A method and application of regulating drug-resistant gene conjugation and transfer using inhibitors of drug-resistant targets
By regulating factors such as the donor bacteria concentration, the recipient bacteria ratio and the inhibitor concentration, and using drug-resistant target inhibitors to regulate the conjugation and transfer of drug-resistant genes, the problem of drug-resistant gene transmission in existing technologies is solved, and effective control of drug-resistant genes is achieved.
Patent Information
- Application Number
- CN202211026870.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-25
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2042-08-25
AI Technical Summary
Existing technologies make it difficult to effectively control the conjugative transfer of drug-resistant genes, especially the spread of resistance genes to β-lactams, polymyxins and aminoglycoside antibiotics.
Inhibitors of drug-resistant targets are used to regulate the conjugation and transfer of drug-resistant genes. By regulating factors such as the donor bacteria concentration, the recipient bacteria ratio, the inhibitor concentration and the action time, donor and recipient bacterial suspensions treated with inhibitors of different concentrations and types are prepared and mixed for culture. The suspensions are then spread on resistance plates for culture, and the conjugation and transfer frequency is calculated.
It has achieved the regulation of the frequency of drug-resistant gene conjugation and transfer, effectively blocking the spread of drug-resistant bacteria at different antibiotic targets, and has targeted control and safety evaluation.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pathogenic microorganism prevention and control, and specifically relates to a method and application of regulating the conjugation and transfer of drug-resistant genes by utilizing inhibitors of drug-resistant targets. Background Art
[0002] The spread of antibiotic resistance can lead to the emergence of multidrug-resistant bacteria, and the transfer of antibiotic resistance genes can also lead to the emergence of multidrug-resistant bacteria. Horizontal gene transfer primarily occurs through conjugation, transformation, transduction, lysogenic conversion, and transposition, with conjugation being the most important. Efficiently controlling the conjugative transfer of resistance genes has become a pressing issue.
[0003] β-lactams, polymyxins, and aminoglycosides are the oldest, most widely used, and most commonly clinically used antibiotics, but the spread of resistance genes has become increasingly serious. β-lactamases, polymyxin resistance proteins, and aminoglycoside-modifying enzymes are the primary causes of bacterial resistance to β-lactams, polymyxins, and aminoglycosides. These resistance-conferring components are known as resistance targets in pathogens. Due to the growing severity of pathogenic resistance, inhibitors targeting these resistance targets are increasingly used in clinical practice. These inhibitors are becoming increasingly important in regulating gene conjugation and transfer. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of existing control of drug-resistant gene conjugation and transfer, and to provide a method and application for regulating drug-resistant gene conjugation and transfer using inhibitors of drug-resistant targets.
[0005] The purpose of the present invention is to provide a method for regulating the conjugation and transfer of drug-resistant genes using inhibitors of drug-resistant targets.
[0006] The present invention also aims to provide application of the method in regulating the conjugation and transfer of drug-resistant genes.
[0007] The present invention provides a method for regulating the conjugative transfer of drug-resistant genes using inhibitors of drug-resistant targets. The method involves inoculating and culturing antibiotic-resistant donor and recipient bacterial strains separately, washing and resuspending them to obtain donor and recipient bacterial suspensions of varying concentrations. Different concentrations and types of drug-resistant target inhibitors are then added for treatment, and the donor and recipient bacterial suspensions are then mixed. The mixture is shaken and cultured in a 37°C constant-temperature shaker for 0.5 to 20 hours to obtain a conjugative transfer solution. The conjugative transfer solution is then applied to a dual-resistance plate and incubated in a 37°C constant-temperature incubator for 24 to 36 hours. The present invention has found that inhibitors of different types of targets can regulate the conjugation and transfer of drug-resistant genes, but the regulatory effects are different; inhibitors of different concentrations of targets have certain differences in the regulatory effects on the conjugation and transfer of drug-resistant genes; different donor bacteria concentrations and different donor-acceptor bacteria ratios have certain differences in the regulatory effects on the conjugation and transfer of drug-resistant genes. The frequency of conjugation and transfer of drug-resistant genes can be regulated by regulating the concentration of donor bacteria, the mixing ratio of donor and acceptor bacteria, the concentration, type, and action time of the inhibitor of the target; the method of the present invention is applicable to the study of conjugation and transfer of different drug-resistant genes (antibiotic-resistant genes), and can achieve the purpose of effectively controlling the spread of drug-resistant genes. The method of the present invention has the advantages of large operability and strong practicality, and can provide a method for controlling the spread of antibiotic-resistant bacteria with different antibiotic action targets in environmental water bodies.
