Method for evaluating drug resistance of microorganism

Abstract

To provide a method for rapidly evaluating the resistance of microorganisms such as bacteria to medicines.SOLUTION: The method includes an extraction step of performing an operation for obtaining, from a sample containing a microorganism, an extract containing a drug-degrading enzyme that can be produced by the microorganism, a mixing step of mixing the extract with a drug to obtain a mixture, an analysis step of subjecting the mixture to mass spectrometry, and an evaluation step of determining whether or not the microorganism has resistance to the drug by detecting the presence or absence of a peak derived from a degradation product of the drug from a mass spectrum obtained in the analysis step.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating drug resistance of a microorganism. [Background technology]

[0002] In recent years, the overuse of antibiotics in patients with infectious diseases has led to an increase in the proportion of bacteria that are resistant to conventional antibiotics (so-called drug-resistant bacteria), which has become a major problem. Rapid identification of the causative bacteria of infectious diseases and evaluation of their resistance to various antibiotics, followed by early prescription of appropriate antibiotics, is important not only for the early recovery of patients but also for preventing the emergence of drug-resistant bacteria.

[0003] One method for identifying the causative bacteria of infectious diseases is to use a matrix-assisted laser desorption / ionization mass spectrometer (MALDI (Matrix Assisted Laser Desorption / Ionization)-MS). In this method, a sample collected from a patient is first treated as needed, mixed with a matrix solution, dried, and then subjected to mass analysis using MALDI-MS. The resulting mass spectral pattern is then compared with the mass spectral patterns of numerous known bacteria stored in a database (including the positions and shapes of peaks characteristic of each bacterium (marker peaks)), thereby identifying the bacteria contained in the sample. Since MALDI-MS measurements are completed in just a few minutes, this method allows for rapid identification of the causative bacteria of infectious diseases.

[0004] On the other hand, one method for assessing bacterial resistance to antibiotics is to detect bacterial metabolites of antibiotics by mass spectrometry. This method typically uses a liquid chromatograph mass spectrometer (LC-MS) for mass analysis. However, if mass analysis of metabolites could be performed using MALDI-MS, as was used to identify the causative bacteria described above, it would be possible to identify the causative bacteria and evaluate their antibiotic resistance using a single instrument. Therefore, a method for assessing bacterial antibiotic resistance by mass spectrometry of antibiotic metabolites using MALDI-MS is being investigated.

[0005] Some types of bacteria are known to exhibit resistance to β-lactam antibiotics (penicillin and cephalosporin antibiotics) due to their ability to produce β-lactamase, an enzyme that degrades drugs (hereinafter referred to as drug-degrading enzyme). β-lactamase inactivates β-lactam antibiotics by hydrolyzing the β-lactam ring. For example, in the method described in Patent Document 1, a solution containing a β-lactam antibiotic is added to bacteria, and the bacterial solution is cultured for several hours at a predetermined temperature. The bacteria are then separated from the cultured bacterial solution, and a solution containing metabolites of the β-lactam antibiotic that may be produced by bacterial metabolism and the remaining β-lactam antibiotic is subjected to mass spectrometry by MALDI-MS. If the intensity of the peak corresponding to the β-lactam antibiotic decreases and a peak corresponding to the antibiotic's metabolic product appears in the resulting mass spectrum, the bacteria can be determined to be resistant to the β-lactam antibiotic used for evaluation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2013-529458 Summary of the Invention [Problem to be solved by the invention]

[0007] The method for evaluating bacterial antibiotic resistance described in Patent Document 1 detects degradation products (metabolites) of antibiotics produced by bacterial metabolism. Therefore, the method described in Patent Document 1 requires the bacteria to be cultured in the presence of antibiotics, which is time-consuming.

[0008] An object of the present invention is to provide a method for rapidly evaluating drug resistance of microorganisms such as bacteria. [Means for solving the problem]

[0009] The method for evaluating drug resistance of a microorganism according to the present invention, which has been achieved to solve the above problems, comprises: an extraction step in which an operation is carried out to obtain an extract containing a drug-degrading enzyme that can be produced by a microorganism from a sample containing the microorganism; a mixing step of mixing the extract and the drug to obtain a mixture; an analyzing step of subjecting the mixture to mass spectrometry; an evaluation step of determining whether or not the microorganism has resistance to the drug by detecting the presence or absence of a peak derived from a decomposition product of the drug from the mass spectrum obtained in the analysis step; It has. [Effects of the Invention]

