JP2008148669A - Medicine for preventing or treating infectious disease, and method for producing the same and evaluation method and screening method for the same and method for evaluating pathogenesis of pathogenic bacterium and method for detecting infectious disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective evaluation method and screening method of a substance having action lowering pathogenesis of pathogenic bacterium having gliding ability, to provide an effective medicine for prophylaxis and treatment of infectious diseases caused by the pathogenic bacterium, and to provide a method for producing the medicine and to provide a method for effectively evaluating pathogenesis of the pathogenic bacterium and a method for detecting infectious diseases caused by the pathogenic bacterium. <P>SOLUTION: The present invention provides an evaluation method and a screening method of a substance, having action for lowering pathogenesis of the pathogenic bacterium by using lowering of gliding activity of a pathogenic bacteria, having gliding ability as index and provides a medicine for prophylaxis and treatment of infectious diseases caused by the pathogenic bacterium, containing a substance having action lowering gliding activity of pathogenic bacteria having gliding ability as an active ingredient and provides a method for producing medication and provides an evaluation method of pathogenesis of the pathogenic bacteria, comprising evaluating the gliding activity of pathogenic bacteria having gliding ability, and a method for detecting infectious diseases caused by the pathogenic bacteria. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is a method for evaluating and screening a substance having an action of reducing the pathogenicity of the pathogenic bacterium, utilizing the inventors' new knowledge about the pathogenicity of the pathogenic bacterium having gliding ability, The present invention relates to a drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria, a method for producing the same, a method for evaluating the pathogenicity of the pathogenic bacteria, and a method for examining an infectious disease caused by the pathogenic bacteria.

For the treatment of infectious diseases caused by pathogenic bacteria, chemotherapy by administration of drugs such as antibiotics is usually performed. However, the conventional use of drugs has caused a problem that pathogenic bacteria have acquired the ability to detoxify these drugs, and resistant bacteria that do not work with the drugs have emerged. In fact, the presence of many drug-resistant bacteria is a problem, especially in the medical field.
In particular, Staphylococcus aureus is known as a causative bacterium of purulent disease, pneumonia, food poisoning, etc., but has acquired resistance to many drugs such as the antibiotic methicillin, which is a methicillin-resistant Staphylococcus aureus (MRSA). The emergence is a major clinical problem. This MRSA is almost harmless to normal healthy people, but it causes infection in the state of reduced resistance, and once it develops, it is resistant to many antibiotics. Is difficult and can lead to death.

Currently, vancomycin, teicoplanin, arbekacin, linezolid and the like are used as typical therapeutic agents for MRSA, but it is generally considered difficult to completely eliminate MRSA. Regarding vancomycin, the appearance of vancomycin-resistant Staphylococcus aureus (VRSA) has already been reported, and it is said that sufficient caution is required for its use.
Furthermore, new drugs have been widely developed for MRSA. For example, a novel antibiotic obtained from a culture solution of actinomycetes belonging to the genus Kineosporia has been proposed ( It is considered that such a new drug still has a risk of causing new resistant bacteria by its use.
Thus, the problem of the emergence of new resistant bacteria against the use of new drugs is the so-called “Weasel Play” state, and there is a long-awaited desire to fundamentally overcome such problems.

JP 2006-176406 A

  An object of the present invention is to solve the above problems and achieve the following object. That is, the present invention can effectively prevent or treat infectious diseases caused by pathogenic bacteria having gliding ability such as Staphylococcus aureus, and does not inhibit the growth of the pathogenic bacteria itself, An efficient evaluation method and screening method for a substance having an action of reducing the pathogenicity of a pathogenic bacterium having a gliding ability, which is useful for developing a drug that is unlikely to cause resistance to pathogenic bacteria. To provide an effective drug for the prevention or treatment of infectious diseases caused by bacterial bacteria and a method for producing the same, and to quantitatively evaluate the level of pathogenicity of the pathogenic bacteria present in a patient, for example This makes it possible to accurately predict whether a patient's symptoms will be serious, and to select a more appropriate treatment method. And to provide a method and an inspection method of infections caused by the pathogenic bacteria.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, it is a finding that there is a close relationship between the gliding activity of pathogenic bacteria having gliding ability such as Staphylococcus aureus and the pathogenicity of the pathogenic bacteria. In recent years, the fact that Staphylococcus aureus has gliding ability was first discovered by the present inventors (for example, 78th Annual Meeting of the Japanese Biochemical Society (2005), Kaito et al., 12th International Symposium on Staphylococcal Infections, 3-6 September 2006, Maastricht, The Netherlands, Poster number: P028), the present inventors have also reported that the level of gliding activity of S. aureus, the level of pathogenicity (for example, the level of hemolytic activity) It was found that there is a close relationship between them (see Examples, later). By utilizing this new knowledge, the present inventors can provide a method for developing a new drug against an infectious disease caused by the pathogenic bacterium, and a new infectious disease caused by the pathogenic bacterium. The present invention has been completed.

Conventionally, the search for drugs for infectious diseases has been mainly performed by using whether or not the candidate drug inhibits the growth of the target pathogenic bacteria as an index. Is capable of effectively preventing or treating infectious diseases, but inhibits the growth of the pathogenic bacteria itself, so that there is a high risk that new resistant bacteria will appear as described above. there were.
In addition, the conventional method for infectious disease testing is to quantitatively evaluate the level of pathogenicity of pathogenic bacteria in a patient affected by the infection and accurately predict whether or not the patient's symptoms are serious. It was difficult.

