Microorganism identification method

Abstract

A microorganism identification method utilizing mass spectrometry is provided. More specifically, a method for identifying a phylotype of Cutibacterium acnes utilizing mass spectrometry is provided. The method includes a) a step for reading out a m/z value of a peak derived from a marker protein on a mass spectrum which is obtained by mass spectrometry of a sample containing microorganisms; and b) a step for judging whether the sample contains Cutibacterium acnes (C. acnes) based on the m/z value.

Claims

1. A microorganism identification method, comprising: a) a step for reading out a m/z value of a peak derived from a marker protein on a mass spectrum, which is obtained by mass spectrometry of a sample containing microorganisms; and b) a step for judging whether the sample contains Cutibacterium acnes (C. acnes) based on the m/z value; wherein the marker protein is one or more proteins selected from a group consisting of ribosomal proteins L7/L12, L9, L18, L28, L29, L30, L31, S8, S15, S19 and S20.

2. A microorganism identification method, comprising: a) a step for reading out a m/z value of a peak derived from a marker protein on a mass spectrum, which is obtained by mass spectrometry of a test microorganism; and b) a step for judging whether the test microorganism is a C. acnes type I, II or III based on the m/z value; wherein the marker protein is a combination of ribosomal proteins L6 and L23, a combination of ribosomal proteins L15 and L23, or a combination of ribosomal proteins L6 and L15.

3. A microorganism identification method, comprising: a) a step for reading out a m/z value of a peak derived from a marker protein on a mass spectrum, which is obtained by mass spectrometry of a test microorganism; and b) a step for judging whether the test microorganism is a phylotype IA1, IA2 or IB of a C. acnes type I based on the m/z value; wherein the marker protein is a combination of a ribosomal protein L13 and an antitoxin.

4. The identification method according to claim 3, wherein the m/z value (m/z) of the peak of the antitoxin on the mass spectrum is 7034.6.

5. The identification method according to claim 1, wherein the marker protein on the mass spectrum further comprises a divalent ion.

6. The identification method according to claim 2, wherein the marker protein on the mass spectrum further comprises a divalent ion.

7. The identification method according to claim 3, wherein the marker protein on the mass spectrum further comprises a divalent ion.

8. A microorganism identification method, comprising: a) a step for reading out a m/z value of a peak derived from a marker protein on a mass spectrum, which is obtained by mass spectrometry of a sample containing microorganisms, and b) a step for judging whether the sample comprises at least one of a C. acnes type I, II or III based on the m/z value; wherein in the step b), on the mass spectrum, in regard to a combination of ribosomal proteins L6 and L23, a combination of ribosomal proteins L15 and L23, or a combination of ribosomal proteins L6 and L15, when there is at least one peak of a m/z value in a case of having mutations specific to the C. acnes types I, II, and III, it is judged that the sample comprises at least one of the C. acnes type I, II or III.

9. A program for making a computer execute each step according to claim 1.

10. A program for making a computer execute each step according to claim 2.

11. A program for making a computer execute each step according to claim 3.

12. A program for making a computer execute each step according to claim 8.

13. An analysis method of skin bacterial flora using the identification method according to claim 1.

14. An analysis method of skin bacterial flora using the identification method according to claim 2.

15. An analysis method of skin bacterial flora using the identification method according to claim 3.

16. An analysis method of skin bacterial flora using the identification method according to claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an overall view of a microorganism discrimination system used in a microorganism discrimination method of the disclosure.

(2) FIG. 2 is a diagram showing a C. acnes type discrimination procedure using the microorganism discrimination method of the disclosure.

(3) FIG. 3 is a list of marker proteins for C. acnes typing. Sequence type is a classification based on Multilocus Sequence Typing (MLST).

(4) FIG. 4 is a diagram showing peaks on a mass spectrum contributing to the C. acnes typing.

(5) FIG. 5 is a diagram showing a peak profile for C. acnes proteo-typing. Sequence type is a classification based on Multilocus Sequence Typing (MLST).

(6) FIG. 6 is a dendrogram showing a classification result of the C. acnes.

