Use of species-specific biomarkers for Staphylococcus bacteria

Abstract

This provides a method for identifying S. aureus and other bacterial species that make up the coagulase-negative staphylococcus group. [Solution] A method for determining the presence or absence of Staphylococcus species in a species-specific manner, wherein the Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis, and comprises a peak detection step of determining whether one or more peaks selected from a group consisting of peaks corresponding to specific m / z values ​​are detected in a mass spectrum obtained using matrix-assisted laser desorption / ionization mass spectrometry on a test sample with respect to S. aureus, and a bacterial species determination step of determining the presence or absence of S. aureus based on the peak detection result of the peak detection step.

Description

Technical Field

[0001] This specification relates to the use of species-specific biomarkers of Staphylococcus bacteria.

Background Art

[0002] Analysis of the human skin microbiome has become possible to perform a detailed analysis of the bacterial flora at the genus level with the spread of next-generation sequencers (NGS) in the latter half of the 2000s, and detailed bacterial flora mapping of human skin has been carried out (Non-Patent Document 1). For example, although it has been found that the bacterial flora on the skin of patients with atopic dermatitis (AD) is characteristic, Staphylococcus aureus (S. aureus), which is considered to be the causative bacterium of AD, is considerably more abundant in healthy individuals, while the group of coagulase-negative Staphylococcus (CNS) (including S. epidermidis, S. capitis, S. caprae, S. hominis, S. lugdunensis), which are regarded as beneficial resident bacteria, is also considered to be considerably more abundant in healthy individuals compared to healthy individuals. Therefore, analysis at the species level is necessary, but analysis using NGS is difficult at the species level. On the other hand, for analysis at the species level, a clone library method using the entire sequence of the 16S rRNA gene is required, which is very laborious (Non-Patent Document 2). Thus, due to technical limitations in flora analysis, analysis at the species and subspecies levels of skin resident bacteria has not been fully approached.

[0003] In recent years, a method (fingerprinting method) has been developed that uses matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify bacteria by comparing their mass spectra with existing databases, and studies have been reported to identify S. aureus and its close relatives (Non-Patent Literature 3). However, fingerprinting methods that do not specify biomarkers have limitations in clearly distinguishing closely related species, and a new breakthrough that can clearly distinguish closely related species was needed. On the other hand, a genomics-proteomics-integrated identification method (S10-GERMS method) has been reported that utilizes the genomic information of approximately 25 ribosomal subunit proteins encoded by the S10-spc-alpha operon, which is common to bacteria. After calculating the theoretical mass, ribosomal subunit proteins with the same number but different masses are selected as biomarkers, and bacteria are identified and distinguished by differences in biomarker mass using MALDI-TOF MS (Non-Patent Literature 4). However, the mass of ribosomal subunit proteins varies at the species and strain level of the target bacteria, making biomarker discovery a technical challenge. Furthermore, ribosomal subunit proteins alone may not be sufficient for identification in some cases (Non-Patent Literature 5). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kong, Trend Mol. Med., 17: 320-328, 2011 [Non-Patent Document 2] Kong et al., Genome Res., 22: 850-859, 2012 [Non-Patent Document 3] Xu et al., Chapter 21B: Subtyping of Staphylococcus spp. Based upon MALDI-TOF MS Data Analysis. In: MALDI-TOF and Tandem MS for Clinical Microbiology, P. 563-578, 2017. ISBN:9781118960257. [Non-Patent Document 4] H. Tamura, Y. Hotta, and H. Sato, Novel accurate bacterial discrimination by MALDI-Time of-Flight MS based on ribosomal proteins coding in S10-spc alpha operon at strain Level S10-GERMS, J. Am. Soc. Mass Spectrom., 24, 1185_1193 (2013) [Non-Patent Document 5] T. Ojima-Kato, N. Yamamoto, M. Suzuki, T. Fukunaga, and H. Tamura, Discrimination of Escherichia coli O157, O26 and O111 from other serovars by MALDI-TOF MS based on the S10-GERMS method, PLOS ONE, 9, e0159730 (2014) [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] In Alzheimer's disease (AD) patients, numerous coagulase-negative staphylococci (CNS) species are commonly present in addition to S. aureus, suggesting that specific CNS species may influence the disease state. Identifying S. aureus and the CNS species is considered useful for the diagnosis, pathogenesis elucidation, and treatment of various diseases, including AD.

[0006] However, because NGS uses approximately 300 bp of the 16S rRNA gene for bacterial species identification, there were cases where species-level differences could not be found when attempting to identify S. aureus and the bacterial species that make up the CNS group.

[0007] Furthermore, if the fingerprints obtained for S. aureus and the CNS group do not match the reference fingerprint, it may be impossible to identify the specific species of S. aureus and the CNS group. In addition, according to the inventors, referring to the dendrogram obtained by the fingerprinting method described above, it was found that multiple species constituting the CNS group may be included in a single type.

[0008] Furthermore, it is unclear to what extent the mass of ribosomal subunit proteins differs at the species and strain level of each bacterium being identified, and it is also assumed that the degree of ionization of ribosomal subunit proteins differs. For these reasons, even the inventors were unsure whether it would be possible to find a biomarker that could determine the presence or absence of S. aureus and the bacterial species constituting the CNS group using only the approximately 25 ribosomal subunit proteins encoded by the S10-spc-alpha operon using the S10-GERMS method.

[0009] This specification aims to provide a biomarker and a method for using the same that can determine, for example, the presence or absence of Staphylococcus species that can constitute the normal skin flora at the species level. [Means for solving the problem]

[0010] The inventors first selected biomarkers that exhibit specific m / z values ​​for S. aureus and individual species constituting the CNS group under certain conditions, targeting the entire genome. However, it was found that it is difficult to determine the presence or absence of Staphylococcus species at the species level using only these biomarkers. Therefore, the inventors further decided to use combinations of proteins that are characteristically detected in specific species among these species, based on the measured mass (m / z) of proteins derived from S. aureus and species constituting the CNS group (S. epidermidis, S. capitis, S. caprae, S. hominis, S. lugdunensis), as additional biomarkers. According to the inventors, it was found that by combining these biomarkers, the presence or absence of each species of Staphylococcus species that can constitute the skin's commensal microbiota can be determined. The following means are provided according to this specification.

