A method for culturing microaerophilic bacteria and its application in drug susceptibility testing

Abstract

This invention provides a method for culturing microaerophilic bacteria and its application in drug susceptibility testing. The method includes the following steps: Step S1, selecting a suitable-sized porous sponge material, sterilizing it at high temperature, and placing it in a culture vessel; the porous sponge material is bamboo fiber, cotton fiber, or polyurethane; Step S2, preparing a liquid culture medium, sterilizing it at high temperature, inoculating it with the microaerophilic bacteria to be cultured, and thoroughly mixing it into a suspension to obtain a liquid culture medium containing the bacteria; Step S3, adding the liquid culture medium containing the bacteria dropwise onto the porous sponge material in the culture vessel; placing the culture vessel in an incubator for 12-72 hours. Using the technical solution of this invention, the results are stable, highly reproducible, simple to operate, and can be used for batch experiments, resulting in better culture effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a culture method of microaerophilic bacteria and application thereof in drug sensitivity test. BACKGROUND

[0002] Microaerophilic bacteria are a kind of bacteria that grow better under the condition of oxygen concentration lower than the normal oxygen level in the atmosphere (about 21%). The oxygen concentration they need is usually between 2% and 10%, and too much oxygen is toxic to them. This kind of bacteria usually lives in low-oxygen environments, such as deep soil, water bottom, animal intestines, etc. Common microaerophilic bacteria include Campylobacter, Helicobacter, Pseudomonas, etc. Microaerophilic bacteria are closely related to human health. The human intestinal tract is a microaerobic environment, and a large number of intestinal probiotics and pathogenic bacteria can only grow under microaerophilic conditions. For example, Helicobacter pylori and Campylobacter jejuni are microaerophilic bacteria, and the research on these bacteria requires strict culture conditions, which is a microaerobic environment of 5% O2, 10% CO2 and 85% N2.

[0003] For microaerophilic bacteria, the current general culture method is solid plate culture, which inoculates bacteria on the surface of solid culture medium and then places it in a low-oxygen environment for culture. This culture method has the following problems: first, the surface area of solid culture medium is limited; second, the diffusion effect of nutrients on solid culture medium is poor, and bacteria cannot continue to grow after consuming the surrounding nutrients; in addition, the operation of solid plate culture is relatively cumbersome and time-consuming. Therefore, it is difficult to collect a large number of microaerophilic bacteria by solid plate culture. Some research reports also use liquid culture method to culture microaerophilic bacteria, but in practice, liquid culture of microaerophilic bacteria has the problem of poor stability, mainly because the dissolved oxygen content in the liquid is affected by many factors such as oxygen concentration in the air, liquid agitation frequency and speed, liquid viscosity, etc. Researchers can only control the oxygen concentration in the air in the culture environment, but cannot accurately control the dissolved oxygen content in the liquid. Too low or too high dissolved oxygen will lead to failure of microaerophilic bacteria culture. Therefore, it is necessary to develop a more efficient culture method for microaerophilic bacteria.

[0004] Antibiotics are the most important drugs for treating bacterial infections. In recent years, drug-resistant bacteria, even superbugs, have emerged continuously, and the clinical treatment urgently needs new antibiotics, which also promotes the screening and drug research of new antibiotics. The conventional antibacterial activity test (also known as drug sensitivity test) generally adopts filter paper disc diffusion method or liquid culture method. The filter paper disc diffusion method is to drop the drug on the filter paper disc, paste it on the surface of the solid culture medium inoculated with bacteria, and culture for a period of time. Then, whether the drug has antibacterial activity is confirmed by measuring the size of the transparent circle (representing no bacterial growth) around the filter paper disc. The filter paper disc method has the disadvantage that it is difficult to control the real concentration of the drug diffused from the paper disc into the culture medium, and it cannot give a clear dose-effect relationship. The liquid culture is to add different concentrations of drugs into the liquid, and quantitatively determine the growth of bacteria, so as to give a clear dose-effect relationship. This method is suitable for conventional aerobic bacteria and anaerobic bacteria, but it is not suitable for the antibacterial test of microaerophilic bacteria such as Helicobacter pylori because the microaerophilic bacteria are unstable in the liquid culture medium. Therefore, it is necessary to develop a new antibacterial test method for microaerophilic bacteria. SUMMARY

[0005] In view of the above technical problems, the present application discloses a culture method for microaerophilic bacteria and its application in drug sensitivity test, which is simple to operate, low in cost and good in culture effect.

