JP7176833B2 - H. influenzae screening method and screening medium - Google Patents

Description

The present invention provides a method for detecting Haemophilus influenzae (hereinafter referred to as Haemophilus influenzae), a medium used therein, and/or Haemophilus influenzae, Haemophilus haemolyticus , and Haemophilus parainfluenzae (hereinafter referred to as Haemophilus parainfluenzae). , called Parainfluenza) and Haemophilus parahaemolyticus , and a medium used therefor. More specifically, a method and a screening medium for easily detecting (screening) the presence or absence of Haemophilus influenzae from a subject that may contain Haemophilus influenzae, and/or Haemophilus influenzae, Haemophilus haemolyticus, and Parainfluenza. The present invention relates to methods and discriminating media capable of discriminating Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus in a specimen in which the bacterium and Haemophilus parahaemolyticus may be present.

Haemophilus influenzae is a Gram-negative bacillus that normally resides in the nasopharynx of humans. typed. Acapsular strains account for the majority of H. influenzae endemic in humans and are often isolated from patients with otitis media, sinusitis, chronic bronchitis, and conjunctivitis. On the other hand, type b capsular strains often cause meningitis and sepsis in children and pneumonia in adults (Non-Patent Document 1).

Usually, for the primary isolation culture of Haemophilus influenzae from test materials, a chocolate agar medium or a chocolate agar medium to which bacitracin is added to improve selectivity is used. Haemophilus influenzae requires sufficient amounts of factor X (Hemin or other porphyrins) and factor V (nicotinamide adenine dinucleotide: NAD, or its phosphate) for growth, so an enzyme that destroys factor V is present in red blood cells. It does not grow on sheep blood agar, which contains sheep blood in abundance. On the other hand, since horse blood contains a small amount of the enzyme that destroys factor V, Haemophilus influenzae grows on the horse blood agar medium. However, the horse blood agar medium is not used for the primary isolation culture of Haemophilus influenzae because the colonies of Haemophilus influenzae that develop are minute. Therefore, a chocolate agar medium rich in factor X and factor V is used for the primary isolation culture of Haemophilus influenzae.
After the primary isolation culture, bacteria are identified by picking colonies suspected of being Haemophilus influenzae from among the grown colonies and conducting a confirmatory test. As a confirmation test, the test bacteria are applied to a nutrient agar medium, placed on a disc for X and V requirement test, and cultured. There is a method of coating and culturing on a medium containing), a method of examining the porphyrin synthesis ability of the test bacteria, and the like (Non-Patent Documents 2 and 3, Patent Documents 1, 2 and 3).

During primary isolation culture, culture using sheep blood agar medium was actually performed in parallel with culture using chocolate agar medium or bacitracin-added chocolate agar medium. A confirmatory test is carried out by catching colonies suspected of being Haemophilus influenzae from colonies grown on a medium or a bacitracin-added chocolate agar medium. However, Haemophilus spp. other than Haemophilus influenzae (Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, etc.) do not grow on sheep blood agar medium, but grow on chocolate agar medium or bacitracin-added chocolate agar medium. Haemophilus influenzae must be identified only by morphology from among the grown Haemophilus spp., and skillful techniques are required for this identification. In addition, in order to pick all colonies suspected of being Haemophilus influenzae from among the numerous colonies grown by the primary isolation culture and conduct confirmation tests, in addition to skilled techniques, complicated work and a large amount of confirmatory test reagents (X, V) are required. disk for factor demand test, 4-compartment medium, porphyrin synthesis ability test drug, etc.) are required. Furthermore, when catching colonies, there is a possibility that Haemophilus influenzae may be caught and missed.

On the other hand, a method for identifying microorganisms based on their color change by using a chocolate agar medium containing an enzyme chromogenic substrate has been disclosed, Mag-phos (5-bromo-6-chloro-3-indolyl phosphate) It has been described that purple Haemophilus influenzae colonies are distinguished from colorless normal sputum flora by using a chocolate agar containing However, even when a chocolate agar medium containing an enzymatic chromogenic substrate is used, skilled techniques are required to distinguish between bacteria exhibiting the same enzymatic resolution as Haemophilus influenzae and Haemophilus influenzae by morphology, resulting in poor work efficiency. However, the problem of high reagent costs still remains.

JP-A-62-155098 Japanese Utility Model Laid-Open No. 59-159300 JP-A-2001-161394 Japanese Patent Application Laid-Open No. 2001-161396

Norichika Arakawa, IASR, 31, 94-95, 2010 Masayoshi Chonan et al., Clinical Laboratory, 58(11), 1362-1365, 2014 Ikuo Yamaguchi, Microbial Inspection Navi, 164-165, 2013

The present invention has been made in view of the above circumstances, and includes a method and a screening medium that can easily and accurately detect (screen) whether Haemophilus influenzae is present in a specimen, and/or Haemophilus influenzae, Haemophilus haemolyticus, Parainfluenza, and Haemophilus parahaemolyticus can be easily and accurately identified, and to provide a medium for identification.

As a result of intensive studies to solve the above problems, the present inventors added β-NAD and an antibacterial agent to horse blood agar medium, which has not been used for primary isolation culture in the past, and added sucrose and sucrose as coloring components. After inoculating a specimen in a medium prepared by adding a pH indicator and culturing it, the strain of Haemophilus spp. can be distinguished by the presence or absence of sucrose decomposition and hemolysis. By using a medium prepared by adding β-NAD and an antibacterial agent to an agar medium and adding sucrose and a pH indicator as color-developing components, it is easy to check the colony color of the grown bacteria and the presence or absence of hemolysis. Haemophilus influenzae (no sucrose decomposition, no hemolysis), Haemophilus haemolyticus (no sucrose decomposition, hemolysis), Parainfluenzae (with sucrose decomposition, no hemolysis), Haemophilus parahaemolyticus (with sucrose decomposition, hemolysis) It was found that the identification of The present inventors also added β-NAD and an antibacterial agent to a horse blood agar medium, and added a galactopyranoside derivative (hereinafter referred to as a Gal derivative) as a color-developing component to prepare a medium. After inoculation and culturing, the species of Haemophilus genus bacteria can be distinguished by the resolution of the Gal derivative and the presence or absence of hemolysis. In addition, by using a medium prepared by adding a Gal derivative as a color-developing component, it is possible to easily confirm the colony color of the grown bacteria and the presence or absence of hemolysis (no decomposition of Gal derivative, no hemolysis). , Haemophilus haemolyticus (no decomposition of Gal derivative, with hemolysis), Haemophilus influenzae (with decomposition of Gal derivative, no hemolysis), and Haemophilus parahaemolyticus (with decomposition of Gal derivative, with hemolysis). Found it. In addition, the addition of titanium oxide to the medium to keep the color of the medium bright improves the visibility of hemolysis and colony color development. The present inventors have found that it is possible to make an accurate identification, and have completed the present invention.

