A method for using extracellular polysaccharide of Escherichia coli to resist biofilm

Abstract

The present invention discloses a method for using extracellular polysaccharide of Escherichia coli to resist biofilms. The method uses extracellular polysaccharide of Escherichia coli to inhibit the formation of bacterial biofilms to reduce the total amount of biofilm formation; and to disperse mature bacterial biofilms to reduce the original total amount of the already mature biofilms; wherein the extracellular polysaccharide of Escherichia coli is composed of the following monosaccharides in mole percentages: mannose 26.79%, galactose 21.42%, glucuronic acid 15.71%, ribose 14.65%, glucose 14.20%, arabinose 4.63%, fucose 1.15%, xylose 0.83%, and the rest is galacturonic acid. In this method, extracellular polysaccharide of Escherichia coli can prevent the formation of bacterial biofilms and disperse mature biofilms, and can enhance the therapeutic effect of antibiotics on bacterial biofilms after being used in combination with antibiotics.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial technology. More specifically, it relates to a method for anti-biofilm using extracellular polysaccharide of Escherichia coli. Background Art

[0002] At present, bacterial infections seriously threaten the safety and health of human life. In addition to the well-known problem of microbial drug resistance, microbial biofilms are an important reason for the difficult radical cure of current clinical infections. In nature, only less than 1% of microorganisms exist in a planktonic state, and biofilms are the main form of existence of microorganisms. Currently, 65 - 80% of infection-related diseases are related to biofilms. During disease treatment, it is found that the resistance of microbial biofilms to antibacterial agents is 100 - 1000 times that of planktonic microorganisms. The main reason is the existence of the extracellular matrix of biofilms. The bacteria in the biofilm are wrapped by the extracellular matrix, so they can avoid being killed by antibacterial agents and recognized by the immune system.

[0003] In nature and clinically, microbial biofilms mainly exist in the form of symbiotic biofilms in which multiple bacteria coexist. Microorganisms use extracellular polysaccharides and autoinducers as signal molecules for interspecies or intraspecies communication. Among them, extracellular polysaccharides mediate cell adhesion by binding to lectins on the surface of microorganisms or mammalian cells. As an important regulator of microbial reproduction and growth and the main component of biofilm composition, extracellular polysaccharides play an important role in the interaction between cells and between cells and surfaces. Currently, studies have shown that extracellular polysaccharides of multiple bacteria can inhibit the formation of heterologous bacterial biofilms and have no inhibitory effect on the growth of bacteria. As a common symbiotic bacterium, Escherichia coli interacts with multiple bacteria in the intestinal tract and competes and communicates with multiple bacteria in the biofilm. Therefore, the use of extracellular polysaccharides of Escherichia coli is expected to become a new method for treating biofilms.

[0004] Antibiotics are still the most common treatment method for bacterial infections in clinical practice at present. However, the widespread use of antibiotics will cause microorganisms to develop drug resistance. Therefore, improving the sensitivity of microorganisms to antibiotics is an urgent problem to be solved at present. If the extracellular polysaccharides of Escherichia coli can be used in combination with antibiotics to improve the sensitivity of microorganisms in biofilms to antibiotics and improve the drug resistance of microorganisms, this will provide new ideas for the treatment of microbial biofilms. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for anti-biofilm using extracellular polysaccharide of Escherichia coli. This method can effectively prevent the formation of bacterial biofilms and disperse mature biofilms. At the same time, after combining with antibiotics, it can enhance the therapeutic effect of antibiotics on mature biofilms.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a method for using extracellular polysaccharide of Escherichia coli to resist biofilms. This method uses the extracellular polysaccharide of Escherichia coli to inhibit the formation of bacterial biofilms to reduce the total amount of biofilm formation; and

[0008] disperse mature bacterial biofilms to reduce the original total amount of the already mature biofilms;

[0009] Among them, the extracellular polysaccharide of Escherichia coli is composed of monosaccharides with the following molar percentages: mannose 26.79%, galactose 21.42%, glucuronic acid 15.71%, ribose 14.65%, glucose 14.20%, arabinose 4.63%, fucose 1.15%, xylose 0.83%, and the rest is galacturonic acid.

