JP6018808B2 - Microorganism test method and test system - Google Patents

Description

  The present invention relates to a microorganism testing method and a microorganism testing system.

  Infections to humans and animals caused by microorganisms are always a major problem in health care and medical care or livestock. In the case of human medicine, a drug is administered to a patient to treat an infection caused by a microorganism. To determine the drug to be administered, two types of information are required: the causative microorganism species and the sensitivity of the causative microorganism to the drug. Therefore, in the clinical microbial test, two tests, an identification test and a drug sensitivity test, are performed. When considering the treatment of patients, it is necessary to obtain the test results as soon as possible, but mainly the time required for culture becomes the rate limit, and the primary culture from the reception of clinical specimens (microbe separation and culture process) It takes 2-3 days to obtain the test result after secondary culture (identification by biochemical property test and sensitivity test for drugs).

  In the biochemical property test, a microbial species is identified by detecting each reaction activity with respect to a metabolic reaction characteristic of each microbial species. However, generally used optical measurement methods have low sensitivity, and it takes 6 to 7 hours to identify them. Depending on the bacterial species, a longer culture time may be required. In recent years, Matrix Assisted Laser Desorption / Ionization-Time of Flight (MALDI-TOF) mass spectrometer is used to derive from major proteins (eg, ribosomal proteins) contained in microorganisms A method of identifying a microbial species by a spectral pattern of mass spectrometry is used. The working time is within 30 minutes because no secondary culture is required for the identification test. However, this method does not provide information on drug sensitivity.

As a method for detecting the growth of microorganisms based on metabolism in a microbiological examination or microbiological experiment, there is a method of measuring the concentration of CO ₂ produced by pH change or respiration of a culture solution (Patent Document 1). Also in this regard, long-term culture is required to detect changes in pH, or changes in pressure or infrared absorption due to CO ₂ . In addition, only a change in pH or the production of CO ₂ provides little information.

In the drug sensitivity test, the sensitivity of microorganisms to various drugs is tested by monitoring the growth of microorganisms in the presence of the drug. There are two main drug susceptibility tests: diffusion and dilution. For example, in the standard method for bacterial drug susceptibility testing established by the Japanese Chemotherapy Society, a 2-fold dilution series (0.06-128 μg / mL) of a drug is prepared using Mueller-Hinton medium, and dispensed into a 96-well plate. Bacteria are planted in each well so that the bacterial cell concentration is 5 × 10 ⁵ CFU / mL, and cultured at 35 ± 1 ° C. for 18 to 24 hours. After incubation, the growth of bacteria in each well was visually determined by the turbidity of the medium. Among the wells in which no bacterial growth was observed, the concentration of the lowest drug concentration was determined as the minimum inhibitory concentration (Minimum Inhibitory Concentration (MIC). Although the method for minimizing the liquid dilution method is adopted in the automated bacteria test apparatus, it takes at least about 8 hours until a result is obtained. Attempts have also been made to conduct drug susceptibility testing of microorganisms by decoding the sequences of genes involved in drug resistance. However, although this method has the merit that secondary culture is not required, known drug resistance can be detected, but unknown drug resistance cannot be detected because the gene involved is unknown. Furthermore, even though information on drug resistance can be obtained, information on drug sensitivity such as which drug shows the growth inhibitory effect at which concentration cannot be obtained.

Patent No. 3010565

  As described above, in conventional microbiological tests, there are methods for judging the growth of microorganisms such as bacteria and fungi by visually and optically determining the turbidity change of the culture solution, and by determining the pH change of the culture solution, etc. It is used, and sufficient growth of microorganisms is required before obtaining a test result. For this purpose, half-day to one-day culture is required, but depending on the microorganism species, further longer culture may be required. In particular, regarding the drug sensitivity test, there is no technology that becomes the decisive factor for speeding up, and speeding up by shortening the culture time is required. Moreover, the microorganism identification test and the drug sensitivity test are performed separately, and time and labor are required.

  Thus, the present invention provides a method and system for rapidly testing microorganisms. Also provided are methods and systems for obtaining detailed information about, for example, microbial species and drug susceptibility.

  In order to solve the above problems, for example, the method and system described in the claims are adopted. Specifically, the present invention cultivates a microorganism in a medium to which a drug and / or a metabolic substrate is added, performs mass spectrometry on a metabolite produced by the microorganism contained in the medium and discharged outside the cell, A microorganism testing method characterized by identifying the type of the microorganism based on the type and amount of the metabolite determined by the mass spectrometry, or determining the influence of the drug and / or metabolic substrate on the microorganism About. The present invention also includes a culture unit including a culture vessel for culturing microorganisms, including a medium to which a drug and / or a metabolic substrate is added, a mass spectrometer for analyzing components of the medium, and the mass Analysis for identifying the type of the microorganism or determining the influence of the drug and / or metabolic substrate on the microorganism based on the type and amount of the metabolite derived from the microorganism in the medium determined by the analyzer And a microorganism testing system.

  The present invention provides a method and system for rapid microbial testing. For example, in drug sensitivity tests, metabolites in microbial cultures are detected by mass spectrometry, metabolism and growth of microorganisms are detected with high sensitivity, and the effects of drugs are determined with high sensitivity in a short period of culture. It is possible. Further, for example, since the components in the medium are measured instead of measuring the metabolites in the microbial cells, it is possible to omit the processing steps such as cell disruption and perform the measurement quickly. Furthermore, in the present invention, a microorganism identification test is performed simultaneously with the drug sensitivity test, and information on the microorganism species is provided together with information on the drug sensitivity.

  Problems, configurations, and effects other than those described above will become apparent from the following description of embodiments.

It is a flowchart which shows an example of a microbe inspection method. It is a schematic diagram showing an example of a microbe inspection system. It is the schematic which shows the structural example of a microbe test | inspection system. It is a graph which shows the influence on the growth by the chloramphenicol density | concentration in colon_bacillus | E._coli, and the pyruvate density | concentration and lactic acid density | concentration in a culture medium. It is a graph which shows the influence on the growth by the concentration of ampicillin in Staphylococcus aureus, and the pyruvic acid concentration and lactic acid concentration in a culture medium. It is a figure which shows the outline | summary of the result determination protocol in a drug sensitivity test. It is a table | surface which shows the result of a drug sensitivity test. It is a graph which shows the result of a drug sensitivity test. It is an interface figure of the result display screen of a drug sensitivity test. In E. coli culture using a stable isotope-labeled ¹³ C ₆ glucose is a graph showing the time course of pyruvate concentration, lactate concentration in the medium. It is a graph which shows the mass spectrometry spectrum by sugar substrate metabolism in colon_bacillus | E._coli. It is a graph which shows the mass spectrometry spectrum by sugar substrate metabolism in S. aureus. It is an interface figure which displays the sugar substrate metabolism pattern in colon_bacillus | E._coli and Staphylococcus aureus.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the contents of the system configuration and processing operation described below are merely examples, and various forms that can be realized by combining or replacing the embodiments with known techniques are also included in the forms of the present invention.

  The environment inhabited by microbial species varies, and culture conditions and metabolism differ accordingly. Among them, a glucose metabolic system can be mentioned as a basic metabolic pathway common to many microbial species. The glucose metabolic system is a central pathway of energy metabolism and intermediary metabolism, and consists of a glycolysis system and a citrate cycle, followed by an oxidative phosphorylation pathway in an electron transport system. The glycolytic system is a metabolic pathway common to many cells, and two pyruvate molecules are synthesized from one glucose molecule taken into the cell. At that time, 2 molecules of adenosine triphosphate (ATP) are produced as energy. The pyruvic acid produced by the glycolysis is further metabolized and becomes lactic acid by ethanol or anaerobic glycolysis by alcohol fermentation under anaerobic conditions. On the other hand, under aerobic conditions, pyruvate enters the citrate cycle via acetyl-CoA, and in the subsequent oxidative phosphorylation pathway of the electron transport system, 30 molecules of ATP are produced per glucose molecule. The glucose metabolism system is not only the center of energy metabolism of microorganisms, but also plays an important role in substance metabolism.

Therefore, the glucose metabolic system serves as an indicator of the metabolic activity of microorganisms, and it is possible to detect proliferation due to cell division by measuring the activity. In the prior art, the metabolite itself is not measured, but the change in pH or turbidity of the culture medium caused by the metabolite, or CO ₂ production caused by respiration is detected. In the present invention, mass spectrometry of the metabolite itself enables rapid and highly sensitive examination, and detailed information on the identification of microbial species, the influence of drugs on microorganisms, and the like can be obtained.