That is, the present invention consists of the following configurations.
(1) After inoculating and culturing the specimen in a medium containing at least the following (a), (b), (c) and (d) in which Haemophilus influenzae can grow, the colony color of the bacteria and the presence or absence of hemolysis are examined. A screening method for Haemophilus influenzae, characterized by confirming.
(a) horse blood,
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d) A coloring component that has the property of making the coloring of the Haemophilus influenzae colony and the coloring of the Parainfluenza colony different.
(2) The screening method for Haemophilus influenzae according to (1), wherein the coloring component is (d1) and/or (d2) below.
(d1) sugars and pH indicators that are not degraded by Haemophilus influenzae but are degraded by Haemophilus influenzae to produce acid;
(d2) Enzymatic chromogenic substrates that are not degraded by Haemophilus influenzae but are degraded by Parainfluenzae to develop color.
(3) the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria;
The sugar that is not decomposed by Haemophilus influenzae but is decomposed by Parainfluenza to generate acid is sucrose,
said pH indicator is selected from aniline blue, bromocresol purple, bromothymol blue, phenol red, neutral red and methyl red;
The screening method for Haemophilus influenzae according to (2), wherein the enzyme chromogenic substrate that is not degraded by Haemophilus influenzae but is degraded by Parainfluenzae to develop a color is a galactopyranoside derivative.
(4) After inoculating and culturing the sample in a medium containing at least the following (a), (b), (c) and (d) in which Haemophilus influenzae can grow, the colony color of the bacteria and the presence or absence of hemolysis are examined. A method for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus, characterized by confirming.
(a) horse blood,
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d) a coloring component, which makes the coloration of the colonies of Haemophilus influenzae and the coloration of the colonies of Haemophilus influenzae different, and makes the coloration of the colonies of Haemophilus influenzae and Haemophilus haemolyticus the same, and Parainfluenza A color-developing component having the property of making the colors of bacteria and colonies of Haemophilus parahaemolyticus the same.
(5) Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza, and Haemophilus parahaemolyticus according to (4), wherein the coloring component is the following (d1) and/or (d2): identification method.
(d1) sugars and pH indicators that are not degraded by Haemophilus influenzae and Haemophilus haemolyticus but are degraded by Haemophilus influenzae and Haemophilus parahaemolyticus to produce acid;
(d2) Enzymatic chromogenic substrates that are not degraded by Haemophilus influenzae and Haemophilus haemolyticus, but are degraded by Parainfluenza and Haemophilus parahaemolyticus to develop color.
(6) the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria;
the sugar that is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but that is degraded by Haemophilus influenzae and Haemophilus parahaemolyticus to produce acid is sucrose;
said pH indicator is selected from aniline blue, bromocresol purple, bromothymol blue, phenol red, neutral red and methyl red;
(5) wherein the enzyme chromogenic substrate that is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but is degraded by Parainfluenza and Haemophilus parahaemolyticus to develop a color is a galactopyranoside derivative; A method for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus and Haemophilus parahaemolyticus described in .
(7) A medium for screening Haemophilus influenzae, characterized by containing the following (a), (b), (c) and (d) in a medium in which Haemophilus influenzae can grow.
(a) horse blood,
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d) A coloring component that has the property of making the coloring of the Haemophilus influenzae colony and the coloring of the Parainfluenza colony different.
(8) The medium for screening Haemophilus influenzae according to (7), wherein the coloring component is (d1) and/or (d2) below.
(d1) sugars and pH indicators that are not degraded by Haemophilus influenzae but are degraded by Haemophilus influenzae to produce acid;
(d2) Enzymatic chromogenic substrates that are not degraded by Haemophilus influenzae but are degraded by Parainfluenzae to develop color.
(9) the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria;
The sugar that is not decomposed by Haemophilus influenzae but is decomposed by Parainfluenza to generate acid is sucrose,
said pH indicator is selected from aniline blue, bromocresol purple, bromothymol blue, phenol red, neutral red and methyl red;
The medium for screening Haemophilus influenzae according to (8), wherein the enzyme chromogenic substrate that is not degraded by Haemophilus influenzae but is degraded by Haemophilus influenzae to develop color is a galactopyranoside derivative.
(10) A medium for Haemophilus influenzae screening, characterized by containing at least the following components per liter of the medium.
(a) horse blood 10-100 mL,
(b) β-NAD 1-100 mg,
(c) bacitracin 1-800 mg and/or vancomycin 0.5-40 mg,
(d) 1-40 g sucrose and 40-400 mg aniline blue, and/or 40-300 mg 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
(11) Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus para, characterized by containing the following (a), (b), (c) and (d) in a medium capable of growing Haemophilus influenzae Haemolyticus identification medium.
(a) horse blood,
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d) a coloring component, which makes the coloration of the colonies of Haemophilus influenzae and the coloration of the colonies of Haemophilus influenzae different, and makes the coloration of the colonies of Haemophilus influenzae and Haemophilus haemolyticus the same, and Parainfluenza A color-developing component having the property of making the colors of bacteria and colonies of Haemophilus parahaemolyticus the same.
(12) Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza, and Haemophilus parahaemolyticus according to (11), wherein the coloring component is (d1) and/or (d2) below. discriminating medium.
(d1) sugars and pH indicators that are not degraded by Haemophilus influenzae and Haemophilus haemolyticus but are degraded by Haemophilus influenzae and Haemophilus parahaemolyticus to produce acid;
(d2) Enzymatic chromogenic substrates that are not degraded by Haemophilus influenzae and Haemophilus haemolyticus, but are degraded by Parainfluenza and Haemophilus parahaemolyticus to develop color.
(13) the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria;
the sugar that is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but that is degraded by Haemophilus influenzae and Haemophilus parahaemolyticus to produce acid is sucrose;
said pH indicator is selected from aniline blue, bromocresol purple, bromothymol blue, phenol red, neutral red and methyl red;
(12) wherein the enzyme chromogenic substrate that is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but is degraded by Parainfluenza and Haemophilus parahaemolyticus to develop a color is a galactopyranoside derivative; A medium for identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus according to .
(14) A medium for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus, characterized by containing at least the following components per liter of the medium.
(a) horse blood 10-100 mL,
(b) β-NAD 1-100 mg,
(c) bacitracin 1-2.5 mg and/or vancomycin 0.5-10 mg,
(d) 1-40 g sucrose and 40-400 mg aniline blue, and/or 40-300 mg 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

According to the present invention, only by confirming the colony color of the bacteria grown in the medium and the presence or absence of hemolysis, Haemophilus influenzae, Haemophilus haemolyticus, Parainfluenza, and Haemophilus parahaemo by primary isolation culture, which was impossible in the past, can be performed. Allows for easy identification of lyticus. This makes it possible to detect (screen) Haemophilus influenzae much more easily and accurately than the conventional primary isolation culture method using a chocolate agar medium. In addition, it is possible to reduce the number of colonies to be caught for the confirmation test after isolation culture, and furthermore, it is not necessary to confirm the presence or absence of hemolysis in the confirmation test, so it is cheaper than the 4-fraction medium. It becomes possible to identify Haemophilus influenzae with only a confirmatory test with the disk for the V-factor demand test. As a result, the time required for the primary isolation culture and confirmation test, the labor involved in the operation, and the cost of the confirmation test (X/V factor requirement test disk, 4-fraction medium, porphyrin synthesis ability test drug, etc.) In addition, it has the effect of eliminating false negatives due to missing bacteria.

