Preparation method of composite nano-enzyme hydrogel and application of composite nano-enzyme hydrogel in preparation of products for preventing and treating bacterial keratitis

Abstract

The invention belongs to the technical field of nano-enzyme materials. In order to solve the problems that gatifloxacin is not strong in binding force with a carrier, burst release may occur, adverse reaction is caused, and the curative effect is reduced, the invention provides the preparation method of the composite nano-enzyme hydrogel and the application of the composite nano-enzyme hydrogel in preparation of a product for preventing and treating bacterial keratitis. According to the preparation method, cerium nitrate and gatifloxacin are utilized to form antibiotic-based nano-enzyme under the action of ammonia water, then the antibiotic-based nano-enzyme is mixed in a hydrogel synthesis system of oxidized xanthan gum and four-arm-polyethylene glycol-amino, and the composite nano-enzyme hydrogel is formed through an aldehyde group-amino Schiff base reaction. The cerium ions and gatifloxacin are subjected to metal organic coordinate bond action, so that burst release can be effectively avoided; the obtained composite nano hydrogel shows a remarkable bactericidal effect on drug-resistant bacteria, and has good repair promotion and prevention and treatment effects on bacterial and drug-resistant bacteria corneal infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoenzyme materials, and particularly relates to a method for preparing a nanoenzyme hydrogel and an application of the nanoenzyme hydrogel in preparing products for preventing and treating bacterial keratitis. Background Art

[0002] Bacterial keratitis is an ocular surface disease caused by bacterial infection with a high rate of blindness, posing a significant threat to human vision. Bacterial keratitis (BK) is primarily caused by Gram-negative and Gram-positive bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Serratia marcescens. Key risk factors for BK include contact lens wear, ocular trauma, ocular surgery, preexisting eye disease, eyelid deformity, dry eye, immunosuppression, and topical steroid use. The local response to bacterial infection of the cornea usually involves an inflammatory process, characterized by the infiltration of inflammatory cells (e.g., macrophages), the production of reactive oxygen species (ROS), the accumulation of proinflammatory cytokines (e.g., tumor necrosis factor-α (TNF-α) and interleukin-1b (IL-1b) 4), and proinflammatory lipid mediators (e.g., prostaglandin E2 and leukotrienes). If not controlled in time, it can cause ulcers to spread throughout the eyeball, while producing an excessive inflammatory response, leading to blindness. Therefore, eliminating pathogens and alleviating excessive inflammatory responses are expected to fundamentally resolve bacterial keratitis. However, broad-spectrum antibiotic eye drops are currently used clinically to eliminate bacteria, but their short residence time on the surface of the eyeball results in low bioavailability and unsatisfactory therapeutic effects. In addition, long-term abuse of antibiotics can lead to the development of bacterial resistance.

