Method for constructing Escherichia coli O157: H7 multi-drug resistance model in vitro and application

Abstract

The invention discloses a method for constructing an escherichia coli O157: H7 multi-drug resistance model in vitro and application, and particularly relates to the technical field of microorganisms. The escherichia coli O157: H7 is induced to generate multi-drug resistance by using a sub-inhibitory concentration in-vitro drug resistance induction method, and the escherichia coli O157: H7 multi-drug resistance model is constructed. The specific construction method comprises the following steps: inoculating Escherichia coli O157: H7 into a nutrient broth culture medium containing 1 / 2MIC florfenicol, culturing in a constant-temperature incubator at 37 DEG C, carrying out transfer inoculation once every 20 hours, carrying out transfer inoculation culture on the nutrient broth culture medium containing the same drug concentration for 4 generations, transferring into a next nutrient broth culture medium with higher florfenicol concentration, and carrying out culture in a constant-temperature incubator at 37 DEG C for 4 generations. And carrying out drug-resistant induction according to the method until the drug-resistant induced strain MIC of florfenicol reaches 64 times that of florfenicol before drug-resistant induction. The method for in-vitro construction of the Escherichia coli O157: H7 multi-drug resistance model and the application of the Escherichia coli O157: H7 multi-drug resistance model are important model tools for drug resistance mechanism research, drug screening, new drug development and breeding epidemic disease prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a method and application of constructing an in vitro multi-drug resistance model of Escherichia coli O157:H7. Background Art

[0002] Escherichia coli O157:H7 is a zoonotic pathogen that can cause death in livestock and humans. Its prevention and control is of great significance in public health.

[0003] E. coli O157:H7 is a zoonotic pathogen that is mainly stored in livestock and poultry hosts such as cattle, sheep, pigs and chickens. E. coli O157:H7 can spread and spread among livestock and poultry and infect humans through foodborne routes. At present, there are also reports of O157:H7 being isolated from diarrhea patients, livestock and poultry feces, commercial meat, and animals such as pigs, pigeons, chickens, and ducks. Since the first case of E. coli O157:H7 infection was reported in China in 1986, sporadic cases of E. coli O157:H7 infection have been found in more than a dozen provinces and cities such as Jiangsu, Shandong, Henan, Anhui, and Beijing, posing a potential threat of an epidemic outbreak. E. coli O157:H7 infection has become a global public health issue due to its characteristics of outbreak trend, strong pathogenicity and lethality. The World Health Organization has listed E. coli O157:H7 as a new foodborne pathogen.

[0004] Antimicrobial treatment is the first choice for preventing and controlling E. coli O157:H7. However, with the widespread and unscientific use of antimicrobial drugs, the drug resistance of E. coli O157:H7 is becoming increasingly serious, especially bacterial multidrug resistance, which can increase its tolerance to several different drugs at the same time, which has brought serious obstacles to disease prevention and treatment.

[0005] Studying the generation and spread mechanism of E. coli O157:H7 multidrug resistance, screening and developing drugs that are sensitive to multidrug-resistant E. coli O157:H7 will provide important reference value for the prevention and treatment of infection by this pathogen. A stable and easy-to-use E. coli O157:H7 multidrug resistance model is the basis for the above research. To this end, it is urgent to build an E. coli O157:H7 multidrug resistance model that is easy to operate, highly stable, has a wide range of applications, is highly scalable, close to clinical conditions, has strong controllability, high reliability, high flexibility, low cost, and a short construction time cycle. Summary of the invention

[0006] The present invention aims to provide a method and application for constructing an in vitro multidrug-resistant model of Escherichia coli O157:H7, and aims to provide an Escherichia coli O157:H7 multidrug-resistant model that is easy to operate, has high stability, a wide range of applications, strong scalability, is close to clinical conditions, has strong controllability, high reliability, high flexibility, low cost, and a short construction time period.

[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a method for constructing a multidrug resistance model of Escherichia coli O157:H7 in vitro, using a sub-inhibitory concentration in vitro resistance induction method to induce Escherichia coli O157:H7 to produce multidrug resistance, and constructing a multidrug resistance model of Escherichia coli O157:H7.

