Selective enrichment culture medium for shiga toxin-producing Escherichia coli and application of selective enrichment culture medium

Abstract

The invention discloses a selective enrichment culture medium for shiga toxin-producing Escherichia coli and application of the selective enrichment culture medium. The culture medium comprises the following components in parts by mass: 18-19 parts of casein tryptone, 3-4 parts of succinic acid, 4.5-5.5 parts of sodium chloride, 3.5-4.5 parts of dipotassium phosphate, 1-2 parts of monopotassium phosphate, 3-4 parts of glucose, 0.000045-16 parts of a selective antibacterial component and 1000 parts of distilled water, the selective antibacterial component is one or a combination of more of magnesium sulfate heptahydrate, nisin and cefixime, and the pH value of the culture medium is 7.0-7.4. The culture medium provided by the invention can reduce the inhibition of selective components on shiga toxin-producing Escherichia coli, improve the inhibition on infectious microbes, shorten the culture time and improve the culture efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of pathogenic bacteria in food, and in particular relates to a pre-enrichment culture medium for detection of pathogenic bacteria in food, specifically a selective enrichment culture medium for Shiga toxin-producing Escherichia coli. Background Art

[0002] Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause a variety of symptoms ranging from mild diarrhea to severe hemorrhagic colitis. In particular, it may cause hemolytic uremic syndrome (HUS) in children and the elderly, manifested as acute renal failure, hemolytic anemia and thrombocytopenia, with high mortality and disability rates. STEC infections are usually transmitted through contaminated food (such as undercooked beef, unpasteurized dairy products) or water, and can cause large-scale outbreaks. Its toxicity comes from the action of Shiga toxins, which can cause multi-organ damage and seriously endanger public health. Therefore, the detection of STEC is one of the very important areas and contents of food safety testing projects. For example, the Chinese national standard GB4789.49-2024 "National Food Safety Standard Food Microbiology Test Shiga Toxin-Producing Escherichia Coli Detection" specifies in detail the detection methods and processes of STEC in food.

[0003] A very important step in the STEC detection method is to use selective culture media to enrich the STEC that may be present in the food sample. Selective enrichment culture is a key step in STEC detection. It is used to enrich the target bacteria from complex samples while inhibiting the growth of other bacteria, which helps to improve the detection sensitivity, especially in environmental samples or low-microbial food samples.

[0004] The culture medium used in GB4789.49-2024 is mTSB medium or EC medium, and the culture conditions are 44℃±1℃ for 18h~24h. Bile salt No. 3 is used in mTSB medium or EC medium. Bile salt No. 3 is a composite bile salt reagent used in microbiological culture medium, often used as a selective inhibitor, especially in the isolation and culture of enteric pathogens. Bile salt No. 3 is a mixture, the main components of which include sodium deoxycholate and sodium cholate, which is the sodium salt of bile acid extracted from animal bile. Bile salt No. 3 can inhibit most Gram-positive bacteria and some non-enteric Gram-negative bacteria, providing a competitive advantage for enteric pathogens (such as STEC, Shigella and Salmonella). However, in the process of using selective culture media with different formulations to proliferate STEC, the actual situation of bacterial growth is not clear, including the inhibition of the proliferation of miscellaneous bacteria and the possible impact on the growth of STEC bacteria. For example, there are currently few literatures mentioning whether bile salt No. 3 has a certain inhibitory effect on the growth of STEC. From this perspective, if the selective culture medium formula can be optimized to minimize the inhibitory effect of the selective components on STEC, while at the same time maximally inhibiting the growth of Gram-positive bacteria and other miscellaneous bacteria, shortening the culture time and improving the culture efficiency, it will be of great significance and value for STEC detection. Summary of the invention

[0005] The object of the present invention is to provide a selective enrichment medium for Shiga toxin-producing Escherichia coli, optimize the selective medium formula, reduce the inhibition of STEC by the selective components, and at the same time improve the inhibition of miscellaneous bacteria, shorten the culture time, and improve the culture efficiency. The object of the present invention is achieved through the following technical solutions:

[0006] A selective enrichment medium for Shiga toxin-producing Escherichia coli, the formula of which is composed of 18-19 parts of tryptic peptone, 3-4 parts of succinic acid, 4.5-5.5 parts of sodium chloride, 3.5-4.5 parts of dipotassium hydrogen phosphate, 1-2 parts of potassium dihydrogen phosphate, 3-4 parts of glucose, 0.000045-16 parts of selective antibacterial components, and 1000 parts of distilled water, wherein the selective antibacterial components can be one or a combination of magnesium sulfate heptahydrate, nisin and cefixime according to actual needs, and the pH of the medium is 7.0-7.4. The combination of three selective components is preferred, and the inhibition of miscellaneous bacteria is maximized.

[0007] The optimization is as follows: the cefixime is 0.000045 to 0.00005 parts; the nisin is 0.09 to 0.12 parts; and the magnesium sulfate heptahydrate is 15 to 16 parts.

[0008] A further optimization scheme is that the selective antibacterial component is a combination of magnesium sulfate heptahydrate, nisin and cefixime, wherein the cefixime is 0.000045-0.00005 parts, the nisin is 0.09-0.12 parts, and the magnesium sulfate heptahydrate is 15-16 parts.

[0009] Furthermore, the formula composition is as follows, by mass: 18.5 parts of tryptic peptone, 3.5 parts of succinic acid, 5 parts of sodium chloride, 4 parts of dipotassium hydrogen phosphate, 1.5 parts of potassium dihydrogen phosphate, 3.5 parts of glucose, 0.00005 parts of cefixime, 0.1 parts of nisin, 15.8 parts of magnesium sulfate heptahydrate, and 1000 parts of distilled water.

[0010] Another optimized solution is that the selective antibacterial component is a combination of nisin and cefixime, the nisin is 0.09 to 0.12 parts, and the magnesium sulfate heptahydrate is 15 to 16 parts.

[0011] Furthermore, the formula composition is as follows, by mass: 18.5 parts of tryptic peptone, 3.5 parts of succinic acid, 5 parts of sodium chloride, 4 parts of dipotassium hydrogen phosphate, 1.5 parts of potassium dihydrogen phosphate, 3.5 parts of glucose, 0.00005 parts of cefixime, 0.1 parts of nisin, and 1000 parts of distilled water.

[0012] The culture medium of the invention is used in the inspection of Shiga toxin-producing Escherichia coli in food and biology.

[0013] A specific application is: take a food-based sample to be tested and add it to the culture medium, the ratio of sample to culture medium is 1g sample / 9mL culture medium, and culture it at 44°C for 6 to 8 hours. The obtained culture solution can be used for subsequent detection of Shiga toxin-producing Escherichia coli. The detection methods that can be used include traditional plate culture methods, nucleic acid-based detection methods, and immune-based detection methods.

[0014] Functions of the main ingredients:

[0015] Peptone provides the nitrogen source.

[0016] Glucose and succinate provide a carbon source (E. coli can utilize succinate as a carbon source and an energy source, and can use succinate as the sole carbon source for reproduction; using succinate as an auxiliary carbon source and an energy source helps the proliferation of E. coli, especially STEC bacteria).

[0017] Nisin has a strong inhibitory effect on many Gram-positive bacteria, including food spoilage bacteria and pathogenic bacteria, but has no obvious inhibitory effect on the growth of Escherichia coli.

[0018] Cefixime is a third-generation cephalosporin antibiotic for oral use. It is highly stable to β-lactamase produced by Gram-negative bacteria, which can inhibit bacterial cell wall synthesis and eventually lead to bacterial death. It has a certain inhibitory effect on non-pathogenic Escherichia coli, but its inhibitory effect on STEC bacteria, especially O157 STEC, is not obvious. Cefixime can promote the growth of STEC bacteria to a certain extent.