In the present invention, at least (a) horse blood, (b) β-NAD or a phosphate thereof, (c) an antibacterial agent, and (d) a coloring component, which are used to color colonies of Haemophilus influenzae and Parainfluenzae A sample is inoculated into the medium of the present invention that contains a color-developing component that has the property of making the coloration of the colonies of different colors, and in which Haemophilus influenzae can grow. After culturing, the colony color of the bacteria and the presence or absence of hemolysis are confirmed. It is characterized by distinguishing Haemophilus species and thereby detecting Haemophilus influenzae. During actual culture, the basal medium contains (a) horse blood, (b) β-NAD or its phosphate, (c) an antibacterial agent, and (d) a coloring component, which is used to color the colony of Haemophilus influenzae. A culture medium to which a color-developing component having the property of making the coloration of a parainfluenza colony differ can be used as the medium of the present invention.

The medium of the present invention contains, as nutrient components, ingredients that allow Haemophilus influenzae to grow and that can determine the presence or absence of hemolysis due to Haemophilus spp. Factor X and factor V are necessary for the growth of Haemophilus influenzae. NADP (nicotinamide adenine dinucleotide phosphate) and β-NADPH (nicotinamide adenine dinucleotide phosphate reduced form) are added. Although factor V is also present in horse blood, in the present invention, β-NAD or its phosphate is added to horse blood for the purpose of promoting the growth of Haemophilus influenzae.

The horse blood contained in the medium of the present invention is also used to determine the presence or absence of hemolysis due to Haemophilus spp. When hemolysin-producing bacteria grow in the medium of the present invention, they hemolyze red blood cells present in the medium to make them transparent, forming transparent hemolytic zones around the colonies. Therefore, when Haemophilus haemolyticus, Haemophilus parahaemolyticus, or the like, which are Haemophilus bacteria that produce hemolysin, grow in the medium of the present invention, it is determined that hemolysis is present. On the other hand, when Haemophilus influenzae, Parainfluenzae, or the like, which do not produce hemolysin, grows in the medium of the present invention, no hemolysis zone is formed around the colony, so it is determined that there is no hemolysis.

The amount of horse blood to be added can be appropriately selected within a range in which Haemophilus influenzae can grow and the presence or absence of hemolysis due to Haemophilus can be determined. . On the other hand, the concentration of factor V to be added can be appropriately selected within a range where Haemophilus influenzae can grow. For example, the concentration of β-NAD to be added is preferably 1 to 100 mg/L. Equivalent amounts of β-NADP and β-NADPH can be added as a substitute for β-NAD.

The medium of the present invention contains a coloring component that has the property of making the coloring of Haemophilus influenzae colonies and the coloring of Haemophilus influenzae colonies different. Specifically, such chromogenic components include (1) sugars and pH indicators that are not degraded by Haemophilus influenzae but are degraded by Haemophilus influenzae to produce acid, and/or (2) not degraded by Haemophilus influenzae. However, an enzymatic chromogenic substrate that is degraded by Parainfluenza to develop color can be used.
Furthermore, the color-developing component contained in the medium of the present invention makes the coloration of the colonies of Haemophilus influenzae and the coloration of the colonies of Haemophilus influenzae different colors, and makes the coloration of the colonies of Haemophilus influenzae and Haemophilus haemolyticus the same color, In addition, it has the property of making the colonies of Parainfluenza and Haemophilus parahaemolyticus the same color. Specific examples of such color-developing components include (1) sugars and pH indicators that are not degraded by Haemophilus influenzae and Haemophilus haemolyticus, but are decomposed by Parahaemophilus influenzae and Haemophilus parahaemolyticus to produce acid, and or (2) an enzymatic chromogenic substrate that is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but degraded by Parahaemophilus and Haemophilus parahaemolyticus to develop color can be used.

The type of sugar used in the medium of the present invention is not particularly limited as long as it is a sugar that is not decomposable by Haemophilus influenzae but is capable of being decomposed by Parainfluenzae. Examples of such sugars include sucrose, mannose, and the like. For example, by using sucrose, Haemophilus influenzae (without decomposition of sucrose) and Haemophilus influenzae (with decomposition of sucrose) can be distinguished with high accuracy. Furthermore, sucrose is a sugar that Haemophilus haemolyticus does not have the ability to decompose and Haemophilus parahaemolyticus has the ability to decompose. (with sucrose decomposition) can also be identified with high accuracy. These take advantage of the difference in sucrose degradability between bacteria. The added concentration of sucrose is in a range that is not degraded by Haemophilus influenzae and is degraded by Haemophilus parahaemolyticus to produce acid, or is not degraded by Haemophilus influenzae and Haemophilus haemolyticus but is degraded by Haemophilus influenzae and Haemophilus parahaemolyticus. It can be selected as appropriate within the range of generating an acid. The concentration of such sucrose can be, for example, 1 to 40 g/L, more preferably 1 to 20 g/L, even more preferably 1 to 15 g/L. In addition, it is also possible to add mannose in addition to sucrose as needed. Mannose is also a sugar that Haemophilus influenzae does not have the ability to decompose, but Parainfluenzae has the ability to decompose mannose.

The type of pH indicator used in the culture medium of the present invention is not particularly limited as long as it changes color with pH change due to acid produced by decomposition of sucrose. For example, pH indicators with different neutral and acid colors, aniline blue, bromocresol purple, bromothymol blue, phenol red, neutral red, methyl red, etc. can be used. Table 1 shows specific examples of types of pH indicators and coloration of colonies when sucrose and pH indicators are added as coloring components. Colonies of bacteria that do not decompose are colorless, while colonies of bacteria that decompose sucrose to produce acid, such as Haemophilus influenzae and Haemophilus parahaemolyticus, are colored based on the color change of the pH indicator. Therefore, by observing the coloration of colonies of bacteria grown in the medium of the present invention, it is possible to determine whether or not sucrose is decomposed by the bacteria. Distinguish between Haemophilus haemolyticus (no sucrose decomposition/no hemolysis), Haemophilus haemolyticus (no sucrose decomposition/haemolysis), Haemophilus parahaemolyticus (with sucrose decomposition/no hemolysis) is.

