KR20220010798A - Composition of antimicrobial complex for animal comprising amphenicols and aminoglycosides - Google Patents

Abstract

The present invention relates to a composition of antimicrobial complex for animal comprising florfenicol and an aminoglycoside-based antibacterial agent as an active ingredient for effectively killing multidrug-resistant pathogenic E. coli. More specifically, the composition containing the amphenicol-based antibacterial agent and the aminoglycoside-based antibacterial agent as active ingredients can be provided as a very effective complex antibacterial composition for animals, as it has been confirmed that combination treatment with an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent for multidrug-resistant E. coli shows an excellently improved antibacterial effect with a very small dose compared to each antibacterial agent previously used alone, and has an effect of reducing the appearance of antimicrobial-resistant mutants.

Description

Compound of antimicrobial complex for animal comprising amphenicols and aminoglycosides as active ingredients

The present invention relates to a complex antimicrobial composition for animals containing florfenicol and an aminoglycoside antibacterial agent as active ingredients for effectively killing multidrug-resistant pathogenic E. coli.

Antibiotics currently used to treat bacterial infections in animals include antibiotics used in humans as well, and important antibiotics to treat human infectious diseases-colistin, erythromycin, and the like. The mainly used antibiotics are tetracycline, macrolide, penicillin, aminoglycoside, a mixture of trimethoprim and sulphonamide, and fluoroquinolone. (fluoroquinolone) and the like.

Most of the prevention and treatment of bacterial infections in animals depend on the use of antibiotics. Development of new antibiotics is required due to the expression and propagation of resistant strains due to the misuse of antibiotics, but the development of new drugs takes considerable time and economical cost and cannot keep up with the acquisition of bacterial resistance. The prevalence of multidrug-resistant E. coli in livestock is gradually increasing, and as the resistance rate to important antibiotics used in humans is also increasing, it is raised as a serious health problem as well as livestock. For example, the cause of antibiotic residues in livestock is causing various problems to the environment and human health.

Recently, interest in developing effective complex antibiotics by selecting antibiotics that have been developed relatively long ago and are of low importance for the treatment of human infectious diseases is increasing. In the case of antibiotics for animals sold in Korea, single agents account for about 74%, whereas complex antibiotics with two or three agents account for 26%. As an example of a combined antibiotic product for animals, tyrosetine-F (Samyang Anipam), chlortetracycline HCL, a product that combines sulfadoxine and tyrosine for the prevention and treatment of digestive and respiratory diseases of livestock ), Dawon CSP, a combined antibiotic of sulfadiazole and penicillin G procaine, and Dawon Linspec (Dawon), a combined antibiotic of lincomycine and spectinomycin. , So far, there has been no report on a combination antibiotic for animals including amphenicols and aminoglycoside antibiotics.

Republic of Korea Patent Publication No. 10-2013-0062187 (published on June 12, 2013)

The present invention is to provide a composite antimicrobial composition for animals comprising an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent.

The present invention provides a composite antimicrobial composition for animals containing an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent as active ingredients.

In addition, the present invention provides an antibacterial injection for animals containing the complex antibacterial composition as an active ingredient.

According to the present invention, the combination treatment of an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent for multidrug-resistant E. coli showed an excellently improved antibacterial effect with a very small dose compared to each antibacterial agent previously used alone, and antimicrobial resistance mutation As it was confirmed that the effect of reducing the appearance of the strain was confirmed, the composition containing the amphenicol-based antibacterial agent and the aminoglycoside-based antibacterial agent as an active ingredient can be provided as a very effective complex antibacterial composition for animals.