[0010] Furthermore, the bacteria are selected from one or more of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

[0011] Furthermore, the extracellular polysaccharide of Escherichia coli is prepared according to the following steps:

[0012] Place Escherichia coli in a culture medium for amplification culture, centrifuge, and take the supernatant;

[0013] Perform alcohol precipitation and centrifugation on the supernatant, collect the precipitate, resuspend the precipitate with Tris-HCl and add proteinase k for incubation. After the incubation ends, perform alcohol precipitation and centrifugation, collect the precipitate, resuspend the precipitate with ultrapure water and perform dialysis, and lyophilize the dialysis solution to obtain the extracellular polysaccharide of Escherichia coli.

[0014] Furthermore, the culture medium is selected from LB culture medium.

[0015] Furthermore, the Escherichia coli is selected from Escherichia coli ATCC 25922.

[0016] Furthermore, the alcohol precipitation uses absolute ethanol, and the alcohol precipitation multiple is 3-5 times.

[0017] Furthermore, the amplification culture is carried out at 37 °C for 18-24 h.

[0018] Furthermore, the conditions for centrifugation are at 8000-10000×g and 4-5 °C for 15-20 min.

[0019] Furthermore, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 8000-14000 kDa for 2-3 days.

[0020] Further, the inhibition of bacterial biofilm formation is achieved by mixing PBS containing extracellular polysaccharide of Escherichia coli with a bacterial suspension and culturing for 24 - 48 h; wherein, the PBS containing extracellular polysaccharide of Escherichia coli and the bacterial suspension are mixed at a volume ratio of 1:1;

[0021] The dispersion of mature bacterial biofilm is carried out by culturing 100 μL - 2 mL of bacterial suspension for 24 - 48 h according to the size of the well plate or substrate, then discarding the culture broth, and then adding PBS containing extracellular polysaccharide of Escherichia coli and incubating for 2 - 12 h.

[0022] Further, the method also includes jointly using extracellular polysaccharide of Escherichia coli and an antibiotic to inhibit the formation of bacterial biofilm and disperse mature bacterial biofilm.

[0023] Further, the inhibition of bacterial biofilm formation is achieved by mixing PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic with a bacterial suspension and culturing for 24 - 48 h; wherein, the PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic and the bacterial suspension are mixed at a volume ratio of 1:1;

[0024] The dispersion of mature bacterial biofilm is carried out by culturing 100 μL - 2 mL of bacterial suspension for 24 - 48 h according to the size of the well plate or substrate, then discarding the culture broth, and then adding PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic and incubating for 2 - 12 h; or by culturing the bacterial suspension for 24 - 48 h, then discarding the culture broth, then adding PBS containing extracellular polysaccharide of Escherichia coli and incubating for 2 - 12 h, discarding the PBS containing extracellular polysaccharide of Escherichia coli, and then adding PBS containing an antibiotic and continuing to incubate for 2 - 12 h.

[0025] Further, the concentration of the bacterial suspension is 10 7 CFU / mL;

[0026] The concentration of the PBS containing extracellular polysaccharide of Escherichia coli is 1 - 200 μg / mL.

[0027] Further, the concentration of extracellular polysaccharide of Escherichia coli in the PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic is 1 - 200 μg / mL.

[0028] Further, when inhibiting the formation of bacterial biofilm, the concentration of the antibiotic in the PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic is 0.5 - 2 MIC;

[0029] When dispersing mature bacterial biofilm, the concentration of the antibiotic in the PBS containing extracellular polysaccharide of Escherichia coli and an antibiotic is 32 - 128 MIC; the concentration of the PBS containing an antibiotic is 32 - 128 MIC.

[0030] The beneficial effects of the present invention are as follows:

[0031] The present invention uses extracellular polysaccharides of Escherichia coli that have no bactericidal ability but have a significant inhibitory effect on biofilms to inhibit the formation of bacterial biofilms and disperse mature bacterial biofilms, showing excellent anti-biofilm effects. At the same time, when the extracellular polysaccharides of Escherichia coli are used in combination with antibiotics widely used clinically, the therapeutic effect of antibiotics on mature biofilms can be enhanced.

