Supramolecular co-assembly antibiotic screening kit based on wound biofilm infected wound fluid as well as preparation method and application of supramolecular co-assembly antibiotic screening kit

Abstract

The invention provides a supramolecular co-assembly antibiotic screening kit based on wound surface biofilm infected wound fluid and a preparation method and application of the supramolecular co-assembly antibiotic screening kit. The antibiotic screening kit comprises various antibiotic solutions with concentration gradient and bionic infected biofilms, the bionic infection biological membrane is prepared from supramolecular precursor peptide powder and wound exudate containing infection pathogenic bacteria, and the sequence of the supramolecular precursor peptide is Pal-VVVAAA-KKKHWGF-NH2. As an in-vitro biological membrane infection model, the method can be used for customized screening of in-vitro antibiotic drugs, and has a very good application effect.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis of biological materials, and particularly to a supramolecular co-assembly antibiotic screening kit based on wound fluid of infected wounds with wound biofilm, and a preparation method and application thereof. Background Art

[0002] A biofilm is a natural bio-polymeric community formed by EPS secreted by microbial cells and wrapping them therein. Due to its special composition mode, the biofilm has extremely strong environmental response ability, and can adjust the microbial cell phenotype and generate different EPSs under different environmental stimuli to adapt to the environment. In the case of in vivo infection, bacteria often accumulate a large amount of EPS and form a high-thickness 3D biofilm to protect the internal bacteria from the action of antibiotics; at the same time, existing studies have found that EPS can help bacteria achieve immune escape by forming pathogen-associated molecular pattern shielding environments on the bacterial surface. In other words, once the biofilm is formed, the drug sensitivity of microorganisms will change greatly, which brings great obstacles to clinical antibiotic treatment, because conventional doses and types of antibiotics can no longer meet the treatment needs of biofilm infections. If early identification and treatment plan adjustment cannot be carried out, it often leads to the patient's condition lingering and even the infection getting worse. Importantly, in vivo biofilm infections are often caused by the simultaneous action of two or more pathogens, which further complicates the clinical use of antibiotics.

[0003] Currently, clinically, in vitro experiments are mainly relied on to culture the obtained infectious clinical samples. After separating the pathogenic bacteria, the antibiotic susceptibility tests are then carried out on single pathogenic bacteria respectively, which can identify the drug sensitivity of the infected bacterial species at an earlier stage and apply it to clinical practice. However, on the one hand, the commonly used in vitro antibiotic screening models in current clinical practice are based on planktonic bacteria or 2D biofilm culture, and cannot reproduce the 3D biofilm structure under in vivo biofilm infection, resulting in deviations in the selected drug categories and doses, and poor treatment effects. On the other hand, due to some limitations of traditional culture models for co-culturing multiple bacteria (such as the rapid invasive suppression growth of Pseudomonas aeruginosa observed when co-culturing Staphylococcus aureus and Pseudomonas aeruginosa in traditional LB liquid medium), the microbial diversity of the multi-microbial community at the in vivo infection site cannot be fully reproduced, making the existing in vitro antibiotic screening models rely on the cumbersome process of isolation culture - single-bacteria drug sensitivity testing, greatly lengthening the timeline for the drug sensitivity report to be handed back to clinicians. In response to this, some studies have fabricated 3D biofilms by bioprinting alginate or bioresin mixed with bacteria, enabling microorganisms to proliferate in 3D. However, the validation tests for co-culturing multiple microorganisms are lacking. More importantly, these biofilm models lack important components in natural biofilms such as host proteins and nucleic acids, and many components have been proven to have a profound impact on the phenotype of biofilms. For example, Staphylococcus aureus has been proven to incorporate fibrin into its EPS through the expression of coagulase to protect bacteria from immune recognition; there are also studies showing that different hormone levels in different individuals of the same species also participate in the phenotypic regulation of infected biofilms, making the infected biofilms have individual specificity. Summary of the Invention

[0004] Aiming at the above deficiencies of the prior art, the present invention provides a supramolecular co-assembly antibiotic screening kit based on wound fluid of a wound infected with a wound biofilm, its preparation method and application. By designing the sequence of the supramolecular precursor peptide, its functionality is endowed, and it can co-assemble with the exudate (i.e., wound fluid) of the patient's infected wound; the co-assembled hydrogel can provide 3D adhesion sites for different microorganisms, further promoting the formation and maintenance of a composite microbial biofilm. At the same time, the co-assembly can incorporate active components such as host proteins in the wound fluid, so as to be able to study the effect of antibiotics on bacterial biofilms under in vivo-like conditions to screen suitable antibiotics.

[0005] The technical solution provided by the present invention: A supramolecular co-assembly antibiotic screening kit based on wound fluid of a wound infected with a wound biofilm, comprising various antibiotic solutions with concentration gradients and a biomimetic infected biofilm, wherein the biomimetic infected biofilm is prepared from a supramolecular precursor peptide and wound exudate containing the infected pathogenic bacteria, and the sequence of the supramolecular precursor peptide is Pal-VVVAAA-KKKHWGF-NH2.

