Testing method for legionella bacteria

Abstract

To provide a testing method for Legionella bacteria that is less susceptible to the influence of microorganisms other than Legionella bacteria, especially acid-fast bacteria, and allows for more accurate detection of Legionella bacteria.SOLUTION: In testing for Legionella bacteria, at least a contact step S1 of bringing a sample into contact with ethambutol or a salt thereof and a culture step S2 of culturing the sample on a selective medium for Legionella bacteria or on a membrane filter attached to the selective medium for Legionella bacteria are performed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting bacteria of the genus Legionella by a culture method. [Background technology]

[0002] Legionella bacteria are aerobic gram-negative rod-shaped bacteria that are naturally found in soil and freshwater, but they are also known to live in artificial water systems such as cooling tower cooling water, pool water, and circulating water for hot spring baths. It is known that inhaling aerosols from water systems inhabited by Legionella bacteria can cause respiratory tract infection with the bacteria, resulting in Legionnaires' disease, such as Legionnaires' pneumonia. Since the first case of Legionnaires' disease was reported in Japan in 1981, the number of reported cases has been increasing year by year.

[0003] Since there have been no reported cases of Legionnaires' disease being transmitted from person to person, proper sanitation management of artificial water systems is considered important as a means of preventing Legionnaires' disease. For example, in bath water in bath facilities, the Ministry of Health, Labor and Welfare's "Guidelines for Water Quality Standards in Public Baths" stipulates that Legionella bacteria must be undetectable (less than 10 CFU / 100 mL). In order to prevent Legionnaires' disease, it is important to accurately grasp the habitat of Legionella bacteria in water systems that may be a source of infection, and high accuracy is required for testing for Legionnaires' disease.

[0004] Conventional methods for detecting Legionella bacteria from artificial water systems such as circulating water in hot spring facilities include, for example, the "smear culture method" in which a specimen (sample water) is concentrated 100-fold by centrifugation or membrane filter filtration, and the concentrated solution is then acid- or heat-treated, and then smeared on a selective medium for Legionella bacteria and cultured to detect Legionella bacteria, and the "filter attachment method" in which an acid- or heat-treated specimen is filtered through a membrane filter, and the membrane filter is attached to a selective medium for culture (see Non-Patent Document 1). In these testing methods, the specimen (sample water) is applied to a selective medium for Legionella bacteria, or a membrane filter through which the specimen (sample water) has been filtered is attached to a selective medium for Legionella bacteria, and the colonies that grow after culture are visually counted.

[0005] In the "smear culture method," 0.1 mL of a 100-fold concentrated solution of the specimen (sample water) is inoculated onto the culture medium, so the lower detection limit is 10 CFU / 100 mL, but because the recovery rate of Legionella bacteria is low during the sample (sample water) concentration and smear procedures, in order to detect Legionella bacteria with even higher sensitivity, it is necessary to inoculate multiple plates of culture medium, which is a factor in reducing the efficiency of the test. In addition, because the "smear culture method" requires many operational steps, there is also the issue that the test results are easily affected by the skill level of the examiner.

[0006] In contrast, the "filter attachment method" does not require the examiner to be as skilled as the smear culture method, since a membrane filter through which a specimen (sample water) has been filtered is attached directly to a selective medium, and the test can be performed with a high recovery rate. In addition, the sensitivity can be freely adjusted by changing the amount of specimen (sample water) to be filtered. However, contaminating microorganisms other than Legionella are likely to grow on membrane filters, and these colonies may make it impossible to detect Legionella (undetectable), in which case measures such as retesting are necessary. In addition, since Legionella does not easily form colonies on membrane filters, the "filter attachment method" also has the problem that Legionella may not be detected.

[0007] Examples of selective media used in the conventional detection methods described above include MWY medium, in which the antifungal agent anisomycin is added to BCYEα medium, a medium capable of culturing Legionella bacteria, in addition to the antibacterial agents glycine, vancomycin, and polymyxin B in order to impart selectivity to Legionella bacteria; GVPCα medium, in which the antifungal agent cycloheximide is added to BCYEα medium in addition to the antibacterial agents glycine, vancomycin, and polymyxin B; and WYOα medium, in which the antifungal agent amphotericin B is added to BCYEα medium in addition to the antibacterial agents glycine, vancomycin, and polymyxin B, and the like. These are known as selective media capable of eliminating the influence of contaminating microorganisms to a certain extent.

[0008] Also, a method for testing Legionella bacteria has been proposed using CATα medium, which is a GVP medium supplemented with cycloheximide, amphotericin B, and thiabendazole (see Patent Document 1). However, even when the selective medium described in Patent Document 1 is used, the effects of coexisting microorganisms may not be completely eliminated. These microorganisms other than Legionella bacteria (contaminating microorganisms) generally form colonies and expand more quickly on selective media than Legionella bacteria, and therefore interfere with counting the colonies of grown Legionella bacteria and further cause obstacles such as preventing the growth of nearby colonies of Legionella bacteria.