  In the present invention, not a metabolite in a microbial cell but a metabolite excreted extracellularly and accumulated in a medium is measured. Measurement of metabolites and constituent components of microorganism cells by mass spectrometry is widely performed for research purposes and the like, but for that purpose, treatment for disrupting the cells is required. Cell disruption includes enzyme treatment, surfactant treatment, mechanical treatment such as ultrasound and pressure, and freeze-thawing. In any method, operations and equipment for crushing cells are required, which increases the time and process for inspection. In addition, in the case of enzyme treatment or treatment with a surfactant, the enzyme or surfactant added for the treatment may hinder subsequent analysis of metabolites by mass spectrometry. Furthermore, the extraction efficiency of the components in the cells varies depending on the degree of cell disruption. Therefore, measuring the metabolite excreted in the medium of the culture solution, not the metabolite in the microbial cell, leads to rapid and simple testing methods.

Therefore, in the microorganism testing method according to the present invention,
Culturing microorganisms in a medium supplemented with drugs and / or metabolic substrates;
Performing mass spectrometry on metabolites produced by the microorganisms contained in the medium and excreted extracellularly;
The type of the microorganism is identified based on the type and amount of the metabolite determined by the mass spectrometry.

Moreover, in the microorganism testing method according to the present invention,
Culturing microorganisms in a medium supplemented with drugs and / or metabolic substrates;
Performing mass spectrometry on metabolites produced by the microorganisms contained in the medium and excreted extracellularly;
Based on the type and amount of the metabolite determined by the mass spectrometry, the influence of the drug and / or metabolic substrate on the microorganism is determined.

  In the present invention, all the microorganisms composed of bacteria and fungi are to be tested, but in particular, microorganisms such as pathogenic bacteria and pathogenic fungi that are to be tested in a hospital laboratory or the like. Pathogenic bacteria include Staphylococcus (specific examples: Staphylococcus aureus, Staphylococcus epidermidis), Micrococcus (Streptococcus) (specific examples: Streptococcus pyogenes, Pneumonia Cocci), Enterococcus (specific examples: Enterococcus faecium, Enterococcus faecalis), Neisseria (specific examples: Phosphorus, Neisseria meningitidis), Moraxella, Escherichia ( Escherichia (specifically, E. coli), Shigella (specifically, Shigella), Salmonella (specifically, Salmonella typhi, Paratyphi A, Enterococcus), Citrobacter ( Citrobacter), Klebsiella (specifically, Klebsiella pneumoniae), Enterobacter, Serratia (Serratia) (specifically, Serratia marcescens), Proteus (Proteus), Providencia, Morganella, Yersinia (specifically, Plasmodium), Vibrio (Specific examples include Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus), Aeromonas, Pseudomonas (specifically, Pseudomonas), Acinetobacter (specifical) , Acinetobacter baumannii), Alcaligenes, Agrobacterium, Flavobacterium, Haemophilus (specifically, Haemophilus), Pasteurella, Francisella (Francisella), Bordetella (Bordetella) (specifically, Bordetella pertussis), Eikenella, Brucella, Streptobacillus, Actinobacillus, Legionella (specific examples Legionella Pneumophila), Bacillus (specific examples: Bacillus anthracis, Bacillus cereus), Corynebacterium (specific examples: Diphtheria), Lactobacillus, Listeria, Eri Erysipelothrix, Nocardia, Actinomyces, Clostridium (specifically, Clostridium perfringens), Bacteroides (specifically, Bacteroides) Fraziris, Fusobacte rium), Mycobacterium (specifically, tuberculosis), Campylobacter, Helicobacter (specifically, Helicobacter), Spirillum, Treponema Bacteria such as Borrelia, Leptospira, and Mycoplasma (specifically, Mycoplasma pneumonia) are included, but microorganisms other than these can also be tested.

  The microorganism to be examined can be prepared by separating and culturing from a sample before carrying out the examination. Alternatively, since the species is not specifically identified at the time of testing and it may be difficult to separate and culture, the microorganism to be tested may be the sample itself. Such a sample can be any sample that contains or is suspected of containing microorganisms. For example, the sample includes a biological sample derived from a living body (for example, human), such as a sample of blood, sputum, tissue, cells, etc., and a sample derived from the environment, such as a sample of seawater, river water, industrial water, soil, etc. Is mentioned. The liquid sample can be used as it is, or diluted or concentrated with a solvent. The solid sample may be suspended in a solvent, homogenized by a pulverizer or the like, or a supernatant obtained by stirring with a solvent may be used. Separation culture of microorganisms from a sample can be performed according to a method known in the art.

  In the present invention, drugs and / or metabolic substrates are added to the medium. As the medium to be used, an appropriate medium can be selected according to the type of microorganism to be examined and the type of sample. For example, a medium containing a natural component (Mueller-Hinton medium, LB medium, blood medium, etc.), a chemically synthesized medium (LeMaster medium, M9 medium, etc.), a semi-synthetic medium with a natural component added to a chemically synthesized medium, or the like may be used. Is possible. For measurement by mass spectrometry, a chemically synthesized medium having completely known medium components and few impurities that prevent ionization is desirable. However, natural components may be required for growth depending on the species of the microorganism.In this case, a semi-synthetic medium in which the minimum natural components necessary for the growth of microorganisms are added to the chemically synthesized medium is used. use.

  The drug (test drug) to be examined in the present invention is an antibacterial drug / antibiotic substance having an antibacterial action mainly used clinically. Antibacterials and antibiotics include penicillins, cephems, cephalosporins, cephamycins, oral cephems, oxacephems, penems ), Carbapenems, carbacephems, monobactams, β-lactamase inhibitors, aminoglycosides, macrolides, ketolides ), Streptogramins, lincomycins, tetracyclines, chloramphenicols, oxazolidinones, polypeptides, glycopeptides , Quinolones, sulfa Drugs such as drugs, antituberculosis drugs, and antifungal drugs are included, but drugs other than these can also be tested.

  One type of test drug may be used, or a plurality of types may be used in combination. In addition, the test drug may be prepared as a medium at a single addition amount / concentration, or may be added to the medium at different addition amounts / concentrations to prepare a plurality of culture media. When different addition amounts / concentrations are used, information on the addition amount (concentration) of the drug sensitivity of microorganisms can also be obtained. For example, it is possible to determine the minimum inhibitory concentration (MIC) of microorganisms.

  The metabolic substrate to be examined is not particularly limited as long as it is a metabolic substrate necessary for the metabolic system. For example, glucose, D-galactose, L-rhamnose, sorbitol, D-mannitol, inositol, D-fructose, arabinose, D-mannose, xylitol, D-xylose, ribose, sucrose, melibiose, trehalose, lactose, Examples of amino acid metabolic substrates such as maltose, raffinose and adonitol include glycine, arginine, lysine and histidine. One kind of metabolic substrate to be added may be used, or a plurality of kinds may be used in combination. This is because the type and / or amount of a metabolite produced by a microorganism is changed by the addition of a different metabolic substrate, and it may be possible to identify the microorganism species based on the change or change pattern.

  A compound labeled with a stable isotope is added to the medium as a metabolic substrate, and a metabolite produced from the compound can be measured. That is, a metabolite produced from a stable isotope-labeled compound contains a stable isotope and can be distinguished from compounds derived from others. This makes it possible to measure only the metabolites generated by a specific metabolic pathway of the microorganism and monitor the metabolism of the microorganism more precisely. In addition, the same component as the metabolite is included in advance as a medium component and may not be distinguished from the metabolite. Even in such a case, it is possible to measure a metabolite by using a stable isotope labeled compound. As described above, by using a stable isotope-labeled compound, it is possible to remove the background and monitor the metabolism and growth of microorganisms more accurately.

  Subsequently, the microorganism is cultured in a medium (sample) to which a test drug and / or a metabolic substrate is added. The culture of microorganisms can be carried out under conditions (temperature and time) conventionally used in the art. Specific culture conditions vary depending on the type of the target microorganism, the type of the sample, the type of the metabolite to be analyzed, and the like, and can be appropriately set by those skilled in the art. The culture time is short-time culture, specifically, 1 to 10 hours, preferably 1 to 5 hours, more preferably about 4 hours or less. The culture time varies depending on the amount and type of microorganism. As a control (standard culture), the microorganism is preferably cultured in a medium (sample) to which a test drug and / or a metabolic substrate is not added.

