KR20200023728A - An infectious eye disease animal model and method of manufacturing the same - Google Patents

Abstract

The present invention relates to an animal model of an infectious eye disease and a method of manufacturing the same, more specifically, to reduce pathogenic resistance caused by antibiotic abuse used for the treatment of an infectious eye disease, and to develop a therapeutic agent and measure a concentration of a therapeutic agent. The present invention relates to the preparation of an infectious eye disease animal model using Enterococcus and Staphylococcus , which can be used for the treatment of multi-drug resistance and treatment of infected patients.

Description

Infectious eye disease animal model and method of manufacturing the same

The present invention relates to an animal model of an infectious eye disease and a method of manufacturing the same, and more particularly, to reduce pathogenic resistance caused by antibiotic abuse used for the treatment of an infectious eye disease, and to develop a therapeutic agent and measure a concentration of a therapeutic agent. The present invention relates to the preparation of an infectious ocular disease animal model using Enterococcus and Staphylococcus , which can be used as a countermeasure against versatility resistance and to treat infected patients.

The eyes are always exposed to the external environment and irritation. Blinking of the eyelids, cleansing the surface of the conjunctiva of the tears and draining of bacteria to maintain homeostasis to the body organs, abundant lymphoid tissue of the conjunctiva and continuous dropping of the conjunctival epithelium, antimicrobial action of immunoglobulins, lysozyme, lactoferrin, and complement in the tear It has many of the same defenses. Depending on the number and pathogenicity of invading bacteria from outside, various infectious eye diseases can occur when this normal defense mechanism is broken down.

 These infectious eye diseases include conjunctivitis, keratitis, endophthalmitis, tear bagitis and tear ductitis, intraocular filling infection, post-traumatic eye rupture, and other eye infections. If the infection is on the eyelids, stye may develop. Enlargement to the iris, ciliary and choroid can lead to uveitis. Infection of the lacrimal glands or the nasolacrimal ducts that cause tears can also lead to nasal obstruction. Infections are the root cause of corneal ulcers, which are difficult to recover from treatment and cause severe vision damage. Infectious eye diseases can cause a variety of serious complications, from cosmetic problems to blindness if not treated quickly and properly.

 Infectious eye disease begins with extensive antibiotic treatment before bacterial culture and then changes the treatment depending on clinical response and bacterial culture. This clinical reality is likely to increase pathogen resistance due to antibiotic abuse. Ophthalmitis is an inflammation that occurs in the eye and is known to cause fatal effects on vision. The rate of endophthalmitis occurring in Korea is maintained similarly each year, and the infection rate is higher with age as there is no gender difference. In particular, the rate is high in the 60s and 80s.

The main cause of endophthalmitis is bacteria known as Staphylococcus epidermidis (SE), which inhabit the skin. Recently, the proportion of Enterococcus faecalis (EF) is increasing. .

Enterococcus faecalis is one of the strains frequently found in clinical specimens as Gram positive bacteria and is known as a cause of many infectious diseases. It has high drug resistance against aminoglycoside family, vancomycin, and fluoroquinolone family widely used for the treatment of endophthalmitis.

In this regard, moxifloxacin is a fourth generation synthetic fluoroquinolone antibacterial agent developed by Bayer AG (BAY 12-8039). The drug is sold under the names Avelox, Avalox and Avelon. Oral medications are also available in parenteral form for intravenous administration in most countries. Moxifloxacin is also marketed as an ophthalmic solution (eye drops) named Vigamox for the treatment of conjunctivitis, but multi-drug resistance, which is resistant to two or more drugs rather than one drug, is occurring. Difficulties arise in treatment.

 This problem is considered to be one of the problems due to the excessive use of antibiotics and the lack of a practical treatment concentration measurement.

Therefore, in order to supplement the above-mentioned problems, the present inventors are urgently required to develop a therapeutic agent that can be used in the eye and to measure the concentration of the therapeutic agent, and to develop an animal model that can help to cope with multi-drug resistance and to treat infected patients. It is recognized that the present invention has been completed.