[0009] For this reason, methods have been developed to suppress the growth of contaminating microorganisms using pretreatment and selective media that do not cause damage to Legionella bacteria and are effective against these microorganisms. One example of an effective pretreatment method against contaminating microorganisms is acid treatment, and a widely used method is to add sterilized water to the specimen (sample water) or the cooled centrifugation sediment of the specimen (sample water) and mix it with an HCl·KCl buffer solution to suppress the growth of contaminating bacteria.

[0010] Also, a test method has been proposed in which an acid treatment is performed by mixing an acidic phosphate buffer with a higher buffering capacity instead of the conventional HCl·KCl buffer (see Patent Document 2). Furthermore, for samples that are severely contaminated with contaminating microorganisms, the influence of the contaminating microorganisms may be suppressed by strengthening the pretreatment. For example, a method of strengthening the pretreatment is a combination of acid treatment and heat treatment, and a method in which the above-mentioned acid treatment is performed on a sample that has been treated at 50°C for about 20 minutes is generally used (see Non-Patent Document 2). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2006-280219 A [Patent Document 2] JP 2005-237275 A [Non-patent literature]

[0012] [Non-Patent Document 1] Toshitaka Nakahara (ed.), "Guidelines for preventing Legionnaires' disease", 4th ed., Japan Building Sanitation Management Education Center, 2017, pp. 32-38, 126, 127 [Non-Patent Document 2] Osamu Kasuga et al., "Study on isolation methods of Legionella spp. from environmental water", Journal of Infectious Diseases, 1999, 73, pp.25-34 Summary of the Invention [Problem to be solved by the invention]

[0013] However, even if measures against contaminating microorganisms are taken by combining pretreatment and selective culture medium, there may be microorganisms that are resistant to the antibacterial or antifungal agents added to the pretreatment or selective culture medium, so the above-mentioned conventional detection methods still have problems such as inability to detect Legionella bacteria and reduced counting accuracy. In particular, when the contaminating microorganism is an acid-fast bacterium, it is difficult to obtain the growth inhibition effect of the acidic buffer solution, selective culture medium, and pretreatment method that have been improved in the past. For this reason, there is a demand for a test method for Legionella bacteria that can inhibit the growth of contaminating microorganisms that are resistant to conventional pretreatment methods without inhibiting the growth of Legionella bacteria.

[0014] Therefore, an object of the present invention is to provide a method for testing Legionella bacteria that is less susceptible to the influence of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, and that can more accurately detect Legionella bacteria. [Means for solving the problem]

[0015] As a result of various investigations conducted by the inventors to solve the above-mentioned problems, they discovered that the growth of acid-fast bacteria can be completely inhibited by using ethambutol or a salt thereof in culture tests for Legionella bacteria, and thus arrived at the present invention.

[0016] That is, the method for testing Legionella bacteria of the present invention includes a contact step of contacting a sample with ethambutol or a salt thereof, and a step of culturing the sample after the contact step on a selective medium for Legionella bacteria or on a membrane filter attached to the selective medium for Legionella bacteria. In the contacting step, the concentration of the ethambutol in the sample may be 0.1 μmol / mL or more and 40 μmol / mL or less. In the contact step, the contact time between the sample and the ethambutol or a salt thereof is, for example, 12 hours or more, and preferably 24 hours or more and 100 hours or less. Effect of the Invention

[0017] According to the present invention, it is possible to perform more accurate testing for Legionella bacteria without being affected by microorganisms other than Legionella bacteria, particularly acid-fast bacteria. [Brief description of the drawings]

[0018] [Figure 1] 1 is a flowchart showing a method for testing for Legionella bacteria according to an embodiment of the present invention. [Diagram 2] 1 is a flowchart showing the steps for detecting bacteria of the genus Legionella by the filter pasting method. [Diagram 3] 1 is a flowchart showing the steps of detecting Legionella bacteria by smear culture method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Note that the present invention is not limited to the following preferred embodiments.

[0020] In a method for testing bacteria of the genus Legionella according to an embodiment of the present invention (hereinafter also simply referred to as a "testing method"), a specimen that has been contacted with ethambutol or a salt thereof is cultured on a selective medium or a membrane filter affixed to a selective medium. FIG. 1 is a flow chart showing the method for testing bacteria of the genus Legionella according to this embodiment. As shown in FIG. 1, the test method of this embodiment includes a contacting step S1 in which a specimen is contacted with ethambutol or a salt thereof, and a culturing step S2 in which the specimen after the contacting step S1 is cultured on a selective medium for bacteria of the genus Legionella or a membrane filter affixed to the selective medium for bacteria of the genus Legionella.

[0021] [Specimen] The "specimen" used in the testing method of this embodiment, i.e., the "specimen" to be tested for Legionella by the testing method of this embodiment, includes not only the water to be tested, but also samples obtained in intermediate steps in the testing process. The form of the "specimen" is not limited to liquid, and may be a solid such as a filtration residue or a mixture of a solid and a liquid.

