WO2009154016A1 - Method of evaluating pathogenicity of pathogenic bacterium, kit for evaluating pathogenicity and gene for evaluating pathogenicity - Google Patents

Abstract

Disclosed are an evaluation method whereby the pathogenicity of a pathogenic bacterium can be quickly and surely evaluated by detecting the genotype characteristic of the bacterium and an evaluation kit therefor. A method of evaluating the pathogenicity of a pathogenic bacterium characterized by comprising confirming the presence or absence of a gene which encodes a protein having a function of controlling the gliding motility of the pathogenic bacterium or the occurrence or non-occurrence of at least one mutation affecting the preceding function of the protein in the gene; a kit for evaluating the pathogenicity of a pathogenic bacterium by using the method of evaluating pathogenicity as described above; and a gene which comprises the following DNA (a) or (b): (a) a DNA comprising the base sequence represented by SEQ ID NO:1, having a function of controlling the gliding motility of a pathogenic bacterium and participating in the pathogenicity of the bacterium; and (b) a DNA being hybridizable under stringent conditions with DNA comprising the base sequence represented by SEQ ID NO:1.

Description

Method for evaluating pathogenicity of pathogenic bacteria, kit for evaluating pathogenicity, and gene for evaluating pathogenicity

Methicillin-resistant Staphylococcus aureus (hereinafter abbreviated as “MRSA”) is a bacterium that requires special attention as a main bacterium for nosocomial infections. This MRSA has been considered to be not particularly strong in pathogenicity other than drug resistance when transported from a hospital to a city such as a home, and to be easily destroyed. However, in recent years, MRSA has been detected in outpatients, and the presence of community-acquired MRSA with high pathogenicity has been reported, and a new response to MRSA with high pathogenicity has been demanded. It was.

In order to appropriately respond to such highly pathogenic MRSA, normal MRSA and highly pathogenic MRSA are clearly identified and each is evaluated, and an appropriate drug is selected according to the evaluation result. It is considered important to select and determine how to use it. Abuse of antibiotics such as vancomycin without considering the high pathogenicity of MRSA isolated from individual patients must be strictly avoided because it will encourage the emergence of resistant bacteria to these. Also, in the development of new therapeutic agents and treatment methods, it is considered important to proceed with development after identifying the high pathogenicity of MRSA.

Usually, a method for identifying a certain bacterium is roughly divided into a method for analyzing the external characteristics of the bacterium, a method for analyzing the biological activity of the bacterium, and a method for analyzing the characteristics of the genotype. So far, pathogenicity has been identified and evaluated by hemolytic activity, vomiting activity, lethal strength, etc. derived from the toxin produced by the bacterium. In addition, the characteristics of genotypes related to pathogenicity have been identified by the presence or absence of genes related to the toxin (for example, Non-Patent Documents 1 to 3).

However, characteristics of many clinically highly pathogenic MRSA genotypes have not been clarified, and there is a current situation that MRSA genotypes are not sufficiently distinguished. That is, depending on the characteristics of the genotype found by the above-described conventional studies, the state of high pathogenicity could not be clearly evaluated.

Recently, the inventor's research has revealed that Staphylococcus aureus, which has been conventionally considered not to have a migration ability on a solid surface, has the ability to slide on a soft agar medium (Non-patent Document 4). . Furthermore, when the present inventor investigated the correlation between the staphylococci of S. aureus and the virulence of S. aureus, it was found that S. aureus having a low slidability has a clearly lower pathogenicity than a higher one. I found it. And based on that knowledge, it was found that the measurement of sliding ability can be used as an unprecedented evaluation method for evaluating the pathogenicity of Staphylococcus aureus, and a patent application has already been filed (Japanese Patent Application No. 2006-342713). ).

Although priority is claimed in this application, at the time of filing the basic application (Japanese Patent Application No. 2008-158176), the above patent application (Japanese Patent Application No. 2006-342713) is not published. Thereafter, the above-mentioned patent application (Japanese Patent Application No. 2006-342713) was published at the time of filing this application with priority claim (Patent Document 1).

However, a gene encoding a factor that controls gliding ability such as Staphylococcus aureus has not been clarified.
JP 2008-148669 A Tatsuo Yamamoto et al., Japanese Society of Chemotherapy, 52 (11), 635-652 (2004) Teruyo Ito et al., Journal of Infectious Diseases, 78 (6), 459-469 (2004) Shigemo Katsuhiko, Food Sanitation, 51 (4), 81-90 (2005) C. Kaito and K. Sekimizu J. Bacteriol., 189 (6), 2553-2557 (2007)

The above-mentioned “method for identifying bacteria based on the external characteristics and biological activity of the bacteria” requires rapid identification because it is necessary to perform the identification process after the bacteria separation process from the specimen, the bacteria culture process, etc. In clinical settings where it is necessary to obtain treatment decisions and to decide on treatment strategies, it is often the case that the patient loses late in terms of dealing with patients and preventing secondary infections. Therefore, if the differences in the genotypes that caused such biological activities, etc. are clarified and analyzed directly, it can be a more definitive and quick identification and evaluation method. It is done.

The present invention has been made in view of the above, and its problem is to find characteristics of a pathogenic bacterial gene that exhibits pathogenicity but has not yet been clarified about the gene, and targets that characteristic. It is an object of the present invention to provide a method for evaluating pathogenicity of pathogenic bacteria, a primer for evaluating pathogenicity, a kit for evaluating pathogenicity including the primer, and the like.

In order to solve the above problems, the present inventor first compared the sliding ability of 10 clinically isolated methicillin-sensitive Staphylococcus aureus (hereinafter abbreviated as “MSSA”) and MRSA 40 strain. As a result, it was confirmed that all the MSSA10 strains slid, whereas MRSA had a decreased sliding ability in 29 of 40 strains, and the remaining 11 strains had high sliding ability.

As a result of intensive studies focusing on the SCC mec region where the mecA gene, which is a gene related to methicillin resistance, is present, known genes such as the mecA gene, mecR1, and mecI gene are not genes that strongly suppress gliding ability. I understood. Therefore, when an open reading frame on this region (hereinafter abbreviated as “ORF”) was examined in detail, a gene encoding 70 amino acids that is not registered as ORF in the genome database of S. aureus N315 (hereinafter referred to as “ORF”). "Fudoh", hereinafter abbreviated as "fudhoh"). And it confirmed that the plasmid which has a fudhoh gene suppressed glide ability.

