阅读说明：本技术 消除大肠杆菌药物耐药性的中药组方及其筛选方法与应用 (Traditional Chinese medicine formula for eliminating drug resistance of escherichia coli and screening method and application thereof ) 是由 张永英 钟翠红 王芝超 石玉祥 郝贺 旷年玲 左丽 于 2021-08-03 设计创作，主要内容包括：本发明提供了一种组成消除耐药中药组方及筛选方法,试验选择4株多重耐药大肠杆菌作为研究对象,分别以亚抑菌浓度(1/2MIC)中药与4株菌分别共培养72h。然后采用凝胶电泳法、双纸片法、荧光定量PCR和生物膜阳性筛选方法检测细菌培养物,根据体外消除细菌耐药效果,确定消除耐药效果较好单味中药组成组方；再通过动物试验,检查各组心肝组织病理学变化,并统计各组治愈率、有效率、死亡率,并验证组方体内消除大肠杆菌耐药的效果。本发明组方山楂黄连组方+抗生素效果优于传统组方三黄汤,说明利用此种方法组成的组方治疗效果优于根据中药辩证论治组成的传统组方。(The invention provides a traditional Chinese medicine formula for eliminating drug resistance and a screening method, wherein 4 strains of multi-drug-resistant escherichia coli are selected as research objects in an experiment and are respectively co-cultured for 72 hours by using a traditional Chinese medicine with sub-inhibitory concentration (1/2MIC) and 4 strains of bacteria. Then detecting the bacterial culture by adopting a gel electrophoresis method, a double-paper-sheet method, a fluorescence quantitative PCR (polymerase chain reaction) method and a biomembrane positive screening method, and determining a formula consisting of a single Chinese medicament with a better drug resistance elimination effect according to the in-vitro drug resistance elimination effect of bacteria; and then, through animal tests, the histopathological changes of heart and liver tissues of each group are checked, the cure rate, the effective rate and the death rate of each group are counted, and the effect of eliminating the drug resistance of escherichia coli in the formula body is verified. The effect of the hawthorn and coptis prescription and the antibiotic of the prescription of the invention is superior to that of the traditional prescription sanhuang decoction, which shows that the treatment effect of the prescription prepared by the method is superior to that of the traditional prescription prepared according to dialectical treatment of traditional Chinese medicines.)

1. A screening method of a traditional Chinese medicine formula for eliminating drug resistance of escherichia coli is characterized by comprising the following steps: the method comprises the following steps:

A. screening single traditional Chinese medicines according to a mechanism for eliminating escherichia coli drug resistance, wherein the drug resistance mechanism mainly comprises R plasmid mediation, ESBLs generation, active efflux system and biofilm formation;

B. screening single Chinese medicine for eliminating R plasmid by gel electrophoresis;

C. screening single Chinese medicine for inhibiting ESBLs activity by using a double-paper sheet method;

D. screening single traditional Chinese medicines for reducing the expression quantity of the efflux system genes Acra-mRNA and Acrb-mRNA by using a fluorescent quantitative PCR method;

E. screening out a single traditional Chinese medicine for reducing the adhesion of the biological membrane by using a biological membrane positive screening method;

F. and C, forming the single traditional Chinese medicine screened in the steps B-E into a traditional Chinese medicine composition.

2. The screening method of traditional Chinese medicine composition for eliminating drug resistance of escherichia coli according to claim 1, wherein the screening method comprises the following steps: the screening method further comprises the following steps:

a-1, preparing a traditional Chinese medicine water extract: soaking the single Chinese medicines for 30min, decocting with slow fire for 2 times, mixing, concentrating, filtering, and sterilizing to obtain single Chinese medicine water extract with concentration of 1g/mL (calculated on crude drugs);

a-2, preparing escherichia coli liquid: inoculating the frozen and preserved bacterium liquid into an LB liquid culture medium, culturing overnight at 37 ℃, streaking and inoculating the activated bacterium liquid into a Macconkey agar plate, performing inverted culture overnight at 37 ℃, selecting a single colony, inoculating the single colony into the LB liquid culture medium, and culturing overnight at 37 ℃ to obtain an Escherichia coli liquid.