[0011] [1] A method for determining the presence or absence of Staphylococcus species in a species-specific manner, The aforementioned Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. For the test samples potentially containing the aforementioned Staphylococcus bacteria, the following biomarkers (a) to (f) were observed in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a) With respect to S. aureus, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42. (b) With respect to S. epidermidis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66. (c) Regarding S. capitis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30. (e) With respect to S. hominis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68. (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45. A peak detection step that determines whether a peak belonging to one or more biomarkers selected from is detected, A bacterial species determination step that determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results of the peak detection step, A method that includes [a certain feature]. [2] The biomarkers (a) to (f) are the following biomarkers (aa) to (ff): (aa) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13172.22, 9494.66, and 12612.01, respectively. (bb) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 6698.82, 9778.08, 8608.22, and 11733.66. (cc)One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12895.81, 14641.36, 6713.46, 9639.61, and 9671.19. (dd)One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 10691.67, 9889.27, and 8526.30. (ee) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12347.54, 12356.57, 11209.97, 7353.67, and 10195.68. (ff) One or more peaks selected from the group consisting of peaks corresponding to m / z values ​​of 11420.09, 12473.98, and 12689.50, respectively. The method according to [1], comprising at least the following: [3] The peak detection step is a step of determining whether peaks belonging to the biomarkers (a), (b), (c), (d), (e) and (f) are detected. The method according to [1] or [2], wherein the bacterial species determination step is a step of determining whether or not there are one or more bacterial species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis and S. lugdunensis as the Staphylococcus bacteria. [4] The peak detection step is a step of determining whether a peak belonging to one or two biomarkers selected from the biomarkers (d) and (f) is detected, The method according to [1] or [2], wherein the bacterial species determination step is a step of determining the presence or absence of one or two bacterial species selected from S. hominis and S. lugdunensis as the Staphylococcus species. [5] The method according to any one of [1] to [4], wherein synapic acid is used as the matrix agent in the measurement of the mass spectrum by the matrix-assisted laser desorption ionization mass spectrometry. [6] The test sample comprises human skin commensal bacteria, as described in any of [1] to [5]. [7] The method according to any one of [1] to [6], wherein the mass accuracy in the peak detection step is 800 ppm or less. [8] The method according to any one of [1] to [7], comprising the step of displaying determination information together with color information previously assigned to the Staphylococcus bacteria whose presence has been confirmed in the bacterial species determination step. [9] A method for evaluating the microbial flora of human skin, The aforementioned human skin commensal bacteria are one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. Regarding the test samples that may contain the aforementioned human skin commensal bacteria, the following biomarkers (a) to (f) were observed in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a) With respect to S. aureus, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42. (b) For S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to m / z of 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66 respectively (c) For S. capitis, one or more peaks selected from the group consisting of peaks corresponding to m / z of 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19 respectively (d) For S. caprae, one or more peaks selected from the group consisting of peaks corresponding to m / z of 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30 respectively (e) For S. hominis, one or more peaks selected from the group consisting of peaks corresponding to m / z of 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68 respectively (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to m / z of 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45 respectively A peak detection step of determining whether a peak belonging to one or more biomarkers selected therefrom is detected A species determination step of determining the presence or absence of one or more species of the human skin commensal bacteria based on the peak detection result of the peak detection step A method comprising the above steps

[10] The method according to [9], for the purpose of diagnosing, elucidating the pathological condition, and treating human skin diseases, by monitoring the flora of the human skin commensal bacteria.

[11] A system for specifically determining the presence or absence of staphylococcal bacteria, wherein the staphylococcal bacteria are one or more selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis, a matrix-assisted laser desorption ionization mass spectrometry unit, and a determination unit for determining the presence or absence of the staphylococcal bacteria based on the mass spectrum of a test sample that may contain the staphylococcal bacteria, obtained in the matrix-assisted laser desorption ionization mass spectrometry unit, comprising: wherein the determination unit, in the mass spectrum, the following biomarkers (a) to (f): (a) For S. aureus, one or more peaks selected from the group consisting of peaks corresponding to m / z of 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42, respectively (b) For S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to m / z of 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66, respectively (c) For S. capitis, one or more peaks selected from the group consisting of peaks corresponding to m / z of 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19, respectively (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30. (e) With respect to S. hominis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68. (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45. A system that detects peaks belonging to one or more biomarkers selected from a set of biomarkers, and determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results.

[12] A system for determining the presence or absence of Staphylococcus species in a species-specific manner, Matrix-assisted laser desorption / ionization mass spectrometry unit, A determination unit that determines the presence or absence of Staphylococcus bacteria based on the mass spectrum of a test sample potentially containing Staphylococcus bacteria obtained in the matrix-assisted laser desorption / ionization mass spectrometry unit, Equipped with, The determination unit determines the following biomarkers (a) to (f) in the mass spectrum: (a) With respect to S. aureus, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66. (c) Regarding S. capitis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 19427.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30. (e) With respect to S. hominis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68. (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45. A system that maintains the above for the purpose of determining existence or non-existence.

[13] A method for determining the presence or absence of Staphylococcus species in a species-specific manner, The aforementioned Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. For the test samples potentially containing the aforementioned Staphylococcus bacteria, the following first biomarkers (a1) to (f1) were observed in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a1) For S. aureus, the peaks corresponding to m / z are 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42 respectively. (b1) For S. epidermidis, the peaks corresponding to m / z values ​​of 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, and 10159.87 respectively. (c1) For S. capitis, the peaks corresponding to m / z values ​​are 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, and 9815.03, respectively. (d1) For S. caprae, the peaks corresponding to m / z values ​​of 13321.27, 9090.38, 194247.91, 6337.79, and 7756.65 respectively. (e1) For S. hominis, the peaks corresponding to m / z values ​​of 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, and 12047.71 respectively. (f1) For S. lugdunensis, the peaks corresponding to m / z values ​​are 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45, respectively. A peak detection step of determining whether a peak belonging to the biomarker of the Staphylococcus bacteria selected from is detected, A bacterial species determination step that determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results of the peak detection step, A method that includes [a certain feature].

[14] Furthermore, in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry, the following second biomarkers (b2) to (e2): (b2) Regarding S. epidermidis, peaks corresponding to 8608.22 and 11733.66 m / z, respectively. (c2) Regarding S. capitis, peaks corresponding to m / z values ​​of 9639.61 and 9671.19, respectively. (d2) For S. caprae, peaks corresponding to 10691.67, 9889.27, and 8526.30 m / z, respectively. (e2) Regarding S. hominis, the peaks corresponding to 7353.67 and 10195.68 m / z, respectively. A peak detection step of determining whether a peak belonging to the biomarker of the Staphylococcus bacteria selected from is detected, The method according to

[13] , comprising a bacterial species determination step of determining the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection result of the peak detection step. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the m / z set used to identify fungal species belonging to S. aureus. [Figure 2] This figure shows the m / z set used to identify bacterial species belonging to S. epidermidis. [Figure 3] This figure shows the m / z set used to identify fungal species belonging to S. capitis. [Figure 4] This figure shows the m / z set used to identify fungal species belonging to S. caprae. [Figure 5] This figure shows the m / z set used to identify bacterial species belonging to S. hominis. [Figure 6] This figure shows the m / z set used to identify fungal species belonging to S. lugdunensis. [Figure 7] This diagram shows an overview of a system for determining the presence or absence of Staphylococcus bacteria. [Figure 8] This diagram shows an example of a flowchart for determining the presence or absence of Staphylococcus bacteria. [Figure 9] This figure shows the results of the bacterial species determination in the example. [Modes for carrying out the invention]

[0013] This specification discloses the determination of the presence or absence of Staphylococcus species in a species-specific manner, and more specifically, the determination of the presence or absence of S. aureus and S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis belonging to the CNS group (hereinafter, these bacteria are collectively referred to as the Staphylococcus species group). More specifically, this specification provides for the use of biomarkers to determine the presence or absence of these bacteria in a species-specific manner.

