Method for distinguishing between subspecies of nontuberculous mycobacterium

Abstract

In the present invention: when there is an m / z peak of 6766 ± allowable error originating from ribosomal protein L30 of M. abscessus subspecies bolletii in the result of mass spectrometry of an M. abscessus test bacterium, it is determined that the test bacterium is of subspecies bolletii (subsp. bolletii) (steps 6 and 7); when there is an m / z peak of 8767 ± allowable error originating from ribosomal protein L29 of M. abscessus subspecies massiliense, it is determined that the test bacterium is of subspecies massiliense (subsp. massiliense) (steps 8 and 9); and when neither of the peaks is present, it is determined that the test bacterium is of M. abscessus subspecies abscessus (subsp. abscessus) (step 10). Through this configuration, it is possible to accurately and easily distinguish between subspecies of Mycobacterium abscessus (Mycobacterium abscessus).

Description

Method for distinguishing subspecies of nontuberculous mycobacteria

[0001] The present invention relates to a method for distinguishing subspecies of nontuberculous mycobacteria.

[0002] Mycobacterium abscessus (hereinafter sometimes abbreviated as M. abscessus), one of the causative bacteria of nontuberculous mycobacteria, exists in three subspecies: Mycobacterium abscessus subsp. massiliense, Mycobacterium abscessus subsp. bolletii, and Mycobacterium abscessus subsp. abscessus. Because M. abscessus subspecies have different susceptibility to antibiotics, early subspecies identification is important clinically.

[0003] Conventional methods for distinguishing mycobacteria (including the nontuberculous mycobacterium M. abscessus) include methods using DNA-DNA hybridization, smear tests, culture tests, or drug susceptibility tests (see, for example, Non-Patent Document 1), and methods using gene sequence analysis (see, for example, Non-Patent Document 2). In recent years, mycobacteria have also been distinguished using mass spectrometry (see, for example, Non-Patent Documents 3 and 4). Examples of mycobacterial distinction methods using mass spectrometry include a so-called fingerprinting method in which a sample containing test bacteria is analyzed by matrix-assisted laser desorption / ionization mass spectrometry (hereinafter referred to as MALDI-MS) and the test bacteria are identified by comparing the obtained mass spectral pattern with a database (see, for example, Non-Patent Document 5), and a distinction method in which machine learning is applied to the mass spectrum of the test bacteria obtained by MALDI-MS (see, for example, Non-Patent Document 6).

[0004] "Handbook of Clinical Microbiology, 5th Edition," edited by Toyoko Oguri, Miwa Shoten, June 17, 2017, pp. 185-192; Adekambi, T. and Drancourt, M., "Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA, and rpoB gene sequencing," International Journal of Systematic and Evolutionary Microbiology (2004), volume 54, issue 6, pp. 2095-2105; Hiromichi Suzuki et al., "A novel cluster of Mycobacterium abscessus complex revealed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)," Diagnostic Microbiology and Infectious Disease, December 2015, volume 83, issue 4, pp. 365-370; Jan Kehrmann et al., "Principal component analysis of MALDI TOF MS mass spectra separates M. abscessus (sensu stricto) from M. massiliense isolates", BMC Microbiology, volume 16, Article number: 24 (2016). Okusu Kiyofumi, "Taxonomy of Medically Important Bacteria: Update on Classification and Identification of Mycobacteria", Modern Media, October 2021, Vol. 67, No. 10, pp. 391-406, 437(1) David Rodriguez-Temporal et al., "Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning", Journal of Clinical Microbiology, 05 January 2023, volume 61, Issue 1, e0111022.

[0005] However, while the above-mentioned DNA-DNA hybridization or fingerprinting methods can distinguish M. abscessus at the species level, they cannot distinguish it at the subspecies level. Furthermore, the above-mentioned genetic sequence analysis method has the drawback of being time-consuming and labor-intensive. Furthermore, the above-mentioned methods using machine learning have the drawback of not providing a clear basis for discrimination, and the possibility of erroneous judgments due to the influence of culture conditions, sample preparation methods, measurement conditions, etc. cannot be eliminated.

[0006] The present invention has been made in view of the above points, and an object of the present invention is to enable accurate and simple identification of subspecies of M. abscessus.