[0006] Further, the wound exudate is the exudative body fluid of the infected or non-infected wound surfaces with or without the pathogenic bacteria to be cultured added arbitrarily on the body surfaces of various organisms.

[0007] Further, the wound exudate is any one of those derived from mice, rabbits, humans or other mammalian sources.

[0008] Another technical solution provided by the present invention is a preparation method of a supramolecular co-assembly antibiotic screening kit based on wound fluid of wound biofilm infection, comprising the following steps:

[0009] S1. Using the Fmoc solid-phase polypeptide synthesis method to synthesize a supramolecular precursor peptide, and separating and purifying the crude supramolecular precursor peptide by reverse high performance liquid chromatography;

[0010] S2. Collecting the wound exudate of the patient with biofilm infection;

[0011] S3. Spreading the wound exudate containing pathogenic bacteria on the cell chamber of a 24-well plate, adding an equal volume of the aqueous solution of the supramolecular precursor peptide to form a co-assembled hydrogel and three-dimensionally incorporating the pathogenic bacteria, and culturing to form a biomimetic infected biofilm;

[0012] S4. Adding various antibiotic solutions with concentration gradients into a 24-well cell culture plate, inserting the biomimetic infected biofilm obtained in step S3 into the various antibiotic solutions with concentration gradients to form an antibiotic screening kit, and determining the minimum inhibitory concentration of different antibiotics on the biomimetic infected biofilm.

[0013] Further, the specific steps of the preparation method are as follows:

[0014] (1) Synthesis and purification of the supramolecular precursor peptide

[0015] a. Using the Fmoc solid-phase polypeptide synthesis method, starting from the resin, according to the amino acid sequence of the supramolecular precursor peptide, coupling amino acids sequentially from the C-terminus to the N-terminus;

[0016] b. Covalently coupling palmitic acid with the amino group on the alanine outside the molecular chain end to obtain palmitoylated polypeptide resin;

[0017] c. After cutting the palmitoylated polypeptide resin with TFA, obtaining the crude supramolecular precursor peptide;

[0018] d. Separating and purifying the crude supramolecular precursor peptide by reverse high performance liquid chromatography;

[0019] (2) Extraction of the infected wound exudate

[0020] a. Prepare sterile forceps and a sterile sealed bag. Remove the surface dressing of the wound to expose the inner wound dressing. Use the sterile forceps to remove the inner dressing soaked with the infected wound fluid and place it in a sterile 50 ml centrifuge tube;

[0021] b. In a biosafety cabinet, use sterile scissors to cut the inner wound dressing in step a into pieces, add normal saline at a volume ratio of 1:1, and incubate at 4°C for 24 h;

[0022] c. After incubation, it can be seen that the originally clear normal saline turns turbid. Use a sterile Pasteur pipette to recover the incubation solution. First, filter it through a 70 μm cell filter to remove residual dressing debris and impurities, which is the wound exudate containing infected pathogenic bacteria. Take a part of the wound exudate containing infected pathogenic bacteria, filter it twice through a syringe and a 0.22 μm filter to sterilize it, which is the sterile wound extract and can be used for the nutritional support of the subsequent co-assembled biomimetic biofilm. The two kinds of wound exudates are used immediately after extraction.

[0023] (3) Preparation of the biomimetic infected biofilm culture kit (kit A)

[0024] Dissolve the synthesized supramolecular precursor peptide powder into a sterile solution with sterile pure water, and use it immediately after sterilization. Spread the extracted wound exudate containing pathogenic bacteria on the bottom of the cell chamber, and add an equal volume of the supramolecular precursor peptide solution to mix the bacteria-carrying wound exudate and the supramolecular precursor peptide solution. When the two solutions come into contact, an interfacial diffusion reaction will occur, gradually forming a co-assembled hydrogel. And when the hydrogel is formed, the supramolecular hydrogel can hierarchically incorporate pathogenic bacteria, providing a 3D growth environment for the formation of subsequent high-thickness biofilms.

[0025] (4) Preparation and use of the antibiotic screening kit (kit B)

[0026] Prepare various antibiotic solutions with concentration gradients using a new 24-well plate. Insert the above-cultured biomimetic infected biofilm directly together with the upper chamber into the antibiotic solution and continue to culture for 24 h. After the culture is completed, the minimum inhibitory concentration of different antibiotics against the biomimetic biofilm can be judged by observing the clarity of the solution in the lower chamber with the naked eye. Further, the killing effect of different antibiotics on the bacteria in the biofilm can be evaluated by counting the CFU of the biofilm.

[0027] Furthermore, the wound exudate in step a of step (2) can be from infectious or non-infectious wounds.

[0028] Furthermore, the wound exudate in step a of step (2) can be from mice, rabbits, humans or other mammalian sources;

[0029] Further, the pathogenic bacteria contained in the wound exudate in step (2), sub-step a, can come from the original wound surface or can be self-added with the pathogenic bacteria to be studied.

[0030] Further, in step (3), sub-step a, the polypeptide powder can be sterilized by ultraviolet sterilization, pasteurization, and filtration sterilization with a 0.22 μm filter membrane.