Republic of Korea Patent Publication No. 10-1784450 Republic of Korea Patent Publication No. 10-1748575

For purposes of the present invention is to reduce the pathogenic immunity appears due to antibiotic abuse which is used for the treatment of ocular disease polychromatic Enterobacter nose kusu (Enterococcus) and Staphylococcus infectious eye diseases in animal models using the (Staphylococcus) and methods for making To provide.

In order to achieve the above object, the present invention provides an infectious eye disease animal model using Enterococcus and Staphylococcus , a manufacturing method thereof and a screening method thereof.

Hereinafter, this specification is demonstrated in detail.

The present invention provides an infectious ocular disease animal model, which is obtained by administering strain Enterococcus and Staphylococcus to normal mammals except humans.

In the present invention, the enterococcus is Enterococcus faecalis , and the Staphylococcus is characterized in that Staphylococcus epidermidis .

In the present invention, the infectious eye disease is characterized by conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, eye rupture or blepharitis after trauma.

The present invention comprises the steps of: (S1) strain Enterococcus and Staphylococcus are instilled into the eyelids of normal mammals except humans; And (S2) suturing the eye of the mammal after the step (S1). It provides a method for producing an infectious eye disease animal model comprising a.

In the present invention, the enterococcus of step (S1) is Enterococcus faecalis, and the Staphylococcus is characterized in that the Staphylococcus epidermidis.

In the present invention, the strain is characterized in that inoculated with 1 × 10 ⁵ to 1 × 10 ⁹ CFU (Colony Forming Unit) per animal individual.

In the present invention, the infectious eye disease is characterized by conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, eye rupture or blepharitis after trauma.

In the present invention, the normal mammal except for the human is characterized in that the rabbit.

The present invention comprises the steps of dropping strains Enterococcus, Staphylococcus and Fluoroquinolone antibiotic eye drops into the eyelids of normal mammals except humans; Suturing the eye of the mammal; And dissecting the sutured eyes to identify the bacteria. The method provides a screening method of an animal model for infectious eye disease, which comprises a.

In the present invention, the enterococcus of step (A1) is Enterococcus faecalis, and the Staphylococcus is characterized in that the Staphylococcus epidermidis.

In the present invention, the fluoroquinolone antibiotic is characterized in that levofloxacin (levofloxacin), ciprofloxacin (ciprofloxacin), moxifloxacin (moxifloxacin), norfloxacin (offloxacin) or ofloxacin (ofloxacin).

All matters mentioned in the infectious eye disease animal model of the present invention, a method of manufacturing the same, and a method of screening thereof are equally applied unless they contradict each other.

And Enterococcus nose kusu (Enterococcus) and Staphylococcus infectious eye diseases in animal models using the kusu (Staphylococcus) of the present invention is to reduce the pathogenic immunity appears due to antibiotic abuse which is used for the treatment of infective eye diseases, therapeutic drug development and The ability to proceed with the measurement of therapeutic concentrations can help to combat multi-drug resistance and to treat infected patients.

Such animal models can be usefully used for diagnosis of infectious eye diseases, development of therapeutic agents, and screening.

1 is an image of an infectious eye disease animal model manufactured according to a manufacturing method according to an embodiment of the present invention.
Figure 2 is an image confirming the colony (colony) for strain lesions of an infectious eye disease animal model according to an embodiment of the present invention.
Figure 3 is an image confirming the antimicrobial effect of bacteria against strain lesions of an infectious eye disease animal model according to an embodiment of the present invention.

The present invention provides an infectious eye disease animal model, a manufacturing method thereof and a screening method thereof.

Infectious Eye Disease Animal Model

The present invention provides an infectious eye disease animal model using Enterococcus and Staphylococcus .

More specifically, the present invention relates to an infectious eye disease animal model produced by administering strain Enterococcus faecalis and Staphylococcus epidermidis to normal mammals except humans.