[0032] The extracellular polysaccharides of Escherichia coli can inhibit the formation of bacterial biofilms by preventing adhesion between bacteria and between bacteria and surfaces, and the lectin acting as the target exists widely in various living organisms. Therefore, the anti-biofilm effect of extracellular polysaccharides has a certain universality. In addition, the extracellular polysaccharides of Escherichia coli can disperse mature biofilms, enabling antibiotics to better treat bacterial infections, reducing the use concentration of antibiotics, and overcoming the problem of drug resistance to a certain extent.

[0033] For antibiotics such as tobramycin, the phenomenon of promoting the formation of bacterial biofilms will occur at low concentrations. After combining tobramycin with the extracellular polysaccharides of Escherichia coli, it is found that the problem of increased biofilm mass caused by tobramycin during the biofilm formation stage can be reversed, so that when using the same concentration of antibiotics, the amount of mature biofilms and the number of bacteria can be significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the infrared spectrogram of the extracellular polysaccharides of Escherichia coli in Example 1 of the present invention.

[0035] Figure 2 It is the high performance liquid chromatogram of the monosaccharide composition of the extracellular polysaccharides of Escherichia coli in Example 2 of the present invention.

[0036] Figure 3 It is the comparison of different test groups in inhibiting the formation of bacterial biofilms in Example 9 of the present invention.

[0037] Figure 4 It is the comparison of different test groups in reducing and killing bacteria in biofilms in Example 10 of the present invention.

[0038] Figure 5 It is the comparison of different test groups in dispersing mature biofilms in Example 11 of the present invention.

[0039] Figure 6 It is the effect of pre-incubation of the extracellular polysaccharides of Escherichia coli on killing bacteria in mature biofilms by tobramycin in Example 12 of the present invention.

[0040] Figure 7 It is the influence of pre-incubation of the extracellular polysaccharides of Escherichia coli on the penetration of tobramycin through mature biofilms in Example 13 of the present invention.

[0041] Figure 8Comparison of different test groups in influencing bacterial gene expression in Example 14 of the present invention, where Figure 8 in (a) gene algD; (b) gene lasI; (c) gene pqsA.

[0042] Figure 9 Research comparison of the mouse implant biofilm infection model in Example 15 of the present invention, where Figure 9 in (a) total number of white blood cells in mouse blood routine; (b) number of bacterial colonies on the surface of mouse implants; (c) H&E stained sections of mouse skin. Detailed implementation mode

[0043] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0044] LB medium: containing 10.0 g / L peptone, 5.0 g / L yeast extract, 5.0 g / L sodium chloride and 1.0 g / L glucose, pH 7.0.

[0045] TSB medium: containing 17.0 g / L tryptone, 3.0 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium chloride and 2.5 g / L glucose, pH 7.3.

[0046] PBS containing extracellular polysaccharide of Escherichia coli refers to dissolving the freeze-dried extracellular polysaccharide of Escherichia coli in 10 mM PBS for subsequent use.

[0047] The bacterial suspension is the general term for the bacterial suspensions obtained by culturing various bacteria that can be used in the present invention. Exemplarily, the Staphylococcus aureus suspension is obtained by culturing Staphylococcus aureus (ATCC 6538) in TSB medium at 37 °C for 18 - 24 hours, and adjusting the concentration of its suspension to 10 7 CFU / mL; the Pseudomonas aeruginosa (PAO1 / ATCC 10145) suspension is obtained by culturing Pseudomonas aeruginosa in LB medium at 37 °C for 18 - 24 hours, and adjusting the concentration of its suspension to 10 7 CFU / mL; the Escherichia coli suspension is obtained by culturing Escherichia coli (ATCC 25922) in LB medium at 37 °C for 18 - 24 hours, and adjusting the concentration of its suspension to 10 7 CFU / mL.