That is, among MRSA11 strains with high gliding ability among MRSA strains, 10 strains had a single base mutation accompanied by amino acid substitution of the fudh gene product, and the remaining 1 strain did not have the fudhoh gene. In addition, it was confirmed that 29 strains having lower sliding ability than those strains had no mutation in the fudh gene.

Furthermore, by examining the gliding ability of the SCCmec occlusion strain and the fudhoh gene-introduced strain, and examining the pathogenicity of these strains against silkworms and mice, the fudh gene suppresses the gliding ability and the pathogenicity also decreases accordingly. It has been confirmed that the present invention has been completed.

That is, the present invention
(1) A method for evaluating the pathogenicity of a pathogenic bacterium, the presence or absence of a gene encoding a protein having a function of controlling the gliding ability of the pathogenic bacterium, or the function of the preceding protein in the gene A pathogenicity evaluation method characterized by confirming whether or not there is at least one mutation which gives
(2) A method for evaluating the pathogenicity of a pathogenic bacterium, wherein the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of the pathogenic bacterium, or the function of the preceding protein in the gene is suppressed. The pathogenicity evaluation method according to (1), wherein whether or not there is at least one mutation to be confirmed is confirmed.
(3) The pathogenicity evaluation method according to (1) or (2), wherein the pathogenic bacterium is methicillin-resistant Staphylococcus aureus.
(4) A method for evaluating the pathogenicity of methicillin-resistant Staphylococcus aureus, the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of methicillin-resistant Staphylococcus aureus, or SEQ ID NO: 1 in the gene The pathogenicity evaluation method according to (3), wherein whether or not there is a mutation in the base sequence corresponding to amino acid number 29 described in (3) is confirmed.
(5) The gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria is a DNA comprising the base sequence set forth in SEQ ID NO: 1 (1) to (4) Evaluation method of pathogenicity.
(6) A kit for evaluating the pathogenicity of pathogenic bacteria, characterized by using the pathogenicity evaluation method according to any one of (1) to (5).
(7) At least a primer for detecting a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria, or a primer for detecting a mutation that suppresses the function of a previous protein in the gene The kit for evaluating pathogenicity of pathogenic bacteria according to (6), which has one.
(8) A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence described in SEQ ID NO: 1 and having a function of suppressing the gliding ability of pathogenic bacteria and encoding a protein involved in the pathogenicity of the fungus;
(B) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1;
(9) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and having a function of suppressing the gliding ability of pathogenic bacteria and involved in the pathogenicity of the fungus.
Etc.

According to the present invention, a method for evaluating pathogenicity by the genotype of drug-resistant bacteria can be provided. That is, the degree of pathogenicity of a pathogenic bacterium, the presence or absence of a new gene that controls the gliding ability of the pathogenic bacterium, or at least one mutation present in the new gene that affects the function of the gene By confirming the presence or absence of “,” it is possible to make a reliable and quick evaluation. As a result, a treatment policy such as selection of an appropriate drug for the patient can be determined, and the spread of infection can be prevented. In addition, since it is possible to accurately evaluate the high pathogenicity of drug-resistant bacteria such as MRSA that have been difficult to identify, it is possible to bring about appropriate use of drugs that are sensitive to MRSA such as vancomycin. is there.

It is the figure which compared the measurement result of the gliding ability on the soft agar medium of MSSA strain 10 strains and 40 MRSA strains which are clinical isolates of Staphylococcus aureus. It is the figure which confirmed the presence or absence of the mecA gene in MRSA clinical isolate 3 strain and its SCCmec area dropout strain. It is a figure which shows the gliding ability of three MRSA clinical isolates and its SCCmec area dropout strain. (A) It is a figure which shows the state of the colony at the time of measuring sliding ability. (B) It is a figure which shows the graph which put together the result of having compared sliding ability. It is a figure which examines the area | region of the gene which controls gliding ability. (A) A diagram showing plasmids introduced with these regions, which were prepared in order to examine in which region in the SCCmec region a gene that suppresses gliding ability exists. (B) It is the figure which showed the state of the colony when the sliding ability of the strain | stump | stock which introduce | transduced each plasmid of FIG. 4 (A) was measured. (C) A graph summarizing the results of comparing the gliding ability of strains into which each plasmid was introduced. It is the figure which compared the base sequence of the fudoh gene in 40 clinically isolated MRSA strains, and the glide ability which each strain | stump | stock shows. It is a figure which shows the gliding ability which the MSSA strain | stump | stock which introduce | transduced the fudoh gene and its mutant gene showed. (A) It is the figure which showed the state of the colony at the time of measuring sliding ability. (B) It is a figure which shows the graph which put together the result of having compared sliding ability. It is a figure which shows the pathogenicity with respect to a silkworm. (A) A diagram comparing the pathogenicity of a SCCmec-dropped MRSA strain with its parent strain. (B) A diagram comparing the pathogenicity of a fudoh-introduced MSSA strain with its parent strain. It is the figure which compared the pathogenicity of fudoh introduction | transduction MSSA strain | stump | stock with respect to a mouse | mouth with respect to the parent strain.

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the technical scope of the present invention.

The present invention confirms the presence or absence of a newly found gene related to the control of gliding ability of a pathogenic bacterium, or whether or not there is a mutation that affects the previous factor in the gene. It is a method for evaluating the high pathogenicity of, and can be carried out by executing a normal gene detection means and a means for detecting the mutated portion thereof.

Therefore, by using a primer capable of amplifying the gene and the like found this time, a gene amplification method such as a polymerase chain reaction (PCR) method, a real-time PCR method, a Loop-Mediated Isothermal Amplification (LAMP) method is executed, The present invention can be carried out by confirming the presence or absence of a gene or a mutation in the gene.

Here, the evaluation of bacterial pathogenicity based on the genotype has been carried out mainly by evaluating the gene related to the “toxin” possessed by the bacteria (see Non-Patent Documents 1 to 3). Evaluation of pathogenicity by a gene relating to “sliding ability” has not been performed conventionally. Therefore, the fact that the pathogenicity of pathogenic bacteria can be evaluated by a gene related to “sliding ability”, which is the basis of the present invention, will be described with respect to the examination content that is the basis thereof.

In the present invention, “sliding ability” refers to the ability of bacteria to move on a soft agar medium, and “size” refers to the size of the colony spread on the soft agar medium under certain conditions. Further, “high pathogenicity” refers to the strength of damage to a host under the condition that the pathogen infects the host, and is evaluated separately from “presence or absence of drug resistance”.

