Culture medium for bacteria

Abstract

The present invention addresses the problem of providing a culture medium having a lower degree of growth inhibition with respect to Escherichia albertii while containing tellurous acid and / or a salt thereof. The aforementioned problem is solved by a culture medium for bacteria, the culture medium containing tellurous acid and / or a salt thereof, and at least two components selected from the group consisting of phosphoric acid and / or salts thereof, non-meat raw-material peptones, polyvalent metal ions, and glucose.

Description

Bacterial culture media

[0001] The present disclosure relates to bacterial culture media and the like.

[0002] In recent years, Escherichia albertii has been attracting attention as one of the bacteria that cause food poisoning. This bacterium, like Escherichia coli O157, has a high possibility of causing intestinal bleeding, and therefore a simple method for specifically detecting E. albertii is needed.

[0003] A selective isolation medium for Escherichia albertii has been reported that contains three sugars, melibiose, rhamnose, and xylose, and a pH indicator whose color changes from neutral to acidic (Patent Document 1). Because Escherichia albertii cannot metabolize these sugars, the pH of the medium is maintained near neutral, and the color of the colonies is in the neutral range (white or colorless). On the other hand, similar bacteria other than Escherichia albertii metabolize these sugars to other compounds, releasing acidic substances, which shifts the pH of the medium toward acidic, and the color of the colonies becomes acidic (red). Therefore, by using this medium, it is possible to isolate Escherichia albertii based on the colony color.

[0004] Japanese Patent Application Laid-Open No. 2019-024418

[0005] The present inventors focused on the use of telluric acid and / or its salts, which are inhibitors of Gram-negative bacteria, to improve the isolation ability of Escherichia albertii. However, when telluric acid and its salts were added to an isolation medium for Escherichia albertii, the growth of Escherichia albertii was also inhibited. In the course of further research, the present inventors found that the addition of telluric acid and / or its salts may not inhibit the growth of Escherichia albertii, depending on the medium composition.

[0006] An object of one aspect of the present invention is to provide a medium that contains telluric acid and / or a salt thereof and yet exhibits a lower degree of growth inhibition of Escherichia albertii.

[0007] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that the above-mentioned problems can be solved by a bacterial culture medium containing telluric acid and / or a salt thereof, phosphoric acid and / or a salt thereof, non-meat peptone, polyvalent metal ions, and at least two components selected from the group consisting of glucose. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present disclosure includes the following exemplary aspects:

[0008] Item 1. A bacterial culture medium comprising tellurous acid and / or a salt thereof, and at least two components selected from the group consisting of phosphoric acid and / or a salt thereof, non-meat-derived peptone, polyvalent metal ions, and glucose.

[0009] Item 2. The bacterial culture medium according to Item 1, containing the phosphoric acid and / or a salt thereof and a non-meat-derived peptone.

[0010] Item 3. The bacterial culture medium according to Item 1 or 2, wherein the non-meat-source peptone is at least one selected from the group consisting of soybean peptone and milk peptone.

[0011] Item 4. The bacterial culture medium according to any one of Items 1 to 3, wherein the content of the phosphoric acid and salts thereof is 0.5 to 10 g / L and the content of the non-meat-source peptone is 2 to 50 g / L.

[0012] Item 5. The bacterial culture medium according to any one of Items 1 to 4, which contains the polyvalent metal ions.

[0013] Item 6. The bacterial culture medium according to any one of Items 1 to 5, wherein the content of the polyvalent metal ions is 0.05 g / L or more.

[0014] Item 7. The bacterial culture medium according to any one of Items 1 to 6, wherein the content of meat-derived peptone is 0 to 10 g / L.

[0015] Item 8. The bacterial culture medium according to any one of Items 1 to 7, wherein the content of tellurous acid and salts thereof is 0.0005 to 0.01 g / L.

[0016] Item 9. The bacterial culture medium according to any one of Items 1 to 8, which contains at least one selected from the group consisting of a gram-positive bacteria inhibitor and a gram-negative bacteria inhibitor.

[0017] Item 10. The bacterial culture medium according to any one of Items 1 to 9, which contains a sugar that cannot be metabolized by Escherichia albertii and a pH indicator.

[0018] Item 11. The bacterial culture medium according to any one of Items 1 to 10, which is a solid culture medium.

[0019] Item 12. The bacterial culture medium according to any one of Items 1 to 11, which is used to culture a bacterial sample that may contain Escherichia albertii.

[0020] Item 13. The bacterial culture medium according to Item 12, which is used for isolating Escherichia albertii.

[0021] Item 14. A method for isolating Escherichia albertii, comprising: (1) culturing a sample using the bacterial medium according to any one of Items 11 to 13; and (2) isolating colonies grown after the step (1).

[0022] Item 15. The isolation method according to Item 14, wherein the bacterial culture medium contains a sugar that cannot be metabolized by Escherichia albertii and a pH indicator, and the colonies are colonies with a color on the neutral side of the pH indicator.

[0023] Item 16. A composition comprising tellurous acid and / or a salt thereof, and at least two components selected from the group consisting of phosphoric acid and / or a salt thereof, a non-meat-derived peptone, a polyvalent metal ion, and glucose.

[0024] Item 17. The composition according to Item 16, which is used for preparing a bacterial culture medium.

[0025] Item 18. The composition according to Item 17, wherein the bacterial culture medium is the bacterial culture medium according to any one of Items 1 to 13.

