Patent ID: 12196752

DESCRIPTION OF EMBODIMENTS

The target of the method for analyzing a microorganism according to the present invention is a sample about which it is previously known that the microorganisms contained in the sample include a kind of bacteria belonging to one of the three groups of serotypes ofSalmonella. The three groups of serotypes are: the group consisting ofAbonyandPakistanwhich are two serotypes (hereinafter called the first group); the group consisting ofMinnesota, InfantisandBrandenburgwhich are three serotypes (hereinafter called the second group); and the group consisting of Schwarzengrund and Montevideo which are two serotypes (hereinafter called the third group). That is to say, the target of the analysis is (a) a sample about which it is previously known that the sample contains a kind ofSalmonellabacteria belonging to the first group but it is unknown which ofAbonyandPakistanis the serotype of the bacteria, or (b) a sample about which it is previously known that the sample contains a kind ofSalmonellabacteria belonging to the second group but it is unknown which ofMinnesota, InfantisandBrandenburgis the serotype of the bacteria, or (c) a sample about which it is previously known that the sample contains a kind ofSalmonellabacteria belonging to the third group but it is unknown which of Schwarzengrund and Montevideo is the serotype of the bacteria.

That is to say, a method for analyzing a microorganism according to the present invention is a method for analyzing a microorganism including:an identification step for determining which ofAbonyandPakistanwhich are two serotypes ofSalmonellabacteria is contained in a sample which contains eitherAbonyorPakistan, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803 and 15990 as well as any combination of these values. In particular, the value (or values) of the predetermined mass-to-charge ratio or ratios should preferably be selected from the group consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803 and 15990 as well as any combination of these values.

The state of the peak detection forAbonyandPakistanwhich are two serotypes corresponding to the previously mentioned mass-to-charge ratios are shown in Tables 1 and 2. In the present invention, the peaks at the mass-to-charge ratios shown in Table 1 or 2 are used as marker peaks, and which of the two serotypesAbonyandPakistanis the actual serotype is determined by checking the state of the detection of those peaks.

TABLE 1Mass-to-Charge Ratio(m/z)S. AbonyS. Pakistan3028.3○x3119.4○x4165.7○x5487.4x○5506.7x○5924.3○x6010.9○x6095.3○x6238.3○x6261.2x○6369.4○x6719.7○x6724.6x○6933.3x○7272.2x○7453.4○x7480.2x○7588.5○x7858.2x○7902.9○x8053.4x○8129.3x○8329.5○x8460.9○x8536.2○x8546.2x○8633.7○x8687.2○x9669.0x○9911.9○x10956.1○x11498.9x○11505.5○x11847.4○x12276.2○x13365.7○x13372.6x○13435.4○x13444.3x○15713.9x○15803.3○x15990.2○x

TABLE 2Mass-to-Charge Ratio(m/z)S. AbonyS. Pakistan3119.4○x4165.7○x5487.4x○6238.3○x6719.7○x7858.2x○8329.5○x8536.2○x8687.2○x9911.9○x12276.2○x15713.9x○15803.3○x15990.2○x

Another method for analyzing a microorganism according to the present invention is a method for analyzing a microorganism including:an identification step for determining which ofMinnesota, InfantisandBrandenburgwhich are three serotypes ofSalmonellabacteria is contained in a sample which containsMinnesota, InfantisorBrandenburg, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838 as well as any combination of these values. In particular, the value or values of the predetermined mass-to-charge ratio or ratios should preferably be selected from the group consisting of 6483, 7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895, 17713 and 17735 as well as any combination of these values.

The state of the peak detection forMinnesota, InfantisandBrandenburgwhich are three serotypes corresponding to the previously mentioned mass-to-charge ratios are shown in Tables 3 and 4. In the present invention, the peaks at the mass-to-charge ratios shown in Table 3 or 4 are used as marker peaks, and which of the three serotypesMinnesota, InfantisandBrandenburgis the actual serotype is determined by checking the state of the detection of those peaks.

TABLE 3Mass-to-ChargeS. MinnesotaS. InfantisS. BrandenburgRatio (m/z)IIIIIII6094.0xx○○○6483.3○○x○○6688.5○○x○○6719.0xx○xx6872.2○○x○○7857.5○xxxx7939.5xx○x○7947.5○○x○x9322.0xxx○○10667.0xxx○x10990.3xx○xx11807.5○○x○○11821.4xx○xx11847.8○○x○○11856.7xx○xx12209.3xx○xx13366.5○x○○x13376.0x○xx○13406.3xxx○x13445.2○○x○○13475.9x○xx○14882.1xxxx○15715.6○xxxx15803.1xx○xx15878.4xx○x○15894.7○○x○x15991.1x○○○○17713.0○○x○○17734.5xx○xx17812.8○○x○○17835.0xx○xx18972.3xx○○○19127.0xx○xx20765.8○○○xx20837.6xxxx○

