene-alt-maleic acid) (PSMA) on Fe 3-δ O 4 nanoparticles. At a concentration higher than that on PSMA-coated on Fe 3-δ O 4 nanoparticles, PSMA did not inhibit spore germination ( Supplementary Fig. 1). In addition, to verify whether Fe 3-δ O 4 nanoparticles promoted or hindered the germination-inducing activity of taurocholate, 10 mM taurocholate was incubated for 20 minutes with different concentrations of Fe 3-δ O 4 nanoparticles and then used to treat spores. The taurocholate incubated with Fe 3-δ O 4 nanoparticles still induced C. difficile spore germination ( Supplementary Fig. 2).
To identify any additional underlying mechanisms that allow Fe 3-δ O 4 nanoparticles to inhibit spore germination, we took advantage of the magnetic property of Fe 3-δ O 4 . Some C. difficile BAA-1805 spores were incubated with 500 μg/mL of Fe 3-δ O 4 nanoparticles for 20 minutes and then separated, using a magnet, into two parts: the nonmagnetic supernatant and the magnetic pellets (Fig. 5A). Each part was collected, and PCR was used to detect the tcdB gene. The tcdB gene was detected only in the supernatant (Fig. 5B) in the spores-alone groups. In the Fe 3-δ O 4 -treated spores, the tcdB gene was also detected in the magnetic pellets.
To clarify the interaction between Fe 3-δ O 4 and spores, the Fe 3-δ O 4 -treated spores were examined under a TEM. The TEM images showed that spores treated with 50 μg/mL of Fe 3-δ O 4 nanoparticles were only spotted with Fe 3-δ O 4 nanoparticles, but that spores treated with 500 μg/mL were almost fully surrounded (Fig. 5C). To reconfirm that Fe 3-δ O 4 nanoparticles were simply attached to the surface of the spores instead of diffused into the spores, the spores were examined using cryo-electron tomography. The rotated dynamic video (Supplementary Video 1) revealed that Fe 3-δ O 4 nanoparticles were directly and dose-dependently bound to the surface of C. difficile spores.  Fig. 4). Therefore, in the mouse experiment, we examined the C. difficile infection pathology 3 days after the mice had been treated with spores. Mice treated with Fe 3-δ O 4 nanoparticles plus spores lost significantly less body weight than did the mice treated with spores alone (P = 0.0119, Student's t test) (Fig. 6A). The cecum weight of the Fe 3-δ O 4 -treated mice was significantly higher than that of the mice treated with spores alone (P = 0.0024, Student's t test) (Fig. 6B), which showed minor inflammation under an in vivo imager (P = 0.0406, Student's t test) (Fig. 6C). The histopathological images also showed that neutrophil infiltration was lower in the mice treated with Fe 3-δ O 4 nanoparticles plus spores ( Fig. 6D) than in the control mice treated with spores alone. A real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of the proinflammatory genes TNF-α, IFN-γ, and IL-1β, compared with the spores-alone groups (Fig. 6E). The level of inflammation caused by the spores was significantly lower (P = 0.0088 for TNF-α, P = 0.0276 for IFN-γ, and P = 0.0097 for IL-1β, Student's t test) at the molecular level in the mice treated with Fe 3-δ O 4 nanoparticles plus spores than in the mice treated with spores alone. To verify whether treatment with 500 μg/mL Fe 3-δ O 4 nanoparticles alone induces any adverse side effects in vivo, we fed the mice with nanoparticles alone and monitored the biochemistry markers ( Supplementary Fig. 5). Based on the ratio of Firmicutes to Bacteroidetes, we found this nanoparticle does not altered the gut microbiota ( Supplementary Fig. 6A), suggesting that this particle did not has the toxicity to the mice.

Discussion
Based on our review of the literature, this is the first study which shows Fe-based nanoparticles that strongly inhibited the germination of C. difficile spores despite the growth inhibitory effect of other type of iron oxide nanoparticles to other bacteria vegetative cells was recently reported.