[0008] Furthermore, the specific construction method is as follows: Escherichia coli O157:H7 is inoculated into a nutrient broth medium containing 1 / 2MIC florfenicol concentration, cultured in a constant temperature incubator at 37°C, and sub-inoculated every 20 hours. After 4 generations of sub-inoculation in a nutrient broth medium containing the same drug concentration, it is transferred to the next nutrient broth medium with a higher florfenicol concentration, and drug resistance is induced according to the above method until the MIC of florfenicol for the resistance-inducing strain reaches 64 times that before resistance induction.

[0009] Furthermore, the Escherichia coli O157:H7 was isolated from a veterinary clinical pig diarrhea feces sample; the strain was cultured in an Escherichia coli O157:H7 chromogenic culture medium to obtain medium-sized, round, pink-purple colonies with smooth and moist edges and a raised center; Gram-negative, small, short rods were observed using a Gram-stained optical microscope.

[0010] Furthermore, the in vitro drug sensitivity test showed that the Escherichia coli O157:H7 was sensitive to florfenicol, amikacin, kanamycin, ceftriaxone and cefotaxime.

[0011] Furthermore, the Escherichia coli O157:H7 grew well in nutrient broth medium and was cultured in a constant temperature shaking incubator at 150 rpm and 37°C for 5-6 hours.

[0012] Furthermore, the Escherichia coli O157:H7 was constructed in vitro by induction of florfenicol gradient concentrations.

[0013] Furthermore, the minimum inhibitory concentration determination method and the KB paper disc method were used to determine the changes in the sensitivity of the Escherichia coli O157:H7 to antimicrobial drugs, evaluate the production of multidrug resistance, and verify the successful construction of the model.

[0014] Furthermore, the induced multidrug-resistant strain of Escherichia coli O157:H7 was continuously subcultured in a nutrient broth medium without antibiotics for 30 generations, and drug sensitivity tests were performed every 4 generations to observe whether the drug resistance could be stably inherited and to evaluate the stability of the model.

[0015] Furthermore, the Escherichia coli O157:H7 multidrug resistance model is resistant to penicillin, amoxicillin, cefazolin, cephradine, cephalothin, cefotaxime, doxycycline, norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin, polymyxin, tetracycline, azithromycin and florfenicol.

[0016] Furthermore, the Escherichia coli O157:H7 multidrug resistance model is resistant to penicillin, amoxicillin, cefazolin, cephradine, cephalothin, cefotaxime, doxycycline, norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin, polymyxin, tetracycline, azithromycin and florfenicol.

[0017] Compared with the prior art, this solution has the following beneficial effects:

[0018] 1. Strong controllability: Experimental conditions such as the type, concentration, and duration of antibiotic action can be precisely controlled to simulate different levels and types of drug-resistant environments and better study the mechanisms and laws of drug resistance. However, the in vivo environment is complex and changeable, making it difficult to precisely control these factors and conduct single variable research.

[0019] 2. Good repeatability: When constructing models in vivo, different animals react differently to bacterial infections, and the state of the immune system also varies, which will have a great impact on the stability of the model. In vitro models are not affected by factors such as individual differences in animals and the immune system. The experimental environment is relatively stable, which can better ensure the consistency and repeatability of experimental results. Under standardized experimental conditions, the model can be repeatedly constructed many times to obtain stable and consistent results, improve the reliability and scientificity of the experiment, and facilitate comparison and verification between different studies.

[0020] 3. High flexibility: According to the research purpose and needs, different methods of introducing or inducing drug-resistant genes can be flexibly selected to construct models with specific drug-resistant phenotypes to meet diverse research needs.

[0021] 4. High flexibility: According to the research purpose and needs, different methods of introducing or inducing drug-resistant genes can be flexibly selected to construct models with specific drug-resistant phenotypes to meet diverse research needs.

[0022] 5. Good stability: The constructed multidrug-resistant Escherichia coli O157:H7 model strain was continuously subcultured for 30 generations in nutrient broth culture medium without antibiotics in vitro, and its resistance to antibiotics did not change, indicating that the constructed Escherichia coli O157:H7 multidrug-resistant model has good stability.

[0023] 6. Low cost: The in vitro construction of a multidrug-resistant model of Escherichia coli O157:H7 does not require the use of a large number of experimental animals, nor does it require complex animal breeding facilities and management procedures, which greatly reduces the experimental costs of animal purchase, breeding, and testing.

