JPH02211899A - Measurement of drug sensibility of bacterium - Google Patents

Abstract

PURPOSE:To rapidly and simply detect a growth state of bacterium by adding an oxidation-reduction indicator to a medium to culture a bacterium having oxidation-reduction ability and optically measuring degree of change of color tone of the indictor or fluorescence intensity. CONSTITUTION:In examining drug sensitivity of bacterium having oxidation- reduction ability, an oxidation-reduction indicator is added to mediums containing a drug having various concentrations. The bacterium is inoculated into the mediums and degree of change of color tone or fluorescence intensity of the mediums during the culture or after the culture is measured. Resazurin is used as the oxidation-reduction indicator, preferably degree of change of color based on reduction of resazurin to resorufine is obtained by measuring absorbance at wavelength in range of 550-600nm or resorufine is irradiated with excitation light and fluorescence intensity at 580-590nm wavelength is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for measuring drug sensitivity of bacteria for selecting effective drugs, for example, in the treatment of bacterial infections.

<Conventional technology> Conventionally, drug susceptibility tests performed in clinical laboratories are based on the minimum inhibitory concentration (hereinafter referred to as rMIC) of a drug against the test bacteria.
J) is measured directly or indirectly, and is broadly divided into dilution method and dispersion method.

There are two types of dilution methods: the agar plate dilution method and the liquid dilution method. Each method involves preparing an agar plate or broth containing serially diluted drugs, inoculating a certain amount of test bacteria onto a medium, and diluting the drug for a certain period of time. This method determines the MIC by visually determining the presence or absence of bacterial growth in the culture medium after culturing.

On the other hand, in the disk diffusion method, a disk (filter paper) containing a certain amount of drug is placed on an agar plate inoculated with test bacteria in advance, and after culturing for a certain period of time, a bacterial growth inhibition zone is formed around the disk. This method determines susceptibility/resistance by measuring the size of (inhibition circle).

However, all of the above methods require culture time for bacteria to grow, so even for fast-growing bacteria such as Escherichia coli, it takes more than 48 hours after specimen submission before the measurement results are known. Test results are not effectively used for initial treatment, which is important for treating infectious diseases.

Furthermore, in order to process a large number of samples, the operating method as well as the method for determining the results are extremely complicated.

In addition, various automated devices that have been developed in recent years determine susceptibility/resistance by optically detecting changes in turbidity or scattered light intensity during bacterial growth in the presence of drugs. However, there are still problems with accuracy and sensitivity, such as being affected by cell expansion (swelling) that occurs during sterilization or the action of cell wall synthesis inhibitors, and not being applicable to slow-growing bacteria. Moreover, the device price and running cost are high, so it has not become widely used.

<Problems to be Solved by the Invention> The present invention has been made in view of the drawbacks of the above-mentioned prior art.
The purpose of the present invention is to provide a method for measuring bacterial drug sensitivity that allows rapid and simple detection of the growth state of bacteria in the presence of various drugs.

〈Means for solving problems〉 Such objects are achieved by the present invention as described below.

That is, the present invention is a method for measuring the drug sensitivity of bacteria, in which bacteria are cultured in a medium containing drugs at various concentrations, and the susceptibility of the bacteria to the drug is measured using the redox ability of the bacteria. A method for measuring drug sensitivity of bacteria, which comprises adding a redox indicator to the medium and optically measuring the degree of color change or fluorescence intensity of the medium, thereby measuring the sensitivity of the bacteria to the drug. It is.

The redox indicator is resazurin, and in order to detect the degree of color tone change due to the reduction of resazurin to resorufin, it is possible to detect the degree of color tone change based on the reduction of resazurin to resorufin by measuring the absorbance at one wavelength in the range of 55e to 600 nm. Preferably, it is a method for measuring the drug susceptibility of bacteria.

Further, the redox indicator is resazurin, and in order to detect the degree of reduction of resazurin to resorufin, bacterial drug sensitivity measurement is performed by irradiating excitation light and measuring fluorescence intensity at a wavelength of 580 to 590 nm. Preferably, it is a method.

Hereinafter, the method for measuring bacterial drug sensitivity of the present invention will be explained in detail.