[0010] According to the above-mentioned method, since the drug-degrading enzyme extracted from the microorganism degrades the drug, there is no need to culture the microorganism during the drug decomposition process, and therefore the presence or absence of drug resistance in the microorganism can be rapidly evaluated. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a flowchart showing the procedure of operations in the extraction step of the method for evaluating drug resistance of microorganisms according to the present embodiment. [Figure 2] In Experimental Example 1, mass spectrum when saline solution was contacted with CTX (Figure 2(a)), mass spectrum when a bacterial suspension of a susceptible strain was contacted with CTX (Figure 2(b)), and mass spectrum when a bacterial suspension of an ESBLs-producing strain was contacted with CTX (Figure 2(c)). [Figure 3] In Experimental Example 1, mass spectrum when CPDX was brought into contact with saline (Figure 3(a)), mass spectrum when CPDX was brought into contact with a bacterial suspension of a susceptible strain (Figure 3(b)), and mass spectrum when CPDX was brought into contact with a bacterial suspension of an ESBLs-producing strain (Figure 3(c)). [Figure 4]In Example 1, mass spectrum when saline was contacted with CPR (Figure 4(a)), mass spectrum when an extract from a susceptible strain was contacted with CPR (Figure 4(b)), and mass spectrum when an extract from an ESBLs-producing strain was contacted with CPR (Figure 4(c)). [Figure 5] FIG. 5(a) shows the Ihydro / Itotal values ​​when an extract from a susceptible strain was contacted with various antibiotics for a specified time in Example 2, and FIG. 5(b) shows the Ihydro / Itotal values ​​when an extract from an ESBLs-producing strain was contacted with various antibiotics for a specified time. [Figure 6] FIG. 6(a) shows the Ihydro / Itotal values ​​when CTX was contacted with extracts prepared using bacterial suspensions adjusted to various bacterial concentrations in Example 3 for 15 minutes (FIG. 6(a)), and FIG. 6(b) shows the Ihydro / Itotal values ​​when the extracts were contacted with CTX for 30 minutes (FIG. 6(b)). [Figure 7] Figure 7(a) shows the Ihydro / Itotal value when CPDX was contacted with an extract prepared using bacterial suspensions adjusted to various bacterial concentrations in Example 3 for 15 minutes (Figure 7(a)), and Figure 7(b) shows the Ihydro / Itotal value when the extract was contacted with CPDX for 30 minutes (Figure 7(b)). [Figure 8] FIG. 8(a) shows the Ihydro / Itotal values ​​when CPR was contacted with extracts prepared using bacterial suspensions adjusted to various bacterial concentrations in Example 3 for 15 minutes (FIG. 8(a)), and FIG. 8(b) shows the Ihydro / Itotal values ​​when CPR was contacted with the extracts for 30 minutes (FIG. 8(b)). [Figure 9] FIG. 9(a) shows the Ihydro / Itotal values ​​when an extract from a urine sample was contacted with various antibacterial agents for 15 minutes in Example 4 (FIG. 9(a)), and FIG. 9(b) shows the Ihydro / Itotal values ​​when an extract from a urine sample was contacted with various antibacterial agents for 30 minutes (FIG. 9(b)). [Figure 10] FIG. 10 shows the characteristics of the specimen used in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments for carrying out the method for evaluating drug resistance of microorganisms according to the present invention will be described. The method for evaluating drug resistance of microorganisms according to this embodiment comprises an extraction step of performing an operation to obtain an extract containing a drug-degrading enzyme that can be produced by the microorganism from a sample containing the microorganism, a mixing step of mixing the extract with a drug, an analysis step of subjecting the mixture obtained in the mixing step to mass spectrometry, and an evaluation step of determining whether the microorganism has resistance to the drug by detecting the presence or absence of peaks derived from degradation products of the drug from the mass spectrum obtained in the analysis step.

[0013] (microorganisms) The microorganisms for which drug resistance is evaluated are typically bacteria, and both gram-positive and gram-negative bacteria can be evaluated in the present invention.

[0014] Examples of gram-positive bacteria include: Listeria monocytogenes, Listeria welshimeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus caprae, Streptococcus pneumoniae, Streptococcus viridans, and Streptococcus pyogenes. pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus licheniformis, Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus larvae, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium ulcerans ulcerans, Mycobacterium kanasasii, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium scrofulaceumscrofulaceam, Mycobacterium microti, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium intracellulare, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium chelonei, Mycobacterium marinum marinum, Nocardia asteroides, and Rhodococcus equi.

[0015] Examples of gram-negative bacteria include Neisseria gonorrhoeae, Neisseria meningitides, Moraxella catarrhalis, Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Pseudomonas putida, Escherichia coli, Proteus mirabilis, Enterobacter cloaceae, Serratia marcescens, and Helicobacter pylori. pylori, Salmonella enteritidis, and Salmonella typhi.

[0016] (sample) The sample contains microorganisms. The sample is, for example, a biological sample (specimen) from a human or animal suffering from an infectious disease, and examples thereof include urine, whole blood, serum, plasma, cerebrospinal fluid, pharyngeal mucus, sputum, or feces. Liquid samples such as urine, whole blood, serum, plasma, and cerebrospinal fluid are preferred because they facilitate the extraction process described below. Furthermore, urine is more preferred because it contains a relatively large amount of microorganisms.

[0017] (extraction process) In the extraction step, an operation is carried out to obtain an extract containing drug-degrading enzymes that can be produced by microorganisms from a sample containing microorganisms. Figure 1 is a flowchart showing the procedure of the extraction step.

