KR102489659B1 - Composition of antimicrobial complex for animal comprising amphenicols and aminoglycosides - Google Patents

Abstract

The present invention relates to a combination antimicrobial composition for animals containing florfenicol and an aminoglycoside-based antibacterial agent as active ingredients for effectively killing multi-drug-resistant pathogenic E. coli, and more particularly, to an amphenicol-based antibacterial agent for multi-drug-resistant E. coli and Combination treatment with aminoglycoside-based antibacterial agents showed significantly improved antibacterial effect with a very small dose compared to each antimicrobial agent previously used alone, and it was confirmed that the antibacterial agent-resistant mutant showed the effect of reducing the appearance, the cancer A composition containing a phenol-based antimicrobial agent and an aminoglycoside-based antimicrobial agent as active ingredients can be provided as a very effective combination antimicrobial agent composition for animals.

Description

Composition of antimicrobial complex for animals comprising amphenicol-based antimicrobial agents and aminoglycoside-based antimicrobial agents as active ingredients

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

Currently, antibiotics used to treat bacterial infections in animals include antibiotics used in humans, and also include a number of important antibiotics such as colistin and erythromycin to treat human infectious diseases. Mainly used antibiotics include tetracycline, macrolide, penicillin, aminoglycoside, mixture of trimethoprim and sulphonamide, and fluoroquinolone. (fluoroquinolones), etc.

Most of the prevention and treatment of bacterial infections in animals rely on the use of antibiotics. The development of new antibiotics is required due to the expression and spread of resistant strains due to the abuse and abuse of antibiotics, but the development of new drugs requires considerable time and economic costs and cannot keep up with the acquisition of resistance by bacteria. The prevalence of multidrug-resistant E. coli in livestock is gradually increasing, and as the resistance rate to important antibiotics used in humans increases, it is being raised as a serious health problem as well as livestock. For example, the residual cause of antibiotics in livestock causes various problems in the environment and human health.

Recently, there is increasing interest in developing effective antibiotics by selecting antibiotics that have been developed relatively long ago and are currently less important for the treatment of human infectious diseases. In the case of veterinary antibiotics sold in Korea, single agents account for about 74%, while complex antibiotics that combine two or three agents account for 26%. As an example of a compound antibiotic product for animals, Tyrosetin-F (Samyang Anipharm), a product combining sulfadoxin and tyrosine for the prevention and treatment of digestive and respiratory diseases in livestock, chlortetracycline HCL ), Dawon CSP, a combination antibiotic of sulfathiazole and penicillin G procaine, and Dawon Linspec (Dawon), a combination antibiotic of lincomycin and spectinomycin. , So far, no combination antibiotics for animals including amphenicols and aminoglycoside antibiotics have been reported.

Republic of Korea Patent Publication No. 10-2013-0062187 (2013.06.12. Publication)

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

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

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

According to the present invention, the combined treatment of an amphenicol-based antibacterial agent and an aminoglycoside-based antibacterial agent against multi-drug-resistant E. coli exhibited a remarkably improved antibacterial effect with a very small dose compared to each antibacterial agent previously used alone, and antimicrobial resistance mutation As it is confirmed that the effect of reducing the appearance of attention is confirmed, the composition containing the amphenicol-based antibacterial agent and the aminoglycoside-based antimicrobial agent as active ingredients can be provided as a highly effective combined antimicrobial composition for animals.

Figure 1 is a result of confirming the survival rate of mice after infecting mice with multidrug-resistant E. coli isolates intraperitoneally and intramuscularly injecting the antibiotic 30 minutes later, and then treating the antibacterial agent alone or in combination in mice infected with E. coli isolates. Figure 1A shows EC2, Mice infected with EC5 and EC9 E. coli isolates were treated with phosphate-buffered saline (PBS, ○), florfenicol (FFL, ▲, 20 mg/kg), amikacin (AMK, ◆, 10 mg/kg), or FFL (20 mg/kg). mg/kg)/AMK (●, 10 mg/kg), and Figure 1B shows PBS (○), FFL (▲, 20 mg/kg), Genta in mice infected with EC1, EC18 and EC29 E. coli isolates. This is the result 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 can provide a combination antibacterial composition for animals containing an amphenicol-based antimicrobial agent and an aminoglycoside-based antimicrobial 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, are slightly soluble in water, and are poorly soluble in alcohol and acetone. Amphenicol antibiotics include chloramphenicol, thiamphenicol, and florfenicol. Among them, chloramphenicol has excellent antibacterial activity against Gram-negative and Gram-positive bacteria, Rickettsia, Chlamydia and Mycoplasma, and is particularly effective against infections caused by Salmonella typhi and Haemophilus influenzae . It is one of the broad-spectrum antibiotics. . Thiamphenicol and florfenicol also have similar broad-spectrum antimicrobial activity. These amphenicol-type antibiotics are rapidly absorbed from the digestive system, and are mainly excreted in the urine or in the bile. However, chloramphenicol is banned as an antibiotic for animals.

The aminoglycoside antibacterial agent is kanamycin, amikacin, tobramycin, dibakacin, gentamicin, sisomicin and netilmicin It may be selected from the group consisting of, and more preferably may be 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. This hexose nucleus is chemically an aminocyclitol, streptidine in streptomycin, and 2-deoxystreptamine in other antibiotics. Therefore, these antibiotics are called aminoglycoside antibiotics simply because they are aminoglycosidic aminocyclitols due to their chemical structure. It shows strong antibacterial action against Gram-negative enterobacteriaceae and Staphylococcus aureus, and Mycobacterium tuberculosis is sensitive to streptomycin and gentamicin. Among Gram-positive bacteria, Staphylococcus aureus is also highly sensitive to aminoglycoside antibiotics. However, anaerobic bacteria are generally resistant.

