JPH04370100A - Microbial drug susceptibility testing method - Google Patents

Abstract

PURPOSE:To carry out test of sensitivity of a microorganism to drug in a remarkably short time and high precision by culturing a microorganism to be examined in a drug-containing medium and then measuring an amount of adenosine triphosphate in microbial cell of the microorganism. CONSTITUTION:A microorganism to be examined is aseptically inoculated into a medium containing a drug such as antibiotic and cultured. Then an amount of adenosine triphosphate(ATP) in microbial cell of the cultured microorganism is measured and the measured ATP amount is compared with reference ATP amount in culture start. Measurement of ATP amount of microbial cell to be examined is preferably carried out by measuring of luminescent amount of luciferin-luciferase system used. A system derived from a firefly is preferably used as the luciferin-luciferase system.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for testing the susceptibility of microorganisms to drugs such as antibiotics in antibacterial chemotherapy.

0002

BACKGROUND OF THE INVENTION The primary purpose of drug susceptibility testing in antibacterial chemotherapy is to quickly and accurately select antibacterial agents that are effective in treating infectious diseases. In other words, it is necessary to measure which drug exhibits an antibacterial effect on the accurately detected causative bacteria and to what extent it has antibacterial activity, which requires both accuracy and speed. On the other hand, it is also applied to epidemiological studies to understand changes in the susceptibility patterns of each bacterial species based on the drug susceptibility results of isolated bacteria.

[0003] Currently used drug susceptibility measurement methods are broadly classified into diffusion methods and dilution methods. As a diffusion method, a disk method using a drug-containing disk is widely used, which aims to be quick and simple.

[0004] The disk method is the most simplified method for testing the drug sensitivity of bacteria, and is currently the
In Japan, the Showa disk (single density disk method), Tori disk (three density disk method), Sensi disk, KB (Kirby-Bauer) disk (single density disk method), etc. are used as sensitive disks in Japan. This method utilizes the property that the drug contained in the disk is dissolved by the moisture in the medium and diffused into the agar medium inoculated with bacteria. The drug begins in the agar layer just below the disk and diffuses toward the periphery. For this reason, a concentration gradient of the drug is created in the medium, and naturally the drug concentration in the medium near the disk is high, and the drug concentration is low in a place far away. Due to differences in the susceptibility of the bacteria inoculated onto the medium plate to the drug, an inhibition circle is formed around the disk where no bacterial growth is observed.

[0005] As a specific example, a culture medium for sensitive disks (
Muller-Hinton medium (based on Muller-Hinton medium) was dispensed into a sterile petri dish to form a plate medium, the test bacteria were evenly spread on it, the drug-containing disk was placed aseptically on the plate, and the plate was incubated at 35 to 37°C. After culturing for 16 to 20 hours, the diameter of the inhibition circle formed when bacterial growth is inhibited is measured, and based on the size, it is determined as sensitive (S), intermediate (I), or resistant (R).

Dilution methods include the agar plate dilution method and the liquid medium dilution method, both of which quantitatively determine the minimum inhibitory concentration (Mi
nimum Inhibitory Concentr
ation:MIC). 1. Muller-Hinton as a medium for sensitivity determination using agar plate dilution method
A medium-based semi-synthetic medium (MH medium) is used. Weigh a drug of known potency and dissolve it in sterile distilled water. Make a 2-fold dilution series of the drug and adjust the temperature of the MH medium to 60℃.
At about this time, mix the drug solution and culture medium in a petri dish to create a drug-containing agar plate. A fixed amount of an overnight culture of the test bacteria or its diluted solution is smeared onto this plate. 3
After culturing at 7° C. for 18-20 hours, the presence or absence of growth is determined, and the lowest concentration at which growth is completely inhibited is evaluated as the MIC compared to the control medium.

