JP7458622B2 - Substances that reduce the resistance of antibiotic-resistant microorganisms and methods for reducing the resistance of antibiotic-resistant microorganisms - Google Patents

Description

The present invention relates to a substance that reduces the antibiotic resistance of antibiotic-resistant microorganisms, and a method for reducing the antibiotic resistance.

The emergence of pathogenic microorganisms that are resistant to conventional antibiotics and other antimicrobial agents is a major problem worldwide. For example, in the early 1940s, penicillin was found to have a sufficient sterilizing effect on Staphylococcus aureus, which causes hospital-acquired infections, but by the late 1960s, more than 80% of Staphylococcus aureus had been killed. It became resistant to penicillin, and by 1972 it was recognized that 2% of Staphylococcus aureus were resistant to methicillin. It is reported that the percentage of methicillin-resistant bacteria continued to rise to 57.1% by 2002.

Regarding such antibiotic-resistant microorganisms, Japanese Patent Application Publication No. 2019-135269 discloses that it is effective against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), Escherichia coli O157:H7, fluoroquinolone-resistant Salmonella typhi, etc. discloses a method for preventing or treating infectious diseases caused by extracellular microorganisms such as bacteria and fungi by administering a gallium compound to a subject (Patent Document 1).

JP 2019-135269 Publication

The same problem regarding antibiotic-resistant microorganisms applies not only to antibiotic-resistant microorganisms for humans and livestock, but also to antibiotic-resistant microorganisms for plant pathogenic bacteria. For example, antibiotics are used as active ingredients in fungicides to treat black rot and rot of vegetables, black spot and black spot of fruit trees, and bacterial blight of rice. Therefore, it is expected that continuous spraying of antibiotics on crops will cause microorganisms present in farms and crops to acquire resistance to antibiotics, resulting in a significant economic burden on farmers. Diseases caused by drug-resistant bacteria become difficult to control with antibiotics due to the emergence of antibiotic-resistant bacteria. Furthermore, developing new antibiotics that are effective against antibiotic-resistant bacteria requires a great deal of time and cost, so there are limits to what a single company or individual can do, and even if a new antibiotic were developed, There is a high possibility that bacteria that are resistant to these new antibiotics will 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 investigated various substances that affect the antibiotic resistance of bacteria, and coincidentally found that manganese (Mn) reduces the resistance of bacteria to antibiotics. I got it.

The present invention was made based on this knowledge, and provides manganese that can be used as a substance for reducing drug resistance in microorganisms.

The present invention also provides the use of manganese for manufacturing a drug for reducing drug resistance against microorganisms.

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

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

FIG. 2 is a diagram showing the results of a streptomycin susceptibility test (Mn-added ASA medium) of peach borehole bacterial pathogens. FIG. 2 is a diagram showing the results of a streptomycin susceptibility test (ASA medium) on peach borehole bacterial pathogens.

The following describes in detail an embodiment of the present invention.

In this embodiment, manganese is used as a substance to reduce drug resistance against microorganisms.

Manganese is an element with an atomic number of 25, and its element symbol is "Mn". Manganese exists in nature in more than 100 minerals in various forms, including sulfides, oxides, carbonates, silicates, phosphates, and borates (NAS-NRC 1973). It is known that the main ores include pyroluthite, psilomelaneite, rhodochrosite, and tephrite. It is widely distributed in the earth's crust, with an average abundance of 950 ppm and 2 μg/l in seawater. Alone, it is a silvery-white metal, but when it contains carbon, it becomes gray. There are four allotropes: α-type, β-type, γ-type, and δ-type, and any of the allotropes can be used in the present invention.

In this embodiment, "manganese" includes manganese compounds (hereinafter, these are collectively referred to as "manganese etc."). Main manganese compounds include, for example, manganese chloride (MnCl ₂ ), manganese sulfate (MnSO ₄ ), manganese oxide (Mn ₃ O ₄ ), and manganese dioxide. Manganese (MnO ₂ ), Potassium permanganate (KMnO ₄ ), Manganese borate (octahydrate) (MnB ₄ O ₇・8H ₂ O), Manganese (II) carbonate ( Manganese carbonate, MnCO ₃ ), Methylcyclopentadienyl manganese tricarbonyl (MMT), C ₉ H ₇ MnO ₃ ), Maneb (C ₄ H ₆ N ₂ S ₄ Mn), Mancozeb, Examples include [C ₄ H ₆ N ₂ S ₄ Mn]x(Zn)y, x:y=10:1).