[0048] Preparation examples

[0049] This preparation example provides a method for extracting extracellular polysaccharide from Escherichia coli. The specific steps are as follows:

[0050] Pick a colony of Escherichia coli (ATCC 25922) and place it in LB medium for amplification. Incubate it in a shaker at 37 °C for 18 - 24 h. Centrifuge the amplified Escherichia coli broth (10000×g, 15 min, 4 °C), discard the bacterial precipitate, and take the supernatant. Slowly add more than 3 times the volume of absolute ethanol to the supernatant, stir at 4 °C for 4 - 12 h to obtain a suspension. Centrifuge the suspension (10000×g, 15 min, 4 °C), discard the supernatant, resuspend the precipitate with Tris-HCl, add proteinase k, incubate in a shaker at 37 °C for 4 h, then place the solution in a 90 °C water bath and incubate for 10 min. After cooling, add more than 3 times the volume of absolute ethanol, stir at 4 °C for 4 - 12 h to obtain a suspension. Centrifuge the suspension (10000×g, 15 min, 4 °C), discard the supernatant, and resuspend the precipitate with ultrapure water. The obtained resuspension is dialyzed using a dialysis bag with a molecular weight cut-off of 8000 - 14000 kDa for 2 - 3 days, freeze-dried to obtain extracellular polysaccharide from Escherichia coli, and dissolved with 10 mM PBS for subsequent use.

[0051] Example 1

[0052] Mix the freeze-dried powder of extracellular polysaccharide from Escherichia coli with dried KBr powder and grind them (mass ratio 1:200). Place the ground extracellular polysaccharide / KBr in a tablet press mold to make a tablet. The prepared KBr tablet is placed in an infrared spectrometer to measure the transmittance at 400 - 4000 cm -1 . The infrared spectrum is as shown in Figure 1 . The broad peak at 3423 cm -1 and the weak peak at 2920 cm -1 are respectively generated by the stretching vibrations of hydroxyl groups and C-H. The peak at ~1642 cm -1 comes from the ring stretching of mannose or galactose. The absorption peaks in the region of 1200 - 1000 cm -1 are attributed to the vibrations of C-O and C-O-C glycosidic bands ( Figure 1 ).

[0053] Example 2

[0054] 10 mg of freeze-dried extracellular polysaccharide of Escherichia coli was added to trifluoroacetic acid solution, transferred to an ampoule and sealed with inert gas, and hydrolyzed at 110 °C for 6 h. Methanol was added and trifluoroacetic acid was removed by a rotary evaporator in a water bath at 50 °C. This step was repeated three times to obtain the sample to be tested, and then pure water was added for dissolution. Standards of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, galactose, xylose, arabinose, and fucose were prepared. NaOH and PMP-methanol were added to the sample to be tested, reacted at 70 °C for 1 h, cooled in cold water for 10 min, neutralized with HCl, and then chloroform was added and vortexed for 1 min, centrifuged at 3000 r / min for 10 min, the supernatant was taken, and extracted 3 times to obtain the monosaccharide derivatization solution for chromatographic analysis. The chromatographic test conditions were as follows: Instrument model: Shimadzu LC-20AD; Chromatographic column: Xtimate C18 4.6*200 mm 5 μm; Column temperature: 30 °C; Flow rate: 1.0 ml / min; Detection wavelength: 250 nm; Injection volume: 20 μl; Mobile phase: 0.05 M potassium dihydrogen phosphate solution. It was determined that the extracellular polysaccharide of Escherichia coli was composed of the following monosaccharides in molar percentages: mannose 26.79%, galactose 21.42%, glucuronic acid 15.71%, ribose 14.65%, glucose 14.20%, arabinose 4.63%, fucose 1.15%, xylose 0.83%, and the rest was galacturonic acid( Figure 2 ).