[0003] Gatifloxacin is one of the fourth-generation fluoroquinolone antibiotics. It is well-known for its excellent antibacterial effect and strong inhibitory activity against Gram-positive bacteria and atypical pathogens. It is a good choice for the treatment of bacterial keratitis. However, compared with other fluoroquinolone antibiotics such as moxifloxacin and levofloxacin, gatifloxacin has relatively poor corneal permeability. Factors such as blinking, tear drainage and conjunctival absorption further shorten the retention time of gatifloxacin on the ocular surface. It is challenging to achieve high utilization through conventional topical administration. High doses or frequent use of gatifloxacin can lead to serious side effects such as dyspnea and arrhythmia. In this regard, some studies have loaded gatifloxacin onto a drug delivery system. For example, CN115671298A discloses a ROS-responsive antibacterial drug nanocarrier and a preparation method thereof, comprising a diblock polymer P(PEGMA 10 -co-PBA2)-b-PMTEMA 25, also provide the antibacterial drug-loaded vesicle based on the above-mentioned nanocarrier, to provide a kind of simultaneously having ROS response and having antioxidant and anti-inflammatory ability drug carrier, when the carrier of ocular anti-inflammatory drug is used, it increases the adhesion of ocular surface drug, increases the retention time of antibacterial drug in ocular surface, and by improving the solubility of antibacterial drug, solves the ocular anti-inflammatory drug in the ocular environment, easily precipitates, stimulates the problem of eye, reduces the dosage and effective antibacterial concentration of antibacterial drug. Treat bacterial keratitis by coating antibiotics, and eliminate the redundant active oxygen of local part, promote the healing process of bacterial keratitis. For another example, CN117959243A discloses a controllable nano hydrogel that can load drug, and the nano hydrogel is borax as catalyst, by the polymerization reaction between polyethylene glycol dimethacrylate (PEGDA) and dithiothreitol (DTT) and the complex reaction between tetrahedron to synthesize polyethylene glycol-dithiothreitol-boric acid (PDB) nano hydrogel. PDB nanohydrogels can serve as an excellent sustained-release system for gatifloxacin, prolonging the drug's duration of action and enhancing its efficacy. Furthermore, PDB nanohydrogels exhibit excellent biocompatibility, significantly improving the efficacy of gatifloxacin in treating bacterial keratitis. These aforementioned methods involve loading gatifloxacin onto pre-synthesized drug delivery systems. However, these methods have the following drawbacks: Simply loading gatifloxacin onto the carriers does not strongly bind gatifloxacin, making burst release more likely. Excessive initial release leads to a sharp increase in drug concentration, exceeding the safe range, and potentially causing adverse reactions or toxic effects. The burst release effect can also result in insufficient drug release in subsequent stages, failing to meet treatment needs and compromising efficacy. Therefore, it is necessary to develop a more robust carrier with a steady drug release rate that meets the following requirements: (I) high antibacterial performance and bioavailability, (II) regulation of excessive inflammatory responses, and (III) biosafety, all while ensuring a safe and convenient method. Summary of the Invention

[0004] In view of the above-mentioned problems that the binding force between gatifloxacin and the carrier in the prior art is not strong enough, a sudden release may occur, resulting in adverse reactions and reducing the efficacy, the present invention provides a method for preparing a composite nanozyme hydrogel and its application in the preparation of a product for preventing and treating bacterial keratitis. The present invention utilizes cerium nitrate and gatifloxacin to form an antibiotic-based nanozyme (CGN) under the action of ammonia water, and then mixes it in a hydrogel synthesis system of oxidized xanthan gum and four-arm-polyethylene glycol-amino (PEG), and forms a composite nanozyme hydrogel (CH) through an aldehyde-amino Schiff base reaction; cerium ions react with the amino group and electron-rich carboxyl group of gatifloxacin to form a metal organic coordination bond, which has a stronger force, and the combined drug and carrier also have a stronger binding force, which can effectively avoid sudden release; the nanozymes are evenly dispersed in the hydrogel, preventing the aggregation of nanozymes, and filling the cornea, and the nanozymes are evenly dispersed in the cornea, improving the therapeutic effect. The resulting composite nanohydrogel shows a significant bactericidal effect on drug-resistant bacteria, and has a good promoting repair and good preventive and therapeutic effect on bacterial and drug-resistant corneal infections.

[0005] The present invention is achieved through the following technical solutions:

[0006] In one aspect, the present invention provides a method for preparing a composite nanoenzyme hydrogel, comprising:

[0007] Step 1: Dissolve cerium nitrate and gatifloxacin completely in water, slowly add ammonia water to the solution and stir to promote the 3+ - Formation of a gatifloxacin complex; centrifugation, collecting the supernatant, and dialysis against distilled water to remove remaining raw materials and by-products, using a dialysis membrane with a molecular weight cutoff of 12 to 14 kD; vacuum freeze-drying the retained solution from the dialysis membrane to obtain the antibiotic nanozyme (CGN);

[0008] Step 2: Dispersing xanthan gum in water, adding NaIO4 to oxidize two pairs of vicinal diols on each repeating unit of the xanthan gum into aldehyde groups, thereby maintaining the water solubility and skeleton stability of the xanthan gum; then adding excess ethylene glycol dropwise to terminate the reaction; collecting the liquid and dialyzing it in distilled water, using a dialysis membrane with a molecular weight cutoff of 12 to 14 kD; and vacuum freeze-drying the retained liquid in the dialysis membrane to obtain oxidized xanthan gum (XPH);

[0009]

[0010] Step 3: Dissolve the obtained oxidized xanthan gum in water, add antibiotic nanozyme, mix well, and then add four-arm-polyethylene glycol-amino. The oxidized xanthan gum and four-arm-polyethylene glycol-amino undergo Schiff base condensation reaction, and the nanozyme is evenly dispersed and suspended in the hydrogel to form a composite nanozyme hydrogel (CH).

