JP2001204492A - Method for assaying proliferation and activity of microorganism, and method for testing sensitivity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly and readily assaying the proliferation and activity of microorganisms without breaking the microorganisms, and further to provide a method for testing the sensitivity for readily determining antimicrobial activity of various kinds of antimicrobial agents to the microorganisms. SOLUTION: This method for assaying the proliferation and/or activity of the microorganisms comprises culturing the microorganisms in the coexistence of a tetrazolium compound and an electron carrier to the tetrazolium compound, and determining the formazan formed by the reduction of the tetrazolium compound by spectroscopy. The method for evaluating the sensitivity of the microorganisms to the various kinds of the antimicrobial agents comprises culturing the microorganisms in the coexistence of the antimicrobial agents, the tetrazolium compound and the electron carrier to the tetrazolium compound, and determining the formazan formed by the reduction of the tetrazolium compound by spectroscopy. The tetrazolium compound and the formazan which is a reduction product thereof are water-soluble.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quickly and simply measuring the growth and activity of a microorganism and a method for testing susceptibility using the method.

[0002]

2. Description of the Related Art A susceptibility test in a microorganism test and a measurement of the activity, growth rate, and survival rate of a microorganism are performed by visually observing the formation of a colony on an agar medium or by measuring the amount of bacteria contained in a liquid medium by measuring the turbidity of the medium. I went by measuring. However, microbiological susceptibility testing using these methods requires long culture times of 18 to 24 hours, which hinders rapid reporting to the clinical setting and hinders treatment (especially determination of the antimicrobial agent to be administered). I'm coming. Under these circumstances, it is strongly desired to develop a quicker and simpler sensitivity test method.

[0003]

As a more rapid test method for drug sensitivity of microorganisms, a tetrazolium reduction method utilizing an enzymatic reduction reaction used for measuring the growth rate of animal cells can be considered. However, tetrazolium used for measuring the growth rate of animal cells, for example, 3-
(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium salt (MTT), etc., most of which are hydrophobic, penetrate into cells, In the form of insoluble formazan. Therefore, in order to measure color development, a process of destroying cells and uniformly dispersing them is necessary, and thereafter, the absorbance is measured. In the case of animal cells, since the cells are relatively easily destroyed, it is relatively easy to uniformly disperse insoluble formazan. However, since microorganisms have a stronger cell membrane than animal cells, it is difficult to treat them against cell destruction, and their growth and activity could not be measured using the tetrazolium reduction method.

[0004] Therefore, there is a demand for the development of a method capable of simply and quickly measuring the growth and activity of microorganisms without performing cell destruction treatment. An object of the present invention is to provide a method capable of quickly and simply measuring the growth and activity of a microorganism without destroying the microorganism. Another object of the present invention is to provide a susceptibility test method that can easily determine the antibacterial activity of various antibacterial agents against microorganisms using the above-described measurement method.

[0005]

According to the present invention, a microorganism is cultured in the presence of a tetrazolium compound and an electron carrier to the tetrazolium compound, and the formazan produced by reduction of the tetrazolium compound is quantified by spectroscopy. And a method for measuring the growth and / or activity of the microorganism, wherein the tetrazolium compound and formazan, which is a reduction product thereof, are water-soluble. Further, the present invention provides an antimicrobial agent for a microorganism,
Cultured in the presence of a tetrazolium compound and an electron carrier to the tetrazolium compound, by quantifying formazan generated by reduction of the tetrazolium compound by spectroscopy, to measure the growth and / or activity of the microorganism, The present invention relates to a method for evaluating the sensitivity of a microorganism to the antibacterial agent, wherein the tetrazolium compound and formazan, which is a reduction product thereof, are water-soluble.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for measuring the growth and / or activity of a microorganism and the method for evaluating susceptibility to an antibacterial agent according to the present invention, a tetrazolium compound is water-soluble and its reduced product, formazan, is water-soluble. Is used. The water-soluble formazan produced by reducing the water-soluble tetrazolium compound is uniformly dissolved in the culture solution, so that the absorbance can be directly measured without destroying the microorganism. The water-soluble tetrazolium compound can be, for example, a disulfonate tetrazolium salt. Further, as the disulfonate tetrazolium salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-
(2,4-disulfophenyl) -2H-tetrazolium salt and 2- (4
-Iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-
Disulfophenyl) -2H-tetrazolium salt. These compounds are known compounds and are commercially available as reagents or can be synthesized by known methods.