On the other hand, using the above knowledge of the present inventors that the level of pathogenicity of pathogenic bacteria having gliding ability has a close relationship with the level of gliding activity of the pathogenic bacteria, It becomes possible to screen the active ingredient of a drug against an infectious disease caused by the pathogenic bacterium by using a decrease in the gliding activity of the pathogenic bacterium as an index, for example, without inhibiting the growth of the pathogenic bacterium. It is also possible to obtain a drug that reduces only the pathogenicity of the pathogenic bacteria. Such a drug can effectively prevent or treat an infectious disease, and does not inhibit the growth of the pathogenic bacterium, so that it can be said that the drug is less likely to cause resistant bacteria. In other words, by using the above knowledge, it is possible to develop a new drug for infectious diseases based on a completely different principle.
Further, using the above findings by the present inventors, for example, the pathogenicity of the pathogenic bacteria present in the patient's body with the level of gliding activity of the pathogenic bacteria isolated from a patient suffering from an infection as an index Can be quantitatively evaluated. This makes it possible to accurately predict whether or not the patient's symptoms are serious, and to select a more appropriate treatment method. That is, if the above knowledge is used, it is possible to provide a new inspection method for infectious diseases that is completely different from the conventional one.

The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A method for evaluating whether a test substance has an action of reducing pathogenicity of pathogenic bacteria having gliding ability,
(A) It is a method characterized by including the process of evaluating whether the said to-be-tested substance reduces the sliding activity of the pathogenic bacteria which have the said sliding ability.
<2> A method for screening a substance having an action of reducing pathogenicity of pathogenic bacteria having gliding ability,
(A) a step of evaluating whether the test substance reduces the gliding activity of the pathogenic bacteria having the gliding ability; and
(B) It is a method characterized by including the process of selecting the substance evaluated as reducing the gliding activity of the pathogenic bacteria which have the said gliding ability in the said process (a).
<3> A method for producing a drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability,
(A) a step of evaluating whether or not the test substance reduces the gliding activity of the pathogenic bacterium having the gliding ability,
(B) a step of selecting a substance evaluated to reduce the gliding activity of the pathogenic bacterium having the gliding ability in the step (a);
(C) generating the substance selected in step (b), and
(D) A production method comprising the step of mixing the substance produced in the step (c) with a pharmaceutically acceptable carrier.
<4> A drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability, comprising as an active ingredient a substance having an action of reducing the gliding activity of the pathogenic bacteria having gliding ability It is a drug characterized by this.
<5> A method for evaluating the pathogenicity of pathogenic bacteria having gliding ability,
(A ′) a method comprising evaluating a gliding activity of a pathogenic bacterium having the gliding ability.
<6> A test method for infectious diseases caused by pathogenic bacteria having gliding ability,
(A ″) a step of collecting a test sample from the test sample;
(B ″) separating the pathogenic bacteria having the gliding ability from the test sample; and
(C ″) a method comprising evaluating the gliding activity of the isolated pathogenic bacterium having the gliding ability.

  According to the present invention, the above-mentioned problems can be solved and the above-mentioned object can be achieved. For example, infectious diseases caused by pathogenic bacteria having gliding ability such as Staphylococcus aureus can be effectively prevented or treated. , Reduce the pathogenicity of pathogenic bacteria having gliding ability, useful for development of drugs that do not inhibit the growth of the pathogenic bacteria themselves, that is, drugs that are unlikely to cause resistance to the pathogenic bacteria To provide an effective evaluation method and screening method for substances having an action, an effective drug for the prevention or treatment of infectious diseases caused by the pathogenic bacteria, and a method for producing the same, and for example, in a patient body It is possible to quantitatively evaluate the level of pathogenicity of the pathogenic bacteria present, thereby accurately predicting the severity of the patient's symptoms and selecting a more appropriate treatment method. To enable inspection method of efficiently the pathogenic pathogenic bacteria evaluation method and infections caused by the pathogenic bacteria can be provided.

(Evaluation method, screening method)
The evaluation method of the present invention is a method for evaluating whether or not a test substance has an action of reducing the pathogenicity of pathogenic bacteria having gliding ability, and includes the following step (a), and if necessary And other steps.
Moreover, the screening method of the present invention is a method for screening a substance having an action of reducing the pathogenicity of pathogenic bacteria having gliding ability, and includes the following steps (a) to (b), as necessary. And other steps.
The evaluation method and the screening method (hereinafter sometimes simply referred to as “method”) include the evaluation or screening of a substance having an action of reducing the pathogenicity of pathogenic bacteria having gliding ability. It is characterized by performing as an index whether to reduce the gliding activity of pathogenic bacteria having gliding ability.

<Process (a)>
In the evaluation method and the screening method, first, it is evaluated whether or not the test substance reduces the gliding activity of the pathogenic bacterium having the gliding ability (step (a)).

-Test substance-
The “test substance” is not particularly limited, and any substance for which it is desired to evaluate whether or not it has an action of reducing the pathogenicity of the pathogenic bacteria having the gliding ability can be used. Product, cell culture supernatant, microbial product, marine organism extract, plant extract, purified protein, crude protein, peptide, non-peptidic compound, synthetic compound, natural compound, existing antibiotic, existing synthetic antibacterial drug Etc.

