An IS26-based drug resistance gene control plasmid, engineering bacteria, bacterial agent and application

Abstract

The present invention discloses an IS26-based resistance gene control plasmid, engineering bacteria, bacterial agent and application, belonging to the technical field of genetic engineering. The nucleotide sequence of the IS26-based resistance gene control plasmid is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The engineering bacteria and bacterial agent containing the resistance gene control plasmid can be used for the control of resistance genes. By specifically destroying the insertion sequence IS26, the number of resistance genes can be fundamentally reduced and the spread of resistance genes can be blocked, providing a precise solution for the treatment of drug resistance. It has broad-spectrum resistance bacteria control performance and can effectively control gentamicin, tetracycline, chloramphenicol and ampicillin-resistant bacteria in wastewater, with the highest control efficiency reaching 83.76%. The plasmid, engineering bacteria and bacterial agent have low preparation costs, can be used for in-situ treatment of wastewater, have low treatment costs and good effects, and have broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a resistance gene plasmid based on IS26, a resistance gene control engineering bacterium, a bacterial agent and applications thereof. Background Art

[0002] With the rapid development of the livestock and poultry breeding industry, the post-treatment and discharge problems of breeding wastewater have become increasingly prominent, especially the problems of antibiotic-resistant bacteria and antibiotic resistance gene pollution in breeding wastewater. The emergence of this phenomenon is closely related to the extensive use of antibiotics in breeding - antibiotics are widely used for preventing and treating diseases of livestock and poultry, promoting growth, etc. After the antibiotics that are not completely metabolized in the livestock and poultry enter the breeding wastewater, they provide a breeding ground for the growth of antibiotic-resistant bacteria and the spread of antibiotic resistance genes.

[0003] Common pathogenic bacteria in breeding wastewater such as Escherichia coli, Salmonella, Klebsiella, Pseudomonas, Acinetobacter, etc. are not only easily resistant to antibiotics such as tetracycline, sulfonamides, quinolones, etc., but also the antibiotic resistance genes they carry can be transmitted between different bacterial species through mobile genetic elements such as plasmids and transposons. Of particular concern is the insertion sequence IS26. A number of studies have shown that as a highly active gene transfer vector, it is often directly linked to a variety of antibiotic resistance genes (such as tetM, blaCTX-M etc.), and promotes the capture and diffusion of antibiotic resistance genes by forming a composite transposon structure, becoming a key driving factor for the spread of antibiotic resistance.

[0004] Existing technologies for treating antibiotic-resistant bacteria in breeding wastewater have significant limitations: Conventional physical and chemical methods such as chlorine disinfection and ultraviolet light can inactivate some bacteria, but cannot degrade free antibiotic resistance gene fragments and may produce toxic by-products; Biological treatment methods such as activated sludge and biofilm methods can remove organic matter and some pathogenic bacteria, but provide a microbial enrichment environment for the horizontal transfer of antibiotic resistance genes; Some emerging treatment methods, such as photocatalytic oxidation and advanced oxidation, although they have a degradation effect on antibiotic resistance genes, their high costs and complex operations limit their large-scale application. These methods all lack the specific blocking ability for mobile elements such as IS26 and are difficult to contain the spread chain of antibiotic resistance genes. Based on the core role of IS26 in the diffusion of antibiotic resistance genes, developing targeted in-situ control technologies has important potential. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a resistance gene control plasmid for controlling the insertion sequence IS26; The second object is to provide a resistance gene control engineering bacterium, a bacterial agent and applications thereof containing the plasmid.

[0006] Technical Solution: The resistance gene control plasmid based on IS26 of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0007] The drug resistance gene control engineered bacteria described in the present invention are donor bacteria containing the aforementioned drug resistance gene control plasmid based on IS26.

[0008] Preferably, the donor bacteria are Escherichia coli S17-1 λpir or Escherichia coli SM10 λpir.

[0009] The drug resistance gene control bacterial agent described in the present invention contains viable bacteria of the aforementioned drug resistance gene control engineered bacteria.