As used herein, the term “infectious eye disease” refers to a generic term for a disease occurring in the eye due to a normal defense mechanism being broken down by bacteria or viruses invading from the outside.

In the present invention, the enterococcus is Enterococcus faecalis (EF), and the Staphylococcus is preferably Staphylococcus epidermidis (SE).

In the present invention, the infectious eye disease may be conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, eye rupture or blepharitis after trauma, but if it is an eye disease caused by an external bacterium or virus, It is not limited.

As used herein, the term "normal mammals except humans" refers to a generic term for animals belonging to the Mammalia, which is a river of vertebrate animals.

In the present invention, the mammal may be preferably a rabbit, but is not limited thereto.

Infectious eye disease animal models using Enterococcus faecalis and Staphylococcus epidermidis of the present invention reduce pathogenic resistance due to antibiotic abuse used for the treatment of infectious eye disease, and develop therapeutic agents and therapeutic agents. By allowing concentration measurements to be carried out, they can be used for countermeasures against versatility and for treating infected patients.

How to make animal model of infectious eye disease

The present invention provides a method for producing an infectious eye disease animal model comprising the following steps.

(S1) dropping strain Enterococcus and Staphylococcus onto the eyelids of normal mammals, except humans; And

(S2) suturing the eye of the mammal after step (S1).

As used herein, the term "comprising" means that it may further include other components, except to exclude other components unless specifically stated otherwise.

In the present invention, the enterococcus of step (S1) is Enterococcus faecalis, the staphylococcus is preferably Staphylococcus epidermidis.

In the present invention, prior to the step (S1) may further comprise the step of anesthetizing the normal mammal except the human. For example, an animal model may be prepared by administering anesthesia to a normal mammal except the human.

As used herein, the term "administration" means a generic term for all actions of taking or injecting a drug.

In the present invention, the administration can be administered orally or non-orally according to the desired method, the external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic administration during non-oral administration It is preferable to select the injection method. The dosage may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion or severity of the disease.

In the present invention, the step (S1) may include dropping strains Enterococcus faecalis and Staphylococcus epidermidis on the eyelids of normal mammals except humans.

In the present invention, the animal model may be an animal model prepared by dropping strain Enterococcus faecalis and Staphylococcus epidermidis on the eyelids of the eye.

More specifically, after lowering the lower eyelids of normal mammals except humans with tweezers or the like, the strain may be instilled using a pipette.

In the present invention, the animal model can be usefully used in that it can easily and simply and reproducibly observe the onset, progression, and response to the therapeutic effect of the drug.

In the present invention, the strain may be inoculated in 1 × 10 ⁵ to 1 × 10 ⁹ CFU (Colony Forming Unit) per animal, but is not limited thereto, and those skilled in the art in view of the type, size and weight of the animal We can judge appropriately. For example, when using rabbits weighing 3 to 4 kg, strains of 2.5 × 10 ⁷ CFU can be instilled.

In the present invention, the step (S2) may include a step of closing the eyes of the mammal after the step (S1).

As used herein, the term "sewing" refers to all of the things that are stitched together to cut or cracked areas.

In the present invention, the suture is preferably sutured in a continuous suture method in order to most effectively prevent the strain is discharged, but if the suture method for preventing the discharge of the strain is not limited thereto.

In the present invention, the infectious eye disease may be conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, eye rupture or blepharitis after trauma, but if the eye disease is caused by an external bacterium or virus It is not limited.

In the present invention, normal mammals, except for humans, include guinea pigs, hamsters, rodents such as mice, cats, dogs, pigs, rabbits, sheep, goats, deer, horses, cattle, baboons, chimpanzees and monkeys. It may be the same non-human primate, preferably a small animal to the extent that the eye disease lesion can be observed in more detail, more preferably a rabbit, but is not limited thereto.