[0019] The present invention uses nisin and cefixime as selective components to inhibit the growth of non-STEC bacteria in the sample to the greatest extent, while not affecting or reducing the impact on the growth of STEC bacteria that may exist in the sample, so as to shorten the culture time and improve the culture efficiency. The simultaneous use of the two can inhibit both Gram-positive bacteria and Gram-negative bacteria.

[0020] Magnesium sulfate and high magnesium ion concentrations have an inhibitory effect on the growth of general bacteria, but Escherichia coli is tolerant to high magnesium ions to a certain extent.

[0021] Beneficial effects of the present invention:

[0022] The selective enrichment medium for Shiga toxin-producing Escherichia coli of the present invention is optimized in formulation compared with the existing Shiga toxin-producing Escherichia coli enrichment medium. By replacing the carbon source, optimizing the selective components, removing the No. 3 bile salt, etc., the inhibition of the selective components on STEC is reduced, and at the same time, miscellaneous bacteria are effectively inhibited, the culture time is shortened, and the culture efficiency is improved. The culture medium has good practical prospects and commercial value. The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the results of each group of formulas in the basic formula selection of the culture medium in Example 1.

[0024] Figure 2 This is a comparison chart of the optimization results of the selective culture medium components in Example 2.

[0025] Figure 3 This is a comparison chart of the applicability of the optimized culture medium in Example 3 to other STEC Escherichia coli.

[0026] Figure 4 This is a comparison chart of the inhibitory effect of formula combination 25 evaluated in Example 4 on common background bacteria. DETAILED DESCRIPTION

[0027] Example 1

[0028] This example is a screening of basic formulas. Formula screening was performed according to Table 1, the preparation volume was 1000 mL, and the pH was adjusted to 7.2 ± 0.2. A 96-well plate was used for the experiment. Escherichia coli O157 was added to the culture medium of the above different formulas as a typical STEC bacterium, and the added concentration was 100 cfu / mL. 100uL was added to a 96-well plate, sealed with a sealing film, and cultured at 44°C for 2, 4, 6, and 8 hours respectively. Use an ELISA reader to read the OD value at a wavelength of 600nm.

[0029] Table 1

[0030] Element Recipe combination 1 Recipe Combination 2 Recipe Combination 3 Recipe Combination 4 Trypticase 18.5g 18.5g 18.5g 18.5g Succinic acid 3.5g 3.5g Sodium chloride 5.0g 5.0g 5.0g 5.0g Dipotassium hydrogen phosphate 4.0g 4.0g 4.0g 4.0g Potassium dihydrogen phosphate 1.5g 1.5g 1.5g 1.5g glucose 3.5g 3.5g lactose 5.0g 5.0g

[0031] The results are as follows Figure 1 As shown in the figure, formula combination 2 has the best support for the growth of E. coli O157. This formula uses a combination of glucose and succinic acid. Compared with the use of glucose or lactose alone, the addition of succinic acid has a significant promoting effect on the growth of E. coli O157. Although E. coli can metabolize lactose, the results show that the use of glucose combined with succinic acid has a significantly better promoting effect on the growth of E. coli O157 than the combination of lactose and succinic acid.

[0032] Example 2

[0033] This example is to screen the selective culture medium components and confirm the usage amount based on the formula combination 2 obtained in Example 1. On the basis of formula combination 2, selective antibacterial ingredients are added according to Table 2, and the formula screening is carried out. The preparation volume is 1000mL, and the pH is adjusted to 7.2±0.2. The experiment was carried out with a 96-well plate. Escherichia coli O157 was added to the culture medium of the above different formulas as a typical STEC bacterium, and the addition concentration was 100cfu / mL. 100uL was added to the 96-well plate, sealed with a sealing film, and cultured at 44°C for 2, 4, 6, and 8 hours respectively. Use an ELISA reader to read the OD value at a wavelength of 600nm.