-Pathogenic bacteria with gliding ability-
The “sliding ability” refers to the ability of bacteria to slide on the surface of a soft agar medium and move by itself, and the “pathogenicity” refers to pathogens such as bacteria infecting other organisms. It refers to the nature that can cause infection. The soft agar medium is an agar medium (for example, containing about 0.24% agar) containing agar at a lower concentration than a normal agar medium (for example, containing about 1.5% agar). Say. Therefore, the “pathogenic bacterium having gliding ability” is particularly a bacterium that has the ability to glide on the surface of the soft agar medium and has the property of infecting other organisms to cause infection. limit is not, can be appropriately selected depending on the intended purpose, e.g., Staphylococcus aureus (Staphylococcus aureus), etc. staphylococci (Staphylococcus), Mycobacterium smegmatis (Mycobacterium smegmatis) Mycobacterium such as (mycobacteria), Streptococcus Mireri (Streptococcus milleri) such as Streptococcus (streptococci), Bacillus subtilis (Bacillus subtilis), E. coli (Escherichia coli) Cholera (Vibrio cholerae), Serratia (Serratia marcescens), and the like. The fact that the S. aureus has the gliding ability is a fact found for the first time by the present inventors in recent years (for example, the 78th Annual Meeting of the Japanese Biochemical Society (2005), Kaito et al., 12th). International Symposium on Staphylococci & Staphylococcal Infections, 3-6 September 2006, Maastricht, The Netherlands, Poster number: P028).

-Evaluation of whether or not to reduce the gliding activity of pathogenic bacteria with gliding ability-
The method for evaluating whether the test substance reduces the gliding activity of the pathogenic bacterium having the gliding ability is not particularly limited and can be appropriately selected depending on the purpose. For example, a soft agar medium Bacterial solid culture technology using can be used. For example, as shown in FIG. 1, a pathogenic bacterium having a high gliding activity (RN4220 in FIG. 1) slides widely on the surface of a soft agar medium during culturing, and shows a widely spread colony state. On the other hand, pathogenic bacteria having low sliding activity (NI-6 to 10 in FIG. 1) show a small colony state without sliding too much on the surface of the soft agar medium. Therefore, for example, when the pathogenic bacterium having the gliding ability is cultured on a soft agar medium under certain conditions except for the presence / absence of the test substance, in the presence of the test substance, When the colony state of the pathogenic bacterium having the sliding ability is small as compared with the absence of the test substance, the test substance is evaluated to reduce the sliding activity of the pathogenic bacterium having the sliding ability. be able to.

As described above, the evaluation method of the present invention can be performed by the step (a).
In the step (a), when the test substance is evaluated to reduce the gliding activity of the pathogenic bacteria having the gliding ability, the test substance has the pathogenicity of the pathogenic bacteria having the gliding ability. It can be evaluated that it has the effect | action to reduce. The type of pathogenicity of the pathogenic bacterium evaluated here is not particularly limited and can be appropriately selected according to the purpose. For example, it is determined using red blood cells such as sheep, rabbits and humans. Hemolysis activity, ability to kill organisms, growth of bacteria in organisms, fever and inflammation, enhancement of immune system determined by increase in white blood cell count, reduction of various organ functions, and the like.
According to the evaluation method, it is possible to efficiently evaluate whether or not the test substance is a substance having an action of reducing the pathogenicity of pathogenic bacteria having gliding ability.

<Step (b)>
In the screening method, a substance evaluated to reduce the gliding activity of the pathogenic bacterium having the gliding ability in the step (a) is further selected (step (b)).
When the evaluation by the step (a) is performed using various test substances, and then, in this step (b), the gliding activity of the pathogenic bacterium having the gliding ability is reduced from the various test substances. By selecting the evaluated substance, a substance having an action of reducing the pathogenicity of the pathogenic bacterium having the gliding ability can be efficiently screened.

  The substance having the action of reducing the pathogenicity of pathogenic bacteria having gliding ability evaluated or screened by the above method can be appropriately generated by a method such as chemical synthesis or separation / purification. The use of the substance is not particularly limited and may be appropriately selected depending on the purpose.For example, it is used as it is for the prevention or treatment of infectious diseases caused by the pathogenic bacteria having the gliding ability. Alternatively, it may be used for basic experiments in the technical field. Moreover, you may use as active ingredients, such as the chemical | medical agent manufactured by the manufacturing method of this invention mentioned later, and the chemical | medical agent of this invention mentioned later.

(Drug manufacturing method)
The method for producing a drug of the present invention (hereinafter sometimes simply referred to as “manufacturing method”) is a method for producing a drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability. Steps (a) to (d) are included, and other steps are further included as necessary.

<Process (a), process (b)>
Step (a) and step (b) in the production method are the same as step (a) and step (b) in the evaluation method and screening method of the present invention described above, respectively. By the steps (a) to (b), a substance having an action of reducing the pathogenicity of the pathogenic bacterium having the gliding ability can be efficiently selected.

<Step (c)>
Next, in the production method, a substance having an action of reducing the pathogenicity of the pathogenic bacterium having the gliding ability selected in the steps (a) to (b) is generated (step (c)).
There is no restriction | limiting in particular as said production | generation means, For example, according to the structure of the said substance, origin, etc., it can select suitably from well-known production | generation means, such as chemical synthesis and isolation | separation purification.

<Step (d)>
In the production method, the substance produced in the step (c) is then mixed with a pharmaceutically acceptable carrier (step (d)).
-Pharmaceutically acceptable carrier-
The pharmaceutically acceptable carrier is not particularly limited and may be appropriately selected depending on, for example, the desired dosage form of the drug to be produced. Moreover, there is no restriction | limiting in particular as said dosage form, For example, the dosage form enumerated by the item of the chemical | medical agent of this invention mentioned later etc. are mentioned.

