JPH10210998A - Test of bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for testing bacteria in a shortened time by culturing the bacteria in the presence of an electron transfer agent and chemiluminescently detecting a reaction in which the electron transfer agent is reduced with a redoxase existing in the bacterial cells in the presence of NADH(P) as a coenzyme. SOLUTION: This method for testing bacteria comprises culturing the bacteria in the presence of an electron transfer agent comprising an oxidation type quinone such as menadione, benzoquinone, naphthoquinone, diphenoquinone or anthraquinone and detecting the reduction reaction of the electron transfer agent with a redoxase existing in the cells of the bacteria in the presence of nicotinamide adenine dinucleotide (NAPH) or nicotinamide adenine dinucleotide phosphate (NADHP) as a coenzyme by a chemiluminescent method using a luminescent reagent comprising an acridine derivative, a luminol derivative, a peroxalate ester, etc. The objective clinically important bacterium test such as an antibacterial agent sensitivity test against bacteria or a bacterium- identification test can be executed in a largely shortened time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting bacteria. More specifically, the present invention relates to an antibacterial drug susceptibility test and an identification test of bacteria by a luminescence reaction.

[0002]

2. Description of the Related Art In a bacterial test, an antimicrobial drug susceptibility test and an identification test of bacteria isolated from a test material are very important. However, in most laboratories, the test results are obtained after 18 to 24 hours or more depending on the target bacteria after the bacteria obtained by the separation and culture are separated using a commercially available microbial test reagent. As is

[0003] Antibacterial agents such as antibacterial agents and antituberculous agents (hereinafter abbreviated as agents) against these bacteria for the purpose of selecting a therapeutic agent, etc. for the disease-causing bacteria isolated from the test material.
Susceptibility tests are routinely performed. The method is
A microfluidic dilution method in which a certain amount of bacteria obtained by separation and culturing is inoculated into a medium containing a concentration of a drug that is diluted stepwise and cultured, and then the minimum concentration (MIC) of the drug that inhibited the growth of the bacteria is determined. In addition, a method of applying the above bacteria on the surface of a designated agar medium, placing a disk containing a certain amount of the drug, and classifying interpretation (sensitivity, moderate sensitivity, resistance) of the inhibition circle that appears after culture. These are roughly divided into the above two methods. However, at present, all methods require a longer culture time depending on the bacterial species for 18 to 24 hours.

[0004] Similarly to drug sensitivity tests, tests for identifying bacteria isolated from clinical materials are also routinely performed. The method is based on the following principle, and most facilities are mechanized by a commercially available bacterial identification system. Bacteria inoculated in an artificial medium grow by enzymatically metabolizing various nutrients in the medium. There are many types of enzymes, some of which are known to be used as indicators for identifying bacteria. Enzymatic reactions are detected by observing changes associated with the consumption of a particular substrate. If the enzyme reaction is substrate-specific, the presence of the enzyme can be confirmed using a medium to which the enzyme substrate has been added. Specifically, for example, an enzyme that metabolizes a carbon source can be regarded as a change in the pH of a medium when an acid is generated by enzymatic decomposition of a saccharide as a substrate. Enzymes that metabolize nitrogen sources also produce p when ammonia is produced along with the decomposition of amino acids as substrates.
H can be regarded as a change. Commercially available bacterial identification systems calculate the possibility of which strain the test strain belongs to based on the combination of enzymes that serve as identification indicators. This method requires growth of the inoculated microorganism. Therefore, in order to obtain reliable results, it takes 18 to 24 hours for culturing for the growth of microorganisms, and more time may be required depending on the species.

Prior to obtaining these drug sensitivity / identification test results, treatments such as drug administration are performed as predictive treatments based on the experience of clinicians, but in some cases this may be accompanied by continuation of the disease and deterioration of the condition. . In particular, the emergence of drug-resistant bacteria due to the frequent use of drugs and the increase of imported infectious diseases due to the increase in overseas travelers have become a problem in recent years, and information for making decisions on treatments that can accurately cure patient conditions has been made. There is a strong demand for the development of a method for testing and identifying bacterial drug susceptibility that can be rapidly provided to clinicians.

