A composition for reducing drug resistance of multi-drug resistant escherichia coli and application thereof

Abstract

The application discloses a composition for reducing drug resistance of multi-drug resistant escherichia coli and application thereof, and belongs to the technical field of antibacterial drug compositions. The composition is composed of Chinese gall and radix scutellariae. The Chinese gall and the radix scutellariae have a synergistic inhibitory effect on the multi-drug resistant escherichia coli, and can inhibit the biofilm formation ability of the multi-drug resistant escherichia coli to a certain extent. In addition, the combination of the two can also reduce the drug resistance of the multi-drug resistant escherichia coli to antibiotics, restore the sensitivity of the bacteria to the antibiotics, and is beneficial to the prevention and treatment of the multi-drug resistant escherichia coli.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug composition technology, specifically relating to a composition for reducing the drug resistance of multidrug-resistant Escherichia coli and its application. Background Technology

[0002] Antibiotics play a vital role in the prevention and treatment of various animal diseases. However, in recent years, the widespread use of antimicrobial drugs has led to a sharp increase in the number of multidrug-resistant bacteria. Long-term inappropriate use of antibiotics can also cause harm such as drug resistance, dysbiosis, and drug residues in the body. *Escherichia coli* is a Gram-negative bacterium belonging to the family Enterobacteriaceae and the genus *Escherichia*. *E. coli* mainly inhabits the intestines of homeothermic animals. Compared to Gram-positive bacteria, Gram-negative bacteria are more prone to developing drug resistance due to their unique cell wall structure. Therefore, during the treatment of *E. coli*, antibiotic resistance easily develops, resulting in poor efficacy of conventional antimicrobial treatments. Increased bacterial resistance leads to weakened antibiotic efficacy and even treatment failure. Therefore, finding new methods to address bacterial resistance, or at least reduce bacterial resistance to antibiotics to some extent, is crucial. Summary of the Invention

[0003] The present invention provides a pharmaceutical composition for inhibiting Escherichia coli, the composition comprising gallnut and scutellaria; wherein gallnut and scutellaria can be selected from decoctions of both.

[0004] In the above composition, the mass ratio of gallnut to scutellaria is selected from 0.12 to 0.49: 7.8 to 62.5.

[0005] In a specific implementation plan, the mass ratio of gallnut to scutellaria can be 0.12:7.8, 0.12:31.25, 0.12:62.5, or 0.49:31.25.

[0006] This invention provides the use of the above composition in the preparation of a drug that synergistically inhibits Escherichia coli.

[0007] In the above applications, the *Escherichia coli* is a multidrug-resistant *Escherichia coli*, preferably resistant to β-lactam, amide, quinolone, aminoglycoside, sulfonamide, tetracycline, polymyxin, or chloramphenicol antibiotics; more preferably, it is resistant to β-lactam antibiotics.

[0008] In the above application, the multidrug-resistant Escherichia coli is a milk-derived multidrug-resistant Escherichia coli.

[0009] This invention provides the use of the above composition in the preparation of a drug that synergistically inhibits the formation of Escherichia coli biofilms.

[0010] This invention provides the use of the above composition in the preparation of drugs that reduce or eliminate drug resistance in Escherichia coli.

[0011] The present invention provides the use of the above composition in the preparation of a medicament for improving the sensitivity of Escherichia coli to antibiotics; wherein the antibiotic is selected from β-lactams, amides, quinolones, aminoglycosides, sulfonamides, tetracyclines, polymyxins or chloramphenicol antibiotics.

[0012] The present invention provides a medicine comprising the above-mentioned pharmaceutical composition composed of gallnut and scutellaria.

[0013] The beneficial effects of this invention are as follows:

[0014] The combined use of Galla chinensis and Scutellaria baicalensis exhibits a synergistic inhibitory effect against multidrug-resistant Escherichia coli (MRC) and can inhibit the biofilm formation ability of MRC to a certain extent. Furthermore, the combined use of these two herbs can reduce the antibiotic resistance of MRC, restoring the bacteria's sensitivity to antibiotics, which is beneficial for the prevention and control of MRC. Attached Figure Description

[0015] Figure 1 The growth curves of Escherichia coli treated with a combination of Galla chinensis and Scutellaria baicalensis;

[0016] Figure 2 The growth curves of Escherichia coli treated with a combination of gallnut and dandelion are shown. Detailed Implementation

[0017] Currently, there is a lack of effective methods on the market to combat infections caused by multidrug-resistant Escherichia coli (MRC). This invention first screened various MRC strains with multidrug resistance using antibiotics, and then treated these MRC strains with a combination of traditional Chinese medicine decoctions. The results showed that the combined use of traditional Chinese medicine had a significant synergistic effect on these drug-resistant bacteria, and could reduce their minimum inhibitory concentration (MIC) in vitro, inhibiting bacterial growth and biofilm formation. This fully demonstrates the high feasibility of using combined traditional Chinese medicine for the prevention and control of such MRC strains.