-Mixed-
The mixing method of the substance produced in the step (c) and the pharmaceutically acceptable carrier is not particularly limited, and may be appropriately selected from, for example, the mixing method of each component in a known pharmaceutical production method. Can do.
Moreover, there is no restriction | limiting in particular as a usage-amount ratio of the said substance and the said pharmaceutically acceptable carrier at the time of the said mixing, According to the objective, it can select suitably.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, the shaping | molding process etc. which shape | mold the mixture obtained at the said process (d) are mentioned.

As mentioned above, the manufacturing method of the chemical | medical agent of this invention can be performed by the said process (a)-process (d), and, thereby, for the prevention or treatment of the infectious disease resulting from the said pathogenic bacteria which have the said gliding ability. The drug can be produced efficiently.
There is no restriction | limiting in particular as a usage form of the chemical | medical agent obtained by the said manufacturing method, According to the objective, it can select suitably, For example, it can use similarly to the chemical | medical agent of this invention mentioned later.

(Drug)
The drug of the present invention is a drug for prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability, and an active ingredient is a substance having an action of reducing the gliding activity of the pathogenic bacteria having gliding ability And other components as necessary. The drug may be a drug manufactured by the above-described manufacturing method of the present invention.

-Substance (active ingredient) having an action of reducing the gliding activity of pathogenic bacteria having gliding ability-
The “pathogenic bacterium having gliding ability” is as described in the item of the evaluation method and screening method of the present invention described above. As shown in the examples described later, there is a close relationship between the high gliding activity of the pathogenic bacteria and the high pathogenicity of the pathogenic bacteria. Therefore, the substance having the action of reducing the gliding activity of the pathogenic bacterium having the gliding ability can reduce the pathogenicity of the pathogenic bacterium having the gliding ability, and the infectious disease caused by the pathogenic bacterium. It can be an active ingredient of a drug for prevention or treatment.
The substance having the action of reducing the gliding activity of the pathogenic bacterium having the gliding ability is not particularly limited and can be appropriately selected according to the purpose. For example, Congo red, crystal violet, safranin, methylene blue, chlorpromazine And berberine chloride. The substance may be a substance that does not inhibit the growth of the pathogenic bacterium and has an action of reducing only the gliding activity of the pathogenic bacterium, or reduces the gliding activity of the pathogenic bacterium. At the same time, it may be a substance that also inhibits the growth of the pathogenic bacteria, but in particular, it does not inhibit the growth of the pathogenic bacteria from the viewpoint of reducing the risk of appearance of new resistant bacteria to the drug. It is preferably a substance that reduces only the gliding activity of the pathogenic bacteria (that is, reduces only the pathogenicity of the pathogenic bacteria without preventing the survival of the pathogenic bacteria).
Moreover, as a substance which has the effect | action which reduces the gliding activity of the pathogenic bacterium which has the said gliding ability, the substance selected by the above-mentioned evaluation method and screening method of this invention can also be used, for example.

The content of the substance (active ingredient) having an action of reducing the gliding activity of the pathogenic bacterium having the gliding ability in the drug is not particularly limited and can be appropriately selected according to the purpose. The drug may be the active ingredient itself.
Moreover, the said active ingredient may be used individually by 1 type, and may use 2 or more types together. There is no particular limitation on the content ratio of each active ingredient in the drug when two or more are used in combination, and the ratio can be appropriately selected according to the purpose.

-Other ingredients-
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, a pharmacologically acceptable carrier etc. are mentioned. The carrier is not particularly limited and may be appropriately selected depending on, for example, the dosage form of the drug described later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said chemical | medical agent, According to the objective, it can select suitably.

-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said chemical | medical agent, According to the objective, it can select suitably, For example, an oral solid agent (a tablet, a coated tablet, a granule, a powder, a capsule etc.), an oral liquid (internal solution) Syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for use), ointments, patches, gels, creams, external powders, sprays, inhaled powders, etc. It is done.

Examples of the oral solid preparation include, for example, excipients and further additives such as binders, disintegrants, lubricants, colorants, flavoring and flavoring agents as necessary, in addition to the active ingredients. Can be manufactured.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding additives such as a flavoring / flavoring agent, a buffering agent and a stabilizer to the active ingredient.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

As the injection, for example, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the active ingredient, and subcutaneous, intramuscular and intravenous use are performed by a conventional method. Etc. can be manufactured.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

The ointment can be produced, for example, by mixing a known base, stabilizer, wetting agent, preservative and the like with the active ingredient and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

−Use−
The drug can be used, for example, by being administered to an infectious disease patient caused by the pathogenic bacterium having the gliding ability.
The animal to which the drug is administered is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, dogs, cats, cows, pigs, monkeys and the like.
There is no restriction | limiting in particular as the administration method of the said chemical | medical agent, According to the dosage form etc. of the said chemical | medical agent, it can select suitably, For example, oral administration, intraperitoneal administration, injection in the blood etc. are mentioned.
The dose of the drug is not particularly limited and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient to be administered. For example, per day administration to an adult The amount of the active ingredient is preferably 1 μg to 10 g, more preferably 1 μg to 100 mg.
There is no restriction | limiting in particular as administration time of the said chemical | medical agent, According to the objective, it can select suitably, For example, you may administer prophylactically with respect to an infectious disease, and you may administer therapeutically.