[0018] In the extraction process, first, a sample solution containing microorganisms is prepared (step S101). For example, the sample solution is prepared by adding physiological saline or a liquid medium (L broth) to a sample. The concentration of microorganisms contained in the sample solution is 103 CFU / mL or more is preferable, and 10 5 CFU / mL is more preferable, and 10 6 It is particularly preferable that the concentration of microorganisms contained in the sample solution is 10 CFU / mL. 3 If the concentration of microorganisms in the sample solution is 10 CFU / mL or more, the presence or absence of drug resistance can be determined with high accuracy. 8 CFU / mL or less is preferable. When preparing a sample solution, it is preferable to prepare it so that the concentration of microorganisms falls within the above range. For example, the microorganisms contained in the sample solution may be cultured. Alternatively, the microorganisms contained in the sample may be isolated and cultured in advance, and physiological saline or the like may be added to the isolated and cultured microorganisms to adjust the microbial concentration, thereby preparing a sample solution. Urine from patients with urinary tract infections often contains a relatively large amount of bacteria, and the bacterial concentration falls within the above range. Therefore, when the sample is a urine specimen, the urine specimen may be used as the sample solution directly without culturing the bacteria contained in the sample.

[0019] Next, the sample solution obtained in step S101 is centrifuged, and the supernatant is removed to obtain a precipitate (step S102). A washing solution is then added to the precipitate obtained in step S102, followed by centrifugation, and the supernatant is removed to obtain a precipitate. This washing procedure is repeated twice (step S103). Phosphate-buffered saline (PBS) or the like can be used as the washing solution.

[0020] Next, a surfactant or acid is added to the precipitate obtained in step S103, and after holding for a predetermined time, the mixture is centrifuged to obtain a supernatant (step S104). The surfactant is preferably a nonionic surfactant or an amphoteric surfactant, in order to prevent denaturation of the enzymes contained in the microorganisms. For example, BugBuster (registered trademark) Protein Extraction Reagent (manufactured by Merck), a protein extraction reagent containing a surfactant, can be used. For example, formic acid can be used as the acid.

[0021] If a solution containing a surfactant is used in step S104, the surfactant is removed from the supernatant obtained in step S104 (step S105). The surfactant can be removed by contacting the supernatant obtained in step S104 with a resin that adsorbs the surfactant. For example, the supernatant obtained in step S104 can be transferred to a spin column filled with a resin that adsorbs the surfactant, followed by centrifugation. If an acid is used in step S104, there is no need to perform step S105. An extract is obtained by the operation of step S104 (if an acid is used) or step S105 (if a surfactant is used).

[0022] Since the precipitate obtained in step S103 contains microorganisms that have been washed, a portion of the precipitate may be used to prepare an identification sample for use in identifying microorganisms by MALDI-MS. In this way, both an evaluation sample for use in evaluating the drug resistance of microorganisms and an identification sample for use in identifying microorganisms can be obtained from a single sample.

[0023] (Mixing process) In the mixing step, the extract prepared in the extraction step described above is mixed with a drug. If the microorganisms contained in the sample are bacteria, the drug is a so-called antibacterial drug.

[0024] Examples of antibiotics include the following: penicillin antibiotics Ampicillin (ABPC) and Piperacillin (PIPC), penicillin antibiotic combinations Sulbactam / Ampicillin (SBT / ABPC), Sulbactam / Cefoperazone (SBT / CPZ) and Tazobactam / Piperacillin (TAZ / PIPC), cephalosporin antibiotics Cefazolin (CEZ), Cefotiam (CTM), Cefotaxime (CTX), Ceftazidime (CAZ), Cefpodoxime (CPDX), Ceftriaxone (CTRX), Cefditoren (CDTR), Cefpirome (CPR) and Cefepime (CFPM), the cephamycin antibiotic Cefmetazole (CMZ), the oxacephem antibiotic Latamoxef (LMOX), the monobactam antibiotic Aztreonam (AZT), the carbapenem antibiotics Imipenem (IPM) and Meropenem (MEPM), the aminoglycosides Amikacin (AMK) and Gentamicin (GM), the new quinolone antibiotics Levofloxacin (LVFX) and Ciprofloxacin (CPFX), the tetracycline antibiotic Minomycin (MINO), and the ST combination drug Sulfamethoxazole-Trimethoprim (ST).

[0025] The method for mixing the extract and the drug is not particularly limited. For example, various antimicrobial drugs are commercially available, which are dispensed into the wells of a microplate and dried, and the extract and the drug may be mixed by adding the extract to the wells of such a microplate.

[0026] In the mixing step, the mixture of the extract and the drug is maintained at a predetermined temperature for a predetermined time under aerobic conditions. The temperature at which the mixture is maintained is preferably a temperature at which the drug-degrading enzymes contained in the extract can easily act, for example, 35°C or higher and 40°C or lower. The time for which the mixture is maintained is preferably 5 minutes or longer, and more preferably 15 minutes or longer, so that the drug-degrading enzymes contained in the extract can act sufficiently on the drug. The time for which the mixture is maintained is preferably 30 minutes or shorter, so that drug resistance can be evaluated quickly.

[0027] (Analysis process) In the analysis step, the mixture obtained in the mixing step is subjected to mass spectrometry. The mass spectrometer is not particularly limited, and for example, MALDI-MS or LC-MS can be used. When MALDI-MS is used as the mass spectrometer, a sample for MALDI mass spectrometry is prepared by dropping the mixture obtained in the mixing step onto a MALDI-MS sample plate, drying it, and then layering a matrix solution on top of it and drying it. A known matrix substance can be used as the matrix solution. An example of a matrix substance is α-cyano-4-hydroxycinnamate (HCCA).