3. A traditional Chinese medicine formula for eliminating drug resistance of escherichia coli is characterized in that: the traditional Chinese medicine composition comprises 1 part of hawthorn and 1 part of coptis chinensis, the traditional Chinese medicine composition is used in combination with an antibiotic, the antibiotic is cefotaxime, and the weight part ratio of the antibiotic to the traditional Chinese medicine composition is (2-5): 10.

4. the application of the traditional Chinese medicine formula is characterized in that: the traditional Chinese medicine formula is used for eliminating drug resistance of chicken-derived escherichia coli beta-lactam drugs.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine formula for eliminating drug resistance of escherichia coli and a screening method thereof.

Background

In recent years, colibacillosis is widely prevalent in domestic poultry farms, the morbidity and mortality are high, the development of poultry industry is seriously threatened, and huge economic loss is caused. For a long time, antibacterial drugs play an extremely important role in the treatment of this disease. However, the wide, continuous and unreasonable use of antibacterial drugs increases the generation and diffusion of drug-resistant escherichia coli, especially escherichia coli producing extended-spectrum beta-lactamase, which causes unsatisfactory drug treatment effect, and has become one of the difficulties hindering the development of poultry industry. It follows that the development of resistance by bacteria is a major cause of the above-mentioned results. Thus, reducing the resistance of E.coli and restoring sensitivity to some or all antibiotics is of great importance for the clinical treatment of bacterial infections and for preventing the spread of bacterial resistance. There are various ways to reduce the drug resistance of bacteria, and low-toxicity and low-cost traditional Chinese medicines and natural plants are concerned, so that the elimination of the drug resistance of chicken-origin escherichia coli by using the traditional Chinese medicines becomes a research direction which can be deeply explored.

The traditional Chinese medicine enables bacteria to recover the sensitivity to antibiotics by eliminating R plasmids, inhibiting the activity of ESBLs, influencing the growth rate and the protein expression quantity of the bacteria, inhibiting an active efflux system, forming a bacterial biofilm and other action mechanisms. Research shows that the traditional Chinese medicine can change the transcription and translation levels of specific drug-resistant genes and non-specific drug-resistant genes of escherichia coli, and fundamentally reverse the drug resistance of bacteria. However, multiple drug resistance mechanisms of bacteria prove that multiple drug resistance is determined by multiple factors, a single reversal agent cannot inhibit all drug resistance mechanisms, and the combination of multiple reversal agents can be more effective for multiple drug resistance mechanisms. By this strategy, combinations of monomeric or complex natural extracts can exert a synergistic antagonistic effect and influence the drug multi-target. At present, the compatibility of traditional Chinese medicines follows the traditional theory of veterinarian in the combination of dialectical treatment of traditional Chinese medicines, and the invention can eliminate multiple drug resistance of bacteria in all directions and at multiple angles aiming at different drug resistance mechanisms and prolong the service life of antibiotics.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a traditional Chinese medicine composition for safely and effectively eliminating drug resistance to chicken-origin escherichia coli beta-lactam drugs and a screening method thereof.

The technical scheme adopted by the invention is as follows: a screening method of a traditional Chinese medicine formula for eliminating drug resistance of escherichia coli comprises the following steps:

A. screening single traditional Chinese medicines according to a mechanism for eliminating escherichia coli drug resistance, wherein the drug resistance mechanism mainly comprises R plasmid mediation, ESBLs generation, active efflux system and biofilm formation;

B. screening single Chinese medicine for eliminating R plasmid by gel electrophoresis;

C. screening single Chinese medicine for inhibiting ESBLs activity by using a double-paper sheet method;

D. screening single traditional Chinese medicines for reducing the expression quantity of the efflux system genes Acra-mRNA and Acrb-mRNA by using a fluorescent quantitative PCR method;

E. screening out a single traditional Chinese medicine for reducing the adhesion of the biological membrane by using a biological membrane positive screening method;

F. and C, forming the single traditional Chinese medicine screened in the steps B-E into a traditional Chinese medicine composition.