[0026] Item 19. The composition according to any one of Items 16 to 18, which is in a solid or liquid form.

[0027] According to one embodiment of the present invention, for example, a medium containing telluric acid and / or a salt thereof can be provided which exhibits a lower degree of growth inhibition of Escherichia albertii.

[0028] The graph shows the correspondence between the growth assessment scores (○, △, ×) in Test Example 2 and the growth state in each evaluation. Photographs showing the growth state (two for each evaluation) are representative examples. The number of colonies grown on each plate medium without dilution, calculated in Test Example 3, is shown. The horizontal axis indicates the components removed (tryptone (Tryp), soy peptone (Soy), glucose (Glu), or dipotassium hydrogen phosphate (PO4)). The bacterial strains used are shown above the graph. The number of colonies grown on each plate medium without dilution, calculated in Test Example 6, is shown. On the horizontal axis, TSA indicates a medium obtained by adding potassium tellurite (final concentration 2.5 μg / mL) to TSA medium, and the others indicate the peptone / tryptone used in the modified TSA medium (Tryp: tryptone (milk-derived peptone), Soy: soybean-derived peptone, Pep: meat-derived peptone).

[0029] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0030] 1. Culture Medium In one aspect, the present invention relates to a bacterial culture medium (sometimes referred to herein as the "culture medium of the present invention") containing at least two components selected from the group consisting of tellurous acid and / or a salt thereof, phosphoric acid and / or a salt thereof, non-meat-derived peptone, a polyvalent metal ion, and glucose. This is described below.

[0031] Tellurite is a compound with the chemical formula H2TeO3.

[0032] The salt of tellurous acid is not particularly limited, but examples thereof include alkali metal salts of tellurous acid and alkaline earth metal salts of tellurous acid, and more specific examples thereof include potassium tellurite, sodium tellurite, magnesium tellurite, calcium tellurite, and barium tellurite.

[0033] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains a salt of tellurous acid, and among such salts, alkali metal salts of tellurous acid are preferred, with potassium tellurite and sodium tellurite being more preferred, and potassium tellurite being particularly preferred.

[0034] The tellurous acid and its salts can be used singly or in combination of two or more.

[0035] The content of tellurous acid and its salts in the medium of the present invention (the content of tellurous acid when only tellurous acid is contained, the content of a salt of tellurous acid when only a salt of tellurous acid is contained, and the total content of both when both are contained) is not particularly limited as long as the components can exert their inhibitory effect on Gram-negative bacteria, and is, for example, 0.0001 to 0.5 g / L. From the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.0002 to 0.1 g / L, more preferably 0.0005 to 0.01 g / L, even more preferably 0.001 to 0.005 g / L, and even more preferably 0.0015 to 0.0035 g / L.

[0036] Examples of phosphoric acid include inorganic phosphoric acids such as H3PO4 (orthophosphoric acid), H4P2O7 (pyrophosphoric acid), and HPO3 (metaphosphoric acid). Among these, orthophosphoric acid is preferred.

[0037] The salt of phosphoric acid is not particularly limited, but examples thereof include alkali metal salts of phosphoric acid and alkaline earth metal salts of phosphoric acid, and more specific examples thereof include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, calcium dihydrogen phosphate, calcium monohydrogen phosphate, tricalcium phosphate, and the like.

[0038] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains a salt of phosphoric acid, and among such salts, alkali metal salts of phosphoric acid or hydrogen phosphate are preferred, alkali metal hydrogen phosphate is more preferred, and dipotassium hydrogen phosphate is particularly preferred.

[0039] The phosphoric acid and salts thereof may be used singly or in combination of two or more.

[0040] When the medium of the present invention contains phosphoric acid and / or a salt thereof, the content of phosphoric acid and a salt thereof in the medium of the present invention (the content of phosphoric acid when only phosphoric acid is contained, the content of a salt of phosphoric acid when only a salt of phosphoric acid is contained, or the total content of both when both are contained) is not particularly limited, and is, for example, 0.1 to 30 g / L, as long as the degree of growth inhibition of Escherichia albertii by telluric acid and / or a salt thereof can be reduced by combining it with non-meat-derived peptone and / or glucose. From the viewpoint of Escherichia albertii separation ability, the content is preferably 0.2 to 15 g / L, more preferably 0.5 to 10 g / L, even more preferably 1 to 5 g / L, and even more preferably 1.5 to 3.5 g / L.

[0041] Non-meat peptone is a component obtained by digesting a protein-containing raw material other than meat with a protease, and contains amino acids, peptides, components derived from the raw material, etc. Meat refers to the meat of vertebrates (e.g., mammals such as cows, pigs, horses, sheep, goats, wild boars, and deer), and is not particularly limited thereto. Examples of protein-containing raw materials other than meat include soybeans, milk (mainly casein), and gelatin (mainly derived from animal bones and skin). Examples of proteases include pepsin, trypsin, papain, and pancreatin. Digestion conditions are not particularly limited as long as they digest the protein in the protein-containing raw material to produce amino acids and peptides to an extent that is effective as a bacterial culture medium component, and conditions according to or similar to known conditions can be adopted.

[0042] As non-meat-based peptones, soybean peptone and milk peptone are particularly preferred from the viewpoint of the ability to separate Escherichia albertii, and soybean peptone is particularly preferred.

[0043] The non-meat raw material peptone may be used alone or in combination of two or more.