[0022] In the testing method of this embodiment, the water to be tested (test water) is not particularly limited, and can be suitably tested for all water systems requiring testing for Legionella bacteria, such as cooling tower cooling water, hot spring water, circulating bath water, and circulating hot water. However, since membrane filters have the property of facilitating the growth of contaminating microorganisms other than Legionella bacteria, when testing is performed by the filter attachment method, it is preferable to test water with a relatively low level of microbial contamination, such as hot water and cold water supply systems.

[0023] [Contact process S1] In the contact step S1, the specimen is contacted with ethambutol or a salt thereof before culturing the Legionella bacteria. The effect of ethambutol or a salt thereof in suppressing the growth of microorganisms other than the Legionella bacteria, particularly acid-fast bacteria, continues even after the specimen is subsequently removed from contact with ethambutol or a salt thereof, thereby improving the accuracy of testing for the Legionella bacteria.

[0024] "Contact" here refers to preparing a sample containing ethambutol or a salt thereof by adding ethambutol or a salt thereof to the water to be tested so that ethambutol or a salt thereof acts bacteriostatically on microorganisms other than Legionella bacteria, particularly acid-fast bacteria, or to dripping water containing ethambutol or a salt thereof onto the filtration residue of the water to be tested remaining on a membrane filter to moisten the membrane filter, or to immersing a solid sample in water containing ethambutol or a salt thereof.

[0025] In the test method of the present embodiment, the specimen is contacted with ethambutol or a salt thereof at a concentration and for a time required to inhibit the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria. For example, when ethambutol or a salt thereof is added to the water to be tested, it is generally preferable to make the ethambutol concentration in the specimen 0.1 to 40 μmol / mL. If the ethambutol concentration in the specimen is less than 0.1 μmol / mL, the effect of sufficiently eliminating the influence of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, may decrease, and even if the ethambutol concentration in the specimen is increased to more than 40 μmol / mL, the effect of inhibiting the growth of microorganisms other than Legionella bacteria cannot be expected to improve, and an effect commensurate with the increased cost cannot be obtained. The ethambutol concentration in the specimen is more preferably 0.3 to 40 μmol / mL. By setting the ethambutol concentration in this range, the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be completely inhibited, and a sufficient effect commensurate with the increased cost can be obtained.

[0026] Similarly, when water containing ethambutol or a salt thereof is dropped onto the filtration residue of the test water remaining on the membrane filter, or when a solid specimen is immersed in water containing ethambutol or a salt thereof, the growth of microorganisms other than Legionella genus, particularly acid-fast bacteria, can be suppressed by using an aqueous solution of ethambutol or a salt thereof in the aforementioned concentration range.

[0027] The method for preparing a sample containing ethambutol or a salt thereof is not particularly limited, and ethambutol or a salt thereof can be added to the sample in the form of an aqueous solution, dispersion, or powder. The form in which ethambutol or a salt thereof is added can be appropriately selected depending on the form of the sample (liquid, solid, mixture of liquid and solid, etc.). For example, the use of an aqueous solution of ethambutol or a salt thereof makes it easy to make the ethambutol concentration in the sample uniform. The concentration of the aqueous solution of ethambutol or a salt thereof used in this case can be appropriately determined depending on the amount added to the sample, etc., within a range that does not affect the viable cell measurement of Legionella genus bacteria.

[0028] In addition, when an aqueous solution of ethambutol or its salts is added to a sample, the sample is diluted by the amount of the aqueous solution, which may affect the accuracy of the Legionella test. In such a case, it is possible to reduce the effect on the accuracy of the test by performing the Legionella test using the filter attachment method and adjusting the sensitivity by changing the amount of sample to be filtered. In addition, when a powder of ethambutol or its salts is used, the concentration can be adjusted without changing the volume of the sample. Furthermore, since the powder is easy to carry, a sample containing ethambutol or its salts can be prepared during the work of collecting the water to be tested, which reduces the effort.

[0029] In addition, the contact time between the sample and ethambutol or its salt is preferably 12 hours or more. If the contact time between the sample and ethambutol or its salt is less than 12 hours, it tends to be difficult to obtain the effect of inhibiting the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria. The upper limit of the contact time between the sample and ethambutol or its salt is determined based on the relationship between the cost increase and the effect to be obtained, but since a contact time that is too long may affect the viable cell measurement of Legionella bacteria, it is preferable to set it to 100 hours or less.

[0030] The contact time between the sample and ethambutol or its salt is more preferably 24 hours or more and 100 hours or less, and particularly preferably 24 hours or more and 72 hours or less. By setting the contact time within this range, it becomes possible to eliminate the influence of acid-fast bacteria and accurately measure the viable cell count of Legionella bacteria. The "contact time" here refers to the time elapsed from the addition of ethambutol or its salt to the sample until the start of culturing Legionella bacteria, or until the ethambutol or its salt is washed away and is no longer in contact with the sample.

[0031] By subjecting a sample that has passed the aforementioned contact time to testing for Legionella bacteria, a sample can be prepared in a state in which the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, is inhibited, even if ethambutol or a salt thereof is not present in or on the culture medium, or in or on the membrane filter during cultivation of the Legionella bacteria, i.e., even if the sample is not in contact with ethambutol or a salt thereof during cultivation.