[0011] Furthermore, in step 1, cerium nitrate and gatifloxacin are dissolved in water at a molar ratio of 0.5 to 1.25:1; further preferably 0.8 to 1:1.

[0012] Furthermore, in step 1, cerium nitrate and gatifloxacin are completely dissolved in water, and ammonia water is slowly added dropwise to the solution while stirring to promote the 3+ -Formation of gatifloxacin complexes.

[0013] Furthermore, the step 1 is stirred at room temperature for 10-14 hours, and further for 12 hours.

[0014] Furthermore, the ammonia water in step 1 has a concentration of 25-28%.

[0015] Furthermore, each pair of vicinal diols in the xanthan gum in step 2 corresponds to one NaIO4 molecule.

[0016] Furthermore, in step 3, the xanthan gum is dispersed in water and heated at 80° C. to 95° C. until dissolved. Furthermore, NaIO 4 is added and stirred in the dark for 20 to 28 hours.

[0017] Another aspect of the present invention provides the use of the composite nanoenzyme hydrogel in preparing products for preventing and treating bacterial corneal infections, particularly drugs for treating drug-resistant bacterial corneal infections.

[0018] Furthermore, the product for preventing and treating bacterial corneal infection is a corneal injection.

[0019] The present invention has the following beneficial effects:

[0020] The present invention uses gatifloxacin as a raw material to synthesize antibiotic nanozymes, and disperses the synthesized nanozymes into a separately prepared hydrogel, which can effectively avoid sudden drug release and has better antibacterial efficiency and antioxidant properties.

[0021] The present invention injects the prepared composite nanozyme hydrogel into the corneal stroma, causing a hybrid corneal cross-linking reaction to form a new corneal antibacterial material. The composite nanozyme hydrogel only needs to be injected once to achieve a good antibacterial effect, is easily degraded, and has good in vitro and in vivo safety.

[0022] The composite nanoenzyme hydrogel prepared by the present invention has very good application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is an optical photograph of CGN.

[0024] Figure 2Figure 3 is the physical characterization of CGN and XPH; Figure A is the scanning electron microscope image of CGN nanozyme; Figure B is the particle size image of CGN; Figures C and F are XPS images of CGN and XPH; Figure D is the electron microscope image of XPH; Figure E is the relationship between the storage modulus (G′) and loss modulus (G′) of XPH and time; Figure G is the Zeta potential image of CGN and XPH; Figure HK is the hydroxyl radical scavenging activity, superoxide anion radical inhibition activity, superoxide dismutase activity and catalase activity of CGN.

[0025] Figure 3 This is an OCT image showing that the corneal thickness returned to normal 5 days after XPH was injected into the cornea.

[0026] Figure 4 To detect the content of CH released into the aqueous humor over time after injection into the cornea.

[0027] Figure 5 This is the in vitro antibacterial CFU experiment of CH, CGN and XPH.

[0028] Figure 6 Bacterial live-dead staining experiments for CH, CGN and XPH.

[0029] Figure 7 This is an electron microscopy picture of CH's antibacterial effect in vitro.

[0030] Figure 8 This is an in vivo antibacterial experiment of CH; images and clinical scores of the mouse corneas after infection with drug-resistant Staphylococcus epidermidis were collected in the PBS, CH, gatifloxacin and levofloxacin groups on days 0, 1, 3, 5 and 7 of treatment.

