JP2021046371A - Antibiotic tolerant microorganism tolerance lowering substance and method for lowering tolerance of antibiotic tolerant microorganism - Google Patents

Abstract

To provide a substance that reduces the tolerance to antibiotics such as streptomycin or a substance that reduces the growth, and to provide an antibiotic-containing agrochemical containing said substance.SOLUTION: By adding manganese or a manganese compound to an agrochemical, a pharmaceutical or an animal drug containing antibiotics such as streptomycin, the manganese or manganese compound lowers the drug tolerance of microorganisms such as phytopathogenic bacteria and enhances the effectiveness of antibiotic-containing drugs even for the case where microorganisms tolerant to the existing antibiotics become actual and are not effective in treatment or control.SELECTED DRAWING: Figure 1

Description

The present invention relates to a substance that reduces antibiotic resistance to an antibiotic-resistant microorganism and a method that reduces the antibiotic resistance.

The emergence of pathogenic microorganisms that are resistant to conventional antibiotics and other antibacterial agents has become a major global problem. For example, Staphylococcus aureus, the cause of hospital infectivity, was found to have a sufficient bactericidal effect by penicillin in the early 1940s, but by the late 1960s more than 80% of Staphylococcus aureus was found. It became resistant to penicillin, and by 1972 2% of Staphylococcus aureus was found to be resistant to mecitillin. It is reported that the proportion of methicillin-resistant bacteria continued to rise to 57.1% by 2002.

For such antibiotic-resistant microorganisms, Japanese Patent Application Laid-Open No. 2019-135269 describes methicillin-resistant Enterococcus faecalis (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), Escherichia coli O157: H7, fluoroquinolone-resistant typhoid bacteria, and the like. Discloses a method for preventing or treating an infectious disease caused by extracellular microorganisms such as bacteria and fungi by administering a gallium compound to a subject (Patent Document 1).

Japanese Unexamined Patent Publication No. 2019-135269

The problem with antibiotic-resistant microorganisms applies not only to antibiotic-resistant microorganisms against humans and domestic animals, but also to antibiotic-resistant microorganisms of phytopathogenic bacteria. For example, antibiotics are used as active ingredients of fungicides such as black rot and rot of vegetables, black rot and black spot of fruit trees, and bacterial blight of rice. Therefore, it is expected that by continuously spraying antibiotics on crops, microorganisms existing in farms and crops will acquire resistance to antibiotics, which will bring a great financial burden to farmers. Diseases caused by drug-resistant bacteria are difficult to control with antibiotics due to the manifestation of antibiotic-resistant bacteria. Furthermore, the development of new antibiotics that are effective against antibiotic-resistant bacteria requires a great deal of time and cost, so there is a limit to the response by one company or one individual, and even if a new antibiotic is developed, Bacteria that are resistant to the new antibiotic are likely to emerge.

Therefore, an object of the present invention is to provide a substance that reduces resistance to antibiotics or a substance that reduces growth.

In order to solve the above problems, the present inventors have studied various substances that affect the resistance of bacteria to antibiotics, and found that manganese (Mn) accidentally reduces the resistance of bacteria to antibiotics. Got

The present invention has been made based on such findings, and provides manganese used as a substance for reducing drug resistance against microorganisms.

The present invention also provides the use of manganese to produce agents to reduce drug resistance to microorganisms.

The present invention further provides a drug containing an antibiotic, which is characterized by containing manganese as a substance that reduces drug resistance to microorganisms.

According to the present invention, when a microorganism resistant to an existing antibiotic becomes apparent and is ineffective in treatment or control, the drug resistance to the microorganism is reduced by treating (administering or spraying) the antibiotic together with manganese. be able to. If this is contained in a drug containing an antibiotic, drug resistance to microorganisms such as bacteria can be reduced, so that even existing antibiotics can be highly effective in treatment and control.

It is a figure which shows the result of the streptomycin susceptibility test (Mn-added ASA medium) of the peach perforated bacterial disease fungus. It is a figure which shows the result of the streptomycin susceptibility test (ASA medium) of the peach perforated bacterial disease fungus.

Hereinafter, embodiments according to the present invention will be described in detail.

In this embodiment, manganese is used as a substance for reducing drug resistance to microorganisms.

Manganese is an element having an atomic number of 25, and the element symbol is "Mn". Manganese is naturally present in more than 100 minerals in various forms such as sulfides, oxides, carbonates, silicates, phosphates and borates (NAS-NRC 1973). It is known that the main ores include pyrolus ore, silomelane ore, rhodochrosite, and tephrosite. It is widely distributed in the crust, has an average abundance of 950 ppm, and is contained in seawater at 2 μg / l. The simple substance is a silver-white metal, but when it contains carbon, it becomes gray. There are four allotropes of α-type, β-type, γ-type, and δ-type, but in the present invention, any allotrope can be used.