Further, the screening method further comprises:

a-1, preparing a traditional Chinese medicine water extract: soaking the single Chinese medicines for 30min, decocting with slow fire for 2 times, mixing, concentrating, filtering, and sterilizing to obtain single Chinese medicine water extract with concentration of 1g/mL (calculated on crude drugs);

a-2, preparing escherichia coli liquid: inoculating the frozen and preserved bacterium liquid into an LB liquid culture medium, culturing overnight at 37 ℃, streaking and inoculating the activated bacterium liquid into a Macconkey agar plate, performing inverted culture overnight at 37 ℃, selecting a single colony, inoculating the single colony into the LB liquid culture medium, and culturing overnight at 37 ℃ to obtain an Escherichia coli liquid.

A traditional Chinese medicine formula for eliminating drug resistance of escherichia coli comprises 1 part of hawthorn and 1 part of coptis chinensis, the traditional Chinese medicine composition is used in combination with an antibiotic, the antibiotic is cefotaxime, and the weight part ratio of the antibiotic to the traditional Chinese medicine composition is (2-5): 10.

the application of a traditional Chinese medicine formula is used for eliminating drug resistance of chicken-derived escherichia coli beta-lactam drugs.

The beneficial effects obtained by the invention are as follows: in the invention, 4 strains of multi-drug-resistant escherichia coli are selected as research objects in the test, the Minimum Inhibitory Concentration (MIC) of 6 traditional Chinese medicine aqueous extracts to 4 strains of drug-resistant escherichia coli is determined by a gradient dilution method, and the sub-inhibitory concentration (1/2MIC) and 4 strains of bacteria are respectively co-cultured for 72 hours; detecting the culture by adopting a gel electrophoresis method, a double-paper-sheet method, a fluorescent quantitative PCR (polymerase chain reaction) method and a biomembrane positive screening method, and determining a formula consisting of a single Chinese medicament with a better medicament resistance elimination effect according to the medicament resistance elimination effect of bacteria in vitro; (Hawthorn, glossogyne; Hawthorn, Coptis); and then, through animal tests, the histopathological changes of the heart and the liver of each group are checked, the cure rate, the effective rate and the death rate of each group are counted, and the effect of eliminating the drug resistance of the escherichia coli in the formula is detected. The hawthorn coptis prescription and cefotaxime screened by the invention have the treatment effective rate of 93.3, the cure rate of 90 percent and the death rate of 6.7 percent; the treatment effect is optimal and is superior to the hawthorn-tongue grass formula, antibiotics and the traditional formula of three-yellow decoction.

Drawings

FIG. 1a is a plasmid electrophoretogram before and after treating the 1 st chicken-origin Escherichia coli with a single Chinese medicine;

FIG. 1b is a plasmid electrophoretogram before and after treating the 2 nd chicken-origin Escherichia coli with a single Chinese medicine;

FIG. 1c is a plasmid electrophoretogram before and after the treatment of 3 rd strain of chicken-origin Escherichia coli with a single Chinese medicine;

FIG. 1d is a plasmid electrophoretogram before and after the 4 th chicken-origin Escherichia coli is treated by a single Chinese medicine;

FIG. 2 shows the results of the analysis of the adhesion of 4 chicken-origin Escherichia coli treated with the aqueous extract of Chinese medicinal materials;

FIG. 3 shows the relative expression quantity (2-), of the efflux pump gene AcrA-mRNA, before and after treatment of 4 strains of chicken-derived Escherichia coli with the aqueous extract of Chinese medicinal materials^△△CT)；

FIG. 4 shows the relative expression amount (2-) -mRNA of efflux pump gene AcrB before and after 4 strains of chicken-derived Escherichia coli are treated with Chinese medicinal aqueous extract^△△CT)；

FIG. 5 is a chicken pathological anatomy caesarean section;

FIG. 6 is a colony morphology of an isolated strain;

FIG. 7 is a bacterial morphology of the isolated strain;

fig. 8 is h.e. staining (10 × 10) of healthy chicken hearts;

fig. 9 is h.e. staining (10 × 10, 20 × 10) of diseased chicken hearts;

fig. 10 is h.e. staining (10 × 10) of healthy chicken liver;

fig. 11 is h.e. staining (10 × 10) of diseased chicken livers;