The concentration of the pH indicator added is not particularly limited as long as it is a concentration that can change the color of the colony of bacteria due to the pH change caused by the acid generated by the decomposition of sucrose. Purple 40-120 mg/L, bromothymol blue 40-80 mg/L, phenol red 40-120 mg/L, neutral red 2-4 mg/L, methyl red 40-80 mg/L, and the like.

The enzymatic chromogenic substrate used in the medium of the present invention is not particularly limited as long as it is not decomposed by Haemophilus influenzae but is decomposed by Parainfluenza to develop color. As an enzyme, for example, galactosidase, which is an enzyme that is not expressed in Haemophilus influenzae but is expressed in Parainfluenza, is selected, and a Gal derivative is used as an enzyme chromogenic substrate. Gal derivative decomposed) can be identified with high accuracy. The Gal derivative is not degraded by Haemophilus haemolyticus, but is degraded by Haemophilus parahaemolyticus and develops color. - Parahaemolyticus (with Gal derivative decomposition) can also be identified with high accuracy.

The Gal derivative used in the medium of the present invention is not particularly limited as long as it is a compound to which a chromophore compound or a fluorescent chromophore compound is bound so as to be hydrolyzed by galactosidase. Specific examples of attached chromophore compounds include 5-bromo-4-chloro-3-indoxyl, 5-bromo-6-chloro-3-indoxyl, 5-bromo-3-indoxyl, 4- indoxyl derivatives such as chloro-3-indoxyl, 6-chloro-3-indoxyl, 6-fluoro-3-indoxyl, 3-indoxyl, 5-iodo-3-indoxyl, N-methylindoxyl; Examples include phenyl derivatives such as 2-chloro-4-nitrophenyl, 2-nitrophenyl, 4-nitrophenyl and 4-aminophenyl, naphthyl derivatives such as 6-bromo-2-naphthyl, and the like. Further, specific examples of the fluorescent chromophore compound include quinoline derivatives such as 8-hydroxyquinoline, coumarin derivatives such as 4-methylumbelliferyl and 7-hydroxycoumarin-6-yl, 7-bromo-N-(2- Methoxyphenyl)-3-hydroxy-2-naphthalenecarboxamide (naphthol AS-BI), naphthyl derivatives such as 1-naphthyl and 2-naphthyl, and resorufin.

Table 2 shows specific examples of types of enzyme chromogenic substrates and coloration of colonies when only a Gal derivative, which is an enzyme chromogenic substrate, is added as a chromogenic component. Colonies of bacteria that do not decompose Gal derivatives such as , are colorless. Color accordingly. When using a Gal derivative to which a fluorescent chromophore compound is bound as an enzyme chromogenic substrate, an ultraviolet lamp or the like is used to irradiate the culture medium with ultraviolet light having a wavelength suitable for the fluorophore compound. , Haemophilus influenzae, and Haemophilus haemolyticus, which do not degrade Gal derivatives, do not emit fluorescence, while colonies of Gal derivative-degrading bacteria, such as Parahaemophilus influenzae and Haemophilus parahaemolyticus, exhibit a fluorescent color. . Therefore, by observing the coloration of colonies of bacteria grown in the medium of the present invention, it is possible to determine whether or not the Gal derivative is decomposed by the bacteria. No derivative decomposition/no hemolysis), Haemophilus haemolyticus (no Gal derivative decomposition/hemolysis), Parainfluenzae (with Gal derivative decomposition/no hemolysis), Haemophilus parahaemolyticus (with Gal derivative decomposition/hemolysis) identification is possible.

In the present invention, the coloration of bacterial colonies can be determined visually, but since the medium contains horse blood, the color of the medium is dark reddish brown, which is lower than when a pH indicator or chromophore compound that exhibits red color is used. Colors of colonies can be easily distinguished when pH indicators or chromophoric compounds exhibiting blue, green, purple, yellow, etc. are used. On the other hand, by further adding an insoluble white compound such as titanium oxide to the medium of the present invention, the color of the medium becomes brighter, and the visibility, including red colonies, improves, making discrimination easier. be able to do it. Although the concentration of titanium oxide to be added is not particularly limited, it can be, for example, 1 to 8 g/L.

In the present invention, an enzymatic chromogenic substrate can be used alone, but two or more enzymatic chromogenic substrates can also be used in appropriate combination depending on the purpose of detection. Further, for example, in addition to the enzymatic chromogenic substrate, hydrolyzed by galactosidase such as isopropyl-β-D-1-thiogalactopyranoside (hereinafter referred to as IPTG). An enzyme substrate that does not develop color can also be used as a hydrolysis reaction accelerator. The concentration of the enzyme chromogenic substrate to be added is not particularly limited as long as the coloration of bacterial colonies due to the chromophoric compound or fluorescent chromophoric compound released by hydrolysis by galactosidase derived from bacteria grown in the medium can be visually identified. . Those skilled in the art can set such a concentration as appropriate. Further, 10 to 200 mg/L of IPTG can be added as a hydrolysis reaction accelerator.

As described above, in the present invention, Haemophilus influenzae, Haemophilus haemolyticus, Parainfluenzae and Haemophilus parahaemolyticus can be obtained by adding sucrose and a pH indicator as color-developing components, or by adding only enzyme color-developing substrates as color-developing components. It is possible to identify dregs and detect Haemophilus influenzae, and by using these as coloring components, it is possible to detect Haemophilus influenzae without overlooking it (Examples 1 and 2 shown later). In addition, as in Example 3 described later, by adding a Gal derivative as an enzyme coloring substrate in addition to sucrose and a pH indicator as coloring components, it becomes possible to distinguish between Haemophilus influenzae and Parainfluenzae with higher accuracy. In the examples, only the combination in which the colonies of bacteria that decompose sucrose and the colonies of bacteria that decompose Gal derivatives are both blue in color was shown. In addition, colonies of bacteria that do not decompose the enzyme chromogenic substrate are colorless, while colonies of bacteria that decompose sucrose and colonies of bacteria that decompose the chromogenic substrate are both colored. Any combination of the indicator and the Gal derivative can distinguish Haemophilus influenzae from other Haemophilus species. For example, in the medium of the present invention containing sucrose, neutral red and 5-bromo-3-indolyl-β-D-galactopyranoside as coloring components, sucrose is not decomposed and 5-bromo-3-indolyl - Colonies of bacteria that do not degrade β-D-galactopyranoside are colorless, colonies of bacteria that degrade sucrose and degrade 5-bromo-3-indolyl-β-D-galactopyranoside Color purple, colonies of bacteria that degrade sucrose but not 5-bromo-3-indolyl-β-D-galactopyranoside Color red, not degrade sucrose but 5-bromo-3-indolyl- Colonies of bacteria that degrade β-D-galactopyranoside are blue, so Haemophilus influenzae (white to grayish white, no hemolysis), Haemophilus haemolyticus (white to grayish white, with hemolysis), Parainfluenzae (purple, red) or blue, no hemolysis), and Haemophilus parahaemolyticus (purple, red, or blue, with hemolysis). Further, in the medium of the present invention containing sucrose, aniline blue and 4-methyl-umbelliferyl-β-D-galactopyranoside as coloring components, sucrose is not degraded and 4-methyl-umbelliferyl - Bacterial colonies that do not degrade β-D-galactopyranoside are colorless (no fluorescence), degrade sucrose, and degrade 4-methyl-umbelliferyl-β-D-galactopyranoside The color of the colony of bacteria is blue + fluorescent color, which decomposes sucrose, but does not decompose 4-methyl-umbelliferyl-β-D-galactopyranoside. The colony color of bacteria is blue (no fluorescence), decomposes sucrose. However, the color of colonies of bacteria that decompose 4-methyl-umbelliferyl-β-D-galactopyranoside is colorless + fluorescent color, so Haemophilus influenzae (white to grayish white, no hemolysis), Haemophilus haemolyticus (white to grayish white, with hemolysis), Parainfluenza (blue + fluorescence, blue (no fluorescence), or no coloring + fluorescence, no hemolysis), Haemophilus parahaemolyticus (blue + fluorescence, blue (fluorescence) (no coloring) or no coloring + fluorescent coloring/hemolysis) can be distinguished.

