JPH07222599A - Method for measuring antimicrobial activity of medicine - Google Patents

Abstract

PURPOSE:To rapidly and simply measure the antimicrobial activity of a medicine such as an antibiotic substance by inoculating a microorganism to be tested into a liquid culture medium containing the medicine, culturing the microorganism for a prescribed time, then measuring the amount of adenosine triphosphate(ATP) in the microbial cell thereof and comparing the resultant amount with the control amount of the ATP. CONSTITUTION:This method for measuring the antimicrobial activity of a medicine is to distribute a liquid culture medium such as a potato dextrose agar(PDA) culture medium containing a medicine such as an antibiotic substance, an antimicrobial agent or an antifungal agent into a microtiter plate, etc., then aseptically inoculate a microorganism to be tested thereinto, culture the microorganism at 25-37 deg.C for a prescribed time, subsequently add an extracting agent for ATP, extract the ATP in the microbial cell thereof, add luciferin-luciferase thereto, measure the luminous quantity, measure the amount of the ATP and compare the resultant amount of the ATP with the amount of the ATP which is the control obtained in the culture medium without containing the medicine. Thereby, the antimicrobial activity of the medicine can rapidly and simply be measured.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring the antimicrobial activity of drugs such as antibiotics.

[0002]

2. Description of the Related Art The greatest purpose of measuring the antimicrobial activity of a drug in antibacterial chemotherapy is to accurately select an antibacterial drug or the like effective for treating infectious diseases in a short time. That is, it is to measure which drug exhibits an antibacterial action and the degree of antibacterial activity of the accurately detected causative bacterium, and accuracy and promptness are required. On the other hand, it is also applied to an epidemiological study to know the transition of the susceptibility pattern of each bacterial species based on the antimicrobial activity of the isolated bacterial drug. Currently used methods for measuring the antimicrobial activity of drugs are roughly classified into the diffusion method and the dilution method. As a diffusion method, a disk method using a drug-containing disk aiming at quickness and simplicity is widely used.

The disk method is the simplest of the methods for examining the drug antimicrobial activity of microorganisms such as bacteria. This method utilizes the property that the drug contained in the disk is dissolved by the water content of the medium and diffuses into the agar medium inoculated with the bacterium. The drug begins in the agar layer beneath the disc and is diffused towards the surroundings. For this reason, a concentration gradient of the drug is formed in the medium, and naturally, the drug concentration in the medium near the disc is high and the distance is thin. Due to the difference in the susceptibility of the bacteria inoculated on the medium plate to the drug, an inhibition circle is formed around the disc where no growth of the bacteria is observed.

The dilution method includes an agar plate dilution method and a liquid medium dilution method, both of which quantitatively have a minimum inhibitory concentration (Mi
nimum Inhibitory Concentr
ation: MIC). The agar plate dilution method uses Muller-Hin as a medium for sensitivity measurement.
A semi-synthetic medium (MH medium) based on ton medium is used. Make a 2-fold dilution series of the drug and keep the MH medium at a temperature of 6
At about 0 ° C, the drug solution and the medium are mixed in a petri dish to prepare a drug-containing agar plate. A certain amount of the overnight culture solution of the test bacteria or a diluted solution thereof is spread and inoculated on this plate. After culturing at 37 ° C. for 18 to 20 hours, the presence or absence of growth is measured, and the minimum concentration at which the growth is completely inhibited as compared with the control medium is evaluated as MIC.

The liquid medium dilution method is a method in which a drug is diluted with a liquid medium, a test bacterium is inoculated, and the MIC for the bacterium is determined. As a general method, a 96-well microtiter plate is aseptically dispensed with a drug solution diluted with a susceptibility measurement medium (CSMH medium), and a certain amount of an overnight culture solution of the test bacterium or a diluted solution thereof is inoculated therein. After culturing the cells at 35 ± 1 ° C. for 18 to 24 hours and confirming the growth in the drug-free medium used as a control, the MIC was determined with the minimum drug concentration in the wells in which the growth of bacteria was not visually recognized. evaluate. In all of these current methods, the growth of microbial cells is directly observed and determined. That is, it is determined as an antimicrobial activity by observing how much the growth of microbial cells can be inhibited and how much the number of cells can be reduced, and it takes about 2 to 5 days to obtain the results. It costs. Therefore, including the culture time of the test bacterium, the result cannot be known until at least about two days have passed since the request for the test was received.