[0032] On the other hand, if the contact time between the sample and ethambutol or its salt is provided, the time required for pretreatment of the sample becomes long. Therefore, it is preferable to add ethambutol or its salt to the collected sample in advance to the above-mentioned concentration when the sample is collected or before the sample is transported to the testing institution. This makes it possible to effectively use the transport time of the sample to the testing institution as the contact time between the sample and ethambutol or its salt, and allows the test to be started immediately after the sample arrives at the testing institution, which is efficient.

[0033] Since Legionella bacteria are natural environmental bacteria, it is safe to assume that they will not die even if they are transported for several days. However, there is a possibility that the live bacteria test for Legionella bacteria will be affected by an increase in microorganisms other than Legionella bacteria or a change in the quality of the sample during transportation. For this reason, it is preferable to shorten the time from water collection to the start of the test as much as possible, and it is preferable to transport the sample to the testing institution immediately after collection. Usually, the sample is transported by courier service, but the transport time in this case is about 14 hours at the earliest. Therefore, when measuring the live bacteria count of Legionella bacteria, it is preferable to transport the sample while storing it at 4 to 10 degrees Celsius.

[0034] The detection method of the Legionella bacteria in the test method of this embodiment is not particularly limited, and a conventionally known method can be applied. Therefore, the test method of this embodiment is effective even if a conventional method is used as is when detecting the Legionella bacteria, and there is no need to change the detection process.

[0035] Fig. 2 is a flow chart showing the detection process of bacteria of the genus Legionella by the filter sticking method, and Fig. 3 is a flow chart showing the detection process of bacteria of the genus Legionella by the smear culture method. In the testing method of this embodiment, the contact step S1, i.e., the addition of ethambutol or a salt thereof, may be performed at any step shown in Figs. 2 and 3 as long as it does not affect the detection of bacteria of the genus Legionella.

[0036] Below, as a specific example of the testing method of this embodiment, a case in which the "filter attachment method" is applied will be described, but the present invention is not limited to this, and any detection method using culture can be used.

[0037] <Sample pretreatment> The sample is pretreated, for example, by the following method: 20 to 100 mL of the sample is mixed with an equal volume of an acidic buffer solution, such as a hydrochloric acid-potassium chloride buffer solution (pH 2.2) or an acidic phosphate buffer solution (pH 2.2, for example, prepared by appropriately mixing a 0.2 mol / L aqueous phosphoric acid solution and a 0.2 mol / L aqueous potassium dihydrogen phosphate solution), and then allowed to stand at room temperature for 10 to 20 minutes.

[0038] In the detection method shown in Figure 2, the sample is pretreated by mixing an equal amount of acidic buffer solution. The purpose of this operation is to lower the pH of the sample to around 2.2 in order to suppress the growth of microorganisms other than Legionella. As long as the pH of the sample can be lowered to around 2.2, the amount of acidic buffer solution mixed does not necessarily have to be the same. The concentration and pH of the acidic buffer solution can be appropriately determined within a range that does not affect the viable measurement of Legionella, depending on the water quality of the sample and the amount of acidic buffer solution mixed with the sample, etc.

[0039] In the acid treatment, the pH of the sample is preferably 1.4 or more and 3 or less. If the pH of the sample is higher than 3, it may be difficult to sufficiently eliminate the effects of microorganisms other than Legionella bacteria, and if the pH is lower than 1.4, the effect on the growth of Legionella bacteria may be large. It is more preferable that the pH of the sample is 1.8 or more and 2.6 or less. By setting the pH of the sample in this range, the growth of microorganisms other than Legionella bacteria can be reliably suppressed without affecting the viable cell measurement of Legionella bacteria.

[0040] The concentration of the acidic phosphate buffer is preferably 0.02 mol / L or more, assuming that all phosphate ions in the buffer are dissociated (in the present invention, this concentration is referred to as the "acidic phosphate buffer concentration"). If the buffer concentration is less than 0.02 mol / L, sufficient pH buffering ability may not be obtained. Here, the lower limit of the pH and the upper limit of the concentration of the acidic phosphate buffer can be appropriately determined within a range that does not affect the viable cell measurement of Legionella genus bacteria, but it is particularly preferable to use an acidic phosphate buffer with a pH of 1.8 to 2.6 and a concentration of 0.1 mol / L to 0.5 mol / L. This allows the influence of microorganisms other than Legionella genus bacteria to be eliminated and the viable cell count of Legionella genus bacteria to be accurately measured.

[0041] The pH and concentration of the acidic phosphate buffer described above are the conditions when an equal amount of the acidic phosphate buffer is mixed with the specimen, and the amount of the acidic phosphate buffer to be mixed does not necessarily have to be equal to that of the specimen as long as the pH of the specimen can be lowered to about 2.2. For example, in cases where an increase in volume is undesirable, a higher concentration acidic buffer may be prepared and added in small amounts to adjust the pH of the test water or a specimen prepared from the test water to 1.8 to 2.6 and the concentration of the acidic phosphate buffer to 0.05 mol / L to 0.25 mol / L.