[0006] Many methods have been published to solve this problem. For example, a method of adding a tetrazolium salt to a medium containing a drug, culturing the bacterium, determining the metabolic activity of the bacterium by coloring by reduction of the tetrazolium salt, and using the results to determine the sensitivity of the drug (Japanese Patent Laid-Open No. 55-39763, JP-A-1-
187097). Also, there is a method in which a fluorogenic substrate such as resazurin is allowed to act during the growth of bacteria, and the drug sensitivity is determined by quantifying the fluorescence intensity of the produced fluorescent substance (Japanese Patent Application Laid-Open No. 2- 211899).

As a commercialized product, there is a Sensititer (trade name) system developed for the purpose of MIC measurement. In this method, the fluorescence intensity of a fluorescent substance produced during the growth of bacteria is measured after 5 hours of culture by causing a fluorogenic substrate to act on the bacteria, and the result is analyzed with dedicated software to calculate the MIC for the bacteria. It is a system that can do it. However, this method was found to be unreliable for slow growing strains (typically several strains of non-glucose non-fermenting bacteria) and those that achieved sufficient fluorescence levels in a very short time. Have been.
As a method of compensating for these disadvantages, drug sensitivity information can be obtained by performing continuous measurements (3 to 15 hours) and analyzing the information obtained therefrom with dedicated software, rather than just a single early measurement. In recent years, a method has been announced (Japanese Patent Publication No. 7-114708). However, these methods still have a problem that a long culture time (15 to 18 hours) is still required particularly for a slow-growing bacterial species.

In a high-sensitivity detection system utilizing color development or fluorescence, menadione is used as an electron transfer agent or a redox accelerator. Since menadione has a growth inhibitory effect on bacteria, its concentration must be carefully set. Japanese Patent Application Laid-Open No. 2-211898 discloses that the optimal concentration of menadione is 1 to 10 μg /
The bacterium is cultured by adding it to a broth in advance together with a fluorescent substrate, resazurin, after defining the concentration to preferably 2.5 to 7.5 μg / ml. Although this method is effective for Gram-negative bacilli described in Examples, sufficient growth results cannot be obtained unless the concentration of Menadione is 1 μg / ml or less for Gram-positive cocci having high sensitivity to menadione.

The present inventors set the concentration of menadione for gram-positive cocci to a concentration that does not hinder growth and disclosed in Japanese Patent Laid-Open No. 2-121898.
A drug sensitivity test was attempted by the method described above. As a result, 10 ⁶ C
It was found that false positives occurred when the test was performed at an inoculum amount of FU / ml or more. Gram-positive cocci have a stronger reducing ability of the cells themselves than gram-negative bacilli, so they are affected by the reducing ability of the cells at the time of inoculation of the cells and give rise to false positives regardless of drug sensitivity. It is considered that the grades become resistant. When one or both of menadione and resazurin were added after cell culture, the control bacterium could not be detected due to insufficient sensitivity, and the drug susceptibility test was impossible.

[0010] Examples of a highly sensitive detection system capable of further reducing the culture time include those utilizing bioluminescence and chemiluminescence. Japanese Patent Application Laid-Open No. 8-103293 discloses a method in which a luminescent reagent of luciferin / luciferase is allowed to act on ATP derived from a living bacterium to perform a drug sensitivity test by a luminescent reaction. The culturing time can be reduced to about 4 to 5 hours. But,
ATP by collecting bacteria on the filter or destroying the cells
The operation is complicated as compared with the conventional method, for example, the operation of extracting Similarly, there is a method in which bacterial cells are destroyed by phage (Japanese Patent Application Laid-Open No. 8-503847). Utilization of phage increases specificity to bacteria, but handling biological material called phage makes operation more complicated than the above-mentioned method.

[0011] In principle, the chemiluminescence method inhibits the growth of bacteria by six.
It is a method having a higher measurement sensitivity than any of the turbidity method for quantification by absorbance at 30 nm, the color development method using a redox indicator such as a tetrazolium salt, and the fluorescence method using a fluorogenic substrate. Therefore, it is considered that the drug sensitivity test method employing this measurement principle can reduce the culture time more than any of the conventionally published methods.