The method for producing an infectious eye disease animal model of the present invention can easily produce an animal model, and can be usefully used for diagnosis, treatment, and screening of an infectious eye disease.

Screening Methods for Infectious Eye Disease Animal Model

The present invention provides a method for screening an animal model of an infectious eye disease comprising the following steps.

A1) dropping strains Enterococcus, Staphylococcus and Fluoroquinolone antibiotic eye drops into the eyelids of normal mammals except humans;

(A2) suturing the eye of the mammal after step (A1); And

(A3) checking the bacteria by dissecting the closed eyes in the step (A2).

As used herein, the term “fluoroquinolone antibiotic” means an antibiotic compound in which fluorine (F) is bonded to the carbon position 6 of quinoline.

In the present invention, the screening of the animal model is a screening method for confirming whether it is effective against antibiotic resistant bacteria.

In the present invention, the enterococcus of step (A1) is Enterococcus faecalis, and the Staphylococcus is preferably Staphylococcus epidermidis.

In the present invention, the strain Enterococcus faecalis (EF), Staphylococcus epidermidis (SE) and fluoroquinolone antibiotic eye drops in the step (A1) at the same time Can be dropped one after the other.

In the present invention, the fluoroquinolone antibiotic may be levofloxacin, ciprofloxacin, cixifloxacin, moxifloxacin, norfloxacin, or ofloxacin, preferably moxibustion. It may be floxacin.

In the present invention, the dosage of the fluoroquinolone antibiotic may vary depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the animal model. .

In the present invention, the step of observing the change of the infectious eye disease lesion is performed by a method commonly used in the art including visual observation, microscopic or endoscopic observation, ultrasound, surgical operation, biopsy or tissue staining. It may be carried out, and can be determined appropriately by those skilled in the art according to the specific purpose. For example, the step of observing the change of the infectious eye disease lesion may be preferably performed by visual observation, endoscope, or ultrasound, but is not limited thereto.

All matters mentioned in the infectious eye disease animal model of the present invention, a method of manufacturing the same, and a method of screening thereof are equally applied unless they contradict each other.

Advantages and features of the present invention and methods of achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Example 1 Preparation for Infectious Eye Disease Animal Model Preparation

1. Anesthetic Manufacturing

Zotiltil anesthetic and Rompun muscle relaxant were used as anesthetics. Anesthetics were prepared to anesthetize normal mammals except humans by mixing 2 ml of muscle relaxant lumps with 5 ml of the zoletil anesthetic.

2. Preparation of Triptic soy broth solution

After the determination of Tryptic soy broth 30 g high-temperature sterilization for 20 minutes at 121 ℃ then mixed in distilled water to 1000 ㎖ were stored at 4 ℃.

3. Manufacture of color detection agar badge

58.12 g of color detection agar commercially available in MB Cell was mixed in 1000 ml of distilled water, sterilized at 121 ° C. for 20 minutes, poured into a petri dish, and then hardened to prepare a medium.

4. Preparation of Enterococcus faecalis and Staphylococcus epidermidis fungus.

Trycotic Soy Broth incubated with single colony of Enterococcus faecalis and Staphylococcus epidermidis for 20 hours, and then cultured for 3 hours after adding 1% of the culture solution in a new Tryptic Soy Broth. Two cultured bacteria were separated using a centrifuge, discarded the supernatant, and 0.85% saline was added to prepare the bacteria. This was dissolved in McFarland standard 0.5 to make a total of 10 ml of bacterial solution, which was then separated by centrifugation. The supernatant was discarded and 0.5 ml of 0.85% saline was added to the residue, followed by 2.0 × 10 ⁹ CFU / mL bacterial solution. Was prepared. 4.0 × 10 ⁹ CFU / mL bacterial solution was prepared by adding 0.25 mL of 0.85% saline solution in the final residue.

