JP6722751B2 - Drug sensitivity test system - Google Patents

Description

The present invention relates to evaluation of bacterial growth. In particular, it relates to a method, device and system for carrying out a drug sensitivity test of bacteria.

With the increase in the number of deaths due to infectious diseases and the emergence of drug-resistant bacteria, attention has been focused on speeding up drug susceptibility testing of infectious disease-causing bacteria. Conventionally, drug susceptibility tests have been carried out based on culture methods. After collecting samples of blood, pharyngeal swab, sputum, etc. from patients with infectious diseases, separate culture is performed overnight to obtain infectious disease-causing bacteria as a single colony from samples containing indigenous bacteria. Further, the bacteria that have formed a single colony are prepared at a fixed concentration, and then distributed in a container in which various concentrations of drugs and antibiotics are placed, and drug-sensitive culture is performed overnight. Then, after culturing, the result of the drug susceptibility test of the infectious disease-causing bacterium is obtained based on the presence or absence of proliferation of the bacterium, and the patient is appropriately dosed based on the result. Therefore, appropriate medication is given to patients with infectious diseases from the third day after the sample collection.

On the other hand, the ATP bioluminescence method, in which the amount of change in ATP (Adenosine Triphosphate) existing as an energy source in the bacterium is used as an index for the growth of the bacterium, is noted as a rapid method for drug sensitivity test Has been done. The ATP method is a method of detecting ATP existing as an energy source in bacteria by using an enzyme luciferase derived from firefly. Luciferase oxidizes the substrate luciferin in the presence of ATP and Mg ²⁺ in the bacterium, and the amount of luminescence generated at that time is proportional to the amount of ATP. Therefore, the growth of the bacterium can be evaluated from the change in the amount of luminescence. As a method for determining the number of bacteria using the ATP method, for example, in Patent Document 1, the number of viable bacteria is counted by ATP measurement, the total number of bacteria is counted by the DNA method, and the number of viable bacteria is subtracted from the total number of bacteria to determine the number of viable bacteria. And a technique for obtaining the number of killed bacteria are disclosed.

Japanese Patent Laid-Open No. 08-304402

In the conventional proliferation determination of bacteria using the ATP method, free ATP in a sample is erased, and only ATP in living cells (viable ATP) is evaluated. In this method, since the growth of bacteria is determined by the increase/decrease of live bacteria ATP with the passage of culture time, it is necessary to carry out multiple measurements of live bacteria ATP during the drug-sensitive culture. It is time-consuming and difficult to obtain drug sensitivity results quickly.

As an aspect of the present invention for solving at least one of the above-mentioned problems, a drug susceptibility testing apparatus includes a measuring unit that measures ATP luminescence of a culture solution containing a drug and a bacterium, and a culture solution measured by the measuring section. And a determination unit that determines whether or not the bacteria are susceptible to the drug, based on the amount of ATP luminescence derived from the dead bacteria.

According to the present invention, an ATP drug susceptibility test can be carried out quickly and conveniently, thereby enabling prompt and proper administration of antibiotics to patients with infectious diseases. Problems, configurations and effects other than those described above will be clarified by the following description of the embodiments.

The figure which shows the structural example of the bacterial culture solution used as the target of a chemical sensitivity test. The figure which shows typically the sample preparation method for measuring live-cell ATP and dead-cell ATP. A table showing ATP which can be measured by each method. The graph which shows an example of the time-dependent change of the live bacterium ATP luminescence amount and dead bacteria ATP luminescence amount in the absence of antibiotics. The graph which shows an example of a time-dependent change of the live-cell ATP luminescence amount and dead-cell ATP luminescence amount in presence of an antibiotic. The flowchart which shows an example of the processing process by a chemical sensitivity test system. The flow figure which shows an example of the measurement process of the killed-bacteria ATP luminescence amount by a chemical susceptibility test system. The figure which shows the structural example of the chemical|medical agent sensitivity test system in a present Example. The figure which shows the internal structure of a measuring device. FIG. 3 is a block diagram showing an internal configuration of a killed bacteria/viable bacteria comparison unit. It is a table which shows the example of the database used as the determination standard of a drug sensitivity test. It is a graph which shows the example of the ATP amount ratio with progress of the culture time of E. coli. It is a graph which shows the example of the ATP amount ratio with progress of the culture time of S. aureuS.

