A chromogenic medium for detecting enteropathogenic escherichia coli

Abstract

The application discloses a chromogenic culture medium for detecting enteropathogenic Escherichia coli, which contains peptone, sodium pyruvate, sodium chloride, D-sorbitol, D-malic acid, bile salt, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide, kaolin, bromothymol blue, crystal violet, polyethylene glycol and agar, and the rest is water. The chromogenic culture medium is simple in composition, convenient to prepare and use, and can be used for identifying enteropathogenic Escherichia coli EPEC according to the color of bacterial colonies, and has the advantages of specificity, high efficiency, rapidness, low cost, simple result judgment and the like, and has a wide application prospect in food safety monitoring.

Description

Technical Field

[0001] This invention relates to a chromogenic culture medium for detecting diarrhea-causing Escherichia coli, and more particularly to a chromogenic culture medium for detecting enteropathogenic Escherichia coli, belonging to the field of food safety microbiological testing and monitoring. Background Technology

[0002] Enteropathogenic Escherichia coli (EPEC) is a diarrheal Escherichia coli that causes adhesion and swabbing damage to the host's intestinal mucosal epithelial cells but does not produce Shiga toxin. This bacterium is one of the important pathogens causing diarrhea in infants and young children and sporadic diarrhea in adults worldwide; it is highly infectious and can lead to death in severe cases. Currently, my country's national food safety standards lack a specific standard detection method for EPEC. The existing national standard method, GB4789.6-2016, first isolates Escherichia coli and then verifies 12 key characteristic genes to determine the specific type of Escherichia coli. Currently, internationally, apart from Enterohemorrhagic Escherichia coli (EHEC) O157, there are no other products that can identify diarrheal Escherichia coli through culture and isolation, directly affecting the detection rate of EPEC and posing a significant risk to food safety.

[0003] The chromogenic medium method, based on traditional culture media, utilizes bacterial biochemical identification techniques such as metabolic differences in specific enzyme substrates to complete culture, isolation, and identification in one step. It is simple to operate, significantly shortens detection time, and improves accuracy through the characteristic identification of target bacteria. Therefore, it has broad application prospects and has been applied in various fields such as clinical medicine, food hygiene, and environmental protection for the detection of bacteria such as Salmonella, Vibrio, Listeria, and EHEC O157. EPEC and EHEC have similar phenotypic characteristics; the key to distinguishing them is whether they produce Shiga toxin. Currently, the principle behind the chromogenic medium for EHEC O157 is that common Escherichia coli has β-D-glucuronidase, while EHEC O157 is negative for this enzyme; EHEC O157 does not ferment sorbitol and is resistant to tellurite. Utilizing these differences, by adding the corresponding enzyme substrate to the culture medium, the difference in phenotypic characteristics can be effectively used to isolate EHEC O157. Two other US patents mention that using α-galactosidase and β-glucosidase substrates combined with potassium tellurite-optimized culture media can not only isolate and identify O157, but also identify Shiga toxin-producing Escherichia coli such as O26, O103, O111, and O145, which demonstrate the feasibility of using chromogenic culture media to isolate and identify EPEC.

[0004] In summary, to address the shortcomings of existing technologies, there is an urgent need to establish a chromogenic culture medium for detecting enteropathogenic Escherichia coli, so as to achieve efficient and accurate separation and detection of EPEC in food. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a chromogenic culture medium suitable for the isolation and detection of enteropathogenic Escherichia coli in food testing samples.

[0006] This invention utilizes the principles of biochemistry, microbiology, and inorganic, organic, and analytical chemistry to screen and optimize different carbon sources, nitrogen sources, inorganic salts, vitamins, pH values, and other factors, resulting in the culture medium of this invention.

