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WO2017171410A4 - Biomarker for predicting susceptibility of staphylococcus aureus and use thereof - Google Patents

Biomarker for predicting susceptibility of staphylococcus aureus and use thereof Download PDF

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Publication number
WO2017171410A4
WO2017171410A4 PCT/KR2017/003449 KR2017003449W WO2017171410A4 WO 2017171410 A4 WO2017171410 A4 WO 2017171410A4 KR 2017003449 W KR2017003449 W KR 2017003449W WO 2017171410 A4 WO2017171410 A4 WO 2017171410A4
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staphylococcus aureus
gene
smp
susceptibility
antibiotics
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French (fr)
Korean (ko)
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WO2017171410A1 (en
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장수진
이윤미
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Institut Pasteur Korea
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Institut Pasteur Korea
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a biomarker for predicting the susceptibility of Staphylococcus aureus and a use thereof, and more particularly, to a composition, a kit, a method for predicting the susceptibility of Staphylococcus aureus to antibiotics using the biomarker, There is provided a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus aureus comprising a screening method of an antibiotic substance susceptible to Staphylococcus aureus and a compound screened therefrom.
  • S. aureus which is the most problematic in the world today, is a Gram-positive bacterium that is detected in about 30% of the total population and is widely known as a pathogenic agent for infectious diseases in hospitals.
  • the bacterium firstly infects skin and soft tissues, causing pyoderma, and has been reported to cause serious life-threatening systemic infections such as osteomyelitis, endocarditis, sepsis and bacteremia.
  • Penicillin and methicillin have been used for several decades as a major therapeutic agent for this infectious disease.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSA MRSA was first reported by the Jevons in the UK in 1961 and has been reported from around the world. Since then, the rate of MRSA has increased by 1-2% in many European countries over the next 20 years, Of the total. In Japan, 59.1-67.3% were isolated in the early 1990s. In Korea, the isolation rate of MRSA gradually increased, reaching an average of 62.1% in 1995, exceeding 50%. In recent years, more than 76% It is showing. The reason for the increase in the MRSA isolation rate is not only the infection in the hospital but also the development of traffic and the use of antibiotics irresponsibly.
  • VRSA Vancomycin-intermediate S. aureus
  • pathogens are obtained by self-defense by acquiring a mutation or an antibiotic resistance gene for resistance to antibiotics.
  • the incidence of these resistant bacteria is increasing with the abuse and abuse of antibiotics.
  • MRSA mec A (mobile genetic element A) resistant gene PCR method to prevent abuse of antibiotics.
  • the PCR method requires about 1 day of reaction time, (2011) Vol. 47, No. 4, pp. 381-385), which can detect only the resistance to methicillin.
  • the inventors of the present invention conducted various studies to overcome the disadvantages of the prior art and developed a novel method for preventing abuse of antibiotics, and found that a virtual small membrane protein (Small), which is encoded by a gene stored only in Staphylococcus aureus Membrane Protein, SMP) is associated with antibiotic susceptibility.
  • Small virtual small membrane protein
  • the present invention provides a biomarker for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance including an SMP (Small Membrane Protein) gene.
  • SMP Small Membrane Protein
  • Another object of the present invention is to provide a composition for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising an agent for measuring the expression level of SMP.
  • It is another object of the present invention to provide a method for producing Staphylococcus aureus comprising: (a) introducing into a Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) treating the Staphylococcus aureus of step (a) with an antibiotic and measuring whether or not the Staphylococcus aureus emits light.
  • the present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance.
  • It is another object of the present invention to provide a method for producing Staphylococcus aureus comprising: (a) introducing into a Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) treating the candidate Staphylococcus aureus of step (a) with a candidate substance and measuring whether or not the staphylococcus aureus emits light.
  • the present invention also provides a screening method for antibiotics susceptible to Staphylococcus aureus.
  • Another object of the present invention is to provide an effective amount of a compound screened by the above-described screening method or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus aureus comprising a pharmaceutically acceptable carrier.
  • the frequency of the expression of resistant bacteria is further increased as the abuse and abuse of antibiotics increases, and it is urgent to develop a method for preventing misuse of antibiotics.
  • the present invention finds that a hypothetical small membrane protein (SMP) encoded by a gene stored only in Staphylococcus aureus is associated with antibiotic susceptibility, and antibiotics including SMP gene
  • SMP small membrane protein
  • the present inventors searched for a solution to the above problem by providing a biomarker for predicting the susceptibility of S. aureus. It is possible to provide treatments for individual patients with Staphylococcus aureus infection and to prevent misuse of antibiotics by appropriate antibiotic administration for each patient.
  • SMP Mal Membrane Protein, SMP
  • SMP Small Membrane Protein
  • SA_13280 gene which is stored only in Staphylococcus aureus, and its function is not yet known.
  • the SMP protein and gene sequences can be obtained from known databases. Specifically, the SMP protein sequence is the NCBI Reference sequence NO.
  • the SMP gene sequence may be the one disclosed in GenBank No. 1, pp. It may be one disclosed in CP013137.1. In one embodiment of the present invention, the SMP gene comprises SEQ ID NO: 1.
  • biomarker of the present invention means a molecule quantitatively or qualitatively related to the presence of a biological phenomenon.
  • the marker may be derived from a genomic nucleotide sequence or from a nucleotide sequence expressed (e.g., from RNA, nRNA, mRNA, cDNA, etc.), or from a coded polypeptide.
  • the term includes nucleic acid sequences that are complementary to or flanked in the marker sequence, such as a probe or a pair of primers that can amplify the marker sequence.
  • the biomarker of the present invention can be an indicator of the susceptibility of Staphylococcus aureus to antibiotics and is excellent in accuracy and reliability as a susceptibility marker of Staphylococcus aureus to antibiotics, It is possible.
  • " susceptibility " in the present invention means whether or not a particular antibiotic substance is effective against Staphylococcal aureus infectious disease in an individual patient.
  • the term " prediction" refers to predicting medical consequences, and for the purpose of the present invention, it is contemplated in advance to determine whether a particular antibiotic has an effect on Staphylococcal Staphylococcal infections in an individual patient it means.
  • the antibiotic substance is a substance produced by microorganisms and other organisms, and can be interpreted as affecting the biological activity of the organism, and mainly represents an antibiotic.
  • These antibiotics may or may not have an effect depending on the type of Staphylococcus aureus.
  • the susceptibility of antibiotics to antibiotics depends on the type of Staphylococcus aureus. According to the present invention, if a patient (a responder) who can expect an effect before initiation of treatment and a patient (a non-responder) who can not expect an effect can be predicted, a treatment method with high efficacy and safety can be realized while preventing misuse of antibiotics .
  • the present invention also provides a composition for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising an agent for measuring the expression level of SMP (Small Membrane Protein).
  • SMP Small Membrane Protein
  • agent for measuring the expression level in the present invention means a molecule that can be used for confirming the expression level of the biomarker gene of the present invention or a protein encoded by these genes, A probe or antisense nucleotide, or an antibody specific for a protein encoded by the biomarker gene.
  • the level of expression can be understood to mean the level of expression, such as DNA level, mRNA level, protein level, and the like.
  • antibody as used herein in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art.
  • an antibody refers to an antibody that specifically binds to a marker of the present invention, which clones each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene , And can be prepared from the obtained protein by a conventional method.
  • partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids.
  • the form of the antibody of the present invention is not particularly limited and any part thereof having a polyclonal antibody, a monoclonal antibody or an antigen-binding property is included in the antibody of the present invention and includes all the immunoglobulin antibodies.
  • the antibodies of the present invention include special antibodies such as humanized antibodies.
  • the antibody against the protein encoded by the biomarker gene of the present invention may be any antibody that can be produced by a method known in the art.
  • an antibody used in the detection of the susceptibility prediction marker of the present invention may comprise a complete form having two full-length light chains and two full-length heavy chains as well as a functional fragment of the antibody molecule.
  • the functional fragment of the antibody molecule refers to a fragment having at least an antigen binding function and may be Fab, F (ab ') 2, F (ab') 2, Fv or the like, but is not particularly limited thereto.
  • primer in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template, and a starting point for copying the template ≪ / RTI >
  • the sensitivity of Staphylococcus aureus to antibiotics can be predicted by performing PCR amplification using the sense and antisense primers of the marker polynucleotide of the present invention to produce a desired product.
  • the PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.
  • a forward primer represented by SEQ ID NO: 2
  • a reverse primer represented by SEQ. ID. NO: 3.
  • probe in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can specifically bind to mRNA and is labeled The presence or absence of a specific mRNA can be confirmed.
  • the probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.
  • the hybridization using the probe complementary to the marker polynucleotide of the present invention can predict the susceptibility of Staphylococcus aureus to antibiotics through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
  • the primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods.
  • Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkers e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • the present invention also provides a kit for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising the aforementioned composition for predicting susceptibility.
  • the kit of the present invention can be used for predicting the susceptibility of Staphylococcus aureus to antibiotics by confirming the mRNA expression level of the biomarker gene for susceptibility prediction or the expression level of the protein encoded by these genes.
  • the kit for measuring the mRNA expression level of the biomarker genes may be a kit containing essential elements necessary for performing RT-PCR.
  • RT-PCR kits can be used to detect and isolate specific primer pairs specific to the marker gene, including test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- DEPC-water, sterile water, and the like.
  • the kit of the present invention may be a kit for detecting a biomarker for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance including essential elements necessary for carrying out a DNA chip.
  • the DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.
  • a protein chip kit for measuring the expression level of a protein encoded by the biomarker gene may be used for immunological detection of the antibody.
  • the kit includes a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, And the like.
  • the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc.
  • the chromogenic enzyme may be peroxidase, Alkaline phosphatase can be used, and fluorescent materials such as FITC, RITC and the like can be used.
  • the coloring substrate liquid is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) and the like can be used.
  • the present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to antibiotics.
  • the term "measurement of mRNA expression level" refers to the measurement of mRNA level and expression level of a biomarker gene in a biological sample in order to predict the susceptibility of Staphylococcus aureus to antibiotics. .
  • RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection method, northern blotting, Or DNA chip technology but the present invention is not limited thereto.
  • the term "measurement of protein expression level" refers to a process for confirming the presence and expression level of a protein expressed in a marker gene in a biological sample to predict the susceptibility of Staphylococcus aureus to antibiotics, The amount of protein can be confirmed using an antibody that specifically binds to the protein expressed in the gene.
  • Analysis methods include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunization Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS (Immunohistochemical staining), immunofluorescence (immunohistochemical staining), immunoprecipitation assays fluorescence activated cell sorter analysis, or protein chip technology, but the present invention is not limited thereto.
  • the term " biological sample isolated from a patient with Staphylococcus aureus infection" refers to all samples obtained from an individual from which expression of the biomarker of the present invention can be detected, including but not limited to patients with Staphylococcus aureus infection Tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.
  • the expression level of the biomarker gene may be measured at an mRNA level or a protein level, and the mRNA or protein may be separated from the biological sample using a known process.
  • the analysis method for measuring the mRNA level and the assay method for measuring the protein level are as described above.
  • the susceptibility to the specific antibiotic of Staphylococcus aureus infected with the patient can be predicted through the expression of the biomarker gene for predicting the susceptibility measured from the sample of the patient who intends to predict the susceptibility to the specific antibiotic substance have. That is, when the biomarker is expressed (when SMP is expressed), the Staphylococcus aureus infected with the patient can be judged as susceptible to the treated antibiotic substance.
  • the susceptibility of Staphylococcus aureus to a specific antibiotic can be accurately predicted, and an appropriate treatment plan can be established according to the patient's suitability according to the result of the susceptibility prediction.
  • the present invention also provides a method for screening antibiotics susceptible to Staphylococcus aureus .
  • the Staphylococcus aureus in the sample can be judged to be susceptible to the candidate substance to be treated.
  • the candidate substance can be determined not only by existing antibiotics, It includes all substances that are intended to measure the inhibition potential of the bacteria.
  • the above screening method can be performed on the same principle as the above-described sensitivity-sensitive prediction method, so redundant contents are omitted in order to avoid excessive complexity of the present specification.
  • strain 4 showed SMP induction with ciprofloxacin, vancomycin and tetracycline in agreement with its antibiotic susceptibility profile, but did not show SMP induction in methicillin treatment (Fig. 5).
  • the present invention also provides a method of producing a Staphylococcus aureus strain comprising the steps of: (a) introducing into a separate Staphylococcus aureus gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to expression by said promoter; And (b) treating the Staphylococcus aureus of step (a) with antibiotics and measuring the emission of Staphylococcus aureus.
  • the present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to antibiotics.
  • the method of introducing the gene construct into Staphylococcus aureus can be carried out by selecting a suitable standard technique known in the art, for example, electroporation, calcium phosphate co-precipitation ), Retroviral infection, microinjection, DEAE-dextran, cationic liposome, and the like, but the present invention is not limited thereto.
  • the Staphylococcus aureus of step (b) When the Staphylococcus aureus of step (b) is luminescent, the Staphylococcus aureus can be judged to be susceptible to the treated antibiotic substance.
  • a gene construct comprising a SMP promoter of SEQ ID NO: 4 and a green fluorescent protein (GFP) gene operably linked to the promoter to be expressed by the promoter is introduced into isolated Staphylococcus aureus, And the sensitivity of Staphylococcus aureus was predicted by measuring the emission of Staphylococcus aureus (Fig. 6).
  • GFP green fluorescent protein
  • a green fluorescent protein (GFP) gene is used as a reporter gene, but any commonly used reporter gene can be used without limitation.
  • a reporter gene may be a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a blue fluorescent protein (BFP) gene, a luciferase gene, and a lactase gene.
  • the time during which the light emission of the step (b) is detected may be different depending on the type of the antibiotic to be treated, specifically the type of the antibiotic, and may vary depending on the mechanism of the antibiotic substance. Can be detected within 1 to 5 hours.
  • the present invention also provides a method of producing a Staphylococcus aureus strain comprising the steps of: (a) introducing into a separate Staphylococcus aureus gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to expression by said promoter; And (b) measuring the presence or absence of the staphylococcus aureus after treating the candidate substance with Staphylococcus aureus in step (a), wherein the method comprises screening for antibiotic substances susceptible to Staphylococcus aureus .
  • the above screening method can be performed on the same principle as the method for predicting the susceptibility of Staphylococcus aureus using the above-described SMP promoter-based reporter system, so redundant contents are omitted in order to avoid the excessive complexity of the present specification.
  • Example 5 an experiment was conducted as described in Example 5 to verify the method for predicting the susceptibility of Staphylococcus aureus or the method for screening antibiotics using the SMP promoter-based reporter system.
  • MIC 50 minimum inhibitory concentration
  • the SMP promoter-based reporter system Compared with conventional culturing-based MIC 50 measurements that take 24 hours, the SMP promoter-based reporter system shortens the assay time with similar accuracy, so that the susceptibility prediction method and the antibiotic screening method using the SMP promoter of the present invention are specific It is a good estimation system for predicting the susceptibility of Staphylococcus aureus to antibiotics and for screening antibiotics against Staphylococcus aureus.
  • the present invention also relates to an effective amount of a compound screened by the screening method or a pharmaceutically acceptable salt thereof; And pharmaceutical compositions for preventing or treating infection by Staphylococcus aureus comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the administration mode.
  • a pharmaceutically acceptable carrier such as sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and any of the above components, And other additives conventionally used, such as buffers.
  • diluents can be added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets,
  • injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets
  • a target organ or tissue specific antibody or other ligand can be used in combination with the carrier.
  • the carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.
  • composition or the mixture may be appropriately administered to a subject according to the conventional method, route of administration, and dose used in the art depending on the purpose or necessity.
  • administration route include oral, non-oral, subcutaneous, Intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally,
  • Non-oral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Appropriate dosages and times of administration may be selected according to methods known in the art, and the amount and frequency of administration will depend on the type of symptoms to be prevented or treated, the route of administration, sex, health status, diet, And weight, and the severity of the disease.
  • the biomarker for predicting the susceptibility of Staphylococcus aureus to antibiotics containing the SMP gene provided by the present invention is excellent in the accuracy and reliability as a susceptibility marker of Staphylococcus aureus against antibiotics, It is possible to provide a therapeutic agent, and appropriate antibiotic administration for each patient can prevent abuse of antibiotics.
  • the SMP promoter-based reporter system provided in the present invention can obtain the results within 1 to 5 hours while maintaining similar accuracy as compared with the conventional 24-hour culture-based MIC 50 measurement, . Therefore, the reporter system of the present invention can promptly and accurately predict the susceptibility of Staphylococcus aureus to a specific antibiotic substance, and can also rapidly and accurately screen antibiotic substances showing susceptibility to Staphylococcus aureus.
  • Figures 1A and 1B are graphs showing that SMP is induced in Staphylococcus aureus in dependence of treatment time and treatment capacity of ciprofloxacin.
  • Fig. 2 is a graph showing RT-qPCR results for measuring SMP gene.
  • Figure 3 is a table showing susceptibility to various antibiotics by testing six different Staphylococcus aureus strains including one pan-susceptible strain and five clinical isolates having different antimicrobial susceptibility profiles.
  • Figures 4 and 5 show the results of RT-qPCR and RT-qPCR after treatment of various antibiotics with six different strains of S. aureus, including one pan-sensitive strain of RT-qPCR and five clinical isolates with different antimicrobial susceptibility profiles. In which SMP induction was measured.
  • FIG. 6 shows a schematic concept of a gene construct containing an SMP promoter and a GFP gene and a reporter system based on the SMP promoter using the same.
  • FIG. 7 shows the results of verifying the method for predicting the susceptibility of Staphylococcus aureus or the screening method for antibiotics using the reporter system of FIG.
  • Staphylococcus aureus was grown in Mueller hinton medium overnight at 37 ° C with shaking at 180 rpm. The culture was re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. Staphylococcus aureus was then diluted to give an absorbance (optical density) of 0.05 at 600 nm in a 125 mL flask. Antibiotics were treated at the indicated concentrations. The flask was incubated at 37 [deg.] C at 180 rpm for the indicated time.
  • Bacteria were harvested by centrifugation at 3300 rpm for 10 min and resuspended in 120 ul of lysis buffer (100 ul of 50 mM EDTA, 10 ul of 10 mg / ml lysozyme, 10 ul of 10 mg / ml resource taffine). Cultured in a 37 ° C water bath for 30 minutes, vortexed every 10 minutes and crushed by sonication. Thereafter, the extraction procedure was performed according to the RNeasy mini kit (Qiagen) protocol and 50 ul of RNA elute was obtained. After extraction of total RNA, the remaining genomic DNA was removed using DNase 1 (Qiagen).
  • the reaction volume was 10 ul and consisted of 1 ul buffer RDD, 0.25 ul of DNas I stock solution and 6 ul of 50 ng / ul RNA. The mixture was incubated for 10 min at room temperature and 40 ul of RNase-free water was added to stop the reaction.
  • RNA sequencing was performed on an Illumina HiSeq 2500 platform using single-ended 50bp sequencing.
  • Sequence data for the standard genome were retrieved from the NCBI database. Quality-filtered reads were sorted according to the standard-genome sequence using Bowtie2. Relative transcript abundance is measured in fractions of a reading per kilobase (FPKM) of exon sequences per million mapped sequence reads.
  • FPKM kilobase
  • EggNOG evolutionary genealogy of genes
  • KEGG Keto Encyclopedia of Genes and Genomes
  • Visualization of the differentially expressed gene (DEG) analysis was performed using the CLRNASeq TM program (ChunLab, South Korea).
  • SA_13280 gene encodes a hypothetical small membrane protein (SMP) as shown in Fig. 1A, and is stored only in S. aureus .
  • Transcript analysis by RNA sequencing of this example also revealed that SMP is induced in S. aureus depending on the treatment time and treatment capacity of ciprofloxacin (Fig. 1B).
  • RT-qPCR of this example shows that SMP induction is induced by ciprofloxacin treatment and that other antibiotics including vancomycin, methicillin and tetracycline also induce SMP.
  • SMP induction was associated with antibiotic susceptibility, and to see SMP induction, one pan-sensitive strain and six other Staphylococcus aureus strains including five clinical isolates with different antimicrobial susceptibility profiles Were tested as follows.
  • the six different Staphylococcus aureus strains were obtained by collecting the affected specimens (blood, wounds, respiratory system, etc.) from infected patients and culturing them in a 5% blood solid medium at 35-37 ° C. agar) or a test (coagulase test, etc.).
  • Staphylococcus aureus ATCC 25923 and MDR strain were grown in Mueller-hint medium by shaking culture at 37 ° C overnight. The cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. The bacteria were then diluted to give an optical density of 0.05 at 600 nm. And the compounds were prepared in sequential two-fold dilutions in Mueller-hint medium. Sequentially diluted compounds and bacteria were loaded three times with 10ul and 40ul, respectively, in 384 microgram plates. Plates were incubated at 37 ° C in 5% CO 2 for 24 hours. Absorbance was measured and analyzed to obtain a 50% minimum inhibitory concentration (MIC 50 ) of antibiotics in the strain.
  • MIC 50 50% minimum inhibitory concentration
  • Staphylococcus aureus was grown in Mueller-hint medium by shaking incubation at 37 ° C overnight at 180 rpm. The cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. Staphylococcus aureus was then diluted in a 125 mL flask to give an optical density of 0.05 at 600 nm. Antibiotics at the indicated concentrations were treated. The flask was incubated at 37 < 0 > C at 180 rpm for the indicated time.
  • Bacteria were harvested by centrifugation at 3300 rpm for 10 min and resuspended in 120 ul of lysis buffer (100 ul of 50 mM EDTA, 10 ul of 10 mg / ml lysozyme, 10 ul of 10 mg / ml resource taffine). Cultured in a 37 ° C water bath for 30 minutes, vortexed every 10 minutes and crushed by sonication. Thereafter, the extraction procedure was performed according to the RNeasy mini kit (Qiagen) protocol and 50 ul of RNA elute was obtained. After extraction of total RNA, the remaining genomic DNA was removed using DNase 1 (Qiagen).
  • the reaction volume was 10 ul and consisted of 1 ul of buffer RDD, 0.25 ul of DNas I stock solution and 6 ul of 50 ng / ul RNA. The mixture was incubated for 10 min at room temperature and 40 ul of RNase-free water was added to stop the reaction.
  • strain 4 showed SMP induction with ciprofloxacin, vancomycin and tetracycline in agreement with its antibiotic susceptibility profile, but did not show SMP induction in methicillin treatment.
  • MRSA Metal-resistant S. aureus
  • MSSA Metal-sensitive S. aureus
  • SMP induction and antibiotic susceptibility predictability were tested for the 50 strains. As a result, it was confirmed that SMP expression could be an excellent biomarker for antibiotic susceptibility prediction.
  • the promoter sequence is S. aureus , Were amplified by PCR using chromosomal DNA as template. The following primer pairs were used for amplification: sense: 5 'TAGGTGGCTAGCTTCTCTCATATATAGGCACTCCC 3' (SEQ ID NO: 5), antisense: 5 'GTTGTTGGTACCTTTCACTCCTAGAACATTTGTTTG 3' (SEQ ID NO: 6) with NheI and KpnI .
  • the PCR product was ligated to pCM29 upstream of the GFP reporter gene and transformed into E. coli Top10 by thermal shock. The transformants were selected in LB supplemented with ampicillin (100 ⁇ g / ml). It was transformed again to the transgenic E.
  • Plasmid Midiprep kit were collected using a (Qiagen) and Dam Dcm methylation prevents the dam - / dcm.
  • the transformants were grown in SOC growth medium at 37 DEG C for 1 hour with shaking at 180 rpm and plated on LB plates containing ampicillin (100 mu g / ml).
  • Electrocomponent cell Electrocompetent Cells
  • Staphylococcus aureus is a Muller-grown by shaking culture overnight at 37 to 180rpm °C medium in question.
  • the cultures were re-inoculated in 75 ml of fresh Mueller hinton medium at 600 nm at 0.05 and grown aerobically at 37 ° C until 3-5 x 10 8 cells / ml (OD 600 0.8-0.85) were reached.
  • Bacteria were chilled on ice for 15 minutes to stop growth and harvested by centrifugation at 3300 rpm at 4 ° C for 5 minutes. Cells were washed 3 times with 10 ml cold ice water and washed with 5 ml cold 10% glycerol.
  • Cells harvested by centrifugation at 4 ° C at 3300 rpm for 5 minutes were resuspended in 5 ml of 10% glycerol. Incubated at room temperature for 5 minutes and harvested by centrifugation at 4 ° C at 3300 rpm for 5 minutes. The collected cells were resuspended in 1 ml of 10% glycerol. The final cell concentration obtained was about 1 x 10 < 10 > cells / ml. Electrocomponent cells were used within 1 hour after manufacture.
  • electroporation 50 ul of electrocomponent cells were mixed with 2 ul of DNA (usually 1 ug) and incubated at room temperature for 30 minutes. The cell suspension-DNA mixture was transferred to a 0.2 second electroporation cuvette and placed in a Shock-Pod. Cells and DNA were electroplated on a Gene Pulser Xcell TM instrument (Bio-Rad Laboratoies) with a capacitance of 25 uF, 100 ohms resistivity and 2.9 kV. The cells were then immediately resuspended in 900 ul of TSA, transferred to a 15 ml Falcon tube and incubated at 37 ° C in 5% CO 2 for 1 h. The cells were then plated on TSA plates containing 10 ug / ml of chloramphenicol and incubated at 37 ° C in 5% CO 2 for 24-48 hours.
  • Staphylococcus aureus comprising the construction of the pCM29-SMP promoter was grown in a Mueller-hint medium supplemented with 10 ug / ml of chloramphenicol by shaking incubation overnight at 37 ° C at 180 rpm.
  • the cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium supplemented with 10 ug / ml of chloramphenicol and aerobically grown at 37 ° C to the mid-logarithmic phase.
  • Cells were harvested by centrifugation at 3300 rpm for 5 minutes and resuspended in 10 ml of fresh Mueller-hint medium. The cells were diluted to give an absorbance of 0.05 at 600 nm.
  • Ciprofloxacin was prepared by sequential double dilution in Mueller-hint medium. Sequentially diluted compounds and bacteria were loaded twice onto 96-well microtiter plates at 40ul and 160ul, respectively. Plates were incubated at 37 ° C in 5% CO 2 for 3 hours or 24 hours. Absorbance (OD 600) or fluorescence (Ex490nm / Em535nm) was measured and analyzed in order to obtain the 50% minimum inhibitory concentration (MIC 50) of ciprofloxacin.
  • MIC 50 minimum inhibitory concentration of Staphylococcus aureus was accurately measured by incubation with ciprofloxacin within 3 hours as shown in FIG.
  • the SMP promoter-based reporter system shortens the assay time with similar accuracy, so that the susceptibility prediction method and the antibiotic screening method using the SMP promoter of the present invention are specific It is a good estimation system for predicting the susceptibility of Staphylococcus aureus to antibiotics and for screening antibiotics against Staphylococcus aureus.
  • the present invention can be provided as a biomarker for predicting the susceptibility of Staphylococcus aureus to antibiotics.
  • the present invention also provides a pharmaceutical composition and a pharmaceutical preparation for preventing or treating infectious diseases caused by Staphylococcus aureus by predicting the susceptibility of Staphylococcus aureus to antibiotics.

