Method for preparing o-antigen sugar chain sample and method for determining e. coli o111

Abstract

To prepare an O antigen sugar chain sample capable of observing clear mass spectrum peaks at equal intervals reflecting the structure of an O antigen sugar chain by a simple operation without using a harmful reagent.SOLUTION: A suspension of bacterial cells of a test bacterium, which is a Gram-negative bacterium, or a solution containing a lipopolysaccharide released from the bacterial cells is prepared, the pH of the suspension or the solution is adjusted to less than 1.6, and then a predetermined treatment including a step of heating the suspension or the solution at 97 °C or more for 13 minutes or more is performed to prepare a sample containing an O antigen sugar chain of the test bacterium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an O-antigen sugar chain sample for serotyping Gram-negative bacteria by mass spectrometry, and a method for distinguishing Escherichia coli O111 using mass spectrometry. [Background technology]

[0002] Enterohemorrhagic Escherichia coli (EHEC) is a pathogenic E. coli bacterium that produces a toxin called verotoxin. Infection with EHEC can cause severe abdominal pain, bloody stools, and hemolytic uremic syndrome (HUS), and can even lead to death. Therefore, when food poisoning or other illnesses occur, it is important to quickly identify whether the pathogen is EHEC. Serotyping, a method based on structural differences in the O-antigen glycans of Gram-negative bacteria, is widely used to identify EHEC. The O-antigen glycan is a partial structure contained in lipopolysaccharide (LPS), a component of the outer membrane of the cell wall of Gram-negative bacteria. It consists of a basic structure consisting of several monosaccharides repeated approximately 40 times. This basic structure is highly diverse even among bacteria of the same species, and the serotype of E. coli is determined based on differences in the basic structure of the O-antigen glycan.

[0003] Conventionally, serotyping tests have used techniques that utilize antigen-antibody reactions. Currently, approximately 180 serotypes of E. coli are known, and approximately 180 types of antibodies are required to identify the serotype of E. coli collected from a sample. However, preparing and comprehensively testing all of the approximately 180 antibodies requires enormous costs and effort, making it unrealistic to target all serotypes. Therefore, in serotyping tests for enterohemorrhagic E. coli infections using antigen-antibody reactions, the determination is limited to a few serotypes, such as O26, O103, O111, O121, O145, O165, and O157, taking into account the frequency of serotypes observed in EHEC.

[0004] However, since serotypes other than those listed above have been reported to produce the pathogenic substance verotoxin, a general-purpose method capable of distinguishing between multiple serotypes using the same reagents and techniques was needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 7365007 [Non-patent literature]

[0006] [Non-Patent Document 1] Shogo Urakami, Hiroshi Hinou, "Development of a rapid identification method for O antigens using MALDI-TOF MS", Journal of the Mass Spectrometry Society of Japan, December 15, 2022, Vol. 70, No. 4, pp. 237-240, [Online], [Retrieved July 29, 2024], Internet<URL:https: / / www.jstage.jst.go.jp / article / massspec / 70 / 4 / 70_S22-60 / _article / -char / ja> [Non-patent document 2] Urakami, S., Hinou, H., "MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria", Sci Rep 14, 12719 (2024), [Online], [Retrieved July 29, 2024], Internet<URL:https: / / doi.org / 10.1038 / s41598-024-62729-1> [Non-patent document 3] Urakami, S., Hinou, H., "MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria" supplementary information, Sci Rep 14, 12719 (2024), [Online], [Retrieved July 29, 2024], Internet <URL: chrome-extension: / / efaidnbmnnnibpcajpcglclefindmkaj / https: / / static-content.springer.com / esm / art%3A10.1038%2Fs41598-024-62729-1 / MediaObjects / 41598_2024_62729_MOESM1_ESM.pdf> Summary of the Invention [Problem to be solved by the invention]

[0007] As a result of intensive research, some of the present inventors discovered that by analyzing LPS isolated from Gram-negative bacteria using mass spectrometry, differences in the structure of the O-antigen glycans contained in the LPS can be observed as a mass spectrum in which successive, equally spaced peaks appear, and that this can be applied to distinguish the serotype of the Gram-negative bacteria, and thus filed a patent application (see Patent Document 1).