[0014] According to the inventors, using a proteotyping method and eMSTAT (statistical analysis software manufactured by Shimadzu Corporation) that they had already developed, they identified biomarkers for determining the presence or absence of each species belonging to the Staphylococcus genus from mass spectrometry using MALDI-TOF MS.

[0015] Firstly, we selected a peak containing ribosomal subunit proteins and possessing an m / z value that is uniquely characteristic of a specific bacterial species as the primary biomarker.

[0016] Secondly, we selected peaks with m / z values ​​that are not characteristic of a specific bacterial species individually but are present in combination of two or three species, thereby distinguishing them from other Staphylococcus species, as a second biomarker.

[0017] By using a combination of these two types of biomarkers, we were able to specifically determine the presence or absence of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunens.

[0018] First, we will describe the set of biomarkers, namely the m / z set that can be used to determine the presence or absence of Staphylococcus bacteria. Next, we will describe the determination of the presence or absence of Staphylococcus bacteria using this m / z set, as well as the system for such determination.

[0019] In this specification, the test sample is not particularly limited as long as it is a sample that may contain Staphylococcus bacteria. For example, a sample taken from the skin surface of a human body can be used. The surface of human skin is home to a flora of commensal bacteria, including Staphylococcus bacteria. To collect the commensal bacteria from the skin, known methods such as the cup scrub method can be used. For example, a physiological saline solution to which a surfactant such as 0.1% polyoxyethylene (20) sorbitan monooleate has been added can be used as the collection solution, and bacteria can be collected by rubbing the surface of the skin on any part of the body, such as the face, forehead, arms, or legs, with a collection material such as a sterile cotton swab impregnated with this collection solution.

[0020] In this specification, the method for detecting ions in MALDI-MS is not particularly limited, but it is useful to use time-of-flight (TOF-MS) because it ionizes and detects high molecular weight proteins. Linear mode or reflectron mode can be used as appropriate during measurement.

[0021] <Biomarkers for specifically determining the presence or absence of Staphylococcus bacteria> Figures 1 to 6 show biomarker sets (a) to (f) as m / z peak sets for specifically identifying Staphylococcus bacteria. Except for biomarker sets (a) and (f), which consist only of the m / z peak of the first biomarker, biomarker sets (b) to (e) include the m / z peaks of both the first and second biomarkers.

[0022] (First biomarker) The primary biomarkers for each bacterium, along with their origins, are shown in Figures 1 to 6. These peaks are based on whole-gene analysis of Staphylococcus bacteria and all originate from proteins encoded not only in the S10-spc-alpha operon but also in all genes.

[0023] The first biomarker is a protein whose amino acid sequence differs due to a difference in base sequence among proteins encoded by the same gene in the aforementioned operon of Staphylococcus bacteria. In other words, the first biomarker is a protein that has a characteristic m / z that differs in a specific bacterial species from other bacterial species. For example, the first biomarker of S. aureus is a protein that is detected with a high detection rate as a specific m / z in S. aureus, while it has a low detection rate m / z in S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis.

[0024] The first biomarkers are all species-specific within the Staphylococcus genus. However, the detection rate may vary depending on the composition of the specific bacterial strains used for marker setting, the instrument used to perform MALDI-MS, and the method of preparing the mass spectrometry sample, such as the selection of the matrix agent. It is anticipated that the detection rate may appear low in some cases. Nevertheless, since it has been confirmed that the biomarkers are species-specific within the Staphylococcus genus based on genomic information, they can be used as first biomarkers.

[0025] In this specification, detection rate (%) refers to the number of simultaneous replications (n) for a single mass spectrometry sample prepared for, for example, one or more strains of any given bacterial species. For example, for a single mass spectrometry sample, it refers to the percentage of spots (%) in which a peak at a specific m / z was detected among multiple spots, such as 4, 6, 8, or 10, prepared on the same sample plate. Typically, the number of spots is 6, 8, 9, 10, or more.

[0026] Furthermore, in this specification, detecting a peak means detecting m / z with a high detection rate of 80% or more, 85% or more, 90% or more, 95% or more, or 100%. A high detection rate typically means a detection rate of 85% or more, 90% or more, 95% or more, or 100%. Not detecting a peak means detecting a peak with a low detection rate, for example, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 10% or less, or 0%. A low detection rate typically means 25% or less, 20% or less, 10% or less, or 0%.

[0027] By detecting a peak corresponding to the m / z of the first biomarker in the mass spectrum obtained from the test sample, the presence or absence of a specific bacterial species in the Staphylococcus genus can be determined with high accuracy and confidence. The first biomarker consists of one or more species. By using multiple first biomarkers, the presence or absence of a specific bacterial species can be determined with even higher accuracy and confidence. Furthermore, when two or more peaks from these first biomarkers can be detected, the presence or absence of a specific bacterial species can be determined with even higher accuracy and confidence.

[0028] The primary biomarker has been selected in a similar manner for other Staphylococcus species.

[0029] (Second biomarker) The second biomarker is indicated in Figures 2 to 5 with the label BM. For S. epidermidis, the second biomarkers are BM1 and BM2 in Figure 2; for S. capitis, they are BM3 and BM4 in Figure 3; for S. caprae, they are BM5, BM6 and BM7 in Figure 4; and for S. hominis, they are BM8 and BM9 in Figure 5.

[0030] Unlike the first biomarker, the second biomarker does not have a characteristic m / z for specific species of Staphylococcus bacteria on its own. However, when two or more of these proteins are combined, they possess an m / z that allows them to be distinguished from other species of Staphylococcus bacteria.

[0031] The second biomarker, as shown in Figure 2, is a protein that is highly detectable in S. epidermidis, but has low detection rates in other species of Staphylococcus bacteria, both of which are not. The same is true for other bacterial species.

[0032] Furthermore, for S. aureus and S. lugdunensis, a second biomarker has not been established because they can be identified using only the first biomarker.