As a detection system utilizing chemiluminescence, there is Japanese Patent Publication No. 6-30628. In this method, oxidized quinone is allowed to act on cells, and hydrogen peroxide generated by the reducing action of a cell membrane enzyme is detected by a luminescent reagent. The examples show that this method gives good results in yeast. However, for large bacteria such as yeast, the amount of enzyme that exhibits a reducing action is sufficient, so that luminescence intensity proportional to the amount of bacterial cells can be obtained.However, bacteria to be subjected to clinical tests are smaller than yeast, and this method lacks sensitivity. And cannot be measured.

[0013] Menadione can be added at the start of culture to eliminate the sensitivity deficiency. However, the growth inhibitory effect of menadione on bacteria and the reduction of bacterial cells in the initial stage of culture in a drug sensitivity test are mentioned. In addition to the effect, peroxidase and catalase derived from bacterial cells during culture,
It must be taken into account that the generated hydrogen peroxide is consumed. The most advantageous chemiluminescence system in terms of sensitivity is also a reaction system via hydrogen peroxide, but a bacterial test method taking advantage of the advantage has not been put to practical use.

As described above, there has been no rapid bacterial test method which has sufficient performance in both sensitivity and accuracy and is applicable to a wide range of clinical bacteria.

[0015]

The problem to be solved by the present invention is to solve a wide range of clinical bacteria (intestinal bacteria, non-glucose nonfermenting bacteria, gram-positive cocci, hemophilus, etc.) utilizing chemiluminescence. (Acid bacteria group).

[0016]

Means for Solving the Problems As a result of intensive studies, the present inventors have completed a test method which can be applied to a wide range of clinical bacteria utilizing chemiluminescence.

The present invention comprises the following bacterial test method or its application. (1) The bacterium is cultured in the presence of an electron transfer agent, and the oxidoreductase present in the cells reduces the electron transfer agent using nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. A bacterial test method characterized by detecting by chemiluminescence. (2) The bacteria test method according to (1), wherein hydrogen peroxide generated by reduction of the electron transfer agent by the oxidoreductase is detected. (3) The method according to (2), wherein the electron transfer agent is an oxidized quinone. (4) The method according to (3), wherein the oxidized quinone is selected from the group consisting of menadione, benzoquinone, naphthoquinone, diphenoquinone, and anthraquinone. (5) The method for testing bacteria according to (4), wherein the oxidized quinone is menadione. (6) The bacteria test method according to (5), wherein the bacteria to be tested are gram-positive cocci. (7) When the concentration of menadione is 0.01 μg / ml or more,
(6) The method for testing a bacterium according to (6), wherein the range is less than μg / ml. (8) The concentration of menadione is 0.5 μg / ml or more and 1 μm
The bacteria test method according to (7), which is less than g / ml. (9) an acridine derivative whose luminescent reagent contains lucigenin,
(1) The method for testing bacteria according to (1), which is selected from the group consisting of a luminol derivative and a peroxalate. (10) The method for testing bacteria according to (9), wherein lucigenin is used as the acridine derivative. (11) Bacteria are cultured in the presence of an antibacterial agent and an electron transfer agent, and the oxidoreductase present in the cells reduces the electron transfer agent using nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. A method of testing antimicrobial drug susceptibility, characterized in that the action of the drug is detected by chemiluminescence. (12) The bacterium is cultured in the presence of the substrate of the substrate-specific enzyme and the electron transfer agent, and the substrate-specific enzyme present in the cells is converted to nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. An identification test method, wherein the action of reducing a transfer agent is detected by chemiluminescence. (13) A kit for performing the antimicrobial drug susceptibility test method according to (11), wherein a luminescent reagent is combined with a nutrient necessary for the growth of bacteria, an antimicrobial agent, and a medium containing an electron transfer agent. (14) A kit for performing the identification test method of (12), wherein a luminescent reagent is combined with a nutrient necessary for the growth of bacteria, a substrate of a substrate-specific enzyme, and a medium containing an electron transfer agent.

Bacteria inoculated in a broth dedicated to each group of bacteria containing a certain amount of the drug take in extracellular nutrients into the cells and start to proliferate by the action of various oxidoreductases present in the cells. . Some of the oxidoreductase reactions acting at that time require the intervention of reduced coenzyme nicotinamide adenine dinucleotide (NADH) or its phosphate compound (NADPH). Therefore, it is presumed that when the substance to be reduced existing outside the cells is reduced by the enzyme present in the cells, dissolved oxygen is simultaneously reduced via the electron transfer agent present in the broth to produce hydrogen peroxide. You.
It is considered that the production amount of hydrogen peroxide increases at an accelerating rate as the bacteria grow (according to the principle of enzyme cycling). The present invention is based on the principle that the produced hydrogen peroxide is quantified by a chemiluminescence method and the test result is obtained more quickly.