[0028] As a MALDI-MS, it is preferable to use a MALDI-TOFMS, which combines a MALDI ion source with a time-of-flight mass spectrometer (TOFMS). When identifying microorganisms by mass spectrometry, relatively high-mass molecules such as proteins, which are components of microorganisms, are analyzed. On the other hand, when evaluating drug resistance, relatively low-mass molecules such as drugs or their degradation products are analyzed. Because MALDI-TOFMS can measure a wide range of mass-to-charge ratios, using a MALDI-TOFMS as a mass spectrometer allows for both drug resistance evaluation and microorganism identification to be performed in a single device.

[0029] (Evaluation process) In the evaluation step, the presence or absence of a peak derived from a drug degradation product is detected from the mass spectrum obtained by mass spectrometry to determine whether the microorganism has drug resistance. If a peak derived from a drug degradation product is detected, it can be determined that the microorganism being evaluated has resistance to the drug. Alternatively, a peak derived from the drug and a peak derived from the drug degradation product may be extracted from the mass spectrum obtained by mass spectrometry, and the presence or absence of drug resistance may be determined based on the peak intensities of these peaks. For example, drug resistance may be determined when the value obtained by dividing the peak intensity of the peak derived from the drug degradation product by the sum of the peak intensities of the peak derived from the drug and the peak derived from the drug degradation product exceeds a predetermined threshold.

[0030] (Microbial drug resistance evaluation kit) A kit suitable for use in the method for evaluating the drug resistance of microorganisms according to this embodiment is provided. For example, the kit for evaluating the drug resistance of microorganisms includes an extraction kit used to extract drug-degrading enzymes from microorganisms to obtain an extract, and an analytical sample preparation kit used to prepare a sample for mass spectrometry using the extract. The extraction kit may include any reagents used in the extraction step, any consumables other than the reagents, and a document describing a protocol for performing the extraction step. For example, the extraction kit may include at least one of a surfactant and an acid. The analytical sample preparation kit may include any reagents used in the mixing step or the analysis step, any consumables other than the reagents, and a document describing a protocol for performing the mixing step or the analysis step. For example, the analytical sample preparation kit may include one or more antimicrobial agents. The analytical sample preparation kit may include a microplate having storage compartments each containing one or more antimicrobial agents. Use of such a kit enables more efficient evaluation of the drug resistance of microorganisms.

[0031] The method for evaluating drug resistance of microorganisms according to the present invention will be explained below with reference to several examples, but these are merely illustrative and the present invention is not limited to these examples.

[0032] The test strains and antibacterial agents used in the following experimental examples and examples are as follows: (Test strain) ·E. coli (NCTC 13462): ESBLs (Extended Spectrum β-Lactamase) producing strain E. coli (ATCC 25922): susceptible strain (Antibacterial drugs) Cefotaxime (CTX) Cefpodoxime (CPDX) Cefpirome (CPR) ·Cefpirome-Clavulate(CPR / CLA)

[0033] (Experimental Example 1) <Measurement of microbial degradation products of antibiotics> Physiological saline was used as the dispersion medium for the test strain. The turbidity of the test strain suspension was adjusted to 1 McFarland turbidity unit. 100 μL of the test strain suspension was added to a well containing 32 μg / mL CTX and 32 μg / mL CPDX on an Eiken DPD-1 dry plate (manufactured by Eiken Chemical Co., Ltd.). After 30 minutes of aerobic incubation at 35°C, 3 μL of the sample supernatant was applied to the target plate of a mass spectrometer and allowed to dry. 1 μL of matrix solution was layered on top and allowed to dry naturally to prepare the analytical sample. The matrix solution was prepared by dissolving α-cyano-4-hydroxycinnamate (HCCA) in 250 μL of a mixture of 50% acetonitrile, 49.9% HPLC-grade water, and 0.1% trifluoroacetic acid. The analytical sample (control sample) was prepared by the same procedure, except that the test strain suspension was replaced with physiological saline. The mass spectrometer is a MALDI-TOFMS (MALDI Biotyper TM (manufactured by Bruker Daltonics) was used.

[0034] Figure 2(a) shows the mass spectrum when saline solution is brought into contact with CTX, Figure 2(b) shows the mass spectrum when a bacterial suspension of a susceptible strain is brought into contact with CTX, and Figure 2(c) shows the mass spectrum when a bacterial suspension of an ESBLs-producing strain is brought into contact with CTX. In Figure 2, the horizontal axis of the mass spectrum is shown in mass (Da) for convenience, but this is synonymous with the mass-to-charge ratio (m / z) when the charge number is 1. This is also true for the mass spectra shown in the following figures. In Figure 2(a), Figure 2(b), and Figure 2(c), the molecular ion peak of the matrix ([2M+H]) is present at 379.1 Da. + , M is a matrix molecule, and H is a hydrogen atom). In Figure 2(a), peaks were observed at 455.9 Da and 477.8 Da, and in Figure 2(b), peaks were observed at 456.0 Da and 478.0 Da. On the other hand, in Figure 2(c), peaks were observed at 370.1 Da, 414.0 Da, and 455.9 Da. Between the peak observed at 370.1 Da and the peak observed at 414.0 Da, the peak observed at 370.1 Da had a greater peak intensity.