1 is a result of confirming the survival rate of mice after infecting a multi-drug-resistant E. coli isolate into the abdominal cavity of a mouse and injecting an antibacterial agent intramuscularly 30 minutes later, and then treating the mice with an antibacterial agent alone or in combination with an E. coli isolate. FIG. 1A is EC2, In mice infected with EC5 and EC9 E. coli isolates, phosphate buffered saline (PBS, ○), florfenicol (FFL, ▲, 20 mg/kg), amikacin (AMK, ◆, 10 mg/kg) or FFL (20 mg/kg)/AMK (●, 10 mg/kg), FIG. 1B shows PBS (○), FFL (▲, 20 mg/kg), genta in mice infected with EC1, EC18 and EC29 E. coli isolates. These are the results of treatment with mycin (GEN, ◆, 20 mg/kg) or FFL (20 mg/kg)/GEN (●, 20 mg/kg).

Hereinafter, the present invention will be described in more detail.

The present invention may provide a composite antimicrobial composition for animals containing an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent as active ingredients.

The amphenicol-based antibacterial agent may be selected from the group consisting of florfenicol, thiamphenicol) and chloramphenicol, and more preferably florfenicol, but is not limited thereto.

Most of the amphenicol-based antibiotics are odorless, white or yellowish-white crystals with a bitter taste, have no odor, and are slightly soluble in water and insoluble in alcohol and acetone. Amphenicol antibiotics include chloramphenicol, thiamphenicol, and florfenicol. In which chloramphenicol is gram is one of the broad-spectrum antibiotics that excellent antibacterial activity this can effectively act on the infection which is caused especially typhoid bacteria (Salmonella typhi), and Haemophilus influenzae (Haemophilus influenzae) with respect to the negative and gram positive bacteria, rikechiah, Chlamydia and Mycoplasma . Thiamphenicol and florfenicol also have similar broad-spectrum antibacterial effects. These amphenicol antibiotics are rapidly absorbed from the digestive system, and are mainly excreted in the urine and sometimes excreted in bile. However, chloramphenicol is prohibited for use as an antibiotic for animals.

The aminoglycoside antibacterial agent is kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin and netilmicin. It may be selected from the group consisting of, more preferably amikacin or gentamicin, but is not limited thereto.

In the aminoglycoside antibiotic, two or more amino-sugars are linked to a central hexose nucleus by a glycosidic linkage. The hexose nucleus is chemically aminocyclitol, which is streptidine in streptomycin and 2-deoxystreptamine in other antibiotics. Therefore, these antibiotics are aminoglycosidic aminocyclitols in chemical structure and are simply called aminoglycoside antibiotics. It shows strong antibacterial action against gram-negative enterobacteriaceae and staphylococcus, and Mycobacterium tuberculosis has sensitivity to streptomycin and gentamicin. Among the Gram-positive bacteria, Staphylococcus aureus is particularly sensitive to aminoglycoside antibiotics. However, anaerobes are generally resistant.

The complex antimicrobial composition may include 20 to 89 parts by weight of the amphenicol-based antibacterial agent and 11 to 80 parts by weight of the aminoglycoside-based antibacterial agent based on 100 parts by weight of the total composition.

In more detail, when the amphenicol-based antibacterial agent and the aminoglycoside antibacterial agent are included below the content range, the desired therapeutic effect cannot be obtained, and when included in excess of the content range, a synergistic effect cannot be obtained for the combined treatment. .

In addition, the composite antimicrobial composition may include an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent in a weight ratio of 1: (0.3 to 1.5), and more specifically, include it in a ratio of 1: (0.5 to 1). , More preferably, florfenicol and amikacin may be included in a ratio of 1:0.5, and florfenicol and gentamicin may be included in a ratio of 1:1. Out of the above ratio range, excellent antibacterial effect cannot be obtained.

The complex antibacterial composition may be to prevent or treat pathogenic E. coli infection in livestock or animals.

The pathogenic E. coli may be uniformly multi-drug resistant.

According to an embodiment of the present invention, the animal may include mammals, poultry and fish, and specifically, as the mammals, pigs, cattle, horses, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets It can be used for animals (eg, dogs, cats), etc., and can be used for chickens, turkeys, ducks, geese, pheasants, and quails as the poultry, and can be used for trout as the fish.

The composition of the present invention may be administered to an animal in various forms in consideration of factors such as the type of animal and the dietary form. Specifically, it may be prepared for oral administration or may be formulated as an injection.