DESCRIPTION OF THE EMBODIMENTS

(7) According to the disclosure, a discrimination can be reproducibly and rapidly made on whether the sample contains the Cutibacterium acnes (C. acnes) which is a representative skin resident bacteria, and on whether the test microorganism is C. acnes type I, II or III. Furthermore, according to the disclosure, a subtype IA1, IA2 or IB of the C. acnes type I can be reproducibly and rapidly identified.

(8) Although the C. acnes is involved in the induction of skin diseases such as acne and the like and skin inflammations, it is considered that the skin diseases and skin inflammations are mainly caused by the C. acnes type I. According to the disclosure, the C. acnes type I in the sample can be rapidly and simply identified, and thus a skin bacterial flora analysis that is important for treatment or prevention of the skin diseases such as acne and the like can be rapidly and simply performed. Furthermore, according to the disclosure, a skin condition evaluation based on the skin bacterial flora analysis can be simply performed, and thus an evaluation of skin care can be easily performed.

(9) FIG. 1 is an overall view of a microorganism discrimination system used in a microorganism identification method of the disclosure.

(10) The microorganism discrimination system roughly consists of a mass spectrometry unit 10 and a microorganism discrimination unit 20. The mass spectrometry unit 10 includes an ionization unit 11 which ionizes molecules and atoms in a sample by matrix assisted laser desorption/ionization mass spectrometry (MALDI), and a time-of-flight mass separator (TOF) 12 which separates various ions emitted from the ionization unit 11 corresponding to a m/z value.

(11) The TOF 12 includes an extraction electrode 13 by which the ions are extracted from the ionization unit 11 and guided to an ion flight space in the TOF 12, and a detector 14 which detects the ions mass separated in the ion flight space.

(12) The entity of the microorganism discrimination unit 20 is a computer such as a workstation, a personal computer and the like; a display unit 23 consisting of a memory 22, a LCD (Liquid Crystal Display) and the like, an input unit 24 consisting of a keyboard, a mouse and the like, and a storage unit 30 consisting of a large-capacity storage device such as a hard disk, a SSD (Solid State Drive) and the like are connected to each other in a CPU (Central Processing Unit) 21 which is a central operation processing device. In the storage unit 30, an OS (Operating System) 31, a spectrum creation program 32, a species determination program 33, and a phylotype determination program 35 (the program of the disclosure) are stored, and a first database 34 and a second database 36 are stored. The microorganism discrimination unit 20 further includes an interface (I/F) 25 for managing a direct connection to external devices and a connection to external devices and the like via a network such as LAN (Local Area Network) and the like, and is connected from the interface 25 to the mass spectrometry unit 10 via a network cable NW (or wireless LAN).

(13) In FIG. 1, a spectrum acquisition unit 37, a m/z readout unit 38, and a phylotype judgment unit 39 are shown as being related to the phylotype determination program 35 (the program of the disclosure). Any one of these units is basically a functional part implemented by software by the CPU 21 executing the phylotype determination program 35. Besides, the phylotype determination program 35 is not necessarily an independent program; for example, the phylotype determination program 35 may be a function incorporated into the species determination program 33 or a part of a program for controlling the mass spectrometry unit 10, and the form of the phylotype determination program 35 is not particularly limited.

(14) In addition, in FIG. 1, the spectrum creation program 32, the species determination program 33, the phylotype determination program 35, the first database 34, and the second database 36 are mounted on a terminal operated by a user; however, at least a part or all of these programs and databases may be arranged in another device connected to the terminal by a computer network, and processing performed by the programs arranged in the another device and/or an access to the databases are/is executed according to instructions from the terminal.

(15) In the first database 34 of the storage unit 30, multiple mass lists related to the known microorganisms are registered. The mass list enumerates m/z values of detected peaks in mass spectra from some microorganism cells. The mass list includes information of the m/z values, taxonomic information (family, genus, species, and the like) of the microorganism. This mass list is desirably created based on data obtained from various microorganisms' cells using the same ionization method and mass separation method as the method performed by the mass spectrometry unit 10.