[0018] The steps for screening test strains with drug-resistant phenotypes and genotypes are as follows:

[0019] First, raw milk samples were collected from pastures in Shandong Province. Following GB 4789.35-2016, 25 mL of raw milk was added to 225 mL of sterilized physiological saline and homogenized. The mixture was then incubated at 37°C for 24 hours. The enrichment broth was streaked onto crystal violet neutral red bile agar (VRBA) and incubated at 37°C for 24 hours. Typical Escherichia coli colonies on VRBA plates are purplish-red with a red bile salt precipitate ring around the colony, and the colony diameter is 0.5 mm or larger. Typical single colonies from the VRBA plates were inoculated onto eosin methylene blue agar (EMB) plates for purification and then incubated for a second time for 24 hours. Typical Escherichia coli colonies on EMB plates are deep purplish-black, smooth, and round with a metallic sheen. Suspected strains were picked for biochemical identification, and DNA was extracted from the enrichment broth for PCR verification. Identified strains were stored at -20°C for subsequent experiments. A total of 27 strains of Escherichia coli were isolated from raw milk through the above experiments, numbered from E01 to E27.

[0020] Then, the resistance phenotypes of these strains to 15 antibiotics in 8 classes (ampicillin, enrofloxacin, tetracycline, penicillin, gentamicin, florfenicol, kanamycin, polymyxin, streptomycin, cephalexin, amoxicillin, chloramphenicol, ceftriaxone, ciprofloxacin, and trimethoprim-sulfamethoxazole) were determined. Antimicrobial susceptibility testing was performed using the CLSI 2019 standard, and resistance phenotypes were determined by the size of the inhibition zone and specific standards for each antibiotic. 100 μL of *E. coli* bacterial suspension (0.5 McFarland turbidity) was mixed in sterile MH medium at approximately 50°C, poured onto plates, and allowed to solidify. Antimicrobial susceptibility test strips were then placed on the plate surface using sterile forceps and incubated at 37°C for 18-24 hours. The diameter of the inhibition zone was observed and measured to determine the sensitivity of the isolated strains to the drugs (resistance: R; intermediate: I; sensitive: S). Each sample was tested three times. The results of antibiotic susceptibility assessment of 27 Escherichia coli isolates are shown in Table 1.

[0021] Table 1. Results of antibiotic susceptibility assessment of Escherichia coli isolates.

[0022]

[0023] Based on the experimental results, the most resistant multidrug-resistant Escherichia coli strains E01, E02, E03, and E04 were selected as subsequent experimental strains, and their resistance phenotypes are shown in Table 2.

[0024] Table 2. Drug resistance phenotypes of multidrug-resistant Escherichia coli isolates

[0025]

[0026]

[0027] The drug resistance profiles of the four multidrug-resistant Escherichia coli isolates screened out are shown in Table 3.

[0028] Table 3 Drug resistance patterns of multidrug-resistant Escherichia coli isolates

[0029] strain Drug resistance spectrum Drug resistance severity E01 AMP+PEN+FLO+S+CN+AMX+C+CTR 8 E02 AMP+ENR+TET+PEN+FLO+S+CN+C+CTR+CIP+SXT 11 E03 AMP+ENR+TET+PEN+GEN+FLO+KAN+S+CN+AMX+C+CTR+CIP+SXT 14 E04 AMP+PEN+PB+S+CN+AMX+CTR 7

[0030] Finally, DNA was extracted from four multidrug-resistant Escherichia coli strains, and the coding of resistance genes for blaTEM, blaCTX-M, blaSHV, tet(A), sul-1, floR, and gyrA was determined by PCR and agarose gel electrophoresis. PCR amplification was performed using a 20 μL system, and the experimental results were obtained using a gel imaging system. The specific encoded resistance genotypes are shown in Table 4.

[0031] Table 4. Encoding of drug resistance genotypes in Escherichia coli strains

[0032] strain Drug-resistant genotypes E01 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA E02 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, gyrA E03 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA E04 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA

[0033] Based on the above experimental results, four multidrug-resistant Escherichia coli strains (E01, E02, E03, and E04) containing the β-lactamase encoding gene and having the highest drug resistance weights were selected from the tested strains as subsequent test strains for drug activity testing.

[0034] The present invention will now be described in further detail with reference to specific embodiments and data. These embodiments are only a part of the embodiments of the present invention, and not all of them. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Other terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art. The following embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention in any way.

[0035] Example 5: Combined use of gallnut and scutellaria baicalensis

[0036] 1. Determination of the combined effects of Gallnut and Scutellaria baicalensis

[0037] The in vitro antibacterial activities of Gallnut and Scutellaria baicalensis, used alone and in combination, were determined, and the procedures are as follows:

[0038] (1) Preparation of Chinese herbal decoction (original solution)

[0039] Accurately weigh 50g each of the Chinese herbs Gallnut and Scutellaria baicalensis, pulverize them, and soak them in 500mL of distilled water for 4 hours. First, bring to a boil over high heat, then simmer over low heat for 30 minutes, and filter with gauze. Add 500mL of distilled water to the dregs and boil for 30 minutes, then filter with gauze. Combine the two decoctions and simmer over low heat to concentrate to 50mL until the final concentration of the decoction is 1000mg / mL. Filter through a 0.22μm filter membrane, autoclave at 110℃ for 20 minutes, and store in a refrigerator at 4℃ for later use.