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Abstract

The present invention relates to a biomarker for predicting the susceptibility of Staphylococcus aureus and a use thereof. More specifically, the present invention provides a composition, a kit and a prediction method for predicting the susceptibility of Staphylococcus aureus to antibiotics by using the biomarker; a method for screening antibiotics having susceptibility to Staphylococcus aureus; and a pharmaceutical composition for preventing or treating infections, caused by Staphylococcus aureus, comprising a compound screened by the method.

Description

황색포도상구균의 감수성 예측용 바이오마커 및 이의 용도Biomarkers for predicting susceptibility of Staphylococcus aureus and uses thereof

본 출원은 2016년 3월 31일 출원된 대한민국출원 제10-2016-0039756호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2016-0039756, filed on March 31, 2016, the disclosure of which is incorporated herein by reference in its entirety.

본 발명은 황색포도상구균(Staphylococcus aureus)의 감수성 예측용 바이오마커 및 이의 용도에 관한 것으로, 더욱 상세하게는 상기 바이오마커를 이용한 항생물질에 대한 황색포도상구균의 감수성 예측용 조성물, 키트, 예측방법, 황색포도상구균에 대해 감수성이 있는 항생물질의 스크리닝 방법 및 이로부터 스크리닝된 화합물을 포함하는 황색포도상구균에 의한 감염증을 예방 또는 치료하기 위한 약학적 조성물을 제공한다.The present invention relates to a biomarker for predicting the susceptibility of Staphylococcus aureus and a use thereof, and more particularly, to a composition, a kit, a method for predicting the susceptibility of Staphylococcus aureus to antibiotics using the biomarker, There is provided a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus aureus comprising a screening method of an antibiotic substance susceptible to Staphylococcus aureus and a compound screened therefrom.