The antibacterial agent contained in the medium of the present invention does not interfere with the detection (screening) of Haemophilus influenzae, the identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus, and does not prevent contaminants other than Haemophilus. It is an antibacterial agent that suppresses growth. Although such an antibacterial agent is not particularly limited, for example, it is preferable to add at least one antibacterial agent having an antibacterial spectrum that inhibits the growth of Gram-positive bacteria. Antibacterial agents with an antibacterial spectrum that inhibits the growth of Gram-positive bacteria are not particularly limited, but specific examples include polypeptide antibacterial agents such as bacitracin (BC), vancomycin (VCM), glycopeptides such as teicoplanin (TEIC). system antibacterial agents and the like. Although only one type of antibacterial agent can be added, it is also possible to add two or more types. It is also possible to add an antibacterial agent other than an antibacterial agent having an antibacterial spectrum that inhibits the growth of Gram-positive bacteria, or to further add an antifungal agent that inhibits the growth of fungi.

For screening of Haemophilus influenzae, the concentration of the antibacterial agent to be added is preferably within a concentration range where Haemophilus influenzae can grow and detection of Haemophilus influenzae is not hindered. On the other hand, for the identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus, it is necessary to identify Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus that are viable and Haemophilus influenzae, It is preferable to add the antibacterial agent in a concentration range that does not interfere with the identification of Haemophilus haemolyticus, Haemophilus parahaemolyticus, and Haemophilus parahaemolyticus. Such a concentration range can be appropriately set by those skilled in the art with reference to the examples of the present invention. For example, when adding bacitracin alone for screening of Haemophilus influenzae, /L, 0.5-40 mg/L if vancomycin is added alone, and 1-800 mg/L bacitracin and 0.5-40 mg/L vancomycin if bacitracin and vancomycin are added together. . In addition, when adding bacitracin for screening of Haemophilus influenzae, it can be 5 to 800 mg / L, more preferably 10 to 800 mg / L, most preferably 10 to 300 mg / L. preferable. When vancomycin is added for screening of Haemophilus influenzae, it can be 1-40 mg/L, more preferably 3-20 mg/L, most preferably 3-10 mg/L. On the other hand, when adding bacitracin alone for identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus, 1 to 2.5 mg/L, when adding vancomycin alone, 0.5 to 10 mg/L, when adding bacitracin and vancomycin together, preferably 1 to 2.5 mg/L bacitracin and 0.5 to 10 mg/L vancomycin; , 0.5-5 mg/L, and when both bacitracin and vancomycin are added, 1-2.5 mg/L bacitracin and 0.5-5 mg/L vancomycin are more preferred.

As described above, the medium of the present invention contains an antibacterial agent for suppressing the growth of contaminating bacteria other than Haemophilus. Even if contaminants grow in spite of this, if the colonies of the grown contaminants are [with sucrose decomposition and/or with hemolysis], it can be easily determined that they are not Haemophilus influenzae. In addition, even if the colony of contaminant bacteria is [no decomposition of sucrose, no hemolysis], if culture using a sheep blood agar medium is performed in parallel with culture using the medium of the present invention, bacteria other than Haemophilus spp. Since the contaminant bacteria of 1 also grow on the sheep blood agar medium, it can be determined that it is not Haemophilus influenzae. Therefore, by confirming the presence or absence of colony color and hemolysis in the bacteria that did not grow on the sheep blood agar medium but grew on the medium of the present invention, detection (screening) of Haemophilus influenzae, Haemophilus influenzae, Haemophilus haemolyticus, Parainfluenzae and Haemophilus parahaemolyticus.

The medium of the present invention contains at least (a) horse blood, (b) β-NAD or a phosphate thereof, (c) an antibacterial agent, and (d) a color-developing component for coloration and paralysis of Haemophilus influenzae colonies. It is a medium in which Haemophilus influenzae can grow, containing a color-developing component having the characteristic of making the coloration of Haemophilus influenzae colonies different colors. The action of the medium of the present invention is that Haemophilus spp. can be identified by the colony color of bacteria grown in the medium and the presence or absence of hemolysis, thereby enabling detection of Haemophilus influenzae. The medium of the present invention containing the above components and having an action can be used for identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus, screening of Haemophilus influenzae, and the like. be. In other words, the medium of the present invention is used as a medium for distinguishing Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus by utilizing the ability to distinguish Haemophilus spp. In addition, it can be used as a screening medium for Haemophilus influenzae by utilizing the effect that Haemophilus influenzae can be detected. That is, the medium of the present invention contains contaminating bacteria other than Haemophilus influenzae (Haemophilus haemophilus, Haemophilus parahaemolyticus, Haemophilus parahaemolyticus, etc.) by adjusting the type, concentration, etc. of the antibacterial agent to be used. ) grows, and the growth of contaminant bacteria other than Haemophilus influenzae is almost suppressed. Therefore, the Haemophilus influenzae screening medium of the present invention includes Haemophilus influenzae detection medium and Haemophilus influenzae selection medium. On the other hand, the medium of the present invention can also be used as a medium for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus.

The basal medium used in the present invention is not particularly limited as long as it is a medium in which Haemophilus bacteria can grow, such as nutrient agar, Muller-Hinton agar, and trypto-soy agar.

If necessary, the medium of the present invention may further contain nutrients, minerals, and other components suitable for the growth of Haemophilus.