[0035] Figure 3(a) shows the mass spectrum when saline and CPDX were contacted, Figure 3(b) shows the mass spectrum when a bacterial suspension of a susceptible strain was contacted with CPDX, and Figure 3(c) shows the mass spectrum when a bacterial suspension of an ESBLs-producing strain was contacted with CPDX. In all of Figures 3(a), 3(b), and 3(c), the molecular ion peak of the matrix ([2M+H]) was observed at 379.1 Da. + ) were observed. In Figures 3(a) and 3(b), peaks were observed at 428.0 Da and 450.0 Da. On the other hand, in Figure 3(c), peaks were observed at 370.1 Da, 414.0 Da, 428.0 Da, and 450.0 Da. Between the peak observed at 370.1 Da and the peak observed at 414.0 Da, the peak observed at 370.1 Da had a greater intensity.

[0036] The peaks at 456 Da and 478 Da in the mass spectrum shown in Figure 2 are thought to be derived from the proton-adduct (456 Da) or sodium ion-adduct (478 Da) of CTX, which exists as a carboxylate anion in saline, during the ionization process. The peaks at 370 Da and 414 Da are thought to be derived from a degradation product (414 Da) generated by cyclocondensation after CTX is hydrolyzed by β-lactamase, and a degradation product (370 Da) generated by further decarboxylation of the degradation product. Similarly, the peaks at 370 Da and 414 Da in the mass spectrum shown in Figure 3 are thought to be derived from degradation products after CPDX is hydrolyzed by β-lactamase.

[0037] Based on the results of Experimental Example 1, in the evaluation of resistance to CTX, the peaks observed at 456 Da and 478 Da were determined as peaks derived from the non-hydrolysate, and the peak observed at 370 Da was determined as peaks derived from the hydrolysate in the following Examples 2 to 4. In addition, in the evaluation of resistance to CPDX, the peaks observed at 428 Da and 450 Da were determined as peaks derived from the non-hydrolysate, and the peak observed at 370 Da was determined as peaks derived from the hydrolysate. [Example]

[0038] In Experimental Example 1, a suspension of the test strain was added to each antibacterial drug, but in Example 1, an extract obtained from the suspension of the test strain was added to each antibacterial drug, and antibacterial drug decomposition products were measured. <Extraction of drug-degrading enzymes> Physiological saline was used as a dispersion medium for the test strain, and 1 mL of the suspension of the test strain, adjusted to 1 McFarland turbidity unit, was centrifuged at 14,000 rpm for 10 minutes. After removing the supernatant, the precipitate was washed with 1 mL of PBS, and the centrifugation process was repeated twice. After removing the supernatant, the precipitate was diluted with BugBuster protein extraction reagent. TM100 μL of Protein Extraction Reagent (Merck) was added to obtain a solution containing the precipitate and the reagent. After allowing the solution to stand for 10 minutes, it was centrifuged at 14,000 rpm for 10 minutes, and the detergent components were removed from the supernatant using Pierce Detergent Removal Spin Columns (Thermo Scientific, Cat. No. 87777) to obtain an extract.

[0039] <Measurement of antibiotic degradation products by drug-degrading enzymes> 50 μL of the extract obtained by the above extraction process was added to a well containing 16 μg / mL CPR of the dry plate 'Eiken' DPE-1CPR (manufactured by Eiken Chemical Co., Ltd.). After 30 minutes of incubation under aerobic conditions at 35°C, 3 μL of the sample supernatant was applied to the target plate of a mass spectrometer and allowed to dry. 1 μL of matrix solution was layered on top of this and allowed to dry naturally to prepare an analytical sample. A similar process was performed using the extract instead of saline to prepare an analytical sample (control sample). The same matrix solution and mass spectrometer as those used in Experimental Example 1 were used.

[0040] <Result> Figure 4(a) shows the mass spectrum when CPR was contacted with saline, Figure 4(b) shows the mass spectrum when CPR was contacted with an extract from a susceptible strain, and Figure 4(c) shows the mass spectrum when CPR was contacted with an extract from an ESBLs-producing strain. In Figures 4(a), 4(b), and 4(c), the matrix molecular ion peak ([2M+H] +) were observed. In Figure 4(a), peaks derived from the non-hydrolyzed CPR were observed at 396.1 Da and 514.9 Da, and in Figure 4(b), peaks derived from the non-hydrolyzed CPR were observed at 396.1 Da and 514.9 Da. In Figure 4(c), peaks derived from the non-hydrolyzed CPR were observed at 396.1 Da and 514.9 Da, and peaks derived from the hydrolyzed CPR were observed at 323.8 Da and 369.9 Da. The peak observed at 369.9 Da had a greater intensity than the peak observed at 323.8 Da.