(Pathogenicity evaluation method)
The pathogenicity evaluation method of the present invention (hereinafter sometimes simply referred to as “evaluation method”) is a method for evaluating the pathogenicity of pathogenic bacteria having gliding ability, and includes the following step (a ′). Further, other steps are included as necessary.

<Process (a ')>
In the evaluation method, first, the gliding activity of the pathogenic bacterium having the gliding ability is evaluated (step (a ′)).
Here, the “pathogenic bacterium having gliding ability” is as described in the item of the evaluation method and the screening method of the present invention, and any one that is desired to evaluate its pathogenicity is selected. Can do.

  The method for evaluating the gliding activity of the pathogenic bacterium having gliding ability is not particularly limited and can be appropriately selected according to the purpose. For example, using a bacterial solid culture technique using a soft agar medium Can do. For example, as described in the evaluation method and screening method of the present invention described above, when the pathogenic bacterium having the gliding ability is cultured on a soft agar medium under certain conditions, the pathogenicity having the gliding ability is used. When the colony state of the pathogenic bacteria is relatively small, it can be evaluated that the gliding activity of the pathogenic bacteria is relatively low, and the colony state of the pathogenic bacteria having the gliding ability spreads relatively large. When it is, it can be evaluated that the gliding activity of the pathogenic bacterium is relatively high.

As described above, the pathogenicity evaluation method of the present invention can be performed by the step (a ′).
In the step (a ′), when the gliding activity of the pathogenic bacterium having the gliding ability is evaluated to be high (low), the pathogenicity of the pathogenic bacterium can be evaluated to be high (low). In addition, there is no restriction | limiting in particular as a pathogenicity type | formula of the said pathogenic bacteria evaluated here, It is as having described in the item of the evaluation method and screening method of above-described this invention.
According to the pathogenicity evaluation method, the pathogenicity of the pathogenic bacterium having the sliding ability can be efficiently evaluated. Further, as shown in the examples described later, since a positive correlation is established between the gliding activity and the pathogenicity of the pathogenic bacteria having the gliding ability, by using the evaluation method, the pathogenic bacteria It is also possible to quantitatively evaluate the pathogenicity of.
The evaluation method can be suitably used, for example, for basic experiments in the technical field, and can also be suitably used in clinical practice, such as the infectious disease inspection method of the present invention described later.

(Test method for infectious diseases)
The infectious disease inspection method of the present invention (hereinafter sometimes simply referred to as “inspection method”) is an infectious disease inspection method caused by a pathogenic bacterium having gliding ability, and includes the following step (a ″). Step (c ″) is included, and other steps are further included as necessary.

<Process (a '')>
In the inspection method, first, a test sample is collected from the test object (step (a ″)).
-Subject-
The subject is not particularly limited as long as it is an organism that can be the subject of the examination, and can be appropriately selected according to the purpose. For example, the subject suffers from an infection caused by the pathogenic bacterium having the gliding ability. Patients, patients suspected of having the infection, and the like. In addition, there is no restriction | limiting in particular also as the biological species of the said test object, According to the objective, it can select suitably.
-Test sample-
The test sample is not particularly limited and may be appropriately selected depending on the intended purpose. For example, nasal mucosa tissue, blood, sputum, stool, catheter tip, tracheal tube, open pus Suitable examples include oral secretions and pharyngeal mucus.
There is no restriction | limiting in particular as a method of extract | collecting the said test sample from the said test object, According to the objective, it can select suitably. In addition, the test sample collected from the subject may be subjected to, for example, the step (b ″) described later as it is, or after the operation such as washing and storage is performed, the step (b ′ ') May be used.

<Process (b '')>
Next, in the test method, the pathogenic bacteria having the gliding ability are separated from the test sample collected in the step (a ″) (step (b ″)).
Here, the “pathogenic bacterium having gliding ability” is as described in the item of the evaluation method and screening method of the present invention described above.
The method for separating the pathogenic bacterium having the gliding ability from the test sample is not particularly limited and can be appropriately selected according to the purpose. For example, it can be performed using a known bacterial culture technique. .

<Process (c '')>
Next, in the test method, the gliding activity of the pathogenic bacterium having the gliding ability separated in the step (b ″) is evaluated (step (c ″)).
Here, the evaluation method of the gliding activity of the pathogenic bacterium having the gliding ability is as described in the item of the pathogenicity evaluation method of the present invention described above.

In the inspection method for infectious diseases, the level of pathogenicity of the pathogenic bacteria present in the subject can be determined based on the evaluation in the step (c ″). For example, in the step (c ″), when it is evaluated that the gliding activity of the pathogenic bacterium isolated from the test sample is high (low), the pathogenic bacterium present in the subject It can be judged that the pathogenicity is high (low). In addition, there is no restriction | limiting in particular as a pathogenicity type | formula of the said pathogenic bacteria evaluated here, It is as having described in the item of the evaluation method and screening method of above-described this invention.
According to the test method of the present invention, for example, efficiently identifying highly pathogenic bacteria among the pathogenic bacteria present in a patient suffering from an infectious disease caused by pathogenic bacteria having gliding ability. This makes it possible to select a more effective treatment method, for example, preferentially treating the highly pathogenic bacteria. In addition, for the prevention or treatment of a patient suffering from an infectious disease determined to have the highly pathogenic bacterium in the body using the test method, for example, the application of the above-described drug of the present invention is effective. Conceivable.
In addition, as shown in the examples described later, since a positive correlation is established between the gliding activity and pathogenicity of the pathogenic bacteria having the gliding ability, according to the infectious disease testing method, the patient It is considered that the pathogenicity of the pathogenic bacterium present in the body can be quantitatively evaluated, and thus whether or not the patient's symptoms are serious can be accurately predicted.