[0007] The technical solution adopted in this invention is as follows:

[0008] A chromogenic culture medium for detecting enteropathogenic Escherichia coli, characterized in that each 1000 mL of the culture medium comprises: 15-20 g peptone, 1-5 g sodium pyruvate, 2-5 g sodium chloride, 5-10 g D-sorbitol, 1-5 g D-malic acid, 1-5 g bile salts, 2-5 g dipotassium hydrogen phosphate, 1-5 g potassium dihydrogen phosphate, 1-3 g sodium hydroxide, 10-15 g kaolin, 0.05-0.08 g bromothymol blue, 0-0.01 g crystal violet, 1-2 g polyethylene glycol, 15-18 g agar, with the remainder being water, and a pH of 7.0 ± 0.2.

[0009] In some embodiments, preferably, the peptone is bacteriological peptone.

[0010] In some embodiments, preferably, the bile salt is bile salt No. 3.

[0011] In some embodiments, the polyethylene glycol is preferably PEG4000.

[0012] This invention differentiates typical enteropathogenic Escherichia coli (tEPEC), atypical enteropathogenic Escherichia coli (aEPEC), and common Escherichia coli (CEC) based on their metabolic differences in D-sorbitol, D-malic acid, and peptone using the indicator bromothymol blue. tEPEC cannot utilize D-sorbitol and D-malic acid, but can only break down peptone to produce alkaloids, resulting in blue-green colonies against a blue background under the action of bromothymol blue. aEPEC cannot utilize D-malic acid, but in the presence of pyruvate, it preferentially breaks down D-sorbitol to produce acid, resulting in yellow colonies under the action of bromothymol blue. However, over time, it gradually breaks down peptone to produce alkaloids, resulting in a green background. Common Escherichia coli can simultaneously break down D-sorbitol, D-malic acid, and peptone, resulting in yellow and blue-green colonies against a blue background under the action of bromothymol blue.

[0013] The present invention adds bile salts and crystal violet to the culture medium, which can inhibit the growth of Gram-positive bacteria in the sample.

[0014] This invention adds kaolin to the culture medium, which makes the components of the culture medium more evenly dispersed, adsorbs the target bacteria on the surface and stimulates their growth by exchanging metal ions. At the same time, it can make the solid culture medium have a milky white background, making the color of the colonies more obvious and prominent.

[0015] The colony characteristics of bacteria in the culture medium of this invention are as follows: tEPEC cannot utilize D-sorbitol and D-malic acid, and appears as blue-green colonies against a blue background; aEPEC cannot utilize D-malic acid, but can preferentially utilize D-sorbitol, and appears as yellow colonies against a green background; Escherichia coli can simultaneously decompose D-sorbitol, D-malic acid and peptone, and appears as yellow and blue-green colonies against a blue background.

[0016] The method for isolating and detecting EPEC using a chromogenic culture medium of the present invention includes the following steps:

[0017] 1) Preparation of chromogenic plates: Add all components of the above chromogenic medium except agar, bacterial peptone, and D-malic acid to 1000 mL of deionized water and stir to dissolve. The solution will turn blue at this point. Add D-malic acid and adjust the pH to 7.0 ± 0.2; the solution will then turn green. Finally, add bacterial peptone and agar to the completely dissolved medium. Heat to boiling, then cool to 50-55°C. Under aseptic conditions, pour the mixture into plates for later use.

[0018] 2) Sample preparation: Weigh 25 g of sample aseptically and add it into a sterile homogenizing bag with a filter containing 225 mL of buffered peptone water (BPW). Homogenize for 1-2 min to prepare a 1:10 sample homogenate and incubate overnight at 41±1℃.

[0019] 3) Acid treatment: Under aseptic conditions, take 0.5 mL of enrichment broth, adjust the pH to 2.0 with hydrochloric acid, and shake to mix for 1-2 hours.

[0020] 4) Incubation: Centrifuge at 8000×g for 2 min, remove the supernatant, add 0.5 mL of pyruvate-modified buffered peptone water (mBPWp), incubate at 36±1℃ for 1~2 h, then streak on crystal violet neutral red agar (VRBA) with a sterile inoculation loop and incubate at 36±1℃ for 18~24 h.