[0007] The method for distinguishing subspecies of nontuberculous mycobacteria of the present invention, which has been developed to solve the above-mentioned problems, uses Mycobacterium abscessus, a nontuberculous mycobacterium, as a test bacterium, and determines whether the test bacterium is Mycobacterium abscessus subsp. boleti based on the m / z value of ribosomal protein L30 in the mass spectrometry results of the test bacterium.

[0008] The non-tuberculous mycobacterial subspecies discrimination device of the present invention, which has been developed to solve the above-mentioned problems, is a device that uses Mycobacterium abscessus, a non-tuberculous mycobacterium, as a test bacterium and discriminates the subspecies of the test bacterium based on the mass spectrometry results of the test bacterium, and has: a mass spectrometry result acquisition unit that acquires data from the mass spectrometry results; a subspecies discrimination unit that discriminates whether the test bacterium is Mycobacterium abscessus subspecies boleti based on the m / z value of ribosomal protein L30 in the mass spectrometry results; and a notification unit that notifies the user of the discrimination results made by the subspecies discrimination unit.

[0009] According to the method for distinguishing subspecies of nontuberculous mycobacteria or the device for distinguishing subspecies of nontuberculous mycobacteria of the present invention, it is possible to accurately and simply distinguish subspecies of M. abscessus.

[0010] 1 is a diagram showing the peak positions of ribosomal protein L30 in the mass spectrum of each subspecies of M. abscessus. 2 is a diagram showing the peak positions of ribosomal protein L29 in the mass spectrum of each subspecies of M. abscessus. 3 is a schematic configuration diagram of a subspecies discrimination system for nontuberculous mycobacteria according to one embodiment of the present invention. 4 is a flowchart showing the processing steps in the subspecies discrimination system.

[0011] Hereinafter, the three subspecies of M. abscessus, namely, Mycobacterium abscessus subsp. bolletii, Mycobacterium abscessus subsp. massiliense, and Mycobacterium abscessus subsp. abscessus, may be abbreviated as subsp. bolletii, subsp. massiliense, and subsp. abscessus, respectively.

[0012] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that the M. abscessus subspecies bolletii can be distinguished from other subspecies (i.e., subspecies massiliense and subspecies abscessus) based on the m / z value of ribosomal protein L30, thereby conceiving the present invention. Furthermore, the present inventors conducted further extensive research and discovered that the M. abscessus subspecies massiliense can be distinguished from other subspecies (i.e., subspecies bolletii and subspecies abscessus) based on the m / z value of ribosomal protein L29, thereby conceiving the present invention. Specifically, the method for distinguishing subspecies of nontuberculous mycobacteria according to the present invention uses ribosomal protein L30 (or L30 and L29) as a biomarker and distinguishes the subspecies of the test bacterium M. abscessus based on the results of mass spectrometry of the test bacterium.

[0013] Figure 1 shows the peak positions of ribosomal protein L30 in the mass spectra of each subspecies of M. abscessus, and Figure 2 shows the peak positions of ribosomal protein L29 in the mass spectra of each subspecies. As shown in Figure 1, the m / z value of ribosomal protein L30 is m / z 6766 in subspecies bolletii and m / z 6796 in subspecies massiliense and subspecies abscessus, meaning that only subspecies bolletii differs from the others. Therefore, subspecies bolletii can be distinguished from other subspecies based on the m / z value of ribosomal protein L30. Also, as shown in Figure 2, the m / z value of ribosomal protein L29 is m / z 8767 in subspecies massiliense and m / z 8781 in subspecies bolletii and subspecies abscessus, meaning that only subspecies massiliense differs from the others. Therefore, subspecies massiliense can be distinguished from other subspecies based on the m / z value of ribosomal protein L29. The m / z values ​​of the peaks in the mass spectra of Figures 1 and 2 are in good agreement with the theoretical masses (observed masses calculated from the amino acid sequences predicted from the DNA base sequences) of the L30 and L29 proteins of each subspecies.

[0014] Modes for carrying out the present invention are described below. The method for distinguishing subspecies of nontuberculous mycobacteria according to this embodiment distinguishes M. abscessus at the subspecies level by using two types of proteins (i.e., ribosomal proteins L30 and L29) that produce subspecies-specific m / z value combinations as biomarkers, as described above. Both of these proteins are housekeeping proteins that are constantly expressed regardless of culture conditions, sample preparation methods, or measurement conditions (hereinafter referred to as culture conditions, etc.). Therefore, by using these proteins as biomarkers, M. abscessus subspecies can be distinguished with high reproducibility without erroneous determinations due to culture conditions, etc. Furthermore, because the method for distinguishing subspecies of nontuberculous mycobacteria according to this embodiment uses mass spectrometry, it can be performed easily and in a relatively short time.