2. Liquid medium dilution method This method involves diluting the drug with a liquid medium, inoculating it with a test bacterium, and determining the MIC for the bacterium. A common method is
Add susceptibility measurement medium to a 96-well microtiter plate (
A drug solution diluted in CSMH medium) was aseptically dispensed, and a certain amount of an overnight culture of the test bacteria or its dilution was inoculated thereto. After confirming growth in the drug-free medium, the minimum drug concentration in wells in which no bacterial growth is visually observed is evaluated as MIC.

[0008] All of these current methods are determined by directly observing the growth of microbial cells. In other words, "sensitivity" is determined by observing to what extent microbial cell growth is inhibited and how much the cell number is reduced, and it takes about one day to obtain the results. Therefore, including the culturing time of the test bacteria, the results cannot be known until at least two days have passed from the time the test request is received.

[0009] As improvements to these current laws, for example,
A method that can be performed more easily and accurately (for example, a bacterial culture tank is filled with a drug-containing medium, and after culturing the test bacteria, a substrate that is an indicator of bacterial growth is added and allowed to react for a certain period of time. A method of detecting the presence or absence of bacterial growth based on a change in color tone by inverting a container and immersing it in a reaction agent containing an indicator or coloring agent for the substrate or substrate decomposition product: JP-A-61-96999 etc.), or improved ones that automatically determine both MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) in practical clinical testing (e.g., using a culture block with a series of wells, Automatically create a 2-fold dilution series, automatically inoculate the test sample, measure the light scattering intensity for each well while culturing to determine the MIC, add an antibacterial inactivator to the required wells, and inoculate the test sample with the same light. Automated methods for determining MBC by measuring scattering intensity have been proposed (e.g., Japanese Unexamined Patent Publication No. 62-25998), but there is still no known technology that can shorten the time required for determination to several hours. do not have.

On the other hand, adenosine triphosphate (ATP) is a type of nucleotide present in the cell body, and is a compound that is involved in many energy metabolisms and plays an important role in acquiring and utilizing energy. ATP is known to be produced by glycolysis and fermentation in anaerobic metabolism, or oxidative phosphorylation reaction in aerobic metabolism.

[0011] It is known that the amount of ATP in microbial cells can be measured mainly by bioluminescence measurement. For example, Japanese Patent Publication No. 62-4120 describes a luminescence measurement method using a firefly-derived luciferin-luciferase system.
It has been described that when a specific surfactant is contained, the amount of ATP in microorganisms can be measured selectively from a mixture of somatic cells and microbial cells without destroying the cell wall or cell membrane. There is no known technique for applying a measurement method to the determination of drug sensitivity, and there is no literature suggesting a correlation between the amount of ATP in microorganisms and drug sensitivity.

0012

[Problems to be Solved by the Invention] Therefore, the problem to be solved by the present invention is to solve the problem of the drug susceptibility testing method according to the current method, which requires at least about one day to measure the ATP amount of microorganisms and make a determination. Rather, the aim is to provide a means that can be implemented in a short period of time.

[0013]

Means for Solving the Invention The present inventors focused on the fact that the increase or decrease in the amount of ATP in active microorganisms is correlated with the growth and proliferation status of the microorganism, and developed We have completed the present invention by discovering that drug sensitivity can be determined in a significantly shorter time than with current methods by measuring the ATP level using a luciferin-luciferase system after inoculating and culturing test bacteria.

That is, in the present invention, a test microorganism is aseptically inoculated into a medium containing a drug at a predetermined concentration, and after culturing,
The present invention provides a method for testing the drug susceptibility of microorganisms, which is characterized by measuring the amount of ATP in microorganism cells and comparing the amount of ATP with the amount of ATP in a control at the start of culture.

[0015] ATP is produced in active microbial cells through glycolysis and fermentation in anaerobic metabolism, or oxidative phosphorylation reaction in aerobic metabolism, and the amount of ATP in cells is In cells that have been inactivated or killed with drugs such as antibiotics, ATP production stops, or it is consumed within the cell and decreases. Therefore, if you compare the amount of ATP when a test bacterium is inoculated and cultured in a medium to which a drug has been added, with the ATP amount measured immediately after the start of culture of a test bacterium inoculated at the same concentration into a medium to which no drug has been added, It is possible to know the degree of antibacterial activity (susceptibility) of the drug against the target microorganism.