Bacteria enzymatically redox and oxidize many organic and inorganic substances, thereby synthesizing necessary substances, metabolizing unnecessary or harmful substances, or obtaining energy necessary for sustaining life. Many oxidoreductases are known to be involved in these reactions, and in particular NAD (H) and HADP (H), which are coenzymes involved in the oxidoreductases that exist in the largest amount in bacterial cells, are converted into electron acceptors ( Numerous enzymes are known (eg, dehydrogenases such as alcohol, lactic acid, glyceraldehyde-3-phosphate, and 3-hydroxysteroids).

The present invention focuses on various oxidoreductase activities that bacteria universally possess, and measures the drug sensitivity of bacteria by observing the growth state of bacteria in the presence of drugs and using the increase in enzyme activity accompanying bacterial growth as an indicator. It is something to do.

In the presence of clinically isolated bacteria and clinically used drugs, a method for measuring the enzymatic activity, that is, redox ability of bacteria, involves adding a redox indicator (hereinafter simply referred to as an indicator) to a medium, particularly a liquid medium. It is preferable that the degree of color tone change or the level of fluorescence intensity is detected by an optical measurement method appropriate for each. Such indicators include, for example, resazurin, tetrazolium salts, or the like.

Hereinafter, a typical case will be described in which resazurin is used as an indicator and a reaction in which bacteria reduce resazurin to produce resorufin is utilized.

Resazurin is reduced to resorufin by various oxidoreductase activities associated with bacterial growth. This reaction formula is shown below.

This reaction is irreversible, and the protons and electrons in the above formula are supplied by protons and electrons exchanged in various redox reactions.

On the other hand, if the bacteria are sensitive to the drug, the reaction will not occur or will be minimized. That is,
By measuring the ability of bacteria to reduce resazurin in the presence of various drugs, the sensitivity of bacteria to drugs can be easily determined.

Resazurin exhibits a blue color under neutral conditions and has a wavelength of 600
It has an absorption maximum near nm. On the other hand, resorufin exhibits a pink color and has an absorption maximum near a wavelength of 570 nm.

Note that resazurin may have a substituent such as an alkyl group, a nitro group, a methoxy group, or a halogen atom.

In the present invention, bacteria are cultured in a medium containing various concentrations of drugs and resazurin, and changes in color (from blue to pink) are measured optically in order to obtain accurate measurement results. . Then, based on predetermined criteria, MI
Determine C, etc.

A specific method of optical measurement will be described below.

A predetermined concentration (preferably 10 to 10,100 IL) of resazurin is added to a medium containing serially diluted drugs (those used clinically), the test bacteria are inoculated, and after culturing for a certain period of time,
The absorbance at one wavelength in the range of 550 to 600 nm is measured.

The reduction of resazurin, that is, the degree of bacterial growth, is determined by: (1) a decrease in absorption at 600 nm, which is the maximum absorption of resazurin, (2) an increase in absorption at 570 nm, which is the maximum absorption of resorufin, or (2) a difference in absorbance between 570 nm and 600 nm [ OD! ? . -OD. ] is preferably detected as an index.

In particular, the above case (2) is useful because it allows measurement to be performed while eliminating the influence of interfering components such as baseline elevation due to scattering of turbidity associated with culture.

In the present invention, the absorbance value can be directly used as an index for drug sensitivity measurement, but in order to make a more detailed determination, the absorbance difference [ODB? . -OD,. . ] It is preferable to determine the growth percentage calculated by the following formula (1) from the value and make the determination based on this.

A: Absorbance difference in the presence of drug [oDst. -OD,. . ] B: Difference in absorbance when no drug is included (resistant control) C: Difference in absorbance when no bacteria are inoculated (susceptibility control) The criteria for determining susceptibility based on growth % differs for each drug, and depends on the mechanism of action of the drug. It is determined based on conditions such as onset, duration of action, and concentration.

Furthermore, in the present invention, the degree of reduction of resazurin to resorufin can be determined by measuring fluorescence intensity.

Resazurin does not have fluorescence, and resorufin has fluorescence. Therefore, the reduction of resazurin to resorufin can be viewed as the production of a fluorescent substance.