Experimental Example 1. Identification of bacterial infection in rabbit eye

The strain used in the experiment was tested using two strains of Enterococcus faecalis and Staphylococcus epidermidis prepared in the above examples, the details are shown in Table 1 below. As mammals used in the experiment, 3 to 3.8 kg male rabbits were used, and all experiments were conducted according to IRB regulations.

TABLE 1

The rabbits used in the experiment were anesthetized by injecting 1.5 ml of the anesthetic prepared in the above example using a 5 ml syringe, and then injecting the rabbit into the hind leg of 3 to 3.8 kg. Then, the lower eyelid of the anesthetized rabbit was lifted up with tweezers, and the fungi of the two Enterococcus faecalis and Staphylococcus epidermidis were dropped into the eye by 12.5 μl, respectively, using a pipette. The colony count of the bacterial solution is 2.5 x 10 ⁷ CFU. After that, the eyes were closed by continuous suture using a surgical thread. After 4 hours, the rabbit was anesthetized by using an inhalation anesthetic, and then the surgical surgical thread was removed using surgical scissors, the lower eyelid was lifted with tweezers, and the cotton swab adhered to the inside of the eyelid while moving. Was taken. The swabs from which the bacteria were collected were put in 1 ml of 0.85% saline solution, extracted with a mixer, and diluted in 10-fold units. The diluted solution was spread by dropping 100 μl in color detection agar (MB Cell) and incubated at 37 ° C. for 24 hours to identify bacterial colonies using colony color. The identified colonies were calculated using the following formula according to the adjusted CFU (Colony Forming Unit).

[formula]

(Ⅰ) 2 x 10 ⁹ If CFU / ml

(Ⅰ) 10 ⁶ Number of colonies diluted to <200

200 CFU ÷ 10 ⁶ = number of colonies diluted, ratio, number of colonies diluted to 10 ⁶ × ratio = adjusted colonies

(Ii) 10 ⁶ Colonies diluted to> 200

(The number of colonies was diluted with 200 ÷ 10 ⁶ CFU) - 1 = ratio, the number of colonies were diluted to 10 ⁶ - (number of colonies were diluted to 10 ⁶ × ratio) = the number of control colonies

(II) 4 x 10 ⁹ If CFU / ml

(Ⅰ) 10 ⁶ When the number of colonies diluted to <400

400 CFU ÷ 10 ⁶ = number of colonies diluted, ratio, ^{6 6} diluted colonies × ratio = adjusted colonies

(Ii) 10 ⁶ Colonies diluted to> 400

(The number of colonies was diluted with 400 ÷ 10 ⁶ CFU) - 1 = ratio, the number of colonies were diluted to 10 ⁶ - (number of colonies were diluted to 10 ⁶ × ratio) = the number of control colonies

The results are shown in the following [Table 2] and FIG.

TABLE 2

[Table 2] as a result of diluting to 10 ² culture, Enterococcus faecalis was found to be 4 hours 234.04, Staphylococcus epidermidis 4 hours 46.86 was confirmed that appear.

In addition, FIG. 2 shows the results of diluting (A) 10, (B) 10 ² , (C) 10 ³ and (D) 10 ⁴ fold, respectively, as a result of incubation with blue Enterococcus faecalis and beige staphyloco Cous epidermidis has been identified and shows results consistent with the results in [Table 2]. Therefore, it can be confirmed from the above results that an infectious eye disease animal model expressed by Enterococcus faecalis and Staphylococcus epidermidis was produced.