[0008] The above-mentioned purpose of the present invention is achieved by the following technical means:
[0009] A method for regulating the conjugation and transfer of drug-resistant genes using an inhibitor of a drug-resistant target comprises the following steps:
[0010] S1. Activating donor and recipient bacteria separately to prepare donor and recipient bacterial suspensions; the donor bacteria contain a gene that confers resistance to an antibiotic, while the recipient bacteria do not contain the gene but instead contain a gene that confers resistance to another antibiotic; the donor bacteria are resistant bacteria whose antibiotic targets are cell walls, cell membranes, or protein synthesis;
[0011] S2. The donor bacterial suspension and the recipient bacterial suspension prepared in step S1 are respectively mixed with an inhibitor of the drug resistance target, and then the two mixed bacterial suspensions are mixed, and the mixture is cultured at a constant temperature of 36-38° C. to obtain a conjugated transfer bacterial solution; the inhibitor of the drug resistance target is an inhibitor that acts on a cell wall drug resistance target, an inhibitor that acts on a cell membrane drug resistance target, or an inhibitor that acts on a protein synthesis drug resistance target; the inhibitor of the drug resistance target is a drug that restores bacterial sensitivity to antibiotics by inhibiting the expression of the drug resistance target of antibiotic-resistant bacteria;
[0012] S3. The conjugative transfer bacterial solution prepared in step S2 is diluted and applied on a resistance plate containing the antibiotic to which the donor bacteria are resistant and another antibiotic to which the recipient bacteria are resistant in step S1. The total recipient bacteria suspension prepared in step S1 is diluted and applied on a resistance plate containing another antibiotic to which the recipient bacteria are resistant in step S1. The concentration of the conjugants in the conjugative transfer bacterial solution and the concentration of the total recipient bacteria are determined. The conjugative transfer frequency is calculated based on the ratio of the conjugant concentration to the total recipient bacteria concentration. The conjugative transfer frequency of the drug-resistant gene is regulated by regulating the donor bacteria concentration, the mixing ratio of the donor and recipient bacteria, and the concentration, type or action time (culture time) of the inhibitor of the drug-resistant target.
[0013] Preferably, in step S1, the donor bacteria are resistant bacteria to cephalosporins, resistant bacteria to polymyxins, or resistant bacteria to aminoglycosides; and the recipient bacteria are resistant bacteria to streptomycin.
[0014] More preferably, the cephalosporin antibiotic is ceftriaxone; the polymyxin antibiotic is polymyxin; and the aminoglycoside antibiotic is gentamicin.
[0015] More preferably, the drug-resistant bacteria is any one of Escherichia coli, Pseudomonas aeruginosa, Salmonella, and Streptococcus.
[0016] More preferably, the drug-resistant bacteria is Escherichia coli.
[0017] Preferably, in step S1, the donor bacteria is Escherichia coli resistant to cephalosporin antibiotics, polymyxin antibiotics or aminoglycoside antibiotics; and the recipient bacteria is Escherichia coli resistant to streptomycin.
[0018] Preferably, in step S1, the concentrations of the donor bacterial suspension and the recipient bacterial suspension are both 10 5 ~10 10 CFU / mL.
[0019] Further preferably, in step S1, the concentration of the bacterial suspension is 10 8 ~10 9 CFU / mL.
[0020] Further preferably, in step S1, the concentration of the bacterial suspension is 10 8 CFU / mL.
[0021] Preferably, in step S2, the donor bacterial suspension and the recipient bacterial suspension are mixed in a volume ratio of (0.25-4):1.
