A contact sterilization surface based on quorum sensing and its preparation method and application
A contact sterilization surface using extracellular polymers and antibiotics adhered to a substrate via quorum sensing addresses surface damage and stability issues, providing effective antibacterial action against bacteria like Staphylococcus aureus and Escherichia coli.
Patent Information
- Application Number
- CN202411203306.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2044-08-30
AI Technical Summary
Current antibacterial strategies for medical devices face challenges such as unstable release rates in non-contact sterilization and potential damage to the surface structure in contact sterilization, while existing methods for preparing antibacterial surfaces require harsh conditions and are prone to water absorption.
A method involving the extraction of extracellular polymers from bacteria, dissolving them in an antimicrobial agent solution, and adhering the mixture to a substrate to create a contact sterilization surface using quorum sensing, which promotes bacterial adhesion and kills bacteria through antibiotics.
The method results in a stable, uniform antibacterial surface that effectively kills bacteria without drug resistance, with a simple and mild preparation process, suitable for implantable medical devices.
Smart Images
Figure CN118718127B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical materials, and in particular to a contact-type sterilization surface based on quorum sensing, and a preparation method and application thereof. Background Art
[0002] Medical device-related infections are mostly caused by bacteria colonizing on the surface to form biofilms. After attaching to the surface, bacteria can secrete extracellular polymers to help further irreversible attachment. Subsequently, the bacteria are affected by the quorum sensing effect to colonize, move, and aggregate on the surface to build a biofilm hundreds of microns thick. The bacteria inside the biofilm will continue to secrete extracellular polymers to provide structural support for the biofilm and protect the bacteria from damage in adverse environments. Therefore, compared with mayfly bacteria, bacteria in biofilms have stronger resistance and recovery capabilities, which is one of the reasons why biofilms are difficult to eliminate and lead to repeated infections.
[0003] The antibacterial strategies that are currently being widely studied include non-contact sterilization and contact sterilization strategies. Non-contact sterilization surfaces generally refer to release-type sterilization surfaces loaded with metal ions or organic antimicrobial agents. Over time, the loaded antimicrobial agents are leached out to kill adhered and adjacent bacteria. This antibacterial method is broad-spectrum and not prone to drug resistance, but the inability to control the release rate will lead to an initial burst release. As time goes on, the release rate decreases, resulting in unstable antibacterial effects. In contrast, contact sterilization surfaces generally anchor antimicrobial agents to the surface of the material through electrostatic adsorption, covalent bonding, etc. This sterilization surface is not affected by the release rate of the antimicrobial agent, can have a uniform killing effect on bacteria adhering to the surface, and will not affect the sterilization effect due to the limited storage capacity of the antimicrobial agent.
[0004] The quorum sensing system is a microbial system that coordinates bacterial cell density-dependent gene expression. Bacteria are able to produce and sense signal molecules, which can accumulate locally to the threshold of activating specific gene transcription after being secreted outside the cell. Once the critical concentration is reached, they can bind to specific receptors and regulate gene expression or physiological activities through a series of cascade reactions, thereby affecting the interaction between microorganisms.
[0005] Publication No. CN106902396A discloses a method for preparing an antibacterial surface on the surface of a medical material, by introducing an atom transfer radical polymerization initiator on the surface of the medical material, first grafting an anti-adhesion polymer, then introducing an azide group, and finally introducing an antibacterial agent through a click reaction, thereby preparing an antibacterial surface co-modified with an anti-adhesion polymer layer and an antibacterial agent layer on the surface of the material. However, this method requires the use of a strong oxidizing solution, which can easily cause great damage to the surface structure of the medical material, and has high requirements on the environment and operators.
[0006] Publication No. CN111298203A discloses an antimicrobial peptide coating, which first uses amine and amino acid anhydride to produce a copolymer by one-pot ring-opening polymerization at room temperature, then dissolves the polypeptide copolymer in water to form a solution, and finally immerses the implant in the solution to obtain the antimicrobial peptide coating. However, since the polypeptide molecules contain many polar groups such as amino, carboxyl, and hydroxyl groups, they produce strong intermolecular hydrogen bonds, making the prepared antimicrobial peptide coating easy to absorb water, which limits its application in some fields. Summary of the invention
[0007] In order to solve the above technical problems, the present invention provides a method for preparing a contact sterilization surface based on quorum sensing, which has a mild preparation process and a simple process.