On the other hand, adenosine triphosphate (ATP) is one of the nucleotides existing in the cell body, and is a compound that is involved in many energy metabolisms and plays an important role in energy acquisition and utilization. A of microbial cells
It is known that the amount of TP can be measured mainly by a bioluminescence measuring method. For example, in a luminescence measurement method using a firefly-derived luciferin-luciferase system, when a specific surfactant is contained, the ATP amount of the microorganism is selectively selected from a mixture of somatic cells and microbial cells without destroying the cell wall or cell membrane. It is known that it can be measured, but the technique of applying these known methods for measuring the ATP amount of microorganisms to the determination of drug antimicrobial activity has not been put into practical use.

[0007]

Therefore, the problem to be solved by the present invention is that it takes 1-2 days for bacteria or yeasts to measure the ATP amount of microorganisms, and 3 for molds. It is to provide a means which can be carried out more simply and in a shorter time than the drug antimicrobial activity test method according to the current method, which requires ~ 7 days.

[0008]

[Means for Solving the Problems] The present inventors focused on the fact that the increase / decrease in the amount of ATP in active microorganisms correlates with the growth / proliferation state of the microorganisms, and the medium containing an agent such as an antibiotic was added to the medium. The present invention was completed by discovering that the drug antimicrobial activity can be determined in a significantly shorter time than the current method by measuring the amount of ATP using luciferin-luciferase after inoculating a test bacterium and culturing, and completed the present invention. That is, the present invention is that a liquid medium containing a drug is inoculated with a test microorganism and cultured for a certain period of time, and then the amount of ATP in microbial somatic cells is measured.
The present invention provides a method for measuring the antimicrobial activity of a drug, which is characterized by comparing with the amount.

ATP is produced in active microbial cells by glycolysis and fermentation in anaerobic metabolism, or by oxidative phosphorylation in aerobic metabolism, and the amount of ATP in the cells is the amount of active microbial cells. Then, it increases with proliferation, and ATP production is stopped in cells inactivated or killed by drugs such as antibiotics, or it is consumed in the cells and decreased. Therefore, if the ATP amount when the test bacterium is inoculated and cultured in the medium to which the drug is added is compared with the ATP amount measured immediately after the start of the culture of the test bacterium inoculated to the medium without the drug at the same concentration, The degree of antibacterial activity of the drug against the target microorganism can be known.

In the present invention, the amount of ATP in the test bacterium can be measured specifically by using a luciferin (luminescent element) -luciferase (luminescent enzyme) system and measuring the amount of luminescence. As the luciferin-luciferase system, any system can be used as long as it requires ATP for light emission. For example, those derived from a known system such as firefly or those derived from a microorganism obtained by cloning can be used. A reagent kit for counting the amount of luminescence of ATP and a device for evaluating (counting) the amount of luminescence using the luciferin-luciferase system are commercially available, and when carrying out the method of the present invention, such a commercially available kit and device are used. The amount of ATP of the test bacterium can be measured by utilizing this. Specifically, a test bacterium is previously cultured in a medium by a conventional method, diluted to a predetermined concentration, and its ATP luminescence is measured using a luciferin-luciferase system, and the value is used as a control value. .

However, since the medium obtained by the conventional method contains a considerable amount of non-microbial-derived ATP, which has a bad influence on the measurement, it is difficult to measure the antimicrobial activity with high sensitivity and accuracy. Therefore, in the present invention, a medium that does not substantially contain ATP, such as LB medium, or P that has been subjected to ATP removal treatment (decomposition / removal with an ATP-degrading enzyme)
DA medium is preferred. On the other hand, the drug-containing measurement medium in which the drug to be used in the evaluation test is adjusted to a predetermined concentration is dispensed into the microtiter plate in a predetermined amount. Then, the pre-cultured test bacterial solution was washed with physiological saline or the like at a predetermined concentration (10 ² C).
FU / well to 2 × 10 ⁶ CFU / well) and inoculate each well of the drug-containing measurement medium. After culturing this plate at 25 to 37 ° C. for a predetermined time (10 seconds to 24 hours), an ATP extractant is added to extract ATP, and then a predetermined amount of luciferin-luciferase is added, and the luminescence amount thereof is measured. This measured value was compared with that of the control, and the concentration at which the decrease in ATP amount was remarkable was determined to be MIC or MB.
Let be C. In other words, by changing the concentration and measuring
MIC or MBC is defined as the drug concentration at which the decrease in TP amount is remarkable.