Regarding the gliding ability of bacteria, there are reports that the movement mechanism is not clear, other than those that move by clear organs such as flagella. Staphylococcus aureus is also a Gram-positive bacterium that does not have flagella, and until recently was thought to have no gliding ability. The present inventors have found that this S. aureus has the ability to slide on a soft agar medium (Non-Patent Document 4). The gliding ability of bacteria with distinct motor organs such as flagella is thought to play an important role in the pathogenicity of the bacteria. However, the relationship between the gliding ability of Staphylococcus aureus found in the soft agar medium discovered by the present inventor and the high pathogenicity of the bacterium has been unknown.

Therefore, the present inventor considers that the ability of S. aureus gliding to play a role in its pathogenicity, and the relationship between the magnitude of the gliding ability of the strain in the soft agar medium and the pathogenicity of the strain against the host. As a result of confirming whether or not there is a silkworm as a host, it has been found that the magnitude of gliding ability correlates well with its pathogenicity, and a patent application has already been filed (Japanese Patent Application No. 2006-342713). The invention according to the already filed application is to evaluate the characteristics that appear on the outer surface among various pathogenicity evaluation methods, whereas the present invention clarifies genes related to the gliding ability of pathogenic bacteria, It is characterized by evaluating pathogenicity according to the characteristics of its genotype.

First, the gliding ability of 10 clinical isolates MSSA and 40 MRSA was compared. (See Test Example 1) As a result, the average value of the running distance of the MSSA strain was 67.7 mm, whereas the average value of the running distance of the MRSA strain was 31.7 mm. In addition, 29 of the 40 MRSA strains had a sliding distance of 35 mm or less, and it was found that most of the MRSA strains reduced the sliding ability (FIG. 1).

Next, in order to examine the effect of the mecA gene, which is a gene that imparts methicillin resistance ability, which is a characteristic for distinguishing MRSA from MSSA by genotype, on the sliding ability of the SCCmec region, the sliding ability is low. The SCCmec region was removed from the MRSA strain and its sliding ability was confirmed (see Test Example 2). The PCR method confirmed that the mecA gene was not present in these strains (FIG. 2), and the resistance to methicillin was reduced. In addition, all of these strains were found to have increased gliding ability (FIGS. 3 (A) and 3 (B)).

In order to examine which gene in the previous SCCmec region suppresses the slidability of Staphylococcus aureus, a plasmid having a region containing the mecA gene, mecR1 gene, and mecI gene in the SCCmec region is methicillin-sensitive yellow. It was inserted into the chromosome of a Newman strain, which is a staphylococcus, and its sliding ability in a soft agar medium was examined (see Test Example 3). As a result, the strain into which the plasmid having the region containing the previous 3 genes and the vicinity thereof had been introduced had a lower glide ability than the strain into which the empty vector was introduced (FIGS. 4B and 4C). ).

Next, it is predicted that any of the three genes above contributes to the decrease in gliding ability, and a plasmid having each gene for which gene contributes to the decrease in gliding ability is created. It examined similarly (FIG. 4 (A)). As a result, the region containing the mecA gene that gave the result of suppressing the gliding ability in the examination using the overexpression system before did not suppress the gliding ability in the examination using the normal expression system (FIG. 4B). FIG. 4 (C)). On the other hand, the region containing both mecR1 and mecI genes suppressed gliding ability (FIG. 4 (B), FIG. 4 (C)).

Therefore, when three types of plasmids were prepared by removing each gene and both genes from the region containing both mecR1 and mecI genes, contrary to expectation, all plasmids suppressed gliding ability (FIG. 4 (B)). FIG. 4 (C)). Therefore, it was confirmed that the gene that suppresses gliding ability is none of the above three genes. Therefore, when the ORF on this region was examined in detail, an ORF encoding 70 amino acids not registered as ORF was found in the genome database of MRSA N315 strain (FIG. 4 (A), rightmost region). And the plasmid which has this gene suppressed gliding ability (FIG.4 (B), FIG.4 (C)). Therefore, the present inventor named this new gene “fudhoh” (SEQ ID NO: 1 in the Sequence Listing).

In order to know whether the high gliding ability of the 11 MRSA strains described above can be explained by the presence or mutation of the fudh gene, the base sequence of the fudh gene possessed by all 40 MRSA strains was determined. As a result, it was revealed that 10 of 11 strains with high gliding ability had a single base mutation (K29R) accompanied by amino acid substitution of the fudh gene product. In addition, the fudoh gene was not present in the remaining strain. Furthermore, no mutation was found in the fudh gene in 29 strains with lower gliding ability than those strains (FIG. 5).

In order to examine whether the mutated fudoh gene has lost the ability to suppress gliding ability, the mutated fudoh gene was introduced into the Newman strain, which is an MSSA strain, and the gliding ability was examined (Test Example 4). As a result, the previous mutant fudoh gene did not inhibit the sliding ability of the Newman strain at all (FIGS. 6A and 6B). Therefore, the previous mutant fudoh gene has lost the ability to suppress gliding ability, and the high gliding ability of 10 out of 11 MRSA strains is explained by the fact that it has lost its function due to mutation of the fudoh gene. It became clear that we could do it.

Accordingly, the present invention is a gene comprising the following DNA (a) or (b).
(A) DNA consisting of the base sequence described in SEQ ID NO: 1 and having a function of suppressing the gliding ability of pathogenic bacteria and encoding a protein involved in the pathogenicity of the fungus;
(B) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1;
The gene “fudh” comprising such DNA is a gene having a function newly found in the present invention.

Next, for the purpose of examining the relationship between S. aureus gliding ability and pathogenicity, NI-5 strain, whose parent strain is MRSA strain, whose gliding ability was increased by dropping of the SCCmec region, and vice versa, The pathogenicity to mice and silkworm larvae (hereinafter abbreviated as “Silkworm”) of the Newman strain whose parent strain is the MSSA strain, whose gliding ability was suppressed by introduction (see Test Example 5), was examined.

The time from the administration of the fungus to the death of the silkworm injected with the SCCmec-dropped NI-5 strain was clearly faster than that of the silkworm injected with the parent strain (FIG. 7 (A)). On the other hand, the death of silkworms injected with the newman-introduced Newman strain was clearly delayed compared to the silkworms injected with the parent strain (FIG. 7B). Furthermore, the death of mice injected with the fudhoh-introduced Newman strain in the mouse sepsis model was also clearly delayed compared to the mice injected with the parent strain (FIG. 8). The above results suggest that the fudoh gene found by the present inventor suppresses the gliding ability of Staphylococcus aureus and at the same time reduces its pathogenicity against silkworms and mice. .