[0040] (2) Preparation of Escherichia coli bacterial culture

[0041] Four multidrug-resistant Escherichia coli strains, E01, E02, E03, and E04, were selected. These resistant strains were streaked onto EMB plates and incubated overnight at 37°C with the plates inverted. Single colonies were then picked and inoculated into LB broth medium and cultured overnight at 37°C with shaking.

[0042] (3) Determination of the minimum inhibitory concentrations of gallnut and scutellaria against Escherichia coli.

[0043] The micro-broth dilution method was used in 96-well plates. A sterile 96-well plate was placed in a laminar flow hood. Different concentrations of gallnut, after serial dilution, were added to the 96-well plate from left to right, from lowest to highest concentration, with 100 μL added to each well. The final concentrations of gallnut in the first to tenth columns were 0.06, 0.12, 0.24, 0.49, 0.98, 1.95, 3.9, 7.8, 15.625, and 31.25 mg / mL, respectively. Similarly, different concentrations of Scutellaria baicalensis, after serial dilution, were added to the 96-well plate from left to right, from lowest to highest concentration, with 100 μL added to each well. The final concentrations of Scutellaria baicalensis in the first to tenth columns were 1.95, 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 mg / mL, respectively. Add 100 μL of bacterial culture to each well from the first to the tenth column, with a final bacterial concentration of 1 × 10⁻⁶ per well. 5 CFU / mL. 200 μL of bacterial suspension was added to column 11 as a positive control, and 200 μL of broth was added to column 12 as a negative control. Three parallel experiments were performed for each bacterium and each traditional Chinese medicine. The mixture was incubated at 37℃ for 16–24 h, with column 11 showing turbidity and column 12 showing clarity. Results were observed and recorded. The lowest drug concentration at which no bacterial growth was observed is the minimum inhibitory concentration (MIC) of the drug for that bacterium.

[0044] (4) Determination of the minimum inhibitory concentration of Galla chinensis combined with Scutellaria baicalensis against Escherichia coli.

[0045] Based on the minimum inhibitory concentration (MIC) results of single-drug agents, six dilution gradients were used for combined drug susceptibility testing. The serially diluted concentrations of *Gnaphalium affine* were added to 96-well plates from left to right, in ascending order, with 50 μL added to each well. The final concentrations of *Gnaphalium affine* in the first to sixth columns were 0.06, 0.12, 0.24, 0.49, 0.98, and 1.95 mg / mL, respectively. The serially diluted concentrations of *Scutellaria baicalensis* were added to 96-well plates from top to bottom, in ascending order, with 50 μL added to each well. The final concentrations of *Scutellaria baicalensis* in the first to sixth rows were 7.8, 15.625, 31.25, 62.5, 125, and 250 mg / mL, respectively. 100 μL of bacterial culture was added to each well in the first to sixth columns to achieve a final bacterial concentration of 1 × 10⁻⁶. 5 The CFU / m³ concentrations of the added drugs are shown in Table 5. 200 μL of bacterial suspension was added to column 7 as a positive control, and 200 μL of broth was added to column 8 as a negative control. The mixture was incubated at 37°C for 16–24 h. The minimum inhibitory concentration (MIC) was recorded when column 7 was turbid and column 8 was clear.

[0046] Table 5. Determination of drug concentrations using the checkerboard method.

[0047]

[0048] (5) Calculate the fractional inhibitory concentration index (FICI).

[0049] The minimum inhibitory concentration of the composition was determined based on the results of the checkerboard method, and the graded inhibitory concentration index for each strain was calculated to classify the antimicrobial synergistic effect of the composition.

[0050] FICI (Fluid-Inhibitory Concentration Index) is used as the basis for interpreting the results of combined drug susceptibility testing. The calculation method for the graded antimicrobial concentration index is: FICI = MIC of Drug A (combination) / MIC of Drug A (single) + MIC of Drug B (combination) / MIC of Drug B (single). Wherein, FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI ≤ 1 indicates additive effect; 1 < FICI ≤ 2 indicates irrelevant effect; FICI > 2 indicates antagonistic effect.

[0051] The experimental results are shown in Table 6:

[0052] Table 6. Results of the synergistic effect between Galla chinensis and Scutellaria baicalensis.

[0053]

[0054] As shown in Table 6, the combined use of Galla chinensis and Scutellaria baicalensis has a synergistic inhibitory effect on all four multidrug-resistant Escherichia coli strains.

[0055] 2. Antibacterial test of combined drugs

[0056] (1) Determination of antibacterial effect

[0057] Based on the results of the checkerboard dilution method, isolate E03 was selected for a drug combination antibacterial test. A combination of 0.12 mg / mL Gallnut and 31.25 mg / mL Scutellaria baicalensis was used. The bacterial suspension of isolate E03 was diluted to 1×10⁻⁶. 5 The combined herbal group was treated with a concentration of CFU / mL of Galla chinensis and Scutellaria baicalensis, with a final concentration of 0.12 mg / mL. The single-drug groups were treated with a final concentration of 1.95 mg / mL Galla chinensis and a final concentration of 125 mg / mL Scutellaria baicalensis, respectively. The positive control group used an equal volume of bacterial suspension, and the negative control group used an equal volume of sterile water. Each herbal group was tested in triplicate. The cultures were incubated at 37°C, and the absorbance at λ=600 nm was measured every 2 hours.