[0034] Table 2

[0035]

[0036] The results are as follows Figure 2As shown, compared with formula combination 2, formula combinations 22, 23, and 24 with added selective ingredients had no clear effect on the growth of E. coli O157. In particular, formula combination 25, which added all the selective antibacterial ingredients, had basically no difference in the growth of E. coli O157 from formula combination 2 and other combinations. On the contrary, in formula combination 21 with added bile salts, the growth of E. coli O157 was significantly slower than that of other combinations. This result shows that the combination of magnesium sulfate + nisin + cefixime selective ingredients has less effect on the growth of E. coli O157 than the traditional culture medium formula using bile salts, and is more conducive to the proliferation of E. coli O157.

[0037] Example 3

[0038] In this embodiment, the optimized formula combination 25 obtained in Example 2 was used for other STEC Escherichia coli to test its applicability. The experiment was conducted using a 96-well plate. STEC bacteria of serotypes O26, O45, O103, O111, O121, O145 and O157 were added to the culture medium of formula combination 25 at a concentration of 100 cfu / mL. 100uL was added to the 96-well plate, sealed with a sealing film, and cultured at 44°C for 2, 4, 6, and 8 hours. The OD value was read using an ELISA reader at a wavelength of 600nm. The results are shown in the figure. Figure 3 shown.

[0039] The results showed that the formula combination 25 had no significant difference in bacterial proliferation rate for the growth of the six serotypes of STEC E. coli tested, compared with the experimental results using E. coli O157 as a typical STEC bacterium, indicating that the formula combination can be widely applicable to STEC E. coli.

[0040] Example 4

[0041] This example is to evaluate the inhibitory effect of formula combination 25 on common background bacteria.

[0042] The experiment was conducted using a 96-well plate. Salmonella, Enterobacter cloacae, Streptococcus lactis, Bifidobacterium lactis, Staphylococcus aureus, and Pseudomonas aeruginosa were added to the culture medium of formula combination 25 at a concentration of 100 cfu / mL. 100uL was added to the 96-well plate, sealed with a sealing film, and cultured at 44°C for 2, 4, 6, and 8 hours. The OD value was read at a wavelength of 600nm using an ELISA reader. Escherichia coli O157 was used as a control. The results are shown in Figure 2. Figure 4 The results show that the growth of the added common background bacteria was significantly inhibited in the formula combination 25, indicating that the selective inhibitory components in the formula combination 25 have a clear inhibitory effect on the growth of non-target common background bacteria.

[0043] Example 5

[0044] This example is to evaluate the use of formula 25 for real sample testing

[0045] Use formula combination 25 as the culture medium. Purchase raw beef from the market and test it to be free of TOP7 STEC bacteria. Take 25g of beef per portion and add it to 225mL of the culture medium of formula combination 25, then add 100uL of Escherichia coli O157 at a concentration of 100cfu / mL. Incubate at 44°C for 2, 4, 6, and 8 hours. After incubation, take 1mL of the above culture solution, add 20uL of Escherichia coli O157 immunomagnetic beads to it, invert and mix at room temperature for 15 minutes, then wash the beads three times and resuspend the beads with 50uL of magnetic bead washing solution. Take 30uL of magnetic beads and use the qPCR detection reagent specific for Escherichia coli O157 to obtain the CT value results.

[0046] Comparative Example 1

[0047] This embodiment is a comparative example of embodiment 5.

[0048] Formula combination 25 was used as the culture medium and mTSB was used as the control culture medium. Other operations and detection methods were the same as those of Example 5. The ingredients of Example 5 and the comparative example are shown in Table 3. The test results are shown in Table 4.

[0049] Table 3

[0050]

[0051] Table 4

[0052]

[0053] The results show that when using formula combination 25 to culture and detect E. coli O157, positive detection is possible after 4 hours of culture, stable positive detection is possible after 6 hours, and strong positive detection is possible after 8 hours. In comparison, when using mTSB, it takes about 6 hours to achieve the possibility of positive detection, and about 8 hours to achieve stable positive detection. This shows that by adding E. coli O157 to real samples and using formula combination 25, faster detection can be achieved.