(16) When the mass list is created from the actually measured data, first, peaks that appear in a prescribed m/z value range are extracted from the mass spectrum acquired as the actually measured data. At this time, the peaks mainly derived from proteins can be extracted by setting the m/z value range to about 3,000-20,000. In addition, undesired peaks (noises) can be excluded by extracting only the peaks of which the peak height (relative intensity) is above a prescribed threshold. Besides, large amount of ribosomal protein groups are expressed in the cells, and thus most of the m/z values described in the mass list can be made to be derived from the ribosomal protein by properly setting the threshold. Then, the m/z value (m/z) of the peaks extracted as described above is listed for each cell, and is registered in the first database 34 after the classification information and the like are added. Besides, desirably, the culture environment is standardized in advance for each microorganism cell used in collection of the actually measured data so as to suppress variations in gene expression caused by culture conditions.

(17) In the second database 36 of the storage unit 30, information related to a marker protein for identifying the known microorganisms by a classification (subspecies, pathotype, serotype, strain and the like) more detail than species is registered. The information related to the marker protein at least includes information of the m/z value (m/z) of the marker protein in the known microorganisms. The marker protein stored in the second database 36 may be a divalent ion. Besides, the m/z value (m/z) described in the specification is a value for monovalent ions unless otherwise stated. In the second database 36 of the embodiment, at least the values of m/z values respectively corresponding to the ribosomal proteins L7/L12, L9, L18, L28, L29, L30, L31, S8, S15, S19 and S20 (L7/L12: m/z 13571.4, L9: m/z 16118.8, L18: m/z 13570.6, L28: m/z 6807.0, L29: m/z 8754.9, L30: m/z 6786.9, L31: m/z 7718.8, S8: m/z 14525.7, S15: m/z 10080.7, S19: m/z 10380.0 and S20: m/z 9570.0) are stored to identify C. acnes strains. Furthermore, in the second database 36, the marker proteins L6, L15 and L23 (L6: m/z 19678.5 or m/z 19706.6, L15: m/z 15384.7 or m/z 15357.6, and L23: m/z 11181.0 or m/z 11200.0) are registered to identify C. acnes type I, II, and III. Here, the information related to the marker proteins L6, L15 and L23 for identifying the type I is L6: m/z 19706.6 and L23: m/z 11200.0, L15: m/z 15384.7 and L23: m/z 11200.0, or L6: m/z 19706.6 and L15: m/z 15384.7. The information related to the marker proteins L6, L15 and L23 for identify the type II is L6: m/z 19678.6 and L23: m/z 11200.0, L15: m/z 15357.6 and L23: m/z 11200.0, or L6: m/z 19678.6 and L15: m/z 15357.6. The information related to the marker proteins L6, L15 and L23 for identify the type III is L6: m/z 19678.6 and L23: m/z 11181.0, L15: m/z 15384.7 and L23: m/z 11181.0, or L6: m/z 19678.6 and L15: m/z 15384.7.

(18) Furthermore, in the second database 36, the marker proteins L6, L15 and L23 (L6: m/z 19678.5 or m/z 19706.6, L15: m/z 15384.7 or m/z 15357.6, and L23: m/z 11181.0 or m/z 11200.0) are registered to identify C. acnes type I, II, and III. Furthermore, in the second database 36, the m/z values (m/z 16153.5, m/z 16167.6 and m/z 16180.7) corresponding to L13 and m/z 7034.6 corresponding to an antitoxin of the C. acnes are registered as the marker protein for subdividing the C. acnes type I into the subtypes IA1, IA2 and IB. The antitoxin means an antitoxic protein coded into chromosome or plasmid DNA of a bacterium. Here, for the information related to the marker protein for subdividing the C. acnes type I into the subtype IA1, L13 is m/z 16167.6 and antitoxin is m/z 7034.6. The marker proteins for subdividing the C. acnes type I into the subtype IA1 are L13 (m/z 16167.6) and antitoxin (m/z 7034.6). C. acnes subtype IA2 is subdivided from type I with L13 (m/z 16167.6). Here, the antitoxin in subtype IA2 is not detected at m/z 7034.6. C. acnes subtype IB is subdivided from type I with L13 (m/z 16153.6). Here, the antitoxin in subtype IB is not detected at m/z 7034.6.