[0058] The measurement results are as follows Figure 1 As shown:

[0059] Compared with the positive control group, the OD in the combined traditional Chinese medicine group was lower. 600 The value decreased significantly, indicating that the combination of gallnut and scutellaria has a very significant inhibitory effect on Escherichia coli.

[0060] (2) Biofilm formation capacity determination

[0061] The biofilm formation ability of bacterial E03 was assessed using crystal violet staining. 100 μL of a 1×10⁻⁶ solution was added to each well of a 96-well flat-bottomed culture plate. 6 The bacterial culture was prepared at CFU / mL. In the combined traditional Chinese medicine group, 100 μL of a combination of 0.12 mg / mL Gallnut and 31.25 mg / mL Scutellaria baicalensis was added. 200 μL of LB broth culture medium without bacterial culture served as a negative control. Each combination was performed in triplicate. The 96-well plates were incubated at 37℃ for 18-24 h. The supernatant airborne bacteria were aspirated, and the plates were washed three times with PBS. After drying, 200 μL of 1% crystal violet staining solution was added to each well. After staining for 30 min, the staining solution was aspirated, and the plates were washed three more times with PBS. After drying, 200 μL of anhydrous ethanol was added to each well for destaining for 20 min. The absorbance at A=590 nm was measured, and the results were repeated three times, with the average value taken.

[0062] The experimental results are shown in Table 7:

[0063] Table 7. Formation of Escherichia coli biofilm

[0064] strain <![CDATA[OD 590 ]]> Biofilm formation ability Untreated strains 0.2653 middle Gallnut + Scutellaria baicalensis 0.2127 weak

[0065] OD in the combined traditional Chinese medicine group 590The value decreased, and the biofilm formation ability changed from medium to weak. Therefore, the combination of gallnut and scutellaria has an inhibitory effect on Escherichia coli biofilm formation.

[0066] 3. Drug resistance elimination test

[0067] The changes in drug resistance phenotypes of strains after elimination by the traditional Chinese medicine combination were compared to further confirm the recovery effect of antibiotic sensitivity after the elimination of drug resistance in *E. coli*. The drug resistance phenotypes of strain E03 treated with the traditional Chinese medicine combination (0.12 mg / mL *Rhus chinensis* + 31.25 mg / mL *Scutellaria baicalensis*) to ampicillin, penicillin, ceftriaxone, cephalexin, amoxicillin, tetracycline, and polymyxin were determined. The changes in drug resistance phenotypes of untreated strain E03 were compared by the size of the inhibition zone and the specific standards of each antibiotic. 100 μL of bacterial suspension (0.5 McFarland turbidity) was mixed in sterile MH medium at approximately 50℃ and poured into plates. After the plates solidified, drug sensitivity test strips were placed on the surface of the plates using sterile forceps. The plates were incubated at 37℃ for 18-24 h, and the diameter of the inhibition zone was observed and measured to determine the drug sensitivity of multidrug-resistant *E. coli* strains (resistance: R; intermediate: I; sensitive: S).

[0068] The drug resistance phenotypic changes of Escherichia coli isolates are shown in Table 8:

[0069] Table 8. Drug resistance phenotypic changes in multidrug-resistant Escherichia coli

[0070]

[0071]

[0072] After treatment with a traditional Chinese medicine combination, the diameter of the inhibition zone of the bacterial strains showed an increasing trend, especially against β-lactamase inhibitors. Therefore, the combined use of Galla chinensis and Scutellaria baicalensis can increase the sensitivity of multidrug-resistant bacteria to antibiotics.

[0073] 4. Drug resistance gene elimination experiment

[0074] DNA was extracted from multidrug-resistant *E. coli* strains treated with a combination of traditional Chinese medicine. The coding of resistance genes for blaTEM, blaCTX-M, blaSHV, tet(A), sul-1, floR, and gyrA was determined by PCR and agarose gel electrophoresis. DNA was extracted from *E. coli* using a boiling method, and PCR amplification was performed in a 20 μL system. The experimental results were obtained using a gel imaging system.

[0075] The specific drug resistance genotype variations are shown in Table 9:

[0076] Table 9. Drug-resistant genotypes of multidrug-resistant Escherichia coli after treatment with traditional Chinese medicine combinations.

[0077] strain Drug-resistant genotypes E01 blaCTX-M, blaSHV, tet(A), sul-1, floR, gyrA E02 blaCTX-M, blaSHV, tet(A), sul-1, gyrA E03 blaCTX-M, tet(A), sul-1, floR, gyrA E04 blaCTX-M, tet(A), sul-1, floR, gyrA

[0078] As shown in Table 8, after treatment with the traditional Chinese medicine combination, the strains had an elimination effect on the genes encoding drug resistance or the bands were weakened. In particular, the resistance genes of β-lactamases were not detected in the strains treated with the traditional Chinese medicine combination.