[0010] Application of the drug resistance gene control engineered bacteria or the drug resistance gene control bacterial agent described in the present invention in in-situ treatment of wastewater drug-resistant bacteria.

[0011] Preferably, the application is for in-situ treatment of drug-resistant bacteria in wastewater from livestock and poultry farms.

[0012] Preferably, the livestock and poultry farm is a pig farm or a cattle farm.

[0013] Preferably, the drug-resistant bacteria are Gram-negative drug-resistant bacteria containing the IS26 insertion sequence.

[0014] Preferably, the steps of the application include: adding the drug resistance gene control engineered bacteria or the drug resistance gene control bacterial agent to the wastewater, fully mixing and then treating for 12 - 36 h, wherein the final concentration of the drug resistance gene control engineered bacteria is 1 - 100 times the bacterial concentration in the wastewater.

[0015] Preferably, the final concentration of the drug resistance gene control engineered bacteria is not less than 10 times the bacterial concentration in the wastewater.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. By specifically destroying the insertion sequence IS26, the number of drug resistance genes can be fundamentally reduced and the spread of drug resistance genes can be blocked, providing a precise solution for drug resistance management; 2. It has a broad-spectrum drug-resistant bacteria control performance and can effectively control drug-resistant bacteria against gentamicin, tetracycline, chloramphenicol, and ampicillin, with the highest control efficiency reaching 83.76%; 3. It can be used for in-situ treatment of wastewater, with low treatment cost and broad application prospects. Description of the Drawings

[0017] Figure 1 It is a graph of the knockout efficiency of different sgRNAs for IS26;

[0018] Figure 2 It is a graph of the escape frequency of different sgRNAs knocking out IS26;

[0019] Figure 3Results of the removal of drug resistance genes in Escherichia coli J53 / pUC57-IS26 by the drug resistance gene control engineering bacteria. Among them, (A) is the control efficiency diagram of drug-resistant Escherichia coli J53 / pUC57-IS26, and (B) is the relative copy number difference diagram of IS26, ampicillin resistance gene bla , plasmid replication origin ori in drug-resistant Escherichia coli J53 / pUC57-IS26 with different treatments of uidA ; (C) is the minimum inhibitory concentration determination result diagram of antibiotics for drug-resistant Escherichia coli J53 / pUC57-IS26 and Escherichia coli J53 with different treatments;

[0020] Figure 4 Results of the removal of drug resistance genes in Klebsiella pneumoniae ATCC13883 / pEX18Gm-IS26 by the drug resistance gene control engineering bacteria. Among them, (A) is the control efficiency diagram of drug-resistant Klebsiella pneumoniae ATCC13883 / pEX18Gm-IS26, and (B) is the relative copy number difference diagram of IS26, gentamicin resistance gene aacC1 , plasmid replication origin ori in drug-resistant Klebsiella pneumoniae ATCC13883 / pEX18Gm-IS26 with different treatments of uidA ; (C) is the minimum inhibitory concentration determination result diagram of antibiotics for drug-resistant Klebsiella pneumoniae ATCC13883 / pEX18Gm-IS26 and Klebsiella pneumoniae ATCC13883 with different treatments;

[0021] Figure 5 Results of the control of drug-resistant bacteria in the pig farm wastewater in-situ treatment. Among them, CK represents untreated;

[0022] Figure 6 Results of the control of drug-resistant bacteria in the cattle farm wastewater in-situ treatment. Among them, CK represents untreated;

[0023] Figure 7 Results of the detection of the abundance of insertion sequence IS26 after the in-situ treatment of the breeding wastewater. Among them, CK represents untreated. Detailed implementation manners

[0024] The technical solutions of the present invention will be further described below.