[0022] Further preferably, in step S2, the donor bacterial suspension and the recipient bacterial suspension are mixed in a volume ratio of (0.75-2):1.
[0023] Further preferably, in step S2, the donor bacterial suspension and the recipient bacterial suspension are mixed in a volume ratio of (1-2):1.
[0024] Further preferably, in step S2, the donor bacterial suspension and the recipient bacterial suspension are mixed in a volume ratio of 1:1.
[0025] Preferably, in step S2, the culturing time is 1 to 20 hours.
[0026] More preferably, in step S2, the culturing time is 4 to 20 hours.
[0027] Further preferably, in step S2, the culturing time is 4 to 8 hours.
[0028] Preferably, in step S2, the concentration of the inhibitor acting on the cell wall drug resistance target in the mixed solution is 1 to 200 mg / L.
[0029] Further preferably, in step S2, the concentration of the inhibitor acting on the cell wall drug resistance target in the mixed solution is 6.25-50 mg / L.
[0030] Further preferably, in step S2, the concentration of the inhibitor acting on the cell wall drug resistance target in the mixed solution is 25-50 mg / L.
[0031] Preferably, in step S2, the concentration of the inhibitor acting on the cell membrane drug resistance target in the mixed solution is 2 to 1024 mg / L.
[0032] Further preferably, in step S2, the concentration of the inhibitor acting on the cell membrane drug resistance target in the mixed solution is 16-128 mg / L.
[0033] Further preferably, in step S2, the concentration of the inhibitor acting on the cell membrane drug resistance target in the mixed solution is 32-64 mg / L.
[0034] More preferably, in step S2, the concentration of the inhibitor acting on the cell membrane drug resistance target in the mixed solution is 64 mg / L.
[0035] Preferably, in step S2, the concentration of the inhibitor acting on the protein synthesis drug resistance target in the mixed solution is 512-1024 mg / L.
[0036] Preferably, in step S2, the inhibitor acting on the cell wall resistance target is any one of tazobactam acid, clavulanic acid or sulbactam; the inhibitor acting on the cell membrane resistance target is any one of pterostilbene, ethanolamine or resveratrol; the inhibitor acting on the protein synthesis resistance target is any one of curcumin, oridonin, garcinol or anacardic acid.
[0037] Further preferably, in step S2, the inhibitor acting on the cell wall resistance target is tazobactam acid; the inhibitor acting on the cell membrane resistance target is pterostilbene; and the inhibitor acting on the protein synthesis resistance target is curcumin.
[0038] The application of the method in regulating the conjugation and transfer of drug-resistant genes is also within the protection scope of the present invention.
[0039] Preferably, the invention is used in the field of regulating environmental microorganisms or water disinfection by conjugation and transfer of drug-resistant genes.
[0040] The action time mentioned in the present invention is the culture time, that is, the culture regulation time.
[0041] Compared with the prior art, the present invention has the following beneficial effects:
[0042] 1. The present invention inhibits the conjugative transfer of drug-resistant genes by using inhibitors of drug-resistant targets. The frequency of conjugative transfer of drug-resistant genes under different conditions is regulated by controlling factors such as the concentration of donor bacteria, the mixing ratio of donor and recipient bacteria, and the concentration, type, or action time (cultivation control time) of the inhibitor of drug-resistant targets. The invention can also specifically evaluate the safety risks of using the inhibitor.
[0043] 2. The present invention utilizes inhibitors of drug-resistant targets to treat drug-resistant bacteria with different antibiotic targets. Compared with other control technologies, it has targeted control and can effectively regulate the spread of drug-resistant genes of drug-resistant bacteria with different targets. At the same time, it can also effectively control the development of drug resistance of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 The frequency of drug-resistant gene conjugation and transfer under treatment with different concentrations of inhibitors acting on cell wall drug-resistant targets in Example 1 of the present invention. DETAILED DESCRIPTION
[0045] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.
[0046] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.