Hereinafter, preferred embodiments of the present invention will be described by showing examples with reference to the drawings. However, the dimensions, materials, shapes, and other relative arrangements of the systems, devices, devices, members, and the like described in this embodiment are limited to the scope of the present invention unless otherwise specified. This is not an intent to do so, but merely an example of explanation.

The bacterial culture solution 1 to be the target of the drug sensitivity test of this example will be described with reference to FIG. In the bacterial culture liquid 1, various bacteria such as live bacteria 2, dead bacteria 3 without damage to the cell membrane, dead bacteria 4 with damage to the cell membrane, etc. are present. In the bacterial culture liquid 1, the live bacteria 2 and the dead bacteria 3 without damage to the cell membrane encapsulate ATP in the cell membrane. On the other hand, in the killed bacterium 4 with damaged cell membrane, ATP leaks from the damaged portion of the cell membrane as the damage of the cell membrane progresses, so that ATP derived from killed bacterium 4 with damaged cell membrane exists as free ATP5 in the culture solution.

Next, a method for measuring ATP derived from live cells and dead cells will be described with reference to FIG. The viable bacterial ATP method of the method (1) eliminates ATP5 outside the bacterial cells, and ATP2000 encapsulated in the cell membrane of viable cell 2 and ATP3000 encapsulated in the cell membrane of dead cell 3 in which the cell membrane is not damaged. It is a method of measuring. The submerged ATP method of the method (2) is a method of measuring the dead bacteria 4 and the free ATP5 in which the cell membrane is damaged in the culture liquid 1 while containing the cells of the live bacteria 2 and the dead bacteria 3 and 4. The killed bacterium 4 is a measurement target because the substrate and the enzyme of the enzyme luciferase reaction, which is a bioluminescent molecule described later, enter from the damaged site. The filtrate ATP method of the method (3) removes the bacterial cells 2, 3 and 4 by filtering the culture broth 1 by centrifugation using a filtering container 12 described later, and free ATP5 contained in the filtrate 1000 is filtered. Is a method of measuring. The whole ATP method of the method (4) includes ATP2000 encapsulated in the cell membrane of live cell 2, ATP3000 encapsulated in the cell membrane of dead cell 3 with no cell membrane damage, and ATP2000 in the cell membrane of killed cell 4 with damaged cell membrane. It is a method of extracting the remaining ATP4000 and the free ATP5 and measuring the combined amounts.

ATP in the culture solution 1 which can be measured by the above methods (1) to (4) is shown in FIG. In the method (1), the ATP encapsulated in the cell membrane of a live bacterium (with cell membrane) and the ATP encapsulated in the cell membrane of a dead bacterium (with cell membrane) can be measured. In the method (2), ATP encapsulated in the cell membrane of dead bacteria (damage to cell membrane) and free ATP derived from dead bacteria (damage to cell membrane) can be measured. In method (3), free ATP derived from dead bacteria (damage to cell membrane) can be measured. In the method (4), ATP encapsulated in the cell membrane of live cells (with cell membrane) and ATP encapsulated in the cell membrane of dead cells (with cell membrane) and ATP entrapped in the cell membrane of dead cells (cell membrane damage) and dead cell Free ATP derived from (cell membrane damage) can be measured.

Thus, since ATP derived from killed bacteria that is present in the culture solution 1 as free ATP5 is included in the methods (2) to (4), it is determined by the methods (2) to (4) based on the amount of free ATP5. Killed bacteria ATP can be measured.

Next, with reference to FIGS. 4a and 4b, an example of the results of measuring the amount of ATP by treating Escherichia coli by methods (1) to (4) is shown. The ATP method is bioluminescence detection of ATP existing as an energy source in bacteria using a firefly-derived enzyme luciferase. Luciferase oxidizes luciferin that is a substrate in the presence of ATP and Mg ²⁺ in the bacterium, and the amount of luminescence generated at that time is proportional to the amount of ATP. Therefore, the growth of the bacterium is evaluated from the change in the amount of luminescence.