[0015] A system for distinguishing subspecies of M. abscessus using a method according to this embodiment will now be described with reference to Figures 3 and 4. Figure 3 is a schematic diagram of the system according to this embodiment. The system according to this embodiment includes a mass spectrometer 100 and a data processing device 200. This data processing device 200 corresponds to the "nontuberculous mycobacterial subspecies discrimination device" of the present invention.

[0016] The mass spectrometer 100 includes an ionization unit 101 that ionizes a sample, a mass separation unit 102 that separates the ions generated by the ionization unit 101 based on their mass-to-charge ratio (m / z), a detector 103 that detects the separated ions, and an analog-to-digital converter (ADC) 104 that digitizes the detection signal output from the detector 103. The ionization unit 101 ionizes the sample using a matrix-assisted laser desorption / ionization (MALDI) method. The mass separation unit 102 may be, for example, a time-of-flight (TOF) type, but is not limited thereto, and may be of other configurations, such as a quadrupole mass filter type or an ion trap type.

[0017] The data processing device 200 includes a mass spectrometry data acquisition unit 210 (corresponding to the mass spectrometry result acquisition unit in the present invention), a peak extraction unit 220, a species discrimination unit 230, a subspecies discrimination unit 240, a discrimination result storage unit 250, and a display control unit 260. Of these, the subspecies discrimination unit 240 further includes a first discrimination unit 241 and a second discrimination unit 242 (details of each of these units will be described later). The functions of the data processing device 200 are realized by a computer including a CPU, memory, a large-capacity storage medium (e.g., a hard disk drive or solid-state drive), and the like. While the computer may be a dedicated computer built into the main body of the mass spectrometer, it is typically a general-purpose computer such as a personal computer. A predetermined program is pre-installed on the computer, and when the CPU executes this program, the functions of the mass spectrometry data acquisition unit 210, the peak extraction unit 220, the species discrimination unit 230, the subspecies discrimination unit 240, and the display control unit 260 are embodied in software. The function of the determination result storage unit 250 is realized by the mass storage medium. An input unit 270 for a user to input various instructions and a display unit 280 for displaying various information are connected to the computer. The display unit 280 is configured, for example, by a liquid crystal display, and the input unit 270 is configured, for example, by a keyboard, a pointing device such as a mouse, or a touch panel attached to the display unit 280. In the system according to this embodiment, the display control unit 260 and the display unit 280 correspond to the notification unit in the present invention.

[0018] The processing procedure in this system will be explained below with reference to the flowchart in Figure 4. In this case, the test bacterium, which is an unknown bacterium, is first identified at the species level, and if the test bacterium is determined to be M. abscessus as a result, it is then determined which subspecies of M. abscessus the test bacterium belongs to.

[0019] First, a user prepares a mixed crystal of a sample containing the test bacterium (e.g., a specimen collected from a patient suspected of having nontuberculous mycobacteria) and a matrix reagent on a sample plate, places the sample plate in the ionization unit 101, and then causes the mass spectrometer 100 to perform mass analysis of the sample. Ions generated in the ionization unit 101 are separated based on m / z in the mass separation unit 102 and then incident on the detector 103. The detector 103 generates a detection signal corresponding to the amount of incident ions, and the ADC 104 converts this detection signal into digital data and sends it to the data processing device 200. In the system according to this embodiment, the ionization unit 101 irradiates the mixed crystal with laser multiple times, and digital data obtained from each of the multiple laser irradiations is sent to the data processing device 200. The mass analysis data acquisition unit 210 of the data processing device 200 acquires the digital data (corresponding to "mass analysis result data" in this specification) obtained for each of the multiple laser irradiations (Step 1). The mass analysis data acquisition unit 210 further integrates the digital data to generate profile data, which is a continuous waveform with the horizontal axis representing time (or m / z) and the vertical axis representing ion intensity.

[0020] Next, the peak extraction unit 220 performs a peak extraction process to detect peaks contained in the profile data and generate a list (peak list) showing the m / z value representing the center of gravity position (or peak top position) of each detected peak and the area or height value of the peak (step 2).