[0008] A method for preparing a contact sterilization surface based on quorum sensing comprises the following steps:
[0009] (1) Extraction of extracellular polymers from bacteria;
[0010] (2) dissolving the extracellular polymer obtained in step (1) in an antimicrobial agent solution to obtain an extracellular polymer solution loaded with an antimicrobial agent;
[0011] (3) The cleaned and dried substrate is soaked and adhered with the antimicrobial agent-loaded extracellular polymer solution obtained in step (2) to obtain a contact sterilization surface based on quorum sensing.
[0012] Extracellular polymers usually refer to macromolecular polymers secreted by bacteria outside the cell, and their main components are polysaccharides, proteins, nucleic acids, lipids, humic acid, etc. Extracellular polymers contain amine functional groups, carboxylates and phosphates, etc., which can promote the adhesion of bacteria on the surface.
[0013] The protein in the extracellular polymer of the present invention has an adsorption effect on antibiotics, and functional groups such as carboxyl, hydroxyl and amine in the protein can form a stable and tight complex with the antibiotics. The extracellular polymer solution loaded with antibacterial agent adheres to the substrate by immersion on the substrate surface to obtain a contact sterilization surface.
[0014] Preferably, in step (1), the bacteria is selected from at least one of Bacillus subtilis and Staphylococcus aureus.
[0015] Further preferably, the Bacillus subtilis is numbered as ATCC 6633, and the Staphylococcus aureus is numbered as ATCC 6538, and both bacteria are from China National Center for Microbiological Culture Collection.
[0016] Preferably, in step (1), the method for extracting extracellular polymers is centrifugation.
[0017] The centrifugation method comprises the following steps: inoculating bacteria in a culture medium, placing the bacteria in a shaking incubator for 16 to 18 hours, taking the bacteria out and centrifuging them, filtering the supernatant, and lyophilizing the liquid after dialyzing the supernatant to obtain extracellular polymers.
[0018] The invention adopts a centrifugal method to extract extracellular polymers, and the polymers on the surface of microorganisms are stripped by the centrifugal force generated during centrifugation. The extraction method has simple steps, will not cause cell rupture and has no reagent pollution.
[0019] Preferably, in step (2), the antimicrobial agent is an antibiotic or a bactericide.
[0020] More preferably, the antibiotic is at least one of levofloxacin, erythromycin and linezolid; and the bactericide is chlorhexidine.
[0021] Preferably, in step (2), the concentration of the antibacterial agent in the extracellular polymer solution loaded with the antibacterial agent is 2-256 μg / mL.
[0022] Further preferably, the concentration of levofloxacin is 2-8 μg / mL; the concentration of erythromycin is 16-256 μg / mL; the concentration of linezolid is 16-256 μg / mL; and the concentration of chlorhexidine is 25-100 μg / mL.
[0023] Preferably, in step (2), the concentration of the extracellular polymer in the extracellular polymer solution loaded with the antibacterial agent is 10-30 mg / mL.
[0024] Preferably, in step (3), the substrate is a silicon wafer.
[0025] Preferably, in step (3), the cleaning step of the substrate is: firstly, ultrasonically cleaning the substrate using anhydrous ethanol, deionized water or ultrapure water, and then drying the surface of the substrate using nitrogen gas.
[0026] Preferably, in step (3), the immersion adhesion is room temperature adhesion, and the time is more than 0.5 h.
[0027] More preferably, the immersion and adhesion time is more than 2 h.
[0028] The present invention also provides a contact-type sterilization surface based on quorum sensing prepared by the above preparation method, which can promote the adhesion of bacteria on the surface and kill them through antibiotics, and has good antibacterial activity.
[0029] Preferably, the contact sterilization surface has activity against Staphylococcus aureus and Escherichia coli.
[0030] The present invention also provides the application of the above-mentioned contact sterilization surface based on quorum sensing in the field of implantable medical devices. The antibacterial agent is compounded on the surface of the substrate through extracellular polymers. It is not affected by the release rate of the antibacterial agent, can adhere to bacteria, and has a uniform killing effect on the bacteria adhered to its surface, thereby preventing infection.
