Bordetella pertussis carbon-free drug sensitivity detection culture medium and application thereof

Abstract

The invention relates to the technical field of microbial culture and drug sensitivity, in particular to preparation and application of a Bordetella pertussis drug sensitivity detection culture medium. The invention provides a culture medium for performing a drug sensitivity test on clinically separated pertussis bordetella. The culture medium is a carbon-free solid agar culture medium. According to the method, the interference of adsorbing antibiotics by carbon particles is avoided, the small inhibition zone caused by overgrowth of bacteria when an eutrophic plate is used for drug sensitivity can be avoided, the influence on the color of the plate is small after whole blood is replaced with serum, and the color of the plate and the color of a common drug sensitive plate MH culture medium tend to be consistent; the method can be used for detecting the drug sensitivity of the bordetella pertussis.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture and drug sensitivity, in particular to a carbon-free drug sensitivity detection culture medium for Bordetella pertussis and application thereof. Background Art

[0002] Pertussis (whooping cough) is an acute respiratory infectious disease caused by infection with Bordetella pertussis (BP). It is highly contagious and can last for weeks or months. Infants and young children are prone to developing severe cases, making prompt antibiotic treatment crucial. Due to adaptive variation in BP and the fading of antibodies after vaccination, cases of pertussis in vaccinated adolescents and adults are increasing. Because of its atypical symptoms, diagnosis and treatment are often delayed, making it a significant source of pertussis infection in infants and young children.

[0003] Macrolide antibiotics remain the preferred choice for the prevention and treatment of pertussis in most countries worldwide. They are crucial for reducing complications and preventing the spread of pertussis. Clinically, sulfamethoxazole, cefoperazone / sulbactam, and piperacillin / tazobactam are used empirically to treat patients infected with drug-resistant pertussis strains. Precise treatment of pertussis based on drug susceptibility testing is of great clinical significance. Appropriate administration of sensitive antibiotics can effectively alleviate symptoms, control disease spread, and reduce the risk of death in severe cases.

[0004] Currently, there is a lack of suitable drug susceptibility testing and culture systems for Bordetella pertussis, making it difficult to screen for effective antibiotics in clinical practice, further exacerbating the emergence of drug-resistant bacteria and creating a vicious cycle. Resistance to macrolide antibiotics has become a major public health concern. In recent years, the in vitro minimum inhibitory concentrations of the preferred macrolide antibiotics for Bordetella pertussis, such as erythromycin and azithromycin, have increased significantly, often exceeding 64 mg / L. The MIC of drug-resistant strains isolated in China for erythromycin has exceeded the maximum detectable concentration of 256 mg / L. The use of chromogenic plates and enrichment solutions for Bordetella pertussis has greatly increased the isolation rate of clinical pathogens, laying the foundation for drug susceptibility testing. Therefore, drug susceptibility testing for Bordetella pertussis is of great clinical significance, enabling comprehensive monitoring of resistance trends to commonly used antibiotics and curbing the emergence of drug-resistant bacteria.

[0005] In recent years, due to the continuous rise in drug resistance rates, the expert consensus recommends drugs such as macrolides, trimethoprim-sulfamethoxazole and β-lactams for the treatment of pertussis. The Chinese Pertussis Diagnosis, Treatment and Prevention Guidelines (2024 Edition) recommends that hospitals with conditions should carry out culture and drug sensitivity testing of Bordetella pertussis to provide a basis for precise clinical drug use. Therefore, it is particularly important for the laboratory to carry out accurate and standardized drug sensitivity testing. The drug sensitivity study of Bordetella pertussis currently mainly uses charcoal agar plates, which are used for the initial isolation of Bordetella pertussis. The plates are rich in nutrients and the charcoal can absorb the toxins produced by the bacteria, which is conducive to growth. However, the use of charcoal agar plates for drug sensitivity testing has significant disadvantages. The charcoal contained in them can non-specifically absorb antibiotics, resulting in deviations in drug sensitivity results and the inability to accurately explain the effect of antibiotics on the growth of Bordetella pertussis. Summary of the Invention

[0006] In view of this, the present invention provides a charcoal-free solid agar medium that allows the growth of Bordetella pertussis in the presence of bacterial-derived toxins. When used in drug susceptibility testing, this plate avoids inaccurate drug sensitivity testing caused by charcoal adsorption of antibiotics. The culture medium for Bordetella pertussis drug susceptibility testing provided by the present invention is easy to prepare, exhibits good bacterial growth when coated at a 0.5 McFarland turbidity, and provides clear, easily identifiable inhibition zones. The present invention significantly improves the accuracy of Bordetella pertussis drug susceptibility testing.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a carbon-free solid culture medium for drug sensitivity testing of Bordetella pertussis, wherein the components of the carbon-free solid culture medium include: sodium L-glutamate, L-proline, sodium chloride, potassium dihydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, Tris, cyclodextrin, and agar powder;

[0009] The components of the carbon-free solid culture medium do not include carbon particles.