TABLE 4Mass-to-ChargeS. MinnesotaS. InfantisS. BrandenburgRatio (m/z)IIIIIII6483.3○○x○○7939.5xx○x○10990.3xx○xx11807.5○○x○○11821.4xx○xx11847.8○○x○○11856.7xx○xx12209.3xx○xx13406.3xxx○x13445.2○○x○○15803.1xx○xx15878.4xx○x○15894.7○○x○x17713.0○○x○○17734.5xx○xx

Another method for analyzing a microorganism according to the present invention is a method for analyzing a microorganism including:an identification step for determining which of Schwarzengrund and Montevideo which are two serotypes ofSalmonellabacteria is contained in a sample which contains either Schwarzengrund or Montevideo, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835 and 19001 as well as any combination of these values. In particular, the value or values of the predetermined mass-to-charge ratio or ratios should preferably be selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12276, 15820 and 15835 as well as any combination of these values.

The state of the peak detection for Schwarzengrund and Montevideo which are two serotypes corresponding to the previously mentioned mass-to-charge ratios are shown in Tables 5 and 6. In the present invention, the peaks at the mass-to-charge ratios shown in Table 5 or 6 are used as marker peaks, and which of the two serotypes Schwarzengrund and Montevideo is the actual serotype is determined by checking the state of the detection of those peaks.

TABLE 5Mass-to-Charge Ratio(m/z)S. SchwarzengrundS. Montevideo5096.4x○6699.2○x6733.1x○6830.1○x9034.4○x12261.9x○12276.1○x13074.0x○15819.5○x15834.5x○19001.0x○

TABLE 6Mass-to-Charge Ratio(m/z)S. SchwarzengrundS. Montevideo5096.4x○6699.2○x6733.1x○6830.1○x9034.4○x12276.1○x15819.5○x15834.5x○

As for the criterion for the peak extraction in the previously described methods for analyzing a microorganism, either the first criterion or second criterion which will be described later may be used, although the criteria are not limited to these examples. Since the second criterion applies stricter conditions to the peak extraction than the first one, it is likely that using the second criterion decreases incorrect determinations. However, since the intensity ratio of the peaks may change depending on the strain or culture conditions, it is preferable to decide the use of the first or second criterion according to the purpose of the determination of the serotype. It is also possible to initially extract peaks according to the first criterion and further extract peaks which satisfy the second criterion among the initially extracted peaks. It is also possible to initially extract peaks according to the second criterion, and if it is still difficult to identify the target serotype, peaks which do not satisfy the second criterion but satisfy the first criterion may subsequently be extracted for identification. The value of the mass-to-charge ratio of each peak should be understood as a rough value and inclusive of a certain range of variation depending on the type of device for mass spectrometry, conditions of the analysis or other factors. Tables 1, 3 and 5 show the states of the peak detection using the first criterion, while Tables 2, 4 and 6 show the states of the peak detection using the second criterion. In each table, the circle indicates that a peak was detected, while the cross indicates that no peak was detected.

(1) First Criterion

In the case of a group consisting of two serotypes, a peak should be extracted (a) if the peak is detected with an S/N value equal to or greater than three in a mass spectrum obtained from a sample containing one of the two serotypes ofSalmonellabacteria, while the same peak in a mass spectrum obtained from a sample containing the other serotype ofSalmonellabacteria is not detected, or is detected with an S/N value not greater than one fifth of the S/N value of the first mass spectrum, or (b) if the peak in one mass spectrum is detected with an S/N value equal to or greater than three, while the same peak in the other mass spectrum is not detected, or is detected with an S/N value not greater than one tenth of the S/N value of the first mass spectrum and is accompanied by, or partially overlaps with, a nearby peak having a close value of the mass-to-charge ratio m/z.

In the case of a group consisting of three serotypes, a peak should be extracted (a) if the peak is detected with an S/N value equal to or greater than three in one of the three mass spectra obtained from three samples respectively containing any one of the three serotypes ofSalmonellabacteria, while the same peak in the two other mass spectra is not detected, or is detected with an S/N value not greater than one fifth of the S/N value of the first mass spectrum, or (b) if the peak in one of the three mass spectra is detected with an S/N value equal to or greater than three, while the same peak on the two other mass spectra is not detected or is detected with an S/N value not greater than one tenth of the S/N value of the first mass spectrum and is accompanied by, or partially overlaps with, a nearby peak having a close value of the mass-to-charge ratio m/z.