A characteristic feature of the present invention is not only that the fudhoh gene has been found, but if the function of fudoh gene is mutated to suppress gliding ability, the gliding ability increases and that pathogenicity In contrast, the presence of the fudhoh gene suppresses gliding ability, thereby reducing pathogenicity.

Therefore, the fudh gene was inactivated by some new mutations such as point mutations, deletions, duplications, inversions, insertions, translocations, etc. other than single nucleotide mutations with amino acid substitutions specifically found by the present invention. In this case, it is possible to easily predict that the function of suppressing the gliding ability is lost and the pathogenicity is thereby increased. That is, it is based on the present invention that a new mutation is found using the gliding ability as a phenotype, a primer for detecting the new mutation is designed, and a target mutation for pathogenicity evaluation is added. Therefore, it is possible to do with common general technical knowledge. Therefore, the present invention is not limited to the above-described single nucleotide mutations specifically described, and these are also included in the scope of the present invention.

The pathogenic bacterium applicable to the present invention completed by the examination described above is a bacterium having gliding ability and a pathogenic bacterium having a gene encoding a protein having a function of controlling gliding ability. Either may be used. Although the present invention was made by using MRSA, it is apparent in principle that pathogenic bacteria are not limited to MRSA from the relationship between gliding ability and pathogenicity. As long as the pathogenic bacterium applicable to the present invention is a bacterium having a gliding ability and having a fudh gene, and the gliding ability is suppressed by the fudh gene, naturally, MRSA and Similarly, the pathogenicity can be evaluated. Examples of such bacterial candidates include staphylococcus epidermidis other than Staphylococcus aureus whose presence of the fudh gene has been clarified by disclosure of genomic information, but the present invention is not limited thereto. is not.

In the present invention, for example, when evaluating the pathogenicity of MRSA, it is confirmed that the bacteria in the clinical specimen are Staphylococcus aureus, the presence or absence of methicillin resistance, the presence or absence of the fudhoh gene found this time and the gene Check if there is a mutation in it. In order to confirm the presence of Staphylococcus aureus, for example, a pair of forward and reverse primers that amplify the protein A gene spa can be used. In order to confirm the presence or absence of methicillin resistance, For example, a pair of primers, mecA-F as a forward primer for amplifying the mecA gene shown in Table 2, and mecA-R as a reverse primer can be used. It is also possible to use other primers and primer sets reported for the above purpose.

That is, the present invention is an evaluation kit for using the above pathogenicity evaluation method, wherein a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria such as methicillin-resistant Staphylococcus aureus is obtained. It is also a “kit for evaluating pathogenicity of pathogenic bacteria” characterized by having at least one primer for detecting or detecting a mutation that suppresses the function of the previous protein in the gene. .

In order to confirm the presence or absence of the fudhoh gene, for example, a pair of fudh-F and fudhoh-R primers shown in Table 2 can be used. In order to confirm the presence or absence of a mutation associated with the substitution of amino acid number 29 in the fudh gene, PCR amplification was performed using a pair of primers S2 and S3 shown in Table 2, and the nucleotide sequence of the PCR product was determined. Determine using S2 and S3 primers. Since the mutation of the fudh gene cannot be detected directly with this primer, the base sequence of the PCR product amplified using this primer is determined using the same primer. As a result, it can be seen whether or not the mutation exists. In addition, if a primer suitable for ARMS (Amplification Refractory Mutation System) to be described later is designed, it should be used as a primer for easily detecting a single base mutation (K29R) at the site shown in FIG. Can do.

Primers used in the present invention for confirming the presence or mutation of the fudhoh gene and its mutation are not limited to the above primers, and detection of the fudhoh gene and loss of gene function in the gene are the same as the above primers. Any material can be used as long as it can be used for the purpose of detecting a mutation that brings about activity. Such primers include DNA in which a part of the previous primer has been modified by deletion, substitution, insertion, addition, or the like as long as the same function is exhibited. Further, in the future, when a new mutation causing inactivation of gene function is found, an invention using a newly designed primer for detecting the mutation is also within the scope of the present invention.

Identification of the bacterial species in the clinical specimen, confirmation of drug resistance, presence of the fudhoh gene, and confirmation of whether or not there is a mutation in the gene may be performed by means of detecting each separately. , All of them may be detected simultaneously.

As a means for detecting each gene separately, each of the previous confirmations may be carried out by ordinary PCR with a set of primers used for each in an independent system. Moreover, it is also possible to use a method in which real-time PCR is performed with a DNA sample extracted from a clinical specimen by a conventional method, and a generated fluorescence signal of a PCR amplification product is detected in real time. In addition, as a means for detecting all at the same time, it is used for confirming that it is Staphylococcus aureus, confirming the presence or absence of drug resistance, and detecting the presence or absence of the fudhoh gene and the mutation in that gene. Multiplex PCR can be performed in which all primer sets are mixed and the PCR reaction is performed in the same reaction system. In this case, the presence or absence of a PCR amplification product corresponding to each gene can be confirmed by confirming the presence or absence of a band obtained by electrophoresis by agarose gel electrophoresis or capillary electrophoresis.

The procedure of the present invention will be described below.
<Nucleic acid extraction>
Nucleic acids are extracted from patient-derived specimens such as blood, sputum, pus, and pharyngeal swabs, or materials such as gauze used for patients, or bacterial fluids obtained by culturing them. Extraction can be performed by heat treatment, enzyme treatment, or using a commercially available DNA extraction kit. In the case of staphylococci, DNA extraction efficiency can be increased by using an enzyme suitable for degradation of staphylococcal membranes such as lysostaphin and achromopeptidase. Moreover, the sensitivity of a test | inspection can be raised by processing the obtained extract by nucleic acid purification methods, such as a phenol process, and also adding concentration operation.

<PCR method>
PCR may be performed under the usual conditions, for example, using Taq DNA polymerase, a heat denaturation step at 92 ° C. for 15 seconds, an annealing step at 60 ° C. to 65 ° C. for 15 seconds, and an extension step at 72 ° C. for 5 seconds to 15 seconds. Can be performed under the condition of repeating the above.