[0008] Furthermore, Non-Patent Documents 1 to 3 describe that samples for serotyping based on O-antigen glycan structures using mass spectrometry (hereinafter sometimes referred to as "O-antigen glycan samples") could be easily prepared by suspending colonies of Escherichia coli O157, O1, and O6 in a 100 mM aqueous hydrochloric acid solution and heating at 90°C for 10 minutes.

[0009] However, for some EHEC serotypes (e.g., O111), mass spectrometry of samples prepared by the methods described in Non-Patent Documents 1 to 3 failed to observe clear mass spectrum peaks with evenly spaced peaks that reflect the structure of the O-antigen glycan. On the other hand, when mass spectrometry was performed on LPS extracted from E. coli of the same serotype by phenol extraction, clear mass spectrum peaks with evenly spaced peaks were observed (see Patent Document 1). However, this method has problems such as the use of phenol, a highly toxic reagent, and the complicated extraction procedure.

[0010] The present invention was made in consideration of the above, and its purpose is to prepare an O-antigen glycan sample that allows observation of clear, evenly spaced peaks reflecting the structure of the O-antigen glycan, using simple procedures without using harmful reagents such as phenol, and to enable the identification of E. coli O111 by mass spectrometry, which has previously been difficult. [Means for solving the problem]

[0011] The method for preparing an O antigen glycan sample according to the present invention, which has been achieved in order to solve the above problems, comprises: preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; A method for preparing a sample containing an O-antigen sugar chain of the test bacterium by subjecting the suspension or the solution to a predetermined treatment, comprising: The predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6, and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer.

[0012] In addition, the method for distinguishing Escherichia coli O111 according to the present invention, which has been achieved in order to solve the above problems, comprises: A method for distinguishing Escherichia coli O111 based on the structure of an O-antigen sugar chain having a repeating structure with a basic unit consisting of multiple monosaccharides, comprising: preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; a sample containing an O-antigen sugar chain of the test bacterium is prepared by subjecting the suspension or solution to a predetermined treatment; subjecting the sample to mass spectrometry; A method for identifying the test bacterium as O111 when the interval between successive equally spaced peaks appearing in the mass spectrum obtained by the mass spectrometry matches, within a predetermined error range, the m / z of an ion generated from a partial basic structure formed by removing the colitose side chain from the basic structure of the O-antigen sugar chain of E. coli O111, The predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6, and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer. [Effects of the Invention]

[0013] According to the method for preparing an O-antigen glycan sample of the present invention having the above-described configuration, an O-antigen glycan sample can be prepared that can be observed in a mass spectrum with clear, evenly spaced peaks that reflect the structure of the O-antigen glycan, without using harmful reagents such as phenol, and through simple operations.

[0014] Furthermore, the method for distinguishing E. coli O111 according to the present invention having the above-mentioned configuration enables the discrimination of E. coli O111 by mass spectrometry, which has been difficult to do in the past. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a flowchart showing an example of a method for preparing an O-antigen glycan sample according to one embodiment of the present invention. [Figure 2] 10 is a flowchart showing another example of the method for preparing an O-antigen glycan sample according to the embodiment. [Figure 3] 1 is a flowchart showing a method for distinguishing Escherichia coli O111 according to another embodiment of the present invention. [Figure 4] FIG. 1 shows a mass spectrum of Test Example 1. [Figure 5] FIG. 1 shows the mass spectrum of Test Example 2. [Figure 6] FIG. 10 shows the mass spectrum of Test Example 3. [Figure 7] FIG. 10 shows the mass spectrum of Test Example 4. [Figure 8] FIG. 10 shows the mass spectrum of Test Example 5. [Figure 9] FIG. 10 shows the mass spectrum of Test Example 6. [Figure 10] FIG. 10 shows the mass spectrum of Test Example 7. [Figure 11] FIG. 10 shows the mass spectrum of Test Example 8. [Figure 12] FIG. 10 shows the mass spectrum of Test Example 9. [Figure 13] 1 shows the mass spectra of Test Examples 10 to 12, with the lower row being Test Example 10, the middle row being Test Example 11, and the upper row being Test Example 12. DETAILED DESCRIPTION OF THE INVENTION

[0016] A method for preparing an O-antigen sugar chain sample according to one embodiment of the present invention will be described with reference to the flowchart of FIG.