That is, the present invention has realized a method for detecting bacteria by a chemiluminescence reaction with the configuration described in (1). The test method of the present invention is not limited to Gram-negative bacteria and Gram-positive bacteria, but can be applied to a wide variety of bacteria. Above all, a major feature of the present invention is that it can be applied to Gram-positive cocci, for which a test based on a conventional chemiluminescence reaction was difficult. The most important feature of the present invention resides in culturing bacteria in the presence of an electron transfer agent. Oxidized quinone can be used as the electron transfer agent. Specifically, menadione, benzoquinone, naphthoquinone, diphenoquinone,
Oxidized quinones such as anthraquinone are known. Among these oxidized quinones, menadione is suitable because it rapidly increases the luminescence intensity after the addition of the luminescence reagent. In addition, menadione can be easily set to a concentration that does not inhibit the growth of various bacteria. For example, even in gram-positive cocci that are sensitive to menadione, the concentration of menadione in the culture environment is 0.01 μg / ml or more,
Less than 1 μg / ml, desirably 0.5 μg / ml or more,
Bacteria can be tested by using less than 1 μg / ml. On the other hand, for bacteria not sensitive to menadione (such as E. coli and K. pneumoniae), 1 μg / ml or more,
It is preferable to use a concentration of less than 10 μg / ml. The oxidoreductase used as an indicator in the test method of the present invention uses a coenzyme as a hydrogen acceptor and is an enzyme known as an indicator of bacterial growth and bacterial species. Such oxidoreductases include sugars and amino acids. Examples include various dehydrogenases such as glucose dehydrogenase and arabinose dehydrogenase that specifically decompose substrates such as organic acids. In the present invention, since the oxidation-reduction reaction is detected by a chemiluminescence reaction, it is not always necessary to cause a change in the medium pH by an enzyme reaction. The coenzymes used in the present invention are combined with those required by the oxidoreductase selected as an indicator. As a general coenzyme,
NAD or NADP can be indicated.

In the present invention, menadione is reduced by an enzyme present in the cells, so that the concentration of menadione and the timing of its addition are important points. Menadione must be added to the medium in advance in order to perform a drug susceptibility test on Gram-negative bacilli, which have a low cell-reducing property. In addition, the concentration of menadione is 0.01 μg / m so as not to hinder the growth of gram-positive cocci having high sensitivity to menadione.
ml or more and less than 1 μg / ml, preferably 0.5 μg / ml.
It is advisable to add the medium to the culture medium in an amount of at least 1 ml / ml and less than 1 μg / ml.

The luminescent reagent used in the present invention is not particularly limited as long as it emits light by hydrogen peroxide and is not affected by drugs or specific enzymes. Such luminescent reagents include acridine derivatives, luminol derivatives, oxalic acid derivatives, and the like. Luminescent reagents of acridine derivatives include lucigenin, various acridinium salts and various acridinium esters, and luminol derivatives include luminol and isoluminol. Examples of the oxalic acid derivative include chloride, ester, and oxamide. When an oxalic acid derivative is used as a luminescent reagent, it is necessary to coexist a fluorescent substance such as perylene or pyrene. In addition, the luminescent reagent may be added with sodium azide for inhibiting the activity of an enzyme consuming hydrogen peroxide, or metal ions, oxides or complexes such as iron, manganese, or nickel for promoting the luminescent reaction. preferable.

In the present invention, the luminescent reagent needs to be added after the culturing of the cells in order to avoid the influence of the reducing properties of the cells at the beginning of the culture. With conventional coloring or fluorescent reagents, if added after cultivation, the sensitivity becomes insufficient and measurement cannot be performed. However, chemiluminescent reagents have high sensitivity and can be sufficiently measured even after addition after culturing.

A known broth can be used as the medium for detecting bacterial growth for carrying out the present invention. The culture medium contains bacterial nutrients and substances, inorganic salts and the like. Substances serving as nutrient sources for cells include glucose, yeast extract, various amino acids, sodium pyruvate, sodium citrate and the like. Examples of the inorganic salts include sodium chloride, monopotassium phosphate, disodium phosphate, magnesium sulfate, calcium chloride and the like. These inorganic salts also function as osmotic pressure adjusting agents and buffers. Specifically, an M70 medium and the like can be mentioned.