[0042] <Sample filtration> Next, the pretreated specimen is suction filtered through a sterilized membrane filter having a pore size of 0.45 μm, 0.20 μm, etc. The membrane filter may be made of nitrocellulose, mixed cellulose ester, polyethersulfone, polycarbonate, polytetrafluoroethylene, polyvinylidene fluoride, or the like.

[0043] After filtration, the inside of the filtration funnel is washed with sterile deionized water, and suction filtration is performed again. If ethambutol or its salt has been added to the specimen beforehand, this operation washes away the ethambutol or its salt. This cuts off contact between the specimen and ethambutol or its salt, but by keeping the time from adding ethambutol or its salt to the specimen to washing it away within the aforementioned contact time range, it is possible to reliably obtain the effect of inhibiting the growth of microorganisms other than Legionella, particularly acid-fast bacteria.

[0044] The color of the membrane filter is not particularly limited and can be, for example, white, green, or black, but since colonies of the Legionella genus are generally pale yellow-gray, it is preferable to use a membrane filter of a dark color such as black, which increases the contrast between the membrane filter and the colonies of the Legionella genus compared to the case of using a membrane filter of a pale color such as white, thereby improving visibility.

[0045] <Contact with liquids containing various components> Next, the filtration surface of the membrane filter through which the specimen has been filtered is brought into contact with a liquid containing (1) a component necessary for the growth of Legionella bacteria, and (2) a component that inhibits the growth of microorganisms other than Legionella bacteria. Here, the "filtration surface of the membrane filter" refers to the surface upstream in the direction of specimen flow during filtration.

[0046] (1) Components necessary for the growth of Legionella Examples of components necessary for the growth of bacteria of the genus Legionella include yeast extract, soluble iron pyrophosphate, and L-cysteine ​​or a salt thereof, and in the test method of this embodiment, these three components can be used as components necessary for the growth of bacteria of the genus Legionella. Note that in the test method of this embodiment, a pH buffer component, other nutrients, and the like may be used in combination as necessary.

[0047] (2) Ingredients that inhibit the growth of microorganisms other than Legionella As a component that inhibits the growth of microorganisms other than Legionella, ethambutol or a salt thereof is used. Note that, in the testing method of this embodiment, conventionally known components that inhibit the growth of microorganisms other than Legionella, such as glycine, vancomycin, polymyxin B, thiabendazole, cycloheximide, amphotericin B, natamycin, or other antibacterial agents, antifungal agents, or antibiotics, may be used in combination as necessary.

[0048] When the membrane filter is contacted with a liquid containing the above-mentioned components (1) and (2), the entire filtration surface of the membrane filter is moistened with the liquid containing these components. For example, when a membrane filter with a diameter of 47 mm is used, about 5 mL of the liquid is dropped onto the filtration surface of the membrane filter, the liquid is brought into contact with the entire filtration surface, and then the membrane filter is suction filtered to make the entire filtration surface moistened with the liquid. (2) The liquid containing a component that inhibits the growth of microorganisms other than Legionella may be an aqueous solution of ethambutol or its salts in which ethambutol or its salts are dissolved in water, or this aqueous solution may be mixed with other antibacterial agents, antifungal agents, and / or antibiotics, etc., as necessary.

[0049] In the above description, (1) a component necessary for the growth of Legionella bacteria and (2) a component that inhibits the growth of microorganisms other than Legionella bacteria are mixed and brought into contact with one liquid on the filtration surface of the membrane filter, but the present invention is not limited to this, and each of (1) and (2) may be divided into several portions and brought into contact. However, from the viewpoint of work efficiency, it is preferable to use a mixture of (1) a component necessary for the growth of Legionella bacteria and (2) a component that inhibits the growth of microorganisms other than Legionella bacteria to form one liquid.

[0050] [Culture process S2] In the testing method of this embodiment, after the above-mentioned contact step S1, the specimen is cultured on a selective medium for bacteria of the genus Legionella or on a membrane filter attached to a selective medium for bacteria of the genus Legionella. Specifically, after the components (1) and (2) are brought into contact with the filtration surface through which the specimen has been filtered, the surface of the membrane filter opposite to the filtration surface is attached to a selective medium for bacteria of the genus Legionella prepared in a plate shape on a petri dish and the dish is covered.

[0051] The petri dish is then turned upside down so that the filtration surface of the membrane filter is facing down, and cultured at 36±1°C for approximately 5 to 10 days. After culture is completed, the number of pale yellow-gray moist colonies suspected of being Legionella bacteria that have grown on the membrane filter is counted. These colonies are then subjected to an L-cysteine ​​requirement confirmation test, and colonies that are L-cysteine ​​auxotrophic are considered to be Legionella bacteria.

[0052] As the selective medium for Legionella bacteria used in the culture step S2, a selective medium containing, as essential components, three components of yeast extract, soluble iron pyrophosphate, and L-cysteine ​​or a salt thereof, among the components necessary for the growth of Legionella bacteria, and at least one component that inhibits the growth of microorganisms other than Legionella bacteria, can be used. Furthermore, if necessary, a component that adsorbs and removes growth inhibitors of Legionella bacteria, such as powdered activated carbon, pH buffer components, other nutrients, etc., may be contained. If these components are not added, the growth of many bacteria species will be extremely reduced or will not grow at all, so in general, conventionally known powdered activated carbon, pH buffer components, other nutrients, etc. are appropriately added at the discretion of a person skilled in the art.