[0041] The results of Example 1 show that even when the extract is brought into contact with an antibiotic, peaks (324 Da and 370 Da) derived from the degradation products of the antibiotic are observed, making it possible to distinguish between susceptible strains and ESBLs-producing strains. In the following Examples 2 to 4, in the evaluation of resistance to CPR, the peaks observed at 396 Da and 515 Da were considered to be peaks derived from the non-hydrolysate, and the peak observed at 370 Da was considered to be peak derived from the hydrolysate. [Example]

[0042] [Contact time between antibiotics and extract] <Measurement of antibiotic degradation products by drug-degrading enzymes> The extract obtained by the same extraction process as in Example 1 was added in an amount of 100 μL to the wells containing CTX: 32 μg / mL and CPDX: 32 μg / mL, and 50 μL to the wells containing CPR: 16 μg / mL on the Eiken DPE-1 dry plate. After incubation at 35°C under aerobic conditions for 5, 15, or 30 minutes, 3 μL of the sample supernatant was applied to the target plate of a mass spectrometer and allowed to dry. 1 μL of matrix solution was layered on top of the well and allowed to dry naturally, preparing a sample for analysis. The matrix solution and mass spectrometer used were the same as those used in Experimental Example 1.

[0043] The measurement results were calculated by dividing the peak intensity of the hydrolysate-derived peak of each antibiotic by the sum of the peak intensity of the non-hydrolysate-derived peak and the peak intensity of the hydrolysate-derived peak (I hydro / Itotal For example, when the antibacterial agent is CTX, the peak intensities of the peaks observed at 370 Da, 456 Da, and 478 Da are expressed as I 370 , I 456 , I 478 Then, I hydro / I total =I 370 / I (370+456+478) =I 370 / (I 370 +I 456 +I 478 ) I hydro / I total A higher value means a higher proportion of hydrolysis products in the sample.

[0044] <Result> Figure 5(a) shows the I when extracts from susceptible strains were contacted with various antimicrobial agents for a specified time. hydro / I total Figure 5(b) shows the I value when extracts from ESBLs-producing strains were contacted with various antimicrobial agents for a specified time. hydro / I total When the antibiotic is CTX, the value of I hydro / I total (I 370 / I (370+456+478) ) was 0.07 to 0.09 for susceptible strains, regardless of contact time, and no effect of contact time was observed. On the other hand, for ESBL-producing strains, hydro / I total The I was 0.73 for 5 minutes of contact, reached the highest value of 0.92 for 15 minutes of contact, and then decreased slightly to 0.87 for 30 minutes of contact. When the antibiotic was CPDX, I hydro / I total (I 370 / I (370+428+450) ) was 0.05 to 0.06 for susceptible strains, regardless of contact time, and no effect of contact time was observed. On the other hand, for ESBL-producing strains, I hydro / I total The I was 0.38 for 5 minutes of contact, reached its highest value of 0.56 for 15 minutes of contact, and then decreased slightly to 0.49 for 3 minutes of contact. When the antibiotic was CPR, I hydro / Itotal (I 370 / I (370+396+515) ) was 0.12-0.16 for susceptible strains, regardless of contact time, and no effect of contact time was observed. On the other hand, for ESBL-producing strains, hydro / I total The I was 0.19 after 5 minutes of contact, which was similar to that of the susceptible strain, but when the contact time was extended to 15 minutes and 30 minutes, hydro / I total were 0.48 and 0.72, respectively, and increased depending on the contact time.

[0045] These results demonstrate that CTX and CPDX can distinguish between susceptible and ESBLs-producing strains by contacting the antibiotic with the extract for 5 minutes or more, while CPR can distinguish between susceptible and ESBLs-producing strains by contacting the antibiotic with the extract for 15 minutes or more. [Example]

[0046] [Consideration of the detection limit] <Extraction of drug-degrading enzymes> Extracts were obtained in the same manner as in Example 1, except that urine samples from healthy individuals were used as dispersions of the test strains, and suspensions of the test strains were prepared to various bacterial concentrations (turbidities). <Measurement of antibiotic degradation products by drug-degrading enzymes> The antimicrobial decomposition products were measured in the same manner as in Example 2.

[0047] <Result> Figure 6 shows the I value when the degradation products of CTX were measured using suspensions of test strains adjusted to various bacterial concentrations for the extraction of drug-degrading enzymes. hydro / I total Figure 6(a) and Figure 6(b) show the I values ​​when the contact time between the extract and the antimicrobial agent was 15 minutes and 30 minutes, respectively. hydro / I total The values ​​of I and IB are shown. The values ​​of susceptible strains are shown with white circles, and the values ​​of ESBLs-producing strains are shown with black circles (the same applies to Figures 7 and 8 below). As shown in Figure 6(a), when the contact time is 15 minutes, Ihydro / I total For susceptible strains, the I was below 0.1 regardless of the bacterial solution concentration. On the other hand, for ESBLs-producing strains, the I hydro / I total The value of increases, 10 5 CFU / mL: 0.18, 10 6 CFU / mL: 0.60, 10 7 In addition, as shown in Figure 6(b), when the contact time was 30 minutes, the I hydro / I total For susceptible strains, the β-glucanase activity was approximately 0.1 regardless of the bacterial solution concentration. 5 CFU / mL: 0.51, 10 6 CFU / mL: 0.85, 10 7 The CFU / mL was 0.98.