[0044] When the medium of the present invention contains a non-meat-source peptone, the content of the non-meat-source peptone in the medium of the present invention is not particularly limited, as long as it can reduce the degree of growth inhibition of Escherichia albertii caused by telluric acid and / or its salts by combining it with phosphoric acid and / or a salt thereof and / or glucose, and is, for example, 0.5 to 100 g / L. From the viewpoint of Escherichia albertii separation ability, the content is preferably 1 to 70 g / L, more preferably 2 to 50 g / L, even more preferably 5 to 25 g / L, even more preferably 5 to 15 g / L, and particularly preferably 7.5 to 12.5 g / L.

[0045] The polyvalent metal ion is not particularly limited as long as it is a metal ion with a valence of 2 or more. The valence of the polyvalent metal ion is, for example, 2 to 6, preferably 2 to 4, and particularly preferably 2 to 3.

[0046] Preferred examples of the polyvalent metal ion include alkaline earth metal ions and transition metal ions. Specific examples of the polyvalent metal ion include calcium ion, iron ion, magnesium ion, manganese ion, cobalt ion, copper ion, zinc ion, and nickel ion, preferably calcium ion, iron ion, and magnesium ion, more preferably calcium ion and iron ion, and particularly preferably calcium ion.

[0047] The polyvalent metal ions can be used singly or in combination of two or more kinds.

[0048] When the medium of the present invention contains polyvalent metal ions, the content of the polyvalent metal ions in the medium of the present invention is not particularly limited, as long as it can reduce the degree of growth inhibition of Escherichia albertii by tellurous acid and / or its salts, and is, for example, 0.05 g / L or more. From the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.05 to 20 g / L, more preferably 0.10 to 10 g / L, even more preferably 0.15 to 5 g / L, and particularly preferably 0.15 to 1 g / L.

[0049] The medium of the present invention containing polyvalent metal ions can be obtained, for example, by adding a salt of the polyvalent metal ion. From this perspective, in one embodiment, the medium of the present invention contains a salt of the polyvalent metal ion.

[0050] When the medium of the present invention contains polyvalent metal ions, the content of the polyvalent metal ions derived from the salts of the added polyvalent metal ions is, for example, 0.05 g / L or more, and from the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.05 to 20 g / L, more preferably 0.10 to 10 g / L, even more preferably 0.15 to 5 g / L, and particularly preferably 0.15 to 1 g / L.

[0051] When the medium of the present invention contains glucose, the glucose content in the medium of the present invention is not particularly limited, as long as the degree of growth inhibition of Escherichia albertii by tellurous acid and / or its salts can be reduced by combining it with phosphoric acid and / or a salt thereof and / or a non-meat-derived peptone, and is, for example, 0.1 to 30 g / L. From the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.2 to 15 g / L, more preferably 0.5 to 10 g / L, even more preferably 1 to 5 g / L, and even more preferably 1.5 to 3.5 g / L.

[0052] On the other hand, if a sugar that cannot be metabolized by Escherichia albertii and a pH indicator that changes color from neutral to acidic are added to the medium of the present invention in order to further enhance the separation ability of Escherichia albertii, the presence of glucose will cause this separation system to malfunction.

[0053] From this viewpoint, the medium of the present invention preferably contains phosphoric acid and / or a salt thereof and a non-meat-derived peptone, and preferably contains a lower glucose content. In this case, the glucose content in the medium of the present invention is, for example, 0 to 1 g / L, preferably 0 to 0.1 g / L, more preferably 0 to 0.01 g / L, even more preferably 0 to 0.001 g / L, still more preferably 0 to 0.001 g / L, and particularly preferably 0 to 0.00001 g / L.

[0054] From the same viewpoint, in one embodiment of the present invention, the medium of the present invention preferably contains a smaller amount of purified glucose. In this case, the amount of purified glucose added during preparation of the medium of the present invention is, for example, 0 to 1 g / L, preferably 0 to 0.1 g / L, more preferably 0 to 0.01 g / L, even more preferably 0 to 0.001 g / L, still more preferably 0 to 0.001 g / L, particularly preferably 0 to 0.00001 g / L, and particularly preferably 0 g / L (no purified glucose added).

[0055] In one embodiment, the medium of the present invention contains polyvalent metal ions in addition to phosphoric acid and / or a salt thereof and non-meat-derived peptone.

[0056] The medium of the present invention preferably contains a lower amount of meat-source peptone, from the viewpoint of minimizing the degree of growth inhibition of Escherichia albertii by tellurous acid and / or its salts. In this case, the content of meat-source peptone in the medium of the present invention is, for example, 0 to 10 g / L, preferably 0 to 5 g / L, more preferably 0 to 2 g / L, even more preferably 0 to 1 g / L, even more preferably 0 to 0.1 g / L, particularly preferably 0 to 0.01 g / L, and particularly preferably 0 g / L. The content of non-meat peptone in the medium of the present invention is, for example, 50 to 100% by mass, preferably 70 to 100% by mass, more preferably 80 to 100% by mass, even more preferably 90 to 100% by mass, even more preferably 95 to 100% by mass, particularly preferably 99 to 100% by mass, particularly preferably 99.9 to 100% by mass, and particularly preferably 100% by mass, relative to 100% by mass of peptone.

[0057] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains a Gram-positive bacteria inhibitor. The Gram-positive bacteria inhibitor is not particularly limited as long as it can inhibit the growth of Gram-positive bacteria, and may be a known component such as bile acid or its salt, bile such as ox bile powder or bile extract, or crystal violet.