(19) Desirably, detected m/z values are compared with the theoretical masses calculated from amino-acid sequence of each marker protein. The marker proteins whose detected masses and calculated masses are matched and selected as the marker proteins stored in the second database 36. As the amino-acid sequence of each marker protein, an amino-acid sequence translated from DNA sequence or an amino-acid sequence registered in a protein DB can be used. In addition, the DNA sequences registered in a public database, for example, the database of NCBI (National Center for Biotechnology Information) can also be used. It is desirable to consider a cleavage of N-terminal methionine residues as a post-translational modification when calculate the theoretical masses from the amino-acid sequences. Specifically, when the second amino-acid residue from the N-terminal is glycine, alanine, serine, proline, valine, threonine or cysteine, the theoretical value is calculated regarding the marker protein as a protein in which the N-terminal methionine is cleaved. In addition, since detected protein ions by MALDI-TOF MS are protonated molecules, mass of proton should be considered in calculating of theoretical masses.

(20) An identification procedure of the C. acnes types I, II and III using the microorganism identification system of the embodiment is described with reference to a flowchart (FIG. 2).

(21) First, the user prepares a sample containing components of the test microorganism, and sets the sample in the mass spectrometry unit 10 to execute mass spectrometry. In this case, a fungus body or cell suspension can be directly used as the sample. However, preferably a cell extract is used, and more preferably a sample obtained by concentration or purification of cell components of the ribosomal protein and the like from the cell extract is used.

(22) The spectrum creation program 32 acquires, via the interface 25, a detection signal obtained from the detector 14 of the mass spectrometry unit 10, and creates the mass spectrum of the test microorganism based on the detection signal (step S101).

(23) Next, the species determination program 33 matches the mass spectrum of the test microorganism with the mass list of the known microorganisms recorded in the first database 34, and extracts the mass list of the known microorganisms having a m/z value pattern similar to the mass spectrum of the test microorganism, for example, a mass list in which many peaks are consistent with the respective peaks in the mass spectrum of the test microorganism in a prescribed error range (preferably 50-500 ppm, more preferably 150-200 ppm) (step S102). Subsequently, the species determination program 33 specifies, by referring to the classification information stored in the first database 34 in association with the mass list extracted in step S102, an organism species to which the known microorganism corresponding to the mass list belongs (step S103). Then, when the organism species is not C. acnes (“No” in step S104), the organism species is output to the display unit 23 as the organism species of the test microorganism (step S113), and the identification processing ends. On the other hand, when the organism species is C. acnes (“Yes” in step S104), the procedure subsequently proceeds to identification processing performed by the phylotype determination program 35. Besides, when it is judged that the sample contains the C. acnes by other methods in advance, the procedure may proceed to the phylotype determination program 35 without utilizing the species determination program using the mass spectrum.

(24) In the phylotype determination program 35, first, the phylotype judgment unit 39 respectively reads out the values of m/z values of the antitoxin and the ribosomal proteins L6, L13, L15, L23 which are marker proteins from the second database 36 (step S105). Subsequently, the spectrum acquisition unit 37 acquires the mass spectrum of the test microorganism created in step S101. Then, the m/z value readout unit 38 selects, on the mass spectrum, the peak that appears in a m/z value range stored in the second database 36 in association with each of the marker proteins as the peak corresponding to each marker protein, and reads out the m/z value thereof (step S106). After that, the phylotype judgment unit 39 compares the m/z value and the value of m/z value of each marker protein read out from the second database 36, and judges whether the two are consistent within a prescribed allowable error range (step S107). When the two are consistent as a result, it is judged that the test microorganism is any one of the C. acnes types I, II or III (step S108), and the fact is output to the display unit 23 as an identification result of the test microorganism (step S113).

(25) Furthermore, the C. acnes classified as the type I in step S108 can be subdivided into the subtype IA1, IA2, or IB by reading out the value of m/z value of L13 and the m/z 7034.6 corresponding to the antitoxin (steps S111 and S112). Besides, when it is judged that the sample includes the C. acnes type I by other methods in advance, the C. acnes type I can be further subdivided into the subtype IA1, IA2, or IB by executing steps S111 and S112 only.

(26) The embodiment of the disclosure is described above with reference to the drawings, but the disclosure is not limited to the above embodiment, and appropriate changes are allowed within the scope of the disclosure.