[0021] 5) Inoculation and culture: Select purple-red single colonies and inoculate them onto a color development plate, and incubate at 33±1℃ for 18~24 h.

[0022] 6) Results analysis: tEPEC showed blue-green colonies against a blue background; aEPEC showed yellow colonies against a green background; common Escherichia coli showed yellow and blue-green colonies against a blue background.

[0023] The beneficial effects of this invention are:

[0024] The chromogenic culture medium of this invention can be used for the isolation and detection of enteropathogenic Escherichia coli, and has the advantages of high specificity, high sensitivity, ease of operation, and simple result interpretation. It is suitable for the detection of food samples and has broad application prospects. The chromogenic culture medium of this invention can not only detect EPEC, but also distinguish between tEPEC and aEPEC. Detailed Implementation

[0025] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0026] Example 1: Specificity experiment of the chromogenic culture medium described in this invention against enteropathogenic Escherichia coli.

[0027] 1) Preparation of solid plates:

[0028] The culture medium was prepared according to the following formula: per 1000 mL, it contained 15 g of bacteriological peptone, 1.5 g of sodium pyruvate, 2.5 g of sodium chloride, 6 g of D-sorbitol, 5 g of D-malic acid, 1.5 g of bile salt No. 3, 5 g of dipotassium hydrogen phosphate, 1.5 g of potassium dihydrogen phosphate, 1.8 g of sodium hydroxide, 15 g of kaolin, 0.08 g of bromothymol blue, 0.001 g of crystal violet, 1 g of PEG4000, and 15 g of agar, with the remainder being water, and the pH being 7.0 ± 0.2. All components of the above colorimetric culture medium, except for agar, bacterial peptone, and D-malic acid, were added to 1000 mL of deionized water and stirred to dissolve; the solution was blue at this point. D-malic acid was then added, and the pH was adjusted to 7.0 ± 0.2; the solution turned green. Finally, bacterial peptone and agar were added to the completely dissolved culture medium. Heat to boiling, then cool to 50-55°C. Perform aseptic processing, pour into plates, and set aside.

[0029] 2) Activation and culture of microbial strains:

[0030] The tested bacteria included common Escherichia coli IQCC10151 (O86) and IQCC30152 (O127), enterohemorrhagic Escherichia coli (EHEC) IQCC50171 (O91) and IQCC50170 (O130), typical enteropathogenic Escherichia coli (tEPEC) IQCC30121 (O127) and IQCC30147 (O111), and atypical enteropathogenic Escherichia coli (aEPEC) IQCC10103 (O157), IQCC10150 (O26), and IQCC50122 (O108). IQCC is an abbreviation for Inspection Quarantine Culture Collection. Each strain underwent whole-genome sequencing verification before the experiment.

[0031] After activating the above-mentioned bacterial strains, they were inoculated onto Columbia blood agar plates and incubated at their respective optimal temperatures for 18–24 hours until distinct colonies appeared. Three to five colonies were then picked from each strain and dispersed in TSB liquid medium to prepare a bacterial suspension. The turbidity of the bacterial suspension was adjusted to a McFarland turbidity standard of 0.5, at which point the concentration of the bacterial suspension was 1.5 × 10⁻⁶. 8 CFU / mL, for later use.

[0032] 3) Inoculation: Take one loopful of the bacterial suspension prepared from the above 10 strains and streak it onto the prepared chromogenic plate. Incubate at 33±1℃ for 18~24h and observe the color development of the colonies.

[0033] 4) Results analysis: Enteropathogenic Escherichia coli (EPEC) grew well on this plate. tEPEC showed blue-green colonies on a blue background; aEPEC showed yellow colonies on a green background; common Escherichia coli showed yellow and blue-green colonies on a blue background; detailed results are shown in Table 1.

