JP7270475B2 - Antibacterial activity evaluation method - Google Patents

Description

The present disclosure relates to methods for evaluating antibacterial activity.

Many antibiotics, substances that inhibit the growth of pathogenic bacteria, are produced by microorganisms. It is said that there are more than 1,000,000 types of microorganisms on the earth, and among them, it is believed that there are many that produce new antibiotics.
Under such circumstances, there is a demand for a system for evaluating the activity of new, yet undiscovered antibiotics produced by microorganisms.
Conventionally, the disc diffusion method is known as a method for evaluating antibacterial activity against pathogenic bacteria (see Non-Patent Document 1). This disk diffusion method is a method in which a paper disk of several millimeters in diameter impregnated with an antibacterial active substance is placed on an agar medium coated with a pathogen solution, and the antibiotic is diffused to evaluate its activity. Bacteria do not proliferate around the paper disc and it becomes transparent. This transparent portion is called a halo. On the other hand, bacteria multiply at a distance away from the paper disc, resulting in a yellowish white turbidity. The area in which bacteria do not proliferate becomes a transparent circle, which is called an inhibition circle, and the antibacterial activity is evaluated by measuring its diameter.

James H. Jorgensen and Mary Jane Ferraro Clinical Infectious Diseases 2009; 49:1749-55

However, in the conventional disk diffusion method, only about 5 samples can be evaluated for one agar medium (for example, a Φ60 mm plate).
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an antibacterial activity evaluation method capable of evaluating a larger number of samples than conventional methods.

The present inventors have developed a novel antibacterial activity evaluation method as a result of extensive research.
And, according to this method, it was found that the problems of the conventional method can be solved, and more samples can be evaluated. Based on this result, the following invention is provided.

[1] An antibacterial activity evaluation method using a microchamber having a plurality of wells,
A step of applying bacteria to an agar medium;
A step of encapsulating an evaluation substance in the well;
placing the microchamber on the agar medium coated with the bacteria so that the evaluation substance is in contact;
a step of keeping the temperature within a predetermined temperature range while the microchamber is placed on the agar medium;
and removing the microchamber from the agar medium;
Observing the state of a cloudy region and a transparent region formed on the agar medium from which the microchamber has been removed, and determining that the evaluation substance contained in the well corresponding to the transparent region has antibacterial properties Activity evaluation method.

According to the antibacterial activity evaluation method of the present invention, by using a microchamber having a plurality of wells and increasing the number of wells, it becomes possible to evaluate a larger number of samples than in the conventional method.

It is a perspective view which shows an example of a microchamber. It is a perspective view showing an example of a well. It is a perspective view which shows an example of an agar medium. 1 is a perspective view showing an example of an agar medium coated with bacteria; FIG. It is a perspective view which shows an example of a microchamber. FIG. 4 is a perspective view showing an example of a microchamber in which a well contains an evaluation substance; FIG. 4 is a cross-sectional view showing a state in which microchambers are placed on an agar medium. FIG. 4 is a cross-sectional view showing a state in which the microchamber is placed on an agar medium after being kept warm within a predetermined temperature range. FIG. 3 is a cross-sectional view showing an agar medium from which microchambers have been removed; FIG. 4 is a plan view showing an agar medium from which microchambers have been removed; It is a stereomicrograph of a microchamber. It is an electron micrograph of a microchamber. It is an image of a phase-contrast microscope in the case of using a microchamber with a well having a square planar shape (length of one side: 300 μm). It is an image of a phase-contrast microscope in the case of using a microchamber with a well having a square planar shape (length of one side: 500 μm). It is an image of a phase-contrast microscope in the case of using a microchamber with a well having a circular planar shape (diameter: 300 μm). It is an image of a phase-contrast microscope in the case of using a microchamber with a well having a circular planar shape (diameter: 500 μm). It is a graph which shows a halo formation rate (%).

A preferred example of the present disclosure will now be presented.
[2] The antibacterial activity evaluation method according to [1], which comprises the step of hydrophilizing the wells of the microchamber.
The substance to be evaluated is usually contained in the wells in the form of an aqueous solution or suspension. By hydrophilizing the wells, the aqueous solution or suspension of the substance to be evaluated can smoothly flow into the wells and be contained therein.

[3] The antibacterial activity evaluation method according to [1] or [2], wherein at least two of the plurality of wells contain different types of substances to be evaluated.
In this way, evaluation substances of different types can be evaluated at once.

The present disclosure will be described in detail below. Note that the notation indicating the range of "x to y" includes x and y unless otherwise specified.