Determination of DNA amplification by PCR is performed by confirming the size of the generated amplified DNA by electrophoresis, or by immobilizing a reaction product on a nitrocellulose membrane or the like and using a labeled probe having a sequence complementary to the sequence of the target amplified DNA. A method of performing hybridization is possible. If you want to determine DNA amplification more easily, perform PCR using a primer with a identifiable label, capture the amplified DNA labeled with digoxigenin, etc. on the solid phase and identify it. Good.

Other methods for capturing and identifying amplified DNA include a method using a solid phase in which a substance that specifically binds to the labeled product of the primer is immobilized, and a capture probe having a sequence complementary to the sequence of the target amplified DNA. There is a method of preliminarily fixing to a solid phase and performing specific binding by hybridization, etc. As the solid phase for that purpose, a microtiter plate, beads, magnetic particles or the like can be used. The use of microtiter plates is preferred when processing multiple samples at once.

When testing a clinical specimen as a direct sample, the specificity and rapidity of the reaction can be enhanced by using hybridization with a capture probe immobilized on a microtiter plate or the like for capturing amplified DNA. Therefore, complementary DNA and DNA fragments that hybridize to the fudoh gene can be used as materials for capture probes and the like, so that the specificity and rapidity of the reaction can be improved.

The combination of the primer for detecting the fudoh gene and the mutation site detecting primer used in the present invention as described above is provided as a primer set for evaluating the pathogenicity of pathogenic bacteria, as well as an enzyme for DNA amplification and reaction. Provided as a kit for evaluating pathogenicity optimized for evaluating pathogenicity by being set with a DNA synthesis reagent such as a buffer, a labeled capture probe, or a microwell plate on which the capture probe is immobilized. it can. Moreover, it is also possible to provide a primer set or kit for performing an amplification method other than normal PCR by separately designing and combining primers suitable for the LAMP method or the like.

As described above, the present invention is useful in that the pathogenicity of pathogenic bacteria can be evaluated from an unconventional viewpoint. In particular, a so-called community-acquired MRSA with high pathogenicity in Japan has not been found a method capable of evaluating the pathogenicity of MRSA with high pathogenicity as a genotype. Under these circumstances, the fudoh gene, which is thought to encode a protein having a function of suppressing the gliding ability of pathogenic bacteria found by the present inventors, and a loss of function due to mutation of the gene, MRSA The fact that the pathogenicity is increased together with the increase in gliding ability makes it possible to clearly distinguish MRSA having high pathogenicity from ordinary MRSA by targeting the fudhoh gene and its mutation.

In addition, it can be used as a research tool for drug development, etc., because it can use simple and rapid detection means based on gene amplification methods that are undergoing rapid technological innovation. It is intended to provide a method for evaluating the pathogenicity of useful pathogenic bacteria that makes it possible to determine a treatment policy.

The “kit for evaluating pathogenicity of pathogenic bacteria” of the present invention is a method for confirming the presence or absence of a marker gene that can be evaluated for pathogenicity of pathogenic bacteria, or a marker gene thereof, It is assembled so that the presence or absence of mutation can be confirmed. That is, whether or not a gene encoding a protein that controls or suppresses the gliding ability of a pathogenic bacterium is present in the pathogenic bacterium to be evaluated, and if that gene exists, It is characterized by being a “kit for evaluating pathogenicity of pathogenic bacteria” that can confirm whether or not there is at least one mutation that affects or suppresses the function of the protein. .

Therefore, the “kit for evaluating pathogenicity of pathogenic bacteria” of the present invention only needs to contain at least a primer set designed for confirming the presence or absence of the marker gene. This is because the presence or absence of a mutation in the marker gene can be confirmed together with the presence of the marker gene by determining the base sequence of the PCR product amplified by the primer set using the same primer. Further, when the mutation to be detected is known, it is preferable that a primer set prepared for confirming the presence of the mutation in the marker gene is in the kit configuration. This is because the presence or absence of mutation can be confirmed more easily.

The above-mentioned “primer set prepared for confirming the presence of the mutation” includes, for example, a primer set compatible with ARMS (Amplification Refractory Mutation System). ARMS is a method that utilizes the fact that the function of PCR as a primer strongly depends on the matching between the 3 'end of the primer and the template DNA. Therefore, it is a feature of ARMS that the primer is designed so that the target mutation site comes to the 3 'end. In addition, when there is one mismatch due to mutation, annealing is often possible and amplification reaction often proceeds. At this time, at least one more upstream of the 3 ′ end of the primer It is preferable to design the primer so as to artificially introduce a mismatch. This is because the annealing cannot be performed well, the amplification reaction is impossible, and the specificity of mutation detection is improved (Kwok S. et al., Nucleic Acids Res 18, 999-1005 (1990)).

Using this phenomenon, a primer that has only an artificially introduced mismatch and can be annealed is created as a normal primer, and the primer introduced with the previous artificial mismatch is mutated in addition to the mismatch due to mutation. When it is prepared as a primer for PCR and a PCR reaction is performed, the presence or absence of mutation can be confirmed depending on whether or not an amplification reaction has occurred.

Specifically, for example, the following operation is performed, but the present invention is not limited to the following specific operation. That is, the test sample is divided into half amounts, PCR is performed by adding a normal primer and its counter primer to one, and PCR is performed by adding a mutation primer and its counter primer to the other. The normal primer hybridizes with the normal gene and PCR proceeds, but does not hybridize with the mutant gene, so the mutant gene is not amplified. Conversely, the mutation primer hybridizes with the mutant gene and proceeds with PCR, but does not hybridize with the normal gene, so the normal gene is not amplified. Therefore, it is possible to determine whether or not there is a target mutation on the DNA of the pathogenic bacterium to be evaluated by examining each band of amplified DNA by performing agarose electrophoresis after PCR.

When the pathogenic bacterium is Staphylococcus aureus, the marker gene encoding a protein that controls or suppresses the gliding ability of the pathogenic bacterium is “fudhoh” found by the present inventors, and its presence is confirmed. For example, a set of fudoh-F and fudoh-R primers shown in Table 2 can be used. In addition, as a mutation that loses the function of suppressing the sliding ability of the fudhoh gene, a single base mutation (K29R) at the site shown in FIG. 5 has been found. If incorporated in a kit, mutations can be detected more easily.