[0054] It should be pointed out that this experiment used immunomagnetic beads as a concentration and purification reagent for E. coli O157 in culture. After using immunomagnetic beads, the detection sensitivity is 2-3 orders of magnitude higher than other methods using nucleic acid extraction. In other words, if other nucleic acid extraction methods are used, the culture time needs to be extended by at least 3-4 hours to detect E. coli O157. It also shows to a certain extent that formula combination 25 is beneficial for improving the efficiency of STEC bacteria detection.

[0055] Example 6

[0056] Formula combination 26 was used as the culture medium. Raw beef was purchased from the market and tested to be free of TOP7 STEC bacteria. 25g of beef was taken per portion and added to 225mL of the culture medium of formula combination 26, and then 100uL of Escherichia coli O157 at a concentration of 100cfu / mL was added thereto. Cultured at 44°C for 2, 4, 6, and 8 hours. After culture, 1mL of the above culture solution was taken, 20uL of Escherichia coli O157 immunomagnetic beads were added thereto, inverted and mixed and incubated at room temperature for 15 minutes, and then the magnetic beads were washed three times and resuspended with 50uL of magnetic bead washing solution. 30uL of magnetic beads were taken and tested using a qPCR detection reagent specific for Escherichia coli O157 to obtain the CT value results. The ingredients of formula combination 26 are shown in Table 5. The results showed that positive detection was possible after 4 hours of culture, stable positive detection was possible after 6 hours, and strong positive detection was achieved after 8 hours.

[0057] Table 5

[0058]

[0059] Example 7

[0060] Formula combination 27 was used as the culture medium. Raw beef was purchased from the market and tested to be free of TOP7 STEC bacteria. 25g of beef was taken per portion and added to 225mL of the culture medium of formula combination 27, and then 100uL of Escherichia coli O157 at a concentration of 100cfu / mL was added thereto. Cultured at 44°C for 2, 4, 6, and 8 hours. After culture, 1mL of the above culture solution was taken, 20uL of Escherichia coli O157 immunomagnetic beads were added thereto, inverted and mixed and incubated at room temperature for 15 minutes, and then the magnetic beads were washed three times and resuspended with 50uL of magnetic bead washing solution. 30uL of magnetic beads were taken and tested using a qPCR detection reagent specific for Escherichia coli O157 to obtain the CT value results. The ingredients of formula combination 27 are shown in Table 6. The results showed that positive detection was possible after 4 hours of culture, stable positive detection was possible after 6 hours, and strong positive detection was achieved after 8 hours.

[0061] Table 6

[0062]

[0063] Example 8

[0064] Formula combination 28 was used as the culture medium. Raw beef was purchased from the market and tested to be free of TOP7 STEC bacteria. 25g of beef was taken per portion and added to 225mL of the culture medium of formula combination 28, and then 100uL of Escherichia coli O157 at a concentration of 100cfu / mL was added thereto. Cultured at 44°C for 2, 4, 6, and 8 hours. After culture, 1mL of the above culture solution was taken, 20uL of Escherichia coli O157 immunomagnetic beads were added thereto, inverted and mixed and incubated at room temperature for 15 minutes, and then the magnetic beads were washed three times and resuspended with 50uL of magnetic bead washing solution. 30uL of magnetic beads were taken and tested using a qPCR detection reagent specific for Escherichia coli O157 to obtain the CT value results. The ingredients of formula combination 28 are shown in Table 7. The results showed that positive detection was possible after 4 hours of culture, and stable positive detection was possible after 6 hours.