When the complex antibacterial agent of the present invention is used for oral administration, the composition of the present invention is mixed with an appropriate amount of auxiliary ingredients and excipients together with pharmaceutically acceptable additives such as lactose, crystalline cellulose, and magnesium stearate. Administer with feed.

In addition, the present invention can provide an antibacterial injection for animals containing the complex antibacterial composition as an active ingredient. When used as an injection, the complex antibacterial agent can be dissolved in distilled water together with dexamethasone to make an injection solution.

The above-mentioned excipients or solubilizers for oral administration or when preparing an injection solution may use other excipients or solubilizers that are acceptable for veterinary drugs, and the dosage may vary depending on whether it is for prophylaxis or treatment.

In addition, the present invention can provide an antibacterial injection for animals containing the complex antibacterial composition as an active ingredient.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are provided to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Bacterial strains

A total of 12 types of multidrug-resistant (MDR) E. coli were isolated.

The multi-drug-resistant E. coli isolate is a strain that exhibits resistance to five or more different classes of antibacterial agents, and from 2011 to 2016, from fecal samples or tissue lesions of diseased animals in the diagnostic laboratory of the Bacterial Disease Department of the Korea Agriculture, Forestry and Livestock Quarantine Headquarters. was collected Twelve representative MDR E. coli isolates were selected based on antimicrobial resistance patterns, antimicrobial minimal inhibitory concentrations (MICs), presence of aminoglycoside modifying enzyme genes, farm geographic location, clinical samples, and animal sources.

The synergistic effect between the antibacterial agents was confirmed using 11 E. coli isolates as shown in Table 1, and the mutation frequency according to the combined treatment with phlorophenicol and aminoglycoside was confirmed using two E. coli isolates EC10 and EC15.

9 and 2 isolates were obtained from pigs and chickens, respectively, and all E. coli isolates were obtained from Korea Veterinary Culture Collection (KVCC).

2. Antimicrobial Susceptibility Testing

The minimum inhibitory concentration (MIC) of 13 antibacterial agents was confirmed using the KRNV4F Sensititre panel (Trek Diagnostic Systems) according to the manufacturer's instructions.

The MICs of amikacin (AMK), gentamicin (KAN) and amoxicillin (AMX) were confirmed by performing the broth microdilution method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) [ Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: Twenty-seventh Informational Supplement M100-S28; CLSI: Wayne, PA, USA, 2018.], Escherichia coli ATCC (American Type Culture Collection) 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains.

When the antimicrobial agent MIC for the quality control strain was within the acceptable range, the antimicrobial susceptibility was confirmed based on the guidelines of the CLSI, and ceftiofur, phlorophenicol and Breakpoints for streptomycin were identified.

3. Polymerase Chain Reaction analysis of aminoglycoside-modifying enzyme gene

Taq DNA polymerase (Bioneer, Daejeon, Republic of Korea) in 2 µL of boiled microorganism suspension, 20 pM primer, 250 µM dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl _{2 and 1.5 U} PCR was performed with an included 20 μL volume.

aac(6')-Ib, aac(3)-IIa, aac(3)-IVa, ant(2")-Ia, ant(3")-Ia, aph(3')-Ia, aph(3" )-Ia and aph(3”)-Ib -specific primers were used to amplify the gene encoding the aminoglycoside-modifying enzyme. PCR conditions were performed by the previously reported method [Belaynehe, KM, et al., FEMS Microbiol. Lett. 2017, 364, 129.].

4. Confirmation of antibacterial synergy

A checkerboard assay was performed in a 96-well microplate to confirm an in vitro synergistic effect.

Preparation of bacteria and antibacterial agents for analysis was performed according to CLSI guidelines.

Briefly, bacteria (about 5 × 10 ⁵ CFUs/mL) were prepared in cation-adjusted Mueller-Hinton broth (Difco) and inoculated into 96-well microplates (100 μL/well).