[0034] Table 1: Experimental results of Example 1.

[0035]

[0036] The experimental results show that this culture medium can effectively distinguish enteropathogenic Escherichia coli (EPEC) from other Escherichia coli.

[0037] Example 2: Experiment on the growth characteristics of enteropathogenic Escherichia coli on the chromogenic culture medium described in this invention.

[0038] 1) Preparation of solid plate: see Example 1.

[0039] 2) Activation and culture of microbial strains: see Example 1.

[0040] 3) Inoculation: Select an appropriate dilution of working bacterial suspension (0.1 mL) and spread it evenly on the chromogenic medium and reference TSA plate. Inoculate two plates for each dilution. The inoculation level per plate is 20–200 CFU. Incubate at 33±1℃ for 18–24 h, record the colony diameter, and calculate the recovery rate.

[0041] 4) Results analysis: The colony diameter d (mm) and recovery rate (PR) results are shown in Table 2.

[0042] Table 2: Experimental results of Example 2.

[0043]

[0044] As shown in Table 2, this culture medium can not only effectively distinguish between enteropathogenic Escherichia coli (EPEC) and other Escherichia coli, but also the recovery rate (PR) of the target bacteria (EPEC) is much higher than the requirement of PR≥0.5 in GB4789.28-2013.

[0045] Example 3: Procedure for using the enteropathogenic Escherichia coli chromogenic culture medium described in this invention.

[0046] 1) Strain selection and activation

[0047] The selected bacterial strains included Escherichia coli IQCC10151 (O86), typical enteropathogenic Escherichia coli (tEPEC) IQCC30147 (O111), atypical enteropathogenic Escherichia coli (aEPEC) IQCC10150 (O26), Staphylococcus aureus IQCC22035, Proteus mirabilis IQCC10602, Proteus vulgaris IQCC10607, Klebsiella pneumoniae IQCC10302, Citrobacter freundii IQCC10204, Citrobacter bromide IQCC10219, Enterobacter cloacae IQCC10401, Enterobacter aerogenes IQCC10447, Cronobacter sakazakii IQCC30460, and Salmonella IQCC30506.

[0048] Thirteen control bacteria were activated and inoculated onto Columbia blood agar plates, and cultured at their respective optimal temperatures for 18–24 hours until distinct colonies appeared. Three to five colonies were picked from each control and dispersed in TSB liquid medium to prepare a bacterial suspension. The turbidity of the bacterial suspension was adjusted to a McFarland standard of 0.5, at which point the concentration of the bacterial suspension was 1.5 × 10⁻⁶. 8 CFU / mL, for later use.

[0049] 2) Culture medium preparation

[0050] Preparation of solid plate: as described in Example 1;

[0051] Preparation of pyruvate-modified buffered peptone water (mBPWp): Each 1000 mL of culture medium contains 3.6 g disodium hydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 5 g sodium chloride, 1.5 g bile salts (No. 3), 1.0 g sodium pyruvate, 10 g peptone, 5 g tryptone, 6 g yeast extract, 10 g lactose, 0.3 g glutamic acid, with the remainder being water. The pH is adjusted to 7.2 ± 0.2. The solution is autoclaved at 121℃ for 15 min, dispensed, and stored for later use.

[0052] Preparation of acid treatment solution: 1 mol / L HCl solution.

[0053] 3) Inoculation and acid treatment

[0054] Take 0.5 mL of the bacterial suspension prepared from the above 13 strains, adjust the pH to about 2.0 (add about 75 μL of 1 mol / L HCl solution), and mix by shaking at room temperature for 1.5 h.

[0055] 4) Incubation

[0056] Centrifuge at 8000×g for 2 min, remove the supernatant, add 0.5 mL of pyruvate-modified buffered peptone water (mBPWp), incubate at 36±1℃ for 1 h, then streak on crystal violet neutral red agar (VRBA) with a sterile inoculation loop and incubate at 36±1℃ for 18~24 h.