[0058] The Gram-positive bacteria inhibitors can be used singly or in combination of two or more.

[0059] When the medium of the present invention contains a Gram-positive bacteria inhibitor, the content of the Gram-positive bacteria inhibitor in the medium of the present invention is not particularly limited, and from the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.0001 to 50 g / L.

[0060] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains bile and / or crystal violet as Gram-positive bacteria inhibitors, and more preferably contains both of these.

[0061] When the medium of the present invention contains a bile, the content of the bile in the medium of the present invention is not particularly limited, and from the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.1 to 50 g / L, more preferably 0.5 to 20 g / L, and even more preferably 1 to 5 g / L.

[0062] When the medium of the present invention contains crystal violet, the content of crystal violet in the medium of the present invention is not particularly limited, and from the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.0001 to 1 g / L, more preferably 0.0002 to 0.1 g / L, and even more preferably 0.0005 to 0.01 g / L.

[0063] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains a gram-negative bacterium inhibitor. The gram-negative bacterium inhibitor is not particularly limited as long as it can inhibit the growth of gram-negative bacteria, and may be a known component, for example, a cephem antibiotic such as cefsulocin, cefixime, or cefotaxime; an aminoglycoside antibiotic such as amikacin; a tetracycline antibiotic such as novosiocin or vancomycin; or a penicillin antibiotic such as piperacillin or carbenicillin. The salt may be, for example, a sodium salt, a potassium salt, or a magnesium salt. Of these, water-soluble salts such as sodium salts and potassium salts are preferred. Among these, cephem antibiotics are preferred, and cefixime is particularly preferred, from the viewpoint of minimal effect on Escherichia albertii.

[0064] The Gram-negative bacteria inhibitors can be used alone or in combination of two or more.

[0065] When the medium of the present invention contains a Gram-negative bacteria inhibitor, the content of the Gram-negative bacteria inhibitor in the medium of the present invention is not particularly limited, and from the viewpoint of the ability to separate Escherichia albertii, the content is preferably 0.000001 to 1 g / L, more preferably 0.00001 to 0.01 g / L, and even more preferably 0.00002 to 0.0002 g / L.

[0066] From the viewpoint of the ability to separate Escherichia albertii, the medium of the present invention preferably contains a sugar that cannot be metabolized by Escherichia albertii and a pH indicator.

[0067] The sugar that cannot be metabolized by Escherichia albertii is a sugar that cannot be metabolized by Escherichia albertii to produce an acidic substance. Such sugars are well known, and examples thereof include xylose, rhamnose, melibiose, etc. Among these, from the viewpoint of the ability to separate Escherichia albertii, it is particularly preferable that the medium of the present invention contains xylose and rhamnose, and it may also contain melibiose.

[0068] The sugars that cannot be metabolized by Escherichia albertii can be used alone or in combination of two or more.

[0069] When the medium of the present invention contains sugars that cannot be metabolized by Escherichia albertii, the content of each sugar is not particularly limited, as long as it is an amount that allows confirmation of a color change in the pH indicator due to acidic substances produced by metabolism of the sugar by bacteria other than Escherichia albertii. The content of each sugar is, for example, 0.1 to 100 g / L, preferably 0.5 to 50 g / L, and more preferably 1 to 20 g / L.

[0070] Although melibiose is desirable from the viewpoint of distinguishing S. Typhimurium from E. albertii, when the growth of S. Typhimurium can be inhibited by tellurite and its salts, or by other Gram-negative bacterium inhibitors (e.g., cephem antibiotics) in the medium of the present invention, the amount of melibiose can be reduced from the viewpoint of cost reduction. In this case, the content of melibiose in the medium of the present invention is, for example, 0 to 5 g / L, preferably 0 to 1 g / L, more preferably 0 to 0.1 g / L, even more preferably 0 to 0.01 g / L, and particularly preferably 0 g / L.

[0071] The pH indicator is added to identify colonies formed by the acid produced by sugar metabolism based on their color. The pH indicator is not particularly limited as long as it changes color from neutral to acidic, and examples include neutral red, phenol red, and bromothymol blue. Neutral red, which is commonly used to detect Escherichia bacteria, is preferred.

[0072] The pH indicators can be used singly or in combination of two or more.

[0073] When the medium of the present invention contains a pH indicator, the content thereof is not particularly limited as long as it is an amount sufficient to confirm a color change of the pH indicator due to an acidic substance produced by the metabolism of the sugar by bacteria other than Escherichia albertii, and the content is, for example, 0.001 to 1 g / L, preferably 0.005 to 0.1 g / L, and more preferably 0.01 to 0.05 g / L.

[0074] In addition to the above components, the medium of the present invention may contain, as necessary, nutritional components, inorganic salts, pH adjusters, etc. Furthermore, the medium of the present invention typically contains water as a solvent. Nutrient components are essential components, being the minimum necessary for bacterial growth. Nutrient components may be, for example, peptone, yeast extract, or enzyme-treated products thereof. Inorganic salts may be inorganic acid metal salts such as sodium chloride, or organic acid metal salts such as sodium citrate. Furthermore, the medium may contain base components for preparing a plate medium as necessary. pH adjusters may be used as necessary to adjust the medium pH to approximately neutral (e.g., 6.8 to 7.8, preferably 7.0 to 7.6, particularly preferably 7.2 to 7.4) during use.