The present invention can be applied to a rapid drug susceptibility test for bacteria. The components required to carry out this method are various nutrients necessary for the growth of bacteria, specified concentrations of drugs, electron transfer agents for protons released from coenzymes, and peroxidation produced. It is a chemiluminescent substrate for quantifying hydrogen. The former three are desirably contained in a stable state in a microtiter plate inoculated with the bacteria to be subjected to the drug sensitivity test. The microtiter plate is inoculated with the bacteria to be tested. After performing the culture specified for each bacterial group, the amount of chemiluminescence at that time is detected while adding a luminescent substrate using a dedicated microtiter plate reader. At that time, since the effect of the drug is not recognized, the plate well in which the bacteria are grown obtains a large luminescence. However, plate wells in which bacterial growth has been inhibited by the drug have luminescence values that are minimal or have no luminescence. Then, based on these measured luminescence values, the minimum growth inhibitory concentration of each drug is determined by a predetermined performance analysis method. The culture time for the drug susceptibility test of each bacterial group is 35 ° C for 4 hours for the enteric bacteria group, non-glucose non-fermenting bacteria group, Gram-positive cocci group, and Haemophilus bacteria group. A good agreement with the results of the method in which the majority of the strains are currently widespread can be obtained.

In the present invention, the following substances can be used as drugs that can be used in a drug sensitivity test. These drugs are usually distributed as salts. In the examples described later, abbreviations in parentheses are used. Reagents (abbreviation) Penicillin G (PCG) Ampicillin (ABPC) Piperacillin (PIPC) Oxacillin (MPIPC) Cefazolin (CEZ) Cefotiam (CTM) Ceftazidime (CAZ) Cefthrosin (CFS) Cefotaxime (CTX) Cefaclor (CCL) Aztreo AZT) Flomoxef (FMOX) Latamoxef (LMOX) Imipenem (IPM) Meropenem (MEPM) Clindamycin (CLDM) Amikacin (AMK) Arbekacin (ABK) Minocycline (MINO) Tobramycin (TOB) Vancomycin (VCM) Eromycin (VCM) Phenicol (CP) Ofloxacin (OFLX) Levofloxacin (LVFX)

The present invention is also applicable to a rapid bacterial identification test method for bacteria, and can be specifically carried out as follows. The components required for conducting the identification test are a substrate for confirming the presence of a specific enzyme, a reaction environment regulator for facilitating the enzymatic reaction to the substrate, and electron transfer of protons released from the coenzyme. It is a luminescent reagent that quantifies the agent and the produced hydrogen peroxide. It is desirable that the former three be contained in a microtiter plate in a stable state in advance similarly to the drug sensitivity test. The microtiter plate is inoculated with the bacteria requiring testing. After incubation at 37 ° C. for 2 hours, the amount of chemiluminescence is measured while adding a luminescent reagent using a dedicated microplate reader. At that time, if no specific enzyme is found in the substrate, the luminescence is almost equal to or less than that of the control well. However, a well in which a substrate-specific enzyme is confirmed can obtain a larger amount of luminescence than a control well. The names of the bacterial species are determined from these measured luminescence values by a predetermined performance analysis method. In the present invention, since the activity of the substrate-specific enzyme is directly measured, the incubation time may be as short as about 120 minutes, and the results obtained by this method are in high agreement with the methods currently used in many facilities. Rate can be obtained.

[0027]