[0053] A selective medium containing ethambutol or a salt thereof can be used as a selective medium containing at least one component that inhibits the growth of microorganisms other than the Legionella genus as an essential component. The selective medium containing ethambutol or a salt thereof may be prepared at home. The method for preparing the medium is not particularly limited, and it can be prepared, for example, by applying an aqueous solution of ethambutol or a salt thereof to a ready-made medium and allowing it to soak in the medium, or by mixing an aqueous solution of ethambutol or a salt thereof with a ready-made medium.

[0054] In addition, when preparing a selective medium containing ethambutol or a salt thereof, a dispersion of ethambutol or a salt thereof in water or a powder of ethambutol or a salt thereof may be used instead of an aqueous solution of ethambutol or a salt thereof. However, in this case, since it is time-consuming to uniformly disperse ethambutol or a salt thereof in the medium, it is preferable to use an aqueous solution of ethambutol or a salt thereof.

[0055] Furthermore, the amount of the aqueous solution of ethambutol or its salt applied to or mixed with the culture medium can be appropriately adjusted within a range that does not affect the culture test of Legionella bacteria. Therefore, the ethambutol concentration in the culture medium can be appropriately adjusted by changing the concentration of the aqueous solution or dispersion of ethambutol or its salt, or the amount of the aqueous solution, dispersion, or powder of ethambutol or its salt applied to or added to the culture medium.

[0056] Examples of easily available selective media include BCYEα agar medium, GVPCα agar medium, WYOα agar medium, CATα agar medium, MWY agar medium, CCVC agar medium, PAC agar medium, PAV agar medium, and GVPNα agar medium, but the present invention is not limited to these, and may be a medium in which the blending amount of each component of these media is adjusted, or a medium to which other components are added within a range that does not impair the effects of the present invention. In addition, the selective medium does not necessarily have to be an agar medium, and a liquid medium may be used by being impregnated with, for example, absorbent cotton or gauze.

[0057] The method for testing Legionella bacteria of this embodiment is not limited to the detection method of Legionella bacteria using the filter attachment method, and other methods, such as smear culture methods, can be appropriately adopted taking into consideration the water quality of the sample and the equipment owned by the testing institution, and in that case, the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be similarly suppressed. The method for testing Legionella bacteria of this embodiment also includes a case where ethambutol or a salt thereof is brought onto the culture medium, and even in that case, the effect of contacting the sample with ethambutol or a salt thereof in advance can be obtained.

[0058] Furthermore, in the method for testing Legionella bacteria of this embodiment, ethambutol or a salt thereof may be heated during the test. The purpose or target of heating is not particularly limited, but generally includes autoclave sterilization (121°C) of a medium for the purpose of sterilizing the medium, heat treatment (50°C) of a sample for the purpose of inhibiting the growth of microorganisms other than Legionella bacteria in the sample, and the like. In the test method of this embodiment, it has been confirmed that the growth inhibitory effect on acid-fast bacteria is not lost even when heat treatment is performed at least for 20 minutes at 121°C, and even if ethambutol or a salt thereof is present in the sample to be heated, the growth inhibitory effect on microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be obtained. Note that the results of a thermal stability test of ethambutol using a medium to which ethambutol or a salt thereof has been added are shown in the examples described below.

[0059] As described above in detail, in the method for testing Legionella bacteria of this embodiment, the specimen is brought into contact with ethambutol or a salt thereof before culturing, and therefore the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be suppressed. As a result, the method for testing Legionella bacteria of this embodiment is less susceptible to the influence of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, and is capable of accurately detecting Legionella bacteria, and can be suitably used in fields where testing for Legionella bacteria is required, such as cooling tower cooling water, hot spring water, circulating bath water, and circulating hot water. EXAMPLES

[0060] The effects of the present invention will be specifically described below with reference to examples and comparative examples. In some of the examples shown below, a filter attachment method is used for detecting Legionella bacteria, but this is for the purpose of evaluating whether a bacteriostatic effect can be obtained by contacting a sample with ethambutol or a salt thereof for a certain period of time alone, and is intended not to bring ethambutol or a salt thereof onto the medium during cultivation.

[0061] <Examples 1 to 7, Comparative Examples 1 and 2> Mycobacterium goodii on the second day of subculture was picked up and suspended in 10 mL of sterile saline to prepare an acid-fast bacterium stock suspension (10 8cells / mL) was diluted with sterile saline to prepare an acid-fast bacillus suspension (10 2 A total of 100 cells / mL was prepared.