[0033] By detecting a peak corresponding to the m / z of the second biomarker in the mass spectrum obtained from the test sample, the performance of the first biomarker can be improved, enabling the determination of the presence or absence of a specific bacterial species with high accuracy and certainty.

[0034] For example, the biomarkers (a) to (f) used to determine the presence or absence of species-specific bacterial species can be, for example, the following biomarkers (aa) to (ff). (aa) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13172.22, 9494.66, and 12612.01, respectively. (bb) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 6698.82, 9778.08, 8608.22, and 11733.66. (cc)One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12895.81, 14641.36, 6713.46, 9639.61, and 9671.19. (dd)One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 10691.67, 9889.27, and 8526.30. (ee) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12347.54, 12356.57, 11209.97, 7353.67, and 10195.68, respectively. (ff) One or more peaks selected from the group consisting of peaks corresponding to m / z values ​​of 11420.09, 12473.98, and 12689.50, respectively.

[0035] <Method for determining the first biomarker> This specification provides a method for determining a first biomarker used to determine the presence or absence of a species-specific Staphylococcus bacteria. This method involves determining that the Staphylococcus bacteria are one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis, and that the following first biomarkers (a1) to (f1): are identified in a mass spectrum obtained using matrix-assisted laser desorption / ionization mass spectrometry of proteins derived from the Staphylococcus bacteria. (a1) For S. aureus, the peaks corresponding to m / z are 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42 respectively. (b1) For S. epidermidis, the peaks corresponding to m / z values ​​of 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, and 10159.87 respectively. (c1) For S. capitis, the peaks corresponding to m / z values ​​are 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, and 9815.03, respectively. (d1) For S. caprae, the peaks corresponding to m / z values ​​of 13321.27, 9090.38, 19247.91, 6337.79, and 7756.65 respectively. (e1) For S. hominis, the peaks corresponding to m / z values ​​of 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, and 12047.71 respectively. (f1) For S. lugdunensis, the peaks corresponding to m / z values ​​are 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45, respectively. The method may include a peak detection step of determining whether a peak belonging to the first biomarker of the Staphylococcus bacteria selected from the above is detected. The determination method may further include a step of determining a first biomarker to be used for species-specific determination from the peaks belonging to the first biomarker of the Staphylococcus bacteria based on the peak detection results of the peak detection step.

[0036] According to this determination method, first, a primary biomarker is prepared for one or more species of Staphylococcus bacteria, and then a biomarker to be used for species-specific determination is determined from these primary biomarkers. This specification also provides a method for species-specifically determining the presence or absence of Staphylococcus bacteria using the primary biomarker determined by this biomarker determination method.

[0037] First, we will explain how to determine the peaks that constitute the first biomarkers (a) to (f). We will obtain amino acid sequence information for proteins encoded by the entire genome, including the S10-spc-alpha operon, of each species of Staphylococcus bacteria, such as S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis, and construct a database of theoretical values ​​for these proteins. Meanwhile, we will obtain mass spectra from multiple strains of each Staphylococcus species using MALDI-MS, such as MALDI-TOF MS. We will then correlate the theoretical values ​​in the database with the peaks in the mass spectra to construct an m / z database for each species.

[0038] (1-1) Obtaining amino acid sequence information for proteins encoded by the entire genome, including the S10-spc-alpha operon. First, if the entire genome of a Staphylococcus bacterium has been sequenced, the nucleotide sequence of the protein encoded by the entire genome, including the S10-spc-alpha operon, is obtained. If the entire genome has not been sequenced, primers are designed based on the nucleotide sequence information in the database, and DNA fragments containing the nucleotide sequence are obtained from each bacterium. The nucleotide sequence information of these DNA fragments is then obtained. The obtained protein nucleotide sequence information is converted into an amino acid sequence. For example, EMBOSS Transeq (www.ebi.ac.uk / Tools / emboss / transeq / index.html) can be used to convert from nucleotide sequence to amino acid sequence.

[0039] (1-2) Obtaining the theoretical mass value of protein We will construct an m / z database of all theoretical mass values ​​(theoretical values) calculated from the amino acid sequence information of proteins of the decoded Staphylococcus bacteria. Theoretical values ​​based on amino acid sequence information will be calculated using, for example, the Compute pI / Mw tool (au.expasy.org / tools / pi_tool.html) on the ExPASy proteomics server.

[0040] (1-3) Selection of peaks based on theoretical m / z values ​​for proteins (Preparation of the first biomarker) By comparing the theoretical m / z values ​​of all proteins across different bacterial species, the m / z peaks of proteins specific to each species are prepared as primary biomarkers.

[0041] (2-1) Obtaining mass spectra from each test strain of Staphylococcus bacteria Multiple test strains (with pre-identified species) belonging to each species of the Staphylococcus genus are used as a group of bacterial species for biomarker determination. Each species in this group includes two or more different test strains, for example, four or more, six or more, eight or more, ten or more, twelve or more, or fourteen or more.

[0042] The WC-MS method (Whole Cell MALDI-TOF MS) is used, in which single colonies on agar plates or strains obtained by centrifugation of liquid cultures of each bacterial strain are directly tested. Furthermore, when preparing samples for mass spectrometry for MALDI, the necessary processes for adjusting bacterial concentration, sonication, centrifugation, matrix selection, matrix additives or matrix agents, matrix solvents, and other mass spectral acquisition can be appropriately determined by referring to JP 2023-05787, JP 2022-130577, JP 2020-60444, JP 2020-54299, JP 2020 / 026353, JP 2019-13811, JP 13810, JP 13809, JP 2018-197662, JP 2018 / 131274, JP 2017 / 168743, and patents 168742, 168741, 168740, etc. as appropriate. Furthermore, when preparing mass spectrometry samples of Staphylococcus bacteria, using sinapic acid as a matrix agent is advantageous in that it allows for the acquisition of stable mass spectra. Mass spectrometry samples prepared from each test strain are spotted into multiple wells on a sample plate, for example, 4, 6, or 8 wells.

[0043] In preparing the matrix agent and pre-coating solution, in addition to referring to the above-mentioned literature, an organic solvent can be selected that optimizes the size and shape of the matrix crystals from the viewpoint of obtaining a mass spectrum with a high signal-to-noise ratio. For example, acetonitrile or ether-based organic solvents with excellent volatility can be used as appropriate.