EXAMPLES Examples of the present invention will be described below, but the examples are for the purpose of detailed explanation and are not intended to limit the inventive concept. Example 1 (rapid drug susceptibility test) Escherichia coli (hereinafter referred to as E. coli) which is one of the precision control strains of the microfluidic dilution method currently used in most facilities.
Sufficiently cultured ATCC25922 colonies were suspended in sterile saline and the concentration was adjusted to McFarland.
An adjustment solution equivalent to the No. 1 turbidity standard was prepared. (Nominal bacterial concentration of about 3 × 10 ⁸ CFU / ml) In order to make the above-mentioned adjusted liquid a nominal concentration of about 3 × 10 ⁶ CFU / ml, it was further diluted 100-fold with sterile physiological saline and used as a test bacterial liquid. . The test bacterial solution was inoculated in an amount of 100 μl / well into a chemiluminescence microtiter plate previously containing a drug and the following medium for bacterial growth in a stable form. [Medium components] Yeast extract 0.5 g Glucose 1.0 g Sodium chloride 5.0 g Monopotassium phosphate 4.0 g Disodium phosphate 2.0 g NAD 0.2 g Menadione 0.5-10 mg Water 1000 ml Finished inoculation of bacteria The microtiter plate was cultured for 4 hours in a special incubator. After the culture, the microtiter plate was set in a special chemiluminescence measurement microplate reader (manufactured by Dynatech: ML-3000), and 65 μl of a luminescent reagent having the following composition was simultaneously dispensed into each well. The amount of luminescence from 0 to 1 second after the addition of the reagent was measured. [Luminescent reagent A] Lucigenin 0.15 g water 1000 ml [Luminescent reagent B] sodium bicarbonate 67.0 g potassium hydroxide 48.0 g water 1000 ml (pH 11.0) Table 1 shows wells containing no drug (PC) And the chemiluminescence values of the wells containing the specified concentration of each drug. (The upper row shows the drug name, the middle row shows the drug concentration (unit: μg / ml), and the lower row shows the measured values.)

[0028]

[Table 1]

In Table 1, P.I. C. The MIC value of each drug calculated based on the amount of luminescence is shown below. In addition, the MIC values obtained by the micro-liquid dilution method currently used in many facilities are also shown below. In the present invention, the MIC was calculated as follows: the sample luminescence value was 20% or more of the positive control luminescence value,
% Was regarded as growth negative (growth inhibition).

[0030]

[Table 2]

Example 2 (Rapid drug susceptibility test) A drug susceptibility test was carried out in the same manner as in Example 1 for bacteria considered to be clinically important isolated from clinical materials from patients. The test bacteria and the culture time are as shown below.

[0032] The culture time is 4 hours for the chemiluminescence method (the present invention) and 20 hours for the microfluidic dilution method (the conventional method). * Because a sufficient increase or growth of luminescence was not observed in some strains, judgment was made after culturing for 6 hours in the chemiluminescence method and after culturing for 45 hours in the trace liquid dilution method.

Table 3 shows a comparison of results with the microfluidic dilution method. The efficacy is reported by the agreement between the results of the microfluid dilution method and the results of the method of the present invention (chemiluminescence method). Regarding the interpretation of the matching rate of the results, the results (MIC values) of both methods were regarded as the results matching up to one tube difference, and the results were mismatched when the difference was two tubes or more. As shown in Table 3, the results of the drug susceptibility test for clinical bacteria according to the present invention were 1/6 to 1/6 of the time for the microfluidic dilution laboratory currently used in many facilities.
Despite being shortened to 1/10, the result is 80
% Or more was consistent.

[0034]

[Table 3]

Example 3 (Rapid Identification Test) The degrading enzyme activities of various bacteria (target enzyme was lysine dehydrogenase) using L-lysine hydrochloride as a substrate were tested by the following method. The following strains, which are separated and stored from clinical materials and whose strains have been identified in advance, are subcultured, and a sufficient number of colonies obtained are sterilized in physiological saline at a concentration of McFarland No. 1 turbidity. Adjusted to be standard. (Nominal bacterial concentration 3 × 10 ⁸ CFU / ml) 1) E. coli 2) C. freundii 3) K. pneumoniae 4) E. aerogenes L-lysine hydrochloride (substrate) 1 mg / ml, M70 medium, menadione 0. In a microtiter plate well containing 8 μg / ml, 100 μl of the above bacterial cell solution was added.
Inoculate. At the same time, microtiter plate wells containing only L-lysine hydrochloride are similarly inoculated. After incubating at 35 ° C. for 120 minutes, the amount of chemiluminescence in each well is measured using a dedicated microplate reader as in the drug sensitivity test. Table 4 shows the measurement results (measured luminescence values). When the luminescence value of the substrate-added well showed a value three times or more the luminescence value of the substrate-free well, it was determined that the substrate was used positively.