Two types of antimicrobial agents were added to each well and serially diluted two-fold horizontally and vertically. The fractional inhibitory concentration index (FICI) value was measured by the following formula.

FICI = FIC _A + FIC _B = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone)

Drug interactions were classified into synergy, partial synergy, additivity, indifference, and antagonistism. Synergistic, partial synergistic, additive, promiscuous and antagonism were defined as FICI 0.5, 0.5 < FICI < 1, FICI = 1, 1 < FICI < 4, and FICI ≥ 4, respectively.

5. E. coli ( E. coli ) to determine the mutation frequency of

Mutation frequencies were determined in two-week E. coli isolates (EC10 and EC15) exposed to florfenicol (FFL), gentamicin (GEN), amikacin (AMK), FFL/GEN, or FFL/AMK.

E. coli EC10 and EC15 isolates are susceptible to FFL, GEN and AMK.

Bacteria (10 ⁹ CFUs) were treated with FFL (32 μg/mL), GEN (16 μg/mL), AMK (64 μg/mL), FFL (32 μg/mL)/GEN (16 μg/mL) and FFL (32 μg/mL)/AMK (64 μg/mL) was plated on a Mueller-Hinton agar plate, and after incubation at 37°C overnight, colonies were counted.

6. Animal Testing

Five-week-old female BALB/c mice (18-20 g) were purchased from Hyochang Science (Daegu, Korea) and bred under aseptic conditions.

Five animals per cage were bred in a laminar flow circulation room, and a temperature of 24 ± 2 °C and a relative humidity of 55% ± 5% were maintained during the experiment. After the mice were kept in cages for at least 5 days, 6-week-old mice were used for the experiment.

Bacterial inoculum was prepared by culturing E. coli overnight on blood agar plates and suspending in PBS.

To determine the fifty percent lethal dose (LD ₅₀ ), 6 mice were intraperitoneally inoculated with E. coli isolates sequentially diluted 10-fold (https://www.aatbio.com/tools/ld50-calculator) ). 5 × LD ₅₀ CFUs of bacteria (6 × 10 ⁶ CFUs/500 μL of EC29 and 2 × 10 ⁸ CFUs/500 L of EC1, EC2, EC5, EC9 and EC18) were injected intraperitoneally.

Thirty minutes after bacterial injection, 50 μL of FFL (20 mg/kg), GEN (20 mg/kg) or AMK (10 mg/kg) alone was injected into the right thigh, and 50 μL of FFL (20 mg/kg) kg) and 50 μL of aminoglycoside (20 mg/kg GEN or 10 mg/kg AMK) were injected into the left and right thighs, respectively, and the control animals were injected with PBS in both thighs. Thereafter, mortality was confirmed for 76 hours.

All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Kyungpook National University (2018-0138).

<Example 1> Confirmation of the effect of combined treatment of amphenicols and aminoglycosides against multidrug-resistant E. coli strains in vitro

As shown in Table 1, checkerboard analysis was performed with 11 types of multidrug-resistant E. coli (MDR E. coli ) isolates to confirm drug interaction between amphhenicol and aminoglycoside or β-lactam. For co-treatment with aminoglycoside and florfenicol, bacteria were isolated from healthy and diseased animals in Korea.

As shown in Table 1, resistance to streptomycin (STR), kanamycin (KAN), gentamicin (GEN) and amikacin (AMK) was confirmed in 11, 8, 5 and 4 isolates, respectively. In addition, various aminoglycoside-modifying enzyme genes were identified from each strain.

First, a checkerboard analysis was performed to confirm the synergistic effect between β-lactam and aminoglycoside, but as shown in Table 2, only the two isolates showed a synergistic effect in the cepharosin (CEF)/GEN combination. In addition, there was no synergistic effect in the CEF/FFL combination.