[0079] 5. Elimination experiment

[0080] E03 was selected for an elimination test of a combination of *Gnaphalium affine* and *Scutellaria baicalensis* (0.12 mg / mL *Gnaphalium affine* + 31.25 mg / mL *Scutellaria baicalensis*). The mixture of *Gnaphalium affine* and *Scutellaria baicalensis* was cultured with E03. E03 was treated with 0.02% SDS as an elimination control, and an equal volume of sterile water was used as a negative control. The culture was incubated at 37℃ for 24 h, 48 h, and 72 h. The cultured bacterial suspension was thoroughly diluted and plated onto LB agar plates, incubated at 37℃ for 12-18 h, yielding numerous single colonies. One hundred single colonies were picked from each plate and spotted onto LB agar plates and LB agar plates containing the traditional Chinese medicine, respectively, and incubated upside down at 37℃ for 12 h. Colonies that did not grow on plates containing the traditional Chinese medicine and those that grew on plates without the traditional Chinese medicine were selected and inoculated again onto a medium containing the traditional Chinese medicine for verification. If no growth was observed, the colony was considered an elimination strain (a strain that had eliminated drug resistance).

[0081] The experimental results are shown in Table 10:

[0082] Table 10 Results of Elimination Tests for Multidrug-Resistant Escherichia coli

[0083]

[0084]

[0085] Within 72 hours after treatment with the combination, the number of drug-resistant Escherichia coli was directly proportional to the culture time, increasing with the extension of culture time. The number of drug-resistant eliminates obtained in the combination with traditional Chinese medicine was significantly higher.

[0086] The combined use of Galla chinensis and Scutellaria baicalensis showed a synergistic inhibitory effect on four strains of multidrug-resistant Escherichia coli, and could also inhibit the biofilm formation ability of multidrug-resistant Escherichia coli to a certain extent. The combined use of Galla chinensis and Scutellaria baicalensis can reduce the antibiotic resistance phenotype and genotype of multidrug-resistant bacteria.

[0087] Comparative Example 1: Single-herb treatment with Gallnut and Scutellaria baicalensis

[0088] 1. Single-drug antibacterial test

[0089] (1) Preparation of Chinese herbal decoction (original solution)

[0090] Accurately weigh 50g each of the Chinese herbs Gallnut and Scutellaria baicalensis, pulverize them, and soak them in 500mL of distilled water for 4 hours. First, bring to a boil over high heat, then simmer over low heat for 30 minutes, and filter with gauze. Add 500mL of distilled water to the dregs and boil for 30 minutes, then filter with gauze. Combine the two decoctions and simmer over low heat to concentrate to 50mL until the final concentration of the decoction is 1000mg / mL. Filter through a 0.22μm filter membrane, autoclave at 110℃ for 20 minutes, and store in a refrigerator at 4℃ for later use.

[0091] (2) Preparation of Escherichia coli bacterial culture

[0092] Multidrug-resistant Escherichia coli E03 was selected. The resistant strain was streaked onto EMB plates and incubated overnight at 37°C with the plates inverted. Single colonies were then picked and inoculated into LB broth medium and cultured overnight at 37°C with shaking.

[0093] (3) Biofilm formation capacity determination

[0094] The biofilm formation ability of bacterial E03 was assessed using crystal violet staining. 100 μL of a 1×10⁻⁶ solution was added to each well of a 96-well flat-bottomed culture plate. 6 CFU / mL bacterial suspension was used. Single-drug treatment groups included *Rhus chinensis* at a final concentration of 1.95 mg / mL and *Scutellaria baicalensis* at a final concentration of 125 mg / mL. 200 μL of LB broth without bacterial suspension served as a negative control. Each treatment was performed in triplicate. The 96-well plates were incubated at 37°C for 18–24 h. The supernatant airborne bacteria were aspirated, and the plates were washed three times with PBS. After drying, 200 μL of 1% crystal violet staining solution was added to each well. Staining was allowed to stand for 30 min, then the staining solution was aspirated. The plates were then washed three times with PBS, dried, and 200 μL of anhydrous ethanol was added to each well for destaining for 20 min. The absorbance at A = 590 nm was measured, and the results were repeated three times, with the average value taken.

[0095] The experimental results are shown in Table 11:

[0096] Table 11. Formation of Escherichia coli biofilm

[0097]

[0098]

[0099] Compared with the untreated strains, the biofilm formation ability of the strains treated with gallnut and scutellaria baicalensis remained essentially unchanged.

[0100] 2. Drug resistance elimination test

[0101] The resistance phenotypes of the E03 strain in the single-drug treatment groups to ampicillin, penicillin, ceftriaxone, cephalexin, amoxicillin, tetracycline, and polymyxin were determined. The changes in resistance phenotypes of the untreated E03 strain were compared by using the size of the inhibition zone and specific standards for each antibiotic. The single-drug treatment groups were gallnut at a final concentration of 1.95 mg / mL and Scutellaria baicalensis at a final concentration of 125 mg / mL. 100 μL of bacterial suspension (0.5 McFarland turbidity) was mixed in sterile MH medium at approximately 50℃, poured onto plates, and allowed to solidify. Then, drug sensitivity test strips were placed on the plate surface using sterile forceps and incubated at 37℃ for 18-24 h. The diameter of the inhibition zone was observed and measured to determine the drug sensitivity of multidrug-resistant Escherichia coli strains (resistance: R; intermediate: I; sensitive: S).