FIG. 12 shows the result of electrophoresis of the amplified product of the isolated strain 16S rDNA gene;

wherein a in FIGS. 1a-1 d: a coptis treated strain; b: a glossogyne spp-treated strain; c: dandelion-treated strain d: rhubarb treated strains; e: a scutellaria treated strain; f: humifuse euphorbia herb treated strain.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Example 1 screening of Single Chinese medicine for eliminating R plasmid of Chicken-derived Escherichia coli

(1) Preparing a traditional Chinese medicine water extract: scutellariae radix, Coptidis rhizoma, radix et rhizoma Rhei, herba Euphorbiae Humifusae, herba Taraxaci, and herba Hedyotidis Diffusae are all purchased from homo-TANG (Handan), soaked for 30min, decocted with slow fire for 2 times, combined and concentrated, filtered to remove bacteria, and the concentration is calculated as crude drug (1 g/mL).

(2) Preparing chicken-origin escherichia coli liquid: the cryopreserved cells were inoculated into LB liquid medium and cultured overnight at 37 ℃. And streaking the activated bacterial liquid on a MacconKa agar plate, and carrying out inverted culture at 37 ℃ overnight. A single colony was picked and inoculated in LB liquid medium, and cultured overnight at 37 ℃.

(3) And (3) MIC determination of the traditional Chinese medicine water extract: the minimum inhibitory concentration of scutellaria baicalensis, coptis chinensis, rheum officinale, humifuse euphorbia herb, dandelion and glossogyne herb on 4 strains of chicken-origin escherichia coli is determined by a two-fold dilution method, and the minimum dilution for aseptic growth is determined as the MIC value.

TABLE 16 MIC determination (mg/mL) of aqueous extracts of Chinese herbs on 4 E.coli strains

According to the results in Table 1, the MIC of the coptis chinensis, the rheum officinale and the scutellaria baicalensis to 4 strains of escherichia coli is 30-60 mg/mL, and the MIC of the dandelion, the glossocadia elata and the humifuse euphorbia herb to 4 strains of escherichia coli is more than 60 mg/mL.

(4) Preparing a traditional Chinese medicine water extract treatment bacterial liquid: at a concentration of 1X 10⁶Adding 1/2MIC traditional Chinese medicine liquid into a cfu/mL bacterial liquid test tube, performing shaking culture at 37 ℃ for 18-24 h, then re-inoculating the bacterial liquid into an LB liquid culture medium, and performing shaking culture at 37 ℃ for 4-4.5 h. The above steps are repeated for 72 hours.

(5) The traditional Chinese medicine water extract has influence on the Escherichia coli R plasmid: extracting plasmids according to the specification of the plasmid extraction kit of Nanjing Nuojingzin Biotechnology Limited, and detecting the elimination condition of the drug-resistant plasmids before and after the treatment of the bacterial liquid by the traditional Chinese medicine water extract. As shown in FIGS. 1a-1d, 4 E.coli strains all carried 2300, 3200 and 21000 plasmid bands, the number of plasmid bands of E-1, E-2, E-3 and E-4 after treatment with Coptidis rhizoma is reduced by 1-3 compared with that before treatment, the number of plasmid bands of E-4 after treatment with Tasmanian Blueflower is reduced by 2, and the elimination range is 2300-21000 bp. The result shows that the effect of eliminating R plasmid is better by the glossogyne incognita and the coptis compared with other traditional Chinese medicines.

Example 2 screening of Single Chinese herbs for inhibiting the Activity of Escherichia coli ESBLs derived from Chicken

The preparation of chicken-derived escherichia coli bacterial liquid, the preparation of traditional Chinese medicine water extract treatment bacterial liquid and the MIC determination of traditional Chinese medicine water extract are the same as those of chicken-derived escherichia coli bacterial liquid

Example 1

Method for determining influence of traditional Chinese medicine water extract on activity of ESBLs (effective systemic lupus erythematosus) by adopting K-B method

And (4) judging a result: if the diameter of the CAZ/CA ratio CAZ inhibition zone is more than or equal to 5mm, or the diameter of the CTX/CA ratio CTX inhibition zone is more than or equal to 5mm, the ESBLs are judged to be positive.

TABLE 2E-1 bacteria inhibition zone for cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid

Note: the shoulder marks represent significant differences, the letters are identical and represent insignificant differences (P <0.05), and the same is used below.