[0017] First, a suspension of Gram-negative bacteria (hereinafter referred to as test bacteria) to be subjected to serotyping by mass spectrometry is heated to 75°C or higher (preferably 80 to 100°C, more preferably 90 to 100°C) (step 11). The suspension can be obtained, for example, by washing test bacteria cells isolated from a specimen or the like and cultured with purified water, and then suspending the cells in a neutral liquid (e.g., purified water). The heating time in step 11 is not particularly limited, but can be, for example, 3 to 30 minutes (preferably 5 to 10 minutes).

[0018] Lipopolysaccharide (hereinafter referred to as LPS) has three substructures: lipid, core sugar chain, and O-antigen sugar chain. In living bacteria, the lipid portion of LPS is embedded in the outer membrane of the cell wall, which is located at the outermost periphery of the bacteria. In step 11, as described above, the test bacteria are heated in a neutral liquid to lyse them, thereby destroying the outer membrane of the cell wall and liberating LPS from the bacteria, allowing it to diffuse into the liquid.

[0019] In step 11, LPS is released from the bacterial cells by heating the bacterial cell suspension under neutral conditions, but the method for releasing LPS is not limited to this. For example, LPS can be released by destroying the outer membrane of the cell wall by adding an acidic substance to the bacterial cell suspension, adding fine particles to the bacterial cell suspension and stirring, freezing and thawing the bacterial cells, or treating the bacterial cells with an enzyme that destroys cell membranes (lysozyme).

[0020] Next, the liquid containing free LPS obtained above (hereinafter sometimes referred to as the "free LPS-containing liquid") is acidified by adding an acidic substance (step 12). The pH of the liquid after the addition of the acidic substance is adjusted to less than 1.6 (preferably 1.3 or less, more preferably 1.1 or less). The liquid is then heated at 97°C or higher (preferably 98°C or higher, more preferably 99°C or higher) for 13 minutes or more (preferably 15 minutes or more) (step 13). While there is no upper limit to the heating time, it is recommended that it be 30 minutes or less (preferably 25 minutes or less, more preferably 20 minutes or less) in consideration of work efficiency. The heating should be performed within a range that does not cause the liquid to disappear due to evaporation.

[0021] The acidic substance may be an inorganic acid, an organic acid, or both. Examples of the inorganic acid include hydrochloric acid, nitric acid, and sulfuric acid. Examples of the organic acid include trifluoroacetic acid (TFA), trichloroacetic acid (TCA), and formic acid. However, the types of inorganic and organic acids are not limited to these.

[0022] After the heating in step 13 is completed, the liquid is centrifuged and the supernatant is collected (step 14). In this manner, the lipid portion and core glycan portion are removed from the LPS composed of the above three partial structures, and the O-antigen glycan portion can be obtained.

[0023] The supernatant obtained as described above, i.e., the O-antigen glycan sample prepared by the method of this embodiment, is subjected to analysis by a mass spectrometer. This makes it possible to observe a clear repeating pattern reflecting the repeating structure of the O-antigen glycan even for serotype Gram-negative bacteria, for which a clear repeating pattern of mass spectrum peaks could not be observed when samples were prepared by conventional methods (i.e., the methods described in Non-Patent Documents 1 to 3).

[0024] The mass spectrometer is not particularly limited, but since glycans are the subject of analysis, it is desirable to use one that ionizes samples using a soft ionization method. A "soft" ionization method refers to an ionization method that is less likely to decompose high-molecular-weight compounds. Examples of soft ionization methods include matrix-assisted laser desorption / ionization (MALDI), electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI). When ionizing a sample using MALDI, the O-antigen glycan sample is mixed with a matrix material for MALDI before being subjected to mass spectrometry.