In the method of the present invention, a microorganism is cultured in the presence of a tetrazolium compound and an electron carrier on the tetrazolium compound, and formazan produced by reduction of the tetrazolium compound directly or via an electron carrier is quantified by spectroscopy. I do. In the method of the present invention for measuring the growth and / or activity of a microorganism, the concentration of the microorganism in the culture solution is, for example, about 10 ⁷ to 10 ⁸ , and the concentration of the tetrazolium compound in the culture solution is 10 to 300 μg / m 2.
It can be about l. Further, nutrient components and the like contained in the culture solution and the temperature of the incubation can be appropriately selected depending on the type of the microorganism (optimal pH and optimal temperature). Further, the culture time can be appropriately determined within a range where the growth and activity of the microorganism can be measured.

[0008] The electron carrier is capable of moving inside and outside the cell, mediating the transfer of electrons between the redox system and the tetrazolium compound in the microbial cell, facilitating the formation of formazan, and the sensitivity of measurement by spectroscopy. Can be mentioned. Examples of the electron carrier include, for example, 1-methoxy
And -5-methylphenazinium methyl sulfate and phenazinium methyl sulfate. These substances are readily available as commercial products. The electron carrier is, for example, 1 to 10 μg / ml in a culture solution.
It is appropriate to add it at a concentration of the order.

The measurement by spectroscopy can be performed as follows. The culture is started by adding the tetrazolium compound and the electron carrier to the culture solution containing the microorganism, and an appropriate amount is sampled from the culture solution at appropriate intervals. The color intensity of the sampling liquid is measured as absorbance. When 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt is used as the tetrazolium compound, the emission of formazan produced is determined by the absorbance at 450 nm. By measuring over time, the growth status and activity of the microorganism can be measured.

[0010] The microorganisms whose growth and / or activity can be measured by the method of the present invention are not particularly limited, and include, for example, enterobacteria, Pseudomonas aeruginosa, H. influenzae, staphylococci, hemolytic streptococci, enterococci. , Bacillus, Bacteroides, Clostridium and the like. The measuring method of the present invention can measure the growth status and activity of a microorganism in about 10 minutes to several hours.

[0011] The susceptibility test method of the present invention is carried out by culturing a microorganism in the above culture solution in the presence of an antibacterial agent. There is no particular limitation on the type of antibacterial agent used. In addition, a drug sensitivity test can be performed from various angles by testing by changing the concentration of the antibacterial agent in the culture solution or by using two or more antibacterial agents in combination. Since the method of the present invention does not require the operation of destroying microorganisms and can evaluate the effect of the drug on microorganisms in a short time as described above, the drug sensitivity test is completed within this time. Conventional drug susceptibility testing of pathogens is based on visual observation of changes in bacterial growth.
It took more than 10 hours. However, in the measurement method of the present invention, the drug sensitivity test can be completed in about 10 minutes to several hours.

Hereinafter, the present invention will be described in more detail with reference to examples. Example 1 (Comparison of absorbance generated by reduction of various tetrazolium by Escherichia coli) Escherichia coli (Eschrichia coli ATCC2592
2) was enriched and cultured in a Mueller Hinton medium, and the turbidity of the cells was adjusted in the same medium so that an absorbance of 0.1 was obtained at a wavelength of 600 nm. After adding various tetrazolium solutions to 2.5 ml of the bacterial suspension, the cells were cultured at 37 ° C., and after 90 minutes, the absorbance at the maximum wavelength was measured over time. In Table 1, WST-1 is 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt, and MTT is 3 -(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium salt. In addition, 1-methoxy-5-methylphenazinium methyl sulfate (1-m), which is an electron carrier that mediates a redox reaction with a tetrazolium compound in the aforementioned bacterial suspension,
ethoxy-5-methyphenazinium-methylsulfate (hereinafter 1-MPM
The mixed solution of S)) was added, and the absorbance was measured by the same operation. The concentration of tetrazolium at this time is 2.5 mM, 1-MPM
The concentration of S was 0.2 mM. In Table 1, (+) indicates a system to which 1-MPMS was added, and (-) indicates a system to which 1-MPMS was not added.

The results are as shown in Table 1. The absorbances at the start of the culture were all about 0.1. In the case of the system to which 1-MPMS was not added, the increase in absorbance with time was the largest in MTT, and the water-soluble tetrazolium WST-1 had the same MTT. On the other hand, in the case of the system to which 1-MPMS of the present invention is added, when WST-1, which is a water-soluble tetrazolium, is used, the absorbance with time is significantly increased as compared with other tetrazolium, and the water-soluble tetrazolium It can be seen that a great effect is exhibited by using the compound and the electron carrier together. As described above, by combining water-soluble tetrazolium such as WST-1 and 1-MPMS as an electron carrier, the growth status and activity of microorganisms can be easily measured within a few hours, and measurement can be performed in a shorter time Becomes

[0014]

[Table 1]

Example 2 (Relationship between initial turbidity of Escherichia coli and reduction reaction rate of WST-1) Escherichia coli (Eschrichia coli ATCC25922) was enriched and cultured in a Mueller Hinton medium. Was adjusted so as to obtain an absorbance of 0.005 to 0.1. 96
After 250 μl of the bacterial suspension was dispensed into the well microplate, 25 μl of a mixture of 2.5 mM WST-1 and 0.2 mM 1-MPMS was added, and the mixture was cultured at 37 ° C. for 30 to 120 minutes. Culture time 30
The absorbance (wavelength 450 nm) and turbidity (600 nm) of formazan were measured for minutes, 60 minutes, 90 minutes and 120 minutes, and the relationship is shown in FIG. When the initial turbidity is 600 nm and the absorbance is 0.1, 90
After the minute culture, the absorbance at 450 nm increases from 0.1 to 1 and W
It means that a large absorbance, which cannot be obtained by conventional turbidity measurement without using ST-1 and 1-MPMS, can be obtained in a short time by using these agents.

Example 3 (Measurement of Escherichia coli growth inhibition by polymyxin B by turbidity method and WST-1 reduction method) Escherichia coli (Eschrichia ATCC2592)
2) was enriched and cultured in a Mueller Hinton medium, and the turbidity of the cells was adjusted in the same medium so that an absorbance of 0.1 was obtained at a wavelength of 600 nm. Cell suspension 250 in a 96-well microplate
After dispensing μl, 25 μl of a solution of polymyxin B adjusted from 2.5 μg / ml to 1 μg / ml was added, and W having the same composition as in Example 2 was added.
Add 25 μl of a mixed solution of ST-1 and 1-MPMS, and add
Turbidity was measured at 00 nm and the results are shown in FIG. Aside from this
The absorbance at 450 nm was also measured and the results are shown in FIG. 2 and 3, the symbols ◇, △ and △ are 1 μg / ml and 2.5 μg / ml, respectively.
Shows the concentration of polymyxin B in ml and 5 μg / ml. □ indicates the case where polymyxin B was not added. 2 and 3, the inhibitory effect of polymyxin B, which inhibits the function of the cell membrane, can be confirmed by culturing for 2 hours in any of the measurement methods. However, the figure between the negative control obtained after 2 hours of culture and the system to which 1 μg / ml of polymyxin B was added was shown.
Compared to the turbidity value (difference) shown in FIG. 2, the value (difference) of the absorbance of WST-1 reduction shown in FIG. 3 is about 10 times larger, and according to the present invention, it is much easier to determine the inhibitory effect. You can see that there is.

Example 4 (Measurement of Escherichia coli Growth Inhibition by Various Antibacterial Agents Using WST-1 Reduction Method)
It was adjusted to obtain an absorbance of 0.1 at a wavelength of 600 nm.
After 250 μl of the bacterial suspension was dispensed into a 96-well microplate, various antibacterial agent solutions (25 μl) were added and cultured. After a certain period of time, 25 μl of a mixed solution of WST-1 and 1-MPMS having the same composition as in Example 2 was added and mixed, and after culturing for 30 minutes, the absorbance at 450 nm was measured. As shown in FIGS. 4 and 5, a DNA synthesis inhibitor (norfloxacin) (FIG. 4), an antimetabolite (trimethoprim) (FIG. 4) and a protein synthesis inhibitor (kentamicin sulfate, clarithromycin, chloramphenico-
(Fig. 5) shows that the absorbance of formazan generated from WST-1 is lower than that of the absence of formazan and can inhibit bacterial growth. Therefore, this measurement method can be used for a sensitivity test for evaluating the antibacterial activity of various antibacterial agents. The concentrations of the antibiotics used in this experiment are as follows. Nofloxacin 0.12 μg / ml, trimethoprim 2 μg / ml, gentamicin sulfate 1 μg / ml, clarithromycin 1 μg / m
l, chloramphenicol 8μg / ml

[Brief description of the drawings]

FIG. 1 shows the initial turbidity of E. coli and the reduction reaction rate of WST-1 determined in Example 2.

FIG. 2 shows the inhibition of polymyxin B growth in Escherichia coli by the turbidity method determined in Example 3.

FIG. 3 shows the inhibition of E. coli growth of polymyxin B by the present invention (WST-1 reduction method) determined in Example 3.

FIG. 4 shows the inhibition of E. coli growth of norfloxacin and trimethoprim by the present invention (WST-1 reduction method) determined in Example 4.

FIG. 5 shows the inhibition of Escherichia coli growth of kentamicin, clarithromycin and chloramphenicol by the present invention (WST-1 reduction method) determined in Example 4.

Continued on the front page (72) Inventor Tsuneo Kaneko Kyoto, Kuseyama-gun, Ohashibetsutsumi Outer Boundary 23, Nikken Biotechnology Research Institute (72) Inventor Kazuya Haruto, Kyoto, Kumiyama-cho, Ohmibetsue Outer Boundary 23, Kyoto F-term in Nikken Biomedical Research Institute (reference) 4B063 QA01 QA06 QA19 QQ05 QQ06 QQ61 QQ98 QR41 QR68 QR74 QR75 QS24 QX01

Claims (4)

[Claims] A microorganism is cultured in the presence of a tetrazolium compound and an electron carrier to the tetrazolium compound, and formazan generated by reduction of the tetrazolium compound is quantified by spectroscopy, whereby the growth and / or Or a method for measuring the activity, wherein the tetrazolium compound and formazan, which is a reduction product thereof, are water-soluble. 2. The microorganism is cultured in the presence of an antibacterial agent, a tetrazolium compound and an electron carrier to the tetrazolium compound, and formazan produced by reduction of the tetrazolium compound is quantified by spectroscopy. A method for evaluating sensitivity to an antibacterial agent, wherein the tetrazolium compound and formazan, which is a reduction product thereof, are water-soluble. 3. The method according to claim 1, wherein the tetrazolium compound is a disulfonate tetrazolium salt. 4. The method according to claim 1, wherein the disulfonate tetrazolium salt is 2- (4-
(Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt or 2- (4-iodophenyl) -3- (2,4-dinitrophenyl)- The method according to claim 3, which is a 5- (2,4-disulfophenyl) -2H-tetrazolium salt. 5. The method according to claim 1, wherein the electron carrier is 1-methoxy-5-methylphenazinium methyl sulfate or phenazinium methyl sulfate.