  A medium from which microorganisms have been removed may be collected from a medium (sample) after culturing microorganisms, and a pretreatment for mass spectrometry may be performed. Removal of microorganisms from the medium can be performed, for example, by filtration using a filter or centrifugation. In addition, as a pretreatment for mass spectrometry, specifically, a process for removing a substance that hinders ionization of components contained in a medium (sample) and a process for concentrating the components of the medium (sample) can be performed.

  The components of the medium (sample) are then mass analyzed for metabolites. A specific method of mass spectrometry is not particularly limited. When the culture is performed in a plurality of culture media (samples), mass spectrometry is performed on each of the metabolites contained in the plurality of culture media. The spectral data obtained by mass spectrometry is analyzed, and the type and amount of metabolites derived from microorganisms are determined from the results. The type of metabolite to be subjected to mass spectrometry is not particularly limited. For example, glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, Dihydroxyacetone phosphoric acid, 1,3-bisphosphoglyceric acid, 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, acetyl CoA, citric acid, isocitric acid, α-ketoglutar Acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, Threonine, valine, tryptophan, tyrosine Acetoin, acetone, butylene glycol, oxalic acid, acetaldehyde, butanol, gluconic acid, lactic acid, ethanol, acetic acid, and formic acid. In the present invention, mass spectrometry is performed on one or more of these metabolites. By determining a plurality of metabolites, it is possible to perform determination with higher accuracy. When a microorganism is cultured in a medium to which a stable isotope-labeled compound is added as a metabolic substrate, the metabolite labeled with the stable isotope is subjected to mass spectrometry.

  Based on the type and amount of the metabolite determined by mass spectrometry as described above, it is possible to identify the type of microorganism or to determine the influence of the drug and / or metabolic substrate on the microorganism. When microorganisms are cultured in multiple media (samples), mass spectrometry is performed on metabolites contained in multiple media, and the types and amounts of metabolites are compared between the multiple media. Based on the results, it is possible to identify the type of microorganism or to determine the effect of drugs and / or metabolic substrates on the microorganism.

  Alternatively, spectral data obtained by performing mass spectrometry may be collated directly with a database composed of spectral data obtained with various microorganisms, and the microorganism species can be identified from the collation result. . Thereby, this inspection method can be implemented more rapidly and simply.

  The identification result of the microorganism species obtained as described above, or the determination result of the influence of the drug and / or metabolic substrate on the microorganism may be displayed, output or stored.

  FIG. 1 shows a flow showing a specific example of the present microorganism testing method, and the outline of the testing procedure will be described below. First, in step 101, a bacterial suspension is prepared by suspending a test target microorganism 109 isolated from a clinical specimen by separation culture or the like in a culture medium. In the next step 102, a sample for culture is prepared from this bacterial suspension. When performing a drug sensitivity test, a dilution series sample 111 to which the drug is added at different concentrations is prepared for a plurality of drugs. In addition, a standard culture sample 110 to which the drug is not added is also prepared for the control of the test. For the bacterial species identification test, a sample 112 to which a metabolic substrate for detecting reactions characteristic of various microorganisms is added is prepared. In the next step 103, the sample is cultured. In the next step 104, a medium is collected from the cultured sample, and in step 105, a pretreatment for mass spectrometry is performed to prepare a sample for mass spectrometry. In the next step 106, mass spectrometry is performed on the prepared sample for mass spectrometry. In the next step 107, data analysis of mass spectrometry is performed, and the test result and the microbial species identification result regarding the drug sensitivity of the test target microorganism are output in step 108. Details of these inspection method flows will be described later in a third embodiment.

The present invention also relates to a system for carrying out the microorganism testing method as described above. Specifically, the microorganism testing system according to the present invention is:
A culture section comprising a culture vessel for culturing microorganisms, comprising a medium supplemented with a drug and / or a metabolic substrate;
A mass spectrometer for analyzing the components of the medium;
Based on the type and amount of the metabolite derived from the microorganism in the medium determined by the mass spectrometer, the type of the microorganism is identified, or the influence of the drug and / or metabolic substrate on the microorganism is determined. An analysis unit.

  The culture unit includes one or preferably a plurality of culture containers for culturing microorganisms. Each culture vessel contains a medium supplemented with drugs and / or metabolic substrates. The added drug and / or metabolic substrate may be one type, or a plurality of types may be combined. Also, the addition amount / concentration of the drug and / or metabolic substrate may be single or different. For example, the culture unit may include a plurality of culture containers including a plurality of culture media having different drug addition amounts / concentrations and / or a plurality of culture media having different types of metabolic substrates. Furthermore, as a control (standard culture), a culture vessel containing a medium (sample) to which a test drug and / or a metabolic substrate is not added may be provided.

  Further, as described above for the microorganism testing method according to the present invention, a stable isotope-labeled compound may be added to the medium as a metabolic substrate. In such a case, the culture unit of the system according to the present invention includes an incubator including a medium containing a stable isotope-labeled compound as a metabolic substrate.

  The mass spectrometer used in the system according to the present invention is not particularly limited as long as it can mass-analyze components contained in the medium (sample), that is, metabolites derived from microorganisms. In general, a mass spectrometer is composed of an ionization unit that ionizes components and a unit that measures the mass-to-charge ratio m / z of the ionized components. A person skilled in the art can appropriately select a mass spectrometer that can be used in the present invention. When a plurality of culture media (samples) are prepared in the culture unit, the mass spectrometer performs mass spectrometry for each of the plurality of culture media (samples).

  The type of metabolite to be analyzed by the mass spectrometer is not particularly limited, and examples thereof include pyruvic acid, lactic acid, citric acid, acetic acid, and ethanol. In the system according to the present invention, mass spectrometry is performed on one or more of these metabolites. In addition, when a stable isotope-labeled compound is added to the culture medium in the culture section, the mass spectrometer generates a metabolite labeled with a stable isotope that is generated from the stable isotope-labeled compound and discharged outside the microorganism cell. The product is mass analyzed.

  The system according to the present invention may include a preprocessing unit that performs preprocessing for mass spectrometry. For example, in the pretreatment section, means for collecting a medium from which microorganisms have been removed from each culture vessel of the culture section (filter, centrifuge, etc.), means for removing substances that interfere with ionization of components, means for concentrating medium components Etc. are included.

  The analysis unit analyzes the spectrum data obtained by the mass spectrometer, determines the type and amount of the metabolite derived from the microorganism in the medium, and identifies the type of the microorganism from the result, or identifies the drug and / or Alternatively, the influence of the metabolic substrate on the microorganism is determined. When a plurality of culture media (samples) are prepared in the culture unit, the analysis unit metabolizes the analysis results of each culture medium (sample) obtained from the mass spectrometer between the plurality of culture media (samples). The type and amount of the product are compared, the type of the microorganism is identified, or the influence of the drug and / or metabolic substrate on the microorganism (antibacterial properties) is determined.

  Or an analysis part may collate the spectrum data obtained with the mass spectrometer with the database comprised from the spectrum data obtained with various microorganisms, and may identify microorganism species from the result.

  The system according to the present invention may include a mechanism for carrying the sample and the medium among the culture unit, the pretreatment unit, and the mass spectrometer.

  Furthermore, the system according to the present invention may include a control unit for controlling and operating the entire system. Further, the system may be provided with means for displaying, outputting or storing the results obtained by the analysis unit. For example, the control unit includes a group consisting of an input unit, a display unit, an output unit, and a storage unit. One or more selected may accompany.

  FIG. 2 shows a specific example of the present microorganism testing system. This is a system for realizing the inspection flow of FIG. This microbiological test system analyzes the data obtained by the culture apparatus 201 for culturing microorganisms, the pretreatment unit 202 for preparing the sample for mass spectrometry, the mass spectrometer 203, and the mass spectrometer 203, and determines the effect of the drug And an analysis unit 204 that performs microbial species identification. The mass spectrometer 203 is mainly composed of an ionization unit 203-1 and a mass analysis unit 203-2. The entire microorganism testing system is controlled and operated by the control unit 206. The control unit 206 is accompanied by an input unit 207, a display unit 208, an output unit 209, and a storage device 210. In addition, the data analysis unit 204 can appropriately refer to the database 205 for information on the types and amounts of metabolites derived from known microorganisms in advance.