[0075] The medium of the present invention may be a solid medium, a liquid medium, or the like. A solid medium allows for more accurate isolation of Escherichia albertii based on the presence or absence of colonies and their color. Even in a liquid medium, it is possible to increase the proportion of Escherichia albertii in the total microorganisms. In the case of a solid medium, its form is not particularly limited and may be a gel, film, or the like. The components used to prepare the solid medium are not particularly limited, and components that can reduce the fluidity of the liquid, such as gelling agents, more specifically, polysaccharides such as agar, can be used. In one embodiment of the medium of the present invention, for example, a solid (e.g., powdered) composition can be used during storage or distribution, and by adding water, gelling agents, etc., when used as a medium, it can be converted into a medium having the composition disclosed herein (the medium of the present invention). The composition can be provided as a kit with instructions for adding the desired composition (including those integrated into the container or packaging of the composition) to prepare the medium. Furthermore, the solid composition can be appropriately prepared by those skilled in the art using existing techniques after reading the description of the present specification. The medium of the present invention can be obtained by diluting and / or concentrating a composition containing at least two components selected from the group consisting of telluric acid and / or its salts, phosphoric acid and / or its salts, non-meat peptone, polyvalent metal ions, and glucose, so that each component has a concentration appropriate for a bacterial medium. The composition can be in a solid form (e.g., powder or gel) or a liquid form.

[0076] Although the medium of the present invention contains tellurous acid and / or a salt thereof, it inhibits the growth of Escherichia albertii to a lesser extent, and therefore can be used for culturing bacterial samples that may contain Escherichia albertii (to increase the proportion of Escherichia albertii in the total organisms) and for isolating Escherichia albertii. In a preferred embodiment of the present invention, the medium of the present invention enables the isolation of Escherichia albertii with high accuracy even from a wide variety of microorganisms.

[0077] 2. Isolation Method In one aspect, the present invention relates to a method for isolating Escherichia albertii (sometimes referred to herein as the "isolation method of the present invention"), which comprises: (1) culturing a sample using the medium of the present invention, which is a solid medium; and (2) isolating colonies grown after step (1). This method is described below.

[0078] The sample is not particularly limited as long as it is usable for bacterial testing, and may be feces or vomit from various animals including humans, various foods such as fresh produce, or environmental water. The sample may be applied directly to the culture medium, or may be dispersed in purified water or physiological saline and then applied. Alternatively, a culture solution pre-cultured using a culture medium for enrichment may be applied.

[0079] The culture is carried out, for example, at 30 to 40°C, preferably 35 to 38°C, for about 8 to 72 hours. In one embodiment of the present invention, colonies grown by culture can be isolated as Escherichia albertii. In a preferred embodiment of the present invention, when the medium of the present invention contains a sugar that Escherichia albertii cannot metabolize and a pH indicator, colonies whose color indicates non-acidity of the pH indicator can be isolated as Escherichia albertii.

[0080] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0081] Test Example 1. Analysis of the effect of Gram-negative bacteria inhibitors on the growth of Escherichia albertii 1. As the bacterial strain, E. albertii JCM17328 T (standard strain) and E. albertii AH-5 (clinical isolate) were used. The strains were harvested and inoculated into 3 mL of LB medium, followed by agitation culture (180 rpm) at 37°C for 12 hours. 20 μL of the bacterial suspension was then inoculated into 3 mL of LB medium or TSB medium, and cefixime (final concentration 0.05 μg / mL) and / or potassium tellurite (final concentration 2.5 μg / mL) was added to the medium, followed by agitation culture (180 rpm) at 37°C for 9 hours. The bacterial suspension was diluted 10-fold with PBS, and the OD 600The turbidity was measured.

[0082] The results are shown in Table 1. In the table, C indicates the case where cefixime was added, T indicates the case where potassium tellurite was added, CT indicates the case where both were added, and blank spaces indicate the case where neither was added.

[0083]

[0084] Cefixime was found to have no effect on the growth of E. albertii. On the other hand, tellurite inhibited the growth of E. albertii. Furthermore, the results for LB and TSB media suggest that the resistance to tellurite may differ depending on the composition of the medium.

[0085] Test Example 2. Analysis of the effect of Gram-negative bacteria inhibitors on the growth of Escherichia albertii 2. As the bacterial strain, E. albertii JCM17328 T (standard strain) and E. albertii AH-5 (clinical isolate) were used. The strains were harvested, inoculated into 3 mL of TSB medium, and incubated overnight at 37°C with agitation (180 rpm). The bacterial suspension was smeared onto plates and incubated at 37°C for 20 hours, after which the growth status was confirmed. The compositions of the two plates used are shown in Table 2 (TSA medium) and Table 3 (XRM Mac medium). Cefixime (final concentration 0.05 μg / mL) and / or potassium tellurite (final concentration 2.5 μg / mL) were added to each plate. The correlation between the growth assessment scores (○, △, ×) and the growth status for each assessment is shown in Figure 1. Photographs showing the growth status are representative examples.

[0086]

[0087]

[0088] The results are shown in Table 4. In the table, C indicates the case where cefixime was added, T indicates the case where potassium tellurite was added, CT indicates the case where both were added, and blank spaces indicate the case where neither was added.