(2) Second Criterion

In the case of a group consisting of two serotypes, a peak should be extracted if the peak is detected with an S/N value equal to or greater than ten in a mass spectrum obtained from a sample containing one of the two serotypes ofSalmonellabacteria, while the same peak in a mass spectrum obtained from a sample containing the other serotype ofSalmonellabacteria is not detected, or is detected with an S/N value not greater than one tenth of the S/N value of the first mass spectrum.

In the case of a group consisting of three serotypes, a peak should be extracted if the peak is detected with an S/N value equal to or greater than ten in one of the three mass spectra obtained from three samples respectively containing any one of the three serotypes ofSalmonellabacteria, while the same peak in the two other mass spectra is not detected, or is detected with an S/N value not greater than one tenth of the S/N value of the first mass spectrum.

In any case, a peak extracted based on the second criterion needs to be an isolated peak accompanied by neither a nearby peak nor partially overlapping peak which leads to an incorrect determination.

In this case, the m/z value should be evaluated, for example, with an accuracy of 800 ppm, or preferably 500 ppm. If a plurality of peaks are present within that accuracy, the peak having the closest m/z value should be selected as a marker peak.

The group to which a microorganism contained in a sample belongs among the first through third groups ofSalmonellabacteria can be identified, for example, by using the values of the mass-to-charge ratios of the peaks originating from the 12 kinds of proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF and SodA) disclosed in Non Patent Literature.

In the method for analyzing a microorganism according to the present invention, the identification of one of the serotypes of bacteria in each group may be based on the presence or absence of a peak at a single mass-to-charge ratio. However, the identification accuracy will be improved if the identification of one of the serotypes of bacteria in each group is based on the presence or absence of the peaks at a plurality of mass-to-charge ratios.

As a mass spectrometer to be used for the method for analyzing a microorganism according to the present invention, a mass spectrometer using a matrix assisted laser desorption/ionization (MALDI) method (MALDI-MS) is preferable. As the MALDI-MS, a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) can preferably be used. Since the MALDI-MS has an extremely wide range of measurable mass-to-charge ratios, a mass spectrum suited for an analysis of high-mass molecules, such as the proteins which are constituents of microorganisms, can be acquired.

Next, one embodiment of the microorganism-analyzing system to be used for the method for analyzing a microorganism according to the present invention is described.

FIG.1shows a schematic overall configuration of the microorganism-analyzing system. This system is roughly divided into a mass spectrometry unit10and a microorganism identification unit20. The mass spectrometry unit10includes an ionization unit11configured to ionize molecules or atoms in a sample by matrix assisted laser desorption/ionization (MALDI) and a time-of-flight mass separator (TOF)12configured to separate various ions, ejected from the ionization unit11, according to their mass-to-charge ratios.

The TOF12includes an extraction electrode13configured to extract ions from the ionization unit11and guide them into an ion flight space within the TOF12, and a detector14configured to detect ions which have been mass-separated within the ion flight space.

The microorganism identification unit20is actually a workstation, personal computer or other types of computers, in which a central processing unit (CPU)21, memory22, display unit23(e.g., a liquid crystal display), input unit24(e.g., a keyboard and mouse), and storage unit30consisting of a large-capacity storage (e.g., a hard disk drive or solid state drive) are connected to each other. Stored in the storage unit30are an operating system (OS)31, spectrum creation program32, species determination program33and serotype determination program35(a program according to the present invention), as well as a first database34and second database36. The microorganism identification unit20further includes an interface (T/F)25for controlling a direct connection to an external device as well as a connection with an external device through a local area network (LAN) or other types of networks. Through this interface25, the microorganism identification unit20is connected with the mass spectrometry unit10by a network cable NW (or wireless LAN).

InFIG.1, a spectrum acquirer37, m/z reader38and serotype identifier39are shown, being linked to the serotype determination program35. Each of those components is basically a functional means implemented at the software level by the CPU21executing the serotype determination program35. The serotype determination program35does not need to be an independent program. There is no specific limitation on its form; for example, it may be a built-in function of the species determination program33or that of a program for controlling the mass spectrometry unit10. As the species determination program33, for example, a program configured to identify microorganisms by a conventional fingerprinting method may be used.

In the configuration inFIG.1, the spectrum creation program32, species determination program33, serotype determination program35, first database34and second database36are installed on a terminal device to be operated by users. Those components may be at least partially, or even entirely, installed on a separate device connected with the aforementioned terminal device via a computer network, with the separate device configured to perform the processing by those programs and/or access to those databases according to commands from the terminal device.

The first database34in the storage unit30holds a large number of mass lists related to known microorganisms. The mass list is a list of the mass-to-charge ratios of ions to be detected in a mass spectrometric analysis of a specific kind of microorganic cell. Along with the information of the mass-to-charge ratios, the list additionally includes at least the information of the classifications (family, genus, species, etc.) to which the microorganic cell belongs (classification information). Those mass lists should preferably be prepared based on actual measurement data obtained beforehand by actually performing mass spectrometric analyses of various kinds of microorganic cells using the same method for ionization and mass separation as used in the mass spectrometry unit10.