[0044] (2-2) Determination of the first biomarker The peaks of the theoretical m / z values ​​for each protein calculated in (3) are compared and analyzed with the measured m / z values ​​of each peak obtained from the mass spectrum obtained by the WC-MS method in (4). That is, one or more primary biomarkers with m / z values ​​characteristic of the bacterial species are determined based on the theoretical m / z peaks and the measured m / z peaks. For example, when a measured m / z peak corresponding to the theoretical m / z peak is detected, that theoretical m / z marker can be designated as the primary biomarker. More specifically, for example, a protein with an m / z value that has a high detection rate in many strains of a specific bacterial species, and a low detection rate in all other bacterial species (sum of detection rates of strains), can be designated as the primary biomarker. When comparing m / z values, attention should be paid to post-translational modifications such as the N-end rule (cleavage of the N-terminal methionine residue), methylation and acetylation, as well as misreading due to annotation errors.

[0045] The determined primary biomarker (peak) may be part of the primary biomarker (theoretical value), or it may represent the entirety of the primary biomarker (theoretical value). The determined peak may also vary depending on the analytical conditions, etc.

[0046] <Method for determining the second biomarker> This specification provides a method for determining a second biomarker used to specifically determine the presence or absence of Staphylococcus species, wherein the Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis, and the following second biomarkers (b2) to (e2) are determined in a mass spectrum obtained using matrix-assisted laser desorption / ionization mass spectrometry of proteins derived from the Staphylococcus species: (b2) Regarding S. epidermidis, peaks corresponding to 8608.22 and 11733.66 m / z, respectively. (c2) Regarding S. capitis, peaks corresponding to m / z values ​​of 9639.61 and 9671.19, respectively. (d2) For S. caprae, peaks corresponding to 10691.67, 9889.27, and 8526.30 m / z, respectively. (e2) Regarding S. hominis, the peaks corresponding to 7353.67 and 10195.68 m / z, respectively. The process may include a step of determining the aforementioned Staphylococcus bacteria as a second biomarker to be used for species-specific determination.

[0047] According to this determination method, a second biomarker can be determined in addition to the first biomarker. That is, this determination method can also be performed after the determination of the first biomarker. The second biomarker can improve and enhance the accuracy and reliability of the bacteria-specific determination of presence or absence using the first biomarker. The second biomarker is determined and used as appropriate as needed. Unlike the first biomarker, the second biomarker does not have a m / z characteristic of a specific species of Staphylococcus bacteria on its own, but is a protein that has an m / z that makes it distinguishable from other species of Staphylococcus bacteria when two or more species are combined within the Staphylococcus genus. According to this specification, a method for species-specific determination of the presence or absence of Staphylococcus bacteria is also provided, using the determined second biomarker in addition to the determined first biomarker.

[0048] As shown in Figures 2 to 5, the second biomarker is a protein peak that is detected at a high rate in specific bacterial species, but at a low rate in other species of the Staphylococcus genus.

[0049] The second biomarker is determined as follows: When it is difficult to determine the presence or absence of a specific bacterial species using only the first biomarker, two or more second biomarkers capable of determining the presence or absence of a specific bacterial species are selected from the mass spectra of all test strains as described in (2-1) above. That is, two or more peaks with a high detection rate for the specific bacterial species and a low detection rate for other bacterial species, for example, two or three peaks with m / z values, are selected as the second biomarker using statistical processing software such as eMSTAT or ClinPro Tools (data analysis software).

[0050] By combining the first biomarker determined in this way with a second biomarker as needed, and using them as biomarkers for specific bacterial species, it becomes possible to determine the presence or absence of bacterial species belonging to the Staphylococcus genus in a species-specific manner.

[0051] A method disclosed herein for species-specific determination of the presence or absence of Staphylococcus species is to use matrix-assisted laser desorption / ionization mass spectrometry to obtain a mass spectrum of a test sample potentially containing Staphylococcus species, in which the following biomarkers are identified: (a) With respect to S. aureus, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66. (c) Regarding S. capitis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30. (e) With respect to S. hominis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68. (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45. The method may include a peak detection step that determines whether a peak belonging to one or more biomarkers selected from the above is detected. Furthermore, the determination method may include a bacterial species determination step that determines, based on the peak detection result of the peak detection step, whether or not one or more species of Staphylococcus bacteria whose presence is confirmed by the one or more biomarkers are present.

[0052] The presence or absence of species-specific Staphylococcus bacteria is determined by obtaining MALDI-MS mass spectra of test samples that may contain Staphylococcus bacteria, using Staphylococcus bacteria as the target for determination, and comparing the m / z detected in the mass spectra with the m / z of a biomarker set including a first biomarker and, if necessary, a second biomarker for Staphylococcus bacteria. When a peak of the biomarker of a specific bacterial species is detected, typically with a higher detection rate than the biomarker set of other bacterial species, the presence of that specific bacterial species is confirmed. Such determination is performed, for example, using the Staphylococcus bacteria presence / absence determination system 100 shown in Figure 7, following the flow shown in Figure 8.

[0053] As shown in Figure 7, the determination system 100 includes, for example, a MALDI-MS unit 10 that performs MALDI-MS, a determination unit 30, and a display unit 50. The MALDI-MS unit 10 can be composed of various mass spectrometers such as conventional MALDI-TOF MS. The MALDI-MS unit 10 is configured to be able to set a sample plate to which a mass spectrometry sample prepared from a test sample is supplied.

[0054] The determination unit 30 acquires information on the mass spectrum obtained by the MALDI-MS unit 10, associates the m / z values ​​in the mass spectrum with the m / z values ​​of a specific bacterial species' biomarker set, and determines the presence or absence of Staphylococcus species in the test sample. The determination unit 30 is configured as a so-called computer, comprising a processor and memory. The processor executes a process to determine the presence or absence of a specific bacterial species in the Staphylococcus species. That is, the processor is configured to execute an application that compares and associates the measured m / z values ​​in the mass spectrum with the m / z values ​​of a predetermined biomarker set.

[0055] The memory stores the application and a set of biomarkers for specific species of Staphylococcus bacteria. Specifically, the following biomarkers (a) to (f): (a) With respect to S. aureus, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.66. (c) Regarding S. capitis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30. (e) With respect to S. hominis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.68. (f) For S. lugdunensis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45. It can be retained for determining whether it exists or not.

[0056] The bacterial species determination process performed by the processor in the determination unit 30 will be explained in the flowchart below. In addition, the determination unit 30 outputs information necessary for measurement, such as the setting of measurement conditions, to the MALDI-MS unit 10. The determination unit 30 may also be configured as a server.

[0057] The display unit 50 displays the output results of the bacterial species determination process performed by the determination unit 30. The display unit 50 is, for example, a display, a printer, etc. The display unit 50 displays the results of the bacterial species determination process in the determination unit 30, for example, the names of the detected Staphylococcus species.

[0058] In the judgment system 100, the judgment unit 30 may be a server installed to be accessible via a communication network such as the Internet. Furthermore, the memory of the judgment unit 30 that stores the marker set may be a database installed to be accessible via a communication network. The judgment system 100 may also take the form of a judgment device, comprising at least the processors of the MALDI-MS unit 10 and the judgment unit 30, among the MALDI-MS unit 10, the judgment unit 30, and the display unit 50. Such a judgment device may include the display unit 50 as a display.