[0006] To this end, the technical scheme adopted by the present application is as follows:

[0007] A culture method for microaerophilic bacteria, comprising the following steps:

[0008] Step S1, selecting a porous sponge material of appropriate size, sterilizing at high temperature, and placing it in a culture vessel; the porous sponge material is bamboo fiber, cotton fiber or polyurethane;

[0009] Step S2, configuring a liquid culture medium, sterilizing at high temperature, inoculating the microaerophilic bacteria to be cultured, thoroughly mixing to form a suspension, and obtaining a liquid culture medium containing the bacteria;

[0010] Step S3, dropping the liquid culture medium containing the bacteria onto the porous sponge material in the culture vessel; placing the culture vessel in a culture box and culturing for 12-72 hours.

[0011] By adopting this technical scheme, the porous sponge structure material is added to the liquid culture medium, which forms a scaffold in the liquid, and the surface tension causes a layer of liquid film to form on the surface of the porous sponge material, which is more conducive to the culture of the bacteria, and the culture effect is significantly better than that of the simple liquid culture method.

[0012] As a further improvement of the present application, the microaerophilic bacteria culture method further comprises step S4: after the bacteria grow to a sufficient amount, the sponge is squeezed, the sponge is flushed with sterile culture medium or water solution, and the liquid is collected, thereby obtaining the desired microaerophilic bacteria.

[0013] As a further improvement of the present application, the porous sponge material has a density of 18-55 g / m 3 . Further, the porous sponge material has a density of 18-45 g / m 3 ; further preferably, the porous sponge material has a density of 30-45 g / m 3 .

[0014] As a further improvement of the present application, the porous sponge material is a polyurethane sponge.

[0015] As a further improvement of the present application, in step S3, the culture box has a three-gas environment of 5% O2, 10% CO2, and 85% N2.

[0016] As a further improvement of the present application, in step S2, the liquid culture medium comprises BHI broth medium.

[0017] The present application further discloses the use of the microaerophilic bacteria culture method as described above in drug sensitivity testing.

[0018] As a further improvement of the present application, the use comprises: after the culture in step S3 is completed, MTT solution is added to the culture vessel, the porous sponge material is squeezed to mix the liquid, and the culture is continued at 37°C for 4 hours;

[0019] Then, anhydrous ethanol is added to the culture vessel to dissolve the blue-purple crystalline formazan formed by MTT metabolism;

[0020] The solution in the culture vessel is taken to a cuvette or an enzyme-labeled plate, and the absorbance at 570 nm is measured using a spectrophotometer or an enzyme-labeled instrument.

[0021] Further, the volume of the added anhydrous ethanol is 2-4 times the volume of the liquid in the culture vessel.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The technical scheme of the present application is that a suitable porous material is placed in a liquid culture medium to form a scaffold structure, liquid forms a film on the surface of the porous material through surface tension, and a stable micro-aerobic liquid environment is formed. The liquid simulates the environment of micro-aerobic bacteria in a biological body, the growth speed of the micro-aerobic bacteria is faster, and the total amount of harvested bacteria is more. Further, different concentrations of drugs can be added in the system according to needs, and the total amount of bacteria can be determined by using the MTT method to determine the bacterial activity, so that the antibacterial activity test can be performed. The method has stable results, high repeatability, simple operation, and can be batched for experiments. The sensitivity to drugs is high, and a clear dose-effect relationship can be given, and the method can be widely applied to the culture and drug sensitivity test of micro-aerobic bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the effect comparison result of culturing Helicobacter pylori by using different types of porous materials in the embodiment 1 of the present application.

[0025] Figure 2 is the effect comparison result of culturing Helicobacter pylori by using different densities of polyurethane sponge in the embodiment 2 of the present application.

[0026] Figure 3 is the effect comparison of culturing Helicobacter pylori by using polyurethane sponge and conventional liquid method in the embodiment 3 of the present application.

[0027] Figure 4 is the effect comparison of culturing Campylobacter jejuni by using polyurethane sponge and conventional liquid method in the embodiment 3 of the present application.

[0028] Figure 5 is the result of using polyurethane sponge and conventional culture method for antibacterial drug screening in the embodiment 4 of the present application. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present application are further described in detail below.