[0031] Further, the gelation sequence in step (3) can be to first add the wound fluid to the bottom of the cell chamber and then add it to the supramolecular precursor peptide solution, or it can be to first add the supramolecular precursor peptide solution to the bottom of the cell chamber and then add the wound fluid.

[0032] Further, the concentration of the polypeptide powder solution in step (3) is 1-4 wt%.

[0033] Further, after the polypeptide powder is configured into a solution in step (3), it is sterilized by filtration with a 0.22 μm filter membrane, avoiding the denaturation effect of ultraviolet rays on proteins.

[0034] Further, the static gelation conditions in step (3) are: 37 degrees, and the static time is 1-2 h.

[0035] As an in vitro biofilm infection model, the present invention can be used for customized screening of in vitro antibiotic drugs and has good application effects.

[0036] Through supramolecular co-assembly, the present invention endows the supramolecular precursor peptide sequence with functionality through design, enables it to co-assemble with the exudate of the infected wound of the patient, reproduces the 3D structure of in vivo biofilm infection, and retains active components such as host proteins. Thus, it can study the effect of antibiotics on bacterial biofilms under in vivo-like conditions to screen suitable antibiotics. In terms of the preparation method, the present invention only needs to collect the wound fluid by extracting the inner dressing of the patient's wound, co-assemble and culture it with the supramolecular precursor peptide for 24 h to form a biomimetic infected biofilm, which can be prepared quickly and in batches for high-throughput antibiotic screening in a timely manner to meet the clinical treatment needs.

[0037] The present invention has carried out co-culture verification for the common infectious bacteria Staphylococcus aureus and Pseudomonas aeruginosa. The supramolecular precursor peptide-wound fluid system has good biocompatibility with both, can promote the 3D growth of bacteria and form a biofilm. At the same time, antibiotic susceptibility tests were carried out on the biofilms cultured by this system, and it was found that their drug resistance was significantly improved, which was more in line with the antibiotic resistance state of in vivo biofilm infection. Description of the Drawings

[0038] Figure 1 It is a flow chart of the preparation method of the GF-wound fluid co-assembled hydrogel biofilm culture scaffold in Example 1 of the present invention.

[0039] Figure 2 Schematic structural diagram of the supramolecular precursor peptide GF molecule in Example 1 of the present invention.

[0040] Figure 3 Results of mass spectrometry (MS) and high performance liquid chromatography analysis of the synthesized supramolecular precursor peptide GF.

[0041] Figure 4 Flow chart for constructing a biomimetic infectious biofilm using a cell chamber (Kit A).

[0042] Figure 5 Mechanism diagram of bacteria participating in the hydrogel during the gelation stage.

[0043] Figure 6 Protein secondary structure of the GF-wound fluid co-assembled hydrogel determined by circular dichroism spectroscopy.

[0044] Figure 7 Scanning electron microscope image of the GF-wound fluid co-assembled hydrogel.

[0045] Figure 8 SDS-PAGE protein band analysis of the GF-wound fluid co-assembled hydrogel.

[0046] Figure 9 Scanning electron microscopy results of Staphylococcus aureus biofilms cultured with GF-wound fluid hydrogel.

[0047] Figure 10 Scanning electron microscopy results of Pseudomonas aeruginosa biofilms cultured with GF-wound fluid hydrogel.

[0048] Figure 11 Scanning electron microscopy results of Staphylococcus aureus-Pseudomonas aeruginosa composite biofilms cultured with GF-wound fluid hydrogel.

[0049] Figure 12 Comparison of vancomycin sensitivity between Staphylococcus aureus biofilms cultured with GF-wound fluid hydrogel and planktonic Staphylococcus aureus.

[0050] Figure 13 Comparison of polymyxin-B sensitivity between Pseudomonas aeruginosa biofilms cultured with GF-wound fluid hydrogel and planktonic Pseudomonas aeruginosa.

[0051] Figure 14 Flow chart for the construction and use of an antibiotic drug screening kit (Kit B).

[0052] Figure 15Photograph of the minimum inhibitory concentration results of susceptibility testing of Staphylococcus aureus - Pseudomonas aeruginosa complex biofilms cultured with GF - wound fluid hydrogel against vancomycin, polymyxin - B, and ciprofloxacin.

[0053] Figure 16 Residual CFU counts of Staphylococcus aureus - Pseudomonas aeruginosa complex biofilms cultured with GF - wound fluid hydrogel after 24 - hour treatment with different concentrations of antibiotics. Detailed implementation mode

[0054] The present invention will be described in detail below in conjunction with embodiments, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope protected by the present invention.

[0055] Example 1: Construction and material characterization method of a bionic infectious biofilm culture kit based on infected wound fluid, and its process is as Figure 1 shown, and the specific preparation process is as follows:

[0056] Under preset conditions, when the bacteria - loaded wound fluid contacts the supramolecular precursor peptide solution, the two trigger co - assembly at the liquid - liquid interface to form a 3D hydrogel structure. At the same time, pathogenic bacteria are three - dimensionally incorporated into the hydrogel during the gelation process, and the hydrogel provides a 3D growth and film - forming environment for pathogenic microorganisms; among them, the wound fluid is the trigger condition for co - assembly. Therefore, in this example, the gelation performance of GF and the wound fluid and the material characterization after gelation are importantly described.