[0102] The test results are shown in Table 12:

[0103] Table 12. Drug resistance phenotypic changes in multidrug-resistant Escherichia coli

[0104]

[0105] After single-drug treatment, the diameter of the inhibition zone of the antibiotic did not change significantly, and it had no effect on increasing the sensitivity of multidrug-resistant bacteria to antibiotics.

[0106] 3. Drug resistance gene elimination experiment

[0107] DNA was extracted from multidrug-resistant *E. coli* strains treated with single drugs. The coding of resistance genes for blaTEM, blaCTX-M, blaSHV, tet(A), sul-1, mcr-1, floR, and gyrA was determined by PCR and agarose gel electrophoresis. DNA was extracted from *E. coli* using a boiling water method, and PCR amplification was performed in a 20 μL system. The experimental results were obtained using a gel imaging system.

[0108] After treatment with a single drug, the strain did not eliminate the genes encoding drug resistance, and all drug resistance genes were detected.

[0109] 4. Elimination experiment

[0110] Eliminator assays were performed using single-drugs of Galla chinensis and Scutellaria baicalensis. Galla chinensis (1.95 mg / mL) and Scutellaria baicalensis (125 mg / mL) were co-cultured with E03. The E03 strain was treated with 0.02% SDS as an elimination control, and an equal volume of sterile water was used as a negative control. The cultures were incubated at 37℃ for 24 h, 48 h, and 72 h. The cultured bacterial suspensions were thoroughly diluted and plated onto LB agar plates, incubated at 37℃ for 12-18 h, yielding numerous single colonies. One hundred single colonies were picked from each plate and spotted onto LB agar plates and LB agar plates containing the herbal medicine, respectively, and incubated upside down at 37℃ for 12 h. Colonies that did not grow on the herbal medicine plate and those that grew on the non-herbal medicine plate were selected and re-inoculated onto the herbal medicine-containing medium for verification. If no growth was observed, the colony was considered an eliminater (a strain that had eliminated resistance).

[0111] The experimental results are shown in Table 13:

[0112] Table 13 Results of Elimination Tests for Multidrug-Resistant Escherichia coli

[0113]

[0114] Based on the combined experimental results, the single-drug treatment group was clearly less effective in eliminating the resistance of multidrug-resistant Escherichia coli than the combined use of the two drugs.

[0115] Comparative Example 2: Gallnut and Dandelion in combination

[0116] 1. Determination of the combined effects of gallnut and dandelion

[0117] The in vitro antibacterial activities of gallnut and dandelion, used alone and in combination, were determined, and the procedures are as follows:

[0118] (1) Preparation of Chinese herbal decoction (original solution)

[0119] Accurately weigh 50g each of the Chinese herbs Gallnut and Dandelion, crush them, and soak them in 500mL of distilled water for 4 hours. First, bring to a boil over high heat, then simmer over low heat for 30 minutes, and filter with gauze. Add 500mL of distilled water to the dregs and boil for 30 minutes, then filter with gauze. Combine the two decoctions and simmer over low heat to concentrate to 50mL until the final concentration of the decoction is 1000mg / mL. Filter through a 0.22μm filter membrane, autoclave at 110℃ for 20 minutes, and store in a refrigerator at 4℃ for later use.

[0120] (2) Preparation of Escherichia coli bacterial culture

[0121] The four multidrug-resistant Escherichia coli strains E01, E02, E03, and E04 were streaked onto EMB plates and incubated upside down at 37°C overnight. Single colonies were picked and inoculated into LB broth medium and cultured overnight at 37°C.

[0122] (3) Determination of the minimum inhibitory concentrations of gallnut and dandelion against Escherichia coli.

[0123] The micro-broth dilution method was used in 96-well plates. A sterile 96-well plate was placed in a laminar flow hood. Different concentrations of gallnut, after serial dilution, were added to the 96-well plate from left to right, from lowest to highest concentration, with 100 μL added to each well. The final gallnut concentrations for the first to tenth columns were 0.06, 0.12, 0.24, 0.49, 0.98, 1.95, 3.9, 7.8, 15.625, and 31.25 mg / mL, respectively. Similarly, different concentrations of dandelion, after serial dilution, were added to the 96-well plate from left to right, from lowest to highest concentration, with 100 μL added to each well. The final dandelion concentrations for the first to tenth columns were 1.95, 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 mg / mL, respectively. Add 100 μL of bacterial culture to each well from the first to the tenth column, with a final bacterial concentration of 1 × 10⁻⁶ per well. 5 CFU / mL. 200 μL of bacterial suspension was added to column 11 as a positive control, and 200 μL of broth was added to column 12 as a negative control. Three parallel experiments were performed for each bacterium and each traditional Chinese medicine. The mixture was incubated at 37℃ for 16–24 h, with column 11 showing turbidity and column 12 showing clarity. Results were observed and recorded. The lowest drug concentration at which no bacterial growth was observed is the minimum inhibitory concentration (MIC) of the drug for that bacterium.

[0124] (4) Determination of the minimum inhibitory concentration of gallnut combined with dandelion against Escherichia coli.