TABLE 3E-2 bacteria inhibition zone for cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid

TABLE 4E-3 bacteria inhibition zone for cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid

TABLE 5E-4 bacteria inhibition zone for cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid

As can be seen in table 2: the diameter of the inhibition zone of the paper sheet containing the enzyme inhibitor is less than 5mm compared with that of a single medicine paper sheet, and the E-1 is judged as ESBLs negative bacteria. As can be seen in table 3: the diameter of the inhibition zone of the E-2 enzyme inhibitor-containing paper sheet is more than or equal to 5 compared with that of a single-medicine paper sheet, the ESBLs positive bacteria are judged to be produced, compared with a control group, the diameters of the inhibition zones of two groups of medicine sensitive paper sheets, namely CAZ and CAZ/CA, CTX and CTX/CA are increased, the difference between the capital letters of the shoulder marks of the coptis chinensis and the control group is obvious, and the fact that the coptis chinensis can inhibit the activity of the ESBLs is shown. As can be seen from table 4: the diameter of the inhibition zone of the paper sheet containing the enzyme inhibitor is less than 5mm compared with that of a single medicine paper sheet, and E-3 is judged as ESBLs-producing negative bacteria. As can be seen from table 5: the diameter of the inhibition zone of the E-4 enzyme inhibitor-containing paper sheet is more than or equal to 5 compared with that of a single-medicine paper sheet, the ESBLs positive bacteria are judged to be produced, compared with a control group, the diameters of the inhibition zones of two groups of medicine sensitive paper sheets, namely CAZ and CAZ/CA, CTX and CTX/CA, are increased, and the shoulder mark letters of the coptis group are different from those of the control group, so that the difference is obvious, and the coptis can inhibit the activity of the ESBLs. From the above results, it can be seen that coptis inhibits ESBLs activity better than other Chinese herbs.

Example 3 Single Chinese medicine screening for influencing adhesion of Chicken-derived Escherichia coli biofilm

The preparation of chicken-derived escherichia coli bacterial liquid, the preparation of traditional Chinese medicine water extract treatment bacterial liquid and the MIC determination of traditional Chinese medicine water extract are the same as those of chicken-derived escherichia coli bacterial liquid

Example 1

Determining the influence of the traditional Chinese medicine water extract on the adhesion of the escherichia coli biological membrane by adopting a biological membrane positive screening method, and calculating and judging the result: the measured value-blank value > 0.12 is judged as the positive of the biological membrane, and the critical value (AC) is the average OD of the blank control group₆₀₀Value +3 × standard deviation; no adhesion (-) A < AC, weak adhesion (+) AC < A < 2AC, moderate adhesion (+ +)2AC < A < 4AC, strong adhesion (+ +++) A > 4 AC.

As shown in FIG. 2, the test value of E-1-blank value <0.12, was judged as a biofilm-negative bacterium; e-2 was changed from medium adhesiveness (++) to weak adhesiveness (+); e-3 is changed from strong adhesiveness (+ +++) to weak adhesiveness (+) -after being treated by coptis chinensis and glossogyne, and is changed into medium adhesiveness (+ +) -after being treated by dandelion and humifuse euphorbia; e-4 changed from medium adhesiveness (+ +) to weak adhesiveness (+) after being treated with Coptidis rhizoma and herba Hedyotidis Diffusae. From the above results, the adhesiveness of the biological films of the coptis chinensis and the glossogyne can be reduced by 4 strains, and the adhesiveness of the biological films of the humifuse euphorbia herb and the dandelion can be reduced by E-3 strains.

Example 4 screening of Single Chinese medicine for inhibiting Chicken-derived Escherichia coli active efflux System

The preparation of chicken-derived escherichia coli bacterial liquid, the preparation of traditional Chinese medicine water extract treatment bacterial liquid and the MIC determination of traditional Chinese medicine water extract are the same as those of chicken-derived escherichia coli bacterial liquid