[0024] 7. Short construction cycle: In vitro model construction can usually be completed in a short period of time, and a large number of strains with stable drug-resistant phenotypes can be quickly obtained, which speeds up the research process. In vivo model construction requires waiting for bacteria to colonize, multiply and develop drug resistance in animals, a process that often takes a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a graph showing the growth morphology of Escherichia coli O157:H7 on the Escherichia coli O157:H7 chromogenic medium in this embodiment;

[0026] Figure 2 is a growth curve diagram of the original strain of Escherichia coli O157:H7 and its multi-drug resistant model strain in this embodiment;

[0027] Figure 3 is a graph showing changes in MIC of florfenicol for the florfenicol-resistant strain of Escherichia coli O157:H7 in this example;

[0028] Figure 4 is a drug resistance stability diagram of the multi-drug resistance model of Escherichia coli O157:H7 in this embodiment;

[0029] Figure 5 This is a floR gene detection diagram of the original strain of Escherichia coli O157:H7 and its multi-drug resistant model strain in this example. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below through specific embodiments:

[0031] Example

[0032] 1. Preparation of materials

[0033] Escherichia coli O157:H7 was isolated, identified and preserved by the Veterinary Institute of Guangxi Zhuang Autonomous Region. Escherichia coli O157:H7 colorimetric medium, nutrient broth medium, nutrient agar medium, and MH medium were purchased from Beijing Aoboxing Biotechnology Co., Ltd., standard drug sensitivity paper was purchased from OXOID, florfenicol powder and dimethyl sulfoxide were purchased from Beijing Solebow Technology Co., Ltd.; PCR primers were synthesized by Shanghai Shenggong Biological Co., Ltd.; PCR Mix and DNA Marker were purchased from Beijing Kangwei Century Co., Ltd.

[0034] 2. Strain recovery and identification

[0035] Take out Escherichia coli O157:H7 from the freezer and thaw it, then inoculate it into nutrient broth medium, and culture it in a constant temperature shaking incubator at 150 rpm and 37°C. After the strain grows, streak it into Escherichia coli O157:H7 chromogenic medium, and culture it in a constant temperature incubator at 37°C for 18-20 hours. Observe whether the growth morphology of the colonies conforms to the growth characteristics of Escherichia coli O157:H7 on the chromogenic medium.

[0036] 3. Prepare florfenicol solution

[0037] Prepare a 10,240 μg / mL solution of florfenicol: accurately weigh 10.24 mg of florfenicol powder using a precision analytical balance and dissolve it in 1 mL of 80% DMSO. Filter through a 0.2 μm filter before use. Prepare the solution before use.

[0038] 4. Determination of the minimum inhibitory concentration (MIC) of florfenicol against Escherichia coli O157:H7

[0039] Escherichia coli O157:H7 was inoculated into nutrient broth medium and cultured in the logarithmic growth phase (OD600nm is 0.6-0.8), and the bacterial solution was adjusted to 0.5 McLaren suspension (1.5×108 cfu / mL). 180 μL of the diluted bacterial solution was added to the 1st to 12th wells of a sterile 96-well disposable culture plate, and then 20 μL of florfenicol solution (1,0240 μg / mL) was added to the 1st well. After mixing with a pipette, 100 μL was transferred to the next well. In this way, continuous dilution was performed to the 10th well, and then 100 μL was aspirated and discarded. 10 μL of bacterial solution was added to each well of the 1st to 11th wells. No drug was added to the 11th well as a positive control, and no bacterial solution and drug solution were added to the 12th well as a negative control. The culture plate was placed in a 37°C constant temperature incubator for 18-24 hours. The lowest drug concentration that inhibited bacterial growth (the culture fluid was transparent and clear) was defined as MIC.

[0040] 5. Construction of a multidrug-resistant model of Escherichia coli O157:H7 in vitro

[0041] The sub-inhibitory concentration in vitro drug resistance induction method was used to induce Escherichia coli O157:H7 to produce multidrug resistance and construct a multidrug resistance model of Escherichia coli O157:H7. Escherichia coli O157:H7 was inoculated in a nutrient broth medium containing 1 / 2MIC florfenicol concentration and cultured in a constant temperature incubator at 37°C. It was inoculated once every 20 hours. After 4 generations of inoculation in a nutrient broth medium containing the same drug concentration, it was transferred to the next nutrient broth medium with a higher florfenicol concentration. Drug resistance was induced in this way until the MIC of florfenicol for the drug-resistant induced strain reached 64 times before drug resistance induction. During the in vitro drug resistance induction process, the bacterial amount, culture medium volume, drug concentration, and culture conditions were precisely controlled. At the same time, the strains were identified by microscopic examination, colorimetric culture medium culture, and other methods to ensure that there was no contamination by foreign bacteria during the construction of the Escherichia coli O157:H7 multidrug resistance model. At the same time, the drug-resistant induced strains were frozen for future use.