In the present embodiment, the light emission amount is represented by integrating the photon count value of 60 seconds, and the unit is the light emission amount (Amount of luminescence (a.u.)). As described above, the light emission amount and the ATP amount are in a proportional relationship, and when the light emission amount is increasing, it is determined that the ATP amount is increasing.

FIG. 4a shows the result of culturing Escherichia coli in the absence of antibiotics, and FIG. 4b shows the result of culturing E. coli in the presence of 8 μg/mL of ampicillin (hereinafter referred to as ABPC) which is an antibiotic. Comparing the results of FIG. 4a and FIG. 4b, in FIG. 4a, the luminescence amount of the live bacterial ATP method of method (1) increases with the culturing time until the culturing time reaches 4 hours, and then the culturing time 5 At the time of 6 hours, the liquid tends to be saturated, while the in-liquid ATP method of the method (2) to (4) all-ATP method shows the same tendency. On the other hand, in FIG. 4b, the luminescence amount of viable ATP of the method (1) decreases with the lapse of culture time (after 3 hrs), whereas the luminescence of the method (2) in-liquid ATP method to (4) total ATP method It shows a tendency that the amount increases or does not change significantly.

This indicates a reaction in which the antibiotic ampicillin acts on Escherichia coli at a culturing time of 4 hours to stop the growth of viable bacteria and cause the bacteria to die over time. ATP derived from killed bacteria leaks out from the damaged portion of the cell membrane as the cell membrane is damaged, so that the ATP in the bacterial culture increases. The reason why the amount of luminescence is constant in the liquid of method (2), the filtrate of (3), and the total ATP of (4) after 4 hours of incubation is that the ATP derived from killed bacteria increases. This is the result showing that

Next, an example of processing steps by the drug susceptibility test system in this example when obtaining live ATP or dead ATP will be described with reference to FIG. The bacterium to be measured in the test is not particularly limited as long as the bacterium contains ATP.

Specimens such as blood, pharyngeal swab, and sputum are collected from patients with infectious diseases (S501), and isolated culture is performed for about 24 hours all day and night to obtain infectious disease-causing bacteria as a single colony from specimens containing indigenous bacteria (S502). After the isolation culture, the bacteria that form a single colony are adjusted to a constant concentration (S503), and then distributed in a container in which various concentrations of drugs and antibiotics are placed (S504), and drug-sensitive culture is performed (S505).

Here, the material and shape of the distribution container used in S505 are not particularly limited, but a plate-shaped one having a plurality of holes (wells) on a plane is desirable, and 96 wells in total of 8 wells×12 wells are integrated. A 96-well microplate, a 384-well microplate in which 16 wells x 24 wells are integrated in a total of 384 wells, a 15-well microplate in which 32 wells x 48 wells are integrated in a total of 1532 wells, and the like are used.

The drug/antibiotic used for the drug-sensitive culture is not particularly limited, but an antibiotic having a bactericidal action such as penicillin series, cephem series, aminoglycoside series, new quinolone series, fosfomycin series is desirable.

After carrying out the drug-sensitive culture for several hours, the viable ATP derived from viable bacteria contained in the culture solution is measured by a viable ATP measurement flow. In addition, killed bacteria ATP derived from killed bacteria in the culture solution is measured by a killed bacteria ATP measurement flow. The drug sensitivity of bacteria is determined based on the measurement results of these measurement flows.

The live bacteria ATP measurement flow ATP includes an ATP elimination step S506, an ATP extraction step S507, and a luminescence measurement S508. In the ATP erasing step S506, the free ATP outside the bacterial cells is removed. The method of removing free ATP is not particularly limited, and examples thereof include a method using an ATP-degrading enzyme such as apyrase, and a method of removing by filter filtration. In the ATP extraction step S507, the cell membrane is crushed and ATP is extracted out of the membrane. The method of crushing the cell membrane is not particularly limited, and examples thereof include a method of crushing by adding a surfactant or the like, an ultrasonic irradiation method, a crushing method using a French press or a homogenizer, and the like. Then, light emission measurement S508 is performed.