[0030] Embodiment 1

[0031] In this embodiment, Helicobacter pylori is cultured by using a liquid culture method and a plurality of porous materials. 1.5x10 8 CFU / mL of bacterial liquid is inoculated, and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial activity is determined by using the MTT method. The specific steps include the following steps:

[0032] 1. Preparation of culture medium and a plurality of porous materials: Columbia blood agar and BHI broth medium are prepared, and a puncher is used to prepare the plurality of porous materials with consistent shapes. The plurality of porous materials are placed in a high-pressure steam sterilization pot and sterilized at 121°C for 20 minutes.

[0033] 2. After dilution, the H. pylori was coated on the Columbia blood agar plate and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial layer was scraped into BHI broth medium, and the concentration of the bacterial solution was determined by the McFarland turbidity method. The solution was diluted to 0.5 McFarland turbidity (1.5 x 10 8 CFU / mL) as the original liquid, and further diluted 10 times with the culture medium as the test solution.

[0034] 3. One 24-well plate was prepared, and different types of sponge materials were added to each well. The experimental groups were as follows: the control group was liquid culture (without adding porous materials), the experimental group 1 was bamboo fiber sponge material, the experimental group 2 was cotton fiber sponge material, and the experimental group 3 was polyurethane sponge. The density of the three sponge materials was 30 g / m 3 . Each well was added with 300 μL of culture medium containing bacteria (test solution), and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0035] 4. The bacterial activity of H. pylori was determined by the MTT method:

[0036] (1) 30 μL of MTT was added to each well of the 24-well plate, and the liquid was mixed and incubated at 37°C for 4 h after squeezing the sponge.

[0037] (2) The liquid in the 24-well plate was transferred to a 1.5 mL EP tube, and 600 μL of anhydrous ethanol was added to each well of the sponge. The blue-purple substance was fully dissolved and extracted by repeated squeezing.

[0038] (3) 600 μL of anhydrous ethanol in the 24-well plate was transferred to the 1.5 mL EP tube in step (2), mixed, and centrifuged at 12000 x g for 5 min. 100 μL of supernatant was transferred to a 96-well enzyme-labeled plate.

[0039] (4) The absorbance value at 570 nm was determined by the enzyme-linked detector, and the specific detection results were as Figure 1 . It can be seen that the use of different types of porous materials (bamboo fiber, cotton fiber, and polyurethane sponge) for culturing H. pylori is better than the traditional liquid culture method, and the culture effect of polyurethane sponge is the best.

[0040] Example 2

[0041] In Example 1, the sponge materials of different materials (bamboo fiber, cotton fiber, polyurethane) have different properties (such as water absorption), thereby affecting the surface tension of the liquid, and further affecting the growth of Helicobacter pylori, but the culture effect is better than the liquid culture method of the prior art. Because the pore size of the sponge material can also affect the surface tension of the liquid, based on Example 1, polyurethane sponge material is selected to investigate the influence of different pore sizes, i.e., different densities of sponge material, on the culture effect of Helicobacter pylori. Specifically, 18 g / m 3 , 30 g / m 3 , 45 g / m 3 , and 55 g / m 3 Four densities of polyurethane sponge are used to culture Helicobacter pylori, and the influence of different densities of polyurethane sponge material on the growth of Helicobacter pylori is compared.

[0042] The specific implementation method and steps are carried out according to Example 1, and the test results are shown in Table 2. Figure 2 As can be seen, the pores of sponges of different densities are different, and the higher the density, the smaller the pore. In the polyurethane sponge of different densities for culturing Helicobacter pylori, in the density range of 18-45 g / m 3 , the higher the density of the polyurethane sponge, the better the culture effect of Helicobacter pylori. Moreover, when the density of the polyurethane sponge reaches 55 g / m 3 , the culture effect of Helicobacter pylori slightly decreases.

[0043] Example 3

[0044] This example is a comparison of the culture effects of Helicobacter pylori and Campylobacter jejuni using polyurethane sponge and conventional liquid method. Mainly, 45 g / m 3 Density of polyurethane sponge is placed or not placed in the well plate, 1.5×10 5 , 1.5×10 6 , 1.5×10 7 , and 1.5×10 8 CFU / mL of Helicobacter pylori or Campylobacter jejuni liquid is inoculated, and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2d, and MTT method is used to determine the activity of bacteria. Specifically, the following steps are included:

[0045] 1. Preparation of culture medium and sponge material: Columbia blood agar and BHI broth medium are prepared, a puncher is used to prepare polyurethane sponge pieces with consistent shape, and the polyurethane sponge pieces are placed in a steam sterilization pot and sterilized at 121°C for 20 min.