When the mass lists are to be prepared from the actual measurement data, the peaks which appear within a predetermined mass-to-charge-ratio range are initially extracted from mass spectra obtained as the actual measurement data. Peaks which mainly originate from proteins can be extracted by setting the aforementioned mass-to-charge-ratio range at approximately 4000-30000, while unwanted peaks (noise) can be excluded by extracting each peak whose height (relative intensity) is equal to or higher than a predetermined threshold. A list of the mass-to-charge ratios (m/z) of the extracted peaks is created for each kind of cell and recorded in the first database34along with the aforementioned classification information and other related information. In order to reduce the variation in gene expression due to the culture conditions, the microorganic cells to be used for collecting the actual measurement data should preferably be cultured under previously normalized conditions.

The second database36in the storage unit30holds information concerning marker proteins for identifying, known kinds of microorganisms, by their serotypes which are classifications lower than the species. The information concerning the marker proteins includes at least the information of the mass-to-charge ratios (m/z) of the marker proteins in the known kinds of microorganisms. The second database36may also hold information concerning marker proteins for identifying known kinds of microorganisms by another sub-classification (e.g., subspecies, pathotype or strain) other than the serotype, or by other criteria.

The second database36in the present embodiment contains the values of the mass-to-charge ratios of peaks originating from 12 kinds of marker proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF and SodA) for determining the serotype of a test microorganism in the case where the microorganism is a kind ofSalmonellabacteria (see Non Patent Literature 1), as well as information related to marker proteins for determining the serotype of a test microorganism whose serotype has been identified as belonging to one of the first through third groups ofSalmonellabacteria. Specifically, this information includes the values of the mass-to-charge ratios of predetermined marker peaks for determining (a) which of the two serotypes belonging to the first group is when the serotype of a test microorganism has been identified as belonging to the first group, or (b) which of the three serotypes belonging to the second group is when the serotype of a test microorganism has been identified as belonging to the second group, or (c) which of the two serotypes belonging to the third group is when the serotype of a test microorganism has been identified as belonging to the third group (specifically, the combinations of the values of the mass-to-charge ratios shown in Tables 1-6).

The values of the mass-to-charge ratios of the marker proteins stored in the second database36should preferably be selected by comparing a calculation mass determined by translating the base sequence of each marker protein into an amino-acid sequence, and a mass-to-charge ratio detected by an actual measurement. The base sequences of the marker proteins may be determined by sequencing method, or they may be retrieved from public databases, e.g., a database at NCBI (National Center for Biotechnology Information). For the determination of the calculation mass from the amino-acid sequence, the cutting of the N-terminal methionine residue should preferably be taken into account as a post-translational modification. Specifically, if the second amino-acid residue to the last is Gly, Ala, Ser, Pro, Val, Thr or Cys, the theoretical value should be calculated on the assumption that the N-terminal methionine will be cut. Additionally, since the molecule to be actually observed with a MALDI-TOF MS is in a protonated form, the addition of the proton should also preferably be taken into account in determining the calculation mass (i.e., a theoretical values of the mass-to-charge ratio of ions to be obtained in an analysis of a protein with a MALDI-TOF MS).

Next, a procedure of the analysis of the serotype ofSalmonellabacteria using the previously described microorganism-analyzing system is described with reference to the flowchart.

Initially, the user prepares a sample containing the constituents of a test microorganism, sets the sample in the mass spectrometry unit10, and operates the same unit to perform the mass spectrometric analysis. The sample may be a cell extract, or cell constituents (e.g., ribosomal proteins) collected from the cell extract and purified. Bacterial bodies or cell suspension may also be used as they are.

The spectrum creation program32receives detection signals from the detector14via the interface25, and creates a mass spectrum for the test microorganism based on the detection signals (Step101).

Next, the species determination program33compares the mass spectrum of the test microorganism with the mass lists of known microorganisms recorded in the first database34, and extracts a mass list of a known microorganism having a similar pattern of mass-to-charge ratios to that of the mass spectrum of the test microorganism, such as a mass list including a considerable number of peaks which coincide with those of the mass spectrum of the test microorganism within a predetermined margin of error (Step102). The species determination program33subsequently searches the first database34for the classification information related to the mass list extracted in Step102, to determine the organism species to which the known microorganism corresponding to the mass list belongs (Step103). If the organism species is notSalmonella, the organism species is displayed on the display unit23as the organism species of the test microorganism (Step114), and the analytical processing is completed. If the organism species isSalmonella, the analysis proceeds to the processing by the serotype determination program35. In the case where it has been previously determined by another method that the sample containsSalmonellabacteria, and the analysis can directly proceed to the serotype determination program35without using the species determination program using the mass spectrum.