The pH range of the medium of the present invention is not particularly limited as long as the Haemophilus bacteria can grow and the pH indicator changes color with the pH change caused by the acid generated by the decomposition of sucrose. Depending on the pH indicator used, for example, when aniline blue is used as the pH indicator, the pH can be about 7-8, preferably 7.0-8.0. Specifically, it can be set to 7.3±0.3, 7.6±0.3, etc., as in Examples shown later.

In the present invention, in order to screen Haemophilus influenzae, specifically, a medium containing the components shown in Table 3 below per 1 L of medium can be used. A medium containing the components shown in Table 4 below is used. is more preferable.

In the medium of the present invention shown in Table 3 above, sodium glutamate (0 to 5 g/L) can be used as nutritional components and mineral components instead of yeast extract and soluble starch. In the medium of , sodium glutamate (0-5 g/L) can be used in place of yeast extract and soluble starch. Further, in the medium of the present invention shown in Table 3, N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (hereinafter referred to as HEPES) is used instead of potassium dihydrogen phosphate and dipotassium hydrogen phosphate. (0 to 10 g/L), 3-morpholinopropanesulfonic acid (hereinafter referred to as MOPS) (0 to 10 g/L), N-(2-acetamido)-2-aminoethanesulfonic acid (hereinafter referred to as ACES) (0 to 10 g/L) of Good's buffer can be used, and in the medium of the present invention shown in Table 4, HEPES (0 to 10 g/ L), MOPS (0-10 g/L), ACES (0-10 g/L), etc. can be used.

On the other hand, in the present invention, in order to identify Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus, specifically, a medium containing the components shown in Table 5 below per 1 L of the medium is used. However, it is more preferable to use a medium containing the components shown in Table 6 below.

In the medium of the present invention shown in Table 5 above, sodium glutamate (0 to 5 g/L) can be used as nutritional components and mineral components instead of yeast extract and soluble starch. In the medium of , sodium glutamate (0-5 g/L) can be used in place of yeast extract and soluble starch. Further, in the medium of the present invention shown in Table 5, instead of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, HEPES (0-10 g / L), MOPS (0-10 g / L), ACES (0-10 g / L), etc., and in the medium of the present invention shown in Table 6, HEPES (0 to 10 g / L), MOPS (0-10 g/L), ACES (0-10 g/L), etc. can be used.

Specimens used in the present invention are not particularly limited as long as the presence of Haemophilus bacteria such as Haemophilus influenzae is suspected. Specific examples include human pharyngeal swab, nasopharyngeal swab, cerebrospinal fluid, otorrhea, and eye discharge.

The form of the medium of the present invention is not particularly limited, as long as it is a form in which the presence or absence of hemolysis can be confirmed, such as a solid medium or a sheet medium. is more preferred.
Examples of solidifying agents for solid media include those commonly used such as agar and agarose.

Examples of the present invention are shown below, but the present invention is not limited to these examples.

(Detection of Haemophilus influenzae using medium containing sucrose and pH indicator as coloring components)
Haemophilus influenzae, Parainfluenzae, and Escherichia coli were inoculated into a medium containing sucrose and aniline blue as color-developing components, and the ability of the medium to detect Haemophilus influenzae was investigated. The strain names used are listed in Table 11.

(1) Preparation of medium Weigh the medium components 1 shown in Table 7 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agent After autoclave, after cooling the medium to 50°C, pre-filter sterilized β-NAD, defibrinated horse blood, bacitracin and vancomycin are added in Table 8 below. Added to the concentrations indicated. After that, 20 mL of the medium was dispensed into petri dishes and solidified.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results Table 11 shows the results. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

(Detection of Haemophilus influenzae using a medium containing a galactopyranoside derivative as a coloring component)
A medium containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as a coloring component was inoculated with Haemophilus influenzae, Parainfluenza, and Escherichia coli, and the ability of the medium to detect Haemophilus influenzae was examined. The strain names used are listed in Table 11.

(1) Preparation of medium Weigh the medium components 2 shown in Table 9 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agent Same as in Example 1.

(3) Inoculation and Culture of Bacteria Same as in Example 1.

(4) Results Table 11 shows the results. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

(Detection of Haemophilus influenzae using medium containing sucrose, pH indicator and galactopyranoside derivative as coloring components)
A medium containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as coloring components was inoculated with Haemophilus influenzae, Parainfluenza and Escherichia coli to detect Haemophilus influenzae in the medium. examined performance. The strain names used are listed in Table 11.

(1) Preparation of medium Weigh the medium components 3 shown in Table 10 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agent Same as in Example 1.

(3) Inoculation and Culture of Bacteria Same as in Example 1.

(4) Results The results are shown in Table 11 below. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

As shown in Table 11, in the medium of Example 1, colonies of all tested Haemophilus influenzae colonies were grayish white indicating no degradation of sucrose, and no hemolysis was observed. In addition, in the medium of Example 2, the colony color of all tested Haemophilus influenzae was grayish white, indicating no degradation of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and hemolysis was observed. In the medium of Example 3, the colony color of all H. influenzae tested showed no degradation of sucrose and no degradation of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. It was an off-white color and no hemolysis. From this, a medium containing only sucrose and aniline blue as coloring components (Example 1) and a medium containing only 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as coloring components (Example 2), using any medium containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Example 3) as coloring components, It was suggested that it is possible to detect Haemophilus influenzae without overlooking it. Furthermore, in the medium of Example 3, the color of all parainfluenza colonies tested indicates the presence of sucrose decomposition and/or the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside decomposition. The colony color of E. coli was blue without hemolysis, and the colony color was blue without hemolysis. It was shown that media containing nosides were free of false positives and could discriminate between Haemophilus influenzae and other bacteria with higher accuracy.

(Distinguishing Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus)
Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus in a medium containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as chromogenic components was inoculated to examine the performance of the medium. The strain names used are listed in Table 14.

(1) Preparation of medium Weigh the medium components 4 shown in Table 12 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agents After autoclaving, the medium was cooled to 50°C and then filtered sterilized β-NAD, defibrinated horse blood, bacitracin (BC) and vancomycin (VCM). was added at concentrations shown in Table 13 below. After that, 20 mL of the medium was dispensed into petri dishes and solidified.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results The results are shown in Table 14 below. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

In media supplemented with bacitracin 2.5 mg/L and/or vancomycin 5 mg/L, Haemophilus influenzae (grey white, no hemolysis), Haemophilus haemolyticus (grey white, no hemolysis), Haemophilus influenzae (blue, no hemolysis), Haemophilus All of the parahaemolyticus (blue, with hemolysis) grew well, and it was shown that these strains can be distinguished by colony color and the presence or absence of hemolysis. On the other hand, in the medium to which 10 mg/L of vancomycin was added alone, Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus could be distinguished by colony color and the presence or absence of hemolysis, but Haemophilus parahaemolyticus The growth of dregs was weak. In addition, as shown in Table 14, it was possible to detect Haemophilus influenzae depending on colony color and the presence or absence of hemolysis in media with any of the concentrations of antibacterial agents examined.