[0046] 2. After dilution and coating of the Columbia blood agar plates with H. pylori and C. jejuni, the plates were incubated in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial lawn was scraped into BHI broth medium, and the concentration of the bacterial solution was determined by the McFarland turbidity method. The solution was diluted to a McFarland turbidity of 0.5 (1.5 x 10 8 CFU / mL).

[0047] 3. Four 24-well plates were prepared, two plates for polyurethane sponge culture and the other two plates for BHI broth liquid culture.

[0048] 4. H. pylori 2 24-well plates were inoculated: one 24-well plate was coated with polyurethane sponge material, and the other 24-well plate did not contain sponge material. The experimental groups in each 24-well plate were as follows: the experimental groups were added to the solution at four concentrations of 1.5 x 10 5 , 1.5 x 10 6 , 1.5 x 10 7 and 1.5 x 10 8 CFU / mL, with four replicate wells in each group. Each well contained 300 μL of liquid. The plates were incubated in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0049] 5. C. jejuni 2 24-well plates were inoculated: one 24-well plate was coated with polyurethane sponge material, and the other 24-well plate did not contain sponge material. The experimental groups in each 24-well plate were as follows: the blank group was BHI liquid medium, and the experimental groups were added to the solution at four concentrations of 1.5 x 10 5 , 1.5 x 10 6 , 1.5 x 10 7 and 1.5 x 10 8 CFU / mL, with four replicate wells in each group. Each well contained 300 μL of liquid. The plates were incubated in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0050] 6. The bacterial activity of H. pylori and C. jejuni was determined by the MTT method described in Example 1. 30 μL of MTT was added to each well, and the liquid was mixed by squeezing the sponge and then incubated at 37°C for 4 hours.

[0051] 7. The liquid in the 24-well plate wells was transferred to a 1.5 mL EP tube, and 600 μL of anhydrous ethanol was added to each well. The liquid was repeatedly squeezed, and the blue-purple substance was fully dissolved and extracted.

[0052] 8. 600 μL of anhydrous ethanol in the 24-well plate was transferred to the 1.5 mL EP tube in the previous step, mixed, and then centrifuged at 12000 x g for 5 minutes. 100 μL of supernatant was transferred to a 96-well enzyme-labeled plate.

[0053] 9. The absorbance at 570 nm was measured using a microplate reader, and the effects of the two methods of culturing H. pylori were compared as shown in FIG. 6. Figure 3 The effects of the two methods of culturing C. jejuni were compared as shown in FIG. 7. Figure 4 As can be seen, the proliferation and activity of H. pylori and C. jejuni were both better in the sponge material culture than in the liquid culture at the four different inoculation concentrations.

[0054] Example 4

[0055] This example compares the sensitivity of H. pylori to drugs using sponge material and liquid culture, respectively. 45 g / m2of polyurethane sponge material was added or not added, 1.5 x 10 3 CFU / mL of bacterial solution was inoculated and different concentrations of positive drugs against H. pylori were added, and the mixture was cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days, and the bacterial activity was determined by the MTT method. The specific steps include the following: 7

[0056] 1. Preparation of culture medium and sponge material: Columbia blood agar and BHI broth medium were prepared, and a puncher was used to prepare multiple sponge pieces with consistent shapes, which were placed in a high-pressure steam sterilization pot and sterilized at 121°C for 20 min.

[0057] 2. Preparation of quadruple drug mother liquor against H. pylori, which contains ① 500 mg / mL amoxicillin, ② 500 mg / mL clarithromycin, ③ 20 mg / mL omeprazole, and ④ 220 mg / mL bismuth citrate potassium. The mother liquor was filtered with a 0.22 μm filter membrane to remove bacteria and stored for later use.

[0058] 3. After H. pylori was diluted and coated on Columbia blood agar plates, the plates were cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days, and the bacterial coating was scraped into BHI broth medium. The bacterial solution concentration was determined by the McFarland turbidity method, and was diluted to 0.5 McFarland turbidity (1.5 x 10 8 CFU / mL).

