Culture medium for detection of listeria

Abstract

Provided is a simple and specific means for detecting Listeria monocytogenes and / or Listeria ivanovii. A culture medium for detecting Listeria monocytogenes and / or Listeria ivanovii contains the following components (A), (B), and (C): (A) a chromogenic substance that develops color by being decomposed with β-glucosidase or a fluorogenic substance that emits fluorescence; (B) a chromogenic substance that develops color by being decomposed with phosphatidylinositol-specific phospholipase C or a fluorogenic substance that emits fluorescence; and (C) a sugar.

Description

Technical Field

[0001] This invention relates to a culture medium and detection method for the simple and specific detection of Listeria monocytogenes and Listeria ivanovii. Background Technology

[0002] Bacteria belonging to the genus Listeria are Gram-positive, facultative anaerobic, non-spore-forming short rod-shaped bacteria. Currently, the following species exist: Listeria monocytogenes (hereinafter referred to as L.), Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria seeligeri, Listeria grayi, Listeria marthii, Listeria rocourtiae, Listeria fleischmannii, Listeria weihenstephanensis, Listeria aquatica, Listeria cornellensis, Listeria floridensis, Listeria grandensis, and Listeria riparia. Of these, only *Listeria monocytogenes* is typically pathogenic to humans, and it is the primary target for food inspection. However, food is frequently contaminated with several other *Listeria* species, including *Listeria innocense*, making differentiation from these bacteria important (Non-Patent Literature 1). Furthermore, *Listeria monocytogenes* is known to cause zoonotic diseases and is a target for environmental inspection in the United States.

[0003] The standard test method for Listeria spp. in Japan includes qualitative and quantitative tests. In the qualitative test, the sample is cultured once in half-Fraser liquid medium, then cultured a second time in Fraser liquid medium, and Listeria spp. is isolated using selective isolation medium containing enzyme substrate (enzyme substrate medium). Simultaneously, the following method is also performed: without secondary enrichment culture, colonies obtained from the first enrichment culture are inoculated onto enzyme substrate medium. On the other hand, in the quantitative test, Listeria spp. are isolated using enzyme substrate medium after resuscitation culture in BPW medium (20±2℃, 1 hour±5 minutes). Methods for identifying Listeria monocytogenes after culturing in enzyme substrate medium in both qualitative and quantitative methods include carbohydrate metabolism assays and cAMP assays, but these require time due to the need for culturing. Therefore, as auxiliary confirmation methods for Listeria monocytogenes, PCR and rapid diagnostic methods using immunodiagnostic methods (Non-Patent Literature 1) are known. For example, as a means of identifying species belonging to the genus Listeria, a detection method based on the LAMP method, which is a type of PCR, is known (Patent Document 1). However, this LAMP method uses four species-specific primers and is not a method that can be used in routine laboratory settings.

[0004] As a method for identifying species belonging to the genus Listeria, as mentioned above, the use of selective isolation media is relatively simple. Such selective isolation media include ALOA (registered trademark) agar (Sysmex-biomerieux Japan Ltd.) and CHROMagar (registered trademark) Listeria (CHROMagar Corporation), which use a chromogenic substance that is broken down by β-glucosidase to produce color. When Listeria monocytogenes is inoculated onto the enzyme substrate medium, typical blue-green colonies with a milky-white halo appear; other Listeria species do not produce a halo. Similarly, Patent Document 2 is known as a medium using a chromogenic substance that is broken down by β-glucosidase to produce color. Furthermore, a medium containing a chromogenic substrate specifically cleaved by phosphatidylinositol-specific phospholipase C (PIPL C) is known (Patent Document 3).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Application Publication No. 2007-61061

[0008] Patent Document 2: Japanese Patent Application Publication No. 2012-130274

[0009] Patent Document 3: Japanese Patent Publication No. 2001-510054

[0010] Non-patent literature

[0011] Non-patent literature 1: Food hygiene inspection guidelines (microbiology edition) 2015, Japan Food Hygiene Association, pp. 340-363 (2015). Summary of the Invention

[0012] The technical problem to be solved by the present invention

[0013] However, using the enzyme substrate culture medium and the selective isolation media of Patent Documents 2 and 3, it is difficult to clearly distinguish between Listeria monocytogenes and Listeria elodeon in the Listeria genus. Therefore, confirmation tests using colonies obtained in the enzyme substrate culture medium are required, which is time-consuming, laborious, and relatively complicated.

