JP2010133975A - Method of screening compound having antimicrobial activity on pathogenic microorganism infecting organism having acquired immune mechanism by using larva of insect having only natural immune mechanism, and method of evaluating antimicrobial activity by using larva of insect having only natural immune mechanism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective model for developing antimicrobial agent for microbe infection in an organism having acquired immune mechanism, wherein the model of microbe infection using the organism having only natural immune mechanism. <P>SOLUTION: Whether the larva of a silkworm is useful or not as an antimicrobial agent evaluation system is investigated. It is found out that the larva of the silkworm is died by infection on the injection of Staphylococcus aureus or Pseudomonas aeruginosa to the larva of the silkworm. The effect of the antimicrobial agent for the larva of the silkworm is surprisingly agrees with the clinical efficacy of these agent on human i.e. an organism having acquired immune mechanism. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for screening a compound having antibacterial activity against a pathogenic microorganism infecting an organism having an acquired immune mechanism using insect larvae having only the innate immune mechanism, and to infect an organism having the acquired immune mechanism. The present invention relates to a method for evaluating antibacterial activity against pathogenic microorganisms using insect larvae having only an innate immune mechanism.

  In the development of new drugs for microbial infections, compounds extracted and purified from various biological resources including molds, radioactive bacteria, marine organisms, etc., chemical substances synthesized by organic chemical methods, or obtained from genetic engineering techniques It is indispensable to confirm the antibacterial effect and safety of these proteins (recombinant drugs) by animal experiments. Conventionally, mammals such as mice and rarely monkeys and pigs have been used as experimental animals.

However, in breeding these experimental mammals, it is necessary to satisfy a condition in which there is no infection with a defined bacterium and virus called SPF (specific pathogen-free). In addition, in order to conduct infection experiments of pathogenic microorganisms with SPF animals, a large amount of money is required for facilities and operations. Furthermore, it has been pointed out that the use of a large number of mammals for drug development is ethically problematic.
Therefore, in the development of new drugs against microbial infections, there is a need to search for experimental animals that can replace these mammals.

  By the way, in the long evolutionary history of living organisms, invasion by pathogens and defense between hosts have led to various immune mechanisms. Vertebrates, including humans, have established an “acquired immune mechanism” by molecules called antibodies that specifically recognize invaders. However, before the emergence of vertebrates, invertebrates already had an “innate immune mechanism” that does not depend on antibodies. Invertebrate insects are one of the most prosperous organisms on the planet, but they prevent invaders from entering by the “natural immunity mechanism”.

  Recent research is revealing that its molecular mechanism is in common with that of humans. For example, in response to bacterial infection, mammals and insects have a commonly conserved signal pathway for expressing defense genes. That is, in mammals, the invasion of a pathogen into the body causes NF-κB expression induction in immune cells via TOLL like receptor (Non-patent Document 1). On the other hand, even in Drosophila melanogaster adults, Dif and Relish which are homologs of NF-κB are induced through dToll and 18-wheeler which are TOLL like receptors, and the antibacterial response system is activated (Non-patent Document 2, Non-patent document 3).

  Recently, several reports have been made on infection models of pathogenic microorganisms using animals other than mammals. For example, analysis of Pseudomonas aeruginosa infection using Caenorhabditis elegans (Non-patent document 4, Non-patent document 5, Non-patent document 6), Arabidopsis thaliana (Non-patent document 7), Analysis of Legionella pheumophila infection using Dictyostelium discoideum Non-patent document 8), Caenorhabditis elegans (non-patent document 9), and Yeast (non-patent document 10) have been reported as research examples of Salmonella typhimurium infection.