병원성 세균 중 현재 전 세계적으로 가장 문제가 되고 있는 황색포도상구균 (S. aureus)은 전체 인구의 약 30%에서 검출되는 그람 양성균으로, 병원 내 감염증을 유발하는 병원체로 널리 알려져 있다. 상기 세균은 일차적으로 피부와 연부조직에 감염을 일으켜 농피증(pyoderma)의 원인이 되고, 골수염, 심내막염, 패혈증과 균혈증 등 생명을 위협하는 중한 전신 감염증을 유발하는 것으로 보고되고 있다. 페니실린 (Penicilin), 메티실린 (methicillin) 등 포도상구균 항균제는 그 효과가 우수하여 지난 수십 년간 본 감염증의 주요 치료약제로 사용되어 왔으나, 1961년 영국에서 메티실린에 내성을 보이는 균주 (methicillin-resistant Staphylococcus aureus: MRSA)가 처음 보고된 이후 전 세계적으로 지속적인 증가 추세를 보이고 있다.Among the pathogenic bacteria, S. aureus , which is the most problematic in the world today, is a Gram-positive bacterium that is detected in about 30% of the total population and is widely known as a pathogenic agent for infectious diseases in hospitals. The bacterium firstly infects skin and soft tissues, causing pyoderma, and has been reported to cause serious life-threatening systemic infections such as osteomyelitis, endocarditis, sepsis and bacteremia. Penicillin and methicillin have been used for several decades as a major therapeutic agent for this infectious disease. However, in 1961, a methicillin-resistant Staphylococcus aureus : MRSA) has been on the rise worldwide since its first reports.

MRSA는 1961년 영국에서 Jevons에 의해 처음 발견된 후 세계 각처에서 보고되기 시작했고, 이후로 20년 동안 MRSA의 비율은 유럽 여러 나라에서 1-2% 비율로 증가하였으며, 북유럽의 스페인, 프랑스, 이탈리아에서는 30-40% 까지 나타났다. 일본에서는 1990년 초에 59.1-67.3 %가 분리되었다고 보고된바 있으며, 국내에서도 MRSA의 분리율이 점차 증가하여 1995년에 50%를 넘어 평균 62.1%로 나타났고 최근 종합병원 환자에게서 76% 이상의 분리율을 보이고 있다. MRSA 분리율의 증가원인은 병원 내 감염뿐만 아니라 교통의 발달과 무분별한 항균제 사용에 있다. 지난 20년간 축산분야와 양식분야가 큰 시설에 집중적으로 이루어지면서 이미 많은 항균제가 사료와 질병의 예방을 목적으로 무분별하게 사용되었고 지속적인 항균제의 노출은 내성세균의 확산을 야기시켰다. 때문에 지역사회에서도 MRSA 감염 환자가 증가하고 있다.MRSA was first reported by the Jevons in the UK in 1961 and has been reported from around the world. Since then, the rate of MRSA has increased by 1-2% in many European countries over the next 20 years, Of the total. In Japan, 59.1-67.3% were isolated in the early 1990s. In Korea, the isolation rate of MRSA gradually increased, reaching an average of 62.1% in 1995, exceeding 50%. In recent years, more than 76% It is showing. The reason for the increase in the MRSA isolation rate is not only the infection in the hospital but also the development of traffic and the use of antibiotics irresponsibly. In the past 20 years, livestock and aquaculture have been concentrated in large facilities, and many antimicrobial agents have been used indiscriminately for the purpose of preventing feed and disease, and continuous exposure of antimicrobial agents has caused the spread of resistant bacteria. Therefore, the number of patients infected with MRSA is increasing in the community.

범세계적으로 MRSA에 의한 병원 감염이 확산되어 반코마이신(vancomycin)이 임상에서 광범위하게 사용되었으나, 1996년도에는 일본에서 반코마이신 중간내성 황색포도상구균(vancomycin-intermediate S. aureus; VISA)이 처음 발견되었으며, 2002년도에는 미국에서 반코마이신 고도내성 황색포도상구균(Vancomycin-resistant S. aureus; VRSA)이 최초로 보고되었다.Vancomycin-intermediate S. aureus (VISA) was first found in Japan in 1996, although vancomycin was widely used in clinical practice due to the spread of hospital infections due to MRSA worldwide. In 1996, vancomycin-intermediate S. aureus Vancomycin-resistant S. aureus (VRSA) was first reported in the United States in the year of vancomycin-resistant Staphylococcus aureus.

이와 같이, 병원균은 자기방어수단으로 항생제에 대한 내성을 돌연변이나 항생제 내성 유전자를 습득함으로써 얻게 되는데, 이러한 내성균의 발현 빈도는 항생제의 오용과 남용이 많아짐에 따라 더욱 증가하고 있다.In this way, pathogens are obtained by self-defense by acquiring a mutation or an antibiotic resistance gene for resistance to antibiotics. The incidence of these resistant bacteria is increasing with the abuse and abuse of antibiotics.

종래에는 항생제 오남용을 막기 위해 mecA(mobile genetic element A) 내성 유전자 PCR법을 이용하여 MRSA를 판독 하였으나, PCR법으로 검출하는 방법은 반응 시간이 약 1일 정도 소요되고, 내성 유전자의 검출은 지정한 유전자만을 검출할 수 있어 메티실린에 대한 내성만 판독할 수 있다는 단점을 가지고 있다(The Korean Journal of Microbiology (2011) Vol. 47, No. 4, pp. 381-385).Conventionally, MRSA was read using the mec A (mobile genetic element A) resistant gene PCR method to prevent abuse of antibiotics. However, the PCR method requires about 1 day of reaction time, (2011) Vol. 47, No. 4, pp. 381-385), which can detect only the resistance to methicillin.

이에, 본 발명자들은 종래 기술의 단점을 극복하면서 항생제의 오남용을 막을 수 있는 신규 방법을 개발하기 위해 다양한 연구를 수행하던 중 황색포도상구균에만 보존되어 있는 유전자에 의해 코딩되는 가상의 작은 막 단백질(Small Membrane Protein, SMP)이 항생제 감수성과 연관되어 있다는 것을 발견함으로써 본 발명을 완성하였다.The inventors of the present invention conducted various studies to overcome the disadvantages of the prior art and developed a novel method for preventing abuse of antibiotics, and found that a virtual small membrane protein (Small), which is encoded by a gene stored only in Staphylococcus aureus Membrane Protein, SMP) is associated with antibiotic susceptibility.

본 발명은 상술한 문제점을 해결하기 위해 안출된 것으로, SMP(Small Membrane Protein) 유전자를 포함하는 항생물질에 대한 황색포도상구균 (Staphylococcus aureus)의 감수성 예측용 바이오 마커를 제공한다.SUMMARY OF THE INVENTION The present invention provides a biomarker for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance including an SMP (Small Membrane Protein) gene.

본 발명의 다른 목적은 SMP의 발현 수준을 측정하는 제제를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising an agent for measuring the expression level of SMP.

본 발명의 또 다른 목적은 상기 감수성 예측용 조성물을 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 키트를 제공하는 것이다.It is still another object of the present invention to provide a kit for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising the composition for predicting susceptibility.

본 발명의 또 다른 목적은 (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 항생물질을 처리한 후 시료 내 SMP의 발현 수준을 측정하는 단계; 및 (b) 상기 (a) 단계에서 측정한 결과에 기초하여 상기 시료 내 황색포도상구균의 항생물질에 대한 감수성을 판정하는 단계를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법을 제공하는 것이다.It is still another object of the present invention to provide a method for the diagnosis of Staphylococcus aureus infection, comprising the steps of: (a) measuring the expression level of SMP in a sample after treating a biological sample isolated from a patient with Staphylococcus aureus infection with antibiotics; And (b) determining a susceptibility of the Staphylococcus aureus in the sample to an antibiotic based on the result of the measurement in the step (a), wherein the susceptibility of Staphylococcus aureus to antibiotics is predicted .

본 발명의 또 다른 목적은 (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 후보물질을 처리한 후 시료 내 SMP의 발현 수준을 측정하는 단계; 및 (b) 상기 (a) 단계에서 측정한 결과에 기초하여 황색포도상구균의 후보물질에 대한 감수성을 판정하는 단계를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질을 스크리닝하는 방법을 제공하는 것이다.It is still another object of the present invention to provide a method of treating a Staphylococcus aureus infection, comprising the steps of: (a) treating a candidate biological material with a biological sample separated from a patient suffering from Staphylococcus aureus infection, and measuring the expression level of SMP in the sample; And (b) determining the susceptibility of the Staphylococcus aureus to a candidate substance based on the result of the measurement in the step (a), wherein the method comprises screening antibiotic substances susceptible to Staphylococcus aureus .

본 발명의 또 다른 목적은 (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게 연결된 리포터 유전자를 포함하는 유전자 구조체를 황색포도상구균에 도입하는 단계; 및 (b) 상기 (a) 단계의 황색포도상구균에 항생물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법을 제공한다.It is another object of the present invention to provide a method for producing Staphylococcus aureus comprising: (a) introducing into a Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) treating the Staphylococcus aureus of step (a) with an antibiotic and measuring whether or not the Staphylococcus aureus emits light. The present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance.

본 발명의 또 다른 목적은 (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게 연결된 리포터 유전자를 포함하는 유전자 구조체를 황색포도상구균에 도입하는 단계; 및 (b) 상기 (a) 단계의 황색포도상구균에 후보물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질의 스크리닝 방법을 제공한다.It is another object of the present invention to provide a method for producing Staphylococcus aureus comprising: (a) introducing into a Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) treating the candidate Staphylococcus aureus of step (a) with a candidate substance and measuring whether or not the staphylococcus aureus emits light. The present invention also provides a screening method for antibiotics susceptible to Staphylococcus aureus.

본 발명의 또 다른 목적은 전술한 스크리닝 방법으로 스크리닝된 화합물 또는 약학적으로 허용가능한 이의 염의 유효량; 및 약학적으로 허용가능한 담체를 포함하는 황색포도상구균에 의한 감염증을 예방 또는 치료하기 위한 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide an effective amount of a compound screened by the above-described screening method or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus aureus comprising a pharmaceutically acceptable carrier.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

상술한 바와 같이, 내성균의 발현 빈도는 항생제의 오용과 남용이 많아짐에 따라 더욱 증가하고 있어 항생제의 오남용을 막기 위한 방법의 개발이 시급한 실정이다. As described above, the frequency of the expression of resistant bacteria is further increased as the abuse and abuse of antibiotics increases, and it is urgent to develop a method for preventing misuse of antibiotics.

이에 본 발명에서는 황색포도상구균(Staphylococcus aureus)에만 보존되어 있는 유전자에 의해 코딩되는 가상의 작은 막 단백질(Small Membrane Protein, SMP)이 항생제 감수성과 연관되어 있다는 것을 발견하고 SMP 유전자를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 바이오 마커를 제공함으로써 상술한 문제의 해결방안을 모색하였다. 이를 이용해 황색포도상구균 감염 환자 맞춤별로 치료제를 제공하는 것이 가능하며, 환자별 적합한 항생제의 투여로 항생제의 오남용을 막을 수 있다.Accordingly, the present invention finds that a hypothetical small membrane protein (SMP) encoded by a gene stored only in Staphylococcus aureus is associated with antibiotic susceptibility, and antibiotics including SMP gene The present inventors searched for a solution to the above problem by providing a biomarker for predicting the susceptibility of S. aureus. It is possible to provide treatments for individual patients with Staphylococcus aureus infection and to prevent misuse of antibiotics by appropriate antibiotic administration for each patient.

본 발명에서 용어 "SMP(Small Membrane Protein, SMP)"는 황색포도상구균에만 보존되어 있는 SA_13280 유전자에 의해 코딩되는 단백질을 지칭하며, 이의 기능에 대해서는 아직까지 알려진 바가 없다. 상기 SMP 단백질 및 유전자 서열은 공지된 데이터베이스에서 얻을 수 있다. 구체적으로 SMP 단백질 서열은 NCBI Reference sequence NO. WP_001574373.1에 개시된 것일 수 있으며, SMP 유전자 서열은 GenBank No. CP013137.1에 개시된 것일 수 있다. 본 발명의 일실시예에서는 상기 SMP 유전자는 서열번호 1로 이루어진다.The term " SMP (Small Membrane Protein, SMP) " in the present invention refers to a protein encoded by SA_13280 gene which is stored only in Staphylococcus aureus, and its function is not yet known. The SMP protein and gene sequences can be obtained from known databases. Specifically, the SMP protein sequence is the NCBI Reference sequence NO. The SMP gene sequence may be the one disclosed in GenBank No. 1, pp. It may be one disclosed in CP013137.1. In one embodiment of the present invention, the SMP gene comprises SEQ ID NO: 1.

본 발명의 용어 "바이오마커"는 생물학적 현상의 존재와 정량적 또는 정성적으로 연관된 분자를 의미한다. 마커는 게놈 뉴클레오티드 서열로부터 또는 발현된 뉴클레오티드 서열로부터(예를 들어, RNA, nRNA, mRNA, cDNA 등으로부터), 또는 코딩된 폴리펩티드로부터 유래될 수 있다. 이 용어는 마커 서열에 상보적이거나 이에 플랭킹된 핵산 서열, 예컨대 마커 서열을 증폭시킬 수 있는 프로브 또는 프라이머 쌍으로 사용된 핵산을 포함한다.The term " biomarker " of the present invention means a molecule quantitatively or qualitatively related to the presence of a biological phenomenon. The marker may be derived from a genomic nucleotide sequence or from a nucleotide sequence expressed (e.g., from RNA, nRNA, mRNA, cDNA, etc.), or from a coded polypeptide. The term includes nucleic acid sequences that are complementary to or flanked in the marker sequence, such as a probe or a pair of primers that can amplify the marker sequence.

본 발명의 바이오 마커는 항생물질에 대한 황색포도상구균의 감수성의 지표가 될 수 있으며, 항생제에 대한 황색포도상구균의 감수성 마커로서의 정확성 및 신뢰도가 탁월하여 황색포도상구균 감염 환자 맞춤별로 치료제를 제공하는 것이 가능하다.The biomarker of the present invention can be an indicator of the susceptibility of Staphylococcus aureus to antibiotics and is excellent in accuracy and reliability as a susceptibility marker of Staphylococcus aureus to antibiotics, It is possible.

본 발명에서 용어 “감수성”은 개개의 환자의 황색포도상구균 감염증에 대하여 특정 항생물질이 효과를 나타내는지 여부를 의미한다.The term " susceptibility " in the present invention means whether or not a particular antibiotic substance is effective against Staphylococcal aureus infectious disease in an individual patient.