[0014] Therefore, the technical problem of the present invention is to provide a simple and specific means for detecting Listeria monocytogenes and / or Listeria escherichiae as a cause of pathogenicity to humans or food contamination.

[0015] Technical means to solve technical problems

[0016] Therefore, the inventors of this application conducted various studies to solve the aforementioned technical problem and discovered that by adding a chromogenic substance or fluorescein substance that is decomposed by β-glucosidase and a chromogenic substance or fluorescein substance that is decomposed by phosphatidylinositol-specific phospholipase C (PIPL C), and simultaneously using sugar, it is possible to clearly identify the color or fluorescence of Listeria monocytogenes and Listeria monocytogenes colonies, rather than the color change around the colonies, thereby completing the present invention.

[0017] That is, the present invention provides the following inventions [1] to [7].

[0018] [1] A culture medium for detecting Listeria monocytogenes and / or Listeria erythrozoonii, comprising the following components (A), (B) and (C):

[0019] (A) A chromogenic substance that develops color by being broken down by β-glucosidase or a fluorescein that emits fluorescence;

[0020] (B) Chromogenic substances that develop color through degradation by phosphatidylinositol-specific phospholipase C, or fluorescein substances that emit fluorescence; and

[0021] (C) Sugar.

[0022] [2] The culture medium for detecting Listeria monocytogenes and / or Listeria eloides as described in [1], wherein the sugar in component (C) is glucose.

[0023] [3] The culture medium for detecting Listeria monocytogenes and / or Listeria eloides as described in [1] or [2] contains sugar of component (C) at a concentration of 2 g / L to 30 g / L at the time of detection.

[0024] [4] A culture medium for detecting Listeria monocytogenes and / or Listeria estradioli according to any one of [1] to [3], wherein component (A) is 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside.

[0025] [5] A culture medium for detecting Listeria monocytogenes and / or Listeria estradiol according to any one of [1] to [3], wherein component (A) is 4-methylumbelliferyl-β-D-glucopyranoside.

[0026] [6] The culture medium for detecting Listeria monocytogenes and / or Listeria estradiolii according to any one of [1] to [5], wherein the medium is supported or stacked on a fibrous absorbent sheet and contains a composition further comprising (D) a gelling agent and (E) bacterial nutrients other than component (C).

[0027] [7] A method for detecting Listeria monocytogenes and / or Listeria erythrozoonii, characterized in that, after inoculating the sample into the culture medium described in any one of [1] to [6] and culturing it, the color or fluorescence of the colonies produced on the culture medium is detected.

[0028] Invention Effects

[0029] By using the culture medium of this invention, bacteria belonging to the genus Listeria can be selectively detected. Among Listeria species, *Listeria monocytogenes* and / or *Listeria escherichiae* can be clearly distinguished from other Listeria species by colony color or fluorescence. Furthermore, since confirmatory tests for *Listeria monocytogenes* and / or *Listeria escherichiae* are not required, the effort and time required for testing can be reduced. Detailed Implementation

[0030] The culture medium for detecting Listeria monocytogenes and / or Listeria erythrozoonii of the present invention contains the following components (A), (B) and (C).

[0031] (A) Chromogenic substances that develop color through degradation by β-glucosidase, or fluorescein substances that emit fluorescence.

[0032] (B) Chromogenic substances that develop color through degradation by phosphatidylinositol-specific phospholipase C, or fluorescein substances that emit fluorescence, and

[0033] (C) Sugar.