[0055] Example 3

[0056] PBS containing extracellular polysaccharide of Escherichia coli was mixed with a suspension of Pseudomonas aeruginosa (PAO1) (volume ratio 1:1). After mixing, three sample groups with extracellular polysaccharide concentrations of 25, 50, and 100 μg / mL of Escherichia coli were obtained respectively, and the control group was 10 mM PBS. Each group of samples was placed in an incubator at 37 °C for 24 h and 48 h, and then the planktonic bacteria were washed with 10 mM PBS, and 2.5% glutaraldehyde solution was added to fix the biofilm, fixed at 4 °C for more than 4 h, and the crystal violet staining method was used to characterize the biofilm amount. The operation steps of the crystal violet staining method were as follows: The fixed biofilm was washed with ultrapure water to remove the glutaraldehyde solution and then dried, 0.1% crystal violet solution was added and incubated for 10 min, and the excess crystal violet dye was washed with ultrapure water and then dried, 30% acetic acid solution was added to elute the crystal violet dye bound to the biofilm, and the absorbance OD was measured using an enzyme-linked immunosorbent assay 620 . The results are shown in Table 1, indicating that the extracellular polysaccharide of Escherichia coli can effectively inhibit the biofilm formation of Pseudomonas aeruginosa.

[0057] Example 4

[0058] Mix the PBS containing extracellular polysaccharide of Escherichia coli with the Staphylococcus aureus suspension (volume ratio 1:1). After mixing, a sample group with a concentration of 100 μg / mL of extracellular polysaccharide of Escherichia coli is obtained, and the control group is 10 mM PBS. Place each group of samples in an incubator at 37 °C for 48 h, then wash the planktonic bacteria with 10 mM PBS, add 2.5% glutaraldehyde solution to fix the biofilm, fix at 4 °C for more than 4 h, and use the crystal violet staining method (the same method as in Example 3) to characterize the biofilm mass. The results are shown in Table 1, indicating that extracellular polysaccharide of Escherichia coli can effectively inhibit the biofilm formation of Staphylococcus aureus.

[0059] Example 5

[0060] Mix the PBS containing extracellular polysaccharide of Escherichia coli with the Escherichia coli suspension (volume ratio 1:1). After mixing, a sample group with a concentration of 100 μg / mL of extracellular polysaccharide of Escherichia coli is obtained, and the control group is 10 mM PBS. Place each group of samples in an incubator at 37 °C for 48 h, then wash the planktonic bacteria with 10 mM PBS, add 2.5% glutaraldehyde solution to fix the biofilm, fix at 4 °C for more than 4 h, and use the crystal violet staining method (the same method as in Example 3) to characterize the biofilm mass. The results are shown in Table 1, indicating that extracellular polysaccharide of Escherichia coli can effectively inhibit the biofilm formation of Escherichia coli.

[0061] Table 1

[0062]

[0063] Note: The calculation formula for the biofilm inhibition rate is: Inhibition rate = (OD 对照组 - OD 样品组 ) / OD 对照组 .

[0064] Example 6

[0065] Culture the Pseudomonas aeruginosa (PAO1) suspension in a constant temperature incubator at 37 °C for 24 h and 48 h, discard the culture broth, add PBS containing extracellular polysaccharide of Escherichia coli with concentrations of 25, 50, and 100 μg / mL respectively to the above-mentioned cultured mature biofilm for incubation, the incubation time is 12 h, denoted as the sample group, and the control group is 10 mM PBS. After incubation, wash the planktonic bacteria with 10 mM PBS, add 2.5% glutaraldehyde solution to fix the biofilm, fix at 4 °C for more than 4 h, and use the crystal violet staining method (the same method as in Example 3) to characterize the biofilm mass. The results are shown in Table 2, indicating that extracellular polysaccharide of Escherichia coli can effectively disperse the mature biofilm of Pseudomonas aeruginosa.

[0066] Example 7

[0067] The Staphylococcus aureus bacterial suspension was cultured in an incubator at 37 °C for 48 h. The cultured bacterial solution was discarded, and PBS containing 100 μg / mL of Escherichia coli exopolysaccharide was added to the above-mentioned cultured mature biofilm for incubation for 12 h, which was designated as the sample group, and the control group was 10 mM PBS. After incubation, the planktonic bacteria were washed with 10 mM PBS, and 2.5% glutaraldehyde solution was added to fix the biofilm, and it was fixed at 4 °C for more than 4 h. The crystal violet staining method (the same method as in Example 3) was used to characterize the biofilm amount. The results are shown in Table 2, indicating that Escherichia coli exopolysaccharide can effectively disperse the mature biofilm of Staphylococcus aureus.