[0057] (1) Preparation of the supramolecular precursor peptide Pal - VVVAAA - KKKHWGF - NH2 (GF)

[0058] a. Synthesize the supramolecular precursor peptide using solid - phase synthesis.

[0059] Swell Fmoc - Glu - Wang resin

[0060] Take 1 g of Fmoc - Glu - Wang resin, load it into a solid - phase synthesizer, transfer 15 mL of DCM (dichloromethane) to the resin and wait for 15 min to fully swell the resin, and then filter off the DCM by suction.

[0061] Remove the amino - protecting group Fmoc

[0062] Add 20% piperidine / 80% DMF, stir and react for 10 min + 10 min, then filter by suction. Wash repeatedly with DCM and isopropanol and drain. Take out a small amount of resin and monitor the reaction progress by the Kaiser method.

[0063] Production of the target peptide

[0064] Dissolve 2 g of Fmoc-aa-Wang resin, DIC, and HOBt in DMF in sequence, add them to a solid-phase synthesizer, add DMF, stir and react for 1.5 h, wash with DCM and isopropanol, and monitor the reaction progress by the Kaiser method.

[0065] Condense Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Trp-OH, Fmoc-His-OH, Fmoc-Lys-OH, Fmoc-Lys-OH, Fmoc-Lys-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Val-OH, and Pal-OH in sequence. The obtained amino acid sequence is PAL-VVVAAA-KKKHWGF (i.e., the GF sequence).

[0066] Swell the synthesized peptide chain-resin with DCM and then filter by suction. Add TFA / EDT / TIS / H2O and react at room temperature for 2 h. Take out the reaction solution, rotary evaporate to dryness, pour it into ice-cold diethyl ether for crystallization. After the ether has completely evaporated, add methanol to dissolve the sample, rotary evaporate to dryness, add ice-cold ether again for crystallization, and take the precipitate and dry it under reduced pressure to obtain the crude polypeptide.

[0067] b. Use reverse high-performance liquid chromatography (RP-HPLC) to separate and purify the crude peptide. After the product is treated by vacuum freeze-drying, its molecular weight is identified by mass spectrometry.

[0068] c. Confirm the quality of the polypeptide by ESI-MS.

[0069] (2) Extraction of infected wound exudate

[0070] a. Prepare sterile forceps and sterile seal bags. Remove the surface dressing of the wound to expose the inner wound dressing. Use sterile forceps to remove the inner dressing soaked with infected wound fluid and place it in a sterile 50 ml centrifuge tube.

[0071] b. In a biosafety cabinet, use sterile scissors to cut the inner wound dressing in step a into pieces, add normal saline at a volume ratio of 1:1, and incubate at 4 °C for 24 h.

[0072] c. After the incubation is completed, it can be seen that the originally clear normal saline turns turbid. Use a sterile Pasteur pipette to recover the incubation solution. First, filter it through a 70-μm cell filter to remove residual dressing debris and impurities, which is the wound exudate containing the infectious pathogen. Take a part of the wound exudate containing the infectious pathogen, and filter it twice through a syringe and a 0.22-μm filter to sterilize it, which is the sterile wound extract and can be used for the nutritional support of the subsequent co-assembled biomimetic biofilm. The two kinds of wound exudates are used immediately after extraction.

[0073] (3) Preparation and use of the biomimetic infectious biofilm culture kit

[0074] a. Dissolve the supramolecular precursor peptide powder synthesized in step (1) into a sterile solution with sterile pure water, and use it immediately after sterilization. Use a 24-well cell culture insert to construct a supramolecular co-assembled biomimetic infectious biofilm culture kit: Add 40 μl of the supramolecular precursor peptide solution to the upper chamber of the cell culture insert, and let it stand to spread the solution over the bottom layer of the upper chamber. Add 1 ml of sterile wound extract to the lower chamber to complete the preparation of the dual-chamber supramolecular co-assembled biomimetic infectious biofilm culture kit. After the kit is prepared, it can be stored at 4 °C for one week and used at any time.

[0075] b. Take 30 μl of the wound exudate containing the infectious pathogen extracted in step (2), and directly add it to the upper chamber of the above kit using a 200-μl pipette tip to mix the bacteria-carrying wound exudate with the supramolecular precursor peptide solution. An interfacial diffusion reaction will occur when the two solutions come into contact, gradually forming a co-assembled hydrogel. And when the hydrogel is formed, the supramolecular hydrogel can hierarchically incorporate the pathogenic bacteria, providing a 3D growth environment for the formation of the subsequent high-thickness biofilm. After standing at room temperature for 1 h until the gelation is completed, place the kit in a 37 °C incubator and culture for 24 h, and the supramolecular hydrogel can form a biomimetic infectious biofilm.