[effect]
According to the evaluation method and the screening method of the present invention, the pathogen of the pathogenic bacterium having gliding ability is efficiently determined using whether or not the test substance decreases the gliding activity of the pathogenic bacterium having gliding ability as an index. It is possible to evaluate or screen a substance having an action of reducing the property. In addition, according to the production method of the present invention, a drug for the prevention or treatment of infectious diseases caused by the pathogenic bacteria having the gliding ability is efficiently produced using the evaluated or screened substance as an active ingredient. be able to. Therefore, the evaluation method, screening method, and production method are very useful for the development of a new drug for infectious diseases.
Moreover, according to the said evaluation method, screening method, and manufacturing method, it becomes possible to obtain the chemical | medical agent which does not inhibit the growth of the said pathogenic bacteria, for example, and reduces only the pathogenicity of the said pathogenic bacteria. Such a drug can effectively prevent or treat an infectious disease and does not inhibit the growth of the pathogenic bacterium, so that it can be an excellent drug with a low possibility of emergence of a new resistant bacterium. Therefore, for example, the evaluation method, screening method, and production method are considered suitable for the development of drugs against multidrug-resistant Staphylococcus aureus such as MRSA and VRSA, which are currently a major clinical problem.

Since the agent of the present invention contains, as an active ingredient, a substance having an action of reducing the gliding activity of pathogenic bacteria having gliding ability, the pathogenicity of the pathogenic bacteria having gliding ability can be lowered. The drug is very useful for the prevention or treatment of infectious diseases caused by the pathogenic bacteria having the gliding ability.
In addition, the agent does not inhibit the growth of the pathogenic bacterium, and uses a substance that reduces only the pathogenicity of the pathogenic bacterium as an active ingredient, thereby preventing the growth of the pathogenic bacterium. It can be an excellent drug that can treat the disease. Such a drug can be said to be an unprecedented new type of drug with a low risk of emergence of new drug-resistant bacteria. Therefore, for example, the drug is considered to be suitable as a drug against multi-drug resistant Staphylococcus aureus such as MRSA and VRSA.

According to the pathogenicity evaluation method and the infectious disease inspection method of the present invention, for example, the gliding of the pathogenic bacteria isolated from the body of a patient suffering from an infectious disease caused by the pathogenic bacteria having gliding ability. By examining the level of activity, it is possible to evaluate the level of pathogenicity of the pathogenic bacteria present in the patient's body, which is very useful for clinical diagnosis of infectious diseases.
In addition, according to the pathogenicity evaluation method and the infectious disease inspection method, the pathogenicity level of the pathogenic bacteria present in the patient can be quantitatively evaluated. Therefore, it is possible to accurately predict the presence or absence of conversion, and to select a more effective treatment method. Therefore, for example, the pathogenicity evaluation method and the infectious disease inspection method are considered suitable for clinical diagnosis of infectious diseases caused by multi-drug resistant Staphylococcus aureus such as MRSA and VRSA.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1: Relationship between gliding activity and hemolytic activity in Staphylococcus aureus)
In order to investigate the relationship between the gliding activity of pathogenic bacteria having gliding ability and the pathogenicity of the pathogenic bacteria, the relationship between gliding activity and hemolytic activity in Staphylococcus aureus was examined as follows. The fact that Staphylococcus aureus has gliding ability is a fact first discovered by the present inventors in recent years (for example, the 78th Annual Meeting of the Japanese Biochemical Society (2005), Kaito et al., 12th International Symposium). on Staphylococci & Staphylococcal Infections, 3-6 September 2006, Maastricht, The Netherlands, Poster number: P028).
<Method>
The MRSA strain (Mesitylin-resistant Staphylococcus aureus) and the MSSA strain (Mesitylin-sensitive Staphylococcus aureus) were distributed from a private university hospital. The RN4220 strain is a laboratory stock of methicillin-sensitive Staphylococcus aureus in the Department of Microbial Drug Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo. Each bacterium was cultured overnight in an LB liquid medium containing 1% NaCl at 37 ° C., and 2 μL of the bacterium was poured into a 10 cm-diameter plastic petri dish and hardened, and 0.24% agar was added. The mixture was spotted on a BHI (beef heart infusion) medium (25 mL) and cultured at 37 ° C. for 10 hours. The diameter of the formed colony was measured with a ruler to determine gliding activity. In addition, each bacterium was cultured overnight at 37 ° C. in a TSB (trypton soy bean) liquid medium, and the released hemolysin activity was measured by the method of Vandensch et al. (Vandensch et al .; J. Bacteriol. 173, 6313 (1991)). ) To determine hemolytic activity.
<Result>
The results are shown in FIGS.
FIG. 1 shows a methicillin-resistant Staphylococcus aureus strain (NI-6, 7, 8, 9, 10; MRSA) and a methicillin-sensitive Staphylococcus aureus strain (RN4220; MSSA) on a soft agar medium. It is the figure which compared the gliding activity in. It can be seen that most strains of clinically isolated MRSA are less glide than MSSA strain RN4220.
FIG. 2A and FIG. 2B are diagrams comparing the gliding activity and the hemolytic activity of clinically isolated MRSA strain (●) and MSSA strain (◯). It can be seen that both the gliding activity and the hemolytic activity of the clinically isolated MRSA strain on the soft agar medium are significantly lower than those of the MSSA strain. Moreover, it can be seen from the graph of FIG. 2A that the level of gliding activity of Staphylococcus aureus and the level of hemolytic activity have a positive correlation.