[0025] As described above, the method for preparing an O-antigen glycan sample according to this embodiment is particularly useful in that it enables the observation of clear repeating patterns for microorganisms of serotypes for which it was difficult to observe repeating patterns using conventional sample preparation methods (e.g., E. coli O111). However, when the method is applied to microorganisms of serotypes for which clear repeating patterns could be observed using conventional methods (e.g., E. coli O157, O103, or O26), clear repeating patterns can also be observed. In other words, the method for preparing an O-antigen glycan sample according to this embodiment is a versatile technique that enables the observation of clear repeating patterns of mass spectral peaks for Gram-negative bacteria of a wide range of serotypes.

[0026] In the above embodiment, an O-antigen glycan sample was prepared through a two-step heating process: a test bacterium cell suspension was heated at 75°C or higher under neutral conditions to lyse the bacterium and liberate LPS (step 11), and the resulting free LPS-containing liquid was then heated at 97°C or higher for 13 minutes or longer at a pH of less than 1.6 (step 13) to generate O-antigen glycans from the LPS (cleaving the lipid and core glycan portions from the LPS). This two-step heating method allows lysis under relatively mild conditions, thereby suppressing the incorporation of contaminants (e.g., protein hydrolysates). However, the present invention is not limited to this; both the release of LPS by lysis and the generation of O-antigen glycans from the LPS can also be performed in a single heating step. This allows sample preparation to be completed in a shorter time than with two-step heating. The method for preparing an O-antigen glycan sample in this case is described with reference to the flowchart in Figure 2.

[0027] First, test bacterial cells are suspended in an acidic aqueous solution with a pH of less than 1.6 (preferably pH 1.3 or less, more preferably pH 1.1 or less) (step 21). By suspending test bacterial cells in an acidic aqueous solution, the outer cell wall membrane of the bacterial cells can be disrupted without heating, liberating LPS. The acidic aqueous solution can be an aqueous solution of either an inorganic acid or an organic acid, or both. Next, the liquid obtained in step 21 is heated at 97°C or higher (preferably 98°C or higher, more preferably 99°C or higher) for 13 minutes or longer (preferably 15 minutes or longer) (step 22). Again, the upper limit of the heating time is not particularly limited, but in consideration of work efficiency, it is desirable to set it to 30 minutes or shorter (preferably 25 minutes or shorter, more preferably 20 minutes or shorter). After heating in step 22 is completed, the liquid is centrifuged and the supernatant is recovered (step 23). This allows the lipid and core glycan moieties to be removed from the LPS liberated in step 21, thereby obtaining the O-antigen glycan moiety.

[0028] Even when O-antigen glycan samples prepared through such a single heating step are subjected to mass spectrometry, mass spectral peaks showing a clear repeating pattern can be observed, just as with O-antigen glycan samples prepared through two heating steps.

[0029] The interval between the repeating patterns of the above-mentioned mass spectrum peaks reflects the basic structure of the O-antigen sugar chain, and the serotype of the test bacterium can be determined based on this repeating interval.

[0030] The basic structure of the O-antigen sugar chain of E. coli O111 is a linear backbone consisting of three monosaccharides to which two side chains (specifically, colitose) (hereinafter referred to as colitose side chains) are attached. However, when an O-antigen sugar chain sample is prepared from E. coli O111 using the method according to this embodiment (i.e., the method shown in the flowchart of FIG. 1 or FIG. 2), it has been found that the two colitose side chains are detached from the basic structure during the preparation process. A method for distinguishing E. coli O111 according to one embodiment of the present invention distinguishes E. coli O111 based on this finding.

[0031] The method for distinguishing E. coli O111 according to this embodiment will be described with reference to the flowchart in Figure 3. First, a test bacterium, which is a Gram-negative bacterium, is treated by the method shown in the flowchart in Figure 1 or Figure 2 to prepare an O-antigen sugar chain sample of the test bacterium (step 31).

[0032] Next, the O-antigen glycan sample is subjected to mass analysis (step 32). The mass spectrometer used here is not particularly limited, but it is desirable to use one that ionizes the sample by the soft ionization method described above.