The killed bacteria ATP measurement flow includes a filter filtration step S509, a filtrate collection step S510, and a luminescence measurement S511. In the filter filtration step S509, bacterial cells having a size larger than the filter pore size are removed, and in the filtrate collection step S510, the filtrate is collected and luminescence is measured (S511) to determine the amount of dead bacteria ATP.

As described above, in the killed bacterium ATP measurement flow illustrated in FIG. 5, ATP in the filtrate of method (3), that is, free ATP5 leaked due to damage to the cell membrane is determined as killed bacterium ATP. However, the method for measuring dead bacterial ATP is not limited to this, and the method for measuring free ATP5 leaked from the bacterial cells in the culture medium using the culture medium as it is in the method (2) of FIG. All ATPs (FIG. 2(4)) may be measured. When all the ATPs are the target of the killed bacteria ATP measurement, the difference between the luminescence measurement S511 and the luminescence measurement S508 is defined as the killed bacteria ATP. When the method (4) is used, a method of adding a surfactant or the like to the culture medium to extract ATP, a method of ultrasonic irradiation, a method of extracting ATP from bacterial cells by a French press or a homogenizer, and the like can be mentioned.

Next, a configuration example of the drug susceptibility testing system in the present embodiment will be shown using FIG. 7. This system comprises an incubator 6, a dispenser 7, a centrifuge 8, a measuring device 9, and a dead/viable bacteria comparison section 10. The incubator 6 cultivates the cells to be subjected to the drug sensitivity test. The dispenser 7 dispenses the bacterial cell liquid into each container of the plate 11 for the drug sensitivity test after culturing the bacterial cells. The dispenser 7 is equipped with a dispensing mechanism capable of dispensing from several microliters to several hundred microliters, and can be arbitrarily changed within the range of 10 microliters to 200 microliters. It is suitable.

Next, the sample in each container in the drug susceptibility test plate 11 for which the drug susceptibility test has been completed is dispensed into the container 12 having the filter at the bottom and the second luminescence measurement container 14. The dispenser 7 is equipped with a dispensing mechanism capable of dispensing from several microliters to several hundred microliters, and dispenses a predetermined sample amount into a container 12 having a filter at the bottom and a second luminescence measuring container 14. .. Then, the second luminescence measuring container 14 for which the process by the pipetting machine 7 is completed is introduced into the measuring device 9. In the centrifuge 8, in the step of preparing dead bacteria ATP, filter filtration for removing bacterial cells from the bacterial culture liquid 1 is performed. The filtering container 12 having the filter at the bottom is installed on the upper part of the first luminescence measuring container 13 and is centrifuged by the centrifuge 8 to filter the solution in the container 12 having the filter at the bottom and to obtain a filtrate. It is obtained in the first luminescence measuring container 13. Then, the first luminescence measuring container 13 is introduced into the measuring device 9.

The measuring device 9 performs the ATP adjustment steps S506, S507, etc., and the killed bacteria ATP luminescence measurements S508, S511. In the killed bacteria/viable bacteria comparison unit 10, from the ATP luminescence amount measured by the measuring device 9, the viable bacterium ATP luminescence amount and the dead bacterium ATP luminescence amount are calculated, and the ratio of the killed bacterium ATP luminescence amount/viable bacterium ATP luminescence amount is calculated. Is determined. The dispenser 7 can also be used in a step (S504) of preparing a single colony-forming bacterium to a constant concentration (S503) and then distributing the bacteria/antibiotics of various concentrations into containers (S504).

FIG. 9 shows a functional block configuration diagram of the dead cell/viable cell comparison unit 10. The killed bacteria/live bacteria ATP calculation unit 901, the live bacteria ATP calculation unit 901, the dead bacteria ATP calculation unit 902, and the dead bacteria ATP/viable bacteria ATP calculation that collectively displays the processing in these 901 and 902 as a final result. It is composed of a unit 903, a database storage unit 904, and a determination unit 905.