[0048] Figure 7 shows the I value when the decomposition products of CPDX were measured using suspensions of test strains adjusted to various bacterial concentrations for the extraction of drug-decomposing enzymes. hydro / I total As shown in Figure 7(a), when the contact time is 15 minutes, I hydro / I total For susceptible strains, the I was below 0.1 regardless of the bacterial solution concentration. On the other hand, for ESBLs-producing strains, the I hydro / I total The value of increases, 10 6 CFU / mL is 0.20 and 10 7 In addition, as shown in Figure 7(b), when the contact time was 30 minutes, the I hydro / I total For susceptible strains, the β-glucanase activity was 0.1 or less regardless of the bacterial solution concentration. 5 CFU / mL: 0.27, 10 6 CFU / mL: 0.65, 10 7 The CFU / mL was 0.95.

[0049] Figure 8 shows the I value when the degradation products of CPR were measured using suspensions of test strains adjusted to various bacterial concentrations for the extraction of drug-degrading enzymes.hydro / I total As shown in Figure 8(a), when the contact time is 15 minutes, I hydro / I total For susceptible strains, the I was approximately 0.1 regardless of the bacterial solution concentration. On the other hand, for ESBLs-producing strains, the I hydro / I total The value of increases, 10 5 ~10 6 CFU / mL is 0.20 and 10 7 In addition, as shown in Figure 8(b), when the contact time was 30 minutes, the I hydro / I total For susceptible strains, the β-glucanase activity was approximately 0.1 regardless of the bacterial solution concentration. 5 CFU / mL: 0.26, 10 6 CFU / mL: 0.32, 10 7 The CFU / mL was 0.86.

[0050] From the above results, when the extract and the antibacterial agent were in contact for 15 minutes, the bacterial solution concentration was 10 6 It was found that if the CFU / mL or higher, it was possible to distinguish between susceptible strains and ESBLs-producing strains. 5 It was found that a CFU / mL or higher level was sufficient to distinguish between susceptible strains and ESBLs-producing strains. [Example]

[0051] [Study using clinical samples] <Extraction of drug-degrading enzymes> Ten mL of urine samples (15 samples) suspected of having a urinary tract infection and showing Enterobacteriaceae-like gram-negative bacilli by Gram staining were centrifuged at 3,400 rpm for 10 minutes, and the supernatant was removed. After the PBS washing step, the procedure was repeated as in Example 1 to obtain an extract.

[0052] <Measurement of antibiotic degradation products by drug-degrading enzymes> Antimicrobial decomposition products were measured in the same manner as in Example 3, except that 25 μL of extract was added to the wells of the dry plate containing CTX, CPDX, and CPR, as well as CPR / CLA (Cefpirome-Clavulanate): 4 μg / mL / 4 ​​μg / mL.

[0053] <Result> The characteristics of the 15 samples used in this example are shown in Figure 10. Of the 15 samples, bla CTX-M Five specimens (E-1 to E-5) contained strains carrying the bla gene (the gene encoding β-lactamase), and E. coli was detected in all specimens. CTX-M Of the 10 specimens (S-1 to S-10) that did not possess type genes, E. coli was detected in five (S-1 to S-4, S-6), K. pneumoniae in two (S-5, S-9), Morganella morganii in one (S-7), and Enterobacter cloacae in one (S-8). In addition, multiple species of E. coli and K. pneumoniae were detected in one specimen (S-10).

[0054] Figure 9 shows the I when extracts from urine samples were brought into contact with various antibacterial agents. hydro / I total The white circles indicate the susceptible strains (bla CTX-M The black circles indicate the values ​​for specimens containing ESBLs-producing strains (strains not carrying bla ). CTX-M As shown in Figure 9(a), in the 10 samples containing susceptible strains, the I hydro / I total The values ​​were 0.09±0.02 for CTX, 0.05±0.03 for CPDX, and 0.11±0.01 for CPR. On the other hand, in the five specimens containing ESBL-producing strains, the I hydro / I totalThe values ​​were 0.96±0.02 for CTX, 0.98±0.02 for CPDX, and 0.87±0.09 for CPR. As shown in Figure 9(b), even after 30 minutes of contact between the extract and the antibiotic, the I hydro / I total The values ​​were similar to those obtained after 15 minutes of contact. hydro / I total The values ​​for both samples were approximately 0.11 after 15 minutes of contact and approximately 0.13 after 30 minutes of contact. CLA (clavulanic acid) is a β-lactamase inhibitor, and β-lactamase was inhibited by CLA, resulting in an I value similar to that of susceptible strains. hydro / I total Furthermore, in samples containing E. coli and E. faecalis (E-5 and S-3 in Figure 10), the signal intensity ratios for CTX, CPDX, and CPR were 1.0, 1.0, and 0.72 for ESBL-producing strains, respectively, and 0.08, 0.05, and 0.11 for susceptible strains, demonstrating that ESBL-producing strains and susceptible strains can be distinguished from each other in clinical samples without being affected by coexisting bacteria.

[0055] [Aspect] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0056] (Item 1) A method for evaluating drug resistance of a microorganism according to one aspect of the present invention comprises: an extraction step in which an operation is carried out to obtain an extract containing a drug-degrading enzyme that can be produced by a microorganism from a sample containing the microorganism; a mixing step of mixing the extract and the drug to obtain a mixture; an analyzing step of subjecting the mixture to mass spectrometry; an evaluation step of determining whether or not the microorganism has resistance to the drug by detecting the presence or absence of a peak derived from a decomposition product of the drug from the mass spectrum obtained in the analysis step; It has.