[0025] Example 1: Design and verification of sgRNA

[0026] 1. Construction of pCas9-sgRNA plasmid

[0027] (1) Design sgRNA1 and sgRNA2 targeting IS26, and design another sgRNA0 that does not target IS26 as a negative control. In order to facilitate subsequent insertion, restriction enzyme cleavage sites are designed at the same time;

[0028] The complete sequences of the forward and reverse strands of the specific sgRNA are as follows. Lowercase letters represent restriction sites, and uppercase letters represent sgRNA:

[0029] sgRNA0-F, as shown in SEQ ID NO: 6: aaacTTCGACGTGAACTTGGTACGg;

[0030] sgRNA0-R, as shown in SEQ ID NO: 7: aaaacCGTACCAAGTTCACGTCGAA;

[0031] sgRNA1-F, as shown in SEQ ID NO: 8: aaacTTCAGCGTGACATCATTCTGg;

[0032] sgRNA1-R, as shown in SEQ ID NO: 9: aaaacCAGAATGATGTCACGCTGAA;

[0033] sgRNA2-F, as shown in SEQ ID NO: 10: aaacCGATCTTTGCCCGTGGCACAg;

[0034] sgRNA2-R, as shown in SEQ ID NO: 11: aaaacTGTGCCACGGGCAAAGATCG;

[0035] (2) It was synthesized by Shanghai Sangon Biotech Co., Ltd. After the obtained forward and reverse strands of sgRNA were incubated at 95 °C for 5 min and then at 50 °C for 1 min, double-stranded sgDNA was obtained;

[0036] (3) The pCas9 plasmid was digested with BsaI endonuclease at 37 °C for 1 h, and the linearized vector was recovered by gel extraction;

[0037] (4) The double-stranded sgRNA and the linearized vector were ligated with T4 ligase at 16 °C for 16 h;

[0038] (5) The ligation product was transformed into Escherichia coli DH5α, spread on an LB plate containing chloramphenicol for screening, and cultured at 37 °C for 14 h. Single colonies were selected for sequencing to ensure the successful insertion of sgRNA.

[0039] 2. Determination of the knockout activity of sgRNA

[0040] (1) Design a pUC57-IS26 plasmid with the sequence shown in SEQ ID NO: 4 and have it synthesized by Nanjing Tsingke Biotechnology Co., Ltd.;

[0041] (2) Transform the pUC57-IS26 plasmid into Escherichia coli J53, screen for Escherichia coli J53 containing the pUC57-IS26 plasmid through the resistance gene tag of the transformed plasmid, and prepare competent cells from Escherichia coli DH5α containing the pUC57-IS26 plasmid;

[0042] (3) Transform pCas9-sg0, pCas9-sg1, and pCas9-sg2 into the above-mentioned competent cells respectively;

[0043] (4) Spread a part of the transformed bacteria on a plate containing chloramphenicol to represent all transformants, and at the same time spread the same volume of bacteria on a plate containing chloramphenicol and ampicillin to screen for bacteria that have not been successfully knocked out;

[0044] (5) Calculate the knockout efficiency according to formula (I),

[0045] Knockout efficiency (%) = [1 - (bacteria that have not been successfully knocked out ÷ total transformants)] × 100 （I） ;

[0046] (6) Calculate the escape rate according to formula (II),

[0047] Escape rate (%) = (bacteria that have not been successfully knocked out ÷ total transformants) × 100 (II);

[0048] The results are as Figure 1 , 2 shown. The editing efficiency of the two sgRNAs targeting IS26 for IS26 is as high as over 99%. By measuring the escape rate, it is found that the escape rates of the two sgRNAs targeting IS26 are extremely low, which are 10 -3.95 and 10 -5.22 respectively. From this, it can be concluded that the designed sgRNAs have strong targeting and high activity for IS26.

[0049] Example 2: Design and synthesis of drug resistance gene control plasmid, preparation and verification of drug resistance gene control engineering bacteria

[0050] 1. Construction of drug resistance gene control plasmid and drug resistance gene control engineering bacteria

[0051] (1) Design the pEdit plasmid with the nucleotide sequence shown in SEQ ID NO: 3, combine with the sgRNAs targeting IS26 designed in Example 1 to obtain drug resistance gene control plasmids with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. According to the results of Example 1, the plasmid with the nucleotide sequence shown in SEQ ID NO: 2 is selected as the drug resistance gene control plasmid for subsequent experiments;

[0052] (2)The drug resistance gene control plasmid designed above was synthesized by Nanjing