[0062] Ethambutol dihydrochloride aqueous solution was added to this acid-fast bacteria suspension, and 10 mL of ethambutol-added acid-fast bacteria suspension was prepared with the following concentrations of ethambutol dihydrochloride: 0.18 μmol / mL (50 μg / mL: Example 1), 0.36 μmol / mL (100 μg / mL: Example 2), 1.8 μmol / mL (500 μg / mL: Examples 3 and 4), and 3.6 μmol / mL (1000 μg / mL: Examples 5 to 7). For comparison, two samples of 10 mL of acid-fast bacteria suspension without the addition of ethambutol dihydrochloride aqueous solution were prepared (Comparative Examples 1 and 2).

[0063] In addition, a test was conducted to examine the effect of the concentration of ethambutol dihydrochloride aqueous solution on the growth of Legionella bacteria, and the results showed that samples containing Legionella bacteria that had been in contact with 36 μmol / L ethambutol dihydrochloride aqueous solution at room temperature for 96 hours had no effect on the growth of Legionella bacteria, compared to samples containing Legionella bacteria that had not been in contact with ethambutol dihydrochloride aqueous solution.

[0064] Thus, specimens with ethambutol dihydrochloride concentrations of 0.18 μmol / mL (Example 1), 1.8 μmol / mL (Example 4), and 3.6 μmol / mL (Example 7) were placed in a 5°C environment and refrigerated for 96 hours. Also, specimens with ethambutol dihydrochloride concentrations of 1.8 μmol / mL (Example 3) and 3.6 μmol / mL (Example 6) were placed in a 5°C environment and refrigerated for 72 hours.

[0065] Similarly, a specimen with an ethambutol dihydrochloride concentration of 0.36 μmol / mL (Example 2), a specimen with an ethambutol dihydrochloride concentration of 3.6 μmol / mL (Example 5), and an acid-fast bacteria suspension (Comparative Example 1) were left to stand in an environment of 5° C. and stored in a refrigerator for 24 hours. On the other hand, the acid-fast bacteria suspension (Comparative Example 2) was filtered through a membrane filter (pore size 0.45 μm) containing glitter immediately after preparation, and then the membrane filter was rinsed by filtering with sterilized deionized water.

[0066] In addition, the eight refrigerated samples (Examples 1 to 7, Comparative Example 1) were returned to room temperature and then filtered in their entirety through a membrane filter, followed by rinsing the membrane filter by filtering with sterilized deionized water. The rinsing of the membrane filter by filtering with sterilized deionized water was performed in anticipation of Legionella testing of actual test water such as hot water supply systems, bathtub water systems, and cooling tower cooling water systems, and is intended to wash away impurities dissolved in the sample, such as chlorine agents and other water treatment chemicals used in the test water system, acidic buffer solutions used in pretreatment for Legionella testing, and dissolved substances derived from the test water system.

[0067] Nine membrane filters treated by the above procedure were each attached to a selective medium for Legionella bacteria and cultured at 36°C for five days (Examples 1 to 7, Comparative Examples 1 and 2). After culture, the growth state of the acid-fast bacteria was observed under each condition, and the results are shown in Table 1 below. In Table 1 below, "+++" indicates that growth occurred over almost the entire surface of the membrane filter, "++" indicates that growth was observed over about half of the membrane filter, "+" indicates that slight growth was observed, and "-" indicates that no growth was observed.

[0068] [Table 1]

[0069] As shown in Table 1 above, in the sample of Comparative Example 2, which was cultured immediately after preparing the acid-fast bacteria suspension, the acid-fast bacteria grew over the entire surface of the membrane filter, whereas in the sample of Comparative Example 1, which was stored refrigerated at 5°C for 24 hours, the growth of the acid-fast bacteria was inhibited and the number of colonies grew to about one-third that of Comparative Example 2.

[0070] In contrast, no growth of acid-fast bacteria was observed in any of the samples of Example 2 and Example 5, in which the ethambutol-added acid-fast bacteria suspension was refrigerated for 24 hours in an environment at 5° C. Furthermore, no growth of acid-fast bacteria was observed in any of the samples of Example 4 and Example 7, in which the ethambutol-added acid-fast bacteria suspension was refrigerated for 96 hours in an environment at 5° C. Among the samples refrigerated for 96 hours in an environment at 5° C., slight growth of acid-fast bacteria was observed in the sample of Example 1, but it was clear that the growth of acid-fast bacteria was suppressed, making it much easier to detect Legionella bacteria.

[0071] It was predicted that ethambutol would be able to inhibit the growth of acid-fast bacteria only when the acid-fast bacteria were in contact with ethambutol or its salts, i.e., when the acid-fast bacteria were in contact with a culture medium or membrane filter containing an effective concentration of ethambutol or its salts. However, in Examples 1 to 7, the ethambutol or its salts contained in the samples were washed away with sterilized deionized water, and acid-fast bacteria did not grow within the five-day cultivation period that is the typical cultivation period for Legionella bacteria.

[0072] It was found that even if contact between the sample and ethambutol or its salts is discontinued during culture testing for Legionella bacteria, the growth of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be temporarily suppressed if the sample has a history of contact with ethambutol or its salts for a certain period of time or more before culture testing. From the above results, it was confirmed that by utilizing this phenomenon, it is possible to reduce the influence of acid-fast bacteria in testing for Legionella bacteria and improve the accuracy of testing for Legionella bacteria.