[0042] 6. Validation test of Escherichia coli O157:H7 multidrug resistance model

[0043] The KB paper disk method was used in vitro to determine the changes in the strain's sensitivity to antimicrobial drugs before and after resistance induction. When Escherichia coli O157:H7 was induced to resist florfenicol in vitro, the induced strain showed resistance to multiple types of drugs that it was originally sensitive to. If it became resistant to more than three types of antimicrobial drugs, it could be determined that the multidrug resistance model of Escherichia coli O157:H7 was successfully constructed.

[0044] 7. Stability test of Escherichia coli O157:H7 multidrug resistance model

[0045] The multidrug-resistant model strain of Escherichia coli O157:H7 was continuously subcultured in a nutrient broth medium without antibiotics for 30 generations. The MIC of florfenicol against the model strain was measured every 5 generations to observe whether the MIC changed and to determine the stability of the model.

[0046] 8. Application of Escherichia coli O157:H7 multidrug resistance model in the study of drug resistance mechanism

[0047] By comparing the differences between the original strain and the multidrug-resistant model strain of Escherichia coli O157:H7 at the gene level, protein level, biofilm formation ability, etc., the mechanism of multidrug resistance of Escherichia coli O157:H7 in gene regulation, efflux pumps, and biofilm formation was revealed.

[0048] 9. Escherichia coli O157:H7 multidrug resistance model for screening new antimicrobial drugs

[0049] Newly synthesized compounds or ingredients extracted from natural Chinese herbal medicines are used to act on multi-drug resistant strains to observe their antibacterial activity. If a compound can inhibit the growth of drug-resistant strains, it may be a new antibacterial drug or drug lead compound.

[0050] 10. The Escherichia coli O157:H7 multidrug resistance model is used to evaluate the effectiveness of combined drug use.

[0051] Using the constructed multidrug-resistant model of Escherichia coli O157:H7, the antibacterial effects of different drug combinations can be tested. For example, a new antibiotic can be used in combination with a traditional antibiotic, or antibacterial drugs and Chinese herbal extracts can be used in combination. By observing indicators such as bacterial growth curves and bactericidal curves, the optimal combination drug regimen can be determined.

[0052] Result analysis:

[0053] 1. Cultivation characteristics of E. coli O157:H7

[0054] Growth morphology of E. coli O157:H7 on E. coli O157:H7 chromogenic medium: The original strain of E. coli O157:H7 and the multidrug-resistant model strain appear as medium-sized, round, pink-purple colonies with smooth and moist edges and a raised center on the chromogenic medium (e.g. Figure 1 coli O157:H7).

[0055] 2. Growth curves of the original strain of E. coli O157:H7 and its multidrug-resistant model strain

[0056] like Figure 2 As shown, the original strain of Escherichia coli O157:H7 and the multidrug-resistant model strain grew well in nutrient broth medium and reached the logarithmic growth phase after culturing in a constant temperature shaking incubator at 150 rpm and 37°C for 5-6 hours.

[0057] 3. Minimum inhibitory concentration of florfenicol against Escherichia coli O157:H7

[0058] Using ATCC25922 as the quality control bacteria, the MIC of florfenicol against Escherichia coli O157:H7 was 4 μg / mL.

[0059] 4. In vitro resistance of Escherichia coli O157:H7 induces horizontal resistance to florfenicol

[0060] After 32 generations of in vitro florfenicol resistance induction, the MIC of florfenicol against Escherichia coli O157:H7 increased from 4 μg / mL to 256 μg / mL (e.g. Figure 3 coli O157:H7 resistance-inducing strains developed high-level resistance to florfenicol.

[0061] 5. Results of in vitro construction of Escherichia coli O157:H7 multidrug resistance model

[0062] The results of in vitro drug sensitivity experiments showed that compared with the original strain of Escherichia coli O157:H7, the constructed Escherichia coli O157:H7 multidrug-resistant model strain was resistant to 12 antimicrobial drugs of 6 categories, and was resistant to amoxicillin, cefazolin, cefadroxil, cephalothin, cefotaxime, doxycycline, norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin, azithromycin and florfenicol (as shown in Table 1 below), and a multidrug-resistant model of Escherichia coli O157:H7 was successfully constructed.