[0034] Component (A) releases a free group present in Listeria spp. that can be detected by β-glucosidase, thereby causing it to develop color and being used to detect Listeria spp. colonies. Examples of this free group include chromogenic compounds and fluorescent compounds. Specific β-glucosidase substrates include 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-β-glucopyranoside, blue), 5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside (MAGENTA-β-glucopyranoside, purple-red), 5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside (purple-red), 6-chloro-3-indolyl-β-D-glucopyranoside (powder), 5-bromo-3-indolyl-β-D-glucopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, o-nitrophenyl- β-D-glucopyranoside, phenyl-β-D-glucopyranoside, 3-nitrophenyl-β-D-glucopyranoside, 4-nitrophenyl-β-D-glucopyranoside, 3-indolephenol-β-glucopyranoside trihydrate, n-heptyl-β-D-glucopyranoside, 5-bromo-4-chloro-3-indole-N-acetyl-β-D-glucosinolate, etc., among which, based on the detectability of Listeria monocytogenes, 5-bromo-4-chloro-3-indole-β-D-glucopyranoside (X-β-glucopyranoside) and 4-methylumbelliferyl-β-D-glucopyranoside are preferred. The concentration of the β-glucosidase substrate having a detectable free group is preferably 0.001 g / L or more and 5 g / L or less, particularly preferably 0.01 g / L or more and 0.25 g / L or less, and even more preferably 0.02 g / L or more and 0.15 g / L or less, as measured at the detection concentration.

[0035] Component (B) releases a free radical present in Listeria bacteria that can be detected by PIPL C, thereby causing it to develop color and being used to detect colonies of Listeria monocytogenes and / or Listeria estradiol. Examples of this free radical include chromogenic compounds and fluorescent compounds. Examples of PIPL C substrates include compounds described in Japanese Patent No. 5711218, preferably phosphoinositol having chromophores such as 5-bromo-4-chloro-1H-indole-3-yl, 5-bromo-6-chloro-1H-indole-3-yl, and 6-chloro-1H-indole-3-yl. These compounds are preferably used in the form of sodium, potassium, or ammonium salts. Preferred commercially available products include Aldol 514 (registered trademark) phosphoinositol ammonium salt (Biosynth Corporation), and Aldol 495 (registered trademark) phosphoinositol ammonium salt (Biosynth Corporation). The concentration of component (B), as measured by the concentration at the time of detection, is preferably 0.001 g / L or more and 5 g / L or less, more preferably 0.05 g / L or more and 2 g / L or less, and even more preferably 0.1 g / L or more and 1 g / L or less.

[0036] The sugar component (C) is not particularly limited as long as it can be utilized by Listeria spp., and is preferably glucose, fructose, galactose, mannose, mannitol, sorbitol, eurythritol, inositol, arabinose, xylose, rhamnose, adapalene, etc. From the perspective of the sugar metabolism capacity of Listeria monocytogenes and Listeria escherichiae, glucose is more preferred. The sugar component (C) has the function of making the color or fluorescence of Listeria monocytogenes and Listeria escherichiae colonies clear, making the identification of Listeria monocytogenes and Listeria escherichiae easy and accurate. From the perspective of making the colony color clear and the growth of Listeria spp. bacteria, the content of component (C), measured at the concentration at the time of detection, is preferably 2 g / L to 30 g / L. From the perspective of making the colony color clear, the concentration of component (C) is more preferably 5 g / L to 30 g / L, and even more preferably 5 g / L to 20 g / L.