(Example 2: Inhibition of S. aureus gliding activity by introduction of mecA gene)
From Example 1, the clinically isolated methicillin-resistant Staphylococcus aureus strain (MRSA) had a relatively low gliding ability compared to the methicillin-susceptible Staphylococcus aureus strain (MSSA). Furthermore, whether or not gliding activity is suppressed by introducing the mecA gene characteristic of MRSA was examined as follows.
<Method>
The Newman strain and the RN4220 strain are laboratory-owned strains of the Department of Microbial Drug Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo. A plasmid (pmecA) in which the mecA gene was inserted into pND50 or pND50 was introduced by electroporation and separated on an agar medium containing chloramphenicol. Bacteria containing each plasmid were cultured overnight at 37 ° C. in TSB medium, 2 μL of the bacteria were spotted on TSB medium containing 0.24% agar, and cultured at 37 ° C. for 10 hours. The diameter of the resulting colony was measured and used as gliding activity.
<Result>
The results are shown in FIG.
FIG. 3 is a diagram showing that the gliding activity of S. aureus was suppressed by introduction of the mecA gene. It can be seen that in the S. aureus strain into which the plasmid (pmecA) having the mecA gene was introduced, the gliding activity was suppressed as compared with the S. aureus strain into which the control vector (pND50) was introduced.

(Example 3: Isolation of high gliding ability from MSSA low gliding ability and evaluation of pathogenicity)
High gliding ability strains were isolated from MSSA low gliding ability strains, and their pathogenicity (haemolytic activity and pathogenicity against silkworm larvae) was evaluated as follows.
<Method>
MSSA1 strain (SP0) is a clinical isolate with low glide ability obtained from Kyushu University Hospital. This strain was cultured overnight at 37 ° C. in a TSB liquid medium containing 0.4% EMS, and further diluted 100-fold in a TSB liquid medium to obtain an overnight culture. When this bacterial solution was spotted on a BHI soft agar plate containing 0.24% agar and cultured at 37 ° C., a part spreading on the soft agar appeared. Bacteria were collected from this portion and named SP1, SP2, SP3, SP4. After culturing each in LB liquid medium overnight, 2 μL of the bacterial solution was spotted on a BHI soft agar plate containing 0.24% agar and cultured at 37 ° C. (FIG. 4). Furthermore, 2 μL of the bacterial solution was added onto a TSB agar plate containing 5% sheep erythrocytes, and cultured at 37 ° C. for 30 hours, and the presence or absence of the appearance of a hemolytic ring was examined (FIG. 5). Furthermore, 50 μL of bacterial solutions of various concentrations diluted with physiological saline (0.9% NaCl) were injected into the blood of silkworm 5th instar larvae and reared at 27 ° C. for 38 hours to increase the survival rate of silkworm larvae. It was investigated (FIG. 6).
<Result>
The results are shown in FIGS.
FIG. 4 is a diagram showing a state in which a high gliding ability mutant strain (SP1 to SP4) is separated from a low gliding ability strain of S. aureus (SP0; MSSA1) by mutagen treatment with EMS.
FIG. 5 shows the hemolytic activity of the isolated high gliding ability mutants (SP1 to SP4). It can be seen that three of the four high-sliding ability mutant strains (SP1 to SP4) have higher hemolytic activity than the parent strain (SP0; MSSA1).
FIG. 6 shows the pathogenicity of the isolated high gliding ability mutants (SP1 to SP4) against silkworm larvae. It can be seen that the isolated high-sliding ability mutants (SP1 to SP4) have increased pathogenicity against silkworm larvae compared to the parent strain (SP0; MSSA1).

(Example 4: Examination of the inhibitory effect of Congo red on the sliding activity and pathogenicity of Staphylococcus aureus)
The inhibitory effect of Congo red on the gliding activity and pathogenicity of Staphylococcus aureus was examined as follows.
<Method>
RN4220 is a methicillin-sensitive Staphylococcus aureus strain possessed by the laboratory of the Department of Microbial Drug Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo. NI-30A, NI-30B, and NI-30C are strains derived from RN4220 and obtained as separate colonies. NI-30 is a clinical isolate of MRSA provided by a private university hospital. The overnight culture solution of each bacterium in LB liquid medium was spread on a BHI agar plate containing 0 to 160 μg / mL Congo red with a platinum loop and cultured at 37 ° C. for 20 hours (FIG. 7). Separately, 2 μL of an overnight culture of each bacterium in LB liquid medium was spotted on a BHI soft agar plate containing 0.24% agar and cultured at 37 ° C. (FIG. 8 and FIG. Only the results from 30 are shown). Further, the overnight culture of RN4220 in TSB liquid medium was diluted 200 times with TSB liquid medium, added with 0-160 μg / mL Congo red, cultured at 37 ° C. for 6 hours, and then centrifuged (3,000 rotations). 5 minutes), and the hemolytic activity of the supernatant was examined. Hemolysis in the presence of saponin (1 mg / mL) was used as a control for 100% hemolysis (FIG. 9). Further, an overnight culture solution of RN4220 in LB medium was diluted 10,000 times with physiological saline (0.9% NaCl), and 50 μL was injected into the blood of 10 silkworm larvae per group. Thereafter, 50 μL of physiological saline containing 0 to 1 mg / mL Congo red (0 to 50 μg as the amount of Congo red) was injected into the blood of silkworm larvae, and the silkworm larvae were reared at 37 ° C. The decrease was examined (Figure 10).
<Result>
The results are shown in FIGS.
FIG. 7 is a diagram showing the influence of Congo Red on the growth of Staphylococcus aureus. It can be seen that Congo Red did not affect the growth of Staphylococcus aureus at the concentration examined (˜160 μg / mL).
FIG. 8 is a diagram showing a state of inhibition of the gliding activity of Staphylococcus aureus by Congo red. It can be seen that Congo red inhibited the gliding activity of Staphylococcus aureus on a soft agar medium.
FIG. 9 is a diagram showing the state of inhibition of the hemolytic activity of S. aureus by Congo Red. It can be seen that the hemolytic activity of Staphylococcus aureus was reduced by Congo Red and 10 μg / mL or more.
FIG. 10 is a diagram showing the therapeutic effect of Congo Red on the infection of S. aureus in silkworm larvae. In silkworm larvae, it can be seen that killing by S. aureus infection was suppressed in a dose-dependent manner with Congo Red.