[0063] Table 1 Results of in vitro drug sensitivity test of Escherichia coli O157:H7 original strain and its multi-drug resistant model strain Strain inhibition zone diameter (mm)

[0064]

[0065]

[0066] 6. Stability test results of Escherichia coli O157:H7 multidrug resistance model

[0067] The constructed E. coli O157:H7 multidrug-resistant strain was subcultured in nutrient broth without antibiotics for 30 generations, and the MIC of florfenicol against it was measured every 5 generations. The results showed that the MIC of florfenicol against the constructed E. coli O157:H7 multidrug-resistant model strain did not change after 30 generations of continuous subculture (e.g. Figure 4 This shows that the model has high stability.

[0068] 7. Results of the study on the changes in the expression of drug-resistant genes using the multidrug-resistant model of Escherichia coli O157:H7

[0069] The floR gene of the original strain of Escherichia coli O157:H7 and the multidrug-resistant model strain of Escherichia coli O157:H7 was detected by LAMP method. The results showed that the floR gene of the multidrug-resistant model strain of Escherichia coli O157:H7 was highly expressed, suggesting that this gene may be involved in the formation of multidrug resistance in Escherichia coli O157:H7 (such as Figure 5 shown).

[0070] The above are only embodiments of the present invention, and the common knowledge such as the known specific structures and / or characteristics in the scheme are not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a multidrug-resistant model of Escherichia coli O157:H7 in vitro, characterized in that: The subinhibitory concentration in vitro resistance induction method was used to induce Escherichia coli O157:H7 to produce multidrug resistance, and a multidrug resistance model of Escherichia coli O157:H7 was constructed.

2. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The specific construction method is as follows: Escherichia coli O157:H7 is inoculated into a nutrient broth medium containing 1 / 2MIC florfenicol concentration, cultured in a constant temperature incubator at 37°C, and sub-inoculated every 20 hours. After 4 generations of sub-inoculation in a nutrient broth medium containing the same drug concentration, it is transferred to the next nutrient broth medium with a higher florfenicol concentration, and drug resistance is induced according to the above method until the MIC of florfenicol for the resistance-inducing strain reaches 64 times that before resistance induction.

3. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The Escherichia coli O157:H7 is isolated from a veterinary clinical pig diarrhea feces sample; the strain is cultured in an Escherichia coli O157:H7 colorimetric culture medium to obtain medium-sized, smooth and moist edges, a raised center, and round pink-purple colonies; Gram-negative, small short rods are observed using a Gram-staining optical microscope.

4. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The in vitro drug sensitivity test showed that the Escherichia coli O157:H7 was sensitive to florfenicol, amikacin, kanamycin, ceftriaxone and cefotaxime.

5. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The Escherichia coli O157:H7 grew well in the nutrient broth medium and was cultured in a constant temperature shaking incubator at 150 rpm and 37° C. for 5-6 hours.

6. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The Escherichia coli O157:H7 is constructed in vitro by induction of florfenicol gradient concentrations.

7. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The minimum inhibitory concentration determination method and the KB paper disk method were used to determine the changes in the sensitivity of the Escherichia coli O157:H7 to antimicrobial drugs, evaluate the production of multidrug resistance, and verify the successful construction of the model.

8. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The induced multidrug-resistant strain of Escherichia coli O157:H7 was continuously subcultured in a nutrient broth medium without antibiotics for 30 generations, and drug sensitivity tests were performed every 4 generations to observe whether the drug resistance could be stably inherited and to evaluate the stability of the model.

9. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The Escherichia coli O157:H7 multidrug resistance model is resistant to penicillin, amoxicillin, cefazolin, cefradine, cephalothin, cefotaxime, doxycycline, norfloxacin, ofloxacin, ciprofloxacin, enrofloxacin, polymyxin, tetracycline, azithromycin and florfenicol.

10. The method for constructing an in vitro multidrug resistance model of Escherichia coli O157:H7 according to claim 1, characterized in that: The Escherichia coli O157:H7 multidrug resistance model is applied to the study of the multidrug resistance mechanism of Escherichia coli O157:H7, the screening of sensitive drugs, the study of cross-resistance of antibacterial drugs and disinfectants, and the screening of probiotics against multidrug-resistant Escherichia coli O157:H7.

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