[0031] Compared with the prior art, the beneficial effects achieved by the present invention are:
[0032] (1) Extracellular polymers extracted from bacteria are selected as active substances that promote bacterial adhesion, and antibacterial agents are loaded into them. The prepared contact sterilization surface has activity against Staphylococcus aureus and Escherichia coli.
[0033] (2) The contact sterilization surface preparation process of the present invention is mild and simple, and does not require complex molecular design and surface pretreatment, and can be achieved by simple immersion.
[0034] (3) The contact sterilization surface based on quorum sensing obtained by the present invention has broad application prospects in the field of medical devices implanted in the body. It can evenly kill bacteria adhering to the surface and prevent infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 This is a digital photo of the extracellular polymers extracted from bacteria in Comparative Example 1.
[0036] Figure 2 The atomic force microscope morphology a and contact angle image b of the surface of the conditional membrane prepared in Comparative Example 1.
[0037] Figure 3 The infrared spectrum test result a of the extracellular polymer loaded with levofloxacin prepared in Example 1 and the extracellular polymer in Comparative Example 1 and the infrared spectrum test result b of the extracellular polymer powder extracted from two bacteria in Comparative Example 1.
[0038] Figure 4 These are SEM images of the effect of the extracellular polymer conditioned membrane prepared in Comparative Example 1 on the adhesion of different bacteria, wherein a-1 is the control group of Staphylococcus aureus, b-1 is the control group of Escherichia coli, c-1 is the control group of Pseudomonas aeruginosa, a-2 is the extracellular polymer conditioned membrane group of Staphylococcus aureus, b-2 is the extracellular polymer conditioned membrane group of Escherichia coli, and c-2 is the extracellular polymer conditioned membrane group of Pseudomonas aeruginosa.
[0039] Figure 5The SEM images of the effect of the extracellular polymer conditioned membrane prepared in Comparative Example 1 on the different adhesion times of Escherichia coli. Among them, a-1 is the control group after 0.5 h of adhesion, b-1 is the control group after 1 h of adhesion, c-1 is the control group after 4 h of adhesion, d-1 is the control group after 8 h of adhesion, e-1 is the control group after 16 h of adhesion, a-2 is the extracellular polymer conditioned membrane group after 0.5 h of adhesion, b-2 is the extracellular polymer conditioned membrane group after 1 h of adhesion, c-2 is the extracellular polymer conditioned membrane group after 4 h of adhesion, d-2 is the extracellular polymer conditioned membrane group after 8 h of adhesion, and e-2 is the extracellular polymer conditioned membrane group after 16 h of adhesion.
[0040] Figure 6 SEM image a, flat plate image b and laser confocal microscope image c of the contact sterilization surface prepared in Example 1 and the extracellular polymer conditioned film prepared in Comparative Example 1 on the adhesion and antibacterial effect of Staphylococcus aureus when the antibacterial time is 2 h.
[0041] Figure 7 SEM image a, flat plate image b and laser confocal microscope image c of the contact sterilization surface prepared in Example 1 and the extracellular polymer conditioned film prepared in Comparative Example 1 on the adhesion and antibacterial effect of Escherichia coli when the antibacterial time is 2 h.
[0042] Figure 8 are the antibacterial rates of the contact sterilization surface prepared in Example 1, respectively, the antibacterial rate a in the suspended bacterial solution and the antibacterial rate b of the contact sterilization surface. DETAILED DESCRIPTION
[0043] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.
[0044] The raw materials used in the present invention are all commercially available.
[0045] Example 1
[0046] (1) Extraction of extracellular polymers:
[0047] Bacillus subtilis (numbered ATCC 6633) or Staphylococcus aureus (numbered ATCC 6538) was inoculated in LB medium or TSB medium, wherein the formula of LB medium was: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 1 L deionized water, and the pH value was 7.2±0.2; the formula of TSB medium was: 17 g / L tryptone, 3 g / L soy peptone, 5 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L glucose, 1 L deionized water, and the pH value was 7.2±0.2. The culture medium inoculated with Bacillus subtilis was placed in a shaking incubator, cultured at 37°C and 140 rpm for 16-18 h, taken out and centrifuged at 10,000 rpm for 30 min, the supernatant was filtered once using 0.22 μm and 0.1 μm filter membranes, the filtered supernatant was placed in a 500 Da dialysis bag, dialyzed with deionized water for 48 h, and the dialyzed liquid was freeze-dried to obtain extracellular polymers.