(Study of pH indicator)
Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae, and Haemophilus parahaemolyticus were inoculated using aniline blue or phenol red as a pH indicator in a medium containing only sucrose and a pH indicator as color-developing components, and the performance of the medium was investigated. rice field. The strain names used are listed in Table 16.

(1) Preparation of medium
The procedure was the same as in Example 1, except that 100 mg/L of aniline blue or 120 mg/L of phenol red was used as the pH indicator.

(2) Addition of β-NAD, horse blood and antibacterial agent After autoclave, after cooling the medium to 50°C, pre-filter sterilized β-NAD, defibrinated horse blood, bacitracin and vancomycin are added in Table 15 below. Added to the concentrations indicated. After that, 20 mL of the medium was dispensed into petri dishes and solidified.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results Results are shown in Table 16 below. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

Both media using aniline blue and phenol red as pH indicators can distinguish between Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus by colony color and the presence or absence of hemolysis. there were.

(Study of antibacterial agents and comparison with conventional methods)
In a medium containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as color-developing components, different types of antibacterial agents were used to treat Haemophilus influenzae, Haemophilus haemolyticus, and parasites. Haemophilus influenzae, Haemophilus parahaemolyticus and other in-house stocks were inoculated to examine the performance of the medium. Furthermore, Poremedia (registered trademark) sheep blood agar medium (Eiken chemical medium), conventional method A medium (Eiken chemical medium) and conventional method B medium (Becton, Bacitracin-added medium manufactured by Dickinson) was prepared, and the same bacteria were inoculated and compared. The strain names used are listed in Table 18.

(1) Preparation of culture medium of the present invention The same as in Example 4 was used.

(2) Addition of β-NAD, horse blood, and antibacterial agent to the medium of the present invention After autoclaving, the medium was cooled to 50°C, and then filtered sterilized β-NAD, horse defibrinated blood, and bacitracin (BC). ) and vancomycin (VCM) were added at the concentrations shown in Table 17 below. After that, 20 mL of the medium was dispensed into petri dishes and solidified.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. After that, the bacterial solution was streaked on the medium prepared in (1) and (2) and each medium for comparison (sheep blood agar medium, conventional method A medium, conventional method B medium), at 35 ° C. for 20 hours, Cultivation was performed in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined.

(4) Results Results are shown in Table 18 below. Results from the medium of the present invention are indicated by the color of the colonies, with the colony colors indicating the presence of hemolysis and the results indicating the presence of hemolysis as -. there is

In the medium of the present invention, when bacitracin alone, when vancomycin alone is used, and when both bacitracin and vancomycin are added as antibacterial agents, Haemophilus influenzae, Haemophilus haemolyticus, and the presence or absence of colony color and hemolysis are observed. , Parainfluenza and Haemophilus parahaemolyticus, and detection of Haemophilus influenzae. On the other hand, the colonies grown on conventional method A medium and B medium were both grayish white, and there was no means for distinguishing between Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus other than morphology. From this, it was shown that the medium of the present invention can detect Haemophilus influenzae remarkably easily and accurately as compared with the conventional method.
In addition, in the medium of the present invention, when bacitracin alone is used as an antibacterial agent, when vancomycin alone is used, and when bacitracin and vancomycin are both added, a certain inhibitory effect against contaminating bacteria other than Haemophilus is used. Among the tested media, the medium of the present invention supplemented with 300 mg/L of bacitracin and 10 mg/L of vancomycin best inhibits the growth of contaminants, and can detect Haemophilus influenzae very easily and accurately. It has been shown to be possible. Some contaminant bacteria were observed to grow in the medium of the present invention, but since the contaminant bacteria also grew on the sheep blood agar medium, it could be easily determined that they were not Haemophilus spp.

(Study using pharyngeal swab sample)
The pharynx of 5 healthy subjects was scraped with a cotton swab and inoculated into the medium of the present invention containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as coloring components. After culturing with , the grown colonies were picked and identified by XV Multidisc 'Eiken' (registered trademark) manufactured by Eiken Chemical Co., Ltd. and API (registered trademark) NH manufactured by Sysmex bioMerieux. In addition, the method of the identification test followed each package insert.

(1) Preparation of culture medium of the present invention The same as in Example 4 was used.

(2) Addition of β-NAD, horse blood and antibacterial agent Same as in Example 1.

(3) Specimen inoculation and culture After scraping the pharynx of 5 healthy subjects with a cotton swab, they were inoculated into the medium prepared in (1) and (2) and cultured at 35°C for 20 hours in a 5% carbon dioxide environment. was performed to examine the color of bacterial colonies and the presence or absence of hemolysis.

(4) Results Table 19 shows the results of identification by Eiken Chemical's XV Multidisc, and Table 20 shows the results of identification by Sysmex bioMerieux's Api NH.

All the grayish-white colonies in the medium of the present invention had the same colony morphology and no hemolysis, and all the blue-colored colonies had no hemolysis (data not shown). Among grayish white colonies without hemolysis and blue colonies without hemolysis, one colony that grew predominantly was picked as a representative and identified by XV Multidisc. It was confirmed to be Haemophilus influenzae because of XV auxotrophy. On the other hand, blue colonies without hemolysis were V-auxotrophic and thus were confirmed to be Parainfluenzae (Table 19). Similarly, among the grayish white colonies with no hemolysis and the blue colonies with no hemolysis, one colony that grew predominantly was identified as a representative by API NH manufactured by Sysmex bioMerieux. It was identified as Haemophilus influenzae with 99.9% confidence, and the blue colored colonies were identified as Haemophilus influenzae with 99.1% confidence (Table 20). From this, it was suggested that the medium of the present invention can distinguish between Haemophilus influenzae and Parainfluenzae with high accuracy by primary isolation culture and detect Haemophilus influenzae.

(Examination of sucrose addition concentration)
Haemophilus influenzae, Parainfluenza, and Haemophilus haemolyticus were inoculated into a medium containing sucrose and aniline blue as color-developing components, and the sucrose concentration capable of distinguishing these strains was examined. The strain names used are listed in Table 23.

(1) Preparation of medium Weigh the medium components 5 shown in Table 21 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agent After autoclave, after cooling the medium to 50°C, pre-sterilized β-NAD, defibrinated horse blood, bacitracin and vancomycin were added to Table 22 below. Added to the concentrations indicated. After that, 20 mL of the medium was dispensed into petri dishes and solidified.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results The results are shown in Table 23. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

As shown in Table 23, it is possible to distinguish between Haemophilus influenzae, Parainfluenzae, and Haemophilus haemolyticus by colony color and the presence or absence of hemolysis in a sucrose concentration range of 1 to 40 g/L. It was possible.