Specifically, after culturing bacteria in the same manner as described above, for example, irradiation with excitation light having a wavelength of 540 to 550 nm, measuring the intensity of fluorescence wavelength of 580 to 590 nm, and measuring the magnitude of the fluorescence intensity. Determine susceptibility by

In this case, the type, wavelength, etc. of the excitation light are not particularly limited.

<Example> (Example 1) Niskerichia coli ATCC 25922 strain was cultured on Mueller-Hinton agar medium at 37°C for 18 hours. 10"/ml).

On the other hand, ampicillin (ABPC) was dissolved in sterile distilled water, and the ABPC solution, which was serially diluted 2 times, was dispensed into a 96-well microplate at 100 μE per well. ABP
After inoculating the bacterial solution, the final C concentration was 0.5.1.2.4.
It was adjusted to have a titer of 8.16.32.64 μg/IIIj.

Next, filter-sterilized Mueller-Hinton broth (pH
7,4) 4.8mff1 to 4. This bacterial solution to which 100 μE of OXlo-3M resazurin aqueous solution was added and 100 μE of the above Niskerichia coli suspension was added was added to the above ABPC.
The solution was inoculated at 100 μm per well in a microplate, and after culturing at 37° C. for 4 hours, the absorbance OD of each well was measured using an absorbance meter UV-160 manufactured by Shimabara Manufacturing Co., Ltd. and OD s. . was measured.

Figure 1 shows ODi. and OD s. . Figure 2 shows the measurement results of the absorbance difference [OD, ? . -OD,. . The growth percentages are shown in Fig. 3 of J1. It can be seen from any of FIGS. 1 to 3 that resazurin reduction is suppressed at an ABPC concentration of 2 Mg/mj. This result is based on the NCCLS
MIC value measured using the MIC measurement method standardized by
= 2 μg/ml).

(Example 2) Various concentrations of ampicillin (A
BPC), cefazolin (CEZ), and gentamicin (GM), the resazurin-reducing ability of each bacterial species shown in Table 1 below was measured, and the drug concentration at which resazurin reduction was suppressed was determined as the MIC.

Table 1 shows the results of the growth percentage calculated based on the above formula (1) from the absorbance measurement results, and the MIC and N
The MIC measured by the CCLS standard method is shown.

In addition, MIC according to the method of the present invention comprehensively considers the correlation between the number of viable bacteria and growth %, the growth % when the number of viable bacteria is constant, etc., and determines the maximum drug concentration at which the growth % is 50% or less. It was set as MIC.

As shown in Table 1, MI by the method of the present invention and the NCCLS method
It can be seen that the C values are consistent for all bacterial species and drugs and are extremely reliable.

<Effects of the Invention> According to the method for measuring bacterial drug susceptibility of the present invention, by using the redox ability of bacteria as an index, compared to conventional measuring methods,
Bacterial drug susceptibility testing can be performed quickly and easily.

In particular, when the degree of reduction of resazurin to resorufin is optically detected, drug sensitivity can be measured with high accuracy.

[Brief explanation of the drawing]

FIG. 1 shows the absorbance of Example 1 at 0 Dsy. and OD,. . This is a graph showing. FIG. 2 shows the absorbance difference [oDsy] in Example 1. -OD,. . ] is a graph showing. FIG. 3 is a graph showing growth percentage in Example 1. (-e-)OL'+ (IQ (-o-)009(IQ FI G: 2 ABPC thick! (P92Ia/ml) FIG, 3

Claims (3)

[Claims] (1) A method for measuring drug sensitivity of bacteria, in which bacteria are cultured in a medium containing drugs at various concentrations, and the susceptibility of the bacteria to the drug is measured using the redox ability of the bacteria, the method comprising: A method for measuring drug sensitivity of bacteria, which comprises adding a reducing indicator to the medium and optically measuring the degree of color change or fluorescence intensity to measure the sensitivity of the bacteria to the drug. (2) The redox indicator is resazurin, and the degree of color tone change due to the reduction of resazurin to resorufin is detected by measuring absorbance at at least one wavelength in the range of 550 to 600 nm. Item 1. The method for measuring bacterial drug susceptibility according to item 1. (3) The redox indicator is resazurin, and the degree of reduction of resazurin to resorufin is detected by irradiating excitation light and measuring fluorescence intensity at a wavelength of 580 to 590 nm. A method for measuring drug susceptibility of bacteria.