In the serotype determination program35, the mass spectrum of the test microorganism is compared with the values of the mass-to-charge ratios of the marker proteins recorded in the second database, to identify the serotype of the test microorganism (Step104). Specifically, the serotype determiner 39 initially reads, from the second database36, the values of the mass-to-charge ratios of the peaks originating from the 12 aforementioned kinds of marker proteins (gns, YaiA, YibT, PPI, L25, L21, S8, L17, L15, S7, YciF and SodA). Subsequently, the spectrum acquirer37obtains the mass spectrum of the test microorganism prepared in Step101. Then, for each marker protein, the m/z reader38selects a corresponding peak present in the mass spectrum within a mass-to-charge-ratio range related to the marker protein in the second database36, and determines the serotype based on the values of the mass-to-charge ratios of the selected peaks.

If the determination result indicates that the serotype of the test microorganism is a serotype belonging to the first group (AbonyorPakistan; Step105), the values of the mass-to-charge ratios of the predetermined marker peaks corresponding to the serotypes belonging to the first group are retrieved from the second database (Step109).

If the determination result indicates that the serotype of the test microorganism is a serotype belonging to the second group (Minnesota, InfantisorBrandenburg; Step106), the values of the mass-to-charge ratios of the predetermined marker peaks corresponding to the serotypes belonging to the second group are retrieved from the second database (Step110).

If the determination result indicates that the serotype of the test microorganism is a serotype belonging to the third group (Schwarzengrund or Montevideo; Step107), the values of the mass-to-charge ratios of the predetermined marker peaks corresponding to the serotypes belonging to the third group are retrieved from the second database (Step111).

If the determination result indicates that the serotype of the test microorganism is a serotype which belongs to none of the first through third groups (Step108), the serotype of the test microorganism is shown on the display unit23as a serotype which is none of the serotypes belonging to the first through third groups (Step114).

After the serotype has been identified as belonging to one of the first through third groups, and the values of the mass-to-charge ratios of the predetermined marker peaks corresponding to the serotypes belonging to each group have been retrieved, the presence or absence of a peak is checked for each of the mass-to-charge-ratio ranges related to those values of the mass-to-charge ratios and stored in the second database36(Step112). Based on the state of the presence or absence of the peaks, the serotype of the test microorganism is determined (Step113), and the determination is shown on the display unit23as the identification result for the test microorganism (Step114).

Example

Hereinafter described is an experiment conducted to prove the effect of the method for analyzing a microorganism according to the present embodiment. It should be noted that the following descriptions are merely illustrative and do not limit the present invention.

1. Culturing ofSalmonellaBacteria

A total of seven kinds ofSalmonellabacteria (Salmonella enterica), i.e., the two kinds ofSalmonellabacteria belonging to the first group, the three kinds ofSalmonellabacteria belonging to the second group, and the two kinds ofSalmonellabacteria belonging to the third group, were cultured at 37 degrees Celsius for 20 hours using LB agar.

The kinds ofSalmonellabacteria belonging to the first through third groups are as follows.

(1) First Group

S. abony, NBRC100797S. pakistan, GTC09493
(2) Second GroupS. minnesota, NBRC15182S. infantis, ATCC BAA-1675S. brandenburg, jfrlSe1402-3
(3) Third GroupS. schwarzengrund, HyogoSO12004S. montevideo, jfrlSe1409-6
2. Preparation of Matrix Solutions

The following two kinds of matrix solutions were prepared.

(2-1) Sinapine acid (SA) as the matrix substance was dissolved in ethanol to obtain a matrix solution (saturated solution) with an SA content of 25 mg/mL. This matrix solution is hereinafter called “SA-1”.

(2-2) SA, methylene diphosphonate (MDPNA) and decyl-β-D-maltopyranoside (DMP) as a surfactant were dissolved in an aqueous solution with an acetonitrile (ACN) content of 50% and trifluoroacetic acid (TFA) content of 0.6% to obtain a matrix solution with an SA content of 25 mg/mL, MDPNA content of 1%, and DMP content of 1 mM. This matrix solution is hereinafter called “SA-2”.

The SA used for the matrix solutions SA-1 and SA-2 was a product of FUJIFILM Wako Pure Chemical Corporation. The MDPNA and DMP were products of Sigma-Aldrich Japan LLC.