[0048] (2) Preparation of extracellular polymer solution loaded with antimicrobial agent:
[0049] 30 mg of freeze-dried extracellular polymer powder was dissolved in 1 mL of 2 μg / mL levofloxacin solution to prepare a levofloxacin-loaded extracellular polymer solution.
[0050] (3) Preparation of contact sterilization surface:
[0051] First, the silicon wafer was ultrasonically cleaned for 5-15 min using anhydrous ethanol and deionized water respectively, and then dried using nitrogen. The cleaned and dried silicon wafer was placed in a well plate, and the extracellular polymer solution loaded with levofloxacin was added to the well plate with the silicon wafer, and adhered at room temperature for 2 h. After the extracellular polymer solution loaded with levofloxacin was sucked out, the silicon wafer was cleaned twice with a phosphate buffer solution with a pH of 7.4 to obtain a contact sterilization surface based on quorum sensing.
[0052] Example 2
[0053] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 4 μg / mL levofloxacin solution.
[0054] Example 3
[0055] The preparation steps were the same as those in Example 1, except that the antibacterial agent used was 8 μg / mL levofloxacin solution.
[0056] Example 4
[0057] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is 16 μg / mL erythromycin solution.
[0058] Example 5
[0059] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is 32 μg / mL erythromycin solution.
[0060] Example 6
[0061] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is 128 μg / mL erythromycin solution.
[0062] Example 7
[0063] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is 256 μg / mL erythromycin solution.
[0064] Example 8
[0065] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 16 μg / mL linezolid solution.
[0066] Example 9
[0067] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 32 μg / mL linezolid solution.
[0068] Example 10
[0069] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 128 μg / mL linezolid solution.
[0070] Embodiment 11
[0071] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 256 μg / mL linezolid solution.
[0072] Example 12
[0073] The preparation steps are the same as those in Example 1, except that the antibacterial agent used is a 50 μg / mL chlorhexidine solution.
[0074] Comparative Example 1: Preparation of an extracellular polymer conditioned membrane, which differs from Example 1 in that no antibacterial agent is loaded.
[0075] (1) Extraction of extracellular polymers:
[0076] Bacillus subtilis (numbered ATCC 6633) or Staphylococcus aureus (numbered ATCC 6538) was inoculated into the culture medium and placed in a shaking incubator, cultured at 37ºC, 140 rpm for 16-18 h, taken out and centrifuged at 10000 rpm for 30 min, the supernatant was filtered once using 0.22 μm and 0.1 μm filter membranes, the filtered supernatant was placed in a 500 Da dialysis bag, dialyzed with deionized water for 48 h, and the dialyzed liquid was freeze-dried to obtain extracellular polymers extracted from Bacillus subtilis or Staphylococcus aureus, respectively.
[0077] (2) Preparation of contact sterilization surface:
[0078] First, the silicon wafer was ultrasonically cleaned for 5-15 min using anhydrous ethanol and deionized water respectively, and then dried using nitrogen. The cleaned and dried silicon wafer was placed in a well plate, and the above-mentioned extracellular polymer was prepared into a 30 mg / mL solution and added to the well plate where the silicon wafer was placed, and adhered at room temperature for 2 h. After the extracellular polymer solution was sucked out, the silicon wafer was washed twice with a phosphate buffer solution with a pH of 7.4 to obtain an extracellular polymer conditioned membrane extracted from Bacillus subtilis or Staphylococcus aureus.
[0079] Sample analysis
[0080] 1. The surface morphology, hydrophilicity and hydrophobicity, infrared spectrum and bacterial adhesion promoting performance of the Staphylococcus aureus extracellular polymer conditioned film prepared in Comparative Example 1 were tested.
[0081] (1) Macroscopic morphology of extracellular polymer powder: Figure 1 As shown in the digital photo, the extracellular polymers extracted from Staphylococcus aureus are in powder form.
