JP7045009B2 - Method for determining silver-resistant or silver-sensitive bacteria, and kit for determining silver-resistant or silver-sensitive bacteria - Google Patents

Description

The present invention relates to a method for determining a silver-resistant bacterium or a silver-sensitive bacterium, and a determination kit for a silver-resistant bacterium or a silver-sensitive bacterium. More specifically, the present invention relates to a technique capable of easily visually determining a silver-resistant bacterium or a silver-sensitive bacterium.

Silver is known to have an antibacterial effect, and has not only bactericidal ability but also antifungal ability. The antibacterial property of silver is mainly due to silver ions (Ag ⁺ ), and silver ions are used for sterilization of bathtub water, sterilization and deodorization of clothes, footwear, etc. (for example, Patent Document 1). reference).

On the other hand, it is known that there are bacteria and molds (fungi) that are resistant to silver. For example, in Non-Patent Document 1, among the bacteria of the genus Bacillus, there are bacteria that are resistant to silver. It is disclosed that there is. Since the antibacterial activity of silver does not act on these bacteria, even if silver is used, it cannot be eradicated.

Here, one of the applicants, as the inventor, previously in Patent Document 2, separated bacteria that produce hydrogen sulfide in the process of culturing Salmonella and the like in a separation medium having an iron source such as DHL agar medium, and TSI. It discloses a technique for easily determining Salmonella with the naked eye using an agar medium or other confirmation medium.

As described above, the technology that can easily determine various bacteria with the naked eye is very useful in the determination of bacteria in tap water, beverages, foods, instruments that come into contact with foods, medical devices, various parts used for these, and the like. ..

Japanese Unexamined Patent Publication No. 3-7201 Japanese Unexamined Patent Publication No. 2013-106587

Gunawan, C .; Teoh, W. Y .; Marquis, C. P .; Amal, R., Induced Adaptation of Bacillus sp. To Antimicrobial Nanosilver. Small 2013, 9 (21), 3554-3560

Conventionally, it has been known that there are bacteria resistant to silver, but there has been no method for easily determining whether or not the bacteria are resistant to silver. In particular, a method that can be easily determined visually is very useful in the field of research on silver-resistant bacteria and silver-sensitive bacteria, and its development has been desired.

Therefore, it is a main object of the present invention to provide a technique capable of easily visually determining a silver-resistant bacterium or a silver-sensitive bacterium.

In the present invention, first, a solution containing silver ions is dropped on an agar medium containing sodium thiosulfate as a sulfur source, an antibacterial portion is added to the dropped portion, a boundary portion is provided around the antibacterial portion, and the boundary portion is formed. The step (I) of forming a non-antibacterial portion on the outside of the silver-resistant portion, the step (II) of growing the target bacterium on the agar plate, and the target bacterium generated inside the boundary portion as silver-resistant bacteria. Provided is a method for determining a silver-resistant bacterium or a silver-sensitive bacterium, which comprises at least performing the step (III) of determining the target bacterium generated outside the boundary portion as a silver-sensitive bacterium.

The present invention also provides a silver-resistant or silver-sensitive bacterium determination kit having at least an agar medium containing sodium thiosulfate as a sulfur source and a solution containing silver ions.

In the present invention, the "silver-resistant bacterium" means a bacterium that is not affected by silver (mainly silver ions). Further, "silver-sensitive bacteria" refers to bacteria affected by silver (mainly silver ions). The influence of silver means, for example, suppression or reduction of bacterial growth, removal of bacterial bacteria, and the like.

According to the present invention, it is possible to easily visually determine a silver-resistant bacterium or a silver-sensitive bacterium. The effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.

It is a drawing substitute photograph which shows the result of Experimental Example 1. FIG. It is a drawing substitute photograph which shows the result of Experimental Example 2. It is a drawing substitute photograph which shows the result of Experimental Example 3. It is a drawing substitute photograph which shows the result of Experimental Example 4. It is a drawing substitute photograph which shows the result of Experimental Example 5. It is a drawing substitute photograph which shows the result of Experimental Example 6. It is a drawing substitute photograph which shows the observation result of the boundary part in Experimental Example 7. It is a drawing substitute photograph which shows the observation result of the antibacterial part in Experimental Example 7. It is a drawing substitute photograph which shows the observation result of the non-antibacterial part in Experimental Example 7.