[0068] Example 8

[0069] The Escherichia coli bacterial suspension was cultured in an incubator at 37 °C for 48 h. The cultured bacterial solution was discarded, and PBS containing 100 μg / mL of Escherichia coli exopolysaccharide was added to the above-mentioned cultured mature biofilm for incubation for 12 h, which was designated as the sample group, and the control group was 10 mM PBS. After incubation, the planktonic bacteria were washed with 10 mM PBS, and 2.5% glutaraldehyde solution was added to fix the biofilm, and it was fixed at 4 °C for more than 4 h. The crystal violet staining method (the same method as in Example 3) was used to characterize the biofilm amount. The results are shown in Table 2, indicating that Escherichia coli exopolysaccharide can effectively disperse the mature biofilm of Escherichia coli.

[0070] Table 2

[0071]

[0072] Note: The calculation formula for the biofilm dispersion rate is: dispersion rate = (OD 对照组 - OD 样品组 ) / OD 对照组 .

[0073] Example 9

[0074] PBS, tobramycin, Escherichia coli exopolysaccharide, and tobramycin diluted with Escherichia coli exopolysaccharide were respectively cultured with Pseudomonas aeruginosa (PAO1) bacterial suspension in an incubator at 37 °C for 24 h, which were respectively designated as the negative control group (concentration 10 mM), the tobramycin group (concentration 0.5 MIC, that is, 0.16 μg / mL), the exopolysaccharide group (concentration 50 μg / mL), and the combined use group (concentration: exopolysaccharide 50 μg / mL + tobramycin 0.5 MIC). After the culture was completed, the planktonic bacteria were washed with 10 mM PBS, and 2.5% glutaraldehyde solution was added to fix the biofilm, and it was fixed at 4 °C for more than 4 h. The crystal violet staining method (the same method as in Example 3) was used to characterize the biofilm amount. The results are shown in Figure 3, compared with the use of antibiotics alone, the combined use of Escherichia coli exopolysaccharide and antibiotics can effectively inhibit the formation of bacterial biofilms.

[0075] Example 10

[0076] PBS, tobramycin, Escherichia coli exopolysaccharide, and tobramycin diluted with Escherichia coli exopolysaccharide were respectively cultured with Pseudomonas aeruginosa (PAO1) bacterial suspension in a 37°C constant temperature incubator for 24 h, and were respectively designated as the negative control group (concentration 10 mM), the tobramycin group (concentration 0.5 MIC, i.e., 0.16 μg / mL), the exopolysaccharide group (concentration 50 μg / mL), and the combined use group (concentration: exopolysaccharide 50 μg / mL + tobramycin 0.5 MIC). After the culture was completed, the planktonic bacteria were washed with 10 mM PBS, and PBS was added to disperse the bacteria in the biofilm into a bacterial suspension. The obtained bacterial suspension was serially diluted, and the bacterial concentration was diluted to 10 3 CFU / mL. 100 μL was taken for plating and cultured overnight for colony counting. The results are shown in Figure 4 , compared with the use of antibiotics alone, the combined use of Escherichia coli exopolysaccharide and antibiotics can effectively reduce and kill the bacteria in the biofilm.