Iron oxide nanoparticles have existed in the earth's stratum and some organisms 35 . Due to the iron oxide nanoparticles are widely used in medical applications. The iron oxide nanoparticles not only can be the MRI T2 contrast material but also be conjugated to other dyes as detection reagents for different imaging systems 35 . The iron oxide nanoparticles could be used as hyperthermia-based cancer therapeutic reagent when combined to radiofrequency. The iron oxide nanoparticles could even be modified with different materials to become the multi-functional nanoparticles, e.g., targeting, drug releasing, and imaging abilities 36 . We performed the new function of iron oxide nanoparticles that could be a C. difficile spore germination inhibition reagent in the study. The 22-nm-Fe 3-δ O 4 nanoparticles presented as unique octahedron single crystals, and the ratio of iron to oxide was 2.966:4, similar to our previously reported 6-nm Fe 3 O 4 nanoparticles 30 . The differences between Fe 3-δ O 4 and Fe 3 O 4 include size, surface chemistry, and the presence of minor non-oxidized Fe voids in the Fe 3-δ O 4 particles. There was no significant difference between the inhibitory ability of the 22-nm and the 14-nm Fe 3-δ O 4 doses. The amount of DPA released from the spores was significantly lower in the Fe 3-δ O 4 -treated group than in the positive controls. The transition of spores from bright to dark can be observed with a phase-contrast microscope after the DPA has been released 37 . In the present study, because Fe 3-δ O 4 nanoparticles inhibited spore germination, there was no transition of spores into phase-dark. However, DPA could be detected, but not in as great abundance as with the taurocholate-treated spores, after the spores had been exposed to Fe 3-δ O 4 nanoparticles. Thus, it is conceivable that there is an underlying mechanism that enables Fe 3-δ O 4 nanoparticles both to induce DPA release and to inhibit spore germination. We hypothesized that taurocholate can be caught by Fe 3-δ O 4 nanoparticles and block the interaction between taurocholate and CspC. CspC is a bile salt germination receptor in C. difficile and transmits the signal to the downstream signaling molecule. However, we later discovered that taurocholate does not bind with Fe 3-δ O 4 nanoparticles, and that taurocholate supernatant without Fe 3-δ O 4 nanoparticles was able to trigger C. difficile spore germination. Therefore, we now hypothesize that Fe 3-δ O 4 nanoparticles inhibit bacterial growth without directly interacting with taurocholate. We also hypothesize that Fe 3-δ O 4 nanoparticles do not penetrate spores based on our cryo-TEM Supplementary Video 1. The details of the molecular mechanisms of Fe 3-δ O 4 nanoparticles require additional study.
Zero-valent iron (ZVI) has recently been reported 38 to be involved in the selective inhibition of cancerous cells while sparing noncancerous cells because it depletes mitochondrial membrane potential. Because mitochondria share distinct structural and biochemical similarities as well as evolutionary links to bacteria, we hypothesize that the nonstoichiometric non-oxidized Fe of Fe 3-δ O 4 also inhibits spore germination. In fact, one study 25 reported  Neutrophils were morphometrically counted in 10 randomly selected fields in the tissue sections. The number a prominent bactericidal effect of ZVI-based nanoparticles on E. coli because they effectively caused oxidative damage to bacterial membranes. Our study showed that Fe 3-δ O 4 -treatment significantly inhibited the germination of C. difficile spores by damaging spore membranes but not the vegetative cells and therefore did not damage the balance of intestinal normal flora. Thus the specific activity to spore germination must go through other yet to be discovered mechanisms.
It is well known that the expression of the tcdA and tcdB genes is closely associated with the virulence of C. difficile infection. We found that Fe 3-δ O 4 nanoparticles inhibited germination in both the tcdA(−)tcdB(−) strain and the more virulent tcdA(+)tcdB(+) strain. In an in vivo C. difficile infection mouse experiment, we found that Fe 3-δ O 4 significantly reduced the inflammation indices in the groups treated with spores +Fe 3-δ O 4 , and that it effectively inhibited C. difficile spore germination, thus controlling in vivo the virulence of C. difficile. Intriguingly, treatment with 500 μg/mL of Fe 3-δ O 4 nanoparticles did not kill vegetative cells.
Because antibiotics are overused everywhere in the world, multidrug-resistant microbes have emerged as a major public health threat 39 . Nanomedicines have inspired various new strategies to control microbes without allowing them to develop drug resistance 40 . Bacterial spores are even more resistant than are the bacteria themselves to harsh environments and antibiotics, but nanoparticle-spore interaction and potential clinical applications are rarely discussed. The germination of Bacillus subtilis spores is greatly compromised by treatment with polyionic polymers and then silica nanoparticles 41 . Such treatment, however, does not affect spore viability. We also found that excess PSMA alone did not significantly inhibit the germination of spores; therefore, PSMA must not be important for inhibiting spore germination. In the present study, 500 μg/mL of Fe 3-δ O 4 nanoparticles were sufficient to significantly, and almost as efficaciously as 3% sodium hypochlorite, inhibit spore germination, which indicated the potential of Fe 3-δ O 4 nanoparticles for clinical development as a novel and effective treatment for managing and perhaps even preventing C. difficile infection.