Hereinafter, suitable embodiments for carrying out the present invention will be described with reference to the drawings. It should be noted that the embodiments described below show an example of a typical embodiment of the present invention, and the scope of the present invention is not narrowly interpreted by this.

1. 1. Judgment method As shown in the drawing substitute photograph of FIG. 1, when the solution containing silver ions was dropleted on the agar medium containing a sulfur source, the present inventors dropped the solution in a circle around the dropped portion. It has been found that an antibacterial portion is formed in a portion, a boundary portion is formed around the antibacterial portion, and a non-antibacterial portion is formed outside the boundary portion. The inventors of the present application have named this phenomenon the "silver moon reaction". Further, since the boundary portion is a silver ring, the inventors of the present application have named it "silver ring".

Further, the inventors of the present application have found that silver-sensitive bacteria do not grow in the antibacterial part, and silver-sensitive bacteria grow in the non-antibacterial part. On the other hand, in the case of silver-resistant bacteria, it was also found that growth in the antibacterial part was observed. Since this state can be observed with the naked eye, it is possible to easily visually determine a silver-resistant bacterium or a silver-sensitive bacterium by using the determination method according to the present invention.

Further, by using the determination method according to the present invention, the colonies (villages) of silver rings, silver-resistant bacteria or silver-sensitive bacteria formed on the medium can be remarkably visually confirmed, so that the accuracy of detection of the bacteria can be confirmed. It is also possible to improve. Therefore, silver-resistant bacteria or silver-sensitive bacteria can be easily isolated at the inspection site of bacteria or fungi, and the inspection efficiency can be significantly improved.

Further, by using the determination method according to the present invention, it is possible to provide a determination kit, a determination device and the like regarding silver-resistant bacteria or silver-sensitive bacteria. As a result, silver-resistant bacteria and silver-sensitive bacteria can be utilized as new biological resources.

Hereinafter, each step in the determination method according to the present invention will be described in detail.

(1) Step (I)
In step (I), a solution containing silver ions is dropped onto an agar medium containing a sulfur source, an antibacterial portion is added to the dropped portion, a boundary portion is provided around the antibacterial portion, and the outside of the boundary portion is not. It is a step of forming an antibacterial portion.

The antibacterial portion, the boundary portion, and the non-antibacterial portion will be described with reference to FIG. 1. The inside of the annulus (silver ring) is the antibacterial portion, the annulus itself is the boundary portion, and the outside of the annulus is the non-antibacterial portion.

Although the antibacterial portion is formed in a substantially circular shape in FIG. 1, in the present invention, the shapes of the antibacterial portion, the boundary portion, and the non-boundary portion are not particularly limited, and the shape of the antibacterial portion is substantially fan-shaped or substantially fan-shaped. It may be semicircular or the like. Further, the boundary portion and the non-boundary portion may also have a deformed shape or the like according to the shape of the deformed antibacterial portion.

The sulfur source is not particularly limited, and examples thereof include one or more sulfur sources selected from sodium thiosulfate and the like. In the present invention, it is particularly preferable to use sodium thiosulfate among these.

The agar medium is not particularly limited as long as it contains a sulfur source, and is, for example, a DHL (Desoxycholate-hydrogen sulfide-lactose) agar medium, an X-SAL agar medium, or an MLCB (Mannitol lysine crystal violet brilliant green) agar medium. , SS-SB (Salmonella-Shigella Sucrose Bromcresolpurple) agar medium and the like. Further, in the present invention, a commercially available agar medium may be used.

The agar medium used in step (I) may contain not only a sulfur source but also one or more iron sources selected from, for example, ammonium ferric citrate.

Further, a nutrient source or the like may be added to the agar medium as long as the effect of the present invention is not impaired, and the nutrient source or the like may be freely used depending on the properties of the bacteria to be cultured. can.