[0059] 4. Two 24-well plates were prepared, one containing sponge material and the other not containing sponge material, for BHI broth liquid culture:

[0060] 5. Inoculation of H. pylori in the two 24-well plates: each 24-well plate was divided into groups as follows:

[0061] The control group was 1.5 x 10 7 ​CFU / mL bacterial solution, experimental group 1 was 0.625 mg / mL amoxicillin + 0.625 mg / mL clarithromycin + 0.025 mg / mL omeprazole + 0.275 mg / mL bismuth citrate, experimental group 2 was 1.25 mg / mL amoxicillin + 1.25 mg / mL clarithromycin + 0.05 mg / mL omeprazole + 0.55 mg / mL bismuth citrate, experimental group 3 was 2.5 mg / mL amoxicillin + 2.5 mg / mL clarithromycin + 0.1 mg / mL omeprazole + 1.1 mg / mL bismuth citrate, experimental group 4 was 5 mg / mL amoxicillin + 5 mg / mL clarithromycin + 0.2 mg / mL omeprazole + 2.2 mg / mL bismuth citrate, 4 replicates in each group. Each well contained 1.5 x 10 7 CFU / mL bacterial solution. Incubate in a 37℃ three-gas incubator (5% O2, 10% CO2, 85% N2) for 2 days.

[0062] 6. MTT method was used to determine the activity of H. pylori bacteria respectively:

[0063] (1) Four 24-well plates of H. pylori were prepared, 30 μL of MTT was added to each well, and the liquid was mixed by squeezing the sponge and then incubated at 37℃ for 4 hours.

[0064] (2) Transfer the liquid in the 24-well plate to a 1.5 mL EP tube, and then add 600 μL of anhydrous ethanol to each well, and repeatedly squeeze to dissolve thoroughly.

[0065] (3) Transfer 600 μL of anhydrous ethanol in the 24-well plate to the 1.5 mL EP tube in the previous step, mix well and centrifuge at 12000 x g for 5 min, and then transfer 100 μL of supernatant to a 96-well enzyme-labeled plate.

[0066] (4) Use an enzyme-labeled instrument to measure the absorbance value at 570 nm, and the specific test results are shown in Figure 5 . It can be seen that, in the conventional liquid culture, antibiotics have a significant inhibitory effect on H. pylori, but there is no significant difference between different concentration treatment groups, and the difference between the treatment group with antibiotics and the control group without antibiotics is small. In contrast, after adding polyurethane sponge material, the bacteria grow better than without sponge, the difference between the treatment group with drugs and the control group without drugs is greater, and the difference between different concentration drug treatment groups is also significant. It is shown that, when using the method of adding polyurethane sponge for drug sensitivity test, H. pylori is more sensitive than ordinary liquid culture method. Therefore, this method can more effectively screen antibacterial drugs, and when using conventional liquid culture method, potential valuable drugs may be missed due to low sensitivity.

[0067] The above is further detailed description of the present application in combination with specific preferred embodiments, and cannot be deemed as limitation of the specific implementation of the present application to these descriptions. For those skilled in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made, and all should be deemed as falling within the protection scope of the present application.

Claims

1. A method for culturing a microaerophile, characterized by: The method comprises the following steps: Step S1, selecting a porous sponge material with a proper size, sterilizing at high temperature, and placing the material in a culture vessel; the porous sponge material is polyurethane; Step S2, configuring a liquid culture medium, inoculating microaerophilic bacteria to be cultured after high-temperature sterilization, fully mixing to form a suspension, and obtaining a liquid culture medium containing the bacteria; the microaerophilic bacteria are Helicobacter pylori or Campylobacter jejuni; Step S3, adding the liquid culture medium containing the bacteria to the porous sponge material in the culture vessel; placing the culture vessel in an incubator for culture for 12-72 hours; The method further comprises Step S4, after the bacteria grow to a sufficient amount, squeezing the sponge, flushing the sponge with sterile culture medium or an aqueous solution, and collecting the liquid to obtain the required microaerophilic bacteria.

2. The culturing method of the microaerophile according to claim 1, characterized by: In Step S3, the environment of the incubator is a three-gas environment of 5% O2, 10% CO2, and 85% N2.

3. The microaerobic culture method according to claim 1, characterized by: In Step S2, the culture medium is BHI broth medium.

4. Application of the microaerophilic bacteria culture method according to any one of claims 1-3 in drug sensitivity testing; after the culture in Step S3 is completed, adding MTT solution to the culture vessel, squeezing the porous sponge material, mixing the liquid, and continuing to culture at 37°C for 4 hours; Then adding anhydrous ethanol to the culture vessel to dissolve the blue-purple crystalline formazan formed after MTT metabolism; Taking the solution in the culture vessel to a cuvette or an enzyme-labeled plate, and using a spectrophotometer or an enzyme-labeled instrument to measure the absorbance at 570 nm.

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