[0089]

[0090] As in Test Example 1, it was found that the growth of E. albertii in the presence of tellurite differed depending on the medium composition. Furthermore, these results suggested that TSA medium contained a positive component for the growth of E. albertii in the presence of tellurite, and that XRM Mac medium contained a negative component for the growth of E. albertii in the presence of tellurite.

[0091] Test Example 3. Search for a positive component for the growth of Escherichia albertii in the presence of tellurite 1. Plate media prepared by adding potassium tellurite (final concentration 2.5 μg / mL) to TSA medium and plates prepared by removing tryptone (Tryp), soy peptone (Soy), glucose (Glu), or dipotassium hydrogen phosphate (PO4) from the composition of the plate media were prepared, and the number of developed colonies was compared. Specifically, the procedure was as follows.

[0092] The strain was E. albertii JCM17328. T (standard strain) and E. albertii AH-5 (clinical isolate) were used. The strains were picked, inoculated into 3 mL of TSB medium, and cultured overnight at 37°C with agitation (180 rpm). 20 μg L of the bacterial solution was taken and inoculated into 3 mL of TSB medium, and cultured at 37°C for approximately 3 hours (OD 600 = 1.0 as a guideline, 10 8 The bacterial solution was serially diluted, and 100 μL of it was dropped onto a plate medium, which was then smeared over the entire surface with a Conn. rod and cultured at 37°C for 20 hours. Petri dishes that had formed 30 to 300 colonies were used, and the number of colonies that would have grown on each plate medium without dilution was calculated from the number of colonies on those dishes and the dilution factor.

[0093] The results are shown in Figure 2. It was suggested that soybean peptone, glucose, and dipotassium hydrogen phosphate were positive components.

[0094] Test Example 4. Search for positive components for the growth of Escherichia albertii in the presence of tellurite 2. Potassium tellurite (final concentration 2.5 μg / mL) was added to XRM Mac medium, and one to three components selected from the group consisting of soy peptone (Soy: final concentration 3 g / L), glucose (Glu: final concentration 2.5 g / L), and dipotassium hydrogen phosphate (PO4: final concentration 2.5 g / L) were added to prepare plate media, and the growth state was evaluated. Specifically, the procedure was as follows.

[0095] The strain was E. albertii JCM17328. T (standard strain) and E. albertii AH-5 (clinical isolate) were used. The strains were picked, inoculated into 3 mL of TSB medium, and cultured overnight at 37°C with agitation (180 rpm). The bacterial suspension was then smeared onto a plate medium and cultured at 37°C for 20 hours, after which the growth status was confirmed.

[0096] The results are shown in Table 5. In the table, T indicates the case where potassium tellurite was added, Soy, Glu, and PO4 indicate the case where the respective component was added, and blank cells indicate the case where none of the three components was added. (Red) indicates the color of the colony, and △ / ◯ evaluations without (red) indicate white (colorless) colonies.

[0097]

[0098] It was found that even the poorly growing standard strain (JCM17328) can grow in the presence of tellurite by using at least two selected from the group consisting of soy peptone, glucose, and phosphate. However, while XRM Mac medium contains sugars that E. albertii cannot decompose and a pH indicator, allowing E. albertii colonies to be identified as white (colorless) and colonies of bacteria that decompose the sugars to be identified as red, the addition of glucose (a sugar decomposed by E. albertii) makes this identification system unusable. Therefore, from the perspective of E. albertii's identification, it is preferable not to use glucose.

[0099] Test Example 5. Search for components that negatively affect the growth of Escherichia albertii in the presence of tellurite 1. Potassium tellurite (final concentration 2.5 μg / mL) was added to TSA medium, and one to three species selected from the group consisting of crystal violet (CV: final concentration 0.001 g / L), neutral red (NR: final concentration 0.03 g / L), and bile salt No. 3 (Bile: final concentration 1.5 g / L) were added to prepare plate media, and the growth state was evaluated. Specifically, the procedure was as follows.

[0100] The strain was E. albertii JCM17328. T (standard strain) was used. The strain was picked, inoculated into 3 mL of TSB medium, and cultured overnight at 37°C with agitation (180 rpm). The bacterial liquid was smeared on a plate medium and cultured at 37°C for 20 hours, after which the growth status was confirmed.

[0101] The results are shown in Table 6. In the table, T indicates the case where potassium tellurite was added, CV, NR, and Bile indicate the cases where the respective components were added, and blank spaces indicate the cases where none of the three components was added.

[0102]

[0103] Crystal violet, neutral red, and bile salts were found to have no negative effect.

[0104] Test Example 6. Search for components that negatively affect the growth of Escherichia albertii in the presence of tellurite 2. Potassium tellurite (final concentration: 2.5 μg / mL) was added to TSA medium, and the number of colonies that grew was compared by adding different tryptone / peptone compositions or types (Table 7).

[0105]

[0106] The strain was E. albertii JCM17328. TThe strain was harvested and inoculated into 3 mL of TSB medium, followed by overnight agitation (180 rpm) at 37°C. 20 μL of the bacterial solution was then inoculated into 3 mL of TSB medium and incubated at 37°C for approximately 3 hours (OD 600 = 1.0 as a guideline, 10 8 The bacterial solution was serially diluted, and 100 μL of it was dropped onto a plate medium, which was then smeared over the entire surface with a Conn. rod and cultured at 37°C for 20 hours. Petri dishes that had formed 30 to 300 colonies were used, and the number of colonies that would have grown on each plate medium without dilution was calculated from the number of colonies on those dishes and the dilution factor.