본 발명에서, 용어 "예측"은 의학적 귀추에 대하여 미리 헤아려 짐작하는 것을 의미하며, 본 발명의 목적상 개개의 환자의 황색포도상구균 감염증에 대하여 특정 항생물질이 효과를 나타내는지 여부를 미리 짐작하는 것을 의미한다.In the present invention, the term " prediction " refers to predicting medical consequences, and for the purpose of the present invention, it is contemplated in advance to determine whether a particular antibiotic has an effect on Staphylococcal Staphylococcal infections in an individual patient it means.

예를 들어, 상기 항생물질은 미생물 및 그 밖의 생물에 의해 만들어지는 물질로서 생물의 생리활성에 영향을 주는 것으로 해석될 수 있으며, 주로 항생제를 나타낸다. 이들 항생제는 황색포도상구균의 종류에 따라 효과를 나타내는 경우와 효과를 나타내지 않는 경우가 있다. 이와 같이 황색포도상구균의 종류에 따라 항생물질이 효과를 나타내는지 여부를 항생물질에 대한 감수성이라고 한다. 본 발명에 따라 치료 개시 전에 효과를 기대할 수 있는 환자(반응자)와 효과를 기대할 수 없는 환자(무반응자)를 예측할 수 있으면, 항생제의 오남용을 막으면서 유효성과 안전성이 높은 치료방법이 실현될 수 있다.For example, the antibiotic substance is a substance produced by microorganisms and other organisms, and can be interpreted as affecting the biological activity of the organism, and mainly represents an antibiotic. These antibiotics may or may not have an effect depending on the type of Staphylococcus aureus. The susceptibility of antibiotics to antibiotics depends on the type of Staphylococcus aureus. According to the present invention, if a patient (a responder) who can expect an effect before initiation of treatment and a patient (a non-responder) who can not expect an effect can be predicted, a treatment method with high efficacy and safety can be realized while preventing misuse of antibiotics .

본 발명은 또한, SMP(Small Membrane Protein)의 발현 수준을 측정하는 제제를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 조성물을 제공한다.The present invention also provides a composition for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising an agent for measuring the expression level of SMP (Small Membrane Protein).

본 발명에서 용어 "발현수준을 측정하는 제제" 란 본 발명의 바이오마커 유전자 또는 이들 유전자에 의해 코딩된 단백질의 발현수준을 확인하기 위하여 사용될 수 있는 분자를 의미하며, 바람직하게는 상기 바이오마커에 특이적으로 결합하는 프라이머 쌍, 프로브 또는 안티센스 뉴클레오티드이거나, 또는 상기 바이오마커 유전자에 의해 코딩되는 단백질에 특이적인 항체일 수 있다. 본 발명에서 상기 발현 수준은 DNA 수준, mRNA 수준, 단백질 수준 등 모든 발현의 수준을 의미하는 것으로 이해될 수 있다.The term " agent for measuring the expression level " in the present invention means a molecule that can be used for confirming the expression level of the biomarker gene of the present invention or a protein encoded by these genes, A probe or antisense nucleotide, or an antibody specific for a protein encoded by the biomarker gene. In the present invention, the level of expression can be understood to mean the level of expression, such as DNA level, mRNA level, protein level, and the like.

본 발명에서 용어, "항체"란 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커에 대해 특이적으로 결합하는 항체를 의미하며, 이러한 항체는 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함되며, 본 발명의 부분 펩티드로는, 최소한 7개의 아미노산, 바람직하게는 9개 아미노산, 더욱 바람직하게는 12개 이상의 아미노산을 포함한다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다. 이러한 본 발명의 바이오마커 유전자에 의해 코딩되는 단백질에 대한 항체는 당업계의 공지된 방법으로 제조될 수 있는 모든 항체가 될 수 있다. 예를 들어, 본 발명의 감수성 예측 마커의 검출에 사용되는 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태 뿐만 아니라 항체 분자의 기능적인 단편을 포함할 수 있다. 상기 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2, Fv 등이 될 수 있으나, 특별히 이에 제한되지는 않는다.The term " antibody " as used herein in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker of the present invention, which clones each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene , And can be prepared from the obtained protein by a conventional method. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and any part thereof having a polyclonal antibody, a monoclonal antibody or an antigen-binding property is included in the antibody of the present invention and includes all the immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. The antibody against the protein encoded by the biomarker gene of the present invention may be any antibody that can be produced by a method known in the art. For example, an antibody used in the detection of the susceptibility prediction marker of the present invention may comprise a complete form having two full-length light chains and two full-length heavy chains as well as a functional fragment of the antibody molecule. The functional fragment of the antibody molecule refers to a fragment having at least an antigen binding function and may be Fab, F (ab ') 2, F (ab') 2, Fv or the like, but is not particularly limited thereto.

본 발명에서 용어, "프라이머"는 짧은 자유 3 말단 수산화기(free 3` hydroxyl group)을 가지는 핵산 서열로 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형의 복사를 위한 시작지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 본 발명에서는 본 발명 마커 폴리뉴클레오티드의 센스 및 안티센스 프라이머를 이용하여 PCR 증폭을 실시하여 원하는 생성물의 생성 여부를 통해 항생물질에 대한 황색포도상구균의 감수성을 예측할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 것을 기초로 변형할 수 있다. 본 발명의 일실시예에서는 서열번호 2로 표시되는 정방향 프라이머; 및 서열번호 3으로 표시되는 역방향 프라이머로 이루어진 프라이머 쌍을 제공한다.The term " primer " in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template, and a starting point for copying the template ≪ / RTI > In the present invention, the sensitivity of Staphylococcus aureus to antibiotics can be predicted by performing PCR amplification using the sense and antisense primers of the marker polynucleotide of the present invention to produce a desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. In one embodiment of the present invention, a forward primer represented by SEQ ID NO: 2; And a reverse primer represented by SEQ. ID. NO: 3.

본 발명에서 용어, "프로브(probe)" 란 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며, 표지(labeling)되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고 뉴클레오티드 프로브, 단일 사슬 DNA(single stranded DNA) 프로브, 이중 사슬 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 본 발명의 마커 폴리뉴클레오티드와 상보적인 프로브를 이용하여 혼성화를 실시하여, 혼성화 여부를 통해 항생물질에 대한 황색포도상구균의 감수성을 예측할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다.The term " probe " in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can specifically bind to mRNA and is labeled The presence or absence of a specific mRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, the hybridization using the probe complementary to the marker polynucleotide of the present invention can predict the susceptibility of Staphylococcus aureus to antibiotics through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

본 발명은 또한, 전술한 감수성 예측용 조성물을 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 키트를 제공한다.The present invention also provides a kit for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising the aforementioned composition for predicting susceptibility.

본 발명의 키트는 상기 감수성 예측용 바이오마커 유전자의 mRNA 발현 수준 또는 이들 유전자에 의해 코딩되는 단백질의 발현수준을 확인함으로써 항생물질에 대한 황색포도상구균의 감수성을 예측하는데 사용할 수 있다. The kit of the present invention can be used for predicting the susceptibility of Staphylococcus aureus to antibiotics by confirming the mRNA expression level of the biomarker gene for susceptibility prediction or the expression level of the protein encoded by these genes.

상기 바이오마커 유전자들의 mRNA 발현수준을 측정하기 위한 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소 및 역전사효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다.The kit for measuring the mRNA expression level of the biomarker genes may be a kit containing essential elements necessary for performing RT-PCR. RT-PCR kits can be used to detect and isolate specific primer pairs specific to the marker gene, including test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- DEPC-water, sterile water, and the like.

본 발명의 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 바이오 마커를 검출하기 위한 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다.The kit of the present invention may be a kit for detecting a biomarker for predicting the susceptibility of Staphylococcus aureus to an antibiotic substance including essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

본 발명에서 상기 바이오마커 유전자에 의해 코딩된 단백질의 발현수준을 측정하기 위한 단백질 칩 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충용액, 발색 효소 또는 형광물질로 표지된 2차 항체 및 발색 기질 등을 포함할 수 있다. 상기에서 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스틸렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase)가 사용될 수 있고, 형광물질은 FITC, RITC 등이 사용될 수 있으며, 발색기질액은 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)) 또는 OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘) 등이 사용될 수 있다.In the present invention, a protein chip kit for measuring the expression level of a protein encoded by the biomarker gene may be used for immunological detection of the antibody. The kit includes a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, And the like. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The chromogenic enzyme may be peroxidase, Alkaline phosphatase can be used, and fluorescent materials such as FITC, RITC and the like can be used. The coloring substrate liquid is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) and the like can be used.

본 발명은 또한, (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 항생물질을 처리한 후 시료 내 SMP의 발현 수준을 측정하는 단계; 및 (b) 상기 (a) 단계에서 측정한 결과에 기초하여 상기 시료 내 황색포도상구균의 항생물질에 대한 감수성을 판정하는 단계를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법을 제공한다.(A) measuring the level of expression of SMP in a sample after treating a biological sample isolated from a patient with Staphylococcus aureus infection with an antibiotic; And (b) determining the susceptibility of the Staphylococcus aureus in the sample to antibiotics based on the result of the measurement in the step (a). The present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to antibiotics.

본 발명에 있어서, 용어, "mRNA 발현수준 측정"이란 항생물질에 대한 황색포도상구균의 감수성을 예측하기 위하여 생물학적 시료에서 바이오마커 유전자의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정함으로써 알 수 있다. 이를 위한 분석 방법으로는 RT-PCR, 경쟁적 RT-PCR(competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(northern blotting) 또는 DNA 칩(DNA chip technology) 등이 있으나, 이에 제한되는 것은 아니다.In the present invention, the term " measurement of mRNA expression level " refers to the measurement of mRNA level and expression level of a biomarker gene in a biological sample in order to predict the susceptibility of Staphylococcus aureus to antibiotics. . RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection method, northern blotting, Or DNA chip technology, but the present invention is not limited thereto.

본 발명에 있어서, 용어, "단백질 발현수준 측정"이란 항생물질에 대한 황색포도상구균의 감수성을 예측하기 위하여 생물학적 시료에서의 마커 유전자에서 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 상기 유전자에서 발현된 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏(western blotting), ELISA(enzyme linked immunosorbentassay), 방사선면역분석(RIA: radioimmunoassay), 방사 면역 확산법(radial immunodiffusion), 오우크테로니면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(rocket immunoelectrophoresis), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complement Fixation Assay), 면역형광법(immunofluorescence), 면역크로마토그래피법(immunochromatography), FACS 분석법(fluorescenceactivated cell sorter analysis) 또는 단백질 칩 방법(protein chip technology) 등이 있으나, 이에 제한되는 것은 아니다.In the present invention, the term " measurement of protein expression level " refers to a process for confirming the presence and expression level of a protein expressed in a marker gene in a biological sample to predict the susceptibility of Staphylococcus aureus to antibiotics, The amount of protein can be confirmed using an antibody that specifically binds to the protein expressed in the gene. Analysis methods include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunization Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS (Immunohistochemical staining), immunofluorescence (immunohistochemical staining), immunoprecipitation assays fluorescence activated cell sorter analysis, or protein chip technology, but the present invention is not limited thereto.

본 발명에서 용어, "황색포도상구균 감염 환자에서 분리된 생물학적 시료"란 본 발명의 바이오 마커의 발현이 검출될 수 있는 개체로부터 얻어지는 모든 시료를 의미하며, 이에 제한되는 것은 아니나, 황색포도상구균 감염 환자의 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨일 수 있다.As used herein, the term " biological sample isolated from a patient with Staphylococcus aureus infection " refers to all samples obtained from an individual from which expression of the biomarker of the present invention can be detected, including but not limited to patients with Staphylococcus aureus infection Tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

본 발명의 감수성 예측방법에 있어서, 상기 바이오마커 유전자의 발현수준을 mRNA 수준 또는 단백질 수준에서 측정할 수 있고, 생물학적 시료에서 mRNA 또는 단백질의 분리는 공지의 공정을 이용하여 수행할 수 있다. mRNA 수준을 측정하기 위한 분석 방법 및 단백질 수준을 측정하기 위한 분석 방법은 상기에서 설명한 바와 같다.In the susceptibility prediction method of the present invention, the expression level of the biomarker gene may be measured at an mRNA level or a protein level, and the mRNA or protein may be separated from the biological sample using a known process. The analysis method for measuring the mRNA level and the assay method for measuring the protein level are as described above.

상기 분석방법들을 통하여, 특정 항생물질에 대한 감수성을 예측하고자 하는 환자의 시료로부터 측정한 감수성 예측용 바이오마커 유전자의 발현 여부를 통해 상기 환자에 감염된 황색포도상구균의 특정 항생물질에 대한 감수성을 예측할 수 있다. 즉, 바이오 마커가 발현되는 경우(SMP가 발현되는 경우), 상기 환자에 감염된 황색포도상구균은 처리된 항생물질에 대해 감수성이 있는 것으로 판단할 수 있다.Through the above analysis methods, the susceptibility to the specific antibiotic of Staphylococcus aureus infected with the patient can be predicted through the expression of the biomarker gene for predicting the susceptibility measured from the sample of the patient who intends to predict the susceptibility to the specific antibiotic substance have. That is, when the biomarker is expressed (when SMP is expressed), the Staphylococcus aureus infected with the patient can be judged as susceptible to the treated antibiotic substance.

따라서, 본 발명에 의하면 특정 항생물질에 대한 황색포도상구균의 감수성을정확하게 예측할 수 있고, 감수성 예측결과에 따라 환자 맞춤별로 적절한 치료계획을 세울 수 있는 이점을 얻을 수 있다.Therefore, according to the present invention, the susceptibility of Staphylococcus aureus to a specific antibiotic can be accurately predicted, and an appropriate treatment plan can be established according to the patient's suitability according to the result of the susceptibility prediction.

본 발명은 또한, (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 후보물질을 처리한 후 시료 내 SMP의 발현 수준을 측정하는 단계; 및 (b) 상기 (a) 단계에서 측정한 결과에 기초하여 황색포도상구균의 후보물질에 대한 감수성을 판정하는 단계;를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질을 스크리닝하는 방법을 제공한다.(A) measuring the level of expression of SMP in a sample after treating a candidate biological material with a biological sample isolated from a patient with Staphylococcus aureus infection; And (b) determining the susceptibility of the Staphylococcus aureus to a candidate substance based on the result of the measurement in the step (a). The present invention also provides a method for screening antibiotics susceptible to Staphylococcus aureus .