[0082] (2) Observation of the morphology and hydrophilicity test of the extracellular polymer conditioned membrane of Staphylococcus aureus: The surface morphology of the extracellular polymer conditioned membrane was observed and photographed by atomic force microscopy (AFM), and the hydrophilicity of the extracellular polymer conditioned membrane was determined by contact angle test.
[0083] Result analysis: Figure 2 As shown in a, compared with the blank silicon wafer, the height of the extracellular polymer conditioned film increased significantly and the roughness also increased, indicating that the extracellular polymer formed a uniform conditioned film on the entire silicon wafer surface. Figure 2 Figure b shows that compared with the blank silicon wafer, the droplet on the extracellular polymer conditioned membrane spreads significantly and the contact angle decreases significantly. In addition, the droplet becomes flatter after 20 seconds and the contact angle decreases further, indicating that the extracellular polymer conditioned membrane is more hydrophilic.
[0084] (3) Infrared spectral analysis: Infrared spectral scanning was performed on the extracellular polymer powder extracted from Bacillus subtilis and the extracellular polymer powder extracted from Staphylococcus aureus to analyze the main components of the extracellular polymer powder extracted from the two bacteria.
[0085] Result analysis: Figure 3 b in the figure shows that 3306 cm -1 and 3310 cm -1 The peaks at 2938 cm-1 come from the stretching vibration of NH and -OH in the protein. -1 The CH stretching vibration of -CH3 is at 1649 cm -1 The stretching vibration of C=O or CN in the secondary structure of the protein is 1541 cm -1 The stretching vibration of CN or the deformation vibration of NH is at 1453 cm -1 and 1400 cm -1 The stretching vibration of C=O in the carboxylic acid group is 1243 cm -1 and 1081 cm -1 The asymmetric stretching vibrations of COC and COH / COR related to the polysaccharide backbone are at 656 cm -1 The results show that the main components of the extracellular polymers extracted from Bacillus subtilis and Staphylococcus aureus are polysaccharides and proteins. Therefore, there is little difference in the performance of the membranes extracted from Bacillus subtilis or Staphylococcus aureus.
[0086] (4) Test of the effect of extracellular polymer conditioned membrane on bacterial adhesion: The test bacteria were Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. First, the silicon wafer adhered with the extracellular polymer conditioned membrane was placed under ultraviolet light for 30 min for sterilization. Then, the bacterial solution of the test bacteria in the logarithmic growth phase was taken and the bacterial concentration was adjusted to 10 6 CFU / mL, the silicon wafer was immersed in the above bacterial solution for co-culture, wherein the blank control group was a silicon wafer without conditional membrane adhesion. After 4 h of incubation at room temperature, the number of bacterial adhesion was observed using a scanning electron microscope to verify the promotion of bacterial adhesion of the extracellular polymer conditional membrane without antibiotic loading prepared in Comparative Example 1.
[0087] Result analysis: Figure 4 It was shown that, compared with the control group, the extracellular polymer conditioned membrane without antibiotic loading prepared in Comparative Example 1 promoted the adhesion of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
[0088] (5) Test on the effect of extracellular polymer conditioned membrane on the adhesion time of Escherichia coli: The test bacteria were Escherichia coli. First, the silicon wafer adhered with the extracellular polymer conditioned membrane was sterilized under ultraviolet light for 30 min. Then, the bacterial solution of the test bacteria in the logarithmic growth phase was taken and the bacterial concentration was adjusted to 10 6 CFU / mL, the silicon wafer was immersed in the bacterial solution for co-culture, wherein the blank control group was a silicon wafer without conditional membrane adhesion. After culturing at room temperature for 0.5, 1, 4, 8, and 16 h, the number of E. coli adhesion was observed using a scanning electron microscope to verify the effect of the antibiotic-free extracellular polymer conditional membrane prepared in Comparative Example 1 on bacteria at different adhesion times.
[0089] Result analysis: Figure 5 The results showed that the extracellular polymer-conditioned membranes promoted the adhesion of Escherichia coli at different time periods, and the adhesion-promoting effect became increasingly obvious as time went on.
[0090] 2. Bacterial adhesion experiment on contact sterilization surface based on quorum sensing.