[0010] In some specific embodiments of the present invention, the components of the carbon-free solid culture medium include:

[0011]

[0012]

[0013] In some specific embodiments of the present invention, the components of the carbon-free solid culture medium further include water; preferably, the water includes distilled water;

[0014] Preferably, the pH value of the carbon-free solid culture medium is 7.3-7.4.

[0015] In some specific embodiments of the present invention, the carbon-free solid culture medium further comprises 10.0% (v / v) serum.

[0016] In some specific embodiments of the present invention, the carbon-free solid culture medium further comprises a growth-promoting additive;

[0017] Preferably, the growth-promoting additive comprises one or more of L-cysteine, niacin, vitamin C, reduced glutathione or ferrous sulfate;

[0018] Preferably, 1L of the carbon-free solid culture medium includes 10mL of a growth-promoting additive solution;

[0019] The growth-promoting additive solution comprises:

[0020]

[0021] In some specific embodiments of the present invention, the method for preparing the carbon-free solid culture medium comprises the following steps:

[0022] Dissolve the formulated amounts of sodium L-glutamate, L-proline, sodium chloride, potassium dihydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, Tris, cyclodextrin, and agar powder in distilled water and adjust the pH to 7.3-7.4 to prepare a mixed culture medium solution A.

[0023] Dissolving the formulated amount of growth-promoting additives L-cysteine, niacin, vitamin C, reduced glutathione, and ferrous sulfate in distilled water to obtain additive solution B;

[0024] The mixed culture medium solution A is sterilized, cooled, mixed with serum and the additive solution B, and packaged.

[0025] In some specific embodiments of the present invention, the cooling temperature is 45°C to 50°C.

[0026] In some specific embodiments of the present invention, 100 mL of serum is added to every 900 mL of mixed culture solution A; and / or

[0027] 10 mL of the additive solution B was added to every 1 L of the mixed culture medium solution A supplemented with serum.

[0028] In some specific embodiments of the present invention, potassium hydroxide (KOH) is used to adjust the pH value.

[0029] In some specific embodiments of the present invention, the sterilization is high-pressure steam sterilization; preferably, the sterilization temperature is 121° C., and the sterilization time is 15 minutes;

[0030] In some specific embodiments of the present invention, the packaging is poured into a 90 cm culture dish so that the thickness everywhere in the dish is 4.0±0.1 mm.

[0031] In a third aspect, the present invention further provides use of the carbon-free solid culture medium or the carbon-free solid culture medium prepared by the preparation method in preparing products for drug sensitivity testing of Bordetella pertussis.

[0032] In a fourth aspect, the present invention further provides use of the carbon-free solid culture medium or the carbon-free solid culture medium prepared by the preparation method in a drug sensitivity test of Bordetella pertussis.

[0033] The present invention provides a solid culture medium for antimicrobial susceptibility testing of Bordetella pertussis. The solid culture medium can be used in accordance with the manual drug sensitivity operation procedure specified by CLSI, with standardized processes and easy-to-read results. At the same time, the adsorption of antibiotics by charcoal is avoided, thereby ensuring the standardization and reliability of drug susceptibility testing of Bordetella pertussis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0035] Figure 1 The figures show the quality control results of drug sensitivity tests of standard strains Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 on MH plates, carbon agar plates and the carbon-free agar plates of the present invention;

[0036] The red arrow points to the MIC value of the quality control. Figure 1 The MIC values ​​of sulfamethoxazole and levofloxacin on the medium carbon agar plate were too large and out of control (*), while the MIC values ​​of the drug sensitivity test on the MH plate recommended by CLSI and the carbon-free agar plate of the present invention were under control;

[0037] Figure 2 The results of susceptibility tests of clinically isolated erythromycin-sensitive strains of Bordetella pertussis on carbon-free agar plates and charcoal agar plates to commonly used antibiotics are compared.

[0038] The red arrow points to the detected MIC value. Figure 2 The MIC values ​​of sulfamethoxazole and levofloxacin for pertussis-sensitive strains on charcoal agar plates were significantly higher than those on the carbon-free agar plates of the present invention.