3. Preparation of Matrix-Microorganism Suspension

(3-1) From each of the 7 kinds ofSalmonellabacteria cultured on LB agar, approximately 1 mg of sample was collected using a microbalance and put in a tube. The matrix solution SA-2 was added to the sample in the tube to obtain a solution with a bacteria concentration of 1 mg/0.075 mL (1×107CFU/μL), and this solution was suspended with a needle.
(3-2) Ultrasonic vibrations were applied to the tube for one minute. The obtained suspension was centrifuged (at 12000 rpm for 5 minutes) to obtain a centrifugation supernatant.
4. Analysis with MALDI-MS
(4-1) The matrix solution SA-1 was dropped into the wells on a MALDI sample plate, at 0.5 μL per one well (precoating).
(4-2) Subsequently, the centrifugation supernatant was dropped into the wells precoated with the matrix solution SA-1, at 1 μL per one well, and was let to naturally dry.
(4-3) The MALDI sample plate obtained in (4-2) was set in a MALDI-MS (AXIMA Performance, manufactured by Shimadzu Corporation), and the measurement was performed in a linear mode (positive ion mode). All measurement data were acquired by a raster analysis. Raster analysis is an automatic measurement function provided in the previously mentioned mass spectrometer. In this technique, the sample in each well of the sample plate is irradiated with a predetermined number of laser shots at a predetermined number of points, to acquire mass spectrum data.
5. Extraction of Peaks

A self-calibration ofSalmonellabacteria was applied to the measurement data (more specifically, a calibration process was performed using, as internal standards, some peaks that have already been assigned to specificSalmonellabacteria), and useful peaks for the identification of the serotype were extracted from the obtained mass spectrum. The values of the mass-to-charge ratios of the peaks originating from the 12 kinds of marker proteins shown in Non Patent Literature 1 were read from the extracted peaks, and the serotype ofSalmonellabacteria to which the microorganism contained in the sample belonged was determined from the read values.

In the determination of the serotype using the 12 kinds of marker proteins, when it was determined that the microorganism was eitherAbonyorPakistanwhich are two serotypes ofSalmonellabacteria, the state of the peak detection on the obtained mass spectrum was checked at one or more values of the mass-to-charge ratios selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803 and 15990.

In the determination of the serotype using the 12 kinds of marker proteins, when it was determined that the microorganism was one ofMinnesota, InfantisandBrandenburgwhich are three serotypes ofSalmonellabacteria, the state of the peak detection on the obtained mass spectrum was checked at one or more values of the mass-to-charge ratios selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13367, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838.

In the determination of the serotype using the 12 kinds of marker proteins, when it was determined that the microorganism was either Schwarzengrund or Montevideo which are two serotypes ofSalmonellabacteria, the state of the peak detection on the obtained mass spectrum was checked at one or more values of the mass-to-charge ratios selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835 and 19001. The m/z values were evaluated with an accuracy of 800 ppm, or preferably 500 ppm. When a plurality of peaks were present within that accuracy, the peak having the closest m/z value was selected as the marker peak.

6. Results

(1) First Group

The state of the peak detection at the previously listed mass-to-charge ratios (m/z values) was checked on the mass spectra obtained from two samples which respectively contained one of the two kinds ofSalmonellabacteria belonging to the first group (S. abonyandS. pakistan). The state of the detection was as shown in Tables 1 and 2. In Tables 1 and 2, the circle indicates that the marker peak at the mass-to-charge ratio concerned was detected, while the cross indicates that the marker peak was not detected.

FIG.3shows the mass spectra, where each mass spectrum covers an m/z range including 4165.7, 5487.4, 6238.3, 7858.2, 8329.5, 9911.9 or 15713.9 among the m/z values of the peaks which satisfy the second criterion (i.e., the m/z values shown in Table 2).FIG.4shows the mass spectra, where each mass spectrum covers an m/z range including 5506.7, 6261.2, 7588.5, 9669.0, 11847.4, 13444.3 or 15990.2 among the m/z values of the peaks which satisfy the first criterion but do not satisfy the second criterion. In bothFIG.3andFIG.4, the upper shows the mass spectrum forS. abony, and the lower shows the mass spectrum forS. pakistan. The arrows inFIGS.3and4indicate the extracted peaks.

As shown inFIGS.3and4, each of the peaks extracted based on the first and second criteria is only present in either the mass spectrum forS. abonyor the mass spectrum forS. pakistan. Therefore, which ofS. abonyandS. pakistanis the serotype of theSalmonellabacteria contained in the sample can be determined by the presence or absence of the peak. That is to say, the serotype of theSalmonellabacteria can be identified. In particular, the use the peaks which satisfy the second criterion enables a more correct identification of the serotype since those peaks have higher S/N ratios than the peaks which satisfy only the first criterion, and since those peaks have no nearby peaks. It should be noted that any one of the peaks at the 42 mass-to-charge ratios (m/z values) shown in Table 1 can be solely used as a marker peak for identifying the serotype of theSalmonellabacteria contained in a sample, although using peaks at a plurality of mass-to-charge ratios (m/z values) as marker peaks enables a more accurate identification of the serotype of theSalmonellabacteria contained in a sample.