1. Antibacterial Activity Evaluation Method The antibacterial activity evaluation method uses a microchamber 3 having a plurality of wells 1 (see FIGS. 1 and 2).
The antibacterial activity evaluation method includes the following steps. 3 to 10 schematically show the concept of one example of the antibacterial activity evaluation method.
[1] A step of applying bacteria 7 to an agar medium 5 (see FIGS. 3 and 4).
[2] A step of encapsulating the evaluation substance 9 in the well 1 (see FIGS. 5 and 6).
[3] A step of placing the microchamber 3 on the agar medium 5 coated with the bacteria 7 so that the evaluation substance 9 is in contact (see FIG. 7).
[4] A step of keeping the microchamber 3 placed on the agar medium 5 within a predetermined temperature range (see FIG. 8).
[5] A step of removing the microchambers 3 from the agar medium 5 (see FIGS. 9 and 10).
The antibacterial activity evaluation method observes the state of the cloudy region 5A and the transparent region 5B formed on the agar medium 5 from which the microchamber 3 has been removed, and the evaluation substance 9 contained in the well 1 corresponding to the transparent region 5B. Judged to have antibacterial properties.
For example, in the antibacterial activity evaluation method shown in FIGS. 2 to 10, the evaluation substance 9 contained in the well 1 corresponding to the transparent region 5B in FIG. 9 is determined to have antibacterial properties.

Each term will be explained in detail below.
(1) Micro chamber 3
A microchamber 3 is a plate-like member having a plurality of recessed wells 1 .
The material of the microchamber 3 is not particularly limited. As the material, for example, silicone resin is preferably used. Although the silicone resin is not particularly limited, polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polymethylhydrogensiloxane, polymethylmethoxysiloxane, polymethylvinylsiloxane, and the like are preferable. These silicone resins may be used singly or in combination of two or more. Polydimethylsiloxane (PDMS) is particularly preferred because it is flexible and elastic and adheres closely to the agar medium when it comes into contact with the agar medium coated with bacteria.

The planar shape and size of the microchamber 3 are not particularly limited.
The planar shape of the microchamber 3 may be rectangular, circular, or the like.
When the planar shape of the microchamber 3 is rectangular, the size of the microchamber 3 is preferably 5 mm × 10 mm or more, more preferably 7 mm × 12 mm or more, and even more preferably 10 mm × 15 mm or more, from the viewpoint of handleability and productivity. . On the other hand, the size of the microchamber 3 is preferably 35 mm×40 mm or less, more preferably 33 mm×38 mm or less, and even more preferably 25 mm×30 mm or less, from the viewpoint of handleability and productivity. From these viewpoints, when the planar shape of the microchamber 3 is rectangular, the size of the microchamber 3 is preferably 5 mm × 10 mm to 35 mm × 40 mm, more preferably 7 mm × 12 mm to 33 mm × 38 mm, and 10 mm × 15 mm to 25 mm. ×30 mm is more preferable.

The thickness of the microchamber 3 is not particularly limited.
From the viewpoint of ensuring a sufficient depth of the well 1, the thickness of the microchamber 3 is preferably 350 μm or more, more preferably 400 μm or more, and even more preferably 450 μm or more. On the other hand, the thickness of the microchamber 3 is preferably 650 μm or less, more preferably 600 μm or less, and even more preferably 550 μm or less, from the viewpoint of handleability and productivity. From these viewpoints, the thickness of the microchamber 3 is preferably 350 μm to 650 μm, more preferably 400 μm to 600 μm, and still more preferably 450 μm to 550 μm.

From the viewpoint that the evaluation substance 9 can be smoothly included in the well 1, the microchamber 3 is preferably hydrophilized. The hydrophilization treatment is not particularly limited, but for example, a hydrophilization treatment using an O ₂ asher is preferably employed.

(2) Well 1
The planar shape and size of the well 1 are not particularly limited.
A planar shape of the well 1 may be, for example, circular or rectangular. The planar shape of the well 1 is preferably circular from the viewpoint that the substance to be evaluated 9 can be smoothly enclosed in the well 1 .
When the planar shape of the well 1 is circular, the diameter 1A (see FIG. 2) is preferably 350 μm or more, more preferably 400 μm or more, more preferably 450 μm or more, from the viewpoint of sensitively evaluating the antibacterial activity. More preferred. On the other hand, as for the size of the well 1, the diameter 1A is preferably 650 μm or less, more preferably 600 μm or less, and even more preferably 550 μm or less, from the viewpoint of evaluating a larger number of evaluation substances 9 at the same time. From these points of view, when the planar shape of the well 1 is circular, the diameter 1A is preferably 350 μm to 650 μm, more preferably 400 μm to 600 μm, and still more preferably 450 μm to 550 μm.