  FIG. 3 shows a configuration example of the present microorganism testing system. This is an example of a system for realizing the inspection flow of FIG. 1, and is a more specific example of the system of FIG. The culture apparatus 301 is composed of a plurality of culture containers 302 for culturing microorganisms. The number of containers varies depending on the number of combinations of drug types and concentrations to be examined or the number of metabolic substrates, but it is usually desirable to have about 100 to 1000 containers. Each culture container has a capacity of 1 mL or less, and in order to perform a large number of cultures with a limited microbial sample, it is desirable that the volume is small, and preferably 100 μL or less. This makes it possible to reduce the size of the culture apparatus and increase the number of combinations of types and concentrations of drugs to be examined and the number of metabolic substrates. The prepared bacterial suspension is introduced into the culture vessel 302 from the bacterial suspension introduction port 311. The entire culture container is in the form of a cartridge, which can be made of plastic or the like and made disposable so that it can be exchanged for each test. Furthermore, it is possible to mount a plurality of cartridges in one inspection system and perform a plurality of inspections simultaneously in parallel. Each culture vessel 302 has a function of stirring a culture solution, a function of controlling temperature, a function of supplying oxygen and medium components necessary for culturing microorganisms, and a function of putting a microorganism suspension in the culture vessel 302 as necessary. , A function of collecting a sample from the culture vessel 302 is provided. The function of stirring the culture solution can be performed by shaking the entire cartridge. Further, the temperature can be controlled by placing the entire cartridge in a temperature-controlled incubator. One of the culture containers 302 of the culture apparatus 301 contains a medium that does not contain a drug. In the case of the drug sensitivity test, the remaining plurality of culture vessels 302 of the culture apparatus 301 contain media containing drugs of different types and combinations of concentrations. In the case of the biochemical property test in the identification test, different metabolic substrates are contained.

  Each culture vessel 302 of the culture apparatus 301 has a mechanism for removing only microbial cells from the culture solution and collecting only the medium. For example, it has a function of capturing microbial cells with a membrane filter and collecting only the medium. This operation is performed by the liquid feeding system 312 provided with the valve 313, and the culture medium can be collected sequentially from each culture vessel 302. Further, this operation can be performed by an automated pipette, or can be performed by another mechanism. In addition, a method such as centrifugation may be used for removing microbial cells. In any case, microorganisms are removed from the culture solution, and only the medium is collected and sent to the next pretreatment unit 303. These mechanisms are accompanied by a cleaning liquid tank 314 having a cleaning function and a system liquid tank 315, and the mechanism is cleaned for each sample to prevent different samples from being mixed with each other. The waste liquid is collected in a waste liquid tank 316. If possible, in order to prevent microorganisms that may have pathogenicity from diffusing into the environment, the work after culturing is performed in a sealed container, and the medium is also collected in the container. It is desirable to do so.

  The pre-processing unit 303 performs pre-processing for applying to the mass spectrometer 304 to the collected culture medium as necessary. The pretreatment to be applied to the mass spectrometer 304 includes a treatment for removing substances that interfere with ionization of a sample during mass analysis, a treatment for concentrating medium components, and the like using a column or a filter. As the pretreatment step, a suitable one is adopted in consideration of the composition of the medium, the metabolite to be measured, and the like.

  The mass spectrometer 304 is composed of a part for introducing a sample and performing ionization, and a part for measuring a mass-to-charge ratio m / z of the ionized sample. Examples of ionization units that perform sample ionization include dielectric barrier discharge (DBD) ionization, electrospray atmospheric pressure ionization (ESI), and matrix-assisted laser desorption ionization (MALDI). The analyzer that separates the ionized sample includes a quadrupole mass spectrometer (QMS), ion trap mass spectrometer (ITMS), time-of-flight mass spectrometer (TOFMS), and multiple quadrupole mass There are methods such as analyzers.

  The data analysis unit 305 performs data processing of the mass spectrometer 304. Furthermore, in the case of a drug sensitivity test, the effect of the drug on microbial growth is determined from microbial metabolite data in the medium obtained by mass spectrometry. In the case of a biochemical property test, the pattern of metabolic results for each metabolic substrate is collated with the database 317 to identify the microbial species. The result of the mass analysis and the test result relating to the effect and species identification of the drug are displayed on the display unit 308 through the control unit 306 and output from the output unit 309, and the data is stored in the storage device 310.

  With the microorganism testing method and the microorganism testing system according to the present invention, rapid microorganism testing can be performed. In particular, in a drug sensitivity test, a metabolite in a microorganism culture solution is detected by mass spectrometry, metabolism and growth of microorganisms are detected with high sensitivity, and the effect of the drug is determined by culturing in a short time. Further, since the components in the medium are measured instead of measuring the metabolites in the microbial cells, it is possible to omit the processing steps such as disruption of the cells and perform the measurement quickly. Since the metabolite of the microorganism is not accumulated inside the cell but is discharged out of the cell body, the concentration in the medium increases with the growth of the microorganism. Therefore, the concentration of the metabolite in the medium shows a correlation with the growth of the microorganism, and the growth of the microorganism can be determined by measuring the concentration. In the present invention, by combining these, the effect of the drug can be determined quickly and with high sensitivity. Furthermore, in the present invention, a microorganism identification test is performed simultaneously with the drug sensitivity test, and information on the microorganism species is provided together with information on the drug sensitivity.

  Hereinafter, specific examples of embodiments of the present invention will be described with reference to the drawings. However, it should be noted that these examples are merely examples for realizing the present invention and do not limit the present invention.

[Example 1]
First, an embodiment of this inspection method will be described. Based on this test method, Escherichia coli ATCC 25922 strain is used as a material to measure the effects of the antibiotic chloramphenicol on growth by measuring glucose metabolites pyruvate and lactic acid in the medium, Shows that it is possible to determine the minimum inhibitory concentration (MIC).

  LeMaster medium, which is a chemically synthesized medium, was used for the culture. A chloramphenicol 2-fold dilution series medium was prepared using LeMaster medium. In addition, a medium containing no chloramphenicol was prepared for standard culture. In total, the following 10 concentrations of chloramphenicol 2-fold dilution series were prepared; 0.0 μg / mL (Sample 1), 0.5 μg / mL (Sample 2), 1 μg / mL (Sample 3) , 2 μg / mL (Sample 4), 4 μg / mL (Sample 5), 8 μg / mL (Sample 6), 16 μg / mL (Sample 7), 32 μg / mL (Sample 8), 64 μg / mL (Sample 9), 128 μg / mL (sample 10).

The E. coli ATCC 25922 strain was planted in 1 mL of LB medium in a 14 mL culture tube using an inoculating loop and subjected to shaking culture at 37 ° C. for about 18 hours to prepare a preculture. Pre-cultured E. coli culture that is considered to have reached stationary phase, 5 mL chloramphenicol 2-fold dilution series placed in a 50 mL conical tube so that the cell concentration is 10 ⁷ CFU / mL It was transferred to LeMaster medium and cultured with shaking at 37 ° C.

  The culture solution was collected 4 hours after the start of the culture. The amount of the collected culture solution was 200 μL, of which 100 μL was used for metabolite measurement with a liquid chromatograph-mass spectrometer and 100 μL was used for counting the number of cells. With respect to 100 μL used for metabolite measurement, filtration sterilization was performed by centrifugation at 3,000 rpm for 3 minutes using a centrifugal filter unit (pore diameter: 0.45 μm), and the cells in the culture solution were removed. The filtrate, that is, the culture medium of the culture solution that has been sterilized, was subjected to metabolite measurement using a liquid chromatograph-mass spectrometer. With respect to 100 μL used for counting the number of cells, colony forming unit (CFU) was measured. About the collected culture solution, a 10-fold dilution series was prepared using physiological saline (0.9% NaCl aqueous solution), plated on LB agar plate medium, and cultured at 37 ° C. for 16 to 24 hours. The colonies formed on the agar plate medium were counted and multiplied by the dilution factor, and the bacterial cell concentration in the culture broth was calculated as CFU / mL.