[0031] Figure 9 The biosafety of CGN and XPH is demonstrated. Figures A and B show slit-lamp images stained with sodium fluorescein at 1, 3, 5, and 7 days after treatment with PBS, CGN, and XPH. Figures C, D and E, F show the viability of human corneal epithelial cells (HCEC) and human corneal stromal cells (HCSC) quantified by the CCK-8 assay after treatment with PBS, CGN, and XPH (0.1 mg / mL and 0.01 mg / mL). Figure G shows hematoxylin and eosin staining of mouse heart, liver, spleen, lung, and kidney (scale bar: 100 μm). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0033] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0034] Example 1

[0035] (1) Add 291.0 mg of cerium nitrate and 250.0 mg of gatifloxacin to a round-bottom flask, add 25 mL of water, and stir on a magnetic stirrer for 15 min until completely dissolved.

[0036] (2) Slowly add 3.0 mL of ammonia solution dropwise to the solution in step (2) and stir at room temperature for 12 h;

[0037] (3) centrifuging the solution obtained in step (2) at 3000 rpm for 10 min, collecting the supernatant, and dialyzing it in distilled water for 48 h, using a dialysis membrane with a molecular weight cutoff of 12 to 14 kD;

[0038] (4) vacuum freeze-drying the retained liquid in the dialysis membrane to obtain the antibiotic nanozyme (CGN);

[0039] (5) Add 3.0 g of xanthan gum and 300 mL of water to a round-bottom flask and gradually heat to 90°C on a magnetic stirrer until dissolved;

[0040] (6) Add 1.8 g of NaIO4 to the round-bottom flask from step (5) and stir in the dark for 24 h;

[0041] (7) 2.0 mL of ethylene glycol was added dropwise to the round-bottom flask of step (6) and the reaction was stopped for 30 min;

[0042] (8) The collected liquid was dialyzed in distilled water for 3 days using a dialysis membrane with a molecular weight cutoff of 12 to 14 kD;

[0043] (9) The retained liquid in the dialysis membrane is vacuum freeze-dried to obtain oxidized xanthan gum (XPH);

[0044] (10) Take 1.0 mg of the oxidized xanthan gum obtained in step (9) and dissolve it in 1.0 mL of water. Add 1.0 mg of the CGN prepared in step (4), mix well, and then add 50.0 mg of four-arm-polyethylene glycol-amino. Mix well to form a composite nanoenzyme hydrogel (CH).

[0045] The obtained materials were used in the following experiments. All experiments used drug-resistant Staphylococcus epidermidis.

[0046] 1. Physical Characterization

[0047] Transmission electron microscopy (TEM) was used to observe the size, shape, and crystal structure of CGN particles. The enzyme activities of CGN were determined using a hydroxyl radical (OH·) assay kit, a superoxide anion radical assay kit, a hydrogen peroxide assay kit, and a total superoxide dismutase (T-SOD) assay kit, respectively. The storage modulus (G') and loss modulus (G") of the XPH hydrogel were measured using a rheometer. CH was injected into the mouse cornea and longitudinally monitored using anterior segment optical coherence tomography (AS-OCT). The corneal thickness was recorded and the biodegradation of CH in the cornea was evaluated. 50.0 μL of aqueous humor was extracted from the limbus of the rabbit eye at each time point, and the gatifloxacin concentration was measured using a mass spectrometer (MS) to evaluate the sustained release characteristics of the drug in vivo.

[0048] like Figure 1 As shown, CGN nanozymes are uniformly dispersed in aqueous solution and appear transparent.

[0049] like Figure 2 As shown, the synthesized CGN particle size is approximately 2 nm (Figure B). Physical characterization of CGN and XPH demonstrates that CGN has hydroxyl radical and superoxide anion (SOD) scavenging activity, as well as hydrogen peroxide scavenging activity (Figures HK). The zeta potentials of CGN and XPH materials (Figure G) are 29.4 mV and -10.3 mV, respectively. Hydrogel properties testing of XPH confirms its hydrogel properties (Figure E).

[0050] like Figure 3 As shown, CH can be well degraded after being injected into the mouse cornea, and the cornea begins to restore corneal thickness at 5 days.

[0051] like Figure 4 As shown, sustained in vivo drug release demonstrated that gatifloxacin concentrations within the CGN hydrogel reached 158.2 ng / mL after 6 hours, rapidly decreasing to approximately 21.9 ng / mL after 24 hours and to 7.8 ng / mL after 48 hours. These data demonstrate that CGNs exhibit sustained-release properties for gatifloxacin. The pharmacokinetic profile suggests an optimized therapeutic strategy, where gatifloxacin provides immediate antibacterial activity.