The well-to-well distance 1B (see FIG. 2) is not particularly limited.
The well-to-well distance 1B is preferably 150 μm or more, more preferably 200 μm or more, and still more preferably 250 μm or more from the viewpoint of suppressing the connection of adjacent halos (transparent regions 5B). On the other hand, the well-to-well distance 1B is preferably 450 μm or less, more preferably 400 μm or less, and even more preferably 350 μm or less, from the viewpoint of simultaneously evaluating a larger number of evaluation substances 9 . From these points of view, the well-to-well distance 1B is preferably 150 μm to 450 μm, more preferably 200 μm to 400 μm, and still more preferably 250 μm to 350 μm.

A depth 1C of the well 1 is not particularly limited.
The depth 1C of the well 1 is preferably 40 μm or more, more preferably 60 μm or more, and even more preferably 80 μm or more, from the viewpoint of encapsulating the evaluation substance 9 sufficient for evaluation in the well 1 . On the other hand, the depth 1C of the well 1 is preferably 160 μm or less, more preferably 140 μm or less, and even more preferably 120 μm or less, from the viewpoint that the well 1 does not contain the evaluation substance 9 excessive for evaluation. From these points of view, the depth 1C of the well 1 is preferably 40 μm to 160 μm, more preferably 60 μm to 140 μm, even more preferably 80 μm to 120 μm.

The number of wells 1 in one microchamber 3 is not particularly limited, and is appropriately selected according to the size of the microchamber 3 and the like.
In the microchamber 3 used for an agar medium plate (Φ60mm), the number of wells 1 in one microchamber 3 is preferably 350 or more, preferably 400 or more, from the viewpoint of simultaneously evaluating a larger number of evaluation substances 9. is more preferred, and 450 or more is even more preferred. On the other hand, the upper limit of the number of wells 1 in one microchamber 3 is not particularly limited, but the microchamber 3 used for an agar medium plate (Φ60mm) is usually 10000 or less.

(3) agar medium 5
The agar medium 5 is a medium using agar, and the concentration of the agar is not particularly limited. The agar medium 5 is preferably a solid medium, more preferably a plate medium, from the viewpoint of ease of handling.

(4) Bacteria 7
The type of bacteria 7 is not particularly limited. Examples of bacteria 7 include pathogenic bacteria.

(5) Evaluation substance 9
Evaluation substance 9 is an evaluation target for antibacterial activity, and can be applied to a wide range of antibacterial substances.
The type of evaluation substance 9 for evaluating antibacterial activity is not particularly limited, and may be one type or two or more types. When the evaluation substance 9 is of one type, the antibacterial activity evaluation method of the present invention can be used to secure the n number (sample size) and to perform antibacterial evaluation more accurately. When there are two or more types of evaluation substances 9, different types of evaluation substances 9 can be evaluated at once by using the antibacterial activity evaluation method of the present invention.

(6) Coating method in step [1] The coating method of the bacteria 7 is not particularly limited, but a coating method using a spin coater is preferably employed from the viewpoint of uniformly coating the bacteria 7 .

(7) Encapsulation method in step [2] The method of encapsulating the evaluation substance 9 is not particularly limited, but for example, a method of encapsulating the evaluation substance 9 by pushing it into the well 1 using a plate-like body is adopted. be. A cover glass or the like is preferably used as the plate-like body.
In this step, only the well 1 of the microchamber 3 is made to contain the substance 9 to be evaluated. Portions other than the well 1 (for example, portions between the adjacent wells 1 and 1) are in a state where the evaluation substance 9 does not exist. This is because if the evaluation substance 9 exists in a portion other than the well 1, adjacent halos (transparent regions 5B) will be connected, making evaluation difficult.
The “encapsulation” in the step [2] of the present disclosure includes not only putting the evaluation substance 9 itself into the well 1, but also putting a microorganism that produces the evaluation substance 9 into the well 1 and culturing it in the well 1. It also means that the evaluation substance 9 is included in the well 1 by

(8) Method of placing microchamber 3 on agar medium 5 in step [3] 3 is preferably pressed onto the agar medium 5 . By doing so, the evaluation substance 9 contained in the well 1 adheres to the agar medium 5, and the antibacterial activity can be evaluated more appropriately.