[0021] Next, the species discrimination unit 230 discriminates the test bacterium at the species level based on the peak list (step 3). The species discrimination can be performed, for example, by comparing the peak list generated in step 2 with a peak list of numerous known bacteria stored in an existing database. Specifically, for each known bacterium stored in the database, a score representing the degree of match between the peak pattern and the test bacterium is calculated, and the species to which the known bacterium with the highest score belongs is determined to be the species to which the test bacterium belongs. Note that in this step, the species discrimination may be performed using the profile data generated in step 1 instead of the peak list generated in step 2. In this case, for example, a score representing the degree of match between the profile data for each known bacterium stored in the database and the profile data for the test bacterium is calculated, and the species to which the known bacterium with the highest score belongs is determined to be the species to which the test bacterium belongs. In this case, steps 2 and 3 may be performed in reverse order. The database used for discrimination at the species level may be any database, such as a public database, a database provided by the manufacturer of the mass spectrometer, or a database previously constructed by the user.

[0022] If the test bacterium is determined to be a bacterium other than M. abscessus in step 3 (i.e., if step 4 in FIG. 4 is answered No), the data processing device 200 determines that the test bacterium is not a target for subspecies discrimination by the system according to this embodiment (step 5) and notifies the user of this (step 11). The notification to the user in step 11 is performed by displaying a predetermined message on the display unit 280 under the control of the display control unit 260. Thereafter, the data processing device 200 stores the discrimination result in step 3 in the discrimination result storage unit 250 (step 12), and ends the series of processes.

[0023] On the other hand, if the test bacterium is determined to be M. abscessus in step 3 (that is, if step 4 in FIG. 4 is Yes), subspecies discrimination is then performed by the subspecies discrimination unit 240 .

[0024] In the subspecies discrimination unit 240, the first discrimination unit 241 first discriminates the subspecies based on the m / z value of ribosomal protein L30. Specifically, it determines whether a peak at m / z 6766 ± the tolerance exists in the peak list generated in step 2 (step 6). The tolerance is preferably within 700 ppm, more preferably within 500 ppm. Furthermore, the tolerance is more preferably within 200 ppm, even more preferably within 100 ppm. However, the tolerance in the present invention is not limited to the above range. If a peak at m / z 6766 ± the tolerance exists in step 6, the first discrimination unit 241 determines that the test bacterium is M. abscessus subsp. bolletii (step 7). The first discrimination unit 241 then notifies the user of this fact (step 11) and stores the result in the discrimination result storage unit 250 (step 12), thereby completing the series of processes.

[0025] On the other hand, if there is no peak at m / z 6766 ± the tolerance in the peak list in step 6, the second discrimination unit 242 then discriminates subspecies based on the m / z value of ribosomal protein L29. Specifically, it is determined whether or not there is a peak at m / z 8767 ± the tolerance in the peak list generated in step 2 (step 8). The tolerance is preferably within 700 ppm, more preferably within 500 ppm. Furthermore, the tolerance is even more preferably within 200 ppm, and even more preferably within 100 ppm. However, the tolerance in the present invention is not limited to the above range. If a peak at m / z 8767 ± the allowable error is present in the peak list (i.e., if Yes in step 8), the second discrimination unit 242 determines that the test bacterium is M. abscessus subsp. massiliense (step 9), notifies the user of this fact (step 11), and stores the result in the discrimination result storage unit 250 (step 12), thereby terminating the series of processes. On the other hand, if a peak at m / z 8767 ± the allowable error is not present in the peak list (i.e., if No in step 8), the second discrimination unit 242 determines that the test bacterium is M. abscessus subsp. abscessus (step 10), notifies the user of this fact (step 11), and stores the result in the discrimination result storage unit 250 (step 12), thereby terminating the series of processes.

[0026] In the above example, the subspecies is identified based on the m / z value of ribosomal protein L30 (step 6) and then the subspecies is identified based on the m / z value of ribosomal protein L29 (step 8), but these steps may be performed in the reverse order.

[0027] If the test bacterium is determined to be M. abscessus in step 4, the content notified to the user in the subsequent step 11, i.e., information on which M. abscessus subspecies the test bacterium is, is used by the user (or another person who receives a report of the notification from the user) to determine a treatment plan (e.g., the selection of antibiotics to use in treating the patient) or for epidemiological investigations, etc. The system according to this embodiment can simultaneously determine the three M. abscessus subspecies based on the results of mass spectrometry of the test bacterium, thereby contributing to the speeding up of treatment plan decisions, epidemiological investigations, etc.