[0059] Next, we will explain an example of a bacterial species identification process for the Staphylococcus genus, using the determination system 100 shown in Figure 7, with reference to the flow chart shown in Figure 8. Steps S20 onward are the processes executed by the determination system 100.

[0060] First, a sample for mass spectrometry is prepared from the test sample that may contain bacterial species belonging to the genus Staphylococcus (step S10). Next, this mass spectrometry sample is spotted onto a sample plate, and this sample plate is set in the MALDI-MS unit 10, and a mass spectrum is acquired under pre-prepared conditions (step S20). At the same time, the MALDI-MS unit 10 outputs the obtained mass spectrum data to the determination unit 30.

[0061] The processor of the determination unit 30 performs smoothing on the acquired mass spectral data as necessary, and then compares the m / z in the mass spectrum with all the biomarker sets of specific bacterial species stored in memory to perform a bacterial species identification process (an example of a bacterial species identification process disclosed herein) to determine which set of biomarker m / z is present (step S30).

[0062] In the bacterial species identification process, all of the biomarker sets (a) to (f) may be compared, or, if necessary, only one or more biomarker sets may be compared.

[0063] For the determination of Staphylococcus species, the presence or absence of the species associated with each biomarker set can be determined based on the peak detection rate and detection pattern of the first and second biomarkers that constitute each set of species-specific biomarker sets (a) to (f). The species determination process provides information about the types (composition) of species contained in the test sample.

[0064] The presence or absence of a specific bacterial species is determined by whether the measured m / z value in the mass spectrum corresponds to a specific m / z value defined in the biomarker set of that specific bacterial species. Here, the mass precision of the measured value can be set to, for example, 1000 ppm or less, 800 ppm or less, 600 ppm or less, or 400 ppm or less. The smaller the mass precision, the closer the measured m / z value is to the true value, and as a result, the presence or absence of the bacterial species can be determined with high accuracy.

[0065] Furthermore, for determining the identity between the measured m / z value and the m / z value of the biomarker set, and for identification processing based on detection rates, appropriate algorithms such as Strain Solution (registered trademark) can be used as appropriate.

[0066] Furthermore, the memory of the determination unit 30 can be pre-set to correspond to specific colors for each species of Staphylococcus bacteria, in other words, to species-specific biomarkers. In this way, the type of biomarker detected along with the species information can be obtained as color information during the species determination process. By obtaining color information, the composition of the bacterial species can be visually indicated as color, or the name of the bacterial species along with the color, in the patient and / or the collection site from which the test sample originates.

[0067] The processor creates judgment information including the patient's ID, the sampling site, bacterial species information, and, if necessary, color information (step S40).

[0068] Next, the processor outputs to the display unit 50, based on the judgment information, the identified bacterial species along with the patient ID and collection site, along with the bacterial species name, and for each collection site, with a colored box, circle, bar, or other marker (step S50), to a display or printed material, and then terminates this process.

[0069] <Method for evaluating the microbiome of commensal bacteria on human skin> The method for identifying Staphylococcus bacteria disclosed herein can also be used as a method for evaluating the commensal flora of human skin. Staphylococcus bacteria constitute the commensal flora of human skin. Each identification step in the identification method can be used as each evaluation step in this evaluation method.

[0070] Furthermore, this evaluation method can be used to monitor the aforementioned microbiota in relation to human skin diseases. For example, monitoring the skin flora of AD patients over time and / or locally is useful for confirming the onset and pathogenesis of AD.

[0071] As described above, by using the first to sixth biomarker sets disclosed herein, it is possible to identify bacterial species of the Staphylococcus genus with high accuracy and precision. This makes it possible to easily understand, for example, the normal skin microbiota, and can contribute to the treatment and research of skin diseases in humans, such as Alzheimer's disease (AD). [Examples]

[0072] The following examples illustrate the disclosures of this specification in more detail. The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosures of this specification. [Examples]

[0073] (Determining the marker set) In the following section, we determined the marker set using a total of 52 strains from six tested bacterial species (S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, S. lugdunensis), including field isolates.

[0074] (1) Construction of a theoretical value m / z database If genomic information was available for each of these test bacteria, the base sequence of the protein encoded by the entire genome, including the S10-spc-alpha operon, was obtained and converted to an amino acid sequence. If the entire genome had not been sequenced, primers were designed based on the relevant base sequence information in the database, DNA fragments were obtained from each bacterium, and converted to the amino acid sequence of the protein. EMBOSS Transeq (www.ebi.ac.uk / Tools / emboss / transeq / index.html) was used for the conversion from base sequence to amino acid sequence.

[0075] A database of all theoretical mass values ​​(m / z) calculated from the amino acid sequence information of proteins of each test bacterium was constructed. The theoretical values ​​based on amino acid sequence information were calculated using the Compute pI / Mw tool (au.expasy.org / tools / pi_tool.html) on the ExPASy proteomics server. The theoretical biomarkers for each bacterial species are shown in Figures 1-6 along with their derived proteins.

[0076] (2) Acquisition of mass spectrum Sample preparation was performed using the ethanol-formic acid extraction method. Specifically, 10 colonies (approximately 1 mm in diameter) from each strain of the genus Staphylococcus, cultured for 16 hours, were suspended in 200 μL of dH2O and mixed with 600 μL of 99.5% ethanol. After centrifugation (10,000 rpm, 4°C, 2 mins), the supernatant was completely removed, and the precipitate was dried in a centrifugal evaporator (MicroVac) for 5 minutes. The precipitate was dissolved in 15 μL of 70% formic acid, allowed to stand at room temperature for 5 minutes, and then 2 μL of this solution was vigorously mixed with 10 μL of matrix material. 0.8 μL of this mixture was spotted into the wells of a sample plate (8 spots / sample) and dried in a clean bench for 30 minutes. In the pre-coating method, first, 0.8 μl of the pre-coating sinapic acid solution was spotted into the wells of the sample plate. After drying, 0.8 μl of the mixture of bacterial suspension and matrix agent, prepared in the same manner as in the premix method, was spotted in the same way (8 spots / sample), and the plates were dried in a clean bench for 30 minutes. The compositions of the matrix agent and the pre-coating sinapic acid solution were as follows.

[0077] Matrix composition (100 μl) 1.4 μl of trifluoroacetic acid Acetonitrile 50.0 μl Water 48.6μl Synapic acid 25.0 mg

[0078] Composition of the sinapic acid solution for pre-coating (100 μl) Acetonitrile 100.0 μl Synapic acid 25.0 mg

[0079] A Shimadzu AXIMA Performance mass spectrometer was used to perform MALDI-TOF MS, and the mass spectra of the test bacteria samples were measured under the following conditions.