Specifically, the result of the luminescence measurement (S508) in FIG. 5 is stored in the viable cell ATP calculation unit 901, and the result of the luminescence measurement (S511) is stored in the dead cell ATP calculation unit 902, and the dead cell ATP/viable cell ATP calculation is performed. In the section 903, assuming that the dead bacterium ATP luminescence amount is x and the live bacterium ATP luminescence amount is y, the dead bacterium ATP luminescence amount/viable bacterium ATP luminescence amount is stored as a value of x/y, and the dead bacterium ATP amount/live It is displayed as the final result of the amount ratio of the bacterial ATP amount. Of course, it is also possible to display the data in a divided manner, and it is also possible to display only the results of S508 and S511.

In addition, the database storage unit 904 stores the judgment criteria stored in the database for each bacterial species based on the calculation result of the luminescence amount ratio (x/y) in the past dead bacteria ATP/viable bacteria ATP calculation unit 903. There is. The determination unit 905 determines the presence or absence of drug sensitivity based on this determination standard. For example, if the bacterial species is identified, the determining unit 905 selects the bacterial species from the database storage unit 904, and determines the ratio of the viable bacterial ATP luminescence amount (S508)/dead bacterial ATP luminescence amount (S511) as the criterion (( The presence/absence of drug sensitivity is determined by reading the threshold value of (x/y) and comparing the calculation result in the dead bacterium ATP/viable bacterium ATP calculation unit 903 with the threshold value. This enables accurate determination based on past test results. However, it is not essential that the determination criteria stored in the database storage unit 904 be based on the calculation result of the dead bacteria ATP/viable bacteria ATP calculation unit 903 in the past, and a predetermined threshold value is set in advance. But it's okay.

FIG. 10 shows an example of determination criteria stored in the database storage unit 904. Killed bacterium luminescence amount (x), viable bacterium luminescence amount (y), dead bacterium ATP luminescence amount / kill bacterium ATP luminescence amount (x/y) showing the ratio of killed ATP to viable ATP for each bacterial species ) Is stored. In the case of bacteria A as an example, the dead bacterial ATP amount/viable bacterial ATP amount (x) calculated based on the dead bacterial luminescence amount (x) and the viable bacterial luminescence amount (y) of the bacterial solution measured after culturing for a certain period of time. If /y) is larger than A, the determination unit 905 determines that the bacterium A is sensitive to the drug. On the contrary, when the amount of killed bacteria ATP/the amount of viable bacteria ATP (x/y) is A or less, the determination unit 905 determines that the bacteria A is not sensitive to the drug, that is, resistant.

FIG. 8 shows an example of the internal configuration of the measuring device 9. The measuring device 9 includes a reagent dispensing mechanism unit 15, a sample preparation mechanism unit 16, and a luminescence detection mechanism unit 17.

The reagent dispensing mechanism unit 15 includes a first electric actuator 18 and a second electric actuator 19 for adjusting a position, a dispensing nozzle 21, a tube pipe 22, a syringe 23, and a third electric actuator 23 for moving the syringe 23. It is composed of an actuator 24, and the dispensing nozzle 21 and the tube pipe 22 are connected using a fixing portion 25.

The reagent is taken in and out by moving the piston of the syringe 23 up and down in conjunction with the vertical movement of the third electric actuator 24 used for pushing and pulling the syringe 23. It is possible to dispense into the luminescence measuring container 28. Further, the position adjusting mechanism 20 can move the dispensing nozzle 21 to an appropriate position on the reagent folder 26 and the luminescence measurement container 28.

The sample preparation mechanism unit 16 includes a stage 29 on which the reagent folder 26 and the luminescence measuring container 28 are installed, a first electric slider 30 and a second electric slider 31 that can move the stage 29 to an arbitrary position. The reagent folder 26 includes the erasing reagent and the extraction reagent used in the live bacteria ATP preparation steps S506 and S507, and the luminescent reagent used in the ATP luminescence measurement steps S508 and S511. In addition to these reagents, necessary reagents and the like can be appropriately mounted on the reagent folder 26. For example, the first cleaning liquid for cleaning the nozzle for preventing cross contamination, the reagent of the dispensing mechanism unit 20, and the like. A second cleaning solution for cleaning the inside of the dispensing nozzle 21, the tube pipe 22, and the syringe 23 that come into contact with each other, and further, a first cleaning solution after cleaning, an empty container for storing the second cleaning solution, etc. can be mounted. Is.