[0016] In the present invention, the amount of ATP in a bacterial cell to be tested can be specifically measured by using a luciferin (luminescent substance)-luciferase (luminescent enzyme) system and measuring the amount of luminescence thereof. Any system that requires ATP for luminescence can be used as the luciferin-luciferase system. For example, those derived from known systems such as fireflies, sea fireflies, lathia, luminescent earthworms, and Renilla can be used, but among these, preferred are those derived from fireflies and cloned microorganisms.

Reagent kits for counting the amount of ATP luminescence using the luciferin-luciferase system and devices for evaluating (counting) the amount of luminescence are commercially available, and when carrying out the method of the present invention, such commercially available kits are used. The amount of ATP in the test microorganism can be measured using the device. Specifically, test bacteria are cultured in a medium in advance using a conventional method, diluted to a predetermined concentration, and the amount of ATP emitted is measured using a luciferin-luciferase system, and this value is used as a control value. .

On the other hand, a predetermined amount of a drug-containing measurement medium containing a drug to be subjected to an evaluation test adjusted to a predetermined concentration is dispensed into a microtiter plate. Next, the pre-cultured test bacterial solution is diluted with physiological saline or the like to a predetermined concentration (104 CFU/
dilute to ~2x106 CFU/ml) and add 100 μl of the drug-containing assay medium to each well of the drug-containing assay medium.
Inoculate aseptically in μl. This plate costs 35~
After culturing at 37° C. for a predetermined time (3 to 5 hours), an ATP release agent is added to extract ATP, a predetermined amount of luciferin-luciferase is added, and the amount of luminescence is measured. This measured value is compared with the value of the control, and those with a significant decrease in ATP amount are determined to be sensitive, those with a small decrease or no change in ATP amount are determined as intermediate, and those with an increase in ATP amount are determined as resistant.

[0019]

Effects of the Invention According to the method of the present invention, the drug susceptibility test, which conventionally required at least about one day of culture time and was conducted by visual observation or absorbance measurement, can be performed in three to five steps. It can be easily carried out in a short time and by mechanically counting the amount of light emitted.

[0020] In addition, conventional methods, such as methods that measure and determine absorbance, include errors in measurement data because they measure not only live bacteria but also dead bacteria, but the method of the present invention, in principle, The drug sensitivity test is characterized by high accuracy because it measures ATP. Therefore, the method of the present invention provides great advantages not only in the treatment of infectious diseases but also in fields that require testing of antibacterial activity of drugs.

[0021]

EXAMPLES Tests showing the correlation between the method of the present invention and the conventional method will be described below. Preculture of Test Bacteria In this test, Escherichia coli NIHJ strain was used as a test bacterium for measuring ATP amount. This test bacterium (Escherichia c
One platinum loopful of Oli NIHJ strain) was aseptically inoculated into 5 ml of a preculture medium having the following composition, and cultured stationary at 35° C. for 20 hours.

Preculture medium (Muller-Hinton Brot
h) Beef infusion 300 g Casamino acids 17.5 g Starch 1.5 g Purified water
1000mlpH 7.2~7
．． 4 (25℃: after sterilization)

Sensitivity test method by measuring ATP amount A medium with the following composition was used as a measurement medium. Measurement medium (Cation Supp)
Muller-Hinton Br.
oth) beef extract
300 g Casamino acids 17.5 g
starch
1.5 g purified water
1000ml
pH 7.2-7.4 (25℃: after sterilization)
Ca ion*5
0mg/l Mg ion*
25mg/l *
It was added aseptically after sterilization.

[0024] A measurement medium containing antibiotics prepared by adjusting the various antibiotics to be tested shown in Table 1 below to a predetermined concentration was added to the above measurement medium in a 96-well microtiter plate (manufactured by Dynatech, white). , 1 per well
00±10 μl was dispensed. In addition to the target antibiotic name and test concentration, M determined in advance by the current method (liquid medium dilution method)
The IC is also shown in Table 1. In addition, the test concentration is MIC
Two points were selected in the range including.