[0052] 2. In vitro antibacterial experiment

[0053] The antibacterial effect was quantitatively assessed by comparing the number of colonies in different treatment groups using plate counts. The CH, CGN, XPH, and gatifloxacin concentrations were 2 mg / mL, respectively. Fluorescence staining, utilizing red / green fluorescent markers, distinguished between live and dead bacteria, which was verified with plate count results. Scanning electron microscopy was used to observe bacterial morphological changes (e.g., wrinkling and surface roughness) to assess the extent of cell structure damage caused by the antimicrobial agents.

[0054] The results are as follows Figures 5 to 7As shown, CGN exhibits excellent in vitro antibacterial activity. While bacterial colony counts did not differ significantly between the PBS and levofloxacin-treated groups, CH, CGN, XPH, and gatifloxacin-treated groups exhibited measurable antibacterial activity, reducing bacterial survival to approximately 4%, 28%, 51%, and 36%, respectively. Bacteria in the CH and CGN groups exhibited significant red fluorescence, while those in the control and levofloxacin groups exhibited significant green fluorescence, consistent with plate count results. Green fluorescence increased in CH, CGN, gatifloxacin, and XPH, while antibacterial activity decreased in that order. Electron microscopy revealed that drug-resistant Staphylococcus epidermidis in the control and levofloxacin groups maintained a characteristic, rounded and smooth morphology, while in the CH-treated group, most bacteria exhibited a rough, wrinkled, and irregular surface, as indicated by the arrow. In the gatifloxacin-treated group, most bacteria exhibited a rounded and smooth morphology, with a few exhibiting a rough, wrinkled surface. This suggests that levofloxacin has no significant antibacterial effect, and that CH exhibits a stronger antibacterial effect than gatifloxacin.

[0055] 3. In vivo antibacterial experiment

[0056] 2 μL (1×10 7 CFU / ml) bacterial solution was injected into the mice to establish a bacterial keratitis model. Then, 12 hours later, the mice were anesthetized again and injected with PBS and drugs in situ for treatment. Slit lamp photography and clinical scoring were performed on days 0, 1, 3, 5, and 7 after treatment.

[0057] like Figure 8 As shown, animal experiments showed that CH has good in vivo antibacterial effect and biosafety.

[0058] The mice were given the drug once 12 hours after infection. The PBS and levofloxacin groups showed the same symptoms, with severe, dense corneal opacity and obvious swelling on the first day of treatment. The infection began to subside on the third day, but the cornea was still severely opaque and swollen. On the seventh day, corneal opacity and swelling were reduced, but a large amount of white opacity remained. Gatifloxacin treatment showed severe, dense opacity and obvious swelling on the first day. The infection began to subside on the third day, and the area of ​​corneal opacity and swelling was smaller than that of the PBS and levofloxacin groups. On the seventh day, the cornea partially recovered its transparency, but some white opacity and swelling remained. Turbidity and swelling began to appear on the first day of CH injection. On the third day, the cornea was mildly opaque and swollen. However, on the seventh day, corneal opacity was significantly reduced. Compared with the PBS, gatifloxacin, and levofloxacin groups, the CH group had smaller corneal opacity and opacity area, confirming the good effect of CH in treating bacterial keratitis.

[0059] Corneal scoring results showed that corneal opacity and inflammatory response were reduced in CH-treated groups compared with those in the PBS, gatifloxacin, and levofloxacin-treated groups.

[0060] 4. Biosafety Experiment

[0061] GCN and XPH (1.0 μL) were injected into the corneal stroma of mice using a microsyringe to evaluate the biosafety of GCN and XPH. The ocular condition was observed and recorded using a slit lamp microscope under cobalt blue light on days 1, 3, 5, and 7. On day 7, 1% sodium fluorescein (10.0 μL) was added to the ocular surface for observation. After 7 days, the mice were euthanized, and tissues such as the heart, liver, spleen, lung, and kidney were collected. The tissues were rinsed with saline, fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for histopathological analysis. The toxicity of CGN and XPH was verified by CCK8 assay and cell live-death assay on human corneal epithelial cells (HCEC) and human corneal stromal cells.