[0091] (1) Infrared spectral analysis: Infrared spectral scanning was performed on the extracellular polymer loaded with levofloxacin prepared in Example 1 and the extracellular polymer prepared in Comparative Example 1 to analyze the effect of loading levofloxacin on infrared absorption.
[0092] Result analysis: Figure 3 As shown in a, after the extracellular polymers were loaded with levofloxacin, the absorption peak position did not change significantly, but the absorbance at the absorption peak related to the protein decreased slightly. This is because the proteins in the extracellular polymers have an adsorption effect on antibiotics, and the functional groups such as carboxyl, hydroxyl and amine in the proteins can form stable and tight complexes with the antibiotics, interfering with the infrared absorption of these groups.
[0093] (2) Effect of contact sterilization surface based on quorum sensing on bacterial adhesion: In vitro antibacterial test was used, and the test bacteria selected were Staphylococcus aureus and Escherichia coli. The silicon wafer was first immersed in an extracellular polymer solution loaded with antibiotics and allowed to adhere at room temperature for 2 h. After cleaning, the silicon wafer was then immersed in a solution with a concentration of 10 7 CFU / mL of the test bacterial strain liquid, and continued to adhere at room temperature for 1, 2, 4, and 8 h. The number of bacteria adhering to the surface was observed by coating, scanning electron microscopy, and laser confocal microscopy, respectively. The control group was the extracellular polymer conditioned film prepared in Comparative Example 1, verifying the antibacterial effect of the quorum sensing-based contact sterilization surface prepared in Example 1.
[0094] The test results of Staphylococcus aureus are as follows: Figure 6 As shown, the results analysis: Figure 6 a and Figure 6Figure b shows that compared with the control group, the number of Staphylococcus aureus adhered to the contact sterilization surface at 1 h and 2 h did not decrease significantly. This may be due to the fact that the antibiotics failed to fully exert their antibacterial effect in a short time and that the bacteria had died but some of the dead bacteria remained on the surface due to the adhesion of extracellular polymers. Figure 6 This phenomenon is also confirmed when the adhesion is 4 h. Figure 6 In b, the number of Staphylococcus aureus adhered to the contact sterilization surface was less than that adhered to the extracellular polymer condition film, but Figure 6 In a, the number of bacteria adhering to the contact sterilization surface is greater than that to the extracellular polymer membrane surface. Figure 6 a and Figure 6 Figure (b) shows that at 8 h, the number of Staphylococcus aureus adhered to the contact sterilization surface was reduced compared with the extracellular polymer-conditioned membrane surface.
[0095] Figure 8 The antibacterial rate of the contact sterilization surface prepared in Example 1 against Staphylococcus aureus is shown, which are the antibacterial rate a in the suspended bacterial solution and the antibacterial rate b on the contact sterilization surface. It can be seen that Figure 8 The antibacterial rate of a against Staphylococcus aureus increased from 16.33% at 1 h to 45.36% at 4 h, but decreased to 2.63% at 8 h. Figure 8 Figure b shows that there is no obvious antibacterial effect at 1 h and 2 h, the antibacterial rate against Staphylococcus aureus reaches 83.68% at 4 h, and drops to 39.63% at 8 h. Considering the characteristic that Staphylococcus aureus is more likely to develop drug resistance, it is speculated that the bacteria have developed a certain degree of drug resistance at this time.
[0096] The test results of Escherichia coli are as follows Figure 7 As shown, where: for Escherichia coli, Figure 7 Figure b shows that compared with the extracellular polymer conditional membrane, the contact sterilization surface showed less bacterial adhesion in each time period, indicating that the contact sterilization surface has a more obvious inhibitory effect on Escherichia coli. Figure 8 a and Figure 8 It can also be reflected in b. Figure 7 In a, at 4 h and 8 h, the amount of bacteria adhered to the contact sterilization surface was similar to that of the extracellular polymer conditional membrane. Figure 7 The laser confocal microscopy results in (c) suggest that some dead bacteria remain on the substrate surface due to the adhesion of extracellular polymers.