In order to prevent the above-mentioned cross-contamination, in consideration of the case where the dispensing nozzle 21 is replaced for each measurement, in consideration of the case where the dispensing nozzle 21 is replaced, a chip group is installed on the stage 29 of the measuring device 9 of the drug sensitivity system of the present invention. A section 27 is provided. The dispensing nozzle 21 can access the chip group setting unit 27 by the position adjusting mechanism 20 and automatically removes and installs the chips.

The bottom of the stage 29 where the luminescence measuring container 28 is installed is transparent or a member is cut out so that the luminescence detection mechanism 17 can detect luminescence. The first electric slider 30 and the second electric slider 31 of the stage 29 and the position adjusting mechanism 20 of the reagent dispensing mechanism unit 15 perform position control in cooperation with each other, and the dispensing nozzle 21 allows an arbitrary position on the luminescence measuring container 28. Dispense the reagent into the target container (well) of.

The method of detecting the light emission in the light emission detection mechanism unit 17 is not particularly limited, but in this embodiment, an example using a photomultiplier tube (hereinafter referred to as PMT) is shown in FIG. The light emission detection mechanism unit 17 includes an optical fiber cable 32 and a PMT 33. The optical fiber cable 32 and the PMT 33 are connected by a fiber coupler, and the light incident on the tip of the optical fiber is transmitted to the light receiving portion of the PMT 33. In the present embodiment of the luminescence measuring container 28, the optic fiber cable 32 having a tip diameter equal to or smaller than each container of the luminescence measuring container 28 can be used to detect luminescence at a specific position on the luminescence measuring container 28. Becomes In addition, the first electric slider 30 and the second electric slider 31 enable luminescence measurement in an arbitrary target container (well) on the luminescence measurement container 28. PMT is preferable in terms of sensitivity, but not only the PMT 33 but also a CCD camera or photodiode can be used as a detector.

So far, the configuration example of the drug susceptibility test system has been described with reference to FIGS. 7, 8 and 9. Here, in FIG. 7, the incubator 6, the dispenser 7, the centrifuge 8, the measuring device 9, and the dead/viable bacteria comparison unit 10 are divided into respective functions, but of course, all of them are It may be an integrated fully automatic system. In that case, the drug susceptibility test plate 11, the container 12 having the filter at the bottom, the first luminescence measuring container 13, the The luminescence measurement container 14 of No. 2 is moved, a robot arm capable of loading is prepared, and the system is configured to be continuously processed.

Further, each functional block described in FIG. 9 may be implemented as a software module or may be realized by hardware. That is, each functional block can be implemented by software by the processor interpreting and executing the program stored in the memory that implements each function in the killed bacteria/viable bacteria comparison unit 10. In addition, each functional block may be realized by hardware, for example, by designing a part or all of the functional block with an integrated circuit. Information such as programs, files, databases, function data, and variable data for realizing each function is, for example, a memory, a hard disk, a recording device such as an SSD (Solid State Drive), an IC card, an SD card, a DVD, or the like. It can also be placed on the recording medium.

Further, as a criterion for the drug sensitivity by the determination unit 905, in addition to the method of using the measurement results of both the live bacterium ATP luminescence amount and the dead bacterium ATP luminescence amount shown in FIG. 5, the dead bacterium ATP luminescence shown in FIG. It is also possible to use only the measurement result of the amount. The measurement of the amount of luminescence in this case is based on the method for measuring the amount of dead bacteria ATP shown in FIG. Specifically, S601 to S605 of FIG. 6 carry out the same operations as S501 to S505 of FIG. In S606 to S608 of FIG. 6, the same operations as S509 to S511 of FIG. 5 are performed.

Hereinafter, an example of determination of drug sensitivity according to the present embodiment will be described in more detail together with experimental contents.