[0033] Next, it is determined whether a repeating pattern of equally spaced peaks is observed in the mass spectrum obtained by mass spectrometry of the O-antigen glycan sample (Step 33). Here, "repeated pattern" refers to a group of peaks consisting of one or more peaks repeatedly observed at equally spaced intervals in the mass spectrum. If no repeating pattern is observed in the mass spectrum (No in Step 33), it is determined that the serotype of the test bacterium cannot be determined, and serotype determination is terminated.

[0034] On the other hand, if a repeating pattern is observed in the mass spectrum (Yes in step 33), it is determined whether the peak spacing between the repeating patterns matches, within a predetermined error range, the m / z of the ions derived from the basic structure of the O-antigen glycan of E. coli O111 minus each colitose side chain (hereinafter referred to as the partial basic structure) (step 34). Here, the "peak spacing between repeating patterns" refers to the spacing between corresponding peaks in adjacent peak groups (e.g., the peaks with the smallest or largest m / z in each peak group) among the peak groups repeatedly observed in the mass spectrum. Note that the magnitude of the allowable error varies depending on the mass accuracy, but is preferably within approximately ±500 ppm.

[0035] The m / z of the ion derived from the partial basic structure is determined by converting the calculated molecular weight of the partial basic structure into the theoretical value (theoretical m / z) of the m / z of the ion derived from the partial basic structure. The calculated molecular weight of the partial basic structure is the sum of the molecular weights of the three monosaccharides constituting the partial basic structure minus the molecular weight of the water molecule lost due to the glycosidic bond. Note that the conversion of the calculated molecular weight of the partial basic structure into the theoretical m / z can be achieved by converting the calculated molecular weight into the theoretical m / z of the ion obtained when ionized by the ionization method used in mass spectrometry in step 32. For example, when a biological sample is analyzed by MALDI-MS, the molecular weight is mainly [M+H] + (M is a molecule, H is a hydrogen atom), [MH] - , or [M+Na] +It is known that molecular weight-related ions such as (Na is a sodium atom) are detected. Therefore, it is relatively easy to convert the calculated molecular weight as described above into the theoretical m / z of the ion.

[0036] If it is determined that the repeat interval of the repeat pattern matches the m / z of the ion derived from the partial basic structure of E. coli O111 within a predetermined error range (Yes in step 34), the test bacterium is determined to be E. coli O111 (step 35).

[0037] On the other hand, if it is determined in step 34 that the repetition interval does not match the m / z of the ion derived from the partial basic structure of E. coli O111 within a predetermined error range (No in this step), the test bacterium is determined not to be E. coli O111, and the serotype determination is terminated. Alternatively, after the result in step 34 is No, the repetition interval may be compared with the m / z of the ion derived from the basic structure of the O-antigen sugar chain in Gram-negative bacteria of another serotype to determine whether the test bacterium is a Gram-negative bacterium of the other serotype. [Example]

[0038] A test example conducted to confirm the effects of the present invention will be described below. In this test example, Escherichia coli (RIMD 0509470), serotype O111 standard strain obtained from the Research Institute for Microbial Diseases, Osaka University, was used as the test bacterium.

[0039] The test bacteria were cultured on LB agar medium (1% peptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) at 37°C for 18 hours. Subsequently, the bacterial cells from five colonies on the medium were suspended in 200 μL of 100 mM hydrochloric acid (HCl) and 25 mM NaCl aqueous solution, and 100 μL of the resulting suspension (hereinafter referred to as the acidic suspension) was dispensed into two 1.5 mL microtubes. One of the two microtubes was heated at 90°C for 10 minutes using a dry block, and the acidic suspension after heating was centrifuged (16,000 g, 90 seconds) to recover the supernatant. Hereinafter, this supernatant is referred to as the O-antigen glycan sample according to Test Example 1. The other of the two microtubes was heated at 100°C for 20 minutes using a dry block, and the acidic suspension after heating was centrifuged in the same manner as above to recover the supernatant. Hereinafter, this supernatant is referred to as the O antigen sugar chain sample according to Test Example 2.