[0125] Based on the minimum inhibitory concentration (MIC) results for each single drug, six dilution gradients were used for combined drug susceptibility testing. The serially diluted concentrations of gallnut were added to 96-well plates from left to right, from lowest to highest, with 50 μL added to each well. The final gallnut concentrations in the first to sixth columns were 0.12, 0.24, 0.49, 0.98, 1.95, and 3.9 mg / mL, respectively. The serially diluted concentrations of dandelion were added to 96-well plates from top to bottom, from lowest to highest, with 50 μL added to each well. The final dandelion concentrations in the first to sixth rows were 15.625, 31.25, 62.5, 125, 250, and 500 mg / mL, respectively. 100 μL of bacterial culture was added to each well in the first to sixth columns to achieve a final bacterial concentration of 1 × 10⁻⁶. 5 The CFU / m³ concentrations of the added drugs are shown in Table 14. 200 μL of bacterial culture was added to column 7 as a positive control, and 200 μL of broth was added to column 8 as a negative control. The mixture was incubated at 37°C for 16–24 h. The minimum inhibitory concentration (MIC) was recorded when column 7 was turbid and column 8 was clear.

[0126] Table 14. Determination of drug concentrations using the checkerboard method.

[0127]

[0128] (5) Calculate the fractional inhibitory concentration index (FICI).

[0129] The minimum inhibitory concentration of the composition was determined based on the results of the checkerboard method, and the graded inhibitory concentration index for each strain was calculated to classify the antimicrobial synergistic effect of the combination.

[0130] The FICI (Fluid-Inhibitory Concentration Index) is calculated as follows: FICI = MIC of Drug A (in combination) / MIC of Drug A (single) + MIC of Drug B (in combination) / MIC of Drug B (single). Where FICI ≤ 0.5 indicates synergistic effect; 0.5 < FICI ≤ 1 indicates additive effect; 1 < FICI ≤ 2 indicates no effect; and FICI > 2 indicates antagonistic effect.

[0131] The experimental results are shown in Table 15:

[0132] Table 15 Results of the synergistic effect between gallnut and dandelion

[0133]

[0134] As shown in Table 15, the combined use of gallnut and dandelion had no effect on the three multidrug-resistant Escherichia coli strains, but had an additive effect on E04.

[0135] 2. Antibacterial test of combined drugs

[0136] (1) Determination of antibacterial effect

[0137] Based on the results of the checkerboard dilution method, isolate E04 was subsequently selected for a drug combination antibacterial test. A combination of 0.12 mg / mL gallnut and 125 mg / mL dandelion was used in the experiment. The bacterial suspension of isolate E04 was diluted to 1×10⁻⁶. 5 The combined herbal group (CFU / mL) consisted of a combination of gallnut (0.12 mg / mL) and dandelion (125 mg / mL). The single-herb groups consisted of gallnut (0.49 mg / mL) and dandelion (250 mg / mL), respectively. The positive control group used an equal volume of bacterial suspension, and the negative control group used an equal volume of sterile water. Each herbal group was tested in triplicate. The cultures were incubated at 37°C, and absorbance at 600 nm was measured every 2 hours.

[0138] The measurement results are as follows Figure 2 As shown:

[0139] Compared with the positive control group, the OD in the combined traditional Chinese medicine group was lower. 600The value decreased slightly; compared with the single-drug group, the OD value in the combined traditional Chinese medicine group was slightly lower. 600 The values ​​were not significantly different from those of the gallnut group, and the combination of gallnut and dandelion did not have a significant inhibitory effect on Escherichia coli.

[0140] (2) Biofilm formation capacity determination

[0141] The biofilm-forming ability of bacterial E04 was assessed using crystal violet staining. 100 μL of a 1×10⁻⁶ solution was added to each well of a 96-well flat-bottomed culture plate. 6 CFU / mL bacterial suspension was used. For the single-drug group, 100 μL of gallnut (final concentration 0.49 mg / mL) and dandelion (final concentration 250 mg / mL) were added. For the combined traditional Chinese medicine group, 100 μL of gallnut (final concentration 0.12 mg / mL) and dandelion (final concentration 125 mg / mL) were added. 200 μL of LB broth culture medium without bacterial suspension served as a negative control. Each sample was performed in triplicate. The 96-well plates were incubated at 37℃ for 18–24 h. The supernatant airborne bacteria were aspirated, and the plates were washed three times with PBS. After drying, 200 μL of 1% crystal violet staining solution was added to each well. After staining for 30 min, the staining solution was aspirated, and the plates were washed three times with PBS. After drying, 200 μL of anhydrous ethanol was added to each well for destaining for 20 min. The absorbance at A=590 nm was measured, and the results were repeated three times, with the average value taken.

[0142] The experimental results are shown in Table 16:

[0143] Table 16. Formation of Escherichia coli biofilm

[0144]

[0145]

[0146] Compared with the single-drug treatment group, the OD in the combined Chinese medicine group was lower. 590 The values ​​remained unchanged or even increased, indicating a medium level of biofilm formation ability. The combination of gallnut and dandelion did not inhibit biofilm formation in Escherichia coli; on the contrary, it enhanced it.

[0147] 3. Drug resistance elimination test

[0148] The changes in drug resistance phenotypes of strains after elimination by the traditional Chinese medicine (TCM) combination were compared to further confirm the recovery effect of antibiotic sensitivity after the elimination of drug resistance in *E. coli*. The drug resistance phenotypes of strain E04 treated with the TCM combination to ampicillin, penicillin, ceftriaxone, cephalexin, amoxicillin, tetracycline, and polymyxin were determined. The changes in drug resistance phenotypes of untreated strain E04 were compared by the size of the inhibition zone and the specific standards of each antibiotic. 100 μL of *E. coli* bacterial suspension (0.5 McFarland turbidity) was mixed in sterile MH medium at approximately 50°C, poured into plates, and after solidification, drug sensitivity test strips were placed on the surface of the plates using sterile forceps. The plates were incubated at 37°C for 18-24 hours, and the diameter of the inhibition zone was observed and measured to determine the drug sensitivity of multidrug-resistant *E. coli* strains (resistance: R; intermediate: I; sensitive: S).

[0149] The drug resistance phenotypic changes of Escherichia coli isolates are shown in Table 17:

[0150] Table 17. Drug resistance phenotypic changes in multidrug-resistant Escherichia coli

[0151]

[0152] Table 17 shows that treatment with the traditional Chinese medicine combination of gallnut and dandelion had little effect on the diameter of the inhibition zone of antibiotics, a slight effect on reducing resistance to penicillin and amoxicillin, and almost no effect on resistance to other antibiotics.

[0153] 4. Drug resistance gene elimination experiment

[0154] DNA was extracted from multidrug-resistant Escherichia coli strains, and the coding of resistance genes for blaTEM, blaCTX-M, blaSHV, tet(A), sul-1, floR, and gyrA was determined by PCR and agarose gel electrophoresis. DNA was extracted from E. coli using a boiling water method, and PCR amplification was performed in a 20 μL system. The experimental results were obtained using a gel imaging system.

[0155] The specific drug resistance genotype variations are shown in Table 18:

[0156] Table 18 Drug-resistant genotypes of multidrug-resistant Escherichia coli after treatment with traditional Chinese medicine combinations

[0157] strain Drug-resistant genotypes E01 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA E02 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, gyrA E03 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA E04 blaCTX-M, blaTEM, blaSHV, tet(A), sul-1, floR, gyrA

[0158] Table 17 shows that after the strain was treated with the traditional Chinese medicine combination of gallnut and dandelion, it did not eliminate the genes encoding drug resistance. All drug resistance genes were still detected.

[0159] 5. Elimination experiment

[0160] E04 was selected for elimination assays using a combination of gallnut and dandelion. Gallnut and dandelion were mixed and cultured together (0.12 mg / mL gallnut + 125 mg / mL dandelion), gallnut (0.49 mg / mL), and dandelion (250 mg / mL) with E04. E04 was treated with 0.02% SDS as an elimination control group, and treated with an equal volume of sterile water as a negative control group. The cultures were incubated at 37℃ for 24 h, 48 h, and 72 h. The cultured bacterial suspensions were thoroughly diluted and plated onto LB agar plates, incubated at 37℃ for 12–18 h, yielding numerous single colonies. One hundred single colonies from each culture were picked and spotted onto LB agar plates and LB agar plates containing the elimination agent, respectively, and incubated upside down at 37℃ for 12 h. Colonies that do not grow on drug-containing plates and colonies that grow on drug-free plates are selected and then inoculated again onto drug-containing culture medium for verification. If no growth occurs, the colony is an eliminateant (a strain that has eliminated drug resistance).

[0161] The experimental results are shown in Table 19:

[0162] Table 19 Results of Elimination Tests for Drug-Resistant Escherichia coli

[0163]

[0164] The experimental results showed that, compared with the control group, the combination of gallnut and dandelion had no effect on biofilm formation, nor did it eliminate the drug-resistant phenotype and genotype of multidrug-resistant Escherichia coli.

[0165] The results above show that the decoctions of Galla chinensis or Scutellaria baicalensis alone have a poor effect on reducing the drug resistance of drug-resistant Escherichia coli, but the combination of the two has a good synergistic effect. The combination of Galla chinensis and Scutellaria baicalensis has a synergistic inhibitory effect on all four strains of multidrug-resistant Escherichia coli, and can inhibit the biofilm formation ability of multidrug-resistant Escherichia coli to a certain extent, reducing the resistance phenotype and genotype of multidrug-resistant bacteria to antibiotics.

[0166] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for synergistically inhibiting Escherichia coli, characterized in that, The composition comprises gallnut and scutellaria; the mass ratio of gallnut to scutellaria is selected from 0.12:7.8, 0.12:31.25, 0.12:62.5 or 0.49:31.25; The Escherichia coli mentioned is a multidrug-resistant Escherichia coli, specifically Escherichia coli resistant to β-lactams, amides, quinolones, aminoglycosides, sulfonamides, tetracyclines, polymyxins, or chloramphenicol antibiotics.

2. The use of the pharmaceutical composition of claim 1 in the preparation of a synergistic inhibitory agent against Escherichia coli.

3. The application according to claim 2, characterized in that, The Escherichia coli in question is a multidrug-resistant Escherichia coli derived from milk.

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