(9) Predetermined temperature range in step [4] The temperature range (predetermined temperature range) for heat retention in step [4] is not particularly limited, but is preferably 20 ° C. to 45 ° C., more preferably 35 to 40. °C. Within this range, the growth of bacteria can be promoted, the cloudy region 5A can be clarified, the contrast between the cloudy region 5A and the transparent region 5B (halo) is enhanced, and the antibacterial activity can be easily evaluated.
In addition, the time for keeping the temperature within the predetermined temperature range is not particularly limited, and can be appropriately changed according to the bacteria 7 and the evaluation substance 9 . The heat retention time is preferably 2 hours to 24 hours, more preferably 3 hours to 18 hours.
In step [4], as shown in FIG. 7, the agar medium 5 is maintained within a predetermined temperature range with the microchambers 3 placed thereon. In this state, the portion 2 between the adjacent wells 1 serves as a partition wall for the evaluation substances 9 contained in the adjacent wells 1, respectively, and mixing of the adjacent evaluation substances 9 is suppressed, so that the antibacterial activity is properly obtained. can be evaluated.

(10) Observation method of agar medium 5 Observation method of agar medium 5 is as follows. It is not particularly limited. For example, an observation method using a phase-contrast microscope can be employed.

2. Effect of the present embodiment According to the antibacterial activity evaluation method of the present embodiment, since the microchamber 3 having a plurality of wells 1 is used, it is possible to evaluate a larger number of samples than the conventional method.

Hereinafter, more specific description will be given with reference to examples.

In the experiments below, all the wells contained the same antibiotic whose antibacterial activity had already been confirmed, as will be described later. In this experiment, when the portion of the agar medium plate that was in contact with the antibiotic contained in the well was transparent and the portion that was not in contact with the antibiotic was opaque, it means the following. That is, when the part in contact with the antibiotic becomes transparent, it means that the growth of the bacteria is inhibited by the antibiotic, and when the part not in contact with the antibiotic becomes opaque, it means that there is no antibiotic. It means that the bacteria are growing and the cloud is cloudy. In other words, the observation of such transparent and opaque areas confirms that the presence of the antibiotic kills the bacteria and that the absence of the antibiotic results in the growth of the bacteria. It is an index that the antibacterial activity of can be evaluated.
In addition, the sensitivity of this antibacterial activity evaluation method can be confirmed by grasping the ratio of the transparent portions to the total number of wells. That is, even if the ratio of the transparent portion (corresponding to the "halo formation rate" described later) is low, if the presence of the transparent portion can be confirmed, the antibacterial activity can be confirmed, so it is useful as an antibacterial activity evaluation method. Furthermore, when the ratio of the transparent portion increases, the antibacterial activity can be confirmed with a high probability, so it can be seen that it is more useful as an antibacterial activity evaluation method.

1. Fabrication of Microchamber (1) Fabrication of Microchamber Mold 1 mL of SU8-50 (resist, manufactured by Nippon Kayaku Co., Ltd.) was placed on a 2.5-inch silicon wafer. After removing air bubbles in SU8-50, SU8-50 was applied to the silicon wafer by rotating the spin coater at 1000 rpm. A silicon wafer coated with SU8-50 was heated at 65° C. for 15 minutes, heated at 95° C. for 40 minutes, and left to cool for 1 hour (cooled to 40° C. or lower) in this order. After that, the silicon wafer coated with SU8-50 was covered with a chromium mask (mold) patterned with microchambers and exposed for 19 seconds using a mask aligner. Then, after post-baking at 65° C. for 2 minutes and 95° C. for 15 minutes, the silicon wafer was washed with SU8 developer and isopropyl alcohol and developed to prepare a 100 μm high SU8 microchamber mold.

(2) Preparation of microchamber PDMS main agent and curing agent were mixed at 10:1, and 5 g of the degassed solution was poured into a SU8 microchamber mold, vacuumed for 1 hour, and cured at 75 ° C. for 2 hours. . A PDMS microchamber was obtained by removing the SU8 template from the hardened PDMS.
The microchamber had a square well shape (length of one side: 300 μm), a square well shape (length of one side: 500 μm), a circular shape (diameter: 300 μm), and a Four types with a circular planar shape (diameter: 500 μm) were prepared. Each microchamber had a well depth of 100 μm, a distance between wells of 300 μm, and 500 wells.
Note that FIG. 11 shows a stereomicroscopic photograph of a microchamber having a well with a circular planar shape (diameter: 500 μm). FIG. 12 shows an electron micrograph (SEM image) of a microchamber with a well having a circular planar shape (diameter: 500 μm).