In addition, the kit of the present invention contains a primer set for identifying pathogenic bacteria, an internal control DNA for confirming that PCR was normally performed, and a primer set for detecting the DNA. Is preferred. For example, when the pathogenic bacterium is Staphylococcus aureus, an amplification primer set targeting a spa gene or the like for distinguishing S. aureus from other staphylococci, or whether S. aureus is methicillin resistant It is preferable to include a primer set for amplifying the mecA gene to determine whether or not. This is because the operation procedure of the entire test can be greatly simplified by adopting a kit configuration capable of performing multiplex PCR using these primer sets simultaneously.

Moreover, incorporating various reagents commonly used in the gene amplification method used, such as various buffer solutions for nucleic acid extraction and reaction, heat-resistant polymerases such as Taq polymerase and KOD polymerase, and dNTP, depending on the method, It is a matter of course in assembling a “kit for evaluating pathogenicity of pathogenic bacteria”. Furthermore, incorporating various solid phases such as a microtiter plate on which a probe for capturing amplified DNA is immobilized into the “kit for evaluating pathogenicity of pathogenic bacteria” of the present invention makes it easy to test, specificity, etc. Is more preferable.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. However, the present invention is not limited to these examples and the like as long as the gist thereof is not exceeded.

<Preparation of test materials, etc.>
Plasmid:
E. coli strain JM109 was used for the preparation of pND50, pCK20 plasmids and their derivatives. E. coli transformed with the above plasmid was cultured at 37 ° C. in LB (Luria-Bertani) liquid medium containing 25 μg / mL chloramphenicol.

S. aureus genomic DNA:
Extraction of S. aureus genomic DNA was performed using QIAamp DNA Blood Kit (Qiagen).

Introduction of plasmid into S. aureus strain:
The electroporation method was used.

Preparation of SCCmec region dropout strain:
The ccrAB gene encoding the SCCmec region excision enzyme was inserted into the pND50 plasmid to obtain a pcccAB plasmid. This plasmid was introduced into the MRSA strains NI-3, NI-4 and NI-5, which have low gliding ability, to obtain a chloramphenicol resistant strain, 5 μL of which was added with 5 mL of chloramphenicol. The cells were cultured overnight in a TSB (Tryptic Soy Broth) liquid medium. After repeating the same operation, a single colony was obtained on a TS (Tryptic Soy) agar medium. This strain was used as a SCCmec region-removed strain in the subsequent studies.

Insertion of SCCmec region into Newman strain:
The Newman strain is a laboratory-owned strain of the University of Tokyo Graduate School of Microbial Medicine Chemistry, which is a MSSA strain and a high gliding ability. A plasmid having regions containing the mecA gene, mecR1 gene, and mecI gene in the SCCmec region was inserted into the chromosome of the Newman strain and used for further studies.

Silkworm:
Incubated from fertilized eggs in the laboratory, and grown to 5th instar larvae with artificial diet silk mate (manufactured by Katakura Kogyo) was used for pathogenicity evaluation of genetically modified strains.

mouse:
Eight-week-old female CD-1 mice (Charles River Laboratories) were used to evaluate the pathogenicity of the strain in the same manner as silkworms.

Strains, plasmids, primers:
Table 1 shows a list of strains and plasmids used in Examples and Test Examples, and Table 2 shows a list of primers.

<Test Example 1> Examination of sliding ability of clinically isolated Staphylococcus aureus strains 10 methicillin-sensitive Staphylococcus aureus (MSSA) strains isolated from clinical specimens and methicillin isolated and provided from clinical specimens at Nihon Medical University Hospital 40 resistant S. aureus (MRSA) strains were used for further studies.

[Measurement of sliding ability]
Each strain was cultured overnight at 37 ° C. in TSB liquid medium, and then 12.5 μg / mL chloramphenicol was added. 2 μL of the previous bacterial solution is spotted on 50 mL of TSB medium containing 0.24% agar that has been dried 20 minutes prior to bacterial cell inoculation in a plastic sterile petri dish with a diameter of 15 cm. After drying for 15 minutes, the cells were cultured at 37 ° C. for 10 hours. The same test was performed twice, the diameter of the colony formed in each test was measured, and the average value was defined as the sliding ability. The obtained results are shown in FIG.

As a result, all 10 MSSA strains were clearly sliding on the soft agar medium, and the average value of the sliding distance (sliding ability) was 67.7 mm. On the other hand, the average value of the gliding ability of 40 MRSA strains was 31.7 mm. In addition, 29 of the 40 MRSA strains had a sliding ability of 35 mm or less, and it was found that most of the MRSA strains decreased the sliding ability. From this result, it was found that the gliding ability was decreased in many MRSA strains compared with the MSSA strain.

<Test Example 2> Glidability study of MRSA from which the SCCmec region has been removed The SCCmec region is a region where the mecA gene, which is a gene conferring methicillin resistance, is present. In order to investigate whether or not the cause of the MRSA strain reducing gliding ability is in the SCCmec region, the SCCmec region was dropped from the MRSA strain, and the gliding ability was examined. In the examination, the ccrAB gene encoding the excision enzyme of the SCCmec region was expressed in NI-3, NI-4, and NI-5 strains with low sliding ability, and the SCCmec region was dropped. The PCR method confirmed that the mecA gene was not present in these strains (FIG. 2), and that the resistance to methicillin was reduced from 4 μg / mL to 2 μg / mL. Furthermore, these strains had increased gliding ability compared with the original strain (FIGS. 3A and 3B). From this result, it was considered that the gliding ability of MRSA was suppressed by the SCCmec region.

<Test Example 3> Examination of genes related to gliding ability suppression in SCCmec region The mecA gene, mecR1 gene, mecI of SCCmec region after pInt plasmid shown in Table 1 on the chromosome of Newman strain which is MSSA strain not having SCCmec region Each plasmid (FIG. 4 (A)) having a region including the vicinity of the gene was inserted, and its sliding ability in the soft agar medium was examined. The examination of the sliding ability was performed in the same manner as in Test Example 1. As a result, the strain into which the region containing three genes and the vicinity thereof (pIntmecAR1I-fudhoh) was introduced had reduced gliding ability compared to the strain into which the empty vector pInt was introduced (FIGS. 4B and 4C). .