  The measurement of E. coli metabolites in the medium was performed by a liquid chromatograph-electrospray ionization triple quadrupole mass spectrometer. For sample separation by high performance liquid chromatography, a polymer column for organic acid analysis was used with Agilent Technologies 1100 series. The mass spectrometer was a Quattro Premier from Waters. The metabolite was quantified based on a calibration curve prepared using a dilution series prepared for each standard medium for each measurement. The results are summarized in Table 1.

At the same time, in parallel with the above work, in order to compare with the standard method defined by the Japanese Society of Chemotherapy, culture using Mueller-Hinton medium was also performed. Using a Mueller-Hinton medium, a 2-fold dilution series medium of chloramphenicol was prepared. In addition, a medium containing no chloramphenicol was prepared for standard culture as a control for comparison. In total, the following 10 concentrations of chloramphenicol 2-fold dilution series medium were prepared; 0.0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL. Transfer the E. coli preculture to 5 mL of chloramphenicol 2-fold diluted Mueller-Hinton medium in a 50 mL conical tube so that the cell concentration is 10 ⁶ CFU / mL, and shake at 37 ° C. Culture was performed. At 24 hours after the start of culture, the change in turbidity of the culture broth was visually determined. For cultures in which Escherichia coli did not grow and turbidity did not increase, the one with the lowest chloramphenicol concentration was defined as the MIC according to the standard method defined by the Japanese Society of Chemotherapy. As a result, the MIC of chloramphenicol was 2 μg / mL.

  From the results shown in Table 1, the cell concentration (CFU / mL), pyruvic acid concentration and lactic acid concentration were plotted against the chloramphenicol concentration, and the graph of FIG. 4 was created. As a result, with respect to chloramphenicol, the growth of E. coli was suppressed at a concentration of 2 μg / mL or more, and no increase in pyruvate concentration or lactic acid concentration in the medium was observed. On the other hand, when the concentration was 1 μg / mL or less, Escherichia coli grew, and the concentration of pyruvate and lactic acid in the medium increased. Therefore, the growth of Escherichia coli and the pyruvic acid concentration / lactic acid concentration in the medium are correlated, and it is possible to determine the MIC by determining the growth of E. coli from the pyruvic acid concentration / lactic acid concentration. That is, among the chloramphenicol concentrations where the pyruvic acid concentration and lactic acid concentration did not increase, the one with the lowest chloramphenicol concentration shows the same value as MIC. In addition, the MIC obtained by turbidity growth determination in 24 hours culture in Mueller-Hinton medium and the MIC obtained by measurement of pyruvic acid and lactic acid in 4 hours culture in LeMaster medium are almost equivalent. Indicated. Therefore, it was shown that the same results as the standard method established by the Japanese Society of Chemotherapy can be obtained by this method in 4 hours of culture.

[Example 2]
Next, the effect of the antibiotic ampicillin on the growth was measured by measuring glucose metabolites pyruvate and lactic acid in the medium using the Staphylococcus aureus ATCC 6538 strain as a material in the same manner as in Example 1. This indicates that it is possible to determine the MIC.

A Staphylococcus aureus ATCC 6538 strain was planted in 1 mL of LB medium in a 14 mL culture tube using an inoculating loop and subjected to shaking culture at 37 ° C. for about 18 hours for pre-culture. For the preculture that is considered to have reached the stationary phase, 5 mL of ampicillin 2-fold diluted LeMaster medium (ampicillin concentration 0.0 μg / mL) placed in a 50 mL conical tube so that the cell concentration is 10 ⁶ CFU / mL (mL, 0.02μg / mL, 0.03μg / mL, 0.06μg / mL, 0.13μg / mL, 0.25μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 4μg / mL) Incubated with shaking.

  The culture solution was collected 4 hours after the start of the culture, and the number of bacterial cells and the metabolites in the medium were measured. Based on the data, the cell concentration (CFU / mL) and the pyruvic acid concentration / lactic acid concentration were plotted against the ampicillin concentration, and the graph of FIG. 5 was created. As a result, growth of Staphylococcus aureus was suppressed at an ampicillin concentration of 0.25 μg / mL or more, and no increase in the concentration of pyruvate in the medium was observed. On the other hand, when the ampicillin concentration was 0.13 μg / mL or less, Staphylococcus aureus proliferated and the pyruvic acid concentration in the medium increased. Therefore, there is a correlation between the growth of S. aureus and the concentration of pyruvate in the medium, and the growth of S. aureus can be determined from the pyruvate concentration to determine the MIC. That is, among the ampicillin concentrations where the pyruvate concentration did not increase, the one with the lowest ampicillin concentration shows the same value as MIC. Simultaneously with this experiment, Staphylococcus aureus was cultured for 24 hours in an ampicillin-diluted series prepared using Mueller-Hinton medium, and the growth was determined by turbidity. The obtained MIC was 0.25 μg / mL. Therefore, it was shown that the same results as the standard method established by the Japanese Society of Chemotherapy can be obtained by this method in 4 hours of culture.

[Example 3]
Next, based on the test method of Example 1, when the drug susceptibility test for microorganisms is performed in the microbe test system, the flow of the microbe test method and the microbe test system shown in FIG. An embodiment will be described. Moreover, the specific structural example of a microbe test | inspection system is shown in FIG. The determination of the effect of a drug on microorganisms such as bacteria and fungi is called a drug sensitivity test. The drug susceptibility test using this microorganism testing system is performed according to the following procedure.

(Bacteria suspension preparation)
When bacteria are to be examined, one or more colonies of the same appearance and shape are collected from bacterial colonies obtained by primary culture of clinical specimens collected from patients on a predetermined agar plate medium. Suspend bacteria in liquid medium. The number of colonies to be picked up is preferably one, but if the amount of bacteria is insufficient, a plurality of colonies are picked up and used. In the case of bacteria, one colony usually contains 10 ⁷ to 10 ⁸ bacteria, so when one colony is suspended in 10 mL of liquid medium, The concentration of bacteria is 10 ⁶ to 10 ⁷ CFU / mL. The concentration of bacteria may be lower or higher, but the higher the concentration of bacteria, the higher the concentration of metabolites excreted in the medium, and the shorter the time it takes to obtain the test results. Can be expected. For example, when 10 mL of a bacterial suspension is prepared, 100 100 μL cultures can be performed. The susceptibility test for fungi is basically performed in the same procedure, although the number of cells is different.

(Culture medium)
Regarding the medium, it is possible to use a medium containing a natural component, a chemically synthesized medium, a semi-synthetic medium obtained by adding a natural component to a chemically synthesized medium, or the like. The medium containing natural components is a medium composed of a meat extract, a yeast extract or the like, and includes Mueller-Hinton medium, LB medium, and the like. A chemically synthesized medium is a medium that is prepared by mixing pure chemical substances, and is a medium in which all components and their concentrations are clear, such as LeMaster medium and M9 medium used for E. coli culture. To do. For measurement by mass spectrometry, a chemically synthesized medium having completely known medium components and few impurities that prevent ionization is desirable. However, natural components may be required for growth depending on the species of the microorganism.In this case, a semi-synthetic medium in which the minimum natural components necessary for the growth of microorganisms are added to the chemically synthesized medium is used. use.

(Stable isotope labeling)
A compound labeled with a stable isotope is added to the medium, and metabolites generated from the compound can be measured. For example, when ¹³ C ₆ glucose in which all 6 carbon atoms are ¹³ C is added to the medium as a carbon source, ¹³ C is contained in the metabolite produced therefrom. For example, in the case of pyruvic acid, ¹³ C ₃ pyruvic acid is produced. Therefore, by measuring ¹³ C ₃ pyruvic acid separately from ¹² C ₃ pyruvic acid composed of normal carbon atoms, it is possible to monitor only the function of the glucose metabolic system. Since the glucose metabolism system is considered to be closely related to the growth of microorganisms, it becomes possible to measure microorganism growth more precisely. Moreover, pyruvic acid, lactic acid, citric acid, etc. may be previously contained in the culture medium as a culture medium component. In such a case, it is possible to distinguish pyruvate, lactic acid, citric acid, etc. contained as a medium component from metabolites generated by metabolism from glucose, and monitor the function of the glucose metabolism system. Is possible. ^{In 13} C ₆ glucose, all 6 carbon atoms are ¹³ C, but glucose having ¹³ C or less can be used. However, when ¹³ C ₆ glucose is used, both of the two molecules of pyruvic acid produced become ¹³ C ₃ pyruvic acid, which is easy to analyze. In addition, since the molecular weight is different from that of normal ¹² C ₃ pyruvic acid, it can be easily distinguished by mass spectrometry. As for molecules other than glucose, those labeled with stable isotopes can be used. However, ¹³ C ₆ glucose is cheaper and more stable than other stable isotope-labeled compounds. Therefore, when putting this microbial test method and microbial test system into practical use, cost and quality assurance can be avoided. This is advantageous.