However, it was completely unknown whether an infection model using organisms having only these innate immune mechanisms could be a model for microbial infection of organisms having acquired immune mechanisms. Therefore, it was also unclear whether or not an infection model using organisms having only the innate immune mechanism can be used in screening antibacterial agents for the treatment of microbial infections of organisms having acquired immune mechanisms.
In particular, with regard to Gram-positive pathogenic bacteria, there are currently no reported examples of infection models using animal individuals other than mammals. Among Gram-positive bacteria, Staphylococcus aureus is a causative bacterium of opportunistic infections in humans, and in recent years, MRSA having multidrug resistance has emerged and has become a clinical problem (Non-patent Document 11). For this reason, development of an antibacterial agent against Staphylococcus aureus is strongly desired.

Medzhitov, R. et al. 1997. Nature 388: 394-397. Lemaitre, B. et al. 1996. Cell 86: 973-983. Bernal, A., and D.A. Kimbell. 2000. Proc. Natl. Acad. Sci. USA 97: 6019-6024. Tan, M.W.et al.1999.Proc.Natl.Acad.Sci.USA.96: 715-20., Tan, M, W., et al. 1999. Proc. Natl. Acad. Sci. USA 96: 2408-13. (Mahjan-Miklos, S. et al., 1999.Cell 96: 47-56.) Reuber, T.L. et al. 1998.Plant J. 16: 473-85. Solomon, J.M.et al. 2000.Infect Immun 68: 2939-47. Aballay, A. et al. 2000. Curr Biol 10: 1539-42. Scherer, C.A. et al. 2000. Mol Microbiol. 37: 1133-45. Speller, DC et al., Lancet 350: 323-325.

  The present invention has been made in view of such a situation, and an object of the present invention is a model of microbial infection using an organism having only an innate immunity mechanism, which is a microbial infection disease in an organism having an acquired immunity mechanism. It is to provide a useful model for the development of antibacterial agents. Another object of the present invention is to provide a method for screening a compound having antibacterial activity against a pathogenic microorganism that infects an organism having an acquired immune mechanism and a method for evaluating the antibacterial activity using these infection models. . In a preferred embodiment of the present invention, an organism belonging to insects is used as the infection model. In another preferred embodiment of the present invention, an organism that can be infected with a Gram-positive pathogenic microorganism is used as the infection model. Another object of the present invention is to reduce experimental costs and experimental space in screening of antibacterial agents and evaluation of antibacterial activity.

  As a result of diligent studies to solve the above-mentioned problems, the present inventors have found that silkworms (arthropods, submaxillary subsidiary) that are quick to change generations, can be easily reared in the laboratory, and have been genetically analyzed. It was focused on the gates, insects and butterflies. Since silkworms have large larvae, C.I. Injection of pathogens and drugs is extremely easy compared to small organisms such as elegans (belonging to the linear phylum, dinosaurs, nematodes, and nematodes) (Okada, E. et al. 1997). J. Seric. Sci. Jpn. 66: 116-122.). For this reason, it is thought that it is very suitable for evaluation of the antibacterial agent with respect to a pathogen. In addition, the use of silkworm larvae is inexpensive, has no ethical problems, and is more useful than mammals.

  Therefore, the present inventors tried to develop an infection model of pathogenic microorganisms using silkworm larvae. First, the present inventors examined whether a silkworm larva was useful as an antibacterial agent evaluation system in an individual. As a result, when silk staphylococcus or Pseudomonas aeruginosa live bacteria were injected into the blood of silkworm larvae, most silkworm larvae died in a short period of time. On the other hand, when autoclaved S. aureus was injected, no silkworm larvae were found dead. When E. coli was injected, most silkworm larvae survived even 5 days after the injection. After injection of Staphylococcus aureus, blood and tissues of silkworm larvae were collected over time, and it was confirmed that Staphylococcus aureus was growing. Anti-S. Immunostaining with an aureus antibody suggested that Staphylococcus aureus was growing in the midgut epithelium. These results indicate that silkworm larvae are killed by infection by injection of S. aureus or Pseudomonas aeruginosa into silkworm larvae.