[0077] Example 11

[0078] After culturing the Pseudomonas aeruginosa (PAO1) bacterial suspension in a 37°C constant temperature incubator for 48 h, the culture broth was discarded, and PBS, tobramycin, Escherichia coli exopolysaccharide, and tobramycin diluted with Escherichia coli exopolysaccharide were respectively added for incubation for 12 h, and were respectively designated as the negative control group (concentration 10 mM), the tobramycin group (concentration 32 - 128 MIC, i.e., 10.24 - 40.96 μg / mL), the exopolysaccharide group (concentration 100 μg / mL), and the combined use group (concentration: exopolysaccharide 100 μg / mL + tobramycin 32 - 128 MIC). After the culture was completed, it was washed with 10 mM PBS and then 2.5% glutaraldehyde was added, and it was fixed at 4°C for more than 4 h, and crystal violet staining was performed to characterize the biofilm amount (the method was the same as in Example 3). The results are shown in Figure 5 , Figure 5 In , "+" represents addition and "-" represents non - addition. Compared with antibiotics at the same concentration, the combined use of Escherichia coli exopolysaccharide and antibiotics can more effectively disperse mature biofilms.

[0079] Example 12

[0080] After culturing the Pseudomonas aeruginosa (PAO1 / ATCC 10145) bacterial suspension in an incubator at 37°C for 48 h, the culture broth was discarded, and the planktonic bacteria were washed with 10 mM PBS. Then, the exopolysaccharide of Escherichia coli was added to the mature biofilm, and the concentration of the exopolysaccharide of Escherichia coli after addition was 100 μg / mL. It was then continuously cultured in an incubator at 37°C for 2 h, which was designated as the combined use group. The control group was added with the same volume of 10 mM PBS. After washing with 10 mM PBS, both groups were added with tobramycin at a concentration of 128 MIC and cultured in an incubator at 37°C for 2 h. After the culture was completed, the planktonic bacteria were washed with 10 mM PBS, and PBS was added to disperse the bacteria in the biofilm into a bacterial suspension. The obtained bacterial suspension was serially diluted, and the bacterial concentration was diluted to 10 3 CFU / mL. 100 μL was taken for plating and cultured overnight for colony counting. The results are shown in Figure 6 . Compared with the same concentration of antibiotics, the pre-incubation of the exopolysaccharide of Escherichia coli can improve the sensitivity of bacteria in the biofilm to antibiotics.

[0081] Example 13

[0082] After culturing the Pseudomonas aeruginosa (PAO1) bacterial suspension in an incubator at 37°C for 48 h, the culture broth was discarded, and the planktonic bacteria were washed with 10 mM PBS. Then, the exopolysaccharide of Escherichia coli was added to the mature biofilm, and the concentration of the exopolysaccharide of Escherichia coli after addition was 100 μg / mL. It was then continuously cultured in an incubator at 37°C for 2 h, which was designated as the combined use group. The control group was added with the same volume of 10 mM PBS. After washing with 10 mM PBS, both groups were added with tobramycin at a concentration of 32 MIC and cultured in an incubator at 37°C for 2 h. After washing with 10 mM PBS, the treated biofilm was fluorescently stained (SYTO9 / PI), and a laser confocal microscope was used to observe the viability of the bacteria in the biofilm. The results are shown in Figure 7 . Compared with the same concentration of antibiotics, the pre-incubation of the exopolysaccharide of Escherichia coli can improve the penetration of antibiotics in the biofilm.

[0083] Example 14

[0084] PBS, tobramycin, Escherichia coli exopolysaccharide, and tobramycin diluted with Escherichia coli exopolysaccharide were respectively cultured with Pseudomonas aeruginosa (PAO1) bacterial suspension in a 37°C constant temperature incubator for 0.5 - 3 h, and were respectively designated as the negative control group (concentration 10 mM), the tobramycin group (concentration 0.5 MIC, i.e., 0.16 μg / mL), the exopolysaccharide group (concentration 100 μg / mL), and the combined use group (concentration: exopolysaccharide 100 μg / mL + tobramycin 0.5 MIC). TRIzol was used to collect the RNA of the treated bacteria (the extraction method refers to the instruction manual of Thermo Fisher Scientific), and the expression of biofilm-related genes was measured by real-time fluorescence quantitative PCR. The results are shown in Figure 8 , the expression of algD, pqsA, and lasI in the bacteria treated with Escherichia coli exopolysaccharide was significantly inhibited.