In this experimental example, Escherichia coli (ATCC25922 strain, hereafter referred to as E. coli) and Staphylococcus aureus (ATCC25923, hereafter referred to as S. aureus) were used as cells, and a drug sensitivity test by the ATP method was performed. Ampicillin and levofloxacin (hereinafter referred to as LVFX) were used as the antibiotics.

First, the cells were cultured overnight on LB agar medium, and the next day, the colonies were suspended in Mueller-Hinton (hereinafter referred to as MH) medium to prepare 0.5 McFarland bacterial suspension. Next, distribute 50 μL of the bacterial suspension diluted 500-fold with MH medium to the plate in which 50 μL of MH medium containing antibiotics at 2 times the final concentration was dispensed, and incubate at 37°C for a certain period of time. A stationary culture was performed to prepare a bacterial suspension. The final antibiotic concentration was 4 μg/mL for ABPC when measuring E. coli, 0.125 μg/mL for LVFX, and 0.25 μg/mL for ABPC when measuring S. aureus. , LVFX was 0.125 μg/mL. In addition, what was cultured in MH medium containing no antibiotic was used as a comparative control.

As for ATP viable bacteria ATP, 18 μL of the above-mentioned bacterial culture solution was mixed with 2 μL of the ATP elimination solution, and the mixture was allowed to stand at room temperature for 30 minutes. After that, 20 μL of the ATP extraction reagent was mixed and allowed to stand at room temperature for 1 minute for preparation. As the killed bacterium ATP, a bacterium culture solution was added on a filter (membrane pore diameter: 0.22 μm), and the filtrate obtained by filtration through centrifugation was used as killed bacterium ATP. A luminescence reagent was added to the prepared live bacterial ATP or dead bacterial ATP, photon counting was performed for 1 minute, and the obtained luminescence amount (Amount of luminescence (a.u.)) was used as an index of the ATP amount.

11 and 12 show the results obtained by obtaining the luminescence amount ratio of the dead bacterium ATP luminescence amount and the live bacterium ATP luminescence amount and calculating it as the ratio of the live bacterium ATP and dead bacterium ATP amounts.

FIG. 11 shows the ratio of live ATP and dead ATP of E. coli. In the medium containing no antibiotics, the amount of killed ATP luminescence/amount of live ATP luminescence, that is, the amount of killed ATP/amount of live ATP remained at about 0.1 or less. On the other hand, in the presence of ABPC or LVFX, the dead bacterial ATP amount/viable bacterial ATP amount increased with the lapse of culture time. If the criteria for determining the effect of antibiotics are killed ATP amount/viable ATP amount=1, the effect of ABPC and LVFX on E. coli can be determined approximately 3 hours after the start of drug-sensitive culture. Was shown.

Next, FIG. 12 shows the ratio of the amount of live cells and dead cells of S. aureus. In the medium containing no antibiotics, the amount of dead bacteria ATP/the amount of viable bacteria ATP remained around 0.5 or less. In the presence of ABPC or LVFX, as in the case of E. coli, the killed ATP amount/viable ATP amount increased with the lapse of culture time. If the criterion for judging the effect of antibiotics is killed ATP amount/viable ATP amount=1, the effect of ABPC on S. aureus is about 2.5 hours after the start of drug-sensitive culture, and the effect of LVFX on S. aureus is: It was shown that it can be judged in about 4 hours.

From the above experiment, as a criterion for the drug susceptibility test by the ATP method, determine the ATP amount of live bacteria and the amount of dead ATP in the reaction system containing the drug by the ATP luminescence amount, and use the ratio of ATP amount of live bacteria to dead bacteria. Thus, it has been clarified that the drug sensitivity can be judged by the same judgment criteria for different bacterial species, and the effect of the drug can be judged more accurately and in a shorter time than the prior art.

That is, it was found that the drug sensitivity test system of the present example can shorten the time required for the drug sensitivity culture in the drug sensitivity test of bacteria using the ATP method and determine the drug effect with a small number of measurements. As a result, the drug susceptibility test can be carried out quickly and simply, whereby the antibiotic can be promptly and appropriately administered to patients with serious infections.