  Furthermore, infection death of silkworm larvae caused by methicillin-susceptible Staphylococcus aureus (MSSA) is suppressed by ampicillin, oxacillin and vancomycin, whereas death death of silkworm larvae caused by methicillin-resistant Staphylococcus aureus (MRSA) is caused by ampicillin and oxacillin. It was not suppressed and was suppressed with vancomycin. Moreover, the death of silkworm larvae caused by MSSA could not be suppressed by the disinfectants EtOH, benzalkonium chloride and povidone iodine. Surprisingly, this result is consistent with the effectiveness of these drugs in human clinical practice. Therefore, a system using silkworm larvae is considered to be extremely effective for screening and evaluation of new antibacterial agents as an infection model for animal individuals. The present invention is the first example in the world in which the effectiveness of an antibacterial agent against pathogenic microorganism infection in an organism having only the innate immune mechanism and the effectiveness of the antibacterial agent against pathogenic microorganism infection in an organism having an acquired immune mechanism have been shown to coincide. It is. Therefore, the present invention is also the first example in the world that has succeeded in developing a system for evaluating antibacterial activity against pathogenic microorganisms infecting organisms having acquired immune mechanisms, using organisms having only innate immune mechanisms. is there.

That is, the present invention relates to screening for compounds having antibacterial activity against pathogenic microorganisms that infect organisms having acquired immune mechanisms and evaluation of the antibacterial activity, using insect larvae having only the innate immune mechanism. Provides the following method.
(1) A method for screening a compound having antibacterial activity against a pathogenic microorganism infecting an organism having an acquired immune mechanism,
(A) a step of administering the pathogenic microorganism and the test sample to an insect larva having only an innate immune mechanism;
(B) a step of detecting the degree of survival of larvae of insects having only the innate immunity mechanism, and (c) an insect having only the innate immunity mechanism as compared with the case where the test sample is not administered (control) Selecting a compound that improves the degree of survival of a class of larvae.
(2) A method for evaluating the antibacterial activity of a test sample against a pathogenic microorganism that infects an organism having an acquired immune mechanism,
(A) administering a pathogenic microorganism and a test sample to an insect larva having only an innate immune mechanism;
(B) detecting the degree of survival of larvae of insects having only the innate immune mechanism, and (c) comparing the test sample with no natural sample (control) Determining whether to improve the degree of survival of insect larvae having only an immune mechanism.
(3) The method according to (1) or (2), wherein the organism having an acquired immune mechanism is a mammal.
(4) The method according to (3), wherein the mammal is a human.
(5) The method according to any one of (1) to (4), wherein the larva is large.
(6) The method according to any one of (1) to (5), wherein the organism belonging to insects is a silkworm.
(7) The method according to any one of (1) to (6), wherein an insect larva having only an innate immune mechanism is infected with a Gram-positive pathogenic microorganism.
(8) The method according to any one of (1) to (7), wherein the pathogenic microorganism that infects an organism having an acquired immune mechanism is a Gram-positive pathogenic microorganism.
(9) The method according to any one of (1) to (8), wherein the pathogenic microorganism that infects an organism having an acquired immune mechanism is a causative bacterium of an opportunistic infection.
(10) Any of (1) to (9), wherein the pathogenic microorganism infecting an organism having an acquired immune mechanism is a pathogenic microorganism selected from the group consisting of Staphylococcus aureus, green bacterium, Vibrio cholerae and pathogenic Escherichia coli The method of crab.

  The present invention is a method for screening a compound having antibacterial activity against a pathogenic microorganism infecting an organism having an acquired immune mechanism using an organism having innate immunity, and evaluating the antibacterial activity using an organism having innate immunity. Provide a way to do it.

  In the method of the present invention, first, a pathogenic microorganism and a test sample are administered to an organism having only an innate immune mechanism (step (a)). In the present invention, the “innate immunity mechanism” means an immune defense mechanism (innate immunity mechanism) that does not depend on an acquired immunity (acquired immunity) mechanism. Vertebrates have an acquired immune mechanism that protects the living body using molecules that specifically recognize invaders such as antibodies against the invasion of pathogens.Invertebrates and plants have such an acquired immune mechanism. do not do.