In the present embodiment, "manganese" includes a manganese compound and a manganese alloy (hereinafter, these are collectively referred to as "manganese and the like"). The main manganese compounds include, for example, manganese chloride (II) (Manganese chloride, MnCl ₂ ), manganese sulfate (II) (Manganese sulfate, MnSO ₄ ), trimanganese tetraoxide (Manganese oxide, Mn ₃ O ₄ ), and manganese dioxide. manganese (manganese dioxide, MnO _2), potassium permanganate (Potasium permanganate, KMnO _4), manganese borate _{(octahydrate) (manganese borate, MnB 4 O} 7 · 8H 2 O), manganese carbonate (II) ( Manganese carbonate, MnCO ₃ ), Methylcyclopentadienyl manganese tricarbonyl (MMT), C ₉ H ₇ MnO ₃ ), Maneb (Maneb, C ₄ H ₆ N ₂ S ₄ Mn), Mancozeb, [C ₄ H ₆ N ₂ S ₄ Mn] x (Zn) y, x: y = 10: 1) and the like can be mentioned.

In the present embodiment, the bacterium may be a bacterium, particularly a phytopathogenic bacterium, specifically, a bacterium of the genus Acidovorax, a bacterium of the genus Burkholderia, or a bacterium of the genus Pseudomonas. , At least one selected from the group consisting of Ralstonia bacterium and Xanthomonas bacterium can be raised, more specifically consisting of Ume scab, Peach perforator bacterium, Kankitsu scab. At least one species selected from the group can be mentioned.

In the present embodiment, the drug may include an antibiotic. Antibiotics are, in a narrow sense, substances produced by microorganisms that inhibit the growth of other microorganisms, and in a broad sense, antibacterial agents and tumor cells that are chemically modified or artificially synthesized from those produced by microorganisms. Includes substances that inhibit the growth and function of cells other than other microorganisms, such as. Examples of the antibiotic include streptomycin, penicillin, actinomycin, rifampicin, fosfomycin, vancomycin, chloramphenicol and the like.

In the present embodiment, "reducing drug resistance" means, in a broad sense, reducing the minimum inhibitory concentration (MIC) of a drug-resistant microorganism to the extent that a drug control effect can be obtained. In a narrow sense, it means that the minimum inhibitory concentration (MIC) of streptomycin against streptomycin-resistant microorganisms is reduced from 18,000 ppm to 500 ppm, and the control effect by streptomycin can be obtained.

The mechanism by which manganese and the like reduce the drug resistance of microorganisms is unknown, but in the process of proliferation of microorganisms that are sensitive to a certain drug, manganese and the like prevent mutations in genes on the plasmid. It is presumed that manganese and the like prevent the uptake of the sex resistance gene, and that manganese and the like prevent the mutagenesis of the resistance plasmid that has acquired drug resistance. In addition, manganese alone has no effect of inhibiting the growth of microorganisms.

In this embodiment, manganese and the like are used to produce a drug for reducing drug resistance to microorganisms.

The drug containing an antibiotic is not particularly limited, and examples thereof include pesticides, pharmaceuticals, and veterinary drugs that can conventionally contain an antibiotic.

The amount (concentration) of manganese or the like added in the production of pesticides is preferably 300 to 3000 ppm. Since the effect of preventing or reducing the acquisition of drug resistance is improved depending on the concentration of manganese or the like, it is difficult to obtain the effect at less than 300 ppm. On the other hand, even if 3000ppm or more is added, there is no adverse effect on the crop, but the effect reaches a plateau, so the upper limit was set to 3000ppm.

In the present embodiment, other components may be blended within a range that does not affect the effect of lowering the drug resistance of the present invention and within a pharmaceutically acceptable range. Examples of the components that can be blended include liquid carriers, solid carriers, surfactants (emulsifiers, dispersants, antifoaming agents, etc.), auxiliary agents, and the like. More specifically, examples of the liquid carrier include a phosphate buffer solution and a carbonic acid buffer solution. Examples of the solid carrier include natural mineral powders such as kaolin, clay, quartz, montmorillonite and diatomaceous earth, and high molecular weight natural products such as crystalline cellulose, corn starch, gelatin and alginic acid, and one or more of these are mixed. Can be used. Examples of the surfactant include polyoxyethylene-fatty acid ester and polyoxine ethylene-fatty alcohol ether. Examples of the auxiliary agent include carboxymethyl cellulose and starch.

Various dosage forms can be selected as the dosage form of the drug, for example, powder, granule, powder or granule, powder, wettable powder, aqueous solvent, emulsion, liquid, oil, aerosol, paste, smoke. Examples thereof include agents, fumigants, coating agents, microcapsules, packing agents, flowable agents and the like.

1. 1. Examination of substances that reduce SM resistance or suppress growth (1) Experimental method As test bacteria, peach foramen (Xanthomonas arboricola pv. Pruni) and citrus bacillus (Xanthomonas) that are resistant to streptomycin (SM) Using citri subsp. Citri) and Xanthomonas syringae (Pseudomonas syringae pv. Morsprunorum), substances that reduce SM resistance or suppress the growth of these phytopathogenic bacteria were investigated.