본 발명의 스크리닝 방법에 있어서, 상기 SMP의 발현이 확인된 경우, 상기 시료 내 황색포도상구균은 처리된 후보물질에 대해 감수성이 있는 것으로 판정할 수 있으며, 상기 후보물질은 기존의 항생제뿐만 아니라 황색포도상구균의 억제 가능성을 측정하고자 하는 모든 물질을 포함한다.In the screening method of the present invention, when the expression of the SMP is confirmed, the Staphylococcus aureus in the sample can be judged to be susceptible to the candidate substance to be treated. The candidate substance can be determined not only by existing antibiotics, It includes all substances that are intended to measure the inhibition potential of the bacteria.

상기 스크리닝 방법은, 전술한 감수성 예측방법과 동일한 원리로 수행될 수 있으므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.The above screening method can be performed on the same principle as the above-described sensitivity-sensitive prediction method, so redundant contents are omitted in order to avoid excessive complexity of the present specification.

도 4 및 5에 나타난 바와 같이 다양한 항생제로 처리된 균주에서 RT-qPCR에 의해 SMP 유도를 측정할 때, 박테리아가 감수성을 나타내는 항생제와 함께 배양했을 때만 현저한 SMP 유도가 관찰되었다. 예를 들어, 1번 균주는 반코마이신에만 감수성을 나타내고, 시프로플록사신, 메티실린 및 테트라사이클린에는 감수성을 나타내지 않았다(도 3). 이러한 감수성 프로파일과 일치하여, SMP는 오직 반코마이신 처리에 의해 1번 균주에서 유도된 반면, 대조균주(항생제 민감성 황색포도상구균)는 테스트된 모든 항생제에 의해 현저하게 SMP를 유도한다(도 4). 유사하게, 4번 균주는 이의 항생제 감수성 프로파일과 일치하여 시프로플록사신, 반코마이신 및 테트라사이클린으로 SMP 유도를 나타내지만 메티실린 처리에서는 SMP 유도를 나타내지 않았다(도 5). 이러한 데이터는 SMP가 황색포도상구균의 항생제 감수성을 예측하기 위한 바이오마커가 될 수 있다는 것을 제안하며, 이로부터 상기 바이오마커의 이용시 특정 항생물질에 대한 황색포도상구균의 감수성 예측 및 황색포도상구균이 감수성을 나타내는 항생물질을 효과적으로 스크리닝할 수 있다는 것을 확인할 수 있다.Significant SMP induction was observed only when bacteria were co-cultured with antibiotics showing susceptibility when measuring SMP induction by RT-qPCR in strains treated with various antibiotics, as shown in Figures 4 and 5. For example, strains No. 1 showed susceptibility to vancomycin only and did not show susceptibility to ciprofloxacin, methicillin and tetracycline (FIG. 3). Consistent with this susceptibility profile, SMP was induced only in strain 1 by vancomycin treatment whereas the control strain (antibiotic sensitive Staphylococcus aureus) induced SMP significantly by all tested antibiotics (FIG. 4). Similarly, strain 4 showed SMP induction with ciprofloxacin, vancomycin and tetracycline in agreement with its antibiotic susceptibility profile, but did not show SMP induction in methicillin treatment (Fig. 5). These data suggest that SMP can be a biomarker for predicting antibiotic susceptibility of Staphylococcus aureus. From this, it is possible to predict the susceptibility of Staphylococcus aureus to specific antibiotics and the sensitivity of Staphylococcus aureus Lt; RTI ID = 0.0 > antibiotics < / RTI >

본 발명은 또한, (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게 연결된 리포터 유전자를 포함하는 유전자 구조체를 분리된 황색포도상구균에 도입하는 단계; 및 (b) 상기 (a) 단계의 황색포도상구균에 항생물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계;를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법을 제공한다.The present invention also provides a method of producing a Staphylococcus aureus strain comprising the steps of: (a) introducing into a separate Staphylococcus aureus gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to expression by said promoter; And (b) treating the Staphylococcus aureus of step (a) with antibiotics and measuring the emission of Staphylococcus aureus. The present invention also provides a method for predicting the susceptibility of Staphylococcus aureus to antibiotics.

상기 유전자 구조체를 황색포도상구균에 도입하는 방법은 당 분야에 공지된 적합한 표준 기술을 선택하여 수행할 수 있으며, 예를 들어, 일렉트로포레이션(electroporation), 칼슘 포스페이트 공동-침전 (calcium phosphate co-precipitation), 레트로바이러스 감염(retroviral infection), 미세주입법 (microinjection), DEAE-덱스트란 (DEAE-dextran), 양이온 리포좀(cationic liposome) 법 등이 있으나, 이로 한정되지 않는다.The method of introducing the gene construct into Staphylococcus aureus can be carried out by selecting a suitable standard technique known in the art, for example, electroporation, calcium phosphate co-precipitation ), Retroviral infection, microinjection, DEAE-dextran, cationic liposome, and the like, but the present invention is not limited thereto.

상기 (b) 단계의 황색포도상구균이 발광되는 경우, 황색포도상구균은 처리된 항생물질에 대해 감수성이 있는 것으로 판정할 수 있다.When the Staphylococcus aureus of step (b) is luminescent, the Staphylococcus aureus can be judged to be susceptible to the treated antibiotic substance.

본 발명의 일실시예에서는 서열번호 4로 이루어진 SMP 프로모터 및 상기 프로모터에 의해 발현되도록 작동가능한게 연결된 녹색형광단백질(GFP) 유전자를 포함하는 유전자 구조체를 분리된 황색포도상구균에 도입한 후 항생물질을 처리하여 황색포도상구균의 발광 여부를 측정함으로써 황색포도상구균의 감수성을 예측하였다(도 6). In one embodiment of the present invention, a gene construct comprising a SMP promoter of SEQ ID NO: 4 and a green fluorescent protein (GFP) gene operably linked to the promoter to be expressed by the promoter is introduced into isolated Staphylococcus aureus, And the sensitivity of Staphylococcus aureus was predicted by measuring the emission of Staphylococcus aureus (Fig. 6).

본 발명에서는 리포터 유전자로 녹색형광단백질(GFP) 유전자를 사용하였으나, 통상적으로 사용되는 리포터 유전자라면 제한없이 사용할 수 있으며, 예를 들어, 리포터 유전자는 녹색형광단백질(GFP) 유전자, 붉은색형광단백질(RFP) 유전자, 푸른색형광단백질(BFP) 유전자, 발광효소단백질(luciferase) 유전자, 및 락타아제(lactase) 유전자로 이루어진 군에서 선택된 어느 하나일 수 있다.In the present invention, a green fluorescent protein (GFP) gene is used as a reporter gene, but any commonly used reporter gene can be used without limitation. For example, a reporter gene may be a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a blue fluorescent protein (BFP) gene, a luciferase gene, and a lactase gene.

상기 감수성 예측방법에 있어서, 상기 (b) 단계의 발광 여부가 검출되는 시간은 처리되는 항생물질의 종류, 구체적으로 항생제의 종류에 따라 다를 수 있고 항생물질의 기작에 따라 다를 수 있으나, 대체로 처리 후 1~5시간 안에 검출될 수 있다.In the susceptibility prediction method, the time during which the light emission of the step (b) is detected may be different depending on the type of the antibiotic to be treated, specifically the type of the antibiotic, and may vary depending on the mechanism of the antibiotic substance. Can be detected within 1 to 5 hours.

본 발명은 또한, (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게연결된 리포터 유전자를 포함하는 유전자 구조체를 분리된 황색포도상구균에 도입하는 단계; 및 (b) 상기 (a) 단계의 황색포도상구균에 후보물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계;를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질의 스크리닝 방법을 제공한다.The present invention also provides a method of producing a Staphylococcus aureus strain comprising the steps of: (a) introducing into a separate Staphylococcus aureus gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to expression by said promoter; And (b) measuring the presence or absence of the staphylococcus aureus after treating the candidate substance with Staphylococcus aureus in step (a), wherein the method comprises screening for antibiotic substances susceptible to Staphylococcus aureus .

상기 스크리닝 방법은, 전술한 SMP 프로모터 기반 리포터 시스템을 이용한 황색포도상구균의 감수성 예측방법과 동일한 원리로 수행될 수 있으므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.The above screening method can be performed on the same principle as the method for predicting the susceptibility of Staphylococcus aureus using the above-described SMP promoter-based reporter system, so redundant contents are omitted in order to avoid the excessive complexity of the present specification.

본 발명의 일 실시예에서는 상기 SMP 프로모터 기반 리포터 시스템을 이용한 황색포도상구균의 감수성 예측방법 또는 항생물질의 스크리닝 방법을 검증하기 위하여 실시예 5에 기재된 바와 같이 실험을 수행하였다. 그 결과, 도 7에 나타난 바와 같이 시프로플록사신과 3시간 내로 배양하여 황색포도상구균의 MIC50(minimum inhibitory concentration)을 정확하게 측정하였다. 24시간이 걸리는 통상적인 배양 기반의 MIC50의 측정과 비교하여, SMP 프로모터 기반 리포터 시스템은 유사한 정확도로 어세이 시간을 단축시키므로 본 발명의 SMP 프로모터를 이용한 감수성 예측방법 및 항생물질의 스크리닝 방법은 특정 항생물질에 대한 황색포도상구균의 감수성 예측 및 황색포도상구균에 대한 항생물질을 스크리닝하기 위한 좋은 어세이 시스템이라는 것을 알 수 있다.In one embodiment of the present invention, an experiment was conducted as described in Example 5 to verify the method for predicting the susceptibility of Staphylococcus aureus or the method for screening antibiotics using the SMP promoter-based reporter system. As a result, MIC 50 (minimum inhibitory concentration) of Staphylococcus aureus was accurately measured by incubation with ciprofloxacin within 3 hours as shown in FIG. Compared with conventional culturing-based MIC 50 measurements that take 24 hours, the SMP promoter-based reporter system shortens the assay time with similar accuracy, so that the susceptibility prediction method and the antibiotic screening method using the SMP promoter of the present invention are specific It is a good estimation system for predicting the susceptibility of Staphylococcus aureus to antibiotics and for screening antibiotics against Staphylococcus aureus.

본 발명은 또한, 상기 스크리닝 방법으로 스크리닝된 화합물 또는 약학적으로 허영가능한 이의 염의 유효량; 및 약학적으로 허용가능한 담체를 포함하는 황색포도상구균에 의한 감염증을 예방 또는 치료하기 위한 약학적 조성물을 제공한다.The present invention also relates to an effective amount of a compound screened by the screening method or a pharmaceutically acceptable salt thereof; And pharmaceutical compositions for preventing or treating infection by Staphylococcus aureus comprising a pharmaceutically acceptable carrier.

본 발명의 약학적 조성물은 투여 방식에 따라 약학적으로 허용가능한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 구체적으로, 식염수 ,멸균수, 링거액, 완충 식염수,덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 상기 성분들 중 어느 하나의 이상의 성분을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 등 통상적으로 사용되는 다른 첨가제를 추가적으로 포함할 수 있다. 또한 투여 목적에 따라 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있고, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 또는 조직 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 상기와 같은 담체, 부형제 또는 첨가제의 종류는 당업계의 통상적인 제제를 모두 포함하며, 상기 예에 의해 사용가능한 담체, 부형제 또는 첨가제의 종류가 제한 되는 것은 아니다.The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the administration mode. Specifically, it is possible to use a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and any of the above components, And other additives conventionally used, such as buffers. In addition, depending on the purpose of administration, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, A target organ or tissue specific antibody or other ligand can be used in combination with the carrier. The carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.

상기와 같은 조성물 또는 혼합물은 목적 또는 필요에 따라 당업계에서 사용되는 통상적인 방법, 투여 경로, 투여량에 따라 적절하게 개체에 투여될 수 있다.투여 경로의 예로는 경구, 비 경구, 피하, 복강 내, 폐 내, 및 비강 내로 투여될 수 있고, 국부적 면역억제 치료를 위해, 필요하다면 병변 내 투여를 포함하는 적합한 방법에 의해 투여된다. 비 경구 주입에는 근육 내, 정맥 내, 동맥 내, 복강 내 또는 피하투여가 포함된다. 또한 당업계에 공지된 방법에 따라 적절한 투여량 및 투여 횟수가 선택될 수 있으며, 조성물의 양 및 투여 횟수는 예방 또는 치료하고자 하는 증상의 종류, 투여 경로, 성별, 건강 상태, 식이, 개체의 연령 및 체중, 및 질환의 중증도와 같은 다양한 인자에 의해 적절하게 결정될 수 있다.The composition or the mixture may be appropriately administered to a subject according to the conventional method, route of administration, and dose used in the art depending on the purpose or necessity. Examples of the administration route include oral, non-oral, subcutaneous, Intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally, Non-oral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Appropriate dosages and times of administration may be selected according to methods known in the art, and the amount and frequency of administration will depend on the type of symptoms to be prevented or treated, the route of administration, sex, health status, diet, And weight, and the severity of the disease.

본 발명에서 제공되는 SMP 유전자를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 바이오 마커는 항생제에 대한 황색포도상구균의 감수성 마커로서의 정확성 및 신뢰도가 탁월하여 이를 이용하여 황색포도상구균 감염 환자 맞춤별로 치료제를 제공하는 것이 가능하며, 환자별 적합한 항생제의 투여로 항생제의 오남용을 막을 수 있다.The biomarker for predicting the susceptibility of Staphylococcus aureus to antibiotics containing the SMP gene provided by the present invention is excellent in the accuracy and reliability as a susceptibility marker of Staphylococcus aureus against antibiotics, It is possible to provide a therapeutic agent, and appropriate antibiotic administration for each patient can prevent abuse of antibiotics.

또한, 본 발명에서 제공되는 SMP 프로모터 기반 리포터 시스템은 기존의 24시간이 걸리는 배양 기반의 MIC50의 측정과 비교하여 유사한 정확도를 유지하면서 1~5시간 안에 결과를 수득할 수 있어 어세이 시간을 현저하게 단축시키는 효과가 있다. 따라서, 본 발명의 리포터 시스템을 이용하여 신속하고 정확하게 특정 항생물질에 대한 황색포도상구균의 감수성을 예측할 수 있을 뿐만 아니라, 신속하고 정확하게 황색포도상구균에 대해 감수성을 나타내는 항생물질을 스크리닝할 수 있다.In addition, the SMP promoter-based reporter system provided in the present invention can obtain the results within 1 to 5 hours while maintaining similar accuracy as compared with the conventional 24-hour culture-based MIC 50 measurement, . Therefore, the reporter system of the present invention can promptly and accurately predict the susceptibility of Staphylococcus aureus to a specific antibiotic substance, and can also rapidly and accurately screen antibiotic substances showing susceptibility to Staphylococcus aureus.