  In the present invention, the organism to which the pathogenic microorganism is administered is an organism belonging to insects. In the present invention, the term “insects” refers to a class of arthropods of the Greater Submaxillary, which is composed of four subclasses of caterpillars, fly insects, wormless insects, and rodent insects. . There is no restriction | limiting in particular as an organism which belongs to the insects used for this invention. It is a larva for convenience of handling. Examples of the larva include, but are not limited to, Lepidoptera (including moths and butterflies) and Coleoptera (including beetles). From the viewpoint of easy administration of pathogenic microorganisms and test samples, the larvae are preferably large. In the present invention, the term “large larva” refers to a larva having a body length of 1 cm or more.

  Organisms that have only the innate immune mechanism include those that are infected by Gram negative bacteria, those that are infected by Gram positive bacteria, and those that are infected by both. Currently, development of therapeutic agents for human infections caused by Gram-positive bacteria such as Staphylococcus aureus is desired. In the development of such therapeutic agents, those infected by Gram-positive bacteria are used. The silkworm larvae used in this example are infected not only by gram-negative bacteria but also by staphylococcus aureus, which is a gram-positive bacterium, and thus can be suitably used for the development of these antibacterial agents.

  In the present invention, “pathogenic microorganism” means a microorganism having an ability to infect a host and cause a disease. In the present invention, a pathogenic microorganism that infects at least one organism having an acquired immune mechanism is used. From the viewpoint of developing antibacterial agents against microbial infections in humans, it is preferable to use those that infect humans as pathogenic microorganisms. Pathogenic microorganisms include both gram negative and gram positive bacteria. Examples of gram-negative bacteria applicable to the present invention include Pseudomonas aeruginosa, Vibrio cholerae, and pathogenic E. coli (O-157), and examples of gram-positive bacteria include Staphylococcus aureus. It is not limited to.

  The test sample to be administered to the host organism is not particularly limited, and a desired sample to be evaluated for antibacterial activity is used. Examples of test samples include cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptidic compounds, synthetic low molecular compounds, natural Examples thereof include, but are not limited to, compounds.

  Administration of the pathogenic microorganism and the test sample to the host can be performed by, for example, intraperitoneal administration, injection into blood, addition to feed (food), injection into the intestine, and the like.

  The dose of the pathogenic microorganism or the test sample to the host varies depending on the pathogenic microorganism, the host, the type of the test sample, and the like. In general, pathogenic microorganisms are administered in a diluted solution from the highest density cultivatable bacterial solution to about 1 / 10,000. When using a larva of an organism belonging to insects as a host, for example, about 0.05 ml of a bacterial solution may be injected into the blood from the leg. For the test sample, the minimum amount that kills the host to be used is determined, and the lower amount is administered. A person skilled in the art will be able to select an appropriate dose according to the pathogenic microorganism, the host, the type of test sample, and the like.

In the present invention, the infectious symptoms or the degree of survival of the organism having the innate immune mechanism to which the pathogenic microorganism and the test sample are administered is then detected (step (b)).
Infectious symptoms to be detected include, for example, [1] increase in the number of pathogenic microorganisms in the host individual, [2] decrease in host weight or inhibition of increase in host weight, [3] antibacterial substances in host blood Examples include reduction of the amount, [4] failure of the host immune function, and [5] reduction of various enzyme activities in the body fluid and body organ of the host. If the host is an insect larva, for example, it may be detected that it does not molt into an old larva or become a pupa or adult. In the present invention, the degree of survival of the host may be detected in addition to the above infection symptoms. Examples of the degree of survival include survival rate and survival period.

  In the screening of the compound having antibacterial activity of the present invention, the infectious symptoms of the organism having the innate immune mechanism are improved or the degree of survival is improved as compared with the case where the test sample is not administered (control). The compound to be selected is selected (step (c)). On the other hand, in the method for evaluating antibacterial activity of the present invention, whether the test sample improves the infectious symptoms of the organism having the innate immune mechanism as compared with the case where the test sample is not administered (control). It is determined whether or not the degree of survival is improved (step (c)).