[0025]

[Table 1]

Next, the pre-cultured test bacterial solution (2×10
8 CFU/ml) was diluted 100 times with physiological saline,
5 μl was aseptically inoculated into each well of a microtiter plate (104 CFU/well). After culturing this plate at 35°C for 4 hours, ATP release agent (trade name:
PICOEX B; manufactured by Packard Japan) 100
After adding μl and extracting ATP, firefly-derived luciferin-luciferase (trade name: Pycozyme;
Packard Japan Co., Ltd.) 40 μl was added. Lumi
nos CT-9000 (manufactured by Chuo Kagaku Kogyo Co., Ltd.)
The amount of luminescence was measured using a voltage of 1050 V and an integration time of 1.0 seconds. Test bacterial solution (2 x 108 CFU/ml)
The same ATP release agent, luciferin-luciferase, was added to 5 μl of 100-fold diluted with physiological saline, and the amount of luminescence was measured and used as a blank value (control). The measurement results are shown in Table 2 and FIG. 1.

[0027]

[Table 2]

Susceptibility testing method according to current method As a comparative test, the antibacterial activity of the same drugs and concentrations as above was investigated using the current liquid medium dilution method. In the frozen plate Eiken Type 1 manufactured by Eiken Scientific Co., Ltd., which is currently used in clinical tests, add 5 μl of the test bacteria solution diluted with physiological saline used in the above ATP measurement method to each well of the frozen plate. (104 CFU/
well). Cultured at 35°C, 0 hours, 4 hours after culture,
After 20 hours, the turbidity of each well was measured by absorbance at 630 nm. The measurement results are shown in Table 3 together with the data of the drug-free control, and the results after 20 hours are shown in FIG.

[0029]

[Table 3]

[0030]

[Discussion] In the current method of determining the presence or absence of microbial growth based on changes in absorbance, almost no change in absorbance is observed up to 4 hours of culture, and the determination is made by measuring absorbance after 20 hours. The change in absorbance after 20 hours using the current method and the increase/decrease in ATP amount after 4 hours of culture using the method of the present invention are shown in Figures 1 and 2. It can be seen that results that are correlated with Figure 2) can be obtained. In other words, in this test, the luminescence amount of ATP amount after 4 hours of culture was measured, and those with a significant decrease in ATP amount were classified as susceptible.
It can be seen that the susceptibility of a drug to microorganisms can be determined by determining resistance if the amount of ATP increases or decreases little.

[Brief explanation of the drawing]

FIG. 1 is a bar graph showing the results of ATP amount measurement after 4 hours according to the method of the present invention.

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

Claims (5)

[Claims] Claim 1: After aseptically inoculating and culturing a test microorganism into a medium containing a drug at a predetermined concentration, the amount of ATP in the microorganism cells is measured, and this ATP amount is compared to that of a control at the start of culture. A drug susceptibility test method for microorganisms characterized by comparing the amount of ATP. 2. The method for testing the drug susceptibility of microorganisms according to claim 1, wherein the amount of ATP is measured using a luciferin-luciferase system. 3. The method for testing the drug susceptibility of microorganisms according to claim 2, wherein the luciferin-luciferase system is selected from systems derived from fireflies and cloned microorganisms. 4. After dispensing a measurement medium containing a drug at a predetermined concentration into a microtiter plate, a test microorganism diluted with physiological saline to a predetermined concentration is aseptically inoculated;
The method for testing microorganisms for drug susceptibility according to any one of claims 1 to 3, wherein after culturing at 5 to 37°C for 3 to 5 hours, an ATP release agent is added, luciferin-luciferase is added, and the amount of luminescence is measured. [Claim 5] Claims 1 to 4, wherein the drug is an antibiotic.
Drug susceptibility testing method described in any of the sections below.