[0047] Cephalosporin-resistant bacteria targeting the cell wall (cephalosporin-resistant E. coli DH5α): Add a resistant plasmid containing 10 μg / μL of the cephalosporin antibiotic ceftriaxone to a pre-cooled competent E. coli DH5α bacterial solution prepared with 0.1 mol / L CaCl2. Place the mixture in an ice-water bath for 25 minutes, then immediately heat-shock the mixture at 42°C for 45 seconds and cool it in an ice-water bath for 2 minutes. Then, add 700 μL of LB sterile broth, resuscitate the resulting mixture in a shaker at 37°C for 1 hour, and then dilute and spread it on a resistance plate containing cefotaxime to screen for resistant bacteria.
[0048] Streptomycin-resistant Escherichia coli: China Center for Type Culture Collection.
[0049] Polymyxin-resistant bacteria with cell membrane as resistance target (polymyxin-resistant E. coli DH5α): add a resistance plasmid containing 20 μg / μl polymyxin-resistant antibiotic polymyxin to a pre-cooled 0.1 mol / LCaCl2 competent E. coli DH5α bacterial solution, place it in an ice water bath for 30 minutes, immediately heat shock the mixture at 42°C for 90 seconds, and cool it in an ice water bath for 2 minutes, add 700 μL LB sterile broth, resuscitate the resulting mixture in a shaker at 37°C for 1 hour, then dilute and spread it on a resistance plate containing polymyxin to screen for resistant bacteria.
[0050] Aminoglycoside-resistant bacteria with protein synthesis as the resistance target (aminoglycoside-resistant E. coli DH5α): Add a resistant plasmid containing 15 μg / μl aminoglycoside to a pre-cooled 0.1 mol / LCaCl2 competent E. coli DH5α bacterial solution, place it in an ice-water bath for 28 minutes, immediately heat-shock the mixture at 42°C for 60 seconds, and cool it in an ice-water bath for 2 minutes. Add 700 μL LB sterile broth and resuscitate the resulting mixture at 37°C on a shaker for 1 hour. Then dilute and spread it on a resistance plate containing aminoglycoside to screen for resistant bacteria.
[0051] Example 1 Effects of Inhibitors of Cell Wall Resistance Targets on Conjugative Transfer of Resistance Genes
[0052] 1. Methods
[0053] Donor bacteria: E. coli DH5α resistant to cephalosporins;
[0054] Recipient bacteria: Streptomycin-resistant Escherichia coli.
[0055] Activation of strains: Remove the donor bacteria and recipient bacteria stored in glycerol in a 1.5 mL centrifuge tube from a -80°C freezer and culture them at 37°C in broth containing 16-256 mg / L of the corresponding antibiotic (cefotaxime or streptomycin) for 1-18 h to obtain activated donor and recipient bacteria.
[0056] Preparation of bacterial suspension: Take the donor bacteria and recipient bacteria that have grown to the logarithmic phase after activation, wash and resuspend them with 0.9% physiological saline to obtain a concentration of 10 8 CFU / mL of donor bacterial suspension and recipient bacterial suspension.
[0057] Conjugative transfer: Tazobactam acid, an inhibitor of cell wall resistance targets, is added to the donor and recipient bacterial suspensions, respectively. The two suspensions are then mixed in a 1:1 volume ratio to achieve inhibitor concentrations of 1, 6.25, 12.5, 25, 50, 100, and 200 mg / L. The mixture is then placed on a shaker and shaken for 1 to 20 hours to obtain the conjugative transfer solution. A certain volume of the conjugative transfer solution is diluted and applied to LB solid medium containing dual antibiotics (cefotaxime and streptomycin). The recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and incubated at 37°C for 24 to 36 hours. The conjugants or recipient bacteria growing on the culture medium are then counted and the conjugant and recipient concentrations are determined. The conjugative transfer frequency of the resistance gene is calculated according to the following formula:
[0058] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0059] 2. Results
[0060] The frequency of drug-resistant gene conjugation transfer under different concentrations of inhibitors acting on cell wall drug resistance targets is as follows Figure 1 shown.