[0065] Table 7

[0066]

[0067] Example 9

[0068] Formula combination 29 was used as the culture medium. Raw beef was purchased from the market and tested to be free of TOP7 STEC bacteria. 25g of beef was taken per portion and added to 225mL of the culture medium of formula combination 29, and then 100uL of Escherichia coli O157 at a concentration of 100cfu / mL was added thereto. Cultured at 44°C for 2, 4, 6, and 8 hours. After culture, 1mL of the above culture solution was taken, 20uL of Escherichia coli O157 immunomagnetic beads were added thereto, inverted and mixed and incubated at room temperature for 15 minutes, then the magnetic beads were washed three times and resuspended with 50uL of magnetic bead washing solution. 30uL of magnetic beads were taken and tested using a qPCR detection reagent specific for Escherichia coli O157 to obtain the CT value results. The ingredients of formula combination 29 are shown in Table 8. The results showed that positive detection was possible after 6 hours of culture, and stable positive detection was possible after 8 hours.

[0069] Table 8

[0070]

[0071] The above-mentioned embodiments are only partial embodiments of the present invention and cannot cover the whole of the present invention. Based on the above-mentioned embodiments and drawings, technical personnel in this field can obtain more implementation methods without paying creative work. Therefore, these implementation methods obtained without paying creative work should be included in the protection scope of the present invention.

Claims

1. A selective enrichment medium for Shiga toxin-producing Escherichia coli, characterized in that: The formula comprises, by weight, 18 to 19 parts of tryptic peptone, 3 to 4 parts of succinic acid, 4.5 to 5.5 parts of sodium chloride, 3.5 to 4.5 parts of dipotassium hydrogen phosphate, 1 to 2 parts of potassium dihydrogen phosphate, 3 to 4 parts of glucose, 0.000045 to 16 parts of a selective antibacterial component, and 1000 parts of distilled water, wherein the selective antibacterial component is one or a combination of several of magnesium sulfate heptahydrate, nisin and cefixime, and the pH value of the culture medium is 7.0 to 7.

4.

2. The selective enrichment medium for Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The cefixime is 0.000045 to 0.00005 parts; the nisin is 0.09 to 0.12 parts; The magnesium sulfate heptahydrate is 15 to 16 parts.

3. The selective enrichment medium for Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The selective antibacterial component is a combination of magnesium sulfate heptahydrate, nisin and cefixime, wherein the cefixime is 0.000045-0.00005 parts, the nisin is 0.09-0.12 parts, and the magnesium sulfate heptahydrate is 15-16 parts.

4. The selective enrichment medium for Shiga toxin-producing Escherichia coli according to claim 3, characterized in that: The formula is composed of 18.5 parts of tryptic peptone, 3.5 parts of succinic acid, 5 parts of sodium chloride, 4 parts of dipotassium hydrogen phosphate, 1.5 parts of potassium dihydrogen phosphate, 3.5 parts of glucose, 0.00005 parts of cefixime, 0.1 parts of nisin, 15.8 parts of magnesium sulfate heptahydrate, and 1000 parts of distilled water.

5. The selective enrichment medium for Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The selective antibacterial component is a combination of nisin and cefixime, wherein the nisin is 0.09-0.12 parts and the magnesium sulfate heptahydrate is 15-16 parts.

6. The selective enrichment medium for Shiga toxin-producing Escherichia coli according to claim 5, characterized in that: The formula composition is as follows, in parts by mass: 18.5 parts of tryptic peptone, 3.5 parts of succinic acid, 5 parts of sodium chloride, 4 parts of dipotassium hydrogen phosphate, 1.5 parts of potassium dihydrogen phosphate, 3.5 parts of glucose, 0.00005 parts of cefixime, 0.1 parts of nisin, and 1000 parts of distilled water.

7. Use of the culture medium according to any one of claims 1 to 6 in the detection of Shiga toxin-producing Escherichia coli in food and biology.

8. The use according to claim 7, characterized in that: Take the food-based sample to be tested and add it to the culture medium. The ratio of sample to culture medium is 1g sample / 9mL culture medium. Culture it at 44°C for 6-8 hours. The obtained culture solution can be used for subsequent detection of Shiga toxin-producing Escherichia coli. The detection methods that can be used include traditional plate culture method, nucleic acid-based detection method, and immune-based detection method.

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