Next, it was considered that any of the above three genes contributed to the gliding ability, and plasmids having the respective genes were prepared and introduced into the Newman strain to examine the gliding ability. In the study using the high expression system, the region containing the mecA gene (pInt mecA) that suppressed the sliding ability did not suppress the sliding ability in the normal expression system in this example. On the other hand, plasmids obtained by removing either mecR1 or mecI from the region including both mecR1 and mecI genes and the vicinity thereof, and plasmids excluding both of them suppressed the gliding ability contrary to expectation (FIG. 4 (B), FIG. 4 (C)). From this result, it was found that the gene that suppresses the gliding ability in the SCCmec region is not any of the above three genes.

<Example 1> Suppression of gliding ability by introduction of fudoh gene Since the result of Test Example 3 suggests the presence of a gene not known so far, the ORF on the SCCmec region was examined in detail. Then, in the genome database of S. aureus N315 strain, an ORF encoding 70 amino acids not registered as ORF was found (upper right right portion of FIG. 4A). Therefore, a plasmid (pInt fudoh) having this gene was introduced into a Newman strain, and its gliding ability was examined. As a result, the Newman strain into which pInt fudoh was introduced had suppressed gliding ability (FIG. 4 (B), FIG. 4 (C) fudoh-introduced strain). The present inventor named the novel gene having a function of suppressing the sliding ability as “fudh”.

<Example 2> Nucleotide sequence of fudoh gene possessed by MRSA clinical isolates 11 out of 40 clinically isolated MRSA strains show high gliding ability. Therefore, in order to know whether or not the high gliding ability can be explained by the mutation of the fudh gene, the nucleotide sequences of the fudh gene of all 40 MRSA strains were determined. The results and the gliding ability shown by each strain are shown in FIG.

[Primer synthesis and labeling]
Primer sets (Table 2, fudhoh-F, fudhoh-R) were designed by a conventional method so that they could be annealed with a specific base sequence contained in the fudhoh gene, and synthesized with a DNA synthesizer.
Producing various labeled primers such as biotin label and dinitrophenyl label by introducing an amino group into the 5 'terminal base of this primer using Aminolink II (Trademark: Applied Biosystems Japan). Can do.

[PCR]
The following reaction solution composition was used.
10 × Buffer for KOD polymerase, 5 μL
25 mM dNTPs, 5 μL
fudh-F primer 10μM stock, 0.75μL
fudh-R primer 10 μM stock, 0.75 μL
5U / μL KOD polymerase, 1μL
25 mM MgCl _{2 2} μL
1 μL of template DNA, 50 ng
Water 34.5μL Total 50μL

[PCR implementation]
The reaction was carried out using a PC-350 gene amplification apparatus manufactured by ASTEC under the following reaction conditions.
DNA denaturation: 1st 94 ° C, 120 sec
From the second time 94 ℃, 15sec
Annealing: 52 ° C, 30 sec
Extension reaction 72 ℃, 60sec
The above reaction was performed for 25 cycles.
Confirmation of the obtained PCR product was performed by agarose gel electrophoresis.

[Confirmation of mutation]
The determination of the base sequence of the fudhoh gene and the presence or absence of mutation were performed using the S2 and S3 primers in Table 2 as sequencing primers.

In 10 out of 11 strains with high gliding ability, a single nucleotide mutation involving amino acid substitution in the portion corresponding to amino acid number 29 of the fudh gene (the amino acid corresponding to amino acid number 29 of SEQ ID NO. It was revealed that a substitution mutation) occurred. In addition, the fudhoh gene was not present in one strain (FIG. 5, NI-15 strain). Furthermore, from these 11 strains (FIG. 5, from NI-26 strain to NI-36 strain), no mutation was found in the fudh gene in the remaining 29 strains with low sliding ability.

In addition, as described above, it is an evaluation kit for using the method for evaluating the pathogenicity of pathogenic bacteria, and a primer for detecting a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria, or It was found that a kit for evaluating the pathogenicity of pathogenic bacteria having at least one primer for detecting a mutation that suppresses the function of the previous protein in the gene can be actually produced.

<Example 3> Gliding ability of Newman strain introduced with mutant fudhoh gene In order to examine whether the previous mutant fudhoh gene (K29Rfudhoh) has lost the ability to suppress gliding ability, K29Rfudhoh was introduced into a Newman strain with high gliding ability. In the same manner as in Example 1, the ability to suppress the sliding ability was confirmed. As a result, K29Rfudhoh did not inhibit the sliding ability of the Newman strain at all (FIGS. 6A and 6B).

From these results and the results of Example 1, it was clarified that the high gliding ability of 11 strains can be explained by the mutation of the fudhoh gene in 10 strains and the absence of the fudhoh gene in 1 strain.

<Example 4> Pathogenicity to silkworms of MRSA clinical isolates in which the SCCmec region has been lost and the fudhoh gene has been lost, and the MSSA strain into which the fudhoh gene has been introduced Next, the relationship between the gliding ability of S. aureus and the pathogenicity to the host For the purpose of studying the silkworm of the NI-5 strain (parent strain MRSA strain) whose gliding ability was increased by dropping the SCCmec region, and conversely, the Newman strain (parent strain MSSA strain) whose gliding ability was suppressed by introduction of the fudh gene. Pathogenicity was examined.

<Example 4-1> Pathogenicity of SCCmec-dropped MRSA strain against silkworms NI-5 strain or SCCmec-dropped NI-5 strain (1.9 × 10 ⁴ each) in which control vector pND50 was introduced into 10 5-year-old silkworms per group 50 μL of cfu / overnight culture was diluted with physiological saline) and injected into the blood. The physiological saline injection group was used as a sterile control. Each silkworm was fasted and kept at 37 ° C., and the survival rate per hour after injection was monitored. The results are shown in FIG. 7A.

The time from the administration of the strain to the death of the silkworm injected with the SCCmec-dropped NI-5 strain was clearly shorter than that of the silkworm injected with the parent strain, and the SCCmec-dropped NI-5 strain was highly pathogenic to the silkworm. SCCmec dropout NI-5 strain has been confirmed to increase gliding ability in Test Example 2 (Fig. 3 (A), Fig. 3 (B)), and the increase in gliding ability due to SCCmec loss increases the pathogenicity against silkworms. The possibility of giving results was suggested.

<Example 4-2> Pathogenicity of fudoh-introduced MSSA strains against silkworms: Newman strain in which empty vector pInt has been introduced into 10 groups of five-year-old silkworms or Newman strains into which fudoh gene has been introduced (each 1.5 × 10 ⁴ cfu) / The overnight culture was diluted with physiological saline) and 50 μL was injected into the blood. The physiological saline injection group was used as a sterility control. Silkworms were fasted and kept at 40 ° C., and the survival rate per hour after injection was monitored. The result is shown in FIG. 7B.