도 1a 및 도 1b는 SMP가 시프로플록사신의 처리 시간 및 처리 용량에 의존적으로 황색포도상구균에서 유도된다는 것을 나타낸 그래프이다.Figures 1A and 1B are graphs showing that SMP is induced in Staphylococcus aureus in dependence of treatment time and treatment capacity of ciprofloxacin.

도 2는 SMP 유전자를 측정하기 위한 RT-qPCR 결과를 나타내는 그래프이다.Fig. 2 is a graph showing RT-qPCR results for measuring SMP gene.

도 3은 1종의 pan-감수성 균주와 서로 다른 항생제 감수성 프로파일을 갖는 5종의 임상적 분리균을 포함하는 6종의 다른 황색포도상구균을 테스트하여 다양한 항생제에 대한 감수성 결과를 나타낸 표이다.Figure 3 is a table showing susceptibility to various antibiotics by testing six different Staphylococcus aureus strains including one pan-susceptible strain and five clinical isolates having different antimicrobial susceptibility profiles.

도 4 및 5는 RT-qPCR 1종의 pan-감수성 균주와 서로 다른 항생제 감수성 프로파일을 갖는 5종의 임상적 분리물을 포함하는 6종의 다른 황색포도상구균에 다양한 항생제를 처리한 후 RT-qPCR에 의해 SMP 유도를 측정한 그래프이다. Figures 4 and 5 show the results of RT-qPCR and RT-qPCR after treatment of various antibiotics with six different strains of S. aureus, including one pan-sensitive strain of RT-qPCR and five clinical isolates with different antimicrobial susceptibility profiles. In which SMP induction was measured.

도 6은 SMP 프로모터와 GFP 유전자를 포함하는 유전자 구조체 및 이를 이용한 SMP 프로모터 기반 리포터 시스템의 개략적인 컨셉을 도시한 것이다.FIG. 6 shows a schematic concept of a gene construct containing an SMP promoter and a GFP gene and a reporter system based on the SMP promoter using the same.

도 7은 도 6의 리포터 시스템을 이용한 황색포도상구균의 감수성 예측방법 또는 항생물질의 스크리닝 방법을 검증한 결과를 나타낸다.FIG. 7 shows the results of verifying the method for predicting the susceptibility of Staphylococcus aureus or the screening method for antibiotics using the reporter system of FIG.

하기의 실시예를 통하여 본 발명을 더욱 구체적으로 설명하기로 하지만, 하기 실시예가 본 발명의 범위를 제한하는 것은 아니며, 이는 본 발명의 이해를 돕기 위한 것으로 해석되어야 할 것이다.The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.

[[ 실시예Example 1] One]

RNA 준비RNA preparation

황색포도상구균은 뮬러-힌트배지(Mueller hinton medium)에서 밤새 37℃에서 180rpm으로 진탕배양하여 성장시켰다. 배양물(culture)은 신선한 뮬러-힌트배지에서 0.02의 600nm에서 재접종 되었고, 중간 대수기(mid-logarithmic phase)까지 37℃에서 호기성으로 성장시켰다. 이후, 황색포도상구균을 희석하여 125mL 플라스크에서 600nm에서 0.05의 흡광도(광학밀도)를 주었다. 표시된 농도로 항생제가 처리되었다. 플라스크는 표시된 시간 동안 37℃에서 180rpm으로 배양되었다. 박테리아는 10분 동안 3300rpm으로 원심분리에 의해 수확되었고, 120ul의 용해 버퍼[100ul의 50mM EDTA, 10ul의 10mg/ml 리소자임, 10ull의 10mg/ml 리소스타핀]에서 재현탁 되었다. 30분 동안 37℃의 water bath에서 배양되었고, 매 10분마다 볼텍싱 하였으며 초음파 분해에 의해 분쇄하였다. 이후, RNeasy mini kit(Qiagen) 프로토콜에 따라 추출 과정을 수행하고 50ul의 RNA elute(용리)를 수득하였다. 총 RNA 추출 후, DNase 1 (Qiagen)을 이용해 남아있는 게놈 DNA를 제거하였다. 반응 부피는 10ul이었고, 1ul 버퍼 RDD, 0.25ul의 DNas I 스톡 용액 및 6ul의 50ng/ul RNA로 구성되었다. 혼합물은 10분 동안 실온에서 배양되었고 40ul의 RNase-free water가 반응을 정지시키기 위해 첨가되었다.Staphylococcus aureus was grown in Mueller hinton medium overnight at 37 ° C with shaking at 180 rpm. The culture was re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. Staphylococcus aureus was then diluted to give an absorbance (optical density) of 0.05 at 600 nm in a 125 mL flask. Antibiotics were treated at the indicated concentrations. The flask was incubated at 37 [deg.] C at 180 rpm for the indicated time. Bacteria were harvested by centrifugation at 3300 rpm for 10 min and resuspended in 120 ul of lysis buffer (100 ul of 50 mM EDTA, 10 ul of 10 mg / ml lysozyme, 10 ul of 10 mg / ml resource taffine). Cultured in a 37 ° C water bath for 30 minutes, vortexed every 10 minutes and crushed by sonication. Thereafter, the extraction procedure was performed according to the RNeasy mini kit (Qiagen) protocol and 50 ul of RNA elute was obtained. After extraction of total RNA, the remaining genomic DNA was removed using DNase 1 (Qiagen). The reaction volume was 10 ul and consisted of 1 ul buffer RDD, 0.25 ul of DNas I stock solution and 6 ul of 50 ng / ul RNA. The mixture was incubated for 10 min at room temperature and 40 ul of RNase-free water was added to stop the reaction.

RNA 시퀀싱 및 분석RNA sequencing and analysis

Ribo-zero rRNA 제거 키트(Epicentre,　USA)는 제조업자의 지시에 따라 리보솜 RNA 고갈(depletion)을 위해 사용하였다. 일루미나 시퀀싱 (Illumina　sequencing)을 위한 라이브러리가 TruSeq　Stranded　mRNA　sample　prep　kit　(Illumina,　USA)와 함께 제조업자의 프로토콜에 따라 제조되었다. RNA 시퀀싱은 싱글-엔드(single-end) 50bp 시퀀싱을 이용하여 Illumina　HiSeq　2500　플랫폼 상에서 수행되었다.The Ribo-zero rRNA removal kit (Epicenter, USA) was used for ribosomal RNA depletion according to the manufacturer's instructions. A library for Illumina sequencing was prepared according to the manufacturer's protocol with the TruSeq Stranded mRNA sample prep kit (Illumina, USA). RNA sequencing was performed on an Illumina HiSeq 2500 platform using single-ended 50bp sequencing.

표준 유전체(reference　genome)를 위한 서열 데이터는　NCBI　데이터베이스로부터 검색하였다.　Quality-filtered　reads는 Bowtie2를 이용하여 표준-유전체 서열에 따라 정렬되었다.　상대적으로 전사가 많은 곳(relative　transcript　abundance)은 백만분의 맵핑된 서열 판독 당 엑손 서열의 킬로베이스 당 판독의 단편(FPKM)에서 측정된다.　Sequence data for the standard genome were retrieved from the NCBI database. Quality-filtered reads were sorted according to the standard-genome sequence using Bowtie2. Relative transcript abundance is measured in fractions of a reading per kilobase (FPKM) of exon sequences per million mapped sequence reads.

EggNOG　(evolutionary　genealogy　of　genes:　Nonsupervised　Orthologous　Groups)　데이터베이스는 유전자를 기능적으로 연관된 그룹으로 모으기 위해 사용되었고　KEGG　(Kyoto　Encyclopedia　of　Genes　and　Genomes)　데이터베이스는 대사경로를 분석하기 위해 사용되었다. CLRNASeqTM　program　(ChunLab,　South　Korea)을 이용하여 맵핑 결과와 차등적으로 발현된 유전자(differentially　expressed　gene, DEG) 분석의 시각화가 수행되었다.EggNOG (evolutionary genealogy of genes) database was used to collect genes into functionally related groups and KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to analyze metabolic pathways. Visualization of the differentially expressed gene (DEG) analysis was performed using the CLRNASeq ™ program (ChunLab, South Korea).

그 결과, 도 1a에 나타난 바와 같이 SA_13280 유전자는 가상의 작은 막 단백질(Small Membrane Protein, SMP)을 코딩하며, 황색포도상구균(S. aureus)에만 보존되어 있다는 것을 확인할 수 있었다. 또한 본 실시예의 RNA 시퀀싱에 의한 전사체 분석은 SMP가 시프로플록사신의 처리 시간 및 처리 용량에 의존적으로 황색포도상구균에서 유도된다는 것을 밝혔다(도 1b).As a result, it was confirmed that SA_13280 gene encodes a hypothetical small membrane protein (SMP) as shown in Fig. 1A, and is stored only in S. aureus . Transcript analysis by RNA sequencing of this example also revealed that SMP is induced in S. aureus depending on the treatment time and treatment capacity of ciprofloxacin (Fig. 1B).

[[ 실시예Example 2] 2]

실시간 real time PCRPCR 어세이Assay

SMP 유전자를 측정하기 위해, 실시간 원스텝 RT-PCR 증폭이 QuantiFast SYBR Green RT-PCR Kit(Qiagen)를 이용하여 수행되었다. 이 과정은 50　ng의 샘플 RNA, 0.4uM의 각 프라이머(sense: 5’- GTTTCAAGTGCAGCCTTTGC -3’(서열번호 2), antisense: 5’- GCGAACACTTTCCCATCTCT -3’(서열번호 3)), 12.5ul의 SYBR Green I PCR master mix 및 0.25ul의 RT mix와 함께 25 μL의 최종 반응 부피에서 3회 수행되었다. PCR은 50°C에서 10분 동안 역전사 개시, 95°C에서 5분 동안 1 사이클, 95°C에서 10초 동안 변성 40 사이클, 58°C에서 10초 동안 어닐링(annealing) 및 72°C에서 20초 동안 Applied biosystems ViiA 7 Real-time PCR System에 의해 연장으로 수행되었다. 그리고 실시간 형광 측정이 이루어졌고, 각 샘플에 대한 threshold cycle (CT) 값은 형광이 threshold limit를 초과하는 포인트를 측정함으로써 계산되었다. CT 값은 항생제가 없는 박테리아와 비교하여 폴드 체인지(fold change)로 변환되었다.To measure the SMP gene, real-time one-step RT-PCR amplification was performed using the QuantiFast SYBR Green RT-PCR Kit (Qiagen). This procedure included 50 ng of sample RNA, 0.4 uM of each primer (sense: 5'- GTTTCAAGTGCAGCCTTTGC-3 '(SEQ ID NO: 2), antisense: 5'- GCGAACACTTTCCCATCTCT- 3' Green I PCR master mix and 0.25 ul RT mix in a final reaction volume of 25 μL. The PCR was carried out for 10 min at 50 ° C for reverse transcription, 1 cycle for 5 min at 95 ° C, 40 cycles of denaturation for 10 sec at 95 ° C, annealing for 10 sec at 58 ° C, and annealing for 20 sec at 72 ° C Lt; RTI ID = 0.0 > ViiA < / RTI > 7 Real-time PCR System. Real-time fluorescence measurements were made and the threshold cycle (C T ) value for each sample was calculated by measuring the point at which fluorescence exceeded the threshold limit. C T values were converted to fold changes compared to bacteria without antibiotics.

본 실시예의 RT-qPCR은 SMP 유도가 시프로플록사신 처리에 의해 유발되며 반코마이신, 메티실린 및 테트라사이클린을 포함하는 다른 항생제도 SMP를 유도한다는 것을 보여준다.The RT-qPCR of this example shows that SMP induction is induced by ciprofloxacin treatment and that other antibiotics including vancomycin, methicillin and tetracycline also induce SMP.

[[ 실시예Example 3] 3]

SMP 유도가 항생제 감수성과 연관되어 있다는 것을 발견하고, SMP 유도를 보기 위해 1종의 pan-감수성 균주와 서로 다른 항생제 감수성 프로파일을 갖는 5종의 임상적 분리물을 포함하는 6종의 다른 황색포도상구균을 하기와 같이 테스트 하였다. 상기 6종의 다른 황색포도상구균은 감염환자의 환부 표본 (혈액, 상처, 호흡기 등) 을 채취하여 5% 혈액 고체배지에 도말 후 35 내지 37 °C에서 배양하여 얻어진 균으로, 선별배지 (mannitol salt agar) 혹은 검사 (coagulase test 등)를 통하여 황색포도상구균임이 확인된 균주이다.We found that SMP induction was associated with antibiotic susceptibility, and to see SMP induction, one pan-sensitive strain and six other Staphylococcus aureus strains including five clinical isolates with different antimicrobial susceptibility profiles Were tested as follows. The six different Staphylococcus aureus strains were obtained by collecting the affected specimens (blood, wounds, respiratory system, etc.) from infected patients and culturing them in a 5% blood solid medium at 35-37 ° C. agar) or a test (coagulase test, etc.).

항균 활성 Antimicrobial activity 어세이Assay

황색포도상구균(ATCC 25923)과 MDR 균주는 밤새 37℃에서 진탕배양함으로써 뮬러-힌트배지에서 성장시켰다. 배양물은 신선한 뮬러-힌트배지에서 0.02의 600nm에서 재접종되었고 중간 대수기(mid-logarithmic phase)까지 37℃에서 호기성으로 성장시켰다. 이후, 박테리아는 600nm에서 0.05의 광학 밀도를 주기 위해 희석되었다. 그리고 화합물은 뮬러-힌트배지에서 순차적 2배 희석으로 제조되었다. 순차적으로 희석된 화합물과 박테리아는 384 미량정량판에 각각 10ul와 40ul로 3회 로딩되었다. 플레이트는 24시간 동안 37℃의 5% CO2에서 배양되었다. 흡광도는 균주에서 항생제의 50% 최소 억제 농도 (MIC50)를 수득하기 위해 측정되고 분석되었다.Staphylococcus aureus (ATCC 25923) and MDR strain were grown in Mueller-hint medium by shaking culture at 37 ° C overnight. The cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. The bacteria were then diluted to give an optical density of 0.05 at 600 nm. And the compounds were prepared in sequential two-fold dilutions in Mueller-hint medium. Sequentially diluted compounds and bacteria were loaded three times with 10ul and 40ul, respectively, in 384 microgram plates. Plates were incubated at 37 ° C in 5% CO 2 for 24 hours. Absorbance was measured and analyzed to obtain a 50% minimum inhibitory concentration (MIC 50 ) of antibiotics in the strain.