[0076] Using sterile wound fluid and supramolecular precursor peptide solution for co-assembly actually generates a biofilm growth scaffold without bacteria, which is convenient for material characterization of the finally generated scaffold material, such as circular dichroism, SDS-PAGE detection, etc. These characterizations will greatly affect the accuracy of the experimental results in the presence of bacteria, so sterile wound fluid is used for material construction and characterization.

[0077] Figure 1 and Figure 2 are respectively the preparation process of the GF supramolecular precursor peptide and the schematic diagram of the molecular structure of the GF supramolecular precursor peptide, Figure 3 The mass spectrometry and high performance liquid chromatography techniques of verified the successful synthesis of the GF supramolecular precursor peptide. Figure 4 is the schematic diagram of the preparation and use of the biomimetic infectious biofilm culture kit in this example, Figure 5 showing the mechanism diagram of the three-dimensional incorporation of pathogenic bacteria into the hydrogel during the gelation process.Figure 6 The circular dichroism measurement verified the transition of the molecular secondary structure before and after assembly, explaining the formation mechanism of the co-assembled hydrogel from the micro level. Figure 7 Scanning electron microscopy showed that the GF-wound fluid co-assembled hydrogel formed a fibrous porous structure mimicking the extracellular matrix, which was beneficial to bacterial adhesion and growth. Figure 8 Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the GF-wound fluid co-assembled hydrogel already contained the protein band part in the wound fluid, indicating that the GF-wound fluid co-assembled hydrogel had the bionic conditions for the nutrient components of the in vivo biofilm.

[0078] Example 2: For the method of constructing an in vitro model of single or composite microbial biofilm infection using a bionic infection biofilm culture kit (Kit A), the specific procedure is as follows:

[0079] (1) Extraction of wound exudate

[0080] a. Prepare sterile forceps and sterile sealable bags. Remove the surface dressing of the wound to expose the inner wound dressing. Use sterile forceps to remove the inner dressing soaked with infected wound fluid and place it in a sterile 50 ml centrifuge tube.

[0081] b. In a biosafety cabinet, use sterile scissors to cut the inner wound dressing in step a into pieces, add normal saline at a volume ratio of 1:1, and incubate at 4 °C for 24 h.

[0082] c. After incubation, it can be seen that the originally clear normal saline turns turbid. Use a sterile Pasteur pipette to recover the incubation solution. First, filter it through a 70 μm cell filter to remove residual dressing debris and impurities. The wound exudate is used immediately after extraction.

[0083] (2) Preparation of bacteria-loaded wound exudate for co-assembly

[0084] In this example, Staphylococcus aureus and Pseudomonas aeruginosa, which are common in infections, were selected to verify Kit A.

[0085] a. Take the Staphylococcus aureus and Pseudomonas aeruginosa strains stored at -80 °C, streak them on an LB plate, and culture them in a 37 °C biochemical incubator for 24 h.

[0086] b. Pick monoclonal colonies from the above-obtained plates and place them in LB medium, and culture them overnight on a 37 °C shaker.

[0087] c. Take the above bacterial solution, centrifuge it at 10000 g for 5 min, discard the supernatant, wash the bacterial pellet three times with PBS. Resuspend the bacterial pellet with WF to prepare a bacterial suspension with a concentration of 10 6 CFU / ml.

[0088] (3) Kit preparation

[0089] a. Sterilize the supramolecular precursor peptide powder GF overnight under ultraviolet ozone, dissolve it in sterile pure water to form a 2 wt% sterile solution, and use it freshly prepared after sterilization.

[0090] b. Use a 24-well cell culture insert to construct a supramolecular co-assembled antibiotic screening kit: Add 40 μl of the supramolecular precursor peptide solution to the upper chamber of the cell culture insert, and let it stand still until the solution covers the bottom layer of the upper chamber. Add 1 ml of sterile wound extract to the lower chamber to complete the preparation of the dual-chamber supramolecular co-assembled antibiotic screening kit.

[0091] (4) Inoculation of the GF-wound exudate supramolecular co-assembled gel biofilm

[0092] a. Take 30 μl of the bacteria-laden wound exudate and drop it into the upper chamber of the kit, allowing the wound exudate to contact the GF solution to trigger co-assembly and form a uniform 3D hydrogel adhering to the bottom of the cell culture insert. At the same time, during the co-assembly process, microorganisms incorporate into the hydrogel for 3D adhesion, providing support for subsequent 3D biofilm growth.

[0093] (5) Culturing the supramolecular co-assembled gel biofilm to maturity

[0094] Place the above-mentioned kit in a 37 °C biochemical incubator and culture for 24 h to form a mature biofilm.

[0095] Figure 9 , Figure 10 Scanning electron microscopy shows that the kit can effectively support the formation of Pseudomonas aeruginosa biofilm and Staphylococcus aureus biofilm. Figure 11 Scanning electron microscopy of the co-culture experiment shows that the kit can further support the growth and maturation of Pseudomonas aeruginosa-Staphylococcus aureus complex biofilm.