[0107] The results are shown in Figure 3. It was found that meat-derived peptone had a negative effect. On the other hand, it was found that peptones derived from non-meat ingredients could be used.

[0108] Test Example 7 Optimization of Medium Composition Based on the results of Test Examples 1 to 6, a plate medium (T-XRM-Soy20-Mac) having the composition shown in Table 8 was prepared.

[0109]

[0110] However, E. albertii JCM17328 was isolated using this medium. T When colonies of the standard strain were observed, they were found to be red. This was thought to be due to the trace amounts of sugar contained in soybean-derived peptone being decomposed by E. albertii, causing the pH indicator to turn red. Therefore, the amount of soy peptone in the T-XRM-Soy20-Mac medium was reduced to 10 g / L or 5 g / L, and colonies were observed. The specific method was the same as in Test Example 2.

[0111] The results are shown in Table 9.

[0112]

[0113] It was found that a concentration of 10 g / L of soy peptone was the condition for favorable growth without causing the colonies to turn red.

[0114] Test Example 8 Evaluation of Escherichia albertii Separation Ability Based on the results of Test Examples 1 to 7, a plate medium (CT-XRM-Soy10-Mac) having the composition shown in Table 10 was prepared.

[0115]

[0116] The growth state of 56 clinical isolates and 8 animal isolates of E. albertii was evaluated in the same manner as in Test Example 2 using XRM Mac medium and CT-XRM-Soy10-Mac medium as plate media.

[0117] The results are shown in Table 11.

[0118]

[0119] Similar to XRM Mac medium, CT-XRM-Soy10-Mac medium was found to be a medium in which various E. albertii strains could grow.

[0120] Similar tests were then performed on bacteria other than E. albertii. The results for bacterial species that formed similar white (colorless) colonies on XRM Mac medium are shown in Table 12, the results for Shigella species that formed similar white (colorless) colonies are shown in Table 13, and the results for bacterial species that formed red colonies are shown in Table 14.

[0121]

[0122]

[0123]

[0124] Test Example 9: Evaluation of the necessity of melibiose XRM Mac medium contains melibiose to distinguish S. Typhimurium from E. albertii (S. Typhimurium produces red colonies). However, the addition of tellurite (or even cefixime) may suppress the growth of S. Typhimurium itself, in which case melibiose is unnecessary. To investigate this, a medium obtained by removing melibiose from XRM Mac medium (XR Mac) and a medium obtained by removing melibiose from CT-XRM-Soy10-Mac medium (CT-XR-Soy-Mac) were prepared, and the growth of S. Typhimurium was evaluated in the same manner as in Test Example 2.

[0125] The results are shown in Table 15.

[0126]

[0127] W means white (colorless) colonies, and R means red colonies.

[0128] Since S. Typhimurium does not grow on a medium obtained by removing melibiose from CT-XRM-Soy10-Mac medium (CT-XR-Soy-Mac), it was found possible to distinguish between S. Typhimurium and E. albertii.

[0129] Test Example 10. Escherichia albertii spike test E. albertii JCM17328 T The standard strain was harvested, inoculated into 3 mL of TSB medium, and cultured overnight at 37°C with agitation (180 rpm). 20 μL of the bacterial solution was inoculated into 3 mL of TSB medium and cultured at 37°C for 2.5 to 3 hours (OD 600 = 1.0 as a guideline, 10 8 The bacterial solution was diluted 10-fold and used as the bacterial solution to be added in the following procedure.

[0130] A negative sample swab of E. albertii was mixed with 900 μL of PBS to prepare a sample solution. 100 μL of the above-mentioned bacterial solution was inoculated into the sample solution. Serial dilutions were made, and the total number of samples was 10.-4 100 μL of the diluted solution was dropped onto a plate medium (XRM Mac or CT-XR-Soy-Mac), smeared over the entire surface with a Conlarge stick, and cultured at 37°C for 16 to 20 hours. The number of grown colonies was counted and their condition was confirmed.

[0131] The results are shown in Table 16. The numbers of red colonies and white (colorless) colonies are shown for each medium with and without spike (with or without added bacterial solution).

[0132]

[0133] When CT-XR-Soy-Mac was used, the ratio of white (colorless) colonies to the total colonies was higher, indicating that E. albertii was easier to isolate.

[0134] Test Example 11. Test to confirm the growth status of Escherichia albertii negative specimens. Each of the 57 swab specimens negative for E. albertii was clouded in 500 μL of PBS, and serially diluted to 10 -4 100 μL of the diluted solution was dropped onto a plate medium (XRM Mac), which was then smeared over the entire surface with a Conlarge stick and cultured at 37°C for 16 to 20 hours. The number of grown colonies was counted and their condition was confirmed.

[0135] The results are shown in Table 17.

[0136]

[0137] Sixteen samples that developed white (colorless) colonies on XRM Mac were tested in the same manner using CT-XR-Soy-Mac as the plate medium. The number of white (colorless) colonies counted for each of these 16 samples is shown in Table 18.

[0138]

[0139] Even in E. albertii-negative samples, many white (colorless) colonies grew as false positives on XRM Mac medium, whereas no white (colorless) colonies grew in E. albertii-negative samples on CT-XR-Soy-Mac medium, demonstrating the superior ability of CT-XR-Soy-Mac medium to isolate E. albertii.