[0037] To further improve the selectivity of Listeria species, the culture medium of the present invention preferably contains one or more selected from moxalactam and colistin sulfate, more preferably containing both moxalactam and colistin sulfate. Based on the selectivity of Listeria species, the moxalactam content, calculated at the concentration at the time of detection, is preferably 0.001 g / L to 0.5 g / L, more preferably 0.005 g / L to 0.3 g / L, and even more preferably 0.01 g / L to 0.2 g / L. Based on the selectivity of Listeria species, the colistin sulfate content is preferably 0.001 g / L to 0.5 g / L, more preferably 0.005 g / L to 0.2 g / L, and even more preferably 0.005 g / L to 0.1 g / L.

[0038] In addition to the aforementioned components, the culture medium of the present invention may be mixed with other cell nutrients, inorganic salts, sugars, and pH adjusters besides component (C). Examples of cell nutrients include peptone, yeast extract, animal meat extract, and fish meat extract.

[0039] Here, as inorganic salts, examples include inorganic acid metal salts such as sodium chloride and sodium thiosulfate; and organic acid metal salts such as ferric ammonium citrate and sodium citrate. Other inorganic salts include bile powder, sodium cholate, and sodium deoxycholate. As sugars, monosaccharides and oligosaccharides can be used, such as lactose, sucrose (white sugar), xylose, cellobiose, and maltose.

[0040] The form of the culture medium of the present invention is not particularly limited. In addition to the usual agar medium, it can also be prepared as a simple sheet-like culture medium (Japanese Patent Application Laid-Open No. 57-502200, Japanese Patent Application Laid-Open No. 6-181741, and, for example, a structure supported on a fibrous absorbent sheet with mesh (Japanese Patent Application Laid-Open No. 9-19282, Japanese Patent Application Laid-Open No. 2000-325072)). Among these, it is preferable to prepare a simple culture medium in which the composition is supported or layered on a fibrous absorbent sheet. In addition to the components (A) to (C), latamoxef and colistin sulfate as needed, the composition also contains (D) a gelling agent and (E) bacterial nutrients other than component (C).

[0041] Here, water-soluble gelling agents can be listed as examples, that is, polymers that are easily dispersed in water and gel. Specifically, pectin, guar gum, xanthan gum, tamarind gum, locust bean gum, gellan gum, gum arabic, ark sylvestris gum, carrageenan gum, carboxymethyl cellulose, etc. Among these, pectin, guar gum, and xanthan gum are preferred, especially xanthan gum, considering factors such as dispersibility in water and gel stability.

[0042] The fibrous absorbent sheets used in this invention need to allow the inoculated test solution to diffuse easily through capillary action. Examples include synthetic fiber nonwoven fabrics such as rayon nonwoven fabrics and natural fiber nonwoven fabrics such as cotton nonwoven fabrics. The shape of these sheets is not particularly limited and can be any of square, rectangular, or circular shapes. Their size is also not particularly limited, but for ease of testing, a major diameter of 1 cm to 15 cm is preferred. Furthermore, the mesh size is preferably 15 to 100 meshes, particularly preferably 20 to 50 meshes, and the thickness is preferably 10 μm to 1000 μm, particularly preferably 50 μm to 600 μm.

[0043] It is preferable to place such a fibrous absorbent sheet on a waterproof plate. The waterproof plate can be made of any waterproof material, such as plastic or glass, but it is preferable to make it transparent for external observation.

[0044] Examples of samples that can be tested using the culture medium of this invention include dairy products, processed meat products, salads, ready-to-eat foods, environmental samples (water, soil, etc.), and clinical samples (human, bovine, mutton, swine, goat, etc.). Alternatively, culture media that have been pre-cultured in Trypto-Soya broth or enrichment media can also be used as samples.

[0045] To detect Listeria monocytogenes and / or Listeria escherichiae using the culture medium of this invention, samples can be inoculated into the medium and cultured, and the staining, fluorescence, and other characteristics of the colonies can be observed. Culture is typically performed at 25–35°C for 24–72 hours.