Examples of the nutrient source include carbon sources such as glucose, fructose, sucrose, lactose, starch, glycerin, dextrin, and lecithin: inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium phosphate, diammonium phosphate, and ammonium chloride. : Organic nitrogen sources such as amino acids and peptones; Inorganic nutrient sources such as sodium, magnesium, potassium, iron, zinc, calcium and manganese; and one or more nutrient sources selected from various other vitamins and the like.

The solution containing silver ions is not particularly limited, and examples thereof include one or more solutions selected from silver nitrate solution, silver fluoride solution, silver ammonia solution, silver citrate solution and the like. In the present invention, among these, it is particularly preferable to use a silver nitrate solution and / or a silver fluoride solution, and it is more preferable to use a silver nitrate solution.

The concentration of the solution containing silver ions is not particularly limited, but a high concentration is preferable because it tends to form a silver ring. More preferably, it is a saturated solution.

The reaction time (time until the antibacterial portion, the boundary portion and the non-antibacterial portion are formed) in the step (I) is not particularly limited, but is preferably 30 minutes or more, more preferably 1 hour or more, and 2 It is more preferable that it is more than an hour.

The reaction temperature in step (I) is preferably about 37 ° C. If the temperature is 37 ° C. or lower, the time until the silver ring is formed becomes long.

(2) Step (II)
Step (II) is a step of growing the target bacteria on the agar medium.

The target bacterium is not particularly limited, and in the determination method according to the present invention, any bacterium including bacteria, fungi and the like can be targeted. In addition, in the present invention, what is called a so-called "bacterial group" including many bacterial species can also be targeted. In the present invention, among these, it is particularly preferable to target bacteria and / or fungi.

The target bacteria include, for example, bacteria belonging to the genus Salmonella, Citrobacter, Proteus, Edwardiella, etc .; Escherichia coli; coliform bacteria including many bacterial species such as Escherichia coli, Citrobacter, Klebsiella, Enterobacter, Proteus; One or more kinds of bacteria selected from Citrobacter and the like can be mentioned.

In the step (II), the conditions for growing are not particularly limited, and the temperature, time and the like can be freely set according to the properties of the growing bacteria and the like. For example, for bacteria, culturing at 37 ° C. for 6 hours, or at 37 ° C. for 24 hours or more, and for fungi (mold), culturing at 25 ° C. (at room temperature) for 1 week or longer.

(3) Step (III)
The step (III) is a step of determining the target bacterium that has developed inside the boundary portion as a silver-resistant bacterium and the target bacterium that has developed outside the boundary portion as a silver-sensitive bacterium.

The definitions of silver-resistant bacteria and silver-sensitive bacteria are as described above. Further, the present invention is characterized in that the determination in the step (III) can be performed with the naked eye.

(4) Other Steps In the determination method according to the present invention, in addition to the above steps (I) to (III), other steps may be performed as long as the effects of the present invention are not impaired.

2. 2. Judgment Kit The present invention also provides a determination kit for silver-resistant or silver-sensitive bacteria having at least an agar medium containing a sulfur source and a solution containing silver ions. Since the solutions containing the sulfur source, the agar medium, and the silver ion are the same as those described above, the description thereof is omitted here.

By using the determination kit according to the present invention, it is possible to easily visually determine a silver-resistant bacterium or a silver-sensitive bacterium. Therefore, it becomes possible to improve the accuracy of detection of bacteria, silver-resistant bacteria or silver-sensitive bacteria can be easily isolated at the inspection site of bacteria or fungi, and the inspection efficiency can be significantly improved.

Hereinafter, the present invention will be described in more detail based on Examples. It should be noted that the examples described below show an example of a typical example of the present invention, and the scope of the present invention is not narrowly interpreted by this.

<Experimental Example 1>
In this experimental example 1, an agar medium was prepared in a circular petri dish having a diameter of 10 cm using 20 mL of a DHL medium (manufactured by Eiken Chemical Co., Ltd.) containing sodium thiosulfate and ammonium iron citrate. Then, 25 μL of a saturated silver nitrate solution was added dropwise to the center of this agar medium, and the mixture was left at 37 ° C. for 5 hours.