Furthermore, in the drug susceptibility test system of the present example, the ATP amount of viable bacteria is determined from the ATP amount contained in the bacterial membrane, and the ATP amount of dead bacteria is determined from the free ATP amount contained in the reaction system. Unlike the method for determining the number of live cells/dead cells, it is possible to count both live cells and dead cells by the ATP method, and since there is no counting error due to the difference in the method, It is possible to count the number of killed bacteria. Further, since the counting method is unified by the ATP method, a simple device configuration can be realized.

ATP6... Incubator, 7... Dispenser, 8... Centrifuge, 9... Measuring device, 10... Dead/live bacteria comparison section, 11... Drug sensitivity test plate, 12... Filtering container, 13... First luminescence measurement Container, 14... Second luminescence measurement container, 15... Reagent dispensing mechanism section, 16... Sample preparation mechanism section, 17... Luminescence detection mechanism section, 18... First electric actuator, 19... Second electric actuator, 20 ... Position adjusting mechanism, 21... Dispensing nozzle, 22... Tube piping, 23... Syringe, 24... Third electric actuator, 25... Fixing part, 26... Reagent folder, 27... Chip group setting part, 28... Luminescence measuring container , 29... Stage, 30... First electric slider, 31... Second electric slider, 32... Optical fiber, 33... PMT

Claims (7)

A culture device for culturing cells in a culture solution,
A distribution container for injecting a drug into a culture solution containing cultured cells,
A dispenser for dispensing the culture solution in which the drug in the distribution container has been injected into the first luminescence measurement container and the second luminescence measurement container, wherein:
A filtering container having a filter at the bottom is provided on the upper part of the first luminescence measuring container, and the first luminescence measuring container is a filtrate obtained by filtering the culture solution in the filtering container. Included in the
The second luminescence measurement container contains therein an ATP extract derived from live cells obtained from cells after erasing extracellular ATP in the culture solution,
Furthermore, a measuring unit for performing ATP luminescence measurement of the filtrate in the first luminescence measurement container and the ATP extract in the second luminescence measurement container, and presence or absence of sensitivity of the bacterial cells to the drug are determined. A testing device having a judging part for judging,
A storage unit for storing the threshold value of the determination, a drug sensitivity test system Ru provided with,
The measuring unit comprises an ATP luminescence amount derived from live bacteria in the ATP extract in the second luminescence measurement container and an ATP luminescence amount derived from dead bacteria in the filtrate in the first luminescence measurement container. And,
The determination unit refers to the threshold value, based on a comparison result of the luminescence amount ratio between the ATP luminescence amount of the live bacterium and the ATP luminescence amount of the dead bacterium and the threshold value, and the bacterial cells. wherein determining the presence or absence of sensitivity to the drug, characterized in that, the drug sensitivity test system. The drug sensitivity test system according to claim 1, wherein
The determination unit refers to the threshold value, the light emission amount ratio and wherein the said sensitivity exists and determines to drugs of bacteria, it is larger than the threshold value, the drug susceptibility testing system. The drug sensitivity test system according to claim 1, wherein
The drug susceptibility test system , wherein the storage unit stores the threshold value based on the luminescence amount ratio that has been previously determined. The drug sensitivity test system according to claim 1, wherein
The determination unit calculates an x/y value, where x is the ATP luminescence amount derived from the dead bacterium measured by the measurement unit, and y is the ATP luminescence amount derived from the live bacterium, and is calculated in the past. The drug susceptibility test system , wherein the presence/absence of susceptibility of the bacterium to the drug is determined with reference to the x/y value of each bacterium stored in the storage unit. The drug sensitivity test system according to claim 1, wherein
The measuring unit performs the measurement at a fixed incubation time interval, characterized in that, the drug sensitivity test system. The drug sensitivity test system according to claim 1, wherein
Said filtration, the first light emitting measuring container and the filtering vessel is introduced into the centrifuge is carried out by centrifugation, characterized in that, the drug sensitivity test system. The drug sensitivity test system according to claim 1, wherein
Said dispensing container, said agent of the drug or more concentrations of plural kinds are micro-well plate to be dispensed, characterized in that, the drug sensitivity test system.

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