[0028] While specific examples of embodiments of the present invention have been described above, the present invention is not limited to these embodiments and can be modified as appropriate within the spirit and scope of the present invention. For example, in the above embodiment, an unknown test bacterium is first identified at the species level (step 3), and if the test bacterium is determined to be M. abscessus as a result, the test bacterium is then identified to which M. abscessus subspecies it belongs (steps 6 to 10). However, if it is known in advance that the test bacterium is M. abscessus, step 3 (and the accompanying steps 4 and 5) can be omitted.

[0029] Furthermore, in the above embodiment, the mass analysis data acquisition unit 210 acquires data (the above-mentioned digital data) of the mass analysis results of the test bacteria from the mass spectrometer 100 directly connected to the data processing device 200. However, the mass analysis result acquisition unit of the present invention is not limited to this, and may acquire profile data (or other forms of mass spectrum) or peak lists obtained by processing data obtained by mass analyzing the test bacteria using a mass spectrometer that is not directly connected to the data processing device 200 using another computer via a network such as a LAN or the Internet.

[0030] In the above embodiment, the m / z values ​​of ribosomal proteins L30 and L29 were used to determine whether a test bacterium is one of the three M. abscessus subspecies. However, the subspecies discrimination method and subspecies discrimination device according to the present invention may be configured to determine only whether a test bacterium is subspecies bolletii based on the m / z value of ribosomal protein L30. Since subspecies bolletii is said to account for approximately 5% to 10% of detected M. abscessus strains, determining whether a test bacterium is subspecies bolletii is of great epidemiological significance. Furthermore, while multidrug combination therapy is the standard treatment for pulmonary MABC disease, an infection caused by the Mycobacterium abscessus complex (the collective term for the three subspecies), it has been reported that subspecies bolletii tends to be multidrug-resistant. Therefore, determining whether a test bacterium is subspecies bolletii is thought to be of great clinical significance.

[0031] Alternatively, subspecies discrimination in the present invention may be performed by a human visually inspecting the results of mass spectrometry of the test bacterium. In this case, the test bacterium is analyzed using a mass spectrometer, and digital data obtained is processed by a computer to generate a mass spectrum (either profile data similar to the above or a mass spectrum in other forms), and the mass spectrum (or a peak list generated based on the mass spectrum) is visually inspected by a human, who then discriminates the subspecies based on the m / z value of the peak of ribosomal protein L30 (or the m / z values ​​of the peaks of L30 and L29) on the mass spectrum (or peak list).

[0032] Using the type strains of the three subspecies of M. abscessus and clinical isolates of M. abscessus, subspecies discrimination was performed based on the m / z values ​​of ribosomal proteins L30 and L29.

[0033] The standard strain was M. abscessus subsp. abscessus GTC 15115, provided by the Microbial Genetic Resources Conservation Center of Gifu University. T, and M. abscessus subsp. bolletii JCM 15297 provided by the RIKEN BioResource Research Center. T and M. abscessus subsp. massiliense JCM 15300 T The clinical isolates used were 20 strains that were mycobacterial culture-positive in clinical laboratories and identified as M. abscessus complex using DNA-DNA hybridization (DDH) or MALDI Biotyper. Of the 20 clinical isolates, 10 were identified as M. abscessus subsp. abscessus and another 10 were identified as M. abscessus subsp. massiliense by whole genome analysis.

[0034] The type strains and clinical isolates were cultured on Middlebrook 7H11 agar medium at 35°C for 3 days and then subjected to MALDI-MS analysis as follows. First, the cells were suspended in Milli-Q water, washed with ethanol, and frozen at -80°C. The cells were then bead-crushed and centrifuged. 1 μL of the resulting supernatant was placed on a sample plate. 1 μL of matrix solution was then placed on top of the sample plate. After drying, the sample plate was placed in a mass spectrometer (Shimadzu Corporation, MALDI-8020) and subjected to mass spectrometry. The matrix solution was prepared by dissolving α-cyano-4-hydroxycinnamic acid (CHCA) at 10 mg / mL in an aqueous solution containing 40% acetonitrile, 10% ethanol, and 1% trifluoroacetic acid. The mass spectrometry analysis was performed five times for each strain.