Thus, which of the two serotypes ofSalmonellabacteria belonging to the first group (S. abonyandS. pakistan) was the actual serotype could be determined by checking the state of the peak detection at the mass-to-charge ratios (m/z values) in Table 1 or 2.

(2) Second Group

The state of the peak detection at the previously listed mass-to-charge ratios (m/z values) was checked on the mass spectra obtained from three samples which respectively contained any one of the three kinds ofSalmonellabacteria belonging to the second group (S. minnesota, S. infantisandS. brandenburg). The state of the detection was as shown in Tables 3 and 4.

FIG.5shows the mass spectra, where each mass spectrum covers an m/z range including 7939.5, 13406.3 or 15878.4/15894.7 among the m/z values of the peaks which satisfy the second criterion (i.e., the m/z values shown in Table 4).

FIG.6shows the mass spectra, where each mass spectrum covers an m/z range including 7947.5, 9322.0 or 13366.5/13376.0 among the mass-to-charge ratios (m/z values) of the peaks which satisfy the first criterion but do not satisfy the second criterion. In bothFIG.5andFIG.6, the upper shows the mass spectrum obtained for the sample containingS. minnesota, the middle shows the mass spectrum obtained for the sample containingS. infantis, and the lower shows the mass spectrum obtained for the sample containingS. brandenburg. The arrows inFIGS.5and6indicate the extracted peaks.

As shown inFIGS.5and6, each of the peaks extracted based on the first and second criteria is present or absent on only one of the mass spectra obtained from the three samples which respectively containedS. minnesota, S. infantisandS. brandenburg. Therefore, which ofS. minnesota, S. infantisandS. brandenburgis the serotype of theSalmonellabacteria contained in the sample can be determined by the presence or absence of the peak. That is to say, the serotype of theSalmonellabacteria can be identified. In particular, the use the peaks which satisfy the second criterion enables a more correct identification of the serotype since those peaks have higher S/N ratios than the peaks which satisfy only the first criterion, and since those peaks have no nearby peaks. It should be noted that any one of the peaks at the 35 mass-to-charge ratios (m/z values) shown in Table 3 can be solely used as a marker peak for identifying the serotype of theSalmonellabacteria contained in a sample, although using peaks at a plurality of mass-to-charge ratios (m/z values) as marker peaks enables a more accurate identification of the serotype of theSalmonellabacteria contained in a sample.

Thus, which of the three serotypes ofSalmonellabacteria belonging to the second group (S. minnesota, S. infantisandS. brandenburg) was the actual serotype could be determined by checking the state of the peak detection at the mass-to-charge ratios (m/z values) in Table 3 or 4.

(3) Third Group

The state of the peak detection at the previously listed mass-to-charge ratios (m/z values) was checked on the mass spectra obtained from two samples which respectively contained one of the two kinds ofSalmonellabacteria belonging to the third group (S. schwarzengrundandS. montevideo). The state of the detection was as shown inFIGS.7and8.

FIG.7shows the mass spectra, where each mass spectrum covers an m/z range including 5096.4, 6830.8, 12276.1 or 15834.5 among the m/z values of the peaks which satisfy the second criterion (i.e., the m/z values shown in Table 6).

FIG.8shows the mass spectra, where each mass spectrum covers an m/z range including 12261.9, 13074.0 or 19001.0 among the m/z values of the peaks which satisfy the first criterion but do not satisfy the second criterion. In bothFIG.7andFIG.8, the upper shows the mass spectrum obtained for the sample containingS. schwarzengrund, and the lower shows the mass spectrum obtained for the sample containingS. montevideo. The arrows inFIGS.7and8indicate the extracted peaks.

As shown inFIGS.7and8, each of the peaks extracted based on the first and second criteria is only present on either the mass spectrum of the sample containingS. schwarzengrundor the mass spectrum of the sample containingS. montevideo. Therefore, which ofS. schwarzengrundandS. montevideois the serotype of theSalmonellabacteria contained in the sample can be determined by the presence or absence of the peak. That is to say, the serotype of theSalmonellabacteria can be identified. In particular, the use the peaks which satisfy the second criterion enables a more correct identification of the serotype since those peaks have higher S/N ratios than the peaks which satisfy only the first criterion, and since those peaks have no nearby peaks. It should be noted that any one of the peaks at the 11 mass-to-charge ratio (m/z) values shown in Table 5 can be solely used as a marker peak for identifying the serotype of theSalmonellabacteria contained in a sample, although using peaks at a plurality of mass-to-charge ratio (m/z) values as marker peaks enables a more accurate identification of the serotype of theSalmonellabacteria contained in a sample.