2. Evaluation of Antibacterial Activity Antibacterial activity was evaluated by performing the following operation using each of the above-described four microchambers.
500 μL of a microbial liquid adjusted to a turbidity of 0.2 was added to an agarose agar medium plate (Φ60 mm), and the plate was evenly coated with a spin coater (1000 rpm). Escherichia coli was used as the microorganism.
A PDMS microchamber was hydrophilized with an _O asher (125 W, 10 sec), and a cover glass (Matsunami Glass Industry Co., Ltd.) with a thickness of 0.3 mm and a length and width of 40 mm × 30 mm was used. was included. In addition, it is known in advance that this antibiotic has antibacterial properties against Escherichia coli.
A microchamber containing an antibiotic was placed on an agarose agar plate coated with the prepared microbial solution.
After allowing to stand at 37° C. for 12 hours in this state, the microchambers were removed from the agarose agar medium plate, halo formation on the agarose agar medium plate was evaluated with a phase-contrast microscope, and the number of halos was counted. Then, the halo formation rate (%) was obtained from the following formula.

Halo formation rate (%) = {(number of halos considered to be halo)/(total number of wells)} x 100

3. Evaluation Results FIG. 13 shows an image of a phase-contrast microscope in the case of using a microchamber with a well having a square planar shape (length of one side: 300 μm). This image is a part of the entire image (the same applies to FIGS. 14 to 16 below). A halo is observed in the portion surrounded by the dashed line (the same applies to FIGS. 14 to 16 below).
FIG. 14 shows an image of a phase-contrast microscope in the case of using a microchamber with a well having a square planar shape (length of one side: 500 μm).
FIG. 15 shows an image of a phase-contrast microscope in the case of using a microchamber with a well having a circular planar shape (diameter: 300 μm).
FIG. 16 shows an image of a phase-contrast microscope in the case of using a microchamber with a well having a circular planar shape (diameter: 500 μm).

Table 1 and FIG. 17 show the halo formation rate (%). Since the formation of halo was confirmed using any microchamber, it was confirmed that the antibacterial activity of the antibiotic could be evaluated in any case.
However, since the halo formation rate is higher in the circular well shape than in the square well shape, it was confirmed that the round well shape enables more sensitive evaluation of the antibacterial activity.
In addition, when the shape of the well is circular, the halo formation rate is higher when the diameter is 500 μm than when the well is 300 μm. Therefore, it was confirmed that when the diameter is 350 μm to 650 μm, the evaluation can be performed with higher sensitivity.

4. Effect of Example According to the antibacterial activity evaluation method of the present example, since a microchamber having a large number of wells is used, it is possible to evaluate a larger number of samples than the conventional method. For example, if the conventional method can evaluate only 5 samples, the antibacterial activity evaluation method of this embodiment can evaluate about 100 times more samples. Moreover, since a larger number of samples can be evaluated at the same time than the conventional method, the cost of evaluation can be significantly reduced.

The foregoing examples are for illustrative purposes only and are not to be construed as limiting the invention. While the present invention has been described by way of examples of exemplary embodiments, it is understood that the words used in describing and illustrating the invention are words of description and illustration, rather than words of limitation. Changes may be made within the scope of the appended claims without departing from the scope or essence of the invention in its form, as detailed herein. Although reference has been made herein to specific structures, materials and embodiments in describing the invention, it is not intended that the invention be limited to the disclosure herein, but rather the invention as set forth in the appended claims. It covers all functionally equivalent structures, methods and uses within its scope.

The present invention is not limited to the embodiments detailed above, and various modifications and changes are possible within the scope of the claims of the present invention.

By using the antibacterial activity evaluation method of the present invention, the activity of novel antibiotics can be evaluated in multiple specimens.

1 ... Well 1A ... Diameter 1B ... Distance between wells 1C ... Depth 2 ... Portion between adjacent wells 3 ... Micro chamber 5 ... Agar medium 5A ... Cloudy region 5B ... Transparent region (halo)
7 ... bacteria 9 ... evaluation substance

Claims (3)

An antibacterial activity evaluation method using a silicone resin microchamber having 350 or more recessed wells,
The distance between each said well is 150 μm to 450 μm,
A step of applying bacteria to an agar medium;
A step of encapsulating an evaluation substance in the well;
placing the microchamber on the agar medium coated with the bacteria so that the evaluation substance is in contact;
a step of keeping the temperature within a predetermined temperature range while the microchamber is placed on the agar medium;
and removing the microchamber from the agar medium;
Observing the state of a cloudy region and a transparent region formed on the agar medium from which the microchamber has been removed, and determining that the evaluation substance contained in the well corresponding to the transparent region has antibacterial properties Activity evaluation method. 2. The antibacterial activity evaluation method according to claim 1, comprising _the step of hydrophilizing the well of the microchamber with an O2 asher. 3. The antibacterial activity evaluation method according to claim 1 or 2, wherein at least two of the plurality of wells contain different types of the substances to be evaluated.

Images