[0073] <Examples 8 and 9> In the culture test of Legionella bacteria, a culture medium that has been sterilized in advance is widely used. A common method for sterilizing a culture medium is, for example, autoclave sterilization, which involves heat treatment at 121°C for about 20 minutes using an autoclave. In addition, a specimen may also be heat-treated in order to suppress the growth of microorganisms other than Legionella bacteria in the specimen. In this case, for example, heat treatment is performed at 50°C for 20 to 30 minutes.

[0074] When ethambutol or its salt is added to a medium or a sample, its inhibitory effect on mycobacteria may be lost by heat treatment depending on its thermal stability. Therefore, in this example, the inhibitory activity against mycobacteria of an aqueous solution of ethambutol dihydrochloride that had been heat-treated (121°C) in an autoclave was investigated.

[0075] Specifically, ethambutol dihydrochloride was dissolved in sterile water to a concentration of 0.36 μmol / mL (100 μg / mL) to prepare an ethambutol dihydrochloride aqueous solution. The prepared ethambutol dihydrochloride aqueous solution was divided into two containers, and one of them was heat-treated at 121°C for 20 minutes using an autoclave. The specimen was prepared by suspending the harvested M. goodii bacteria in sterile saline and 8 The concentration was adjusted to cells / mL.

[0076] After inoculating 0.2 mL of this sample into a petri dish, 9.8 mL of standard agar medium was poured in, mixed, and cooled to solidify. In Example 8, an aqueous solution of ethambutol dihydrochloride that had been heat-treated at 121°C (abbreviation: heat-treated) was impregnated into a paper disk, and in Example 9, an aqueous solution of ethambutol dihydrochloride that had not been heat-treated (abbreviation: non-heat-treated) was impregnated into a paper disk, which was then placed on the medium and cultured at 36°C for 6 days to observe the growth of acid-fast bacteria (M. goodii).

[0077] The results are shown in Table 2 below. In Table 2 below, "+" indicates the presence of an inhibition zone (a region in which the growth of acid-fast bacteria was inhibited).

[0078] [Table 2]

[0079] As shown in Table 2 above, inhibition zones were observed in both the ethambutol dihydrochloride aqueous solution that had been heat-treated at 121° C. for 20 minutes (Example 8) and the ethambutol dihydrochloride aqueous solution that had not been heat-treated (Example 9). It was predicted that if ethambutol or its salts were thermally decomposed by heating, the inhibition zones would become extremely small or would not be observed at all, but the respective inhibition zones were sufficiently large and had almost the same diameter, confirming that ethambutol or its salts were not decomposed by heat treatment at 121° C. in an autoclave and that their growth inhibitory activity against mycobacteria (M. goodii) was not inactivated.

[0080] Of course, even at 50°C, the same temperature as when the sample is heat-treated, ethambutol or its salts do not decompose, and the growth inhibitory activity against mycobacteria (M. goodii) is not inactivated. Therefore, the timing of adding ethambutol or its salts to the sample is not particularly limited, and it was confirmed that the growth inhibitory effect against mycobacteria can be obtained even if ethambutol or its salts are added to the sample in advance. When the sample contains water treatment agents such as chlorine agents, sodium thiosulfate is sometimes added as a chlorine neutralizer at the time of collection, and similar results are obtained in this case as well.

[0081] From the above results, it was confirmed that according to the present invention, the effects of microorganisms other than Legionella bacteria, particularly acid-fast bacteria, can be suppressed, and Legionella bacteria can be detected more accurately.

[0082] The present invention can also have the following configuration. [1] a contacting step of contacting a sample with ethambutol or a salt thereof; Cultivating the sample after the contact step on a selective medium for Legionella bacteria or on a membrane filter attached to the selective medium for Legionella bacteria; A method for testing for Legionella bacteria, comprising the steps of: [2] The method for testing Legionella bacteria described in [1], wherein the contact step is performed to adjust the concentration of ethambutol in the sample to 0.1 μmol / mL or more and 40 μmol / mL or less. [3] The method for testing Legionella bacteria according to [1] or [2], characterized in that the contact time between the sample and the ethambutol or a salt thereof is 12 hours or longer. [4] The method for testing Legionella bacteria according to any one of [1] to [3], wherein the contact time between the sample and the ethambutol or a salt thereof is from 24 hours to 100 hours.

Claims

1. a contacting step of contacting a sample with ethambutol or a salt thereof; Cultivating the sample after the contact step on a selective medium for Legionella bacteria or on a membrane filter attached to the selective medium for Legionella bacteria; A method for testing for Legionella bacteria, comprising the steps of:

2. 2. The method for testing for Legionella bacteria according to claim 1, wherein the concentration of ethambutol in the sample is set to 0.1 μmol / mL or more and 40 μmol / mL or less in the contact step.

3. The method for testing for Legionella bacteria according to claim 2, wherein the contact time between the sample and the ethambutol or a salt thereof is 12 hours or longer.

4. 4. The method for testing Legionella bacteria according to claim 3, wherein the contact time between the sample and the ethambutol or a salt thereof is from 24 hours to 100 hours.

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