Example 1

The influence of the traditional Chinese medicine water extract on an active efflux system (Acra-mRNA, Acrb-mRNA) of the escherichia coli is detected by a fluorescent quantitative PCR method. As shown in figure 3, compared with the traditional Chinese medicine before treatment, the coptis can reduce the expression level of 4 strains of AcrA-mRNA, and the difference is significant (P < 0.05). After the tongue grass treatment, the expression level of E-3 and E-4AcrA-mRNA is reduced, and the difference is obvious (P < 0.05). The expression level of E-1 and E-4AcrA-mRNA is reduced after the humifuse euphorbia herb is treated, and the difference is obvious (P < 0.05). As shown in figure 4, compared with the Chinese medicine before treatment, the coptis can reduce the expression level of AcrB-mRNA of 4 strains, and the differences of E-2, E-3 and E-4 are obvious (P is less than 0.05). The expression level of E-3 and E-4AcrB-mRNA is reduced after the action of the glossolalia and the difference is obvious (P < 0.05). After the euphorbia humifusa is treated, the expression level of E-1AcrB-mRNA is reduced, and the difference is obvious (P < 0.05). In conclusion, the coptis chinensis, the glossogyne herb and the humifuse euphorbia herb can reduce the expression quantity of drug-resistant genes of the chicken-derived escherichia coli active efflux system.

Example 5 determination of formula of traditional Chinese medicine for eliminating chicken-derived escherichia coli beta-lactam medicines

In summary, the following steps: the effect of eliminating the drug-resistant Escherichia coli R plasmid is good by using a single traditional Chinese medicine (coptis chinensis and glossogyne); the effect of inhibiting the activity of drug-resistant escherichia coli ESBLs by using a single traditional Chinese medicine (coptis) is good; the effect of inhibiting the active efflux system of the drug-resistant escherichia coli by using a single traditional Chinese medicine (coptis chinensis, glossogyne herb and humifuse euphorbia herb) is good; the effect of reducing the adhesion of the drug-resistant escherichia coli biological membrane by using a single traditional Chinese medicine (coptis chinensis, glossocadia officinalis, dandelion and humifuse euphorbia herb) is good. Earlier studies prove that the hawthorn has the best effect of eliminating drug resistance of escherichia coli beta-lactam drugs in 7 traditional Chinese medicines (hawthorn, scutellaria baicalensis, coptis chinensis, rheum officinale, humifuse euphorbia herb, dandelion and glossogyne). Therefore, two groups of compositions are formed, and a test group I: hawthorn, coptis (1: 1), test group two: haw, tongue grass (1: 1)

(1) FIC verification formula compatibility effect

The chessboard method is adopted to detect the combined action of the traditional Chinese medicine and the western medicine. Respectively measuring traditional Chinese medicines of rhizoma Coptidis and fructus crataegi; herba Hedyotidis Diffusae and fructus crataegi; cefotaxime, hawthorn and coptis; cefotaxime, hawthorn and glossogyne; the effect of the combined medication of cefotaxime and Sanhuang decoction.

Table 6 measurement results (mg/mL) of FIC of Chinese medicinal water extract

From Table 6, Hawthorn fruit, Coptis chinensis; hawthorn and glossogyne; cefotaxime and hawthorn tongue grass; the judgment results that the FIC index is more than 0.5 and less than or equal to 1.0 between the cefotaxime and the Sanhuang decoction are additive effects. The FIC index between the cefotaxime and the hawthorn coptis root formula is less than or equal to 0.5, and the result is a synergistic effect, which indicates that the effect of each medicine is better in compatibility.

(2) Establishment of Escherichia coli animal model

Selecting 120 egg chicks of 1 day old, normally feeding for 6 days, and selecting serotype O at 7 days old₇₈And the toxicity of the cefotaxime-resistant chicken-origin escherichia coli is attacked. The mortality rate within 48h was recorded and half the lethal dose was calculated according to modified kouyama method. The dead chicks were examined by dissecting and the pathological changes of the organs were observed. And c, performing cesarean inspection on the challenged egg chicks, performing aseptic operation to separate bacteria from the diseased viscera, performing biochemical test, determining the bacterial species, identifying the serotype through a slide agglutination test, and detecting whether the separated strain and the challenged strain are consistent. Histopathological examination: and C, performing a dissecting examination on the dead chickens, recording pathological changes, taking heart and liver tissues, making pathological sections, and recording results.