[0096] Experiment III: Conduct antibiotic susceptibility testing on the in vitro models of Pseudomonas aeruginosa microbial biofilm and Staphylococcus aureus microbial biofilm infections using the antibiotic screening kit (Kit B). The specific procedure is as follows:

[0097] (1) Extraction of wound exudate

[0098] a. Prepare sterile forceps and a sterile sealed bag. Remove the surface dressing of the wound to expose the inner wound dressing, and use sterile forceps to remove the inner dressing soaked with infected wound fluid and place it in a sterile 50 ml centrifuge tube.

[0099] b. In a biosafety cabinet, use sterile scissors to cut the inner wound dressing in step a into small pieces, add normal saline at a volume ratio of 1:1, and incubate at 4 °C for 24 h.

[0100] c. After the incubation, it can be seen that the originally clear normal saline turns turbid. Use a sterile Pasteur pipette to recover the incubation solution. First, filter it through a 70-μm cell filter to remove residual dressing debris and impurities. The wound exudate is used immediately after extraction.

[0101] (2) Preparation of the bacteria-loaded wound exudate for co-assembly

[0102] a. Take the Staphylococcus aureus and Pseudomonas aeruginosa strains stored at -80 °C, streak them on an LB plate, and culture them in a 37 °C biochemical incubator for 24 h.

[0103] b. Pick monoclonal colonies from the above-mentioned obtained plates and place them in LB medium, and culture them overnight on a shaker at 37 °C.

[0104] c. Take the above bacterial solution, centrifuge it at 10,000 g for 5 min, discard the supernatant, and wash the bacterial pellet three times with PBS. Resuspend the bacterial pellet with WF to prepare single-bacterial suspensions of Staphylococcus aureus and Pseudomonas aeruginosa respectively, with a concentration of 10 7 CFU / ml.

[0105] (3) Preparation and use of the bionic infection biofilm culture kit (Kit A)

[0106] a. Take the supramolecular precursor peptide powder, dissolve it in sterile pure water to form a sterile solution, and use it immediately after sterilization; use a 24-well cell culture insert to construct a supramolecular co-assembly bionic infection biofilm culture kit: add 40 μl of the supramolecular precursor peptide solution to the upper chamber of the cell culture insert, and let it stand to cover the bottom layer of the upper chamber; add 1 ml of sterile wound extract to the lower chamber to complete the preparation of the double-chamber supramolecular co-assembly bionic infection biofilm culture kit. After the kit is prepared, it can be stored at 4 °C for one week and used at any time.

[0107] b. Take 30 μl of the bacteria-loaded wound fluid prepared in step (2), and directly add it to the upper chamber of the above kit using a 200-μl pipette tip to mix the bacteria-loaded wound exudate with the supramolecular precursor peptide solution. An interfacial diffusion reaction will occur when the two solutions come into contact, gradually forming a co-assembled hydrogel. And when the hydrogel forms, the supramolecular hydrogel can hierarchically incorporate pathogenic bacteria, providing a 3D growth environment for the formation of subsequent high-thickness biofilms. After standing at room temperature for 1 h until the gelation is completed, place the kit in a 37 °C constant temperature incubator and culture it for 24 h, and the supramolecular hydrogel can form a bionic infection biofilm.

[0108] (4) Preparation and use of the antibiotic screening kit (Kit B)

[0109] The preparation and use process of Kit B is as Figure 14As shown. In summary, use MH medium to configure a concentration gradient of the antibiotic to be detected in a well plate. In this example, vancomycin sensitive to Gram-positive bacteria and polymyxin-B sensitive to Gram-negative bacteria were used to test Staphylococcus aureus and Pseudomonas aeruginosa respectively:

[0110] a. Weigh 21 g of MH medium powder and dissolve it in 1000 ml of pure water. After autoclaving, cool it to room temperature for standby.

[0111] b. Use a precision balance to accurately weigh 10.24 mg of vancomycin and polymyxin-B into an EP tube, add 1 ml of MH medium to dissolve them respectively, and prepare the antibiotic stock solution. The stock solution can be stored at 4 °C for up to one week.

[0112] c. Dilute the antibiotic stock solution tenfold with MH medium to obtain the antibiotic test culture solution with the highest concentration; then serially dilute the antibiotic test culture solution with the highest concentration to obtain the antibiotic test solution with a concentration gradient. Configure vancomycin and polymyxin-B-MHB solutions with concentrations of 1024, 512, 256, 128, 64, and 32 μg / ml respectively in this way.

[0113] d. Add the two antibiotic concentration gradient solutions obtained in (c) to each well of a 24-well plate, and the volume of the solution in each well is 1 ml.

[0114] e. Insert the Pseudomonas aeruginosa biofilm cultured in (3) into the above polymyxin-B-MH solution with a concentration gradient, and insert the Staphylococcus aureus biofilm into the above vancomycin-MH solution with a concentration gradient, and culture them in a 37 °C biochemical incubator for 24 hours. Directly observe whether the peripheral solution is clear to evaluate the inhibitory effect of the antibiotic on the in vitro model of biofilm wound infection at different concentrations.