[0035] The m / z values ​​of the ribosomal proteins L29 and L30 of each strain were determined by the above mass spectrometry and are shown in Table 1. In the table, "SE" stands for standard error.

[0036]

[0037] As shown in Table 1, the average m / z value of the L29 protein in the type strain of subspecies abscessus was 8781.0, and the average m / z value of the L30 protein was 6796.5. The average m / z value of the L29 protein in the type strain of subspecies bolletii was 8784.5, and the average m / z value of the L30 protein was 6767.4. Furthermore, the average m / z value of the L29 protein in the type strain of subspecies massiliense was 8768.1, and the average m / z value of the L30 protein was 6797.3. These m / z values ​​were all within the acceptable error range of the m / z values ​​of the biomarkers for each subspecies, confirming that the method of the present invention can distinguish each subspecies. Furthermore, as shown in Table 1, similar results were obtained for the 20 clinical isolates.

[0038] These results were also obtained when 5% sheep blood agar was used instead of the Middlebrook 7H11 agar medium, confirming that the subspecies discrimination method of the present invention is applicable regardless of the culture conditions.

[0039] Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0040] (Item 1) A method for distinguishing subspecies of nontuberculous mycobacteria according to one embodiment of the present invention uses Mycobacterium abscessus, a nontuberculous mycobacterium, as a test bacterium, and determines whether the test bacterium is Mycobacterium abscessus subsp. bolletii based on the m / z value of ribosomal protein L30 in the mass spectrometry results of the test bacterium.

[0041] (Item 2) The method for distinguishing the subspecies of nontuberculous mycobacteria according to item 2 is the method for distinguishing the subspecies of nontuberculous mycobacteria according to item 1, further comprising determining whether the test bacterium is Mycobacterium abscessus subsp. massiliense based on the m / z value of ribosomal protein L29 in the mass spectrometry results of the test bacterium, and determining that the test bacterium is Mycobacterium abscessus subsp. massiliense if the test bacterium is determined to be neither Mycobacterium abscessus subsp. boletii nor Mycobacterium abscessus subsp. massiliense.

[0042] (Item 3) The method for distinguishing subspecies of nontuberculous mycobacteria according to item 3 is the method for distinguishing subspecies of nontuberculous mycobacteria according to item 1 or 2, in which the test bacterium is distinguished as Mycobacterium abscessus subsp. boleti when the peak of the ribosomal protein L30 is present at m / z 6766 ± within the range of the allowable error in the mass spectrometry results.

[0043] (4) The method for distinguishing subspecies of nontuberculous mycobacteria according to paragraph 4 is the method for distinguishing subspecies of nontuberculous mycobacteria according to paragraph 2, wherein the test bacterium is distinguished as Mycobacterium abscessus subsp. massiliens when the peak of the ribosomal protein L29 is present at m / z 8767 ± within the range of the allowable error in the mass spectrometry results.

[0044] (Item 5) The non-tuberculous mycobacterial subspecies discrimination device according to item 5 is a device that uses Mycobacterium abscessus, a non-tuberculous mycobacterium, as a test bacterium and discriminates the subspecies of the test bacterium based on the mass spectrometry results of the test bacterium, and has: a mass spectrometry result acquisition unit that acquires data on the mass spectrometry results; a subspecies discrimination unit that discriminates whether the test bacterium is Mycobacterium abscessus subsp. bolletii based on the m / z value of ribosomal protein L30 in the mass spectrometry results; and a notification unit that notifies a user of the discrimination results made by the subspecies discrimination unit.

[0045] (Item 6) The subspecies discrimination device for nontuberculous mycobacteria according to item 6 is the subspecies discrimination device for nontuberculous mycobacteria according to item 5, wherein the subspecies discrimination unit further discriminates whether the test bacterium is Mycobacterium abscessus subsp. massiliense based on the m / z value of ribosomal protein L29 in the mass spectrometry results of the test bacterium, and if the test bacterium is determined to be neither Mycobacterium abscessus subsp. boletii nor Mycobacterium abscessus subsp. massiliense, the test bacterium is determined to be Mycobacterium abscessus subsp. abscessus.

[0046] (Item 7) The non-tuberculous mycobacterial subspecies discrimination device according to item 7 is the non-tuberculous mycobacterial subspecies discrimination device according to item 5 or 6, wherein the subspecies discrimination unit discriminates the test bacterium as Mycobacterium abscessus subsp. boletii when the peak of the ribosomal protein L30 is present at m / z 6766 ± the allowable error range in the mass spectrometry results.