[0085] Example 15

[0086] In this example, a mouse implant biofilm infection model was used to verify the use of Escherichia coli exopolysaccharide for the treatment of biofilm infections.

[0087] Implants were prepared using KANGDAONING Sylgard 184 PDMS, and biofilms were cultured in the implants placed in a bacterial solution diluted with LB broth for 30 - 36 h. BALB / c mice were selected and divided into 4 groups for the experiment. After the mice were anesthetized, the hair on the back was removed, and a 3 - 5 mm wound was cut. The implant was pushed in through the wound and placed between the skin and the muscle, and the wound was sutured. The implant in the negative control group was sterile PDMS, and the implants in the positive control group, the tobramycin group, and the combined use group were PDMS with biofilms.

[0088] The following corresponding drugs were injected at the wound and intraperitoneally at 24 h, 48 h, and 72 h respectively:

[0089] Negative control group: 60 μL of normal saline was injected at the wound, and 100 μL of normal saline was injected abdominally;

[0090] Positive control group: 60 μL of normal saline was injected at the wound, and 100 μL of normal saline was injected abdominally;

[0091] Tobramycin group: 60 μL of normal saline was injected at the wound, and 100 μL of tobramycin (9.2 mg / mL) was injected abdominally;

[0092] Combined use group: 60 μL of Escherichia coli exopolysaccharide (200 μg / mL) was injected at the wound, and 100 μL of tobramycin (9.2 mg / mL) was injected abdominally.

[0093] The mice were sacrificed after 96 h, and the implants were taken out and placed in physiological saline for ultrasound. The bacteria were counted by the dilution plating method. It can be seen that the use of exopolysaccharide significantly reduced the number of colonies in the biofilm on the PDMS surface ( Figure 9 a) in Figure 9 b) in Figure 9 c). Blood was taken from the mice for routine blood tests. It can be seen that the use of Escherichia coli exopolysaccharide avoided systemic infections caused by implant biofilm infections (

[0094] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Use of extracellular polysaccharide of Escherichia coli in the preparation of a medicament for inhibiting the formation of bacterial biofilms and dispersing mature bacterial biofilms, characterized in that, The Escherichia coli is ATCC 25922, the bacteria are Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli, the drug inhibits the formation of bacterial biofilms to reduce the total amount of biofilm formation; and disperses mature bacterial biofilms to reduce the original total amount of the already mature biofilms; Among them, the extracellular polysaccharide of Escherichia coli is prepared according to the following steps: Pick the Escherichia coli ATCC 25922 colony and place it in LB medium for amplification, culture in a shaker at 37 °C for 18 - 24 h, centrifuge the amplified Escherichia coli bacterial solution at 10000 ×g and 4 °C for 15 min, discard the bacterial precipitate, take the supernatant, slowly add more than 3 times its volume of absolute ethanol to the supernatant, stir at 4 °C for 4 - 12 h to obtain a suspension, after centrifuging the suspension at 10000 ×g and 4 °C for 15 min, discard the supernatant, resuspend the precipitate with Tris-HCl, add protease k and incubate in a shaker at 37 °C for 4 h, then place the solution in a 90 °C water bath and incubate for 10 min, after cooling, add more than 3 times its volume of absolute ethanol, stir at 4 °C for 4 - 12 h to obtain a suspension, centrifuge the suspension at 10000 ×g and 4 °C for 15 min, discard the supernatant, resuspend the precipitate with ultrapure water, dialyze the obtained resuspension using a dialysis bag with a molecular weight cut-off of 8000 - 14000 kDa for 2 - 3 days, and freeze-dry to obtain the extracellular polysaccharide of Escherichia coli; The extracellular polysaccharide of Escherichia coli is composed of the following monosaccharides in mole percentages: mannose 26.79%, galactose 21.42%, glucuronic acid 15.71%, ribose 14.65%, glucose 14.20%, arabinose 4.63%, fucose 1.15%, xylose 0.83%, and the rest is galacturonic acid; The extracellular polysaccharide of Escherichia coli shows peaks at 3423, 2920, 1642 and 1066 cm in the infrared spectrum -1 respectively.

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