Many studies showed their materials existed excellent antibacterial activity, but sometimes the impact of the materials on the normal flora and biocompatibility were rarely discussed. The guts normal flora plays a vital role in animals, because it attend the metabolism and pathogenic colonization prevention in the host 29 . Therefore, we should have to understand the potential adverse effect of Fe 3-δ O 4 nanoparticles on normal flora and biocompatibility. We found that Fe 3-δ O 4 nanoparticles at a concentration of 500 μg/mL inhibited spore germination but did not damage their vegetative cells. Another study 42 reported that a high concentration (3 mg/mL) of Fe 3 O 4 nanoparticles inhibited the growth of Staphylococcus aureus. After mice were fed with Fe 3-δ O 4 nanoparticles for 72 hours, RT-qPCR was used to analyze the results of normal flora population changes. The two main phyla of bacteria in the gut are Firmicutes and Bacteroidetes 43 . We found that the Fe 3-δ O 4 nanoparticles did not change the ratio of Firmicutes to Bacteroidetes in the mice (Supplementary Fig. 6A). Bacteroides fragilis and Enterococcus faecalis are the common bacteria in the large intestine 44,45 . Our results showed that Fe 3-δ O 4 nanoparticles did not cause damage to the two bacteria ( Supplementary Fig. 6B). According to our results we hypothesize that Fe 3-δ O 4 nanoparticles do not cause imbalance of gut microbiota. The Fe 3-δ O 4 nanoparticles will not be an issue in the normal flora destruction after administering to human and other animals. We also found excellent biocompatibility between Fe 3-δ O 4 nanoparticles and a variety of colorectal cells. We previously reported 30, 31 that Fe 3-δ O 4 nanoparticles used with MRI scans and cancer therapy were safe. Although the safe dosages are different in these three studies, we have shown that Fe 3-δ O 4 nanoparticles are biocompatible and safe in vivo.
Metronidazole and vancomycin are antibiotics frequently used to treat patients with C. difficile infection, but the some clinical isolates are metronidazole-resistant 46 . Antibiotics unselectively damage pathogens as well as normal flora and might trigger a highly contagious state of super-shedding C. difficile spores, thus increasing the risk of infection 47 . Spores can survive for many years in harsh environments and wait to be ingested by animals 48 . The imbalance of normal flora and spore germination might be the major cause of a C. difficile infection relapse. Moreover, bowel inflammation has long been recognized as an important index for C. difficile infection severity and prognosis. Therefore, we quantitatively assessed C. difficile infection by directly injecting mice with spores, which is most relevant to the pathogenesis of clinical C. difficile infection. Transgenic mice with NF-κB-dependent luciferase reporter enabled us to visualize bowel inflammation using the IVIS imaging system. Because of their unique ability to selectively kill spores without damaging normal flora, mammalian cells, or the mouse gastrointestinal tract, Fe 3-δ O 4 nanoparticles offer a new strategy for controlling infectious clinical diseases. Fe 3-δ O 4 nanoparticle treatment not only inhibited the germination of different clinical strains of C. difficile spores in vitro, but also reduced the virulence of spores that induced C. difficile infection in vivo. We also report the first relatively low-dose but efficacious Fe-based nanoparticle inhibition of spore germination, one which uses a novel mechanism that does not interfere with the growth of healthy vegetative flora, which is an important clinical consideration in C. difficile infection management. We hypothesize that coating the surfaces of medical devices with Fe 3-δ O 4 nanoparticles will reduce the threat of spores in hospitals. Additional research on improving the design of Fe 3-δ O 4 nanoparticles should help nanoparticle-based therapeutics become safer and more effective, and should contribute to the development of new therapeutics for C. difficile infection and other emerging infectious diseases. of neutrophils in the control group was 16 ± 5 per 170 m × 130 µm field; the number in the Fe 3-δ O 4 nanoparticles-treated group was 2 ± 1 per 170 m × 130 µm field. (E) A reverse transcription-quantitative polymerase chain reaction (RT-QPCR) shows that the total RNA extracted from the Fe 3-δ O 4 -nanoparticletreated spores expressed significantly lower levels of proinflammatory genes than did the spores in the control group. (*P < 0.05, **P < 0.01; Student's t test) (n = 6).