[0040] Subsequently, mass spectrometry was performed on each of the O-antigen glycan samples according to Test Examples 1 and 2 obtained above. Specifically, first, 1 μL of a 1.25 mg / mL 2,5-dihydroxybenzoic acid solution (solvent: 50% acetonitrile, 50% purified water) was dropped into each of two wells of a target plate as a matrix solution for MALDI and allowed to dry. Then, 1 μL of the O-antigen glycan sample according to Test Example 1 was dropped into one of the two wells and allowed to dry. Furthermore, 1 μL of the O-antigen glycan sample according to Test Example 2 was dropped into the other of the two wells and allowed to dry. The target plate was then set in a mass spectrometer (Shimadzu Corporation, MALDI-8030), and mass spectrometry was performed in positive linear mode.

[0041] The mass spectrum obtained for the O-antigen glycan sample of Test Example 1 (hereinafter referred to as the mass spectrum of Test Example 1) is shown in Figure 4. The mass spectrum obtained for the O-antigen glycan sample of Test Example 2 (hereinafter referred to as the mass spectrum of Test Example 2) is shown in Figure 5.

[0042] The mass spectrum of Test Example 2 (Figure 5) shows a series of equally spaced peaks starting from m / z 567.6 with an m / z interval of approximately 528, indicating that the O-antigen glycan was observed. Furthermore, the m / z interval between peaks such as m / z 1,420.3, 1,948.1, and 2,475.6 is also approximately 528. These peak intervals (m / z 528) correspond to the dehydrated molecular weight of the partial basic structure in which the colitose side chain is removed from the repeating unit (i.e., basic structure) of the O111 O-antigen glycan.

[0043] On the other hand, the above-mentioned equally spaced peaks were not observed in the mass spectrum of Test Example 1 (Figure 4). Although peaks presumably derived from the O-antigen glycan, such as m / z 2,476.4, were also observed, it was extremely difficult to determine that the peak spacing was m / z 528. The reason why the equally spaced peaks reflecting the structure of the O-antigen glycan of E. coli O111 could not be clearly observed may be that the lipid moiety and core glycan moiety from LPS were insufficiently removed, or that impurities contained in the sample were not hydrolyzed sufficiently.

[0044] As described above, the O-antigen glycan sample of Test Example 1 (i.e., the O-antigen glycan sample prepared by the conventional methods described in Non-Patent Documents 1 to 3) did not allow for the production of a mass spectrum that clearly reflected the evenly spaced peaks that reflect the O-antigen glycan structure of E. coli O111, whereas the O-antigen glycan sample of Test Example 2 (i.e., the O-antigen glycan sample prepared by the method of the present invention) was confirmed to provide a mass spectrum of a quality that allows for the serotype discrimination of O111. [Example]

[0045] Next, a test example in which the heating time of the acidic suspension was changed will be described. In this test, the same serotype O111 standard strain of Escherichia coli (RIMD 0509470) as in Test Example 1 was used as the test bacterium.

[0046] First, the test bacteria were cultured on the same LB agar medium as above, and the cultured bacteria were suspended in 500 μL of a [100 mM HCl, 25 mM NaCl] solution to an OD of 2 or greater. Then, 125 μL of the resulting acidic suspension was dispensed into each of four microtubes, and each microtube was heated at 100°C using a dry block. One of the four microtubes was heated for 10 minutes, another for 15 minutes, another for 20 minutes, and the remaining for 30 minutes. The acidic suspension in each microtube after heating was then centrifuged (16,000 g, 90 seconds), and the supernatant was recovered. Hereinafter, the supernatants recovered from each microtube are referred to as the O-antigen glycan sample according to Test Example 3, the O-antigen glycan sample according to Test Example 4, the O-antigen glycan sample according to Test Example 5, and the O-antigen glycan sample according to Test Example 6, in order of shortest heating time.