[0036]

[Table 4]

From the results shown in Table 4, the activity of the enzyme for degrading the present substrate was observed in E. coli, K. pneumoniae and E. aerogenes, but not in C. freundii. Therefore, by using L-lysine hydrochloride as a substrate and measuring the enzymatic activity for decomposing it, it was possible to identify only C. freundii in a short period of time from the above bacterial groups.

Example 4 (Rapid Identification Test) When citric acid was used as a substrate, various bacterial degrading enzyme activities (the target enzyme was citrate dehydrogenase) were tested by the same method as described above. The method for preparing the bacterial solution and the test strain were the same as in Example 3. The target substrate was citric acid (1 mg / ml), and the method of adding the necessary components to other microtiter plates and the method of inoculation were performed in the same manner as in Example 3. Incubation and measurement conditions were performed in the same manner as in Example 3. Table 5 shows the measurement results.

[0039]

[Table 5]

From the results shown in Table 5, the activity of the enzyme for degrading the present substrate was observed in C. freundii, K. pneumoniae and E. aerogenes but not in E. coli. Therefore, by measuring the enzymatic activity of decomposing it using citric acid as a substrate, it is possible to quickly
Only E.coli could be identified.

[0041]

As described above, the present invention is an invention for a method of clinical bacterial examination. Bacterial testing methods currently used in most facilities use bacteria that have been isolated and cultured from clinical materials of patients for 18 to 24 hours or more depending on the type of bacteria to obtain test results. .
However, when the test method according to the present invention is used, most bacteria considered to be clinically important are cultured for 4 hours in a drug susceptibility test (some bacteria require 6 hours in rare cases).
In the identification test, the results can be obtained within about 2 hours (incubation time varies depending on the target substrate), which is almost the same as that of the method currently used. Reducing the time spent on examinations would allow for more appropriate patient treatment. Further, a wide application range is also a feature of the present invention. For example, the present invention can be applied to bacterial species such as gram-positive cocci, for which chemiluminescent reaction could not be applied by conventional methods. Therefore,
The present invention can greatly contribute to the research and development in the medical field and the development of the actual patient treatment field.

Claims (14)

[Claims] 1. A method of culturing bacteria in the presence of an electron transfer agent, wherein an oxidoreductase present in the cells reduces the electron transfer agent using nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. A bacterial test method, wherein the bacteria are detected by chemiluminescence. 2. The method according to claim 1, wherein hydrogen peroxide generated by reduction of the electron transfer agent by the oxidoreductase is detected. 3. The method according to claim 2, wherein the electron transfer agent is an oxidized quinone. 4. The method according to claim 3, wherein the oxidized quinone is selected from the group consisting of menadione, benzoquinone, naphthoquinone, diphenoquinone, and anthraquinone. 5. The method according to claim 4, wherein the oxidized quinone is menadione. 6. The method according to claim 5, wherein the bacteria to be tested are gram-positive cocci. 7. The method according to claim 6, wherein the menadione concentration is in the range of 0.01 μg / ml or more and less than 1 μg / ml. 8. The method according to claim 7, wherein the concentration of menadione is in the range of 0.5 μg / ml or more and less than 1 μg / ml. 9. The method according to claim 1, wherein the luminescent reagent is selected from the group consisting of acridine derivatives, luminol derivatives, and peroxalates. 10. The method according to claim 9, wherein lucigenin is used as the acridine derivative. 11. A bacterium is cultured in the presence of an antibacterial agent and an electron transfer agent, and the oxidoreductase present in the cells is nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. A method for testing sensitivity to antibacterial drugs, which comprises detecting an action of reducing lipase by chemiluminescence. 12. A bacterium is cultured in the presence of an electron transfer agent and a substrate of a specific enzyme which serves as an indicator for identifying the bacterium, and the enzyme present in the cell is nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. An identification test method comprising detecting, by chemiluminescence, an action of reducing an electron transfer agent by using as a coenzyme. 13. A kit for carrying out the method for testing antimicrobial drug susceptibility according to claim 11, wherein a luminescent reagent is combined with a nutrient necessary for the growth of bacteria, an antimicrobial agent, and a medium containing an electron transfer agent. 14. The identification test method according to claim 12, wherein a luminescent reagent is combined with a nutrient necessary for the growth of bacteria, a substrate of a specific enzyme serving as an indicator of bacterial identification, and a medium containing an electron transfer agent. Kit to do.