[0062] like Figure 9 As shown, animal experiments demonstrated that CGN and XPH possessed good in vitro and in vivo biosafety. Slit-lamp observation revealed that the corneas remained transparent and showed no signs of edema in all groups after 7 days of administration. Fluorescein staining revealed intact corneal epithelium 7 days after administration, further confirming the absence of corneal epithelial damage (Figure A). Live-dead cell staining assessed the toxicity of CGN and XPH. CGN and XPH at concentrations of 0.1 mg / mL and 0.01 mg / mL, respectively, did not cause cell death in human corneal epithelial cells or human corneal stromal cells (Figures C and E). CCK-8 cytotoxicity assays revealed that CGN and XPH at concentrations of 0.1 mg / mL (P>0.05) and 0.01 mg / mL (P>0.05) were nontoxic to human corneal epithelial cells and stromal cells (Figures B and D). HE staining of mouse visceral sections revealed that compared with normal mice, no significant degeneration, necrosis, cell regeneration, inflammatory cell infiltration, or tissue fibrosis was observed in the heart, liver, spleen, lung, and kidney of the four groups (Figure F).

[0063] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for preparing a composite nanozyme hydrogel, characterized in that: The following steps are involved: Step 1: Completely dissolve cerium nitrate and gatifloxacin in water to prepare Ce 3+ - gatifloxacin complex; centrifugation, collecting the supernatant, and dialyzing against distilled water to remove remaining raw materials and by-products, with the dialysis membrane having a molecular weight cutoff of 12-14 kD; vacuum freeze-drying the retained solution in the dialysis membrane to obtain the antibiotic nanozyme; Step 2: Disperse xanthan gum in water, add NaIO4 to oxidize two pairs of vicinal diols on each sugar unit of the xanthan gum into aldehyde groups, then add excess ethylene glycol dropwise to terminate the reaction, collect the liquid and dialyze it in distilled water using a dialysis membrane with a molecular weight cutoff of 12 to 14 kD; and freeze-dry the retained liquid in the dialysis membrane under vacuum to obtain oxidized xanthan gum. Step 3: Dissolve the obtained oxidized xanthan gum in water, add antibiotic nanozyme, mix well, and then add four-arm-polyethylene glycol-amino. The oxidized xanthan gum and four-arm-polyethylene glycol-amino undergo Schiff base condensation reaction, and the nanozyme is evenly dispersed and suspended in the hydrogel to form a composite nanozyme hydrogel.

2. The preparation method according to claim 1, characterized in that In the step 1, cerium nitrate and gatifloxacin are dissolved in water at a molar ratio of 0.5 to 1.25:

1.

3. The preparation method according to claim 2, characterized in that In the step 1, cerium nitrate and gatifloxacin are dissolved in water at a molar ratio of 0.8 to 1:

1.

4. The preparation method according to claim 1, characterized in that In the step 1, cerium nitrate and gatifloxacin are completely dissolved in water, and 25-28% ammonia water is slowly added dropwise to the solution and stirred at room temperature for 10-14 hours.

5. The preparation method according to claim 1, characterized in that In step 2, the oxidized xanthan gum is dispersed in water and heated at 80° C. to 95° C. until dissolved, and NaIO 4 is added, and stirred in the dark for 20 to 28 hours.

6. The preparation method according to claim 1, characterized in that Each pair of vicinal diols in the xanthan gum in step 2 corresponds to one NaIO4 molecule.

7. Use of the composite nanoenzyme hydrogel according to claim 1 in the preparation of products for preventing and treating bacterial corneal infections.

8. The use according to claim 7, characterized in that The product for preventing and treating bacterial corneal infection is a corneal injection.

9. The use according to claim 7 or 8, characterized in that The bacterial corneal infection is a drug-resistant bacterial infection.

10. The use according to claim 9, characterized in that The drug-resistant bacterial infection is drug-resistant Staphylococcus epidermidis corneal infection.

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