[0061] Figure 1 The results showed that the frequency of drug-resistant gene conjugation transfer in the bacterial solution obtained by regulating the conjugation for 4 hours under different concentrations of inhibitors was 7.28×10 -6 Compared with the case where no inhibitor was added (concentration of 0 mg / L), the frequency of drug-resistant gene conjugation transfer decreased by 3.74×10 -6 When the inhibitor concentration was 12.5 mg / L, the frequency of drug-resistant gene conjugation transfer decreased by 4.8×10 -6 When the inhibitor concentration was 25 mg / L, the frequency of drug-resistant gene conjugation transfer decreased by 6.19×10 -6At an inhibitor concentration of 50 mg / L, the frequency of conjugative transfer of resistance genes was 0, indicating complete inhibition. This suggests that tazobactam acid, an inhibitor of cell wall resistance targets, can achieve excellent control of gene conjugation and transfer at concentrations of 6.25 to 50 mg / L, effectively preventing the spread of cefotaxime-resistant genes and mitigating the development of resistance to cephalosporin antibiotics.
[0062] Example 2 Effects of Different Culture Control Times on Drug-Resistance Gene Conjugation and Transfer
[0063] 1. Methods
[0064] The donor bacteria and recipient bacteria are the same as in Example 1.
[0065] The activation of the strains was the same as in Example 1.
[0066] The preparation of the suspension was the same as in Example 1.
[0067] Conjugative transfer: Tazobactam acid, an inhibitor of cell wall resistance targets, is added to the donor and recipient bacterial suspensions, respectively. The two suspensions are then mixed at a volume ratio of 1:1 to a concentration of 25 mg / L of the inhibitor in the mixture. The mixture is then placed on a shaker and shaken for 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, and 20 h to obtain conjugative transfer bacterial solutions. A certain volume of the conjugative transfer bacterial solution is diluted and applied to LB solid medium containing dual antibiotics (cefotaxime and streptomycin). The recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and incubated at 37°C for 24 to 36 h. The conjugants or recipient bacteria growing on the culture medium are then counted and the conjugant concentration and recipient bacteria concentration are determined. The conjugative transfer frequency of the resistance gene is calculated according to the following formula:
[0068] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0069] 2. Results
[0070] The test results showed that compared with no inhibitor added (concentration of 0 mg / L), the frequency of conjugative transfer of drug-resistant genes decreased by 30% to 41% after 1-2 hours of regulation; the frequency of conjugative transfer of drug-resistant genes decreased by 60% to 85% after 4-8 hours of regulation; and the frequency of conjugative transfer of drug-resistant genes decreased by 55% to 57% after 16-20 hours of regulation. This shows that the incubation control time has a significant effect on the inhibitor's inhibition of drug-resistant gene conjugative transfer, and a incubation control time of 4-8 hours can effectively control the conjugative transfer of drug-resistant genes.
[0071] Example 3 Effects of different donor bacteria concentrations on conjugative transfer of drug-resistant genes
[0072] 1. Methods
[0073] The donor bacteria and recipient bacteria are the same as in Example 1.
[0074] The activation of the strains was the same as in Example 1.
[0075] Preparation of suspension: According to the preparation method of Example 1, a suspension with a concentration of 10 10 CFU / mL of donor bacterial suspension and a concentration of 10 8 CFU / mL of recipient bacterial suspension; the donor bacterial suspension was diluted in a 10-fold ratio to obtain a concentration of 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL of bacterial suspension.