On the other hand, in the combination of amphenicol and aminoglycoside, 10 and 6 isolates showed a synergistic effect in the combination treatment of FFL/AMK (Table 3) and FFL/GEN (Table 4), respectively, and CHL/GEN (Table 3). 5) and CHL/AMK (Table 6) showed a synergistic effect in 6 and 8 isolates, respectively, in the combined treatment. On the other hand, isolates showing a synergistic effect were not identified in the FFL/CEF combination, and a partial synergistic effect was observed only in 3 weeks (Table 7).

Next, to determine whether the FFL/GEN or FFL/AMK combination could reduce the occurrence of resistant bacteria compared to a single antimicrobial agent, two E. coli isolates, FFL and GEN-sensitive EC10 and FFL and AMK-sensitive EC15 Alternatively, culture was performed on Mueller-Hinton agar plates containing two antibacterial agents, and the frequency of mutant colonies was checked after 24 hours.

As a result, as shown in Table 8, the E. coli EC10 isolate is a strain isolated from pig feces, and the FFL/GEN combination treatment slightly reduced the occurrence of mutant colonies in the EC10 isolate. However, mutant colonization of EC15 isolates was not observed in FFL/AMK co-treatment.

From the above results, it was confirmed that the FFL/AMK combination treatment showed very good activity against various multidrug-resistant E. coli strains, and it was confirmed that the antibacterial agent combination treatment could prevent the generation of resistant bacteria in vitro.

<Example 2> Confirmation of the effect of florfenicol and aminoglycoside combination treatment on multidrug-resistant E. coli strains in vivo

In order to confirm whether the combined treatment of aminoglycoside and florfenicol in vivo can exhibit activity against multidrug-resistant E. coli strains, E. coli was infected intraperitoneally in mice, and then an antibacterial agent was intramuscularly injected.

For the combination treatment, three E. coli isolates (EC2, EC5 and EC9 for FFL/AMK combination and EC1, EC18 and EC29 for FFL/GEN combination) that showed a synergistic effect in the checkerboard analysis of each group were selected.

As a result, all control mice treated with PBS as shown in FIG. 1A died within 48 hours. On the other hand, mice treated with FFL/AMK combination after infection with one of the three E. coli isolates had very good survival rates ( 60%-100%).

In addition, as shown in FIG. 1B , the survival rate (100%) of mice infected with EC18 and EC29 E. coli isolates was the same in the FFL/GEN combination treatment group compared to the GEN alone treatment group (100%).

Compared with the combination treatment of FFL/AMK (60%-100%) or FFL/GEN (80%-100%), the survival rate (20%-80%) of mice infected with the E. coli isolate for 6 weeks was decreased in the FFL-only treatment group. Confirmed.

From the above results, it was confirmed that the FFL/AMK combination treatment showed a superior antibacterial effect in mice infected with the multidrug-resistant E. coli strain than the FFL or AMK alone treatment.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A complex antibacterial composition for animals containing an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent as active ingredients. The method according to claim 1,
The amphenicol-based antibacterial agent is a compound antimicrobial composition for animals, characterized in that it is selected from the group consisting of florfenicol, thiamphenicol and chloramphenicol. The method according to claim 1,
The aminoglycoside antibacterial agent is kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin and netilmicin. Complex antibacterial composition for animals, characterized in that selected from the group consisting of. The method according to claim 1,
The complex antimicrobial composition for animals, characterized in that it comprises 20 to 89 parts by weight of an amphenicol-based antibacterial agent and 11 to 80 parts by weight of an aminoglycoside antibacterial agent, based on 100 parts by weight of the total composition. The method according to claim 1,
The complex antibacterial composition for animals, characterized in that the amphenicol-based antibacterial agent and the aminoglycoside antibacterial agent are included in a weight ratio of 1: (0.3 to 1.5). The method according to claim 1,
The complex antibacterial composition for animals, characterized in that for preventing or treating pathogenic E. coli infection in livestock or animals. 7. The method of claim 6,
The pathogenic E. coli is a complex antimicrobial composition for animals, characterized in that the multi-drug-resistant bacteria. An injection for animal antibacterial use comprising the complex antibacterial composition of any one of claims 1 to 7 as an active ingredient.

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