In this embodiment, the microorganism can include bacteria, particularly plant pathogenic bacteria, and specifically, bacteria of the genus Acidovorax, bacteria of the genus Burkholderia, bacteria of the genus Pseudomonas. , at least one selected from the group consisting of bacteria of the genus Ralstonia and bacteria of the genus Xanthomonas, and more specifically, the bacteria consisting of canker bacterium, peach borer bacterium, and citrus canker bacterium. At least one selected from the group can be mentioned.

In this embodiment, the drug may include antibiotics. Antibiotics, in a narrow sense, refer to substances produced by microorganisms that inhibit the growth of other microorganisms, and in a broader sense, they refer to substances produced by microorganisms that are chemically modified or artificially synthesized, antibacterial agents, and tumor cells. Contains substances that inhibit the growth and function of other non-microbial cells, such as Examples of antibiotics include streptomycin, penicillin, actinomycin, rifampicin, fosfomycin, vancomycin, chloramphenicol, and the like.

In the present embodiment, "reducing drug resistance" in a broad sense means lowering the minimum inhibitory concentration (MIC) of microorganisms with drug resistance to an extent that the control effect of the drug can be obtained, In a narrow sense, this means that the minimum inhibitory concentration (MIC) of streptomycin against streptomycin-resistant microorganisms has been lowered from 18,000 ppm to 500 ppm, allowing streptomycin to have a control effect.

The mechanism by which manganese and other substances reduce the drug resistance of microorganisms is unknown, but manganese and other substances prevent the mutation of genes on chromosomes during the proliferation of microorganisms that are sensitive to certain drugs. The reasons for this are thought to be that manganese and the like prevent the incorporation of sexual resistance genes, and that manganese and the like prevent the conjugative transfer of resistance plasmids that have acquired drug resistance. Note that 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 against microorganisms.

There are no particular limitations on drugs containing antibiotics, and examples thereof include agricultural chemicals, pharmaceuticals, veterinary drugs, etc. that can conventionally contain antibiotics.

The amount (concentration) of manganese etc. added in the production of agricultural chemicals is preferably 300 to 3000 ppm. The effect of preventing or reducing the acquisition of drug resistance increases depending on the concentration of manganese, etc., so it is difficult to obtain an effect at less than 300 ppm. On the other hand, although adding more than 3000ppm will not have any negative effects on crops, the effect will reach a plateau, so the upper limit was set at 3000ppm.

In this embodiment, other components can also be blended within a range that does not affect the effect of reducing drug resistance of the present invention and within a pharmaceutically acceptable range. Examples of components that can be blended include liquid carriers, solid carriers, surfactants (emulsifiers, dispersants, antifoaming agents, etc.), adjuvants, and the like. More specifically, liquid carriers include phosphate buffers and carbonate buffers. Examples of solid carriers include natural mineral powders such as kaolin, clay, quartz, montmorillonite, and diatomaceous earth, and polymeric natural products such as crystalline cellulose, cornstarch, gelatin, and alginic acid. can be used. Examples of the surfactant include polyoxin ethylene-fatty acid ester, polyoxin ethylene-fatty alcohol ether, and the like. Examples of auxiliary agents include carboxymethyl cellulose and starch.

Various dosage forms can be selected as the dosage form of the drug, such as powder, granule, powder, powder, wettable powder, aqueous solution, emulsion, liquid, oil, aerosol, paste, and smoke. agent, fumigation agent, coating agent, microcapsule agent, pack agent, flowable agent, etc.