[0076] Conjugative transfer: Tazobactam acid, an inhibitor of cell wall resistance targets, is added to the donor and recipient bacterial suspensions, respectively. The two bacterial suspensions are then mixed at a volume ratio of 1:1 to a concentration of 12.5 mg / L of the inhibitor in the mixture. The mixture is then placed on a shaker and shaken for 4 hours to obtain conjugative transfer bacterial solutions. A certain volume of conjugative transfer bacterial solution is diluted and applied to LB solid medium containing dual antibiotics (cefotaxime and streptomycin). The recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and cultured at 37°C for 24 to 36 hours. The conjugants or recipient bacteria grown on the culture medium are then counted and the conjugant concentration and the recipient bacteria concentration are determined. The conjugative transfer frequency of the resistance gene is calculated according to the following formula:
[0077] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0078] 2. Results
[0079] The test results showed that the concentration of the donor bacterial suspension before mixing was 10 8 CFU / mL of conjugative transfer bacterial solution, the conjugative transfer frequency of drug-resistant genes was 2.48×10 -6 Compared with the case where no inhibitor was added (concentration of 0 mg / L), the frequency of drug-resistant gene conjugation transfer decreased by 66%; compared with the case where no inhibitor was added (concentration of 0 mg / L), the concentration of the donor bacterial suspension before mixing was 10 5 CFU / mL, the frequency of drug-resistant gene conjugation transfer decreased by 49%, and the donor bacterial suspension before mixing was 10 9 , 10 10 CFU / mL, and the frequency of drug-resistant gene conjugation transfer decreased by 56% and 45% respectively. In summary, the concentration of the donor bacterial suspension before mixing has a significant effect on the inhibitor's inhibition of drug-resistant gene conjugation transfer. 8 ~10 9CFU / mL can effectively control the conjugation transfer of drug-resistant genes. The concentration of the donor bacterial suspension before mixing is 10 8 CFU / mL has a better control effect.
[0080] Example 4 Effects of different donor-recipient ratios on conjugative transfer of drug-resistant genes
[0081] 1. Methods
[0082] The donor bacteria and recipient bacteria are the same as in Example 1.
[0083] The activation of the strains was the same as in Example 1.
[0084] The preparation of the suspension was the same as in Example 1.
[0085] Conjugative transfer: Tazobactam acid, an inhibitor of cell wall resistance targets, is added to the donor and recipient bacterial suspensions, respectively. The donor and recipient bacterial suspensions are then mixed at volume ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, and 4:1, respectively, to achieve an inhibitor concentration of 25 mg / L. The mixture is then shaken on a shaker for 4 hours to obtain the conjugative transfer solution. A certain volume of the conjugative transfer solution is diluted and applied to LB solid medium containing dual antibiotics (cefotaxime and streptomycin). The recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and incubated at 37°C for 24-36 hours. The conjugants or recipient bacteria growing on the culture medium are then counted and the conjugant concentration and recipient bacterial concentration are determined. The conjugative transfer frequency of the resistance gene is calculated according to the following formula:
[0086] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0087] 2. Results
[0088] The experimental results showed that the inhibitor had an inhibitory and control effect on the conjugation transfer of drug-resistant genes at different mixing ratios of donor and recipient bacteria. The best control effect occurred when the volume ratio of donor bacteria to recipient bacteria was 1:1. At this time, the conjugation transfer frequency of drug-resistant genes was 1.09×10 -6 . When the mixing volume ratio is lower than or higher than 1:1, the inhibitor's inhibitory effect on the conjugation transfer of drug-resistant genes is reduced. The regulatory effect of the conjugation transfer bacterial solution with a mixing volume ratio of 0.25:1 between the donor bacteria and the recipient bacteria is the weakest, only 51% of the 1:1 ratio. This shows that different mixing ratios of the donor and recipient bacterial suspensions have a significant effect on the inhibitor's inhibition of the conjugation transfer of drug-resistant genes. A mixing volume ratio of 1:1 between the donor bacteria and the recipient bacteria can effectively control the conjugation transfer of drug-resistant genes.
[0089] Example 5 Effects of Inhibitors of Cell Membrane Drug Resistance Targets on Drug Resistance Gene Conjugation and Transfer
[0090] 1. Methods
[0091] Donor bacteria: E. coli DH5α resistant to polymyxin antibiotics;
[0092] Recipient bacteria: Streptomycin-resistant Escherichia coli.
[0093] Activation of strains: Remove the donor bacteria and recipient bacteria stored in glycerol in a 1.5 mL centrifuge tube from a -80°C freezer and culture them at 37°C in broth containing 2-8 mg / L of the corresponding antibiotic (polymyxin or streptomycin) for 1-18 h to obtain activated donor and recipient bacteria.