The inventors of the present application have visually observed that the saturated silver nitrate solution is dipped and then diffused in a substantially circular shape around the drip site to form a silver ring (silver ring) at the end as shown in FIG. confirmed.

<Experimental Example 2>
In Experimental Example 2, Salmonella and Escherichia coli were inoculated into the agar medium on which the silver ring was formed, which was prepared by the method described in Experimental Example 1, and grown at 37 ° C. for 24 hours.

As shown in FIG. 2, the inventors of the present application confirmed that Salmonella and Escherichia coli did not grow and colonies (villages) were not formed in the antibacterial portion inside the silver ring (boundary portion). On the other hand, these bacteria grew only in the non-antibacterial part on the outside, and colony formation was observed.

<Experimental example 3>
In Experimental Example 3, the agar medium on which the silver ring was formed produced by the method described in Experimental Example 1 was left uncovered for one day to promote the invasion of falling bacteria (mainly fungi). It was left at room temperature of 25 ° C. for 1 week.

As shown in FIG. 3, the inventors of the present application confirmed that fungi (mold) did not grow and no colonies were formed in the antibacterial portion inside the silver ring (boundary portion). On the other hand, fungi grew only in the non-antibacterial part on the outside, and colony formation was observed. Furthermore, from the results of Experimental Example 3, it was inferred that the silver ring itself had no antibacterial action or had a weaker antibacterial action than the antibacterial portion inside the silver ring.

<Experimental Example 4>
In Experimental Example 4, an agar medium containing only sodium thiosulfate, which is a sulfur source, was prepared in a circular petri dish having a diameter of 10 cm. In addition, an agar medium containing only ammonium iron citrate, which is an iron source, was prepared in a circular petri dish having a diameter of 10 cm. Then, 25 μL of a saturated silver nitrate solution was added dropwise to the center of these agar media, and the mixture was allowed to stand at 37 ° C. for 5 hours.

FIG. 4 is a drawing substitute photograph showing the results of Experimental Example 4, in which the left side shows an agar medium containing only sodium thiosulfate, and the right side shows an agar medium containing ammonium iron citrate. As a result, the inventors of the present application confirmed that the generation of the silver ring (boundary portion) is a phenomenon caused not by the iron source of ammonium ferric citrate but by the sulfur source of sodium thiosulfate.

<Experimental Example 5>
In Experimental Example 5, Salmonella was inoculated into an agar medium containing only sodium thiosulfate, which is a sulfur source, on which a silver ring produced by the method described in Experimental Example 4 was formed, and grown at 37 ° C. for 24 hours. I let you.

As shown in FIG. 5, the inventors of the present application confirmed that Salmonella did not grow and no colonies were formed in the antibacterial portion inside the silver ring (boundary portion). On the other hand, Salmonella grew only in the non-antibacterial part on the outside, and colony formation was observed.

<Experimental Example 6>
In this experimental example 6, the silver ring produced by the method described in the above experimental example 4 was formed and dropped on an agar medium containing only sodium thiosulfate, which is a sulfur source, without a lid for one day. After promoting the invasion of bacteria (mainly fungi), the cells were left at room temperature of 25 ° C. for 1 week.

FIG. 6 is a drawing-substituting photograph showing the results of Experimental Example 6, in which the left side shows the growth of a silver-sensitive fungus and the right side shows the growth of a silver-resistant fungus. As shown in FIG. 6, the inventors of the present application confirmed that silver-sensitive fungi do not grow on the antibacterial portion inside the silver ring (boundary portion), but silver-resistant fungi grow.

From the results of Experimental Examples 1 to 6, when the solution containing silver ions was dropleted on an agar medium containing a sulfur source, the inventors of the present application had antibacterial action on the portion dropped in a circle centered on the dropped portion. It has been found that a portion, a boundary portion (silver ring) around the antibacterial portion, and a non-antibacterial portion outside the boundary portion are formed.