SCieNtifiC REPORTS | 7: 8124 | DOI:10.1038/s41598-017-08387-y Methods Ethics statement and animals. All NF-κB-dependent reporter mice (FVB/NJNarl genetic background) were obtained from the National Laboratory Animal Center in Taiwan and housed in a pathogen-free barrier facility. All animal experiments were approved and in accordance with the relevant guidelines and regulations required by the Institutional Animal Care and Use Committee of National Cheng Kung University (NCKU-IACUC-102-296).
Bacterial culture and spore purification. C. difficile CCUG 37780 (tcdA − , tcdB − ) and CCUG 19126 (tcdA + , tcdB + ) were purchased from the Culture Collections of the University of Göteborg (Göteborg, Sweden), and BAA-1805 (tcdA + , tcdB + ) from American Type Culture Collection (Manassas, VA). All strains were incubated in brain-heart infusion-supplemented medium (BHIS; BD Difco, Franklin Lakes, NJ), with 0.5% yeast extract (BD Difco) and 0.1% L-cysteine (Amresco, Solon, OH), at 37 °C under anaerobic conditions. The spores were prepared and purified as previously described 32 with a slight modification. Briefly, C. difficile in BHIS medium was diluted in fresh BHIS medium to an optical density (OD) (600 nm) of 0.2. The diluted bacterial suspension (900 μL) was added to a 6-well dish with BHIS agar, and then the dish was incubated at 37 °C in an anaerobic jar (Thermo Fisher, Oxoid Ltd., Basingstoke, England) for 4 days. The whole cells were washed 5 times with ice-cold sterile water, the bacteria were resuspended with 3 mL of ice-cold sterile MQ water. The suspension was spread on top of a 10-mL 50% (wt/vol) sucrose solution (J.T. Baker Chemical, Phillipsburg, PA) in a centrifuge tube, and then centrifuged at 3500 g for 20 minutes to separate spores from vegetative cells. The purified spores at the bottom of the centrifuge tube were washed 5 times with ice-cold sterile water to remove sucrose, and then stored at 4 °C. Optical density based spore germination test. Spores were germinated as previously described 32 .

Preparation of the nanoparticles.
Before the germination experiments, the spore suspension was incubated at 60 °C for 30 minutes. The heat-treated spores were then moved to ice. The C. difficile spores (concentration: OD 600 0.5) were co-incubated with various nanoparticles or at different concentrations (5 to 500 μg/mL) in BHIS in a 96-well plate for 20 minutes. The concentration of Fe 3-δ O 4 nanoparticles was measured based on the total particle weight per 1 mL of solution. Fe 3-δ O 4 (22-nm) nanoparticles were used here. Spores treated with 3% bleach (Wako, Osaka, Japan) were the positive controls to inhibit germination, and spores treated with BHIS only were the negative controls. After the spore + nanoparticle co-incubation, the spores were treated with 10 mM taurocholate (Sigma-Aldrich) to induce germination. The OD 600 of treated spores was kinetically determined at 1 minute intervals using a spectrophotometer (TECAN, Grödig, Austria) at room temperature for 12 minutes. The OD at different time points was used to plot the spore germination curve.
Phase-contrast microscopy observation of spore germination. The images of spore germination were recorded using phase-contrast optical microscopy described elsewhere 49 . The purified spores were incubated at 60 °C for 30 minutes. The spores were co-incubated for 20 minutes with 500 μg/mL of Fe 3-δ O 4 nanoparticles or with buffer alone. Taurocholate was then added to the samples to a final concentration of 10 mM in BHIS. Five microliters of samples were then dropped onto the 0.7% agarose surface on the glass slide. After 15 minutes, phase-contrast images of the spores were captured (Nikon Eclipse 80i; Tokyo, Japan). Six random fields of view were obtained for each experimental group.