[0039] Figure 3 The results of susceptibility tests of erythromycin-resistant strains of Bordetella pertussis isolated from clinic to common antibiotics on carbon-free agar plates and charcoal agar plates are shown.

[0040] The red arrow points to the detected MIC value. Figure 3 The MIC values ​​of sulfamethoxazole and levofloxacin against pertussis-resistant strains on charcoal agar plates were significantly higher than those on the carbon-free agar plates of the present invention. DETAILED DESCRIPTION

[0041] The present invention discloses a Bordetella pertussis drug sensitivity detection culture medium and a preparation method thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The technical solutions of the present invention are as follows:

[0043] The present invention provides a carbon-free solid agar culture medium for drug susceptibility testing of Bordetella pertussis. In a specific embodiment, the culture medium includes sodium L-glutamate, L-proline, sodium chloride, potassium dihydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, Tris, cyclodextrin, and agar powder. The present invention does not particularly limit the sources of the above components; conventional commercial products in the field can be used.

[0044] In some specific embodiments of the present invention, the culture medium also includes a commercially available animal serum preparation. Serum serves as a nutrient and antidote in the charcoal-free solid agar medium of the present invention. It can serve as a substitute for defibrinated sheep blood and charcoal. It does not adsorb antibiotics, allowing the antibiotics in the culture medium to fully contact and interact with bacteria. Furthermore, the culture medium of the present invention is substantially transparent, consistent in appearance with commonly used drug sensitivity plates, facilitating the measurement of inhibition zones. In specific implementations of the present invention, the serum is added at a level of 10.0%, and the serum is selected from fetal bovine serum.

[0045] In some specific embodiments of the present invention, the culture medium further comprises formulated additives.

[0046] In a specific embodiment of the present invention, the culture medium additives include L-cysteine, niacin, vitamin C, reduced glutathione, and ferrous sulfate.

[0047] In the present invention, the pH of the carbon-free culture medium can be adjusted to 7.2 to 7.6, preferably to about 7.3 to 7.4. Acidic conditions will result in poor growth of Bordetella pertussis. In the embodiment of adjusting the pH, the preferred pH adjuster is potassium hydroxide (KOH).

[0048] The carbon-free solid culture medium for Bordetella pertussis drug sensitivity test is characterized by being prepared in the following order:

[0049] (1) Dissolve the required amounts of sodium L-glutamate (10.70 g / L), L-proline (0.24 g / L), sodium chloride (2.50 g / L), potassium dihydrogen phosphate (0.50 g / L), potassium chloride (0.20 g / L), magnesium chloride (0.10 g / L), calcium chloride (0.02 g / L), Tris (6.10 g / L), cyclodextrin (1.00 g / L), and agar powder (15 g / L) in distilled water, and adjust the pH to 7.3-7.4 with potassium hydroxide (KOH) to obtain a mixed culture medium solution A.

[0050] (2) The required amount of growth-promoting additives L-cysteine ​​0.03 g, niacin 0.002 g, vitamin C 0.01 g, reduced glutathione 0.05 g, and ferrous sulfate 0.005 g were dissolved in 20 mL of distilled water and mixed to obtain additive solution B.

[0051] (3) Sterilize the mixed culture medium solution A with high pressure steam at 121°C for 15 min. After the culture medium cools to 45°C-50°C, add serum at a ratio of 10% and mix well. Then add the prepared additive solution B (10 mL / L). Pour the mixture into the culture dish to a thickness of 4.0 ± 0.1 mm at all locations in the dish. After the culture medium solidifies, store it at 2-8°C.

[0052] The present invention provides a charcoal-free solid agar medium that allows the growth of Bordetella pertussis in the presence of bacterial-derived toxins. When used in drug susceptibility testing, this plate avoids inaccurate drug sensitivity testing caused by charcoal adsorption of antibiotics. The medium for drug susceptibility testing of Bordetella pertussis provided by the present invention is easy to prepare, exhibits good bacterial growth when coated at a 0.5 McFarland turbidity, and provides clear, easily identifiable inhibition zones. The present invention significantly improves the accuracy of drug susceptibility testing of Bordetella pertussis.