RNA 준비RNA preparation

황색포도상구균은 밤새 37℃에서 180rpm으로 진탕배양함으로써 뮬러-힌트배지에서 성장시켰다. 배양물은 신선한 뮬러-힌트배지에서 0.02의 600nm에서 재접종하였고 대수기(mid-logarithmic phase)까지 37℃에서 호기성으로 성장시켰다. 이후, 황색포도상구균은 125mL 플라스크에서 600nm에서 0.05의 광학 밀도를 주기 위해 희석시켰다. 표시된 농도의 항생제를 처리하였다. 플라스크는 37℃에서 180rpm으로 표시된 시간 동안 배양되었다. 박테리아는 10분 동안 3300rpm에서 원심분리하여 수확되었고 120ul의 용해 버퍼 [100ul의 50mM EDTA, 10ul의 10mg/ml 라이소자임, 10ull의 10mg/ml 리소스타핀]에서 재현탁 되었다. 30분 동안 37℃의 water bath에서 배양되었고, 매 10분 마다 볼텍싱 하였으며 초음파 분해에 의해 분쇄하였다. 이후, RNeasy mini kit(Qiagen) 프로토콜에 따라 추출 과정을 수행하고 50ul의 RNA elute(용리)를 수득하였다. 총 RNA 추출 후, DNase 1 (Qiagen)을 이용해 남아있는 게놈 DNA를 제거하였다. 반응 부피는 10ul이었고, 1ul의 버퍼 RDD, 0.25ul의 DNas I 스톡 용액 및 6ul의 50ng/ul RNA로 구성되었다. 혼합물은 10분 동안 실온에서 배양되었고 40ul의 RNase-free water가 반응을 정지시키기 위해 첨가되었다.Staphylococcus aureus was grown in Mueller-hint medium by shaking incubation at 37 ° C overnight at 180 rpm. The cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium and aerobically grown at 37 ° C to the mid-logarithmic phase. Staphylococcus aureus was then diluted in a 125 mL flask to give an optical density of 0.05 at 600 nm. Antibiotics at the indicated concentrations were treated. The flask was incubated at 37 < 0 > C at 180 rpm for the indicated time. Bacteria were harvested by centrifugation at 3300 rpm for 10 min and resuspended in 120 ul of lysis buffer (100 ul of 50 mM EDTA, 10 ul of 10 mg / ml lysozyme, 10 ul of 10 mg / ml resource taffine). Cultured in a 37 ° C water bath for 30 minutes, vortexed every 10 minutes and crushed by sonication. Thereafter, the extraction procedure was performed according to the RNeasy mini kit (Qiagen) protocol and 50 ul of RNA elute was obtained. After extraction of total RNA, the remaining genomic DNA was removed using DNase 1 (Qiagen). The reaction volume was 10 ul and consisted of 1 ul of buffer RDD, 0.25 ul of DNas I stock solution and 6 ul of 50 ng / ul RNA. The mixture was incubated for 10 min at room temperature and 40 ul of RNase-free water was added to stop the reaction.

실시간 real time PCRPCR 어세이Assay

SMP 유전자를 측정하기 위해, 실시간 원스텝 RT-PCR 증폭이 QuantiFast SYBR Green RT-PCR Kit(Qiagen)를 이용하여 수행되었다. 이 과정은 50　ng의 샘플 RNA, 0.4uM의 각 프라이머(sense: 5’- GTTTCAAGTGCAGCCTTTGC -3’(서열번호 2), antisense: 5’- GCGAACACTTTCCCATCTCT -3’(서열번호 3)), 12.5ul의 SYBR Green I PCR master mix 및 0.25ul의 RT mix와 함께 25 μL의 최종 반응 부피에서 3회 수행되었다. PCR은 50°C에서 10분 동안 역전사 개시, 95°C에서 5분 동안 1 사이클, 95°C에서 10초 동안 변성 40 사이클, 58°C에서 10초 동안 어닐링(annealing) 및 72°C에서 20초 동안 Applied biosystems ViiA 7 Real-time PCR System에 의해 연장으로 수행되었다. 그리고 실시간 형광 측정이 이루어졌고, 각 샘플에 대한 threshold cycle (CT) 값은 형광이 threshold limit를 초과하는 포인트를 측정함으로써 계산되었다. CT 값은 항생제가 없는 박테리아와 비교하여 폴드 체인지(fold change)로 변환되었다.To measure the SMP gene, real-time one-step RT-PCR amplification was performed using the QuantiFast SYBR Green RT-PCR Kit (Qiagen). This procedure included 50 ng of sample RNA, 0.4 uM of each primer (sense: 5'- GTTTCAAGTGCAGCCTTTGC-3 '(SEQ ID NO: 2), antisense: 5'- GCGAACACTTTCCCATCTCT- 3' Green I PCR master mix and 0.25 ul RT mix in a final reaction volume of 25 μL. The PCR was carried out for 10 min at 50 ° C for reverse transcription, 1 cycle for 5 min at 95 ° C, 40 cycles of denaturation for 10 sec at 95 ° C, annealing for 10 sec at 58 ° C, and annealing for 20 sec at 72 ° C Lt; RTI ID = 0.0 > ViiA < / RTI > 7 Real-time PCR System. Real-time fluorescence measurements were made and the threshold cycle (C T ) value for each sample was calculated by measuring the point at which fluorescence exceeded the threshold limit. C T values were converted to fold changes compared to bacteria without antibiotics.

그 결과, 도 4 및 5에 나타난 바와 같이 다양한 항생제로 처리된 균주에서 RT-qPCR에 의해 SMP 유도를 측정할 때, 박테리아가 감수성을 나타내는 항생제와 함께 배양했을 때만 현저한 SMP 유도가 관찰되었다. 예를 들어, 1번 균주는 반코마이신에만 감수성을 나타내고, 시프로플록사신, 메티실린 및 테트라사이클린에는 감수성을 나타내지 않았다(도 3). 이러한 감수성 프로파일과 일치하여, SMP는 오직 반코마이신 처리에 의해 1번 균주에서 유도된 반면, 대조균주(항생제 민감성 황색포도상구균)는 테스트된 모든 항생제에 의해 현저하게 SMP를 유도한다(도 4). 유사하게, 4번 균주는 이의 항생제 감수성 프로파일과 일치하여 시프로플록사신, 반코마이신 및 테트라사이클린으로 SMP 유도를 나타내지만 메티실린 처리에서는 SMP 유도를 나타내지 않았다. 이러한 데이터는 SMP가 황색포도상구균의 항생제 감수성을 예측하기 위한 바이오마커가 될 수 있다는 것을 제안한다(도 5).As a result, when SMP induction was measured by RT-qPCR in strains treated with various antibiotics as shown in Figs. 4 and 5, remarkable SMP induction was observed only when bacteria were cultured with an antibiotic exhibiting sensitivity. For example, strains No. 1 showed susceptibility to vancomycin only and did not show susceptibility to ciprofloxacin, methicillin and tetracycline (FIG. 3). Consistent with this susceptibility profile, SMP was induced only in strain 1 by vancomycin treatment whereas the control strain (antibiotic sensitive Staphylococcus aureus) induced SMP significantly by all tested antibiotics (FIG. 4). Similarly, strain 4 showed SMP induction with ciprofloxacin, vancomycin and tetracycline in agreement with its antibiotic susceptibility profile, but did not show SMP induction in methicillin treatment. These data suggest that SMP can be a biomarker to predict antibiotic susceptibility of Staphylococcus aureus (Fig. 5).

동일한 방법으로 수득한 30 개의 MRSA(Methicillin-resistant S. aureus) 균주 및 20개의 MSSA(Methicillin-sensitive S. aureus) 균주에 대해 실험하였다.30 MRSA (Methicillin-resistant S. aureus) strains and 20 MSSA (Methicillin-sensitive S. aureus) strains obtained by the same method were tested.

상기 50개 균주에 대해 SMP 유도와 항생제 감수성 예측 가능성에 대해 실험하였고, 그 결과 마찬가지로 SMP 발현은 항생제 감수성 예측을 위한 뛰어난 바이오마커가 될 수 있다는 점을 확인할 수 있었다. SMP induction and antibiotic susceptibility predictability were tested for the 50 strains. As a result, it was confirmed that SMP expression could be an excellent biomarker for antibiotic susceptibility prediction.

[[ 실시예Example 4] 4]

SMPSMP 프로모터를 포함하는  Containing a promoter pCM29의pCM29 구성 Configuration

프로모터 서열은 황색포도상구균(S. aureus) 염색체 DNA를 주형으로 사용하여 PCR에 의해 증폭되었다. 다음의 프라이머 쌍은 증폭을 위해 사용되었다: NheI 및 KpnI와 함께 sense: 5’ TAGGTGGCTAGCTTCTCTCATATATAGGCACTCCC 3’(서열번호 5), antisense: 5’ GTTGTTGGTACCTTTCACTCCTAGAACATTTGTTTG 3’(서열번호 6). PCR 산물은 GFP 리포터 유전자의 상부(upstream)에서 pCM29에 연결되었고 열 충격 과정에 의해 E. coli　Top10에 형질전환 되었다. 형질전환체는 암피실린 (100　μg/ml)으로 보충된 LB에서 선별되었다. SMP 프로모터를 포함하는 pCM29의 구성은 Plasmid Midiprep kit (Qiagen)를 이용하여 수집되었고 Dam과 Dcm 메틸레이션이 없도록 dam-/dcm- 형질전환성 E. coli에 다시 형질전환 되었다. 형질전환체는 37℃의 SOC 성장 배지에서 1시간 동안 180rpm으로 진탕배양하여 성장시켰고 암피실린 (100　μg/ml)을 포함하는 LB 플레이트에 도말하였다.The promoter sequence is S. aureus , Were amplified by PCR using chromosomal DNA as template. The following primer pairs were used for amplification: sense: 5 'TAGGTGGCTAGCTTCTCTCATATATAGGCACTCCC 3' (SEQ ID NO: 5), antisense: 5 'GTTGTTGGTACCTTTCACTCCTAGAACATTTGTTTG 3' (SEQ ID NO: 6) with NheI and KpnI . The PCR product was ligated to pCM29 upstream of the GFP reporter gene and transformed into E. coli Top10 by thermal shock. The transformants were selected in LB supplemented with ampicillin (100 μg / ml). It was transformed again to the transgenic E. coli - configuration of pCM29 containing SMP promoter Plasmid Midiprep kit were collected using a (Qiagen) and Dam Dcm methylation prevents the dam - / dcm. The transformants were grown in SOC growth medium at 37 DEG C for 1 hour with shaking at 180 rpm and plated on LB plates containing ampicillin (100 mu g / ml).

일렉트로컴포넌트Electrocomponent 세포( cell( ElectrocompetentElectrocompetent Cells)의 준비 Cells

황색포도상구균(S. aureus )은 뮬러-힌트배지에서 밤새 37℃에서 180rpm으로 진탕배양하여 성장시켰다. 배양물은 75ml의 신선한 Mueller hinton 배지에 0.05의 600nm에서 재접종되었고 3~5 x 108 cells/ml(OD600 0.8-0.85)에 도달할 때까지 37℃에서 호기성으로 성장시켰다. 박테리아는 성장을 멈추기 위해 15분 동안 얼음에서 냉각시켰고 5분 동안 3300rpm으로 4℃에서 원심분리로 수확하였다. 세포는 10ml의 찬 얼음물로 3회 세척하였고 5ml의 차가운 10% 글리세롤로 세척하였다. 5분 동안 3300rpm으로 4℃에서 원심분리에 의해 수확된 세포는 5ml의 10% 글리세롤에 재현탁시켰다. 실온에서 5분 동안 배양되었고 5분 동안 3300rpm으로 4℃에서 원심분리에 의해 수확되었다. 수집된 세포는 1ml의 10% 글리세롤에 재현탁되었다. 수득된 최종 세포 농도는 약 1 x 1010 cells/ml였다. 일렉트로컴포넌트 세포는 제조 후 1시간 이내에 사용되었다.Staphylococcus aureus (S. aureus) is a Muller-grown by shaking culture overnight at 37 to 180rpm ℃ medium in question. The cultures were re-inoculated in 75 ml of fresh Mueller hinton medium at 600 nm at 0.05 and grown aerobically at 37 ° C until 3-5 x 10 8 cells / ml (OD 600 0.8-0.85) were reached. Bacteria were chilled on ice for 15 minutes to stop growth and harvested by centrifugation at 3300 rpm at 4 ° C for 5 minutes. Cells were washed 3 times with 10 ml cold ice water and washed with 5 ml cold 10% glycerol. Cells harvested by centrifugation at 4 ° C at 3300 rpm for 5 minutes were resuspended in 5 ml of 10% glycerol. Incubated at room temperature for 5 minutes and harvested by centrifugation at 4 ° C at 3300 rpm for 5 minutes. The collected cells were resuspended in 1 ml of 10% glycerol. The final cell concentration obtained was about 1 x 10 < 10 > cells / ml. Electrocomponent cells were used within 1 hour after manufacture.

전기 천공법 과정Electric Perforation course

전기 천공을 위해, 50ul의 일렉트로컴포넌트 세포가 2ul의 DNA(보통 1ug)와 혼합되었고 30분 동안 실온에서 배양되었다. 그리고 세포 현탁-DNA 혼합물을 0.2초의 전기 천공 큐벳으로 옮기고 Shock-Pod에 두었다. 세포와 DNA는 Gene Pulser XcellTM 장치(Bio-Rad Laboratoies)에서 25uF의 커패시턴스, 100 ohms 저항력 및 2.9kV로 전기 천공되었다. 이후 세포는 즉시 900ul의 TSA에 재현탁되었고, 15ml 팔콘 튜브(Falcon tube)로 옮겨져 1시간 동안 37℃의 5% CO2에서 배양되었다. 이후 세포는 10ug/ml의 클로람페니콜을 포함하는 TSA 플레이트에 도말되었고 37℃의 5% CO2에서 24~48시간 동안 배양되었다.For electroporation, 50 ul of electrocomponent cells were mixed with 2 ul of DNA (usually 1 ug) and incubated at room temperature for 30 minutes. The cell suspension-DNA mixture was transferred to a 0.2 second electroporation cuvette and placed in a Shock-Pod. Cells and DNA were electroplated on a Gene Pulser Xcell TM instrument (Bio-Rad Laboratoies) with a capacitance of 25 uF, 100 ohms resistivity and 2.9 kV. The cells were then immediately resuspended in 900 ul of TSA, transferred to a 15 ml Falcon tube and incubated at 37 ° C in 5% CO 2 for 1 h. The cells were then plated on TSA plates containing 10 ug / ml of chloramphenicol and incubated at 37 ° C in 5% CO 2 for 24-48 hours.