(Microorganisms subject to inspection)
In the present microorganism testing method and microorganism testing system, the target of inspection is microorganisms such as bacteria and fungi that are generally inspected in a hospital laboratory or the like. It is possible to inspect microbial species without pre-determining. Estimate or identify microbial species based on microscopic observation of patient symptoms and stained specimens, based on the type of agar plate used for primary culture, appearance and shape of colonies, base sequence information of microbial genes, etc. It is possible. Furthermore, it is possible to identify a species by an inspection method using a MALDI-TOF mass spectrometer. If the microbial species can be estimated or identified, it is possible to conduct a drug susceptibility test focusing on drugs suitable for the target microbial species. Test targets include not only microorganisms that infect humans but also microorganisms that infect livestock and pet animals.

(Drug dilution series)
After the prepared bacterial suspension is set in the present microorganism inspection system, the bacterial suspension is sent to each culture container 302 from the bacterial suspension inlet 311 as shown in FIG. Each culture container contains different amounts of drugs in advance, and samples containing different concentrations of drugs are prepared. This is prepared for a plurality of types of drugs. At the same time, a sample containing no drug (referred to as standard culture) is also prepared as a control. The type and concentration of the drug are selected according to the microorganism to be examined or its candidate species. The concentration of the drug can be a dilution series, or can be limited to a predetermined concentration for determination of drug-resistant bacteria. In addition, after adding a chemical | medical agent, it is also possible to send to each culture container. By preparing a plurality of samples containing drugs with different concentrations in this way and acquiring drug sensitivity information according to the concentrations, it is possible to evaluate the effects of the drugs on the bacteria according to the concentrations.

(Culture and incubation time)
The prepared sample is cultured at a constant temperature in the culture apparatus 301. The temperature is set as necessary, but in the case of clinical specimens, for example, it is often set at, for example, 32 to 40 ° C., usually 37 ° C. The measurement of the metabolite by culture and mass spectrometry can be performed by appropriately collecting a medium while culturing. It is also possible to carry out after culturing for a certain time. The incubation time can be determined in advance according to the microorganism species to be examined. It is also possible to determine from the growth in a standard culture containing no drug. In such a case, the culture medium can be collected at regular intervals, measured by the present microorganism testing system, and the culture can be terminated when a predetermined component (metabolite) reaches a predetermined concentration.

(Preprocessing)
After culturing the microorganisms, the medium is collected, sample preparation is performed by the pretreatment unit 303, and then measurement is performed by the mass spectrometer 304. In collecting the medium, microorganisms are removed from the culture solution using a membrane filter having a pore size of 0.22 μm or 0.45 μm, and only the medium is collected. However, when ionization and measurement in the mass spectrometer 304 are not hindered, it is also possible to perform measurement using a culture solution containing microorganisms without removing the microorganisms.

(Mass spectrometry)
The influence of the drug on the growth of microorganisms is performed by the following procedure. The concentration of a predetermined microbial metabolite in the medium is measured by mass spectrometry of the medium collected from each culture solution. Metabolites derived from microorganisms to be measured are selected according to the type of microorganism. For example, it is possible to use pyruvic acid, lactic acid, citric acid, acetic acid, ethanol, and the like that are produced by adding glucose to the medium, metabolizing glucose, and excreted outside the cells. Whether other saccharides are metabolized depends on the bacterial species, but when metabolized, pyruvic acid, lactic acid, citric acid, acetic acid, ethanol and the like may be generated as metabolites. Other metabolites can also be used. As for the concentration of metabolites, absolute quantification is desirable, but it is not always necessary. It is only necessary to be able to compare the amount of metabolites between different samples. Relative to the peak area of the spectrum obtained by mass spectrometry It may be quantitative.

(Judgment protocol)
A specific example of a protocol for determining the effect of a drug in the data analysis unit 305 from the metabolite concentration obtained by mass spectrometry is shown in FIGS. When the effect of chloramphenicol in E. coli was measured with this test system, the MIC was determined based on the pyruvate concentration data in the medium relative to the chloramphenicol concentration in E. coli measured in Example 1. . Note that the same operation is possible even if a metabolite other than pyruvic acid (for example, lactic acid or citric acid) is used, and a suitable one is selected from various metabolites and the determination is made.

  For the pyruvic acid concentration measured as described in the section of mass spectrometry, as shown in FIG. 6A, between the data 501 of the standard culture sample 1 containing no drug and the data 502 of the culture sample 2 containing the drug. Comparison 503 is performed. 6A to 6C, only the sample 2 is numbered, but the comparison operation with the data of the sample 1 is similarly performed for the samples 3 to 10 below. FIG. 6B summarizes the results of the comparison, and FIG. 6C is a graph plotting the relative concentration of pyruvic acid against the chloramphenicol concentration. As a result of the comparison, it is judged that the growth or metabolism of Escherichia coli is suppressed or the Escherichia coli is dead due to the effect of the drug for the culture having a lower pyruvic acid concentration than the standard culture. In this case, the effect of the drug can be determined by the ratio of pyruvic acid concentration. That is, assuming that the metabolite concentration 504 in the standard culture is 100, the metabolite concentration ratio 505 in the sample 2 containing chloramphenicol, that is, the relative concentration is calculated. Hereinafter, the relative concentrations of metabolites are similarly calculated for the other samples 3 to 10. When this relative concentration is lower than a certain threshold value 506, it is determined that there is a drug effect. The relative concentration serving as the threshold is determined according to the type of microorganism or the type of drug. When culturing E. coli in the presence of different concentrations of drugs using a dilution series of drugs, the lowest drug concentration among the drug concentrations at which only E. coli-derived metabolites were detected was detected as the minimum growth inhibitory concentration. (MIC) 507. This time, the chloramphenicol concentration in sample 4 is 2 μg / mL.

  The background level of the metabolite concentration described above can be determined from the slope of the straight line of the graph connecting the metabolite concentrations at each drug concentration. For example, in the graph of FIG. 6C in which the horizontal axis represents the chloramphenicol concentration, the straight line connecting the pyruvic acid concentration at each drug concentration is almost horizontal, and the absolute value of the slope is less than a certain threshold value. A state in which a change in pyruvic acid concentration hardly occurs between the concentrations can be determined as the background level. In the graph of FIG. 6C, a slope 509 of a straight line 508 connecting the pyruvic acid concentration 504 in the standard culture and the pyruvic acid concentration 505 in the sample 2 containing chloramphenicol is calculated. Hereinafter, the slope of the graph is also calculated between other samples 2 to 10 adjacent to each other. As a result, in sample 4 (chloramphenicol concentration 2 μg / mL) and later, the straight line of the graph is almost horizontal, the absolute value of the slope is small, and it is judged that there is almost no change in the pyruvic acid concentration. Therefore, the pyruvate concentration at these concentrations was determined to be the background level, and in the samples with chloramphenicol concentrations of 0.0 μg / mL, 0.5 μg / mL, and 1 μg / mL, the pyruvate concentration was higher than this. Proliferation has occurred.

(Judgment result display)
The above determination results are displayed on the display unit 308. FIG. 7 shows the interface of the display screen. When the concentration of pyruvate in the standard culture is set to 100, the relative concentration of pyruvate in each sample to which chloramphenicol was added at different concentrations was calculated and displayed in five stages as shown in FIG. went. Each stage has a relative density of 0 to less than 5, Stage 1, 5 to less than 10, Stage 2, 10 to less than 20, Stage 3, 20 to less than 50, Stage 4, and 50 to Stage 5, each with a different color And display it on the interface. This is displayed by an interface as shown in FIG. In addition, as a result of the MIC of chloramphenicol in E. coli ATCC 25922, “Sample 4” and its drug concentration “2 μg / mL” are displayed. It should be noted that with respect to this stage division, it is possible to increase the number of stages or change the numerical value as a break.