Further, the inventors of the present application have found that silver-sensitive bacteria do not grow in the antibacterial part, and silver-sensitive bacteria grow in the non-antibacterial part. On the other hand, in the case of silver-resistant bacteria, it was also found that growth in the antibacterial part was observed. Since this state can be observed with the naked eye, it has been found that silver-resistant bacteria or silver-sensitive bacteria can be easily visually determined by using the determination method according to the present invention.

<Experimental Example 7>
Each part of the antibacterial part, the boundary part (silver ring), and the non-antibacterial part was observed by TEM (Transmission Electron Microscope) using an electron microscope.

Specifically, the experiment was conducted as follows.
1. 1. Each sample of the antibacterial part, the boundary part (silver ring), and the non-antibacterial part was cut into an appropriate size (3 mm × 5 mm) together with the medium using a cutter.
2. 2. Each cut sample was placed on a filter paper to remove water from the medium.
3. 3. Each sample from which water was removed was sandwiched between a Si substrate and glass, and fixedly bonded with Araldite.
4. Each sample was cut into a size of 3 mm × 1 mm using a diamond cutter.
5. The thickness of each sample was polished to 0.1 mm with water-resistant abrasive paper (# 1200).
6. Each sample was prepared and processed for TEM observation using an ion slicer.
7. A cross-sectional TEM observation was performed using an electron microscope (manufactured by JEOL; model: 2000EX2, manufactured by TOPCON; model: 002B).

7 to 9 are drawing substitute photographs showing the observation results in Experimental Example 7. As shown in FIG. 7, at the boundary portion, the size of the silver particles differed depending on the location, and silver particles of about 10 nm to 1 μm were distributed as a whole. Further, as shown in FIG. 8, in the antibacterial portion, the variation in the size of the silver particles is small, and the silver particles of about 10 nm to 500 nm are distributed as a whole, and the ratio of the medium to the whole sample is not so large. rice field. Further, as shown in FIG. 9, in the non-antibacterial portion, the variation in the size of the silver particles was small, the silver particles of about 10 nm were distributed as a whole, and the ratio of the medium to the whole sample was large.

From this, silver particles (mostly of a size of several nm to 50 nm in diameter and 10 nm in diameter) were dispersed in the entire agar medium, and silver particles were densely packed at the boundary portion (silver ring). It was also found that the antibacterial part had a large proportion of silver particles and the non-antibacterial portion had a small proportion of silver particles. In addition, as silver nitrate becomes silver nanoparticles, the concentration of silver decreases toward the non-antibacterial part, and the density of silver nanoparticles decreases. Therefore, the density of silver nanoparticles that exert a sufficient antibacterial effect decreases. Therefore, it was considered that the non-antibacterial part had no antibacterial effect.

Therefore, as a result of electron microscopic observation, it was inferred that the silver ions in the solution became silver nanoparticles in the agar medium, thereby imparting antibacterial properties to the medium. The inventors of the present application have confirmed that the same result can be obtained even when the silver fluoride aqueous solution is used instead of the silver nitrate aqueous solution.

By using the determination method according to the present invention, it is possible to easily visually determine a silver-resistant bacterium or a silver-sensitive bacterium. Therefore, silver-resistant bacteria or silver-sensitive bacteria can be detected easily, quickly, and with high accuracy in tap water, beverages, foods, instruments that come into contact with foods, medical devices, various parts used for these, and the like.

Claims (1)

A solution containing silver ions was dropped onto an agar medium containing sodium thiosulfate as a sulfur source, and an antibacterial portion was added to the dropped portion, a boundary portion was provided around the antibacterial portion, and a non-antibacterial portion was provided outside the boundary portion. And the step (I) of forming
The step (II) of growing the target bacteria on the agar medium and
The step (III) of determining the target bacterium that has developed inside the boundary portion as a silver-resistant bacterium and the target bacterium that has developed outside the boundary portion as a silver-sensitive bacterium.
A method for determining silver-resistant bacteria or silver-sensitive bacteria.

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