  From the results of the above Examples, the high level of gliding activity of pathogenic bacteria having gliding ability, such as Staphylococcus aureus, and the high level of pathogenicity of the pathogenic bacteria (for example, hemolytic activity and ability to kill organisms) It was shown that there is a close relationship between the two. Further, the substance having the action of reducing the gliding activity of the pathogenic bacterium can reduce the pathogenicity of the pathogenic bacterium, that is, an excellent preventive or therapeutic agent for infectious diseases caused by the pathogenic bacterium. It was shown that it can be. Such a substance can reduce only the pathogenicity of the pathogenic bacteria without inhibiting the growth of the pathogenic bacteria. Therefore, it is possible to effectively prevent or treat infectious diseases caused by the pathogenic bacteria, and since it does not inhibit the growth of the pathogenic bacteria, it can be an excellent drug with a low possibility of causing new resistant bacteria. It is thought that it has sex.

  The evaluation method and screening method of the present invention, the drug and the production method thereof, the pathogenicity evaluation method and the infectious disease test method include the development of a drug for infectious diseases caused by pathogenic bacteria having gliding ability, It can be suitably used for clinical diagnosis of infectious diseases.

FIG. 1 is a diagram comparing the gliding activity on a soft agar medium between clinically isolated MRSA strains (NI-6, 7, 8, 9, 10) and MSSA strain (RN4220). FIG. 2A is a diagram comparing the gliding activity and the hemolytic activity of clinically isolated MRSA strain (●) and MSSA strain (◯). FIG. 2B is a diagram comparing gliding activity and hemolytic activity between clinically isolated MRSA strains and MSSA strains. FIG. 3 is a diagram showing that the gliding activity of S. aureus was suppressed by introduction of the mecA gene. FIG. 4 is a view showing a state in which a high gliding ability mutant strain (SP1 to SP4) is separated from a low gliding ability strain of S. aureus (SP0; MSSA1) by mutagen treatment. FIG. 5 shows the hemolytic activity of the isolated high gliding ability mutants (SP1 to SP4). FIG. 6 shows the pathogenicity of the isolated high gliding ability mutants (SP1 to SP4) against silkworm larvae. FIG. 7 is a diagram showing the influence of Congo Red on the growth of Staphylococcus aureus. FIG. 8 is a diagram showing a state of inhibition of the gliding activity of Staphylococcus aureus by Congo red. FIG. 9 is a diagram showing the state of inhibition of the hemolytic activity of S. aureus by Congo Red. FIG. 10 is a diagram showing the therapeutic effect of Congo Red on the infection of S. aureus in silkworm larvae.

Claims (6)

A method for evaluating whether a test substance has an action of reducing the pathogenicity of pathogenic bacteria having gliding ability,
(A) A method comprising evaluating whether or not the test substance reduces the gliding activity of the pathogenic bacterium having the gliding ability. A method for screening a substance having an action of reducing pathogenicity of pathogenic bacteria having gliding ability,
(A) a step of evaluating whether the test substance reduces the gliding activity of the pathogenic bacteria having the gliding ability; and
(B) A method comprising the step of selecting a substance evaluated to reduce the gliding activity of the pathogenic bacterium having the gliding ability in the step (a). A method for producing a drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability,
(A) a step of evaluating whether or not the test substance reduces the gliding activity of the pathogenic bacterium having the gliding ability,
(B) a step of selecting a substance evaluated to reduce the gliding activity of the pathogenic bacterium having the gliding ability in the step (a);
(C) generating the substance selected in step (b), and
(D) A production method comprising the step of mixing the substance produced in the step (c) and a pharmaceutically acceptable carrier.   A drug for the prevention or treatment of infectious diseases caused by pathogenic bacteria having gliding ability, characterized in that a substance having an action of reducing the gliding activity of the pathogenic bacteria having gliding ability is an active ingredient Drug. A method for evaluating the pathogenicity of pathogenic bacteria having gliding ability,
(A ′) A method comprising evaluating the gliding activity of a pathogenic bacterium having the gliding ability. A test method for infectious diseases caused by pathogenic bacteria having gliding ability,
(A ″) a step of collecting a test sample from the test sample;
(B ″) separating the pathogenic bacteria having the gliding ability from the test sample; and
(C ″) A method comprising evaluating the gliding activity of the isolated pathogenic bacterium having the gliding ability.