  If the administered test sample improves the infection symptoms of the host organism or improves the degree of survival compared to the control, the test sample has antibacterial activity against pathogenic microorganisms administered to the host organism. On the other hand, if the administered test sample does not improve the infectious symptoms of the host organism or improve the degree of survival compared to the control, the test sample is It can be determined that it has no antibacterial activity against pathogenic microorganisms administered to the host organism. Samples determined to have antibacterial activity are potential candidates for antibacterial agents against pathogenic microorganisms administered to the host organism.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to a following example.

  The Staphylococcus aureus RN4220 used in this example was distributed by Dr. Juntendo Hiramatsu. Staphylococcus aureus MSSA and MRSA used clinical isolates at Kyushu University Hospital (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother 43: 3042-3043.). The Staphylococcus aureus Smith strain and the Escherichia coli NIHJ strain were provided by Dr. Hamada from the Institute for Microbial Chemistry. Laboratory-preserved strains were used for Pseudomonas aeruginosa (S24), Escherichia coli W3110 strain and K12-3 strain. Staphylococcus aureus was cultured on an agar medium in Mannit salt medium (Eiken Chemical Co., Ltd.), Pseudomonas aeruginosa on NAC medium (Eiken Chemical Co., Ltd.), and DOC medium (Eiken Chemical Co., Ltd.). Single colonies of these bacteria were cultured overnight in LB liquid medium and used.

  In addition, fertilized eggs of silkworm larvae were purchased from Memoto Sericulture Industry Co., Ltd. and fed with artificial feed (Silk Mate: Nippon Sericulture Industry Co., Ltd.) at room temperature.

[Example 1]
Infection and death of silkworm larvae caused by Staphylococcus aureus and Pseudomonas aeruginosa Staphylococcus aureus and Pseudomonas aeruginosa are causative organisms for human opportunistic infections. The present inventors examined whether or not these bacteria killed silkworm larvae.
0.05 ml of a bacterial solution or antibacterial solution of Staphylococcus aureus and Pseudomonas aeruginosa was injected into the first abdominal leg of a 5-year-old silkworm larva, and hemostasis was carried out for 10 seconds by finger pressure. The injection needle used was 27G x 3/4 (Terumo Corporation), and the syringe used 1 ml (Terumo Corporation). The number of surviving individuals over time after injection was examined (FIG. 1).

When a dilution of medium without bacteria was injected, all individuals survived for more than 5 days. In contrast, when 3 × 10 ⁷ Staphylococcus aureus were injected, 90% or more of silkworm larvae died within 2 days after injection in any of the four strains (RN4220, Smith, MSSA, MRSA) (FIG. 1). . The silkworm larvae injected with Staphylococcus aureus gradually ceased to eat after the injection, moved slowly, and died on the second day with a light brown epidermis. On the other hand, the survival rate of silkworm larvae when injected with autoclaved S. aureus (3 × 10 ⁸ cells) was 80% or more even after 5 days from injection (data not shown). The results of investigating 20 lines of clinically isolated MRSA (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother 43: 3042-3043.) Showed no particularly toxic strain compared to MSSA. There was a difference in the number of cells required to cause infection death (data not shown). Furthermore, when the number of S. aureus (MSSA) to be injected was 3 × 10 ⁶ cells, all individuals died 5 days after the injection, but in 3 × 10 ⁵ cells, the survival rate after 5 days from the injection was 50%. (Data not shown). On the other hand, when 3 × 10 ⁷ Pseudomonas aeruginosa (S24) were injected, all individuals died one day later (FIG. 1). Silkworm larvae injected with Pseudomonas aeruginosa also ceased to eat food after the injection and died with a black epidermis. On the other hand, when 3 × 10 ⁷ Escherichia coli were injected, the survival rate after 5 days of injection in any of the three strains examined (NIHJ, K12-3, W3110) was 90% or more, It did not show pathogenicity.