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

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

In addition, the combined antimicrobial agent composition may include an amphenicol-based antimicrobial agent and an aminoglycoside-based antimicrobial agent in a weight ratio of 1: (0.3 to 1.5), and more specifically, 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 combined antimicrobial agent composition may prevent or treat pathogenic Escherichia coli infection of livestock or animals.

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

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

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

When the combined antibacterial agent of the present invention is used for oral administration, the composition of the present invention is combined with pharmaceutically acceptable additives such as lactose, crystalline cellulose, and magnesium stearate with appropriate amounts of auxiliary components and excipients. Administer with feed.

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

Excipients or solubilizers used when preparing the above-mentioned oral administration or injection solutions may use other excipients or solubilizers acceptable for animal drugs, and the dosage may vary depending on whether they are for prevention or treatment.

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

Hereinafter, an embodiment will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but 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 skilled in the art.

<Experimental example>

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

1. Bacterial Strains

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

The multidrug-resistant E. coli isolates are strains exhibiting antimicrobial resistance of five or more different series, and from 2011 to 2016 from fecal samples or tissue lesions of diseased animals in the diagnostic laboratory of the Bacterial Disease Division of the Korea Agriculture, Forestry and Livestock Quarantine Headquarters. has been 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, geographic location of farms, clinical samples and animal sources.

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

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

2. Antimicrobial Susceptibility Testing

The minimum inhibitory concentration (MIC) of 13 antimicrobial 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.], E. coli ATCC (American Type Culture Collection) 25922 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) ATCC 27853 were used as quality control strains.

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

3. Polymerase Chain Reaction Analysis of Aminoglycoside Modifying Enzyme Genes

2 μL of boiled microbial 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 of Taq DNA polymerase (Bioneer, Daejeon, Republic of Korea) PCR was performed with the 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 as previously reported [Belaynehe, KM, et al., FEMS Microbiol. Lett. 2017, 364, 129.].

4. Confirm antibacterial synergistic effect

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

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

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

Two types of antimicrobial agents were added to each well and serially diluted 2-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 antagonism. Synergistic, partial synergistic, additive, promiscuous and antagonistic were defined as FICI 0.5, 0.5 < FICI < 1, FICI = 1, 1 < FICI < 4, and FICI ≥ 4, respectively.

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

Mutation frequencies were determined in 2-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 analyzed by 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). μg/mL)/AMK (64 μg/mL) were plated on Mueller-Hinton agar plates, incubated overnight at 37°C, and colonies were counted.

6. Animal Testing

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

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

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

To confirm the fifty percent lethal dose (LD ₅₀ ), 6 mice were sequentially inoculated with 10-fold diluted E. coli isolates intraperitoneally (https://www.aatbio.com/tools/ld50-calculator ). 5 x LD ₅₀ CFUs of bacteria (6 x 10 ⁶ CFUs/500 μL of EC29 and 2 x 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, followed by 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 control animals were injected with PBS into both thighs. Mortality was checked for 76 hours thereafter.

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

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

A checkerboard analysis was performed on 11 multidrug-resistant E. coli (MDR E. coli ) isolates as shown in Table 1 to confirm drug interactions between amphenicol-based and aminoglycoside-based or β-lactams. For the combined 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 two isolates showed a synergistic effect in the combination of Cepharosin (CEF) / GEN. In addition, no synergistic effect was observed in the combination of CEF/FFL.

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

Next, two E. coli isolates, EC10 sensitive to FFL and GEN and EC15 sensitive to FFL and AMK, were isolated to determine whether the combination of FFL/GEN or FFL/AMK could reduce the occurrence of resistant bacteria compared to single antimicrobial agents. Alternatively, the cells were cultured on a Mueller-Hinton agar plate containing two antibiotics, and the frequency of mutant colonies was confirmed 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 combined treatment slightly reduced the occurrence of mutant colonies of the EC10 isolate. However, FFL/AMK co-treatment did not show mutant colonization of the EC15 isolate.

From the above results, it was confirmed that the FFL/AMK combined treatment exhibited very good activity against various multidrug-resistant E. coli strains, and that the antibacterial agent combined treatment could prevent the occurrence of resistant bacteria in the test tube.

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

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

For the combined treatment, three E. coli isolates (EC2, EC5, and EC9 for the FFL/AMK combination and EC1, EC18, and EC29 for the 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 died within 48 hours, as shown in FIG. 1A. On the other hand, mice treated with FFL/AMK combination after infection with one of the three E. coli isolates had a very good survival rate ( 60%-100%).

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

Compared to FFL/AMK (60%-100%) or FFL/GEN (80%-100%) combination treatment, the FFL alone treatment group showed a decrease in the survival rate (20%-80%) of mice infected with E. coli isolates at 6 weeks. Confirmed.

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

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, 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 composite antimicrobial composition for animals against multidrug-resistant pathogenic Escherichia coli infection, containing florfenicol and amikacin as active ingredients. delete delete The method of claim 1,
The combined antimicrobial composition for animals, characterized in that it comprises 20 to 89 parts by weight of florfenicol antimicrobial agent and 11 to 80 parts by weight of amikacin, based on 100 parts by weight of the total composition. The method of claim 1,
The combined antimicrobial composition is a combination antimicrobial composition for animals, characterized in that florfenicol and amikacin are included in a weight ratio of 1: (0.3 to 1.5). delete delete An antibacterial injection for animals against multidrug-resistant pathogenic Escherichia coli infection, comprising the combination antimicrobial composition of any one of claims 1, 4, and 5 as an active ingredient.

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