[0047] (Item 8) The non-tuberculous mycobacterial subspecies discrimination device according to item 8 is the non-tuberculous mycobacterial subspecies discrimination device according to item 6, wherein the subspecies discrimination unit discriminates the test bacterium as Mycobacterium abscessus subsp. massiliens when the peak of the ribosomal protein L29 is present at m / z 8767 ± within the range of the allowable error in the mass spectrometry results.

[0048] According to the method for distinguishing subspecies of nontuberculous mycobacteria relating to any one of paragraphs 1 to 4, or the device for distinguishing subspecies of nontuberculous mycobacteria relating to any one of paragraphs 5 to 8, it becomes possible to accurately and easily distinguish subspecies of Mycobacterium abscessus.

[0049] DESCRIPTION OF SYMBOLS 100 Mass spectrometer 200 Data processing device 210 Mass analysis data acquisition unit 220 Peak extraction unit 230 Species discrimination unit 240 Subspecies discrimination unit 241 First discrimination unit 242 Second discrimination unit 250 Discrimination result storage unit 260 Display control unit 270 Input unit 280 Display unit

Claims

1. A method for distinguishing subspecies of nontuberculous mycobacteria, comprising using Mycobacterium abscessus, a nontuberculous mycobacterium, as a test bacterium, and determining whether the test bacterium is Mycobacterium abscessus subsp. bolletii based on the m / z value of ribosomal protein L30 in the mass spectrometry results of the test bacterium.

2. The method for distinguishing subspecies of nontuberculous mycobacteria described in claim 1, further comprising determining whether the test bacterium is Mycobacterium abscessus subsp. massiliense based on the m / z value of ribosomal protein L29 in the mass spectrometry results of the test bacterium, and determining that the test bacterium is Mycobacterium abscessus subsp. abscessus if the test bacterium is determined to be neither Mycobacterium abscessus subsp. boleti nor Mycobacterium abscessus subsp. massiliense.

3. A method for distinguishing subspecies of nontuberculous mycobacteria as described in claim 1, wherein the test bacterium is identified as Mycobacterium abscessus subsp. boleti when the peak of the ribosomal protein L30 is present at m / z 6766 ± within the range of acceptable error in the mass spectrometry results.

4. The method for distinguishing subspecies of nontuberculous mycobacteria described in claim 2, wherein the test bacterium is identified as Mycobacterium abscessus subsp. massiliens when the peak of the ribosomal protein L29 is present at m / z 8767 ± within the range of acceptable error in the mass spectrometry results.

5. An apparatus for discriminating the subspecies of a test bacterium, Mycobacterium abscessus, based on the results of mass spectrometry of the test bacterium, comprising: a mass spectrometry result acquisition unit for acquiring data on the mass spectrometry results; a subspecies discrimination unit for discriminating whether the test bacterium is Mycobacterium abscessus subsp. bolletii based on the m / z value of ribosomal protein L30 in the mass spectrometry results; and a notification unit for notifying a user of the discrimination result made by the subspecies discrimination unit.

6. The device for discriminating subspecies of nontuberculous mycobacteria as described in claim 5, wherein the subspecies discrimination unit further discriminates whether the test bacterium is Mycobacterium abscessus subsp. massiliense based on the m / z value of ribosomal protein L29 in the mass spectrometry results of the test bacterium, and discriminates the test bacterium as Mycobacterium abscessus subsp. abscessus if the test bacterium is determined to be neither Mycobacterium abscessus subsp. boletii nor Mycobacterium abscessus subsp. massiliense.

7. The device for discriminating subspecies of nontuberculous mycobacteria described in claim 5, wherein the subspecies discrimination unit discriminates that the test bacterium is Mycobacterium abscessus subsp. boleti when the peak of the ribosomal protein L30 is present at m / z 6766 ± within the range of acceptable error in the mass spectrometry results.

8. The device for discriminating subspecies of nontuberculous mycobacteria described in claim 6, wherein the subspecies discrimination unit discriminates that the test bacterium is Mycobacterium abscessus subsp. massiliens when the peak of the ribosomal protein L29 is present at m / z 8767 ± within the range of the allowable error in the mass spectrometry results.

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