Dipicolinic acid (DPA) release assay. The DPA release was estimated by modified method from a previous study 50 . Briefly, the spores were heated as described and washed with distilled water then resuspended in spore germination buffer (10 mM Tris [pH 7.5], 150 mM NaCl, and 100 mM glycine). The spores were treated with MQ water or 500 μg/mL of Fe 3-δ O 4 nanoparticles for 20 minutes in a black 96-well plate before taurocholate (10 mM) and terbium (III) chloride (TbCl 3 ) (100 μM) were added. The total DPA of the spores was extracted by boiling the samples for 30 minutes. The spores treated with TbCl 3 only were negative controls. The DPA-Tb signal was monitored immediately in real-time using a microplate reader (FlexStation 3 Multi-Mode; Molecular Devices, CA) with excitation/emission at 270 nm and 545 nm, and a cutoff at 530 nm. Spore viability analysis. After the spore germination curves had been recorded, each aliquot was removed from the 96-well plate and plated on BHIS agar dishes and serially diluted. The plate-counts (CFU/mL) at each dilution were done after 48 hours of incubation in an anaerobic environment. The inhibition rates were calculated using the following formula: SCieNtifiC REPORTS | 7: 8124 | DOI:10.1038/s41598-017-08387-y Nanoparticle-spore binding analysis. C. difficile BAA-1805 spores at a concentration of OD 600 0.5 were co-incubated with Fe 3-δ O 4 nanoparticles (500 μg/mL) in BHIS in a 96-well plate for 20 minutes. The spore samples without Fe 3-δ O 4 treatment were controls. The samples were placed next to a magnet for 5 minutes and then all supernatant was removed to other tubes. The magnet-attracted parts and supernatant were washed 3 times with 1× phosphate-buffered saline. All samples were resolved in distilled deionized water, and the tcdB DNA in samples was detected using a polymerase chain reaction (PCR). Spore and nanoparticle images were captured using a transmission electron microscope (TEM) (JEM-1400; JEOL, Tokyo, Japan).

In vivo analysis of C. difficile infection.
To directly monitor the colonic inflammation, we infected mice with C. difficile spores in a previously generated NF-κB-dependent reporter mouse model containing the luciferase transgene under the transcriptional control of NF-κB (NF-κB-RE-luciferase) 51 . Before the mice were fed the spores with NF-κB reporter, they were given an antibiotic cocktail (0.4 mg/mL of kanamycin, 0.035 mg/mL of gentamicin, and 0.057 mg/mL of colistin) in their drinking water for 48 hours and a refreshed cocktail every 24 hours for 2 days. The mice were gavaged with a 200-μL proton pump inhibitor (PPI) (2 mg/mL) every 12 hours for 2 days before the C. difficile spore infection. C. difficile CCUG 19126 spores (2 × 10 5 CFUs) were co-incubated with or without 500 μg/mL of Fe 3-δ O 4 nanoparticles for 20 minutes before they were gavaged with 100 L of sample solution. While the spores and Fe 3-δ O 4 nanoparticles were incubating, all the mice were gavaged with a 50-μL PPI (2 mg/mL) and then intraperitoneally injected with clindamycin (4 mg/kg). The antibiotic cocktail water was replaced with normal water after the C. difficile infection. All mice were monitored for C. difficile infection symptoms, e.g., diarrhea, weight loss, hunched posture, and death. Seventy-two hours post-infection, the mice were intraperitoneally injected with 150 mg/kg of luciferin (PerkinElmer, Waltham, MA) to show NF-κB activation-mediated luminescence. The mice were anesthetized with isoflurane and oxygen, and then images were collected for 5 minutes using an imaging system (Xenogen IVIS ® Spectrum; Advanced Molecular Vision, Grantham, Lincolnshire, UK). Data were analyzed (Xenogen Living Image ® ), and luciferase activity was presented in photons/sec/cm 2 /steradian (p/s/cm 2 /sr). After the IVIS images had been obtained, a reagent (TRI; Sigma-Aldrich) was used to extract RNA from colon tissue samples. The levels of inflammatory gene expression were estimated using a real-time PCR assay (StepOnePlus; Applied Biosystems).
Histopathology examination. Histopathological analysis was used to evaluate C. difficile infection-induced mucosal damage and inflammation. Resected colon tissue samples were fixed in 4% formaldehyde buffered with PBS and then embedded in paraffin. Deparaffinized 6-μm-thick sections were stained with hematoxylin and eosin. The results were captured using optical microscopy. Neutrophils were randomly counted in 10 fields for both the spores-alone group and the Fe 3-δ O 4 -treated-spores group.
Statistical analysis. GraphPad Prism 5.01 was used for all statistical analyses. All experiments were done in triplicate. Data are means ± standard error of the mean (SEM) from three independent experiments. One-way analysis of variance (ANOVA) and then Tukey's Multiple Comparison test were used for spore germination curve analyses, and Student's t test was used in the other CFU inhibition test.