Experimental Example 2. Confirmation of antimicrobial effect of bacteria in rabbit eye

The rabbits used in the experiment were anesthetized by injecting 1.5 ml of the anesthetic prepared in the above example using a 5 ml syringe, and then injecting the rabbit hind leg of 3 to 3.8 kg. Then, the lower eyelid of the anesthetized rabbit was lifted up with tweezers, and the fungi of the two Enterococcus faecalis and Staphylococcus epidermidis were dropped into the eye by 12.5 μl, respectively, using a pipette. The colony count of the bacterial solution is 2.5 x 10 ⁷ CFU. Moxifloxacin was added as an antibiotic. After that, the eye was closed by continuous suture using a surgical thread. After 4 hours, the rabbit was anesthetized by using an inhalation anesthetic, and then the surgical surgical thread was removed using surgical scissors, the lower eyelid was lifted with tweezers, and the cotton swab adhered to the inside of the eyelid while moving. Was taken. The swabs from which the bacteria were collected were put in 1 ml of 0.85% saline solution, extracted with a mixer, and diluted in 10-fold units. The diluted solution was dispersed in 100 μl of color detection agar (MB Cell), incubated at 37 ° C. for 24 hours, and colonies were identified using colony color. Is shown in the following [Table 3].

TABLE 3

The table 3 is reference to a result of the culture was diluted tenfold ^2, as compared to animal models into an animal model and moxifloxacin that does not contain moxifloxacin as antibiotic, in an animal model into the moxifloxacin as antibiotics It can be seen that Enterococcus faecalis and Staphylococcus epidermidis strains are less present. The above results can be seen that confirmed through [FIG. 3].

From the above results, it can be confirmed that the infectious eye disease animal model of the present invention can be usefully used for diagnosis, treatment, and screening of inflammatory eye disease.

From the above description, it will be understood by those skilled in the art that the present invention may be implemented in other specific forms without changing the technical spirit or essential features of the present invention. In this regard, the embodiments described above are illustrative in all respects and should be understood as not limiting.

Claims (11)

Strain Enterococcus nose kusu (Enterococcus) and Staphylococcus (Staphylococcus) the infective eye disease animal model, characterized in that obtained by administration of a normal non-human mammal. The method of claim 1,
The enterococcus is Enterococcus faecalis (EF), the staphylococcus is Staphylococcus epidermidis (SE) characterized in that the animal model of infectious eye disease characterized in that. The method of claim 1,
The infectious eye disease is conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, post-traumatic eye rupture or blepharitis animal model. (S1) strains Enterobacter nose kusu (Enterococcus) and Staphylococcus (Staphylococcus) a step of dripping of the eyelid of a normal non-human mammal; And
(S2) after the step (S1) of suturing the eye of the mammal; method of producing an infectious eye disease animal model comprising a. The method of claim 4, wherein
The enterococcus of step (S1) is Enterococcus faecalis (EF), and the Staphylococcus is Staphylococcus epidermidis (SE), characterized in that an infectious eye disease animal model . The method of claim 4, wherein
The strain is 1 × 10 ⁵ to 1 × 10 ⁹ CFU (Colony Forming Unit) per animal individual, characterized in that the method for producing an infectious eye disease animal model. The method of claim 4, wherein
The infectious eye diseases are conjunctivitis, keratitis, endophthalmitis, tear bagitis, tear ductitis, intraocular filling infection, post-traumatic eye rupture or blepharitis, characterized in that the production method of an animal model of infectious eye disease. The method of claim 4, wherein
The normal mammal except the human is a method for producing an infectious eye disease animal model, characterized in that the rabbit. (A1) strains Enterobacter nose kusu (Enterococcus), Staphylococcus (Staphylococcus) and quinolone (Fluoroquinolone) fluoroalkyl step of dripping the antibiotic eye drops on the eyelid of the normal non-human mammal;
(A2) suturing the eye of the mammal after step (A1); And
(A3) identifying the bacteria by dissecting the closed eye in the step (A2); screening method for an infectious eye disease animal model comprising a. The method of claim 9,
The enterococcus of step (A1) is Enterococcus faecalis (EF), and the Staphylococcus is Staphylococcus Epidermidis (SE), characterized in that the animal model of infectious eye disease . The method of claim 9,
The fluoroquinolone antibiotics are levofloxacin, ciprofloxacin, cixifloxacin, moxifloxacin, norfloxacin, or ofloxacin, a screening method for an animal model of infectious eye disease .

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