EXAMPLES

(27) A selection procedure of the marker proteins in the disclosure and an experiment conducted to demonstrate the effect of the disclosure are described below, but the scope of the disclosure is not limited hereto.

(28) (1) Strain and Culture Condition

(29) 22 strains of the C. acnes are used to construct a protein mass database. These strains are purchased from Japan Collection of Microorganisms (JCM) of Bioresource Research Center in Institute of Physical and Chemical Research (Tsukuba City, Japan). A modified GAM medium or a GAM medium of Nissui Pharmaceutical CO., LTD. is used in cultivation of the C. acnes.

(30) (2) Construction of Protein Mass Database

(31) An amino acid sequence of ribosomal subunit protein is obtained from the database of National Center for Biotechnology Information (NCBI) in the U.S. Compute pI/Mw tool of ExPASy proteomics server provided by Swiss Institute of Bioinformatics is used in calculation of the calculation mass of each protein. At this time, when the second amino acid residue from the N-terminal is glycine, alanine, serine, proline, valine, threonine or cysteine, the calculation mass is calculated regarding the protein as a protein in which the N-terminal Methionine is cleaved.

(32) (3) Measurement Using MALDI-TOF MS

(33) Bacterial cells on an agar medium or fungus recycled from a liquid medium by centrifugation are used in measurement. The bacterial cells are suspended with distilled water (OD610=1). The bacterial cell suspension (500 μL) is crushed using bead-beating at 5000 rpm three times at 60 seconds each. Crushed bacterial cell is centrifuged to remove debris (15000 g, 5 minutes), and a cell-lysate is obtained as a supernatant. Ribosomal fraction is obtained by ultrafiltration (Nominal molecular weight limit: 30 kDa, 14000 g, 10 minutes) as a captured fraction. Sinapinic acid (SA) or α-cyano-4-hydroxycinnamic acid (CHCA) is used as a matrix reagent. SA or CHCA matrix solution at a concentration of 15 mg/mL in 50% acetonitrile (ACN) with 1% trifluoroacetic acid (TFA) is used for MALDI-MS sample preparation. 10 μlof matrix solution is mixed with 1 μL of ribosome fraction, and 1 μL of the mixture is dropped on a sample plate and air dried.

(34) The MALDI-MS measurements are performed using an AXIMA Performance™ mass spectrometer in the positive ion linear mode. The assignment of detected peaks is judged from mass differences within 200 ppm compared between the calculated masses as [M+H].sup.+ ions and the detected masses in the MALDI mass spectra.

(35) Each of following ribosomal protein, L7/L12, L9, L18, L28, L29, L30, L31, S8, S15, S19 and S20, are clearly detected from all the sample strains at the same m/z values.

(36) Detected m/z values of biomarker proteins which contribute to the phylotype identification are shown in FIG. 3 and FIG. 4. The following is shown based on FIG. 3 and FIG. 4.

(37) By focusing on L23, the type III can be discriminated (type III: m/z 11180.9, types I and II: m/z 11200.0).

(38) By focusing on L15, the type II can be discriminated (type II: m/z 15357.6, types I and III: m/z 15384.7).

(39) By focusing on L6, the type I can be discriminated (type I: m/z 19706.6, types II and III: m/z 19678.5).

(40) By focusing on L13, the phylotype IB of the type I can be discriminated (IB: m/z 16153.5, IA1 and IA2: m/z 16167.6).

(41) By focusing on the peak (antitoxin) of m/z 7034.6, the IA1 of the type I can be discriminated.

(42) By focusing on L13 and the peak (antitoxin) of m/z 7034.6, the IA1 or IA2 and IB of the type I can be discriminated.

(43) (4) Cluster Analysis

(44) All the sample strains are confirmed as C. acnes by MALDI-MS. The judgement results for each biomarker protein are summarized in the table, in which scores used either 1 or 0 (FIG. 5). “1” means that a biomarker peak is detected, and “0” means that biomarker peak is not detected. The data is used for cluster analysis to create a dendrogram (FIG. 6). As shown in FIG. 6, C. acnes strains are successfully classified into the types I, II and III. Furthermore, the type I related to skin inflammation can be further divided into subtype IA1, IA2 and IB. Accordingly, an analysis method of skin bacterial flora is provided.