Further, the antimicrobial activity of the drug can be known by changing the culture time and measuring the amount of ATP. In the case of an antifungal agent, it can be judged in about 10 seconds to 1 hour, in yeast it can be judged in 1 to 6 hours, and in mold it can be judged in 10 to 24 hours.

[0013]

EFFECTS OF THE INVENTION According to the method of the present invention, at least about 1 day of culture time is required until the determination, and the drug antimicrobial activity measurement which has been performed by visual observation or measurement of absorbance can be performed in a short time. Moreover, the amount of light emission can be mechanically counted and easily implemented. Further, in the conventional method, for example, in the method of determining by measuring the absorbance, the measurement data contains an error because not only live cells but also dead cells are determined, but in the method of the present invention, in principle, Since ATP is measured, the drug antimicrobial activity test is characterized by high accuracy. Therefore, the method of the present invention brings great advantages not only as a drug antimicrobial activity but also as a drug susceptibility test method in the treatment of infectious diseases.

[0014]

【Example】

Example 1 In this test, C was used as a test bacterium for measuring the amount of ATP.
andida albicans ATCC10231
Strains were used. One platinum loop amount of this test bacterium is the standard P for fungi
A DA ground plate was inoculated and statically cultured at 35 ° C. for 24 hours. The following measuring medium was used for measuring the antimicrobial activity by measuring the amount of ATP. The following composition indicates the composition in 1 liter of medium. Tryptone 5 g Yeast extract 3 g Salt 1 g The above measurement medium was supplemented with Amphoter as an antifungal agent.
icin B and Benzalkonium chl
Oride was added to a 96-well microtiter plate (Dynal
ech: white), 100 ± 10μ per well
It was dispensed in liters.

Next, the pre-cultured test bacterial solution (2 × 10
⁶CFU / ml) diluted 100 times with physiological saline,
Aseptic 10 μl in each well of the icrotiter plate
Vaccination. After incubating this plate at 35 ° C for 6 hours,
ATP extractant (Brand name: Shiro Shiro ATP antibacterial test kit
ATP manufactured by Toyo Ink Mfg. Co., Ltd.
Luciferin derived from firefly-lucifera
(Product name: Bushiro ATP Antibacterial Test Kit) 100
μl was added. Lumat LB9501 (Beltold
(Manufactured by the same company) was used to measure the amount of light emission at an integration time of 10 seconds. Cover
Test liquid (2 x 10 ⁶CFU / ml) with saline 10
ATP extraction was also performed on 10 μl diluted 0 times.
Agent, luciferin-luciferase, and measure the amount of luminescence.
Was set as a blank value (control). The measurement results are shown in Fig. 1 and
And shown in FIG.

The following equation was used to calculate the inhibition rate. [(Blank value-Amount of luminescence at each antifungal agent concentration) / Blank value] x 100 On the other hand, the result of counting the viable cell count (CFU) of the luminescence measurement sample by culturing at 35 ° C for 48 hours on a conventional standard agar medium The suppression rate is also shown in FIGS. 1 and 2. In each figure, the vertical axis represents the inhibition rate (%) and the horizontal axis represents the drug concentration. From FIG. 1 and FIG. 2, it can be seen that this measuring method has a high correlation with the conventional standard agar culture method and is sufficiently practical. In addition, because the suppression rate drops sharply, MI
C can be quantitatively determined. It should be noted that this determination does not require a visual determination by a skilled person, and anyone can easily perform the measurement. Example 2 [Preculture of test bacterium] In this test, Escherichia coli N was used as a test bacterium for measuring the amount of ATP.
The IHJ strain was used. This test bacteria (Escherich
IA coli NIHJ strain) in an amount of 1 platinum loop is aseptically inoculated into 5 ml of the preculture medium having the following composition,
The culture was allowed to stand for a period of time. [Preculture medium (Muller-Hinton Bro
th)] Beef pouring solution 300 g Casamino acid 17.5 g Starch 1.5 g Purified water 1000 ml pH 7.2 to 7.4 (25 ° C: after sterilization) [Antimicrobial activity test method by ATP amount measurement] Measurement medium As the medium, the medium having the following composition was used.