[0080] Mass spectroscopy measurement conditions Laser intensity: 90 Profile 100 Shot 5 Voltage 2680V Range, m / z 2000~30000 Tolerance: 800 ppm (mass accuracy)

[0081] The m / z values ​​obtained from mass spectra (a total of 8 spots) for each strain of all bacterial species were compared with a theoretical m / z database. Specifically, first, a protein specific to each bacterial species was prepared as a theoretical biomarker. Furthermore, for each of the 8 spots of all bacterial strains of each species, it was checked whether the selected theoretical biomarker had a peak at a specific theoretical m / z value. The m / z peak that was detected in all 8 spots of all strains (100% detection rate) and not detected in other bacterial species (0% detection rate of other bacterial species) was selected as the first candidate biomarker. Subsequently, the second and subsequent candidates were determined based on factors such as the percentage of strains with 100% detection rate (the ratio of strains with 100% spot detection rate to the total number of strains of the same bacterial species), the overall detection rate of the bacterial species (the ratio of spots where the peak was detected / the total number of spots of the bacterial species), and the low detection rate of other bacterial species for each bacterial species. From the candidate group, including the first candidate, the m / z peaks of biomarkers derived from species-specific proteins (the first biomarker in this specification) were determined. As a result, the biomarkers selected for each bacterial species are shown in Table 1.

[0082] [Table 1]

[0083] Furthermore, for S. epidermidis, S. capitis, and S. caprae, in addition to the biomarkers already determined, we used eMSTAT® to identify biomarkers with m / z values ​​that distinguish them from other bacterial species (these are the second biomarkers in this specification). Specifically, we identified protein combinations with m / z values ​​that are detected in all of the specific bacterial species, but are not detected at all or are detected only in some of the other bacterial species. For these bacterial species, the specificity of the determined biomarkers was as shown in Figures 2 to 5.

[0084] The origin of the second species-specific biomarker protein determined as described above is unknown, but the combination of species-specific biomarkers is shown in Table 1. The m / z values ​​of these biomarkers were registered in Strain Solution. [Examples]

[0085] In this example, mass spectra were obtained for 14 clinically isolated strains derived from the skin's commensal microbiota obtained in clinical settings, under the same conditions as described above. Species identification was performed using Strain Solution, which registered the m / z values ​​of the new biomarkers determined in Example 1. The results are shown in Figure 9. In this software, species that were detected with a high detection rate and number of detections of the first biomarker were given priority and identified as species confirmed by the first biomarker, with the second biomarker used as a supplementary tool.

[0086] As shown in Figure 9, it was found that all of the following species—S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis—could be correctly identified. Specifically, one strain of S. aureus, five strains of S. epidermidis, two strains of S. capitis, two strains of S. caprae, two strains of S. hominis, and two strains of S. lugdunensis were identified. Furthermore, strains that could not be identified by commercially available identification software (SARAMIS) (one strain of S. caprae and two strains of S. lugdunensis) were also successfully identified.

[0087] For example, in strain J49, the primary biomarker CR009 of S. caprae was detected in 7 / 8 cases and the secondary biomarker or BM007 in 1 / 8 cases. However, one primary biomarker and two secondary biomarkers of S. capitis were detected in 8 / 8 cases, so the presence of S. capitis can be confirmed. Similarly, in strain J53, the primary biomarker CR009 of S. epidermidis was detected in 7 / 8 cases, but two primary biomarkers and one secondary biomarker of S. caprae were detected in 8 / 8 cases, so the presence of S. caprae can be confirmed. In strain J51, the secondary biomarkers of S. epidermidis and S. capitis were detected, but one primary biomarker of S. lugdunensis was detected in 8 / 8 cases, and two other biomarkers were detected in 7 / 8 and 7 / 8 cases respectively, so the presence of S. lugdunensis can be confirmed. Regarding strain J54, while secondary biomarkers for S. epidermidis and S. capitis were detected, the presence of S. lugdunensis can be confirmed as three primary biomarkers for S. lugdunensis were detected in 8 out of 8 samples. Regarding strain J58, while one secondary biomarker for S. epidermidis was detected in 8 out of 8 samples, the presence of S. hominis can be confirmed as one primary biomarker for S. hominis was detected in 8 out of 8 samples. Regarding strain J71, while one primary biomarker for S. caprae was detected in 8 out of 8 samples, the presence of S. capitis can be confirmed as one primary biomarker and one secondary biomarker for S. capitis were detected in 8 out of 8 samples. In the case of strain J72, two secondary biomarkers for S. caprae were detected in 8 out of 8 samples, while one primary biomarker for S. hominis was detected in 6 out of 8 samples, and simultaneously, two secondary biomarkers were detected in 8 out of 8 samples, thus confirming the presence of S. hominis. In the case of strain J102, two primary biomarkers for S. caprae were detected in 8 out of 8 samples, thus confirming the presence of S. caprae.

[0088] As is clear from the results above, the use of the first and second biomarkers allowed for the identification of bacterial species with high accuracy. Furthermore, for example, the combination of the second biomarker was effective for S. capitis and S. hominis.

Claims

1. A method for determining the presence or absence of Staphylococcus species in a species-specific manner, The aforementioned Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. For the test samples potentially containing the Staphylococcus species, the following biomarkers (a) to (f) were observed in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. aureus: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.

42. (b) With respect to S. epidermidis, one or more peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.

66. (c) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. capitis: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.

19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30 m / z, respectively. (e) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. hominis: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.

68. (f) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. lugdunensis: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.

45. A peak detection step that determines whether a peak belonging to one or more biomarkers selected from is detected, A bacterial species determination step, which determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results of the peak detection step, A method that includes [a certain feature].

2. The aforementioned biomarkers (a) to (f) are, respectively, the following biomarkers (aa) to (ff): (aa) One or more peaks selected from the group consisting of peaks corresponding to m / z values ​​of 13172.22, 9494.66, and 12612.01, respectively. (bb) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 6698.82, 9778.08, 8608.22, and 11733.66, respectively. (cc) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12895.81, 14641.36, 6713.46, 9639.61, and 9671.

19. (dd) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 13321.27, 9090.38, 10691.67, 9889.27, and 8526.

30. (ee) One or more peaks selected from the group consisting of peaks corresponding to the m / z values ​​of 12347.54, 12356.57, 11209.97, 7353.67, and 10195.

68. (ff) One or more peaks selected from the group consisting of peaks corresponding to m / z values ​​of 11420.09, 12473.98, and 12689.50, respectively. The method according to claim 1, comprising at least the following:

3. The peak detection step is a step of determining whether peaks belonging to the biomarkers (a), (b), (c), (d), (e), and (f) are detected. The method according to claim 1, wherein the bacterial species determination step is a step of determining whether or not one or more bacterial species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis are present as Staphylococcus bacteria.