[0046] To detect Listeria monocytogenes and / or Listeria escherichiae using the simplified culture medium of this invention, the test solution is inoculated onto the surface of the medium. In this case, the test solution readily diffuses through the capillary action of the three-dimensional voids between the pores, causing swelling and gelation. Listeria monocytogenes and Listeria escherichiae in the test solution are captured, their free movement is inhibited, and colonies form through culture. Therefore, the formed colonies can be easily observed simply by observing the surface of the culture medium. By quantitatively inoculating the sample onto the simplified culture medium, the bacterial count can be easily calculated by counting the colonies that appear after culture.

[0047] In addition, bacterial cell inoculation is usually carried out using a pipette or similar method to inoculate a certain amount, but it can also be done by stamping individuals with high water content or by immersing them in the sample solution. Furthermore, the culture after inoculation can be carried out statically or during transportation.

[0048] Example

[0049] The following examples illustrate the invention in detail, but the invention is not limited to these examples.

[0050] Example 1 (Agar medium containing chromogenic substrate)

[0051] The composition of the culture medium is shown in Table 1. The composition of this culture medium is added to 1 liter of purified water, autoclaved at 121°C for 15 minutes, cooled to about 50°C, and then aseptically dispensed into 20 mL of each plastic petri dish (90 mm in diameter). The mixture is allowed to stand until the culture medium solidifies to prepare the culture medium of the present invention.

[0052] In addition, culture media with glucose concentrations of 0, 2, 5, 20, and 30 g / L as shown in Table 1 were prepared in the same manner.

[0053] [Table 1]

[0054] composition g / L meat peptone 18.00g Casein peptone 6.00g yeast extract 10.00g Anhydrous magnesium sulfate 0.50g Magnesium glycerol phosphate 1.0g Sodium chloride 5.0g Lithium chloride 10.0g disodium hydrogen phosphate 2.50g Colistin sulfate 0.01g Latamoxef 0.02g Agar 15.00g silicon dioxide 10.00g <![CDATA[Aldol514 TM Inositol phosphate, ammonium salt (Biosynth) 0.30g 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside (Biosynth) 0.05g glucose 10.00g

[0055] The test strains were pre-cultured in sheep blood agar for 24 hours, and the colonies were suspended in sterile physiological saline to prepare bacterial suspensions. Each bacterial suspension was then inoculated by streak plating and inoculation loop. The color development of the colonies after incubation at 37°C for 44 hours is shown in Table 2.

[0056]

[0057] According to Table 2, by using agar medium containing 2 g / L to 30 g / L glucose (C) in addition to components (A) and (B), it is possible to clearly distinguish the colonies of Listeria monocytogenes and Listeria eeris and other Listeria species.

[0058] Example 2 (Agar medium containing fluorescent substrate)

[0059] The composition of the culture medium is shown in Table 3. The composition of this culture medium is added to 1 liter of purified water, autoclaved at 121°C for 15 minutes, cooled to about 50°C, and then aseptically dispensed into 20 mL of each of the following: plastic petri dishes (90 mm in diameter). The mixture is allowed to stand until the culture medium solidifies to prepare the culture medium of the present invention.

[0060] [Table 3]

[0061] composition g / L meat peptone 18.00g Casein peptone 6.00g yeast extract 10.00g Anhydrous magnesium sulfate 0.50g Magnesium glycerol phosphate 1.0g Sodium chloride 5.0g Lithium chloride 10.0g disodium hydrogen phosphate 2.50g Colistin sulfate 0.01g Latamoxef 0.02g Agar 15.00g silicon dioxide 10.00g <![CDATA[Aldol514 TM Inositol phosphate, ammonium salt (Biosynth) 0.30g 4-Methylumbelliferone-β-D-glucopyranoside (Biosynth) 0.1g glucose 10.00g

[0062] The test strains were pre-cultured in sheep blood agar for 24 hours, and the colonies were suspended in sterile physiological saline to prepare bacterial suspensions. Each bacterial suspension was then inoculated by streak plating and inoculation loop. The color development and fluorescence of the colonies after incubation at 37°C for 44 hours are shown in Table 4.