Thus, which of the two serotypes ofSalmonellabacteria belonging to the third group (S. schwarzengrundandS. montevideo) was the actual serotype could be determined by checking the state of the peak detection at the mass-to-charge ratios (m/z values) in Table 5 or 6.

Modes of Invention

A person skilled in the art can understand that the previously described illustrative embodiments are specific examples of the following modes.

(Clause 1) A method for analyzing a microorganism according to one mode is a method for analyzing a microorganism including:an identification step for determining which ofAbonyandPakistanwhich are two serotypes ofSalmonellabacteria is contained in a sample which contains eitherAbonyorPakistan, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 3028, 3119, 4166, 5487, 5507, 5924, 6011, 6095, 6238, 6261, 6369, 6720, 6725, 6933, 7272, 7453, 7480, 7589, 7858, 7903, 8053, 8129, 8330, 8461, 8536, 8546, 8634, 8687, 9669, 9912, 10956, 11499, 11506, 11847, 12276, 13366, 13373, 13435, 13444, 15714, 15803 and 15990 as well as any combination of these values.

(Clause 2) In the method for analyzing a microorganism described in Clause 1, the value or values of the predetermined mass-to-charge ratio or ratios are specifically selected from the group consisting of 3119, 4166, 5487, 6238, 6720, 7858, 8330, 8536, 8687, 9912, 12276, 15714, 15803 and 15990 as well as any combination of these values.

According to the method for analyzing a microorganism described in Clause 1, if it is previously known that the serotype of theSalmonellabacteria contained in a sample is one of the two serotypes (AbonyandPakistan), it is possible to determine which ofAbonyandPakistanis the actual serotype. According to the method for analyzing a microorganism described in Clause 2, which ofAbonyandPakistanis the actual serotype can be more correctly determined.

(Clause 3) A method for analyzing a microorganism according to another mode is a method for analyzing a microorganism including:an identification step for determining which ofMinnesota, InfantisandBrandenburgwhich are three serotypes ofSalmonellabacteria is contained in a sample which containsMinnesota, InfantisorBrandenburg, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 6094, 6483, 6689, 6719, 6872, 7858, 7940, 7948, 9322, 10667, 10990, 11808, 11821, 11848, 11857, 12209, 13366, 13376, 13406, 13445, 13476, 14882, 15716, 15803, 15878, 15895, 15991, 17713, 17735, 17813, 17835, 18972, 19127, 20766 and 20838 as well as any combination of these values.

(Clause 4) In the method for analyzing a microorganism described in Clause 3, the value or values of the predetermined mass-to-charge ratio or ratios are specifically selected from the group consisting of 6483, 7940, 10990, 11808, 11821, 11848, 11857, 12209, 13406, 13445, 15803, 15878, 15895, 17713 and 17735.

According to the method for analyzing a microorganism described in Clause 3, if it is previously known that the serotype of theSalmonellabacteria contained in a sample is one of the three serotypes (Minnesota, InfantisandBrandenburg), it is possible to determine which ofMinnesota, InfantisandBrandenburgis the actual serotype. According to the method for analyzing a microorganism described in Clause 4, which ofMinnesota, InfantisandBrandenburgis the actual serotype can be more correctly determined.

(Clause 5) A method for analyzing a microorganism according to still another mode is a method for analyzing a microorganism including:an identification step for determining which of Schwarzengrund and Montevideo which are two serotypes ofSalmonellabacteria is contained in a sample which contains either Schwarzengrund or Montevideo, based on the presence or absence of a peak (or peaks) at a predetermined mass-to-charge ratio (or ratios) in a mass spectrum obtained by a mass spectrometric analysis of the sample,where the value or values of the predetermined mass-to-charge ratio or ratios are selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12262, 12276, 13074, 15820, 15835 and 19001 as well as any combination of these values.

(Clause 6) In the method for analyzing a microorganism described in Clause 5, the value or values of the predetermined mass-to-charge ratio or ratios are specifically selected from the group consisting of 5096, 6699, 6733, 6830, 9034, 12276, 15820 and 15835 as well as any combination of these values.

According to the method for analyzing a microorganism described in Clause 5, if it is previously known that the serotype of theSalmonellabacteria contained in a sample is one of the two serotypes (Schwarzengrund and Montevideo), it is possible to determine which of Schwarzengrund and Montevideo is the actual serotype. According to the method for analyzing a microorganism described in Clause 6, which of Schwarzengrund and Montevideo is the actual serotype can be more correctly determined.

(Clause 7) The second mode is a program for making a computer execute the step described in one of Clauses 1-6.