Next, it was examined whether the injected S. aureus was growing in the silkworm larvae. The number of bacteria in the silkworm larvae was measured as follows.
Cut the tail leg of silkworm larvae injected with fungus, collect body fluid, dilute with 0.6% NaCl, apply on Mannit medium (Eiken Chemical Co., Ltd.), and culture at 37 ° C overnight Thereafter, the number of colonies that appeared was counted, and the number of bacteria in the silkworm body fluid was calculated. For fungi in the tissue, silkworm larvae were opened on a paper towel, and after removing body fluids, suspended in 0.9% NaCl, applied onto mannitol medium, and the number of colonies that appeared was counted. The number of bacteria in was calculated. The body fluid and tissue volume of silkworm larvae were calculated as 1.5 ml and 1 ml, respectively.

As a result, a significant increase in the number of bacteria after injection was observed in both silkworm larva tissues and body fluids (FIG. 2), and the total number of bacteria in silkworms reached 1 × 10 ^{8 on the} second day after injection.

Next, the growth site of Staphylococcus aureus in the silkworm larvae tissue was examined using a fluorescent antibody method against a paraffin tissue section. Silkworm larvae were fixed with Carnoy's solution (EtOH: chloroform: CH ₃ COOH = 6: 3: 1) at room temperature for 10 hours. Thereafter, it was embedded in paraffin and sliced with a thickness of 10 μm. After the deparaffinized tissue section was subjected to a blocking operation with 1% BSA (Sigma), S. aureus monoclonal antibody (15702 QED Bioscience Inc.) was reacted. Thereafter, the sample was washed three times with PBS and reacted with a fluorescently labeled secondary antibody (FluoroLink ^™ Cy ^™ 2 labeled goat anti-mouse IgG (H + L), Amersham). Further, the sample was washed 3 times with PBS and observed with a fluorescence microscope (Olympus BH2).

  FIG. 3 is a histological image obtained by cutting the midgut of the silkworm larvae 40 hours after injection with Staphylococcus aureus perpendicularly to the body axis. In the silkworm larvae injected with S. aureus, clear fluorescence was observed in the midgut epithelium. This fluorescence was not observed in silkworm larvae that were not injected with S. aureus (FIG. 3A) or when no primary antibody was used (data not shown). The above results suggest that the death of silkworm larvae due to Staphylococcus aureus is the growth of Staphylococcus aureus in silkworm larvae body fluid and the death of infection due to invasion and proliferation of midgut tissue.

[Example 2]
Effects of Antibiotics and Disinfectants on Infection Death of Silkworm Larvae Caused by Staphylococcus aureus Furthermore, the present inventors investigated whether or not infection death of silkworm larvae caused by Staphylococcus aureus can be suppressed by antibiotics. The clinical isolates of S. aureus were injected into silkworm larvae, then ampicillin, oxacillin and vancomycin were injected, and the number of surviving silkworm larvae was counted over time.

  When injected with MSSA alone, all silkworm larvae died after 2 days. At this time, when ampicillin (200 μg / body), oxacillin (200 μg / body), and vancomycin (200 μg / body) were injected, the survival rate was 90% or more even on the fourth day of injection (FIG. 4A). That is, all three antibiotics suppressed the death of silkworm larvae caused by MSSA.

  When MRSA alone was injected, 90% silkworm larvae died after 1 day. At this time, even when ampicillin (200 μg / body) and oxacillin (200 μg / body) were injected, all silkworm larvae died on the second day of injection. In contrast, when vancomycin (200 μg / body) was injected, the survival rate was 80% or more even on the fourth day of injection (FIG. 4B). Therefore, it was found that the death of silkworm larvae caused by MRSA cannot be suppressed by ampicillin or oxacillin but can be suppressed by vancomycin.