[0057] 5) Cultivation

[0058] Select a single purple-red colony and inoculate it onto a chromogenic plate. Incubate at 33±1℃ for 18~24 h and observe the color development of the colony.

[0059] 6) Results Analysis

[0060] After acid treatment, only Escherichia coli survived on VRBA. EPEC was then identified based on the characteristic colonies of the chromogenic medium. Detailed results are shown in Table 3.

[0061] Table 3: Experimental results of Example 3.

[0062]

[0063] Example 4: Detection of enteropathogenic Escherichia coli in beef samples.

[0064] 1) Preparation of relevant culture media:

[0065] Preparation of solid plates: Plates were prepared according to Examples 1 and 2;

[0066] Preparation of pyruvate-modified buffered peptone water (mBPWp) and acid treatment solution: Refer to Example 3 for preparation.

[0067] 2) Preparation of artificially contaminated samples: High-temperature sterilized beef samples were prepared according to 10... 2 At an inoculation level of CFU / 25g, samples were inoculated with common Escherichia coli IQCC10151 (O86), typical enteropathogenic Escherichia coli (tEPEC) IQCC30147 (O111), and atypical enteropathogenic Escherichia coli (aEPEC) IQCC10150 (O26), and were numbered Sample 1, Sample 2, and Sample 3, respectively; the sample without additives was numbered Sample 4.

[0068] 3) Sample preparation: Add 4 samples to 225ml BPW, homogenize and mix, enrich at 41±1℃ for 8~10h, then take 0.5ml of enrichment broth from each sample, adjust the pH to about 2.0, and mix by shaking at room temperature for 2h.

[0069] 4) Inoculation and culture: Centrifuge at 8000×g for 2 min, discard the supernatant, add 0.5 mL mBPWp, incubate at 36±1℃ for 1 h, then streak onto VRBA with a sterile inoculation loop and incubate at 36±1℃ for 18~24 h. Select purple-red single colonies and inoculate them onto chromogenic plates, incubate at 33±1℃ for 18~24 h, and observe the color development of the colonies.

[0070] 5) Results analysis: This method can detect enteropathogenic Escherichia coli (EPEC) in food. Detailed results are shown in Table 4.

[0071] Table 4: Experimental results of Example 4.

[0072]

[0073] The embodiments described above are illustrative rather than limiting. Several embodiments may be listed according to the defined scope. Therefore, any changes and modifications made without departing from the overall concept of the present invention should be within the protection scope defined by the present invention.

Claims

1. The application of a chromogenic culture medium in the preparation of reagents for detecting enteropathogenic Escherichia coli, characterized in that, Each 1000 mL of culture medium comprises: 15–20 g of bacteriological peptone, 1–5 g of sodium pyruvate, 2–5 g of sodium chloride, 5–10 g of D-sorbitol, 1–5 g of D-malic acid, 1–5 g of bile salts, 2–5 g of dipotassium hydrogen phosphate, 1–5 g of potassium dihydrogen phosphate, 1–3 g of sodium hydroxide, 10–15 g of kaolin, 0.05–0.08 g of bromothymol blue, 0–0.01 g of crystal violet, 1–2 g of polyethylene glycol, 15–18 g of agar, with the remainder being water, and a pH of 7.0 ± 0.2; wherein the enteropathogenic Escherichia coli includes typical enteropathogenic Escherichia coli and atypical enteropathogenic Escherichia coli.

2. The application as described in claim 1, characterized in that, The bile salts are at least one of sodium deoxycholate, porcine bile salts, mixed bile salts, bovine bile salts, and bile salt No.

3.

3. The application as described in claim 1, characterized in that, The kaolin is of pharmaceutical or cosmetic grade, with a mesh size of 5000 mesh or higher.

4. The application as described in claim 1, characterized in that, The polyethylene glycol is at least one of PEG4000, PEG6000 and PEG8000.

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