Measurement medium (Cation Supple
Mented Muller-Hinton Brot
h) Beef extract 300 g Casamino acid 17.5 g Starch 1.5 g Purified water 1000 ml pH 7.2 to 7.4 (25 ° C .: after sterilization) Ca ion * 50 mg / l Mg ion * 25 mg / l * Sterile after sterilization Was added.

A 96-well microtiter plate (manufactured by Sumitomo Chemical Co., Ltd .: white) was added to the above-mentioned measuring medium, and the measuring medium containing various antibiotics to be tested shown in Table 1 below was adjusted to a predetermined concentration. ), 100 per well
Aliquots of ± 10 μl were dispensed. MIC determined in advance by the current method (liquid medium dilution method) in addition to the target antibiotic name and test concentration
Is also shown in Table 1. As the test concentration, two points within the range including MIC were selected. Then, the pre-cultured test bacterial solution (2 × 10 ⁸ CFU / m 2
1) was diluted 100 times with physiological saline, and 5 μl of each well of the microtiter plate was aseptically inoculated (10 ⁴ CFU / well). After culturing the plate at 35 ° C for 4 hours, the ATP releasing agent (trade name: PICOEX
B: manufactured by Packard Japan Co., Ltd.)
After extracting TP, 40 μl of firefly-derived luciferin-luciferase (trade name: Pycozyme: manufactured by Packard Japan) was added. Luminos CT-
Using 9000 (Chuo Kagaku Kogyo Co., Ltd.), voltage 10
The amount of light emission was measured at 50 V and an integration time of 1.0 second. A test extract (2 × 10 ⁸ CFU / ml) was diluted 100 times with physiological saline, and 5 μl of the same was added with the ATP extractant, luciferin-luciferase, and the amount of luminescence was measured to obtain a blank value (control). . The measurement results are shown in Table 2. In the present test, the amount of luminescence of the ATP amount after culturing for 4 hours was measured, and it can be determined that the antibacterial activity is high when the decrease in the ATP amount is remarkable.

[Brief description of drawings]

FIG. 1 is a graph showing the results of measuring the amount of ATP after 6 hours by the method of the present invention.

FIG. 2 is a graph showing the results of an antimicrobial activity test of a drug based on the absorbance after 20 hours according to the current method.

Claims (7)

[Claims] 1. An AT in a microbial cell after inoculating a test microorganism into a liquid medium containing a drug and culturing for a certain period of time.
A method for measuring antimicrobial activity of a drug, which comprises measuring the amount of P and comparing the amount of ATP with the amount of ATP of a control in a medium containing no drug. 2. The method for measuring the antimicrobial activity of a drug according to claim 1, wherein the medium is a medium which does not substantially contain ATP or a medium which has been subjected to a preliminary ATP removal treatment. 3. The method for measuring the antimicrobial activity of a drug according to claim 1 or 2, wherein the amount of ATP is measured using luciferin-luciferase. 4. A measurement medium containing a drug is dispensed into a microtiter plate, and then a test microorganism is aseptically inoculated and cultured at 25 to 37 ° C. for 10 seconds to 24 hours, followed by ATP.
The method for measuring the antimicrobial activity of a drug according to any one of claims 1 to 3, wherein an extractant is added, and luciferin-luciferase is added to measure the amount of luminescence. 5. The method for measuring the antimicrobial activity of a drug according to claim 1, wherein the drug is at least one selected from antibiotics, antibacterial agents and antifungal agents. 6. The method for measuring the antimicrobial activity of a drug according to any one of claims 1 to 5, wherein the antimicrobial activity measurement of the drug is a minimum inhibitory concentration (MIC) or a minimum bactericidal concentration (MBC). 7. The method for measuring the antimicrobial activity of a drug according to claim 1, wherein the liquid medium is a potato dextrose agar (PDA) medium.