[0053] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0054] The present invention will be further described below in conjunction with the embodiments:

[0055] Example 1 Preparation of Bordetella pertussis drug sensitivity test culture medium

[0056] Weigh 10.70 g of sodium L-glutamate, 0.24 g of L-proline, 2.50 g of sodium chloride, 0.50 g of potassium dihydrogen phosphate, 0.20 g of potassium chloride, 0.10 g of magnesium chloride, 0.02 g of calcium chloride, 6.10 g of Tris, 1.00 g of cyclodextrin, and 15 g of agar powder and dissolve them in 1 L of distilled water to obtain a mixed culture medium solution A.

[0057] The pH of solution A was adjusted to 7.3-7.4 using potassium hydroxide (KOH).

[0058] The required amounts of growth-promoting additives, including 0.03 g of L-cysteine, 0.002 g of niacin, 0.01 g of vitamin C, 0.05 g of reduced glutathione, and 0.005 g of ferrous sulfate, were weighed and dissolved in 20 mL of distilled water and mixed to obtain additive solution B.

[0059] Filter the additive solution B using a filter with a pore size of 0.22 μm, and store in aliquots at -20°C.

[0060] The mixed culture medium solution A was sterilized by high pressure steam sterilization at 121°C for 15 min.

[0061] After the culture medium is cooled to 45°C-50°C, add 100 mL of serum to 900 mL of solution A at a volume ratio of 9:1 between the total volume of culture medium and serum to obtain a culture medium mixture; add 10 mL of the prepared additive B at a ratio of 10 mL to 1000 mL of the culture medium mixture and mix thoroughly.

[0062] Pour the mixed carbon-free culture medium into a 90 cm sterile culture dish to make the thickness of the dish 4.0 ± 0.1 mm.

[0063] After the culture medium is cooled and solidified, it is stored at 2-8°C and can be used for drug sensitivity testing of Bordetella pertussis.

[0064] Example 2 Application of Bordetella pertussis drug sensitivity test culture medium

[0065] Prepare a Bordetella pertussis suspension with a turbidity of 0.5 McFarland, dip a sterile cotton swab into the bacterial solution, gently remove it, rotate and press the tube wall above the bacterial solution to remove excess liquid in the cotton swab until the bacterial solution does not flow out on its own, apply the bacterial suspension to the prepared drug sensitivity test plate, rotate the plate 60 degrees each time, repeat three times to ensure that the bacterial solution is evenly distributed; finally, apply a circle on the edge of the agar plate.

[0066] After the plate is coated with the Bordetella pertussis liquid, an antimicrobial drug test strip is applied within 3 to 5 minutes, and the plate is placed in a high-humidity constant temperature incubator at 35-37°C. The MIC value is read after aerobic culture for 4-5 days.

[0067] The quality control strains were ATCC29213 and ATCC25922. Erythromycin susceptibility test strips were controlled using ATCC29213, while other antibiotics were controlled using ATCC25922. Quality control was performed by coating the strips on conventional carbon agar medium and the carbon-free drug sensitivity test medium of the present invention, respectively, according to CLSI operating procedures and quality control ranges.

[0068] Comparison of the results of drug susceptibility testing of Bordetella pertussis using conventional carbon agar medium (OXOID, UK) and the carbon-free agar medium of the present invention.

[0069] The drug sensitivity test was performed on MH plates, carbon-free agar plates and carbon agar plates using standard strains of Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213. The quality control results were as follows: Figure 1 The MIC value of the commonly used anti-pertussis drug cotrimoxazole on the carbon agar plate was significantly increased, and it was out of control at the same time as levofloxacin; the drug sensitivity results of the carbon-free agar plate and the MH plate of the present invention were both under control.

[0070] Standard strains of Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were used for quality control.

[0071] Quality control was performed in accordance with CLSI standards, with the results on MH agar plates as the reference standard. The MIC values ​​of each antibiotic on carbon agar plates and carbon-free agar plates and the quality control results are shown in Table 1:

[0072] Table 1 Quality control results of drug sensitivity test on MH plate, carbon agar plate and non-carbon agar plate

[0073]

[0074] The drug sensitivity test of 3 erythromycin-sensitive strains (No. 1-3) and 10 erythromycin-resistant strains (No. 4-13) of Bordetella pertussis was carried out on carbon-free agar plates and carbon agar plates respectively. The results are as follows Figure 2 and Figure 3 The MIC values ​​of cotrimoxazole and levofloxacin on carbon agar plates were larger than those on non-carbon agar plates, and the carbon-free agar plates of the present invention can significantly avoid the antibiotic adsorption effect of charcoal.