[0094] Preparation of bacterial suspension: Prepare the suspension according to the method of Example 1 with a concentration of 10 8 CFU / mL of donor bacterial suspension and recipient bacterial suspension.
[0095] Conjugative transfer: Pterostilbene, an inhibitor of cell membrane resistance targets, is added to the donor and recipient bacterial suspensions respectively, and then the two bacterial suspensions are mixed at a volume ratio of 1:1 so that the concentration of the inhibitor in the mixed solution is 2-1024 mg / L; the mixed solution is then placed on a shaker and shaken for 4 hours to obtain the conjugative transfer bacterial solution. A certain volume of the conjugative transfer bacterial solution is diluted and applied to LB solid medium containing double resistance (polymyxin and streptomycin), and the recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and cultured at 37°C for 24-36 hours. The conjugants or recipient bacteria grown on the culture medium are then counted and the conjugant concentration and the concentration of the recipient bacteria are determined. The conjugative transfer frequency of the resistance gene is calculated according to the following formula:
[0096] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0097] 2. Results
[0098] The results showed that the inhibitor, pterostilbene, could inhibit the formation of zygotes at concentrations ranging from 2 to 1024 mg / L, effectively suppressing the conjugative transfer of drug-resistant genes. The inhibitory effect first increased and then decreased with increasing inhibitor concentration, reaching its peak at 64 mg / L.
[0099] Compared with the absence of an inhibitor (0 mg / L), concentrations of 16 and 128 mg / L of pterostilbene, an inhibitor of cell membrane resistance targets, reduced the frequency of resistance gene conjugation by 43% and 35%, respectively. Concentrations of 32-64 mg / L reduced the frequency of resistance gene conjugation by 71-82%. This suggests that different concentrations of pterostilbene have a significant effect on the inhibitor's ability to inhibit resistance gene conjugation. Pterostilbene concentrations of 16-128 mg / L effectively inhibit resistance gene conjugation, while concentrations of 32-64 mg / L are even more effective, preventing the spread of polymyxin-resistant genes.
[0100] Example 6 Effects of Inhibitors of Protein Synthesis Drug Resistance Targets on Drug Resistance Gene Conjugation and Transfer
[0101] 1. Methods
[0102] Donor bacteria: E. coli DH5α resistant to aminoglycoside antibiotics;
[0103] Recipient bacteria: Streptomycin-resistant Escherichia coli.
[0104] Activation of strains: The donor and recipient bacteria stored in glycerol in 1.5 mL centrifuge tubes were taken out of the -80°C freezer and cultured at 37°C in broth containing 8-128 mg / L of the corresponding antibiotic (gentamicin or streptomycin) for 1-18 h to obtain activated donor and recipient bacteria.
[0105] Preparation of bacterial suspension: Prepare the suspension according to the method of Example 1 with a concentration of 10 8 CFU / mL of donor bacterial suspension and recipient bacterial suspension.
[0106] Conjugative transfer: Curcumin, an inhibitor of protein synthesis resistance targets, is added to the donor and recipient bacterial suspensions, respectively. The two bacterial suspensions are then mixed at a volume ratio of 1:1 to give an inhibitor concentration of 2 to 1024 mg / L in the mixture. The mixture is then placed on a shaker and shaken for 4 hours to obtain conjugative transfer bacterial solutions. A certain volume of conjugative transfer bacterial solution is diluted and applied to LB solid medium containing dual antibiotics (gentamicin and streptomycin). The recipient bacterial suspension is diluted and applied to LB solid medium containing streptomycin and cultured at 37°C for 24 to 36 hours. The conjugants or recipient bacteria grown on the culture medium are then counted and the conjugant concentration and the recipient bacteria concentration are determined. The frequency of conjugative transfer of drug-resistant genes is calculated according to the following formula:
[0107] The frequency of conjugative transfer = the concentration of conjugates screened on the double antibody / the total concentration of recipient bacteria.
[0108] 2. Results
[0109] The results showed that the inhibitor curcumin at low concentrations of 2-256 mg / L not only had no inhibitory effect on the conjugative transfer of drug-resistant genes, but actually promoted it. Curcumin inhibitor concentrations of 512-1024 mg / L were able to significantly inhibit the formation of conjugates, demonstrating a significant inhibitory effect on the conjugative transfer of drug-resistant genes.