[[ 실시예Example 5] 5]

형광 리포터 시스템Fluorescent Reporter System

pCM29-SMP 프로모터의 구성을 포함하는 황색포도상구균은 10ug/ml의 클로람페니콜로 보충된 뮬러-힌트배지에서 밤새 37℃, 180rpm으로 진탕배양하여 성장시켰다. 배양물은 10ug/ml의 클로람페니콜로 보충된 신선한 뮬러-힌트배지에서 0.02의 600nm에서 재접종되었고 대수기(mid-logarithmic phase)까지 37℃에서 호기성으로 성장시켰다. 세포는 3300rpm으로 5분 동안 원심분리에 의해 수집되었고 10ml의 신선한 뮬러-힌트배지에 재현탁되었다. 세포는 600nm에서 0.05의 흡광도를 주기 위해 희석되었다. 시프로플록사신은 뮬러-힌트배지에서 순차적 2배 희석으로 제조되었다. 순차적으로 희석된 화합물과 박테리아는 96 미량정량판(microtiter plate)에 각각 40ul 및 160ul로 2회 로딩되었다. 플레이트는 3시간 또는 24시간 동안 37℃의 5% CO2에서 배양되었다. 흡광도(OD600) 또는 형광(Ex490nm/Em535nm)은 시프로플록사신의 50% 최소억제농도(MIC50)를 수득하기 위해 측정 및 분석되었다. Staphylococcus aureus comprising the construction of the pCM29-SMP promoter was grown in a Mueller-hint medium supplemented with 10 ug / ml of chloramphenicol by shaking incubation overnight at 37 ° C at 180 rpm. The cultures were re-inoculated at 600 nm at 0.02 in fresh Mueller-hint medium supplemented with 10 ug / ml of chloramphenicol and aerobically grown at 37 ° C to the mid-logarithmic phase. Cells were harvested by centrifugation at 3300 rpm for 5 minutes and resuspended in 10 ml of fresh Mueller-hint medium. The cells were diluted to give an absorbance of 0.05 at 600 nm. Ciprofloxacin was prepared by sequential double dilution in Mueller-hint medium. Sequentially diluted compounds and bacteria were loaded twice onto 96-well microtiter plates at 40ul and 160ul, respectively. Plates were incubated at 37 ° C in 5% CO 2 for 3 hours or 24 hours. Absorbance (OD 600) or fluorescence (Ex490nm / Em535nm) was measured and analyzed in order to obtain the 50% minimum inhibitory concentration (MIC 50) of ciprofloxacin.

그 결과, 도 7에 나타난 바와 같이 시프로플록사신과 3시간 내로 배양하여 황색포도상구균의 MIC50(minimum inhibitory concentration)을 정확하게 측정하였다. 24시간이 걸리는 통상적인 배양 기반의 MIC50의 측정과 비교하여, SMP 프로모터 기반 리포터 시스템은 유사한 정확도로 어세이 시간을 단축시키므로 본 발명의 SMP 프로모터를 이용한 감수성 예측방법 및 항생물질의 스크리닝 방법은 특정 항생물질에 대한 황색포도상구균의 감수성 예측 및 황색포도상구균에 대한 항생물질을 스크리닝하기 위한 좋은 어세이 시스템이라는 것을 알 수 있다.As a result, MIC 50 (minimum inhibitory concentration) of Staphylococcus aureus was accurately measured by incubation with ciprofloxacin within 3 hours as shown in FIG. Compared with conventional culturing-based MIC 50 measurements that take 24 hours, the SMP promoter-based reporter system shortens the assay time with similar accuracy, so that the susceptibility prediction method and the antibiotic screening method using the SMP promoter of the present invention are specific It is a good estimation system for predicting the susceptibility of Staphylococcus aureus to antibiotics and for screening antibiotics against Staphylococcus aureus.

본 발명은 항생물질에 대한 황색포도상구균의 감수성 예측용 바이오 마커로 제공될 수 있다. 또한 본 발명은 항생물질에 대한 황색포도상구균의 감수성 예측을 통하여 황색포도상구균에 의한 감염증을 예방 또는 치료하기 위한 약학적 조성물 및 제제를 제공할 수 있다.The present invention can be provided as a biomarker for predicting the susceptibility of Staphylococcus aureus to antibiotics. The present invention also provides a pharmaceutical composition and a pharmaceutical preparation for preventing or treating infectious diseases caused by Staphylococcus aureus by predicting the susceptibility of Staphylococcus aureus to antibiotics.

Claims (25)

SMP(Small Membrane Protein) 유전자를 포함하는 항생물질에 대한 황색포도상구균 (Staphylococcus aureus)의 감수성 예측용 바이오 마커.A biomarker for predicting the susceptibility of Staphylococcus aureus to antibiotics containing SMP (Small Membrane Protein) gene. 제1항에 있어서, 상기 SMP 유전자는 서열번호 1로 이루어진 것을 특징으로 하는 바이오 마커.2. The biomarker of claim 1, wherein the SMP gene comprises SEQ ID NO: 1. SMP(Small Membrane Protein)의 발현 수준을 측정하는 제제를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 조성물.A composition for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising an agent for measuring the expression level of SMP (Small Membrane Protein). 제3항에 있어서, 상기 발현 수준을 측정하는 제제는 mRNA에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 포함하는 것을 특징으로 하는 조성물.4. The composition of claim 3, wherein the agent that measures the expression level comprises an antisense oligonucleotide, a primer pair, or a probe that specifically binds mRNA. 제4항에 있어서, 상기 프라이머 쌍은 (a) 서열번호 2로 표시되는 정방향 프라이머; 및 (b) 서열번호 3으로 표시되는 역방향 프라이머로 이루어진 것을 특징으로 하는 조성물.5. The primer set of claim 4, wherein the primer pair comprises: (a) a forward primer represented by SEQ ID NO: 2; And (b) a reverse primer represented by SEQ ID NO: 3. 제4항에 있어서, 상기 발현 수준을 측정하는 제제는 단백질에 특이적으로 결합하는 항체, 펩타이드 또는 뉴클레오티드를 포함하는 것을 특징으로 하는 조성물.5. The composition of claim 4, wherein the agent that measures the expression level comprises an antibody, peptide, or nucleotide that specifically binds to the protein. 제3항 내지 제6항 중 어느 한 항의 조성물을 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측용 키트.A kit for predicting the susceptibility of Staphylococcus aureus to antibiotics comprising the composition of any one of claims 3 to 6. 제7항에 있어서, 상기 키트는 RT-PCR 키트, 경쟁적 RT-PCR 키트, 실시간 RT-PCR 키트, DNA 칩 키트 또는 단백질 칩 키트인 것을 특징으로 하는 키트.8. The kit according to claim 7, wherein the kit is an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit or a protein chip kit. (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 항생물질을 처리한 후 시료 내 SMP 유전자의 mRNA 또는 이의 단백질 발현 수준을 측정하는 단계; 및(a) measuring the mRNA of the SMP gene or the protein expression level thereof in the sample after treating the biological sample isolated from the patient with Staphylococcus aureus infection with antibiotics; And (b) 상기 (a) 단계에서 측정한 결과에 기초하여 상기 시료 내 황색포도상구균의 항생물질에 대한 감수성을 판정하는 단계;(b) determining the susceptibility of Staphylococcus aureus in the sample to antibiotics based on the result of the measurement in the step (a); 를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법.A method for predicting the susceptibility of Staphylococcus aureus to antibiotics. 제9항에 있어서, 상기 SMP 유전자의 mRNA 또는 이의 단백질 발현이 확인된 경우, 상기 시료 내 황색포도상구균은 처리된 항생물질에 대해 감수성이 있는 것으로 판정하는 것을 특징으로 하는 예측방법.10. The method according to claim 9, wherein when the mRNA of the SMP gene or protein expression thereof is confirmed, the Staphylococcus aureus in the sample is judged to be susceptible to the treated antibiotic substance. 제9항에 있어서, 상기 mRNA 수준을 측정하는 방법은 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사효소 중합효소반응(real time quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Northern blotting) 또는 DNA 칩 방법(DNA chip technology)인 것을 특징으로 하는 예측방법.10. The method according to claim 9, wherein the mRNA level is measured by RT-PCR, competitive RT-PCR, real-time quantitative RT- PCR, RNase protection method, Northern blotting, or DNA chip technology. 제9항에 있어서, 상기 단백질의 수준을 측정하는 방법은 웨스턴 블랏(western blotting), ELISA(enzyme linked immunosorbentassay), 방사선면역분석(RIA: radioimmunoassay), 방사 면역 확산법(radial immunodiffusion), 오우크테로니면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(rocket immunoelectrophoresis), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complement Fixation Assay), 면역형광법(immunofluorescence), 면역크로마토그래피법(immunochromatography), FACS 분석법(fluorescenceactivated cell sorter analysis) 또는 단백질 칩 방법(protein chip technology)인 것을 특징으로 하는 예측방법.10. The method of claim 9, wherein the level of the protein is measured by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemical staining, An immunochromatography, a fluorescence activated cell sorter analysis (FACS) analysis, or a protein chip technology. (a) 황색포도상구균 감염 환자에서 분리된 생물학적 시료에 후보물질을 처리한 후 시료 내 SMP의 발현 수준을 측정하는 단계; 및(a) measuring the level of expression of SMP in a sample after treating a candidate biological material with a biological sample isolated from a patient suffering from Staphylococcus aureus infection; And (b) 상기 (a) 단계에서 측정한 결과에 기초하여 황색포도상구균의 후보물질에 대한 감수성을 판정하는 단계;(b) determining the susceptibility of Staphylococcus aureus to a candidate substance based on the result of the measurement in the step (a); 를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질을 스크리닝하는 방법.≪ / RTI > wherein the antibiotic is susceptible to Staphylococcus aureus. 제13항에 있어서, 상기 SMP의 발현이 확인된 경우, 상기 시료 내 황색포도상구균은 처리된 후보물질에 대해 감수성이 있는 것으로 판정하는 것을 특징으로 하는 스크리닝방법.14. The screening method according to claim 13, wherein when the expression of the SMP is confirmed, the Staphylococcus aureus in the sample is judged to be susceptible to the candidate substance to be treated. (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게연결된 리포터 유전자를 포함하는 유전자 구조체를 분리된 황색포도상구균에 도입하는 단계; 및(a) introducing into the isolated Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) 상기 (a) 단계의 황색포도상구균에 항생물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계;(b) treating the Staphylococcus aureus of (a) with an antibiotic and measuring whether the staphylococcus aureus emits light; 를 포함하는 항생물질에 대한 황색포도상구균의 감수성 예측방법.A method for predicting the susceptibility of Staphylococcus aureus to antibiotics. 제15항에 있어서, 상기 (b) 단계의 황색포도상구균이 발광되는 경우, 황색포도상구균은 처리된 항생물질에 대해 감수성이 있는 것으로 판정하는 것을 특징으로 하는 예측방법.16. The method according to claim 15, wherein when the Staphylococcus aureus of step (b) is luminescent, the Staphylococcus aureus is determined to be susceptible to the treated antibiotic. 제15항에 있어서, 상기 SMP 프로모터 서열은 서열번호 4로 이루어진 것을 특징으로 하는 예측방법.16. The method of claim 15, wherein the SMP promoter sequence is SEQ ID NO: 4. 제15항에 있어서, 상기 리포터 유전자는 녹색형광단백질(GFP) 유전자, 붉은색형광단백질(RFP) 유전자, 푸른색형광단백질(BFP) 유전자, 발광효소단백질(luciferase) 유전자, 및 락타아제(lactase) 유전자로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 예측방법.16. The method of claim 15, wherein the reporter gene is selected from the group consisting of a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a blue fluorescent protein (BFP) gene, a luciferase gene, Wherein the prediction method is one selected from the group consisting of: 제15항에 있어서, 상기 (b) 단계의 발광 여부는 후보물질을 처리한 뒤 1~5시간 안에 검출되는 것을 특징으로 하는 예측방법.16. The method according to claim 15, wherein the light emission of step (b) is detected within 1 to 5 hours after the candidate substance is treated. (a) SMP의 프로모터 서열 및 상기 프로모터에 의해 발현되도록 작동가능하게연결된 리포터 유전자를 포함하는 유전자 구조체를 분리된 황색포도상구균에 도입하는 단계; 및(a) introducing into the isolated Staphylococcus a gene construct comprising a promoter sequence of SMP and a reporter gene operably linked to be expressed by said promoter; And (b) 상기 (a) 단계의 황색포도상구균에 후보물질을 처리한 후 황색포도상구균의 발광 여부를 측정하는 단계;(b) measuring whether or not the Staphylococcus aureus is luminescent after treating candidate Staphylococcus aureus in step (a); 를 포함하는 황색포도상구균에 대해 감수성이 있는 항생물질의 스크리닝 방법.≪ / RTI > wherein the antibiotic is susceptible to Staphylococcus aureus. 제20항에 있어서, 상기 (b) 단계의 황색포도상구균이 발광되는 경우, 황색포도상구균이 처리된 후보물질에 대해 감수성이 있는 것으로 판정하는 것을 특징으로 하는 스크리닝 방법.21. The screening method according to claim 20, wherein when the Staphylococcus aureus of step (b) emits light, the Staphylococcus aureus is judged to be susceptible to the candidate substance to be treated. 제20항에 있어서, 상기 SMP 프로모터 서열은 서열번호 4로 이루어진 것을 특징으로 하는 스크리닝 방법.The screening method according to claim 20, wherein the SMP promoter sequence is SEQ ID NO: 4. 제20항에 있어서, 상기 리포터 유전자는 녹색형광단백질(GFP) 유전자, 붉은색형광단백질(RFP) 유전자, 푸른색형광단백질(BFP) 유전자, 발광효소단백질(luciferase) 유전자, 및 락타아제(lactase) 유전자로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 예측방법.21. The method according to claim 20, wherein the reporter gene is selected from the group consisting of a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a blue fluorescent protein (BFP) gene, a luciferase gene, Wherein the prediction method is one selected from the group consisting of: 제20항에 있어서, 상기 (b) 단계의 발광 여부는 후보물질을 처리한 뒤 1~5시간 안에 검출되는 것을 특징으로 하는 스크리닝 방법.21. The screening method according to claim 20, wherein the light emission of step (b) is detected within 1 to 5 hours after the candidate substance is treated. 제13항 또는 제20항의 방법으로 스크리닝된 화합물 또는 약학적으로 허용가능한 이의 염의 유효량; 및 약학적으로 허용가능한 담체를 포함하는 황색포도상구균에 의한 감염증을 예방 또는 치료하기 위한 약학적 조성물.An effective amount of a compound screened by the method of claims 13 or 20 or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for preventing or treating infection by Staphylococcus aureus comprising a pharmaceutically acceptable carrier.
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