[0075] Table 2 Comparison of drug sensitivity test results of Bordetella pertussis sensitive strains (3 strains) on carbon agar plates and non-carbon agar plates

[0076]

[0077] Table 2 shows the results of antimicrobial susceptibility testing of erythromycin-susceptible strains of Bordetella pertussis on carbon-free and carbon-based agar plates. Piperacillin showed no difference in MIC values ​​on the two agar plates. Co-trimoxazole, affected by carbon adsorption, resulted in a 5-6 concentration difference between the two agar plates. Similarly, levofloxacin showed a 5-concentration difference between the two agar plates. Erythromycin showed a 1-concentration difference between the two agar plates, essentially meeting the consistency requirements for comparison. In antimicrobial susceptibility testing of erythromycin-susceptible strains of Bordetella pertussis, both co-trimoxazole and levofloxacin were significantly affected by carbon-based agar adsorption.

[0078] The statistical results of the differences in the MIC values ​​of each antibiotic for erythromycin-resistant strains of Bordetella pertussis on carbon-free agar plates and carbon agar plates are shown in Table 3:

[0079] Table 3 Comparison of drug sensitivity test results of Bordetella pertussis resistant strains (10 strains) on carbon agar plates and non-carbon agar plates

[0080]

[0081] Table 3 shows the results of antimicrobial susceptibility testing of erythromycin-resistant strains of Bordetella pertussis on carbon-free and carbon-based agar plates. The MIC values ​​of erythromycin and piperacillin on the two agar plates were similar. However, the adsorption effect of carbon on co-trimoxazole resulted in a 5-6 concentration difference between the two agar plates. Similarly, the MIC values ​​of levofloxacin differed by 5 concentrations between the two agar plates for the same reason. In antimicrobial susceptibility testing of erythromycin-resistant strains of Bordetella pertussis, both co-trimoxazole and levofloxacin were significantly affected by the adsorption effect of carbon-based agar plates.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A carbon-free solid culture medium for Bordetella pertussis drug sensitivity test, characterized in that: The components of the carbon-free solid culture medium include: sodium L-glutamate, L-proline, sodium chloride, potassium dihydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, Tris, cyclodextrin and agar powder; The components of the carbon-free solid culture medium do not include carbon particles.

2. The carbon-free solid culture medium according to claim 1, wherein The components of the carbon-free solid culture medium include:

3. The carbon-free solid culture medium according to claim 1 or 2, wherein The components of the carbon-free solid culture medium also include water; preferably, the water includes distilled water; Preferably, the pH value of the carbon-free solid culture medium is 7.3-7.

4.

4. The carbon-free solid culture medium according to any one of claims 1 to 3, wherein 10.0% (v / v) serum was also included.

5. The carbon-free solid culture medium according to any one of claims 1 to 4, wherein Also included are growth-promoting additives; Preferably, the growth-promoting additive comprises one or more of L-cysteine, niacin, vitamin C, reduced glutathione or ferrous sulfate; Preferably, 1L of the carbon-free solid culture medium includes 10mL of a growth-promoting additive solution; The growth-promoting additive solution comprises:

6. The method for preparing a carbon-free solid culture medium according to any one of claims 1 to 5, wherein: The steps include: Dissolve the formulated amounts of sodium L-glutamate, L-proline, sodium chloride, potassium dihydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, Tris, cyclodextrin, and agar powder in distilled water and adjust the pH to 7.3-7.4 to prepare a mixed culture medium solution A; Dissolving the formulated amount of growth-promoting additives L-cysteine, niacin, vitamin C, reduced glutathione, and ferrous sulfate in distilled water to obtain additive solution B; The mixed culture medium solution A is sterilized, cooled, mixed with serum and the additive solution B, and packaged.

7. The preparation method according to claim 6, wherein The cooling temperature is 45°C to 50°C.

8. The preparation method according to claim 6 or 7, characterized in that Add 100 mL of serum to every 900 mL of mixed culture medium solution A; and / or 10 mL of the additive solution B was added to every 1 L of the mixed culture medium solution A supplemented with serum.

9. The preparation method according to any one of claims 6 to 8, characterized in that The sterilization is high-pressure steam sterilization; preferably, the sterilization temperature is 121°C and the sterilization time is 15 minutes; The thickness of the carbon-free solid culture medium after subpackaging is 4.0±0.1 mm.

10. Use of the carbon-free solid culture medium according to any one of claims 1 to 5 or the carbon-free solid culture medium prepared by the preparation method according to any one of claims 6 to 9 in preparing a product for drug sensitivity testing of Bordetella pertussis.

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