Furthermore, it was investigated whether the death of silkworm larvae caused by MSSA and MRSA can be suppressed by various disinfectants. First, the MIC values for S. aureus and LD ₅₀ values for silkworm larvae in agar media of various disinfectants were compared (Table 1).

As a result, it was found that EtOH and povidone iodine had lower LD ₅₀ values than MIC and could not be expected to have a therapeutic effect. On the other hand, the MIC value of benzalkonium chloride was smaller than the LD ₅₀ value, but the death of silkworm larvae caused by MSSA was not suppressed even by an amount of benzalkonium chloride close to LD ₅₀ . That is, it has been found that treatment with a disinfectant is not effective for the death of silkworm larvae caused by Staphylococcus aureus.

In addition, ampicillin (Ashiyu Pharmaceutical Co., Ltd.), oxacillin (Sigma), vancomycin (Shionogi Pharmaceutical Co., Ltd.), benzalkonium chloride (Yoshida Pharmaceutical Co., Ltd.), and povidone iodine (Meiji Seika Co., Ltd.) used in this experiment And diluted with 0.6% NaCl. The MIC against Staphylococcus aureus for each of the antimicrobial substance, diluted overnight culture of bacteria on LB10 agar medium containing antimicrobial agent of various concentrations ^(1x10 3 cells / ml) in 1 of 10 ³ minutes, the 1 μl was spread with ase and cultured at 37 ° C. for 72 hours (Akimitsu, N., et al. 1999. Antimicrob Agents Chemother. 43: 3042-3043.). In the case of obtaining the IC ₅₀ for the infected silkworm larvae, 5 × 10 ⁶ S. aureus were injected into the silkworm larvae blood and then 0.05 ml of an antibacterial substance solution having various concentrations was injected. The concentration of the antibacterial substance was calculated so that the number of surviving individuals became half after 4 days from the injection. The IC ₅₀ value was calculated by setting the body fluid of silkworm larvae to 1.5 ml. The LD ₅₀ for silkworm larvae was injected at a concentration at which half of the larvae were killed on the first day after injection by injecting 0.05 ml of an antibacterial solution of various concentrations into 5 larvae.

  According to the present invention, a model of pathogenic microorganism infection for an organism having an acquired immune mechanism using an organism having only an innate immune mechanism is provided. The effect of antibacterial agents using the infection model of the present invention is expected to correspond to the effectiveness of these agents in organisms with acquired immune mechanisms. Therefore, the infection model of the present invention can be an infection model of various pathogens against organisms having acquired immune mechanisms including humans, and is useful for screening antibacterial agents for infections caused by these pathogens. By using the infection model of the present invention as a pre-stage of infection experiments of pathogenic microorganisms using mammals, it can be expected to contribute to the efficiency of the development of antibacterial agents that can be applied to human clinical applications. In particular, the silkworm infection model is effective in developing antibacterial agents against opportunistic infections caused by Staphylococcus aureus, for example, because it is infected by Gram-positive pathogenic bacteria.

In addition, when the infection model of the present invention is used, in the screening of drugs, unlike the case of using conventional mammals, it is possible to greatly reduce the acquisition cost, breeding cost, and experimental space per individual. . For example, the space required to raise 1000 mice in an SPF environment ([1] filter air and [2] keep temperature and humidity constant) is approximately 25 m ² In addition to this, backup facilities such as a cage cleaning room and an autoclave room are required. On the other hand, with silkworm larvae, if an incubator is installed at 1 m ² , 1000-10000 animals (depending on the age) can be raised, and in addition to keeping the temperature constant (eg, 30 ° C.), cage washing No special backup equipment such as a room or autoclave room is required (the number of animals, the required number of areas, etc. is an approximate number and varies depending on various conditions).

The silkworm infection model of the present invention is C.I. Compared with small organisms such as elegans, it is extremely easy to inject pathogens and drugs, and can be said to be suitable for evaluation of antibacterial agents against pathogens.
Invertebrates and plants including silkworms do not have antibody-mediated immune immunity, but have innate immune immunity in common with humans. The infection model of the present invention is also effective for elucidating the innate immune mechanism against pathogen infection at the molecular level using genetic techniques.