Apple juice _{40.0 ml, Ca (NO 3)} 2 · 4H 2 O 0.5 g, Na 2 HPO 4 · 12H 2 O2.0g, Peptone 5.0 g, Agar15.0 g, of distilled water 960.0Ml ASA medium (Moriyama et al., In 2016), SM-sensitive bacteria and resistant bacteria (MIC: 500 and> 16000 ppm) were cultured at 25 ° C. for 3 days in a medium mixed with SM at Mn 0.3, 1.5 and 3.0 g / L to investigate their growth.

(2) Results Table 1 shows the measurement results of the minimum inhibitory concentration (ppm) of streptomycin against phytopathogenic bacteria in manganese-added ASA medium. In Table 1, "streptomycin resistance" means bacteria with low susceptibility to SM at MIC: 500 and> 16000 ppm, and "streptomycin sensitivity" means low MIC such as 15.6 ppm or less, and SM is generally effective. Means highly sensitive bacteria. In addition, each was carried out for a plurality of bacteria having different strain numbers.

The MIC of SM-resistant bacteria in ASA medium mixed with SM and Mn at 3.0 g / L decreased from 125 to 500 ppm for peach perforated bacteria, and from> 2000 ppm to 2000 ppm for worm and citrus bacilli. Mixing only Mn did not affect the growth of the test bacteria.

From the above results, it was speculated that Mn may affect the expression of the strA gene (Scholz et al., 1989) involved in SM resistance.

A similar test was conducted using Zn, ZnO, and a manzeb agent ((C4H6MnN2S4) xZny) instead of Mn. As a result, Zn and ZnO were 3.0 g / L, and manzeb was a single substance at a spray concentration (1333 ppm). The growth of all test bacteria was suppressed regardless of SM sensitivity. Manzeb may be effective in controlling not only filamentous fungal diseases of peach but also perforated bacterial diseases, as well as scabs of plums and citrus fruits.

2. Effect of the amount of manganese added on the resistance of streptomycin-resistant phytopathogenic bacteria (1) Experimental method As a test bacterium, a peach perforated bacterium (Xanthomonas arboricola pv. Pruni) having resistance to streptomycin (SM) was used. The effect of the amount of manganese added on the minimum inhibitory concentration (ppm) of streptomycin was investigated.

ASA media supplemented with Mn at 0.3, 1.5 and 3.0 g / L were autoclaved and then streptomycin was added to these media at 50 ° C. at 1.95, 3.90, 7.80, 15.6, 31.3, 62.5, 125, 250 and 500. , 1000, 2000, 4000, 8000 and 16000 ppm, respectively, to prepare a plate medium. Bacterial suspension was prepared by suspending the test bacteria cultured in this plate medium for 24 to 48 hours ^{in sterile distilled water so as to have a concentration of about 10 8 cfu / ml.} This bacterial suspension was applied to the above-mentioned plate medium with a sterilized cotton swab and cultured at 25 ° C. for 3 days to investigate the presence or absence of bacterial growth.

(2) Results The results are shown in FIGS. 1 and 2. FIG. 1 shows the results of culturing at each SM concentration in the ASA medium supplemented with Mn 3.0 g / l, and FIG. 2 shows the results of culturing at each SM concentration in the ASA medium without Mn added. In the ASA medium without streptomycin added at 0.3, 1.5 and 3.0 g / L, respectively (upper left in Fig. 1), the test bacteria grew at the same level as the ASA medium without streptomycin and Mn (upper left in Fig. 2). there were. Mn did not affect the growth of the test bacteria. On the other hand, in the medium to which streptomycin was added together with Mn (Fig. 1), the MIC of streptomycin against the test bacteria decreased.

Similar experiments were carried out using peach perforated bacterial disease bacteria and Xanthomonas campatai disease bacteria. As a result, in the medium supplemented with 3.0 g / L of Mn, the MIC of streptomycin-resistant bacteria (MIC: 500 and> 16000 ppm) was 125 to 500 ppm in the peach perforated bacterial disease, and the MIC was also obtained in the worm and citrus fungus. It decreased from> 2000 ppm to 2000 ppm.

Claims (9)

Manganese used as a substance to reduce drug resistance to microorganisms. Use of manganese to produce drugs to reduce drug resistance to microorganisms. A drug that contains antibiotics
A drug characterized by containing manganese as a substance that reduces drug resistance to microorganisms. The drug according to claim 3, wherein the microorganism is a bacterium. The agent according to claim 3 or 4, wherein the microorganism is a phytopathogenic bacterium. At least the microorganism selected from the group consisting of Acidovorax bacteria, Burkholderia bacteria, Pseudomonas bacteria, Ralstonia bacteria and Xanthomonas bacteria. The agent according to any one of claims 3 to 5, which is one. The agent according to any one of claims 3 to 6, wherein the microorganism is at least one selected from the group consisting of plum scab, peach perforated bacterium, and Xanthomonas campis. The agent according to any one of claims 3 to 7, wherein the antibiotic is streptomycin. A method for reducing the resistance of an antibiotic-resistant microorganism, which comprises reducing the drug resistance to a microorganism by adding manganese to a drug containing an antibiotic.

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