4. The peak detection step is a step of determining whether a peak belonging to one or two biomarkers selected from the biomarkers (d) and (f) is detected. The method according to claim 1, wherein the bacterial species determination step is a step of determining whether or not one or two bacterial species selected from S. hominis and S. lugdunensis are present as Staphylococcus bacteria.

5. The method according to claim 1, wherein synapic acid is used as the matrix agent in the measurement of the mass spectrum by the matrix-assisted laser desorption / ionization mass spectrometry method.

6. The method according to claim 1, wherein the test sample includes human skin commensal bacteria.

7. The method according to claim 1, wherein the mass accuracy in the peak detection step is 800 ppm or less.

8. The method according to any one of claims 1 to 7, further comprising the step of displaying determination information together with color information previously assigned to the Staphylococcus bacteria whose presence has been confirmed in the bacterial species determination step.

9. A method for evaluating the microbiome of commensal bacteria on human skin, The aforementioned human skin commensal bacteria are one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. Regarding the test samples that may contain the aforementioned human skin commensal bacteria, the following biomarkers (a) to (f) were observed in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. aureus: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.

42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.

66. (c) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. capitis: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.

19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30 m / z, respectively. (e) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. hominis: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.

68. (f) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. lugdunensis: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.

45. A peak detection step that determines whether a peak belonging to one or more biomarkers selected from is detected, A bacterial species determination step that determines the presence or absence of one or more species of commensal bacteria of human skin based on the peak detection results of the peak detection step, A method that includes [a certain feature].

10. The method according to claim 9, for the purpose of monitoring the microbial flora of human skin for the purpose of diagnosing, elucidating the pathogenesis of, and treating human skin diseases.

11. A system for determining the presence or absence of Staphylococcus species in a species-specific manner, The aforementioned Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. Matrix-assisted laser desorption / ionization mass spectrometry unit, A determination unit that determines the presence or absence of Staphylococcus bacteria based on the mass spectrum of a test sample that may contain Staphylococcus bacteria, obtained in the matrix-assisted laser desorption / ionization mass spectrometry unit, Equipped with, The determination unit determines the following biomarkers (a) to (f) in the mass spectrum: (a) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. aureus: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.

42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.

66. (c) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. capitis: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.

19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to 13321.27, 9090.38, 19247.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30 m / z, respectively. (e) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. hominis: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.

68. (f) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. lugdunensis: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.

45. A system that detects peaks belonging to one or more biomarkers selected from a list, and determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results.

12. A system for determining the presence or absence of Staphylococcus species in a species-specific manner, Matrix-assisted laser desorption / ionization mass spectrometry unit, A determination unit that determines the presence or absence of Staphylococcus bacteria based on the mass spectrum of a test sample potentially containing Staphylococcus bacteria obtained in the matrix-assisted laser desorption / ionization mass spectrometry unit, Equipped with, The determination unit determines the following biomarkers (a) to (f) in the mass spectrum: (a) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. aureus: 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.

42. (b) With respect to S. epidermidis, one or two peaks selected from the group consisting of peaks corresponding to the following m / z values: 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, 10159.87, 8608.22, and 11733.

66. (c) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. capitis: 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, 9815.03, 9639.61, and 9671.

19. (d) With respect to S. caprae, one or more peaks selected from the group consisting of peaks corresponding to 13321.27, 9090.38, 19427.91, 6337.79, 7756.65, 10691.67, 9889.27, and 8526.30 m / z, respectively. (e) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. hominis: 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, 12047.71, 7353.67, and 10195.

68. (f) One or more peaks selected from the group consisting of peaks corresponding to the following m / z values ​​for S. lugdunensis: 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.

45. A system that maintains the above for the purpose of determining existence or non-existence.

13. A method for determining the presence or absence of Staphylococcus species in a species-specific manner, The aforementioned Staphylococcus species is one or more species selected from the group consisting of S. aureus, S. epidermidis, S. capitis, S. caprae, S. hominis, and S. lugdunensis. For the test sample potentially containing the Staphylococcus aureus, the following first biomarkers (a1) to (f1) were observed in the mass spectrum obtained using matrix-assisted laser desorption / ionization mass spectrometry: (a1) Regarding S. aureus, the peaks corresponding to m / z values ​​of 13172.22, 9494.66, 12612.01, 8892.78, 12602.72, 10485.83, 13611.92, 5934.24, 9725.28, and 9592.42 respectively. (b1) For S. epidermidis, the peaks corresponding to m / z values ​​of 6698.82, 9778.08, 13705.84, 18595.84, 15839.52, 15068.34, 6948.14, 9212.47, 14505.70, and 10159.87 respectively. (c1) For S. capitis, peaks corresponding to m / z values ​​of 12895.81, 14641.36, 6713.46, 11700.64, 11905.61, 7833.28, 7631.05, 6386.33, 10705.53, and 9815.03 respectively. (d1) Regarding S. caprae, peaks corresponding to 13321.27, 9090.38, 194247.91, 6337.79, and 7756.65 m / z respectively (e1) For S. hominis, the peaks corresponding to m / z values ​​of 12347.54, 12356.57, 11209.97, 10370.24, 16084.29, 18509.38, 17469.61, 13660.98, 12742.24, and 12047.71 respectively. (f1) For S. lugdunensis, the peaks corresponding to m / z values ​​of 11420.09, 12473.98, 12689.50, 14279.09, 13453.45, 6320.02, 9512.77, 10868.49, 8819.59, and 3843.45 respectively. A peak detection step of determining whether a peak belonging to the biomarker of the Staphylococcus bacteria selected from is detected, A bacterial species determination step, which determines the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection results of the peak detection step, A method that includes [a certain feature].

14. Furthermore, in the mass spectra obtained using matrix-assisted laser desorption / ionization mass spectrometry, the following second biomarkers (b2) to (e2): (b2) Regarding S. epidermidis, the peaks corresponding to 8608.22 and 11733.66 m / z, respectively (c2) Regarding S. capitis, peaks corresponding to m / z values ​​of 9639.61 and 9671.19, respectively. (d2) Regarding S. caprae, peaks corresponding to 10691.67, 9889.27, and 8526.30 m / z, respectively. (e2) Regarding S. hominis, peaks corresponding to 7353.67 and 10195.68 m / z, respectively A peak detection step of determining whether a peak belonging to the biomarker of the Staphylococcus bacteria selected from is detected, The method according to claim 13, further comprising a bacterial species determination step of determining the presence or absence of one or more species of Staphylococcus bacteria based on the peak detection result of the peak detection step.

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