It is a figure which shows the survival rate of the silkworm larva injected with Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. An overnight culture of Staphylococcus aureus (RN4220, Smith, MSSA, MRSA), Pseudomonas aeruginosa (S24), Escherichia coli (K12-3, W3110, NIHJ) was diluted 10-fold with 0.6% NaCl. .05 ml (3 × 10 ⁷ cells) was injected into the blood of 10 silkworm 5th instar larvae. Surviving individuals were counted over time. It is a figure which shows the proliferation of Staphylococcus aureus in the silkworm larva body. An overnight culture of Staphylococcus aureus (MSSA) was diluted 10-fold with 0.6% NaCl, 0.05 ml (3 × 10 ⁷ cells) was injected into silkworm larvae, and body fluid of silkworm larvae over time (A) The tissue (B) was collected, suspended in 0.6% NaCl, applied to a mannitol medium, cultured at 37 ° C. overnight, and the number of colonies that appeared was counted. It is a microscope picture which shows the localization of Staphylococcus aureus in the midgut of a silkworm larva. 0.9% NaCl (A) or Staphylococcus aureus (MSSA) (3 × 10 ⁷ cells) (B) was injected into silkworm larvae, and after 40 hours, paraffin-embedded tissue sections of the midgut were prepared, and S. aureus antibodies Indirect fluorescent antibody method was performed. The left side of the figure is the body surface side, and the right side is the inside of the intestine. It is a figure which shows the effect of various antibiotics with respect to the infection death of the silkworm larva by Staphylococcus aureus. Staphylococcus aureus (3 × 10 ⁷ cells / 0.05 ml) was injected into 10 silkworm larvae, followed by antibiotics (0.2 mg / 0.05 ml), and the number of surviving individuals was counted over time. Ampicillin, oxacillin and vancomycin were used as antibiotics. A, MSSA B, MRSA.

Claims (10)

A method for screening a compound having antibacterial activity against a pathogenic microorganism infecting an organism having an acquired immune mechanism,
(A) a step of administering the pathogenic microorganism and the test sample to an insect larva having only an innate immune mechanism;
(B) a step of detecting the degree of survival of larvae of insects having only the innate immunity mechanism, and (c) an insect having only the innate immunity mechanism as compared with the case where the test sample is not administered (control) Selecting a compound that improves the degree of survival of a class of larvae. A method for evaluating the antibacterial activity of a test sample against a pathogenic microorganism infecting an organism having an acquired immune mechanism,
(A) administering a pathogenic microorganism and a test sample to an insect larva having only an innate immune mechanism;
(B) detecting the degree of survival of larvae of insects having only the innate immune mechanism, and (c) comparing the test sample with no natural sample (control) Determining whether to improve the degree of survival of insect larvae having only an immune mechanism.   The method according to claim 1 or 2, wherein the organism having an acquired immune mechanism is a mammal.   4. The method according to claim 3, wherein the mammal is a human.   The method according to any one of claims 1 to 4, wherein the larva is large.   The method according to any one of claims 1 to 5, wherein the organism belonging to insects is a silkworm.   The method according to any one of claims 1 to 6, wherein an insect larva having only an innate immune mechanism is infected by a Gram-positive pathogenic microorganism.   The method according to claim 1, wherein the pathogenic microorganism that infects an organism having an acquired immune mechanism is a Gram-positive pathogenic microorganism.   The method according to any one of claims 1 to 8, wherein the pathogenic microorganism that infects an organism having an acquired immune mechanism is a causative bacterium of an opportunistic infection.   The method according to any one of claims 1 to 9, wherein the pathogenic microorganism infecting an organism having an acquired immune mechanism is a pathogenic microorganism selected from the group consisting of Staphylococcus aureus, green bacterium, Vibrio cholerae and pathogenic Escherichia coli. .