1. Examination of substances that reduce SM resistance or inhibit growth (1) Experimental method The test bacteria used were the peach bore bacteria (Xanthomonas arboricola pv. pruni), the citrus canker bacteria (Xanthomonas citri subsp. citri), and the plum canker bacteria (Pseudomonas syringae pv. morsprunorum), all of which are resistant to streptomycin (SM), and substances that reduce the SM resistance or inhibit the growth of these plant pathogenic bacteria were examined.

ASA _medium ( _{Moriyama et al . ,} (2016), SM-sensitive and resistant bacteria (MIC: 500 and >16000 ppm) were cultured at 25°C for 3 days in a medium mixed with SM at 0.3, 1.5, and 3.0 g/L of Mn to investigate their growth.

(2) Results Table 1 shows the measurement results of the minimum growth inhibitory concentration (ppm) of streptomycin against plant pathogenic bacteria on the ASA medium supplemented with manganese. In Table 1, "streptomycin resistant" refers to bacteria with a low MIC of MIC: 500 and >16000 ppm, to which SM is not effective, and "streptomycin sensitive" refers to bacteria with a low MIC of 15.6 ppm or less, and in which SM is generally not effective. Denotes highly susceptible bacteria. In addition, experiments were conducted on multiple bacteria with different strain numbers.

The MIC of SM-resistant bacteria on ASA medium containing SM and Mn at 3.0 g/L was 125 to 500 ppm for peach borer fungi, and further decreased from >2000 ppm to 2000 ppm for plum and citrus fungi. Mixing only Mn had no effect on the growth of the test bacteria.

These results suggest that Mn may affect the expression of genes such as strA (Scholz et al., 1989), which are involved in SM resistance.

In addition, when a similar test was conducted using Zn, ZnO, and mancozeb agent ((C4H6MnN2S4)xZny) instead of Mn, it was found that Zn and ZnO were 3.0 g/L, and mancozeb was each alone at the spray concentration (1333 ppm). The growth of all tested bacteria was inhibited regardless of SM susceptibility. Mancozeb may be effective in controlling not only filamentous fungal diseases of peaches, but also boring bacterial diseases, as well as canker diseases of plums and citrus.

2. Effect of the amount of manganese added on the resistance of streptomycin-resistant phytopathogenic bacteria (1) Experimental method As the test bacterium, peach borer fungus (Xanthomonas arboricola pv. pruni), which is resistant to streptomycin (SM), was used. The effect of the amount of manganese added on the minimum growth inhibitory concentration (ppm) of streptomycin was investigated.

ASA medium supplemented with 0.3, 1.5, and 3.0 g/L Mn was sterilized by autoclaving, and then heated to 50°C. Streptomycin was added to these media at concentrations of 1.95, 3.90, 7.80, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, and 16000 ppm to prepare plate media. Test bacteria were cultured on these plate media for 24 to 48 hours, and suspended in sterile distilled water to a concentration of approximately 10 ⁸ cfu/ml to prepare a bacterial suspension. This bacterial suspension was applied to the aforementioned plate media with a sterile cotton swab, and the media was cultured at 25°C for 3 days to examine whether the bacteria grew or not.

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

Similar experiments were conducted using peach borer fungi and citrus canker fungi. As a result, in a 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 for peach borer fungi, and the MIC for plum canker and citrus fungi was 125 to 500 ppm. It decreased from >2000 ppm to 2000 ppm.

Claims (3)

The use of manganese for the production of a drug for reducing the drug resistance of drugs containing antibiotics against microorganisms,
The microorganism is at least one selected from the group consisting of a plum canker, a peach borer, and a citrus canker,
the antibiotic is streptomycin;
Use of said manganese. A drug characterized by containing manganese as a substance that reduces the drug resistance of drugs containing antibiotics against microorganisms,
The microorganism is at least one selected from the group consisting of a plum canker, a peach borer, and a citrus canker,
the antibiotic is streptomycin;
Said drug. A method for reducing drug resistance of an antibiotic-resistant microorganism, comprising adding manganese to a drug containing an antibiotic, thereby reducing drug resistance of the microorganism,
The microorganism is at least one selected from the group consisting of Xanthomonas prunus, Bacillus orbiculare prunus, and Xanthomonas campestris prunus,
The antibiotic is streptomycin.
The method .