[0047] Mass spectra obtained by performing mass spectrometry on each of the O-antigen glycan samples from Test Examples 3 to 6 in the same manner as in Example 1 are shown in Figures 6 to 9. As can be seen from these figures, in the mass spectrum from Test Example 4 (heating time: 15 minutes) (Figure 7), the mass spectrum from Test Example 5 (heating time: 20 minutes) (Figure 8), and the mass spectrum from Test Example 6 (heating time: 30 minutes) (Figure 9), peaks appearing at intervals of about m / z 528 can be easily found by focusing on peaks with high intensity. On the other hand, in the mass spectrum from Test Example 3 (heating time: 10 minutes) (Figure 6), there are many contaminating peaks, so it is not easy to find the peaks appearing at intervals of about m / z 528 unless one refers to the mass spectra from Test Examples 4, 5, or 6. The peaks appearing in the mass spectrum in Figure 6 with a peak interval of approximately m / z 130 are presumed to be derived from colitose side chains that remained uncleaved from the basic structure of the O-antigen glycan of E. coli O111. The random presence of these uncleaved colitose side chains in the basic structure is thought to be the cause of the complexity of the mass spectrum in the figure. [Example]

[0048] Next, a test example using serotype O26 E. coli will be described. Here, the O26 standard strain E. coli (RIMD 0509624) was used as the test bacterium, and an O-antigen glycan sample was prepared by the conventional method and by the method of the present invention.

[0049] Specifically, the test bacteria were first cultured on the same LB agar medium as above, and the cultured cells were suspended in 250 μL of a [100 mM HCl, 25 mM NaCl] solution to an OD of 2 or greater. Subsequently, 125 μL of the resulting acidic suspension was dispensed into each of two microtubes. The two microtubes were then heated using a dry block. One of the two microtubes was heated at 90°C for 10 minutes, and the other was heated at 100°C for 20 minutes. The acidic suspension in each microtube after heating was then centrifuged (16,000 g, 90 seconds), and the supernatant was recovered from each microtube. Hereinafter, the supernatant recovered from the microtube heated at 90°C for 10 minutes, i.e., the O antigen glycan sample prepared by the conventional method, is referred to as the O antigen glycan sample according to Test Example 7. The supernatant recovered from the microtube heated at 100°C for 20 minutes, i.e., the O antigen glycan sample prepared by the method according to the present invention, is referred to as the O antigen glycan sample according to Test Example 8.

[0050] Mass spectra obtained by performing mass spectrometry on each of the O-antigen glycan samples from Test Examples 7 and 8 described above in the same manner as in Example 1 are shown in Figures 10 and 11. As can be seen from these figures, for O26, whether the sample was prepared by the conventional method (Test Example 7; Figure 10) or the method of the present invention (Test Example 8; Figure 11), consecutive peaks appearing at intervals around m / z 537 were observed, demonstrating that the O-antigen glycan was observed. This peak interval (m / z 537) coincides with the dehydrated molecular weight of the repeating unit (i.e., the basic structure) of the O-antigen glycan of O26. [Example]

[0051] Next, a test example will be described in which 1% trifluoroacetic acid (TFA) was used as the acidic substance in preparing the acidic suspension instead of the 100 mM hydrochloric acid. The test bacteria used here were the same as in Test Example 1, serotype O111 standard strain Escherichia coli (RIMD 0509470).

[0052] First, the test bacteria were cultured on the same LB agar medium as above. The cultured bacteria were then suspended in 125 μL of a [1% TFA, 25 mM NaCl] solution to an OD of 2 or higher, thereby preparing an acidic suspension of the bacteria. The acidic suspension was then heated at 100°C for 20 minutes using a dry block, and then centrifuged (16,000 g, 90 seconds) to recover the supernatant. The supernatant thus obtained is hereinafter referred to as the O-antigen glycan sample according to Test Example 9.

[0053] The mass spectrum obtained by performing mass spectrometry on the O-antigen glycan sample of Test Example 9 in the same manner as in Example 1 is shown in Figure 12. As shown in the figure, it was confirmed that clear, evenly spaced peaks reflecting the structure of the O-antigen glycan could be observed even when TFA was used as the acidic substance. [Example]

[0054] Next, a test example in which the pH of the above-mentioned acidic suspension was changed will be described. Note that, as in Test Example 1, the test bacteria used here was the serotype O111 standard strain of Escherichia coli (RIMD 0509470).