[0063] [Table 4]

[0064] strain Colony staining Fluorescence Listeria monocytogenes ATCC15313 red + Listeria monocytogenes NS5168 red + Listeria monocytogenes NS5169 red + Listeria monocytogenes NS5170 red + Listeria monocytogenes JCM7681 red - Listeria eluti NS5167 red - Listeria innocosis NS5166 White + Listeria welris NS5172 White +

[0065] According to Table 4, by using an agar medium containing 10 g / L glucose (C) in addition to components (A) and (B), it is possible to clearly distinguish the colonies of Listeria monocytogenes and Listeria eeriens and other Listeria species.

[0066] Example 3 (Simple culture medium containing chromogenic substrate)

[0067] The composition of the culture medium is shown in Table 5. The culture medium composition was added to 1000 mL of ethanol solution to prepare a suspension. One mL of the ethanol suspension was aseptically dispensed in portions into a container (50 mm in diameter) containing absorbent fibrous sheets (cotton nonwoven fabric, 50 mm in diameter). The containers were then allowed to stand overnight in a closed space to dry, ensuring they did not overlap. The container was then capped to prepare the simplified culture medium of this invention.

[0068] [Table 5]

[0069] composition g / L meat peptone 18.00g Casein peptone 6.00g yeast extract 10.00g Anhydrous magnesium sulfate 0.50g Magnesium glycerol phosphate 7.5g Sodium chloride 5.0g Lithium chloride 10.0g disodium hydrogen phosphate 2.50g Colistin sulfate 0.01g Latamoxef 0.02g Xanthan Gum 20.00g silicon dioxide 10.00g <![CDATA[Aldol514 TM Inositol phosphate, ammonium salt (Biosynth) 0.30g 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside (Biosynth) 0.05g glucose 10.00g

[0070] The test strains were pre-cultured in sheep blood agar for 24 hours, and the colonies were then suspended in sterile physiological saline and appropriately diluted to prepare bacterial suspensions. 1 mL of each suspension was inoculated onto a prepared simple culture medium. The color development of the colonies after incubation at 37°C for 44 hours is shown in Table 6.

[0071] [Table 6]

[0072] strain Colony staining Listeria monocytogenes ATCC15313 Red (surrounded by blue) Listeria monocytogenes NS5168 Red (surrounded by blue) Listeria monocytogenes NS5169 Red (surrounded by blue) Listeria monocytogenes NS5170 Red (surrounded by blue) Listeria monocytogenes JCM7681 red Listeria eluti NS5167 red Listeria innocosis NS5166 blue Listeria welris NS5172 blue

[0073] According to Table 6, by using a simple culture medium containing 10 g / L glucose (C) in addition to components (A) and (B), it is possible to clearly distinguish the colonies of Listeria monocytogenes and Listeria escherichiae and other Listeria species.

Claims

1. A culture medium for identifying and detecting Listeria monocytogenes and Listeria elodeonii, comprising the following components (A), (B) and (C): (A) A chromogenic substance that develops color by being broken down by β-glucosidase or a fluorescein that emits fluorescence; (B) A chromogenic substance that develops color through degradation by phosphatidylinositol-specific phospholipase C; and (C) Glucose with a concentration of 5 g / L or more but less than 30 g / L at the time of detection; in, Component (A) is 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside or 4-methylumbelliferyl-β-D-glucopyranoside.

2. The culture medium for identifying and detecting Listeria monocytogenes and Listeria elodeonum according to claim 1, wherein a composition further comprising (D) a gelling agent and (E) bacterial nutrients other than component (C) is supported or stacked on a fibrous absorbent sheet.

3. A method for identifying and detecting Listeria monocytogenes and Listeria elodeon, characterized in that, After inoculating the sample into the culture medium described in claim 1 or 2 and culturing it, the color or fluorescence of the colonies produced on the culture medium is detected. The identification and detection method is used for non-diagnostic purposes.

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