[0115] Mature biofilms are more resistant to drugs than planktonic bacteria. In this example, the MIC drug sensitivity test of planktonic bacteria was used as a control group to illustrate that the biofilm resistance constructed by this model was significantly increased. As Figure 12 , shown in Figure 13, the minimum inhibitory concentration of vancomycin for planktonic Staphylococcus aureus is 4 μg / ml, and the minimum inhibitory concentration for Staphylococcus aureus biofilm has increased to 1024 μg / ml. The drug resistance has increased significantly; the minimum inhibitory concentration of polymyxin-B for planktonic Pseudomonas aeruginosa is 4 μg / ml, and the minimum inhibitory concentration for Pseudomonas aeruginosa biofilm has increased to 1024 μg / ml. The drug resistance has increased significantly, indicating that the biomimetic biofilm cultured by this kit successfully simulated the high drug resistance of the in vivo biofilm.

[0116] Implementation 4: Antibiotic drug screening for an in vitro model of wound infection with a Pseudomonas aeruginosa and Staphylococcus aureus complex microbial biofilm using an antibiotic screening kit (Kit B):

[0117] (1) Extraction of wound exudate

[0118] a. Prepare sterile forceps and sterile sealable bags. Remove the surface dressing of the wound to expose the inner wound dressing. Use sterile forceps to remove the inner dressing soaked with infected wound fluid and place it in a sterile 50 ml centrifuge tube;

[0119] b. In a biosafety cabinet, use sterile scissors to cut the inner wound dressing from step a into pieces, add normal saline at a 1:1 volume ratio, and incubate at 4 °C for 24 h;

[0120] c. After incubation, the originally clear normal saline becomes turbid. Use a sterile Pasteur pipette to recover the incubation solution. First, filter it through a 70 μm cell filter to remove residual dressing debris and impurities, and then filter it twice through a syringe and a 0.22 μm filter to sterilize it, obtaining a sterile wound extract. The wound exudate is used immediately after extraction.

[0121] (2) Preparation of the bacteria-loaded wound exudate for co-assembly

[0122] a. Take the Staphylococcus aureus and Pseudomonas aeruginosa strains stored at -80 °C, streak them on an LB plate, and culture them in a 37 °C biochemical incubator for 24 h.

[0123] b. Pick monoclonal colonies from the above-obtained plates and place them in LB medium, and culture them overnight on a 37 °C shaker.

[0124] c. Take the above bacterial solution, centrifuge it at 10000 g for 5 min, discard the supernatant, and wash the bacterial pellet three times with PBS. Resuspend the bacterial pellet with WF to prepare a Staphylococcus aureus - Pseudomonas aeruginosa dual-bacteria suspension, such that the final concentration of both bacteria is 10 7 CFU / ml.

[0125] (3) Preparation of Kit A

[0126] a. Sterilize the supramolecular precursor peptide powder GF overnight under ultraviolet ozone, dissolve it in sterile pure water to a 2 wt% sterile solution, and use it immediately after sterilization;

[0127] b. Use a 24-well plate cell chamber to construct a supramolecular co-assembly biomimetic biofilm preparation kit: Add 40 μl of the supramolecular precursor peptide solution to the upper chamber of the cell chamber, and let it stand to cover the bottom layer of the upper chamber; add 1 ml of sterile wound extract to the lower chamber.

[0128] (4) Inoculation of GF - wound exudate supramolecular co - assembled gel biofilm

[0129] a. Take 30 μl of the Staphylococcus aureus - Pseudomonas aeruginosa double - bacterium suspension prepared in (3), drop it into the upper chamber of the kit, so that the wound exudate contacts the GF solution, trigger co - assembly, and gel at room temperature for 1 h to form a uniform 3D hydrogel adhering to the bottom of the cell chamber.

[0130] (5) Culturing and maturing of the supramolecular co - assembled gel biofilm

[0131] Place the above - mentioned kit in a 37 - degree biochemical incubator and culture for 24 h to form a mature biofilm.

[0132] (6) Preparation and use of the antibiotic screening kit (Kit B)

[0133] Prepare antibiotic solutions with concentration gradients to be detected in the well plate using MH medium. In this example, ciprofloxacin, vancomycin, and polymyxin - B are taken as examples:

[0134] f. Weigh 21 g of MH medium powder, dissolve it in 1000 ml of pure water. After autoclaving, cool it to room temperature for standby.

[0135] g. Precisely weigh 0.64 mg of ciprofloxacin, 10.24 mg of vancomycin, and polymyxin - B into an EP tube, add 1 ml of MH medium to each to dissolve, and prepare antibiotic stock solutions. The stock solutions can be stored at 4 degrees for up to one week.

[0136] h. Dilute the antibiotic stock solutions ten - fold with MH medium to obtain the highest - concentration antibiotic test culture solutions; then serially dilute the highest - concentration antibiotic test culture solutions to obtain antibiotic test solutions with concentration gradients. Prepare ciprofloxacin - MHB solutions with concentrations of 64, 32, 16, 8, 4, 2 μg / ml, and vancomycin, polymyxin - B - MHB solutions with concentrations of 1024, 512, 256, 128, 64, 32 μg / ml in this way. i. Add the three kinds of antibiotic concentration - gradient solutions obtained in (c) to each well of a 24 - well plate, and the volume of the solution in each well is 1 ml.