[0055] First, the test bacteria were cultured on the same LB agar medium as described above. Then, the cultured bacteria were suspended in 125 μL each of a [100 mM HCl, 25 mM NaCl] solution (hereinafter referred to as the first solution), a [50 mM HCl, 25 mM NaCl] solution (hereinafter referred to as the second solution), and a [25 mM HCl, 25 mM NaCl] solution (hereinafter referred to as the third solution) to an OD of 2 or higher to prepare acidic suspensions of the bacteria. The pH of the first solution was approximately 1, the pH of the second solution was approximately 1.3, and the pH of the third solution was approximately 1.6. Each acidic suspension was then heated at 100°C for 20 minutes using a dry block, followed by centrifugation (16,000 g, 90 seconds) to recover the supernatant. In the above, of these supernatants, the one obtained using the first solution will be referred to as the O antigen glycan sample of Test Example 10, the one obtained using the second solution will be referred to as the O antigen glycan sample of Test Example 11, and the one obtained using the third solution will be referred to as the O antigen glycan sample of Test Example 12.

[0056] Figure 13 shows mass spectra obtained by performing mass spectrometry on each of the O-antigen glycan samples from Test Examples 10 to 12 in the same manner as in Example 1. As shown in the figure, peaks appearing at intervals of approximately m / z 528 (peaks at m / z approximately 568, 1096, 1624, 2151, and 2678) were observed in the mass spectrum of the O-antigen glycan sample from Test Example 10 (lower part of Figure 13). On the other hand, only three such peaks were observed in the mass spectrum of the O-antigen glycan sample from Test Example 11 (middle part of Figure 13), and no such peaks were observed in the mass spectrum of the O-antigen glycan sample from Test Example 12 (upper part of Figure 13).

[0057] [Aspect] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0058] (Item 1) A method for preparing an O-antigen glycan sample according to one aspect of the present invention comprises: preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; A method for preparing a sample containing an O-antigen sugar chain of the test bacterium by subjecting the suspension or the solution to a predetermined treatment, comprising: The predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6, and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer.

[0059] (2) The method for distinguishing Escherichia coli O111 according to paragraph 2 is as follows: A method for distinguishing Escherichia coli O111 based on the structure of an O-antigen sugar chain having a repeating structure with a basic unit consisting of multiple monosaccharides, comprising: preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; a sample containing an O-antigen sugar chain of the test bacterium is prepared by subjecting the suspension or solution to a predetermined treatment; subjecting the sample to mass spectrometry; A method for identifying the test bacterium as O111 when the interval between successive equally spaced peaks appearing in the mass spectrum obtained by the mass spectrometry matches, within a predetermined error range, the m / z of an ion generated from a partial basic structure formed by removing the colitose side chain from the basic structure of the O-antigen sugar chain of E. coli O111, The predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6, and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer.

Claims

1. preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; A method for preparing a sample containing an O antigen sugar chain of the test bacterium by subjecting the suspension or the solution to a predetermined treatment, comprising: the predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6 and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer; Method for preparing an O-antigen glycan sample.

2. A method for distinguishing Escherichia coli O111 based on the structure of an O-antigen sugar chain having a repeating structure in which a basic structure consisting of multiple monosaccharides is used as one unit, comprising: preparing a suspension of test bacteria that are gram-negative bacteria or a solution containing lipopolysaccharide liberated from the test bacteria; a sample containing an O-antigen sugar chain of the test bacterium is prepared by subjecting the suspension or solution to a predetermined treatment; subjecting the sample to mass spectrometry; A method for identifying the test bacterium as O111 when the interval between successive equally spaced peaks appearing in the mass spectrum obtained by the mass spectrometry matches, within a predetermined error range, the m / z of an ion generated from a partial basic structure formed by removing the colitose side chain from the basic structure of the O-antigen sugar chain of E. coli O111, the predetermined treatment includes a step of adjusting the pH of the suspension or the solution to less than 1.6 and then heating the suspension or the solution at 97°C or higher for 13 minutes or longer; Method for distinguishing Escherichia coli O111.

Images