[0140] Test Example 12. Spiking Test and Colony Hybridization Test: 10 g of commercially available chicken meat negative for E. albertii and 10 CFU and 100 CFU of E. albertii AH5 strain were added to 90 mL of stomaching bag containing TSB. The stomaching bag was stomached for 45 seconds and then incubated at 37°C for 18–20 hours. The culture was serially diluted 10-fold with PBS (pH 7.4), and 100 μL was inoculated into XRM-Mac medium or CT-XR-Soy-Mac medium. XRM-Mac medium was incubated at 37°C for 20 ± 1 hour, and CT-XR-Soy-Mac medium was incubated at 37°C for 24 ± 1 hour. After incubation, the number of red and white (colorless) colonies was counted. White (colorless) colonies were identified by hybridization using an Eacdt-specific gene probe to confirm their identity as E. albertii. Specifically, white (colorless) colonies obtained from each medium were inoculated onto LB agar plates with a nitrocellulose membrane and cultured for 4–6 hours at 37°C. The nitrocellulose membrane was then treated with alkali (0.5 N NaOH for 10 minutes, 1 M Tris-HCl [pH 8.0] for 1 minute three times, 1 M Tris-HCl [pH 8.0] 1.5 M NaCl for 10 minutes), dried, and then treated with UV light to immobilize the DNA on the nitrocellulose membrane. 32A P-labeled Eacdt gene probe was prepared, and hybridization was performed on a nitrocellulose membrane under high-stringency conditions (50% formamide, 5X SSC, 0.1% SDS, 1 mM EDTA, 1X Denhardt's solution, 10 mg / mL heat-denatured herring sperm DNA) at 42°C for 18 ± 2 hours. The membrane was then rinsed once with a wash solution containing 2X SSC and 0.1% SDS, followed by another wash at 65°C for 1 hour. After rinsing with the same wash solution, the membrane was dried and the radioactivity on the membrane was measured using a BAS FLA-3000 to determine whether white colonies were E. albertii.

[0141] The results are shown in Table 19.

[0142]

[0143] Test Example 13: Evaluation of Polyvalent Metal Ions. A medium was prepared by adding ammonium ferric citrate (a salt of iron (III) ion) to CT-XR-Soy-Mac medium to a final concentration of 0.1% (CT-XR-Soy-Mac + 0.1% AFC). A medium was also prepared by adding calcium chloride (a salt of calcium (II) ion) to CT-XR-Soy-Mac to a final concentration of 0.05% (CT-XR-Soy-Mac + 0.05% CaCl2). The iron ion concentration (AFC) in CT-XR-Soy-Mac + 0.1% AFC medium was approximately 0.21 g / L, and the calcium ion concentration (CaCl2) in CT-XR-Soy-Mac + 0.05% CaCl2 medium was approximately 0.18 g / L. Various strains of E. albertii and non-E. albertii strains were cultured in CT-XR-Soy-Mac medium, CT-XR-Soy-Mac + 0.1% AFC medium, or CT-XR-Soy-Mac + 0.05% CaCl2 medium, and colony sizes were measured. The results are shown in Table 20. The symbols in the table mean the following: No growth (-); Less growth <0.5 mm colony size (Δ); Growth 0.5 mm colony size (+); Good growth >0.5 mm colony size (++).

[0144]

Claims

1. A bacterial culture medium comprising tellurous acid and / or a salt thereof, and at least two components selected from the group consisting of phosphoric acid and / or a salt thereof, non-meat-derived peptone, polyvalent metal ions, and glucose.

2. The bacterial culture medium according to claim 1, containing the phosphoric acid and / or a salt thereof and a non-meat-derived peptone.

3. The bacterial culture medium according to claim 2, wherein the non-meat-source peptone is at least one selected from the group consisting of soybean peptone and milk peptone.

4. The bacterial culture medium according to claim 2, wherein the content of the phosphoric acid and its salts is 0.5 to 10 g / L, and the content of the non-meat-source peptone is 2 to 50 g / L.

5. The bacterial culture medium according to claim 2, which contains said polyvalent metal ions.

6. The bacterial culture medium according to claim 5, wherein the content of said polyvalent metal ions is 0.05 g / L or more.

7. The bacterial culture medium according to claim 1, wherein the content of meat peptone is 0 to 10 g / L.

8. The bacterial culture medium according to claim 1, wherein the content of the tellurous acid and its salts is 0.0005 to 0.01 g / L.

9. The bacterial culture medium according to claim 1, which contains at least one selected from the group consisting of gram-positive bacteria inhibitors and gram-negative bacteria inhibitors.

10. The bacterial culture medium according to claim 1, which contains a sugar that cannot be metabolized by Escherichia albertii and a pH indicator that changes color as the pH changes from neutral to acidic.

11. The bacterial culture medium according to claim 1, which is a solid culture medium.

12. The bacterial culture medium according to any one of claims 1 to 11, which is used to culture a bacterial sample that may contain Escherichia albertii.

13. The bacterium culture medium according to claim 12, which is used for isolating Escherichia albertii.

14. A method for isolating Escherichia albertii, comprising: (1) culturing a sample using the bacterial culture medium described in claim 11; and (2) isolating colonies grown after step (1).

15. The isolation method according to claim 14, wherein the bacterial culture medium contains a sugar that cannot be metabolized by Escherichia albertii and a pH indicator that changes color as the pH changes from neutral to acidic, and the colonies are colonies whose color indicates non-acidity according to the pH indicator.

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