  Next, FIG. 7C shows an example of an interface that displays the results of drug sensitivity tests on a plurality of drugs. As an example, glucose is added to the medium as a carbon source, and three types of drugs, drug A, drug B, and drug C, are examined, and the results of sensitivity tests at concentrations 1 to 10 are shown. Moreover, the ratio of the pyruvic acid concentration in the other samples was calculated by setting the sample without the drug as a standard culture with a drug concentration of 0 and setting the concentration of pyruvate as a glucose metabolite in this sample as 100. The interface was displayed in five stages as shown in FIG. As a result, in drug A, pyruvic acid was not detected at a concentration of 6 or higher, and pyruvic acid was detected at a concentration of 5 or lower. Therefore, glucose metabolism did not occur at a concentration of 6 or higher and growth was suppressed. Is shown. In drug B, pyruvic acid was not detected at a concentration of 8 or higher, and pyruvic acid was detected at a concentration of 7 or lower, indicating that glucose metabolism did not occur at a concentration of 8 or higher and growth was suppressed. . In drug C, pyruvic acid was not detected at a concentration of 4 or higher, and pyruvic acid was detected at a concentration of 3 or lower, indicating that glucose metabolism did not occur at a concentration of 4 or higher and growth was suppressed. . Therefore, the MIC for drug A was determined to be 6; the MIC for drug B was determined to be 8; the MIC for drug C was determined to be 4; drug C was the most effective. In addition, it is not necessary to prepare the standard culture with a concentration of 0 in C in FIG. 7 for each drug, and one standard culture may be prepared.

[Example 4]
Next, specific effects when ¹³ C ₆ glucose labeled with a stable isotope is used as a carbon source will be described. In Example 1, the LeMaster medium contains 1% glucose. As this glucose, normal ¹² C ₆ glucose and ¹³ C ₆ in which all 6 carbon atoms are labeled with a stable isotope ¹³ C are used. The case of using glucose was compared. In the same manner as in Example 1, using a medium containing normal ¹² C ₆ glucose as a carbon source and a medium containing stable isotope-labeled ¹³ C ₆ glucose, E. coli ATCC 25922 strain was cultured, respectively, The time course of concentration and concentration of pyruvic acid and lactic acid in the medium was measured. ^{When 12} C ₆ glucose was used, ¹² C ₃ pyruvic acid and ¹² C ₃ lactic acid were produced, so ¹² C ₃ pyruvic acid and ¹² C ₃ lactic acid were measured. In this case, however, ¹² C ₃ pyruvic acid and ¹² C ₃ lactic acid derived from other than glucose are also measured. On the other hand, when ¹³ C ₆ glucose is used, ¹³ C ₃ pyruvic acid and ¹³ C ₃ lactic acid are produced. Therefore, ¹³ C ₃ pyruvic acid and ¹³ C ₃ lactic acid were measured. In this case, only pyruvic acid and lactic acid derived from glucose are measured.

The actual measurement results are shown in FIG. FIG. 8B is an enlarged scale of the vertical axis showing the pyruvate concentration / lactic acid concentration in the graph of FIG. 8A, and shows the rise of the pyruvate concentration and the lactic acid concentration in an easy-to-understand manner. Comparing the data of ¹² C ₃ pyruvate and ¹³ C ₃ pyruvate, ¹² C ₃ pyruvate has a background from the beginning of the culture and the increase in concentration is not clear, whereas for ¹³ C ₃ pyruvate, It was possible to detect that the pyruvic acid concentration rose and increased 1 to 2 hours after the start of culture without the background. This is considered to be due to the fact that peaks overlapping in the spectrum were avoided by using ¹³ C ₆ glucose and pyruvic acid derived from other than glucose was excluded. Thus, by using a stable isotope labeled compound as a metabolic substrate, it is possible to determine the growth of microorganisms in a shorter time of culture.

[Example 5]
Next, a specific embodiment of the flow of the microorganism testing method of the present invention shown in FIG. 1 will be described in the case where a microorganism species is identified by conducting a biochemical property test of microorganisms in the present microorganism testing system. Moreover, the specific structural example of a microbe test | inspection system is shown in FIG. The biochemical property test using this microorganism testing system is performed according to the following procedure.

  A bacterial suspension 101 is prepared in the same manner as in Example 3. After the prepared bacterial suspension is set in the culture apparatus 301 of the present microorganism inspection system, the bacterial suspension is sent to each culture vessel 302 from the bacterial suspension introduction port 311. Each culture container contains different metabolic substrates in advance, and samples containing different metabolic substrates are prepared. At the same time, a sample for comparison (referred to as standard culture) is also prepared. There are various sugars and amino acids as substances (metabolic substrates) to be added to the medium for identification of the microorganism species, and they are selected according to the microorganism species to be examined or its candidate species. Regarding the medium, as long as the effect of metabolism can be determined, similarly to Example 3, a chemically synthesized medium, a medium containing a natural component, a semi-synthetic medium obtained by adding a natural component to a chemically synthesized medium, or the like can be used. is there. In addition, a compound labeled with a stable isotope can be added to the medium, and metabolites generated from the compound can be measured. In the present microorganism testing method and microorganism testing system, the target of inspection is microorganisms such as bacteria and fungi that are generally inspected in a hospital laboratory or the like. Microorganisms can be inspected in advance, but as a result of microscopic observation of patient symptoms and stained specimens, the type of agar plate used for primary culture, colony appearance / shape, It is desirable to estimate or identify based on the information on the base sequence of the gene, the test results using a MALDI-TOF mass spectrometer, etc., and to perform the test focusing on an appropriate metabolic substrate according to the candidate microorganism.

  Next, the culture is performed in the same manner as in Example 3, the medium is collected, pretreated by the pretreatment unit 303, and then measured by the mass spectrometer 304. The influence of each metabolic substrate on the growth of microorganisms is performed by measuring the concentration of a predetermined microbial metabolite in the medium by mass spectrometry of the medium collected from each culture solution, as in Example 3. The metabolite to be measured is selected according to the microorganism species. For example, it is possible to use pyruvic acid, lactic acid, citric acid, acetic acid, ethanol, and the like that are produced by adding glucose to the medium, metabolizing glucose, and excreted outside the cells. Whether other saccharides are metabolized differs depending on the microorganism species, but when metabolized, pyruvate, lactic acid, citric acid, acetic acid, ethanol and the like may be produced as metabolites. Other metabolites can also be used. As for the concentration of metabolites, absolute quantification is desirable, but it is not always necessary. It is only necessary to be able to compare the amount of metabolites between different samples. Relative to the peak area of the spectrum obtained as a result of mass spectrometry, etc. It may be quantitative.

[Example 6]
In the biochemical property test, a specific example of a protocol for determining the effect of each metabolite by data analysis from the metabolite concentration obtained by mass spectrometry is shown below.

  In E. coli Escherichia coli ATCC 25922 strain and Staphylococcus aureus ATCC 6538 strain, the metabolism of various sugar substrates is determined and the metabolic patterns are different. This makes it possible to distinguish between E. coli and S. aureus. The LeMaster medium used in Example 1 contained 1% glucose, but instead of this glucose, various sugar substrates were added to a concentration of 1%. Glucose, sorbitol, L-rhamnose, D-arabinose, sucrose, and D-raffinose were selected as E. coli sugar metabolism substrates. In addition, glucose, trehalose, D-mannitol, mannose, lactose, and D-raffinose were selected as sugar metabolism substrates for Staphylococcus aureus. For each of the above sugar substrates, a LeMaster medium containing each sugar substrate instead of glucose was prepared. A medium not containing a sugar substrate was also prepared. Escherichia coli and Staphylococcus aureus were cultured using these media, and the media were collected 6 hours after the start of the culture, and the pyruvate concentration and lactic acid concentration in the media were measured by mass spectrometry.

  The spectra of the mass spectrometry obtained for these samples are shown in FIGS. 9A and 9B. FIG. 9A is a spectrum obtained by metabolism of various sugar substrates in E. coli. FIG. 9B is a spectrum obtained by metabolism of various sugar substrates in Staphylococcus aureus. Based on these spectral data, the concentration ratio of pyruvic acid and lactic acid in the sample containing each sugar substrate, that is, the relative concentration was calculated with the concentration of pyruvic acid and lactic acid in the sample cultured in a medium containing glucose as 100. . Relative concentrations were also calculated for cultures that did not contain a sugar substrate. The results were displayed in five stages shown in FIG. Each stage has a relative density of 0 to less than 20, stage 1, 20 to less than 40, stage 2, 40 to less than 60, stage 3, 60 to less than 80, stage 4, and 80 or more to stage 5, each with a different color And display it on the interface. As a result, the result in E. coli is as shown in FIG. Moreover, the result in S. aureus is as shown in FIG. These metabolic patterns are considered to be characteristic for each bacterial species. Therefore, this metabolic pattern is measured for each bacterial species, and a database of metabolic patterns is constructed. The bacterial species can be identified by referring to this database with a metabolic pattern obtained in a microorganism whose bacterial species are unknown. In that case, the accuracy of identification can be improved by increasing the types of metabolic substrates to be used and detecting metabolic reactions characteristic of each bacterial species.