The death of silkworms injected with the Fudhoh-introduced Newman strain was clearly delayed compared to silkworms injected with the parent strain, confirming that pathogenicity against silkworms was reduced. In Test Example 3, it was confirmed that when fudoh was introduced into the Newman strain, the gliding ability was suppressed (FIGS. 4B and 4C), and the reduction of gliding ability due to the introduction of fudhoh is a pathogenicity against silkworms. It was suggested that this may have led to a decrease in From this result, it is considered that the pathogenicity of the result of Example 4-1 was increased by the removal of fudhoh in the SCCmec region.

<Example 5> Pathogenicity of fudoh-introduced MSSA strains to mice (sepsis model): Newman strain in which empty vector pInt was introduced into CD-1 mice of 5 weeks / group 8 mice or newman strain into which fudoh was introduced (2. 9 × 10 ⁷ cfu / overnight culture diluted with PBS) 100 μL was injected into the tail vein. The PBS injection group was used as a sterile control. Mice were raised according to a normal mouse breeding protocol, and the survival rate per hour after the injection was monitored. As a result, the death of the mice injected with the fudhoh-introduced Newman strain was clearly delayed compared to the mice injected with the parent strain (FIG. 8). This result shows that fudhoh acts on the pathogenicity of Staphylococcus aureus to mice as well as silkworms.

Based on the above results, the newly discovered fudoh gene has a function of suppressing the ability of Staphylococcus aureus to slide, and is also involved in the high pathogenicity of Staphylococcus aureus. On the other hand, it was confirmed that it resulted in a decrease in the pathogenicity of both mice and mice. This indicates that the high pathogenicity of the pathogenic bacteria to the host can be evaluated by performing a means for detecting the presence or absence of fudhoh in the pathogenic bacterial genome.

In addition, as shown in Example 3, since S. aureus having a mutated fudoh gene has increased gliding ability, it is considered that S. aureus having a mutated fudoh gene has increased pathogenicity to the host. It is done. Performing a means for detecting the presence or absence of a single base mutation that results in substitution of the amino acid of the 29th amino acid part of fudhoh found in the present invention is similar to performing a means for detecting the presence or absence of fudoh itself. It is clear that this is a method for evaluating the high pathogenicity of pathogenic bacteria to a host. Similarly, even when a new mutation is found in fudoh, using the phenotype “sliding ability” as a clue, it is easy to detect the mutation, and the detection of the mutation The pathogenicity of pathogenic bacteria can be evaluated.

Furthermore, the present invention is not limited to the invention relating to the specific “fudhoh gene”, but extends to the invention relating to “a gene encoding a protein having a function of controlling the gliding ability of pathogenic bacteria”. It is also clear from the basic inventive properties of the invention.

The pathogenicity evaluation method for pathogenic bacteria of the present invention evaluates the high pathogenicity of pathogenic bacteria that could not be evaluated for the high pathogenicity depending on the genotype related to conventional toxins and drug resistance. Therefore, it is widely used in fields where new drugs and treatment methods for pathogenic bacteria are being developed. In addition, by clarifying the genes used for pathogenicity assessment and the mutations in those genes, it is possible to use any gene amplification method targeting them, so clinical trials that require rapid and accurate result determination are required. Since an evaluation kit based on the principle of a method for evaluating a pathogenic bacterium or a gene amplification method can be provided at the site, it is widely used in the clinical field.

This application is based on Japanese Patent Application No. 2008-158176 filed on Jun. 17, 2008, the entire contents of which are incorporated herein by reference as the disclosure of the specification of the present invention. It is what

Claims (11)

A method for evaluating the pathogenicity of pathogenic bacteria, the presence or absence of a gene encoding a protein having a function of controlling the gliding ability of the pathogenic bacteria, or a mutation that affects the function of the preceding protein in the gene A pathogenicity evaluation method characterized by checking whether or not there is at least one. A method for evaluating the pathogenicity of a pathogenic bacterium, wherein the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of the pathogenic bacterium, or a mutation that suppresses the function of the preceding protein in the gene The method for evaluating pathogenicity according to claim 1, wherein whether or not there is at least one is confirmed. The method for evaluating pathogenicity according to claim 1, wherein the pathogenic bacterium is methicillin-resistant Staphylococcus aureus. 3. The method for evaluating pathogenicity according to claim 2, wherein the pathogenic bacterium is methicillin-resistant Staphylococcus aureus. A method for evaluating the pathogenicity of methicillin-resistant Staphylococcus aureus, the presence or absence of a gene encoding a protein having a function of suppressing the gliding ability of methicillin-resistant Staphylococcus aureus, or the sequence described in SEQ ID NO: 2 The method for evaluating pathogenicity according to claim 4, wherein it is confirmed whether or not there is a mutation in the base sequence corresponding to amino acid number 29. The gene encoding a protein having a function of suppressing the gliding ability of a pathogenic bacterium is a DNA comprising the base sequence set forth in SEQ ID NO: 1, or any one of claims 4, 4 and 5 The pathogenicity evaluation method according to claim. An evaluation kit for using the pathogenicity evaluation method according to claim 1 or 2, wherein the primer detects a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria. Or a kit for evaluating the pathogenicity of pathogenic bacteria, comprising at least one primer for detecting a mutation that suppresses the function of the previous protein in the gene. An evaluation kit for using the pathogenicity evaluation method according to claim 4 or 5, for detecting a gene encoding a protein having a function of suppressing gliding ability of methicillin-resistant Staphylococcus aureus. Or at least one primer for detecting a mutation that suppresses the function of the previous protein in the gene. An evaluation kit for using the pathogenicity evaluation method according to claim 6, comprising a primer for detecting a gene encoding a protein having a function of suppressing the gliding ability of pathogenic bacteria, or the A kit for evaluating the pathogenicity of pathogenic bacteria, comprising at least one primer for detecting a mutation that suppresses the function of a previous protein in a gene. A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the base sequence described in SEQ ID NO: 1 and having a function of suppressing the gliding ability of pathogenic bacteria and encoding a protein involved in the pathogenicity of the fungus;
(B) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1; A gene consisting of the amino acid sequence set forth in SEQ ID NO: 2 and having a function of suppressing the gliding ability of pathogenic bacteria and encoding a protein involved in the pathogenicity of the fungus.

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