[0097] Examples 2-12 can prepare contact-type sterilization surfaces based on quorum sensing with similar surface morphology to that of Example 1. The difference is that although the antibiotics loaded in Examples 2 and 3 are the same as those in Example 1, which are third-generation quinolone antibiotics levofloxacin, the concentrations are different. The concentration of levofloxacin loaded in Example 2 is 4 μg / mL, and the concentration of levofloxacin loaded in Example 3 is 8 μg / mL. Through Examples 1-3, sterilization surfaces loaded with different concentrations of levofloxacin were prepared to obtain the minimum inhibitory concentration of levofloxacin.
[0098] The macrolide antibiotic erythromycin was loaded in Examples 4 to 7, wherein the erythromycin concentration in Example 4 was 16 μg / mL, the erythromycin concentration in Example 5 was 32 μg / mL, the erythromycin concentration in Example 6 was 128 μg / mL, and the erythromycin concentration in Example 7 was 256 μg / mL. Different concentrations of erythromycin were loaded in Examples 4-7 because theoretically, Staphylococcus aureus has good sensitivity to erythromycin, but in actual experiments, erythromycin at the minimum inhibitory concentration (16 μg / mL) did not show ideal antibacterial performance against Staphylococcus aureus, so an attempt was made to observe the antibacterial effect by increasing the erythromycin concentration.
[0099] The antibiotics loaded in Examples 8 to 11 are the synthetic oxazolidinone antibiotic linezolid, wherein the linezolid concentration loaded in Example 8 is 16 μg / mL, the linezolid concentration loaded in Example 9 is 32 μg / mL, the linezolid concentration loaded in Example 10 is 128 μg / mL, and the linezolid concentration loaded in Example 11 is 256 μg / mL. Different concentrations of linezolid are loaded in Examples 8-11 because theoretically Staphylococcus aureus has good sensitivity to linezolid, but in actual experiments, the minimum inhibitory concentration (16 μg / mL) of linezolid does not show ideal antibacterial performance against Staphylococcus aureus, so an attempt is made to observe the antibacterial effect by increasing the concentration of linezolid.
[0100] Theoretically, the three antibiotics have good inhibitory activity against Staphylococcus aureus, but the antibacterial rate decreases in actual application. This may be related to the thick cell wall of Staphylococcus aureus, which is difficult for antibiotics to penetrate, and the production of drug-resistant genes in the bacteria during reproduction.
[0101] The bactericide loaded in Example 12 is the cationic surfactant chlorhexidine, and the concentration is 50 μg / mL. The prepared contact sterilization surface exhibits good bactericidal performance.
[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for preparing a contact sterilization surface based on quorum sensing, characterized in that: The following steps are involved: (1) Extraction of extracellular polymers from bacteria; (2) dissolving the extracellular polymer obtained in step (1) in an antimicrobial agent solution to obtain an extracellular polymer solution loaded with an antimicrobial agent, wherein the concentration of the extracellular polymer is 10 to 30 mg / mL; (3) The cleaned and dried substrate is soaked and adhered with the antimicrobial agent-loaded extracellular polymer solution obtained in step (2) to obtain a contact sterilization surface based on quorum sensing.
2. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (1), the bacteria is selected from at least one of Bacillus subtilis and Staphylococcus aureus.
3. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (1), the method for extracting the extracellular polymer is a centrifugation method; the steps of the centrifugation method are: inoculating bacteria in a culture medium, placing it in a shaking incubator for 16 to 18 hours, taking it out and centrifuging it, filtering the supernatant, and lyophilizing the liquid after dialyzing the supernatant to obtain the extracellular polymer.
4. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (2), the antibacterial agent is at least one of levofloxacin, erythromycin, linezolid and chlorhexidine.
5. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (2), the concentration of the antibacterial agent in the extracellular polymer solution loaded with the antibacterial agent is 2-256 μg / mL.
6. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (3), the substrate is a silicon wafer.
7. The method for preparing a contact sterilization surface according to claim 1, characterized in that: In step (3), the immersion adhesion is room temperature adhesion, and the time is more than 0.5 h.
8. A contact sterilization surface based on quorum sensing prepared according to the method for preparing a contact sterilization surface according to any one of claims 1 to 7.
9. Application of the contact sterilization surface based on quorum sensing according to claim 8 in the field of implantable medical devices.