  Note that the spectrum pattern obtained by mass spectrometry of a medium when culturing for a predetermined time using a specific medium is characteristic of each microorganism species, and identification of the microorganism species based on this spectrum pattern Is considered possible. 9A and 9B are spectral patterns for pyruvate and lactic acid produced by sugar substrate metabolism in E. coli and S. aureus, respectively. Even in the spectrum derived from the same glucose, the pattern is different between Escherichia coli ((2) in FIG. 9A) and Staphylococcus aureus ((2) in FIG. 9B). If this is applied to more metabolic substrates and metabolites, it is possible to identify bacterial species by their patterns. For this purpose, the peak pattern of the spectrum obtained by mass spectrometry of the medium is measured for each microorganism species, and its database is constructed. By referring to this database with reference to the peak pattern of the spectrum obtained for a microorganism whose species is unknown, it is possible to easily identify the species.

  The above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

101: Bacteria suspension preparation step
102: Culture sample preparation step
102-1: Step of preparing culture sample for drug sensitivity test
102-2: Preparation of culture sample for biochemical property test
103: Culture step
104: Culture solution (medium) collection step
105: Pretreatment (sample preparation for mass spectrometry) step
106: Mass spectrometry step
107: Data analysis step
108: Inspection result display / output step
109: Microorganisms to be tested
110: Standard culture sample without added drug
111: Samples with different concentrations of drug added
112: Samples with different metabolic substrates added
201, 301: Culture equipment
202, 303: Preprocessing section
203, 304: Mass spectrometer
203-1: Mass spectrometer ionization section
203-2: Mass spectrometer
204, 305: Data analysis section
205, 317: Species identification database
206, 306: Control unit
207, 307: Input section
208, 308: Display section
209, 309: Output section
210, 310: Storage device
302: Culture container
311: Bacteria suspension inlet
312: Liquid delivery system
313: Liquid supply system valve
314: Cleaning liquid tank
315: System liquid tank
316: Waste liquid tank

Claims (15)

The microorganism is cultured in a medium to which a drug and / or a metabolic substrate is added, the metabolite produced by the microorganism contained in the medium is subjected to mass spectrometry, and the metabolite determined by the mass spectrometry is determined. based on the type and amount, it is those you identify the type of the microorganism,
The method of testing microorganisms, wherein the metabolite is any one or both of pyruvic acid and lactic acid. 2. The microorganism testing method according to claim 1, wherein the influence of the drug and / or metabolic substrate on the microorganism is further determined based on the type and amount of the metabolite determined by the mass spectrometry. The microorganisms are cultured in a plurality of media with different amounts of the drug added, mass spectrometry is performed on metabolites contained in the media, and the types and amounts of the metabolites are determined between the media. 3. The microorganism testing method according to claim 2 , wherein the antibacterial property of the drug given to the microorganism is determined based on the comparison result. Prepare each sample consisting of a sample with no test drug added to the separately cultured microorganism and a plurality of samples with one or more test drugs added at different concentrations to the separately cultured microorganism. Process,
Culturing each of the samples;
Collecting a medium from which the microorganisms have been removed from each of the cultured samples and performing a pretreatment for mass spectrometry;
Mass spectrometric analysis of the pre-treated medium components;
By analyzing the spectral data obtained by the mass spectrometry, determining the type and amount of metabolites derived from the microorganism, and comparing the plurality of samples with different concentrations of the test drug in the microorganism. 3. The microorganism testing method according to claim 2 , further comprising a step of determining an influence exerted. Preparing each sample of a plurality of samples obtained by adding different metabolic substrates to each separately cultured microorganism;
Culturing each of the samples;
Collecting a medium from which the microorganisms have been removed from each of the cultured samples and performing a pretreatment for mass spectrometry;
Mass spectrometric analysis of the pre-treated medium components;
Analyzing the spectral data obtained by the mass spectrometry, determining the type and amount of metabolites derived from the microorganism, and comparing the samples to identify the microbial species. 2. The microorganism testing method according to claim 1, wherein Each sample contains a stable isotope-labeled compound as a metabolic substrate, and a metabolite labeled with a stable isotope produced from the stable isotope-labeled compound and discharged outside the cells of the microorganism 6. The microorganism testing method according to claim 4 or 5 , wherein analysis is performed. The spectral data obtained by the mass spectrometry, against the database constructed from the spectrum data obtained in various microorganisms, to claim 1 or 5, wherein the identification of the microbial species from the result The microbe inspection method as described. A culture section comprising a culture vessel for culturing microorganisms, comprising a medium supplemented with a drug and / or a metabolic substrate;
A mass spectrometer for analyzing the components of the medium;
Based on the type and amount of metabolites derived from the microorganism in the culture medium determined by said mass spectrometer, comprising an analysis unit you identify the type of the microorganism,
The metabolite is any one or both of pyruvic acid and lactic acid. 9. The microorganism testing system according to claim 8, wherein the analysis unit further determines an influence of the drug and / or metabolic substrate on the microorganism. The culture unit includes a plurality of culture vessels including a plurality of culture media having different amounts of the drug added and / or a plurality of culture media having different types of the metabolic substrates, and the mass spectrometer is configured to include the plurality of culture vessels. Wherein the analysis unit compares the types and amounts of the metabolites among the plurality of media to identify the types of the microorganisms, or the drugs and / or metabolic substrates. 10. The microorganism testing system according to claim 8 or 9 , wherein an antibacterial property given to the microorganism is determined. A culture containing each sample consisting of a sample to which no test drug is added and a plurality of samples to which one or a plurality of types of test drugs are added at different concentrations for culturing separated microorganisms A culture section equipped with a container;
A pretreatment unit for collecting a medium from which the microorganisms have been removed from each of the culture containers of the culture unit and performing a pretreatment for mass spectrometry;
A mass spectrometer for analyzing the components of the medium processed in the pretreatment unit;
A mechanism for transporting each sample and the medium between the culture unit, the pretreatment unit, and the mass spectrometer;
Analyzing the spectral data obtained by the mass spectrometer, determining the type and amount of the metabolite derived from the microorganism, and comparing the type and amount of the metabolite between the samples, An analysis unit for determining the influence of the test drug on the microorganism;
10. The microorganism testing system according to claim 9 , further comprising a control unit for controlling and operating the entire system. A culture section including a culture container containing each sample of a plurality of samples each having a different metabolic substrate added thereto, for culturing the separately cultured microorganisms;
A pretreatment unit for collecting a medium from which the microorganisms have been removed from each of the culture containers of the culture unit and performing a pretreatment for mass spectrometry;
A mass spectrometer for analyzing the components of the medium processed in the pretreatment unit;
A mechanism for transporting each sample and the medium between the culture unit, the pretreatment unit, and the mass spectrometer;
Analyzing spectral data obtained by the mass spectrometer, determining the type and amount of the metabolite derived from the microorganism, and comparing the type and amount of the metabolite between the samples An analysis unit for identifying
9. The microorganism testing system according to claim 8 , further comprising a control unit for controlling and operating the entire system. The culture unit includes an incubator including a medium containing a stable isotope-labeled compound as a metabolic substrate, and the mass spectrometer is stably generated from the stable isotope-labeled compound and discharged out of the microbial cell. 13. The microorganism testing system according to claim 11 or 12 , wherein the metabolite labeled with an isotope is subjected to mass spectrometry. Claim 8 wherein the analyzing unit, the spectral data obtained by the mass spectrometer, against the database constructed from the spectrum data obtained in various microorganisms, and identifying the microbial species from the result Or the microbe inspection system of 12 . 13. The microorganism testing system according to claim 8 , further comprising means for displaying, outputting, or storing a result obtained by the analysis unit.

Images