[0110] Compared with no inhibitor (0 mg / L), curcumin, an inhibitor of protein synthesis resistance targets, increased the frequency of resistance gene conjugation by 85% at a concentration of 32 mg / L, demonstrating a promoting effect on resistance genes. However, curcumin, an inhibitor at a concentration of 512 mg / L, exhibited a significant inhibitory effect on resistance gene conjugation. This suggests that not all concentrations of curcumin can inhibit resistance gene conjugation, and exploring target inhibitors to regulate resistance gene conjugation is essential. Only inhibitors at appropriate concentrations can regulate resistance gene conjugation. Curcumin, an inhibitor of protein synthesis resistance targets, effectively inhibited resistance gene conjugation at concentrations of 512 to 1024 mg / L, effectively preventing the spread of aminoglycoside-resistance genes.
[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. A method for regulating the conjugation and transfer of drug-resistant genes using an inhibitor of a drug-resistant target, characterized in that: The following steps are involved: S1. The donor bacteria and the recipient bacteria are activated to prepare a donor bacterial suspension and a recipient bacterial suspension; the donor bacteria contain an antibiotic-resistant gene, the recipient bacteria do not contain the antibiotic-resistant gene, and contain another antibiotic-resistant gene; S2. The donor bacterial suspension and the recipient bacterial suspension prepared in step S1 are mixed with the inhibitor of the drug resistance target, respectively. The two mixed bacterial suspensions are then mixed and the mixture is cultured at a constant temperature of 36 to 38°C to obtain a conjugated transfer bacterial solution. The donor bacteria are resistant to cefotaxime, the recipient bacteria are resistant to streptomycin, and the inhibitor of the drug resistance target is tazobactam acid; The donor bacteria are resistant to polymyxin antibiotics, the recipient bacteria are resistant to streptomycin, and the inhibitor of the drug resistance target is pterostilbene; The donor bacteria are resistant to aminoglycoside antibiotics, the recipient bacteria are resistant to streptomycin, and the inhibitor of the drug resistance target is curcumin; S3. The conjugative transfer bacterial solution prepared in step S2 is diluted and applied on a resistance plate containing the antibiotic to which the donor bacteria are resistant and another antibiotic to which the recipient bacteria are resistant in step S1. The total recipient bacterial suspension prepared in step S1 is diluted and applied on a resistance plate containing another antibiotic to which the recipient bacteria are resistant in step S1. The concentration of conjugants in the conjugative transfer bacterial solution and the concentration of total recipient bacteria are determined. The conjugative transfer frequency is calculated based on the ratio of the conjugant concentration to the total recipient bacteria concentration. The conjugative transfer frequency of the drug-resistant gene is regulated by regulating the donor bacteria concentration, the mixing ratio of the donor and recipient bacteria, and the concentration, type or action time of the inhibitor of the drug-resistant target.
2. The method according to claim 1, characterized in that The drug-resistant bacteria is any one of Escherichia coli, Pseudomonas aeruginosa, Salmonella, and Streptococcus.
3. The method according to claim 2, characterized in that The drug-resistant bacteria is Escherichia coli.
4. The method according to claim 1, wherein In step S1, the concentrations of the donor bacterial suspension and the recipient bacterial suspension are both 10 5 ~10 10 CFU / mL.
5. The method according to claim 1, wherein In step S2, the donor bacterial suspension and the recipient bacterial suspension are mixed at a volume ratio of (0.25-4):
1.
6. The method according to claim 1, characterized in that In step S2, the concentration of tazobactam acid in the mixed solution is 1-200 mg / L, the concentration of pterostilbene is 2-1024 mg / L, and the concentration of curcumin is 512-1024 mg / L.
7. The method according to claim 1, characterized in that The culturing time in step S2 is 1 to 20 hours.
8. Use of the method according to any one of claims 1 to 7 in regulating the conjugative transfer of drug-resistant genes.