TABLE 7 determination of optimal challenge dose for E.coli (cfu/mL)

	Blank group	1×109	1×108	1×107	Physiological saline group
						Number of starts (only)	15	15	15	15	15
Death number (only)	0	12	7	4	1
						Survival number (only)	15	3	8	11	14

As can be seen from Table 7, LD50 ═ lg-1[ Xm-i (. sigma.p- (3-Pm-Pn)/4) according to the modified Kouzhi method]Obtaining Escherichia coli O₇₈For chicks LD50 ═ 1.102 × 10⁸cfu/mL. I.e. 1.102 x 10⁸cfu/mL and 0.5mL are the optimal dose for counteracting toxic substances.

After the autopsy, yellow cellulosic exudates were present on the heart and liver surfaces, and the capsule was thick and easily peeled off, and adhered to the surrounding tissues (fig. 5). Aseptically inoculating the above disease material to Macconkey agar plate, and culturing in 37 deg.C incubator for 24h to form medium-sized, protruding, smooth and moist round pink colony (FIG. 6). Under a microscope, gram-negative Brevibacterium with blunt round ends and pink color were observed (FIG. 7).

H.e. stained cardiac tissue sections results are shown in fig. 8, 9. The heart fiber tissue structure of the sick egg chicks is sparse, the gap is enlarged, and edema appears among tissues (figure 9 Ba); there was inflammatory infiltration of the epicardial, local tissue of the myocardium (fig. 9Ba, Bb). As shown in FIG. 11, the lobular structure of the liver of the dead chicken is unclear, the clearance of the liver cell cord is enlarged, and edema appears.

TABLE 8 Biochemical reactions of isolated strains

Results of biochemical experiments showed 99.8% closeness of E.coli (Table 8). Through PCR verification, clear bands appear on the 1467bp position of the separated strain (figure 12), a PCR amplification product of the separated strain is sent to Shanghai biological engineering limited company for sequencing, the sequencing result is subjected to sequence comparison on GenBank, the homology is more than 99.9 percent, the separated strain is escherichia coli, and a slide agglutination test shows that an obvious agglutination phenomenon appears, so that the separated strain and an attacking strain are the same strain, and the establishment of an escherichia coli pathogenic model is successful.

(3) Observation of clinical application effect of traditional Chinese medicine

120 egg chicks of 1 day old are selected, randomly divided into 6 groups and adaptively raised for 6 days. At the age of 7 days, the concentration is 1.102X 10⁸cfu/mL O₇₈Type and challenge against cefotaxime escherichia coli. The treatment mode of drinking water administration is adopted from the inoculation time. The medicine is artificially administrated for chickens with serious clinical symptoms and incapable of normally drinking water, and the dosage is 20mLKg, 2 times per day. Recording the morbidity of the test group chickens, and counting the cure rate, the effective rate and the mortality.

TABLE 9 clinical treatment effect of Chinese medicinal composition on chicken colibacillosis

As can be seen from Table 9, the combination of the Chinese medicinal formulation and the antibiotic enhanced the therapeutic effect of the antibiotic on Escherichia coli, and the therapeutic effect was shown to different degrees among the test groups. The cure rate of the positive control group 1 (cefotaxime) for treating the chicken flocks is 40%, the effective rate is 66.6%, and the death rate is 33.3%; the cure rate of the positive control group 2 (cefotaxime + Sanhuang decoction) for treating chicken flocks is 66.6%, the effective rate is 80%, and the death rate is 20%. Test group 1, namely the chicken flocks treated by hawthorn and coptis combined with antibiotic cefotaxime, has the cure rate of 90 percent, the effective rate of 93.3 percent and the death rate of 6.7 percent; the test group 2, namely the chicken flocks treated by hawthorn and glossogyne combined with antibiotic cefotaxime, has the cure rate of 80 percent, the effective rate of 83.3 percent and the death rate of 13.3 percent. In conclusion, compared with other test groups, the combination of hawthorn, coptis and cefotaxime has the best effect.

The results show that the effect of the hawthorn and coptis prescription is superior to that of the traditional prescription (three-yellow decoction) and the hawthorn and glossogyne prescription, and the hawthorn and coptis prescription has the best effect of treating the chicken-origin drug-resistant colibacillosis.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.