[0137] j. Directly insert the biofilm cultured in (5) into the above 24 - well plate with gradient antibiotics and culture for 24 hours. Directly observe whether the peripheral solution is clear to evaluate the inhibitory effects of different antibiotics at different concentrations on the in - vitro model of biofilm - wound infection. Further compare the curative effects of different antibiotics through the gradient - dilution plate - coating CFU experiment to obtain the best drug regimen.

[0138] As Figure 15As shown, compared with vancomycin and polymyxin-B, ciprofloxacin can inhibit the growth of the complex biofilm at a lower concentration, while vancomycin and polymyxin-B generally have only a mediocre inhibitory effect on the complex biofilm even at higher concentrations; Figure 16 The determination of the remaining CFU of the biofilm Figure 16 shows that vancomycin can only have a bactericidal effect (greater than 99%) on Pseudomonas aeruginosa at a high concentration (1024 μg / ml); while polymyxin-B does not have an obvious inhibitory effect on Staphylococcus aureus even at a high concentration (1024 μg / ml). Ciprofloxacin can effectively kill the two pathogenic bacteria in the complex biofilm (greater than 99.9%) at a low concentration (64 μg / ml). To sum up, in the Pseudomonas aeruginosa-Staphylococcus aureus complex biofilm, the effectiveness of the three antibiotics from strong to weak is ciprofloxacin, vancomycin, and polymyxin-B.

[0139] As mentioned above, this is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A supramolecular co-assembly antibiotic screening kit based on wound fluid infected with wound biofilm, characterized in that, Various antibiotic solutions including a biomimetic infectious biofilm and a concentration gradient, wherein the biomimetic infectious biofilm is prepared from a supramolecular precursor peptide and wound exudate containing infectious pathogenic bacteria, and the sequence of the supramolecular precursor peptide is Pal-VVVAAA-KKKHWGF-NH2.

2. The supramolecular co-assembly antibiotic screening kit based on wound fluid of wound biofilm infection according to claim 1, wherein The wound exudate is exudative body fluid from infected or non-infected wounds on the body surface of various organisms, with or without the addition of pathogenic bacteria to be cultured.

3. The supramolecular co-assembly antibiotic screening kit based on wound fluid of wound biofilm infection according to claim 2, wherein The wound exudate is any one of mouse-derived, rabbit-derived, human-derived or other mammalian-derived.

4. The preparation method of the supramolecular co-assembly antibiotic screening kit based on wound fluid of wound biofilm infection according to claim 1, characterized in that, It includes the following steps: (1) Using the Fmoc solid-phase peptide synthesis method to synthesize the supramolecular precursor peptide, and separating and purifying it using reverse high-performance liquid chromatography; (2) Collect the wound exudate from the patient with a biofilm infection; (3) Spread the wound exudate containing pathogenic bacteria in a 24-well cell chamber, add an equal volume of an aqueous solution of the supramolecular precursor peptide to form a co-assembled hydrogel and three-dimensionally incorporate the pathogenic bacteria, and form a biomimetic infectious biofilm after cultivation; (4) Add various antibiotic solutions with a concentration gradient to a 24-well cell culture plate, insert the biomimetic infectious biofilm obtained in step (3) into different antibiotic solutions with a concentration gradient to form different antibiotic screening kits, and determine the minimum inhibitory concentration of different antibiotics against the biomimetic infectious biofilm.

5. The preparation method of the supramolecular co-assembly antibiotic screening kit based on wound fluid of wound biofilm infection according to claim 4, characterized in that, When collecting the patient's wound exudate in step (2), the specific method is as follows: a. Use sterile forceps to remove the wound surface dressing, place the inner gauze in a sterile sealed bag for recovery, and store it frozen at -80 °C in a refrigerator; b. Cut the frozen inner gauze in step a into small pieces and place them in a sterile centrifuge tube, add normal saline at a volume ratio of 1:1, incubate at 4 °C for 24 h, recover the supernatant, and filter it through a 70-μm filter to obtain the wound exudate.

6. The preparation method of the supramolecular co-assembly antibiotic screening kit based on wound fluid of infected wounds with wound biofilm according to claim 4, wherein, The specific method of step (3) is as follows: a. Dissolve the purified supramolecular precursor peptide powder in step (1) with sterile pure water to form a sterile peptide solution, and use it immediately after sterilization; b. Spread the wound exudate containing pathogenic bacteria extracted in step (2) on the bottom of the cell chamber, add it to an equal volume of the supramolecular precursor peptide solution, and let it stand to form a co-assembled hydrogel and support the growth of the biofilm.

7. The preparation method of the supramolecular co-assembly antibiotic screening kit based on wound fluid of infected wounds with wound biofilm according to claim 6, characterized in that, The concentration of the sterile peptide solution is 1-4 wt%, and the conditions for standing to form the co-assembled hydrogel are: 37 °C, and the standing time is 1-2 h.

8. Use of a supramolecular co-assembled antibiotic screening kit based on wound fluid of a wound infected with a wound biofilm, characterized in that, As an in vitro biofilm infection model, it is used for customized screening of in vitro antibiotic drugs.

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