(Examination of aniline blue addition concentration)
Haemophilus influenzae, Parainfluenza, and Haemophilus haemolyticus were inoculated into a medium containing sucrose and aniline blue as color-developing components, and the concentration of aniline blue capable of distinguishing these strains was examined. The strain names used are listed in Table 25.

(1) Preparation of medium Weigh the medium components 6 shown in Table 24 below, add β-NAD, horse blood and antibacterial agents in the following (2), and add purified water so that the medium volume becomes 1 L. , and autoclaved at 121°C for 15 minutes.

(2) Addition of β-NAD, horse blood and antibacterial agent Same as in Example 8.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results Table 25 shows the results. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

As shown in Table 25, it was possible to distinguish between Haemophilus influenzae, Haemophilus influenzae, and Haemophilus haemolyticus by colony color and the presence or absence of hemolysis in the range of aniline blue addition concentrations of 40 to 400 mg/L. was possible.

(Examination of buffering agent)
In a medium containing sucrose, aniline blue and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside as chromogenic components, phosphate buffer, HEPES, MOPS or ACES as buffer components, influenza The performance of the medium was investigated by inoculating the bacteria, Parainfluenzae and Haemophilus haemolyticus. The strain names used are listed in Table 26.

(1) Preparation of culture medium The same as in Example 4, except that the buffer components are potassium dihydrogen phosphate 1 g/L, dipotassium hydrogen phosphate 4 g/L, HEPES 5 g/L, MOPS 5 g/L, or ACES 5 g/L. and

(2) Addition of β-NAD, horse blood and antibacterial agent Same as in Example 1.

(3) Inoculation and Cultivation of Bacteria Each precultured strain was suspended in sterilized physiological saline to prepare a McFarland No. 1 bacterial solution. Thereafter, the bacterial solution was streaked on the medium prepared in (1) and (2), cultured at 35° C. for 20 hours in a 5% carbon dioxide gas environment, and the colony color of the bacteria and the presence or absence of hemolysis were examined. .

(4) Results Table 26 shows the results. The presence of hemolysis is indicated by +, and the absence of hemolysis is indicated by -, which are indicated together with colony color.

In media containing any of phosphate buffer, HEPES, MOPS and ACES as buffer components, Haemophilus influenzae, Haemophilus influenzae, and Haemophilus haemolyticus can be well distinguished by colony color and the presence or absence of hemolysis. Bacteria were well detectable.

The method for detecting Haemophilus influenzae and the medium used therefor of the present invention merely confirms the colony color of the bacteria grown in the medium and the presence or absence of hemolysis, and detects Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus para by primary isolation culture. Allows easy identification of haemolyticus. Compared to conventional methods, it has become possible to detect Haemophilus influenzae much more easily and accurately. Significant reduction is possible. Therefore, the method and culture medium of the present invention are useful in clinical practice, epidemiological research, and a wide range of other fields.

Claims (14)

After inoculating and culturing the specimen in a medium capable of growing Haemophilus influenzae containing at least all of the following (a), (b), (c) and (d1), the bacterial colony color and hemolysis A method for screening Haemophilus influenzae by primary isolation culture , characterized by confirming the presence or absence of the strain.
(a) non-hemolyzed horse blood;
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d1) sucrose and aniline blue . 2. The screening method for Haemophilus influenzae according to claim 1, wherein said medium further contains (d2).
(d2) 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-3-indolyl-β-D-galactopyranoside and 3-indolyl-β-D-galacto An enzymatic chromogenic substrate selected from the group consisting of pyranosides . 3. The screening method for Haemophilus influenzae according to claim 2, wherein the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria. Inoculate the specimen into a medium containing at least all of the following (a), (b), (c) and (d1) in which Haemophilus influenzae can grow, and after culturing, confirm the colony color of the bacteria and the presence or absence of hemolysis. A method for identifying Haemophilus influenzae, Haemophilus haemolyticus , Haemophilus parahaemolyticus and Haemophilus parahaemolyticus by primary isolation culture .
(a) non-hemolyzed horse blood;
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d1) sucrose and aniline blue . 5. The method for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus according to claim 4, wherein said medium further contains (d2).
(d2) 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-3-indolyl-β-D-galactopyranoside and 3-indolyl-β-D-galacto An enzymatic chromogenic substrate selected from the group consisting of pyranosides . 6. The method for distinguishing Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus according to claim 5, wherein the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria. A primary isolation culture medium for screening Haemophilus influenzae, characterized by containing all of the following (a), (b), (c) and (d1) in a medium in which Haemophilus influenzae can grow.
(a) non-hemolyzed horse blood;
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d1) sucrose and aniline blue . 8. The primary isolation culture medium for screening Haemophilus influenzae according to claim 7, further comprising (d2).
(d2) 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-3-indolyl-β-D-galactopyranoside and 3-indolyl-β-D-galacto An enzymatic chromogenic substrate selected from the group consisting of pyranosides . 9. The primary isolation culture medium for screening Haemophilus influenzae according to claim 8, wherein the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria. A primary isolation culture medium for screening Haemophilus influenzae, characterized by containing at least all of the following components per liter of the medium.
(a) 10-100 mL of non-hemolyzed horse blood,
(b) β-NAD 1-100 mg,
(c) bacitracin 1-800 mg and/or vancomycin 0.5-40 mg,
(d) 1-40 g sucrose and 40-400 mg aniline blue, or 1-40 g sucrose, 40-400 mg aniline blue and 40-300 mg 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza and Haemophilus parahaemolyticus, characterized by containing all of the following (a), (b), (c) and (d1) in a medium in which Haemophilus influenzae can grow Primary isolation culture medium for identification of dregs.
(a) non-hemolyzed horse blood;
(b) β-NAD or its phosphate;
(c) an antimicrobial agent;
(d1) sucrose and aniline blue . 12. The primary isolation culture medium for identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus according to claim 11, further comprising (d2).
(d2) 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-bromo-3-indolyl-β-D-galactopyranoside and 3-indolyl-β-D-galacto An enzymatic chromogenic substrate selected from the group consisting of pyranosides . Primary isolation culture for identification of Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus influenzae and Haemophilus parahaemolyticus according to claim 12, wherein the antibacterial agent is an antibacterial agent that suppresses the growth of Gram-positive bacteria. Culture medium. A primary isolation culture medium for identifying Haemophilus influenzae, Haemophilus haemolyticus, Haemophilus parainfluenza, and Haemophilus parahaemolyticus, characterized by containing at least all of the following components per liter of the medium.
(a) 10-100 mL of non-hemolyzed horse blood,
(b) β-NAD 1-100 mg,
(c) bacitracin 1-2.5 mg and/or vancomycin 0.5-10 mg,
(d) 1-40 g sucrose and 40-400 mg aniline blue, or 1-40 g sucrose, 40-400 mg aniline blue and 40-300 mg 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.