[0057] According to the method for evaluating drug resistance of a microorganism according to paragraph 1, it is possible to quickly evaluate whether or not a microorganism has drug resistance.

[0058] (2) In the method for evaluating drug resistance of a microorganism according to (1), The drug-degrading enzyme may be β-lactamase.

[0059] According to the method for evaluating drug resistance of a microorganism according to the second aspect, it is possible to evaluate whether or not a microorganism has resistance to a drug that is decomposed by β-lactamase.

[0060] (Item 3) In the method for evaluating drug resistance of a microorganism according to item 1 or 2, In the extraction step, the extract can be obtained by contacting the microorganism with a surfactant.

[0061] According to the method for evaluating the drug resistance of a microorganism according to the third aspect, it is possible to carry out an operation for extracting the drug-degrading enzyme while suppressing denaturation of the drug-degrading enzyme.

[0062] (Item 4) In the method for evaluating drug resistance of a microorganism according to any one of Items 1 to 3, In the extraction step, the operation can be carried out without culturing the microorganism.

[0063] According to the method for evaluating drug resistance of a microorganism according to item 4, it is possible to more quickly evaluate whether or not a microorganism is drug-resistant.

[0064] (Item 5) In the method for evaluating drug resistance of a microorganism according to any one of Items 1 to 4, The sample may be urine.

[0065] According to the method for evaluating drug resistance of microorganisms relating to paragraph 5, a sample containing a relatively large amount of microorganisms can be obtained, and therefore, the presence or absence of drug resistance of microorganisms can be evaluated without culturing the microorganisms.

[0066] (Item 6) In the method for evaluating drug resistance of a microorganism according to any one of Items 1 to 5, The extraction step may further include a step of collecting an isolated product of the microorganism from the sample, performing an operation to obtain the extract using a portion of the isolated product, and preparing an identification sample using a portion of the isolated product to identify the microorganism contained in the sample.

[0067] According to the method for evaluating drug resistance of microorganisms according to item 6, both evaluation of drug resistance of microorganisms and identification of the microorganisms can be performed from a single sample.

[0068] (Item 7) In the method for evaluating drug resistance of a microorganism according to any one of Items 1 to 6, The mass spectrometry may be matrix-assisted laser desorption ionization mass spectrometry.

[0069] According to the method for evaluating drug resistance of microorganisms in accordance with paragraph 7, both evaluation of drug resistance of microorganisms and identification of microorganisms can be performed using a single device.

[0070] (Item 8) A kit for evaluating the drug resistance of a microorganism according to one embodiment of the present invention is a kit for carrying out the method for evaluating the drug resistance of a microorganism according to items 1 to 7, and may include a surfactant.

[0071] (Item 9) A kit for evaluating the drug resistance of a microorganism according to one aspect of the present invention is a kit for carrying out the method for evaluating the drug resistance of a microorganism according to items 1 to 7, and may include one or more antibacterial agents.

[0072] According to the kit for evaluating drug resistance of a microorganism according to item 8 or 9, drug resistance of a microorganism can be efficiently evaluated.

Claims

1. an extraction step in which an operation is carried out to obtain an extract containing a drug-degrading enzyme that can be produced by a microorganism from a sample containing the microorganism; a mixing step of mixing the extract and the drug to obtain a mixture; an analyzing step of subjecting the mixture to mass spectrometry; an evaluation step of determining whether or not the microorganism has resistance to the drug by detecting the presence or absence of a peak derived from a decomposition product of the drug from the mass spectrum obtained in the analysis step; A method for evaluating drug resistance of microorganisms having

2. The method for evaluating drug resistance of a microorganism according to claim 1, wherein the drug-degrading enzyme is β-lactamase.

3. The method for evaluating drug resistance of a microorganism according to claim 1 or 2, wherein the extraction step is carried out by contacting the microorganism with a surfactant.

4. The method for evaluating drug resistance of a microorganism according to claim 1 or 2, wherein the extraction step is performed without culturing the microorganism.

5. The method for evaluating drug resistance of a microorganism according to claim 1 or 2, wherein the sample is urine.

6. The method for evaluating the drug resistance of bacterial microorganisms described in claim 1 or 2, wherein the extraction step further comprises a step of collecting an isolated product of the microorganism from the sample, performing an operation to obtain the extract using a portion of the isolated product, and preparing an identification sample using a portion of the isolated product to identify the microorganism contained in the sample.

7. The method for evaluating drug resistance of a microorganism according to claim 1 or 2, wherein the mass spectrometry is matrix-assisted laser desorption / ionization mass spectrometry.

8. A kit for evaluating the drug resistance of a microorganism, which is used to carry out the method for evaluating the drug resistance of a microorganism according to claim 1 or 2, and which comprises a surfactant.

9. A kit for evaluating the drug resistance of a microorganism, which is used to carry out the method for evaluating the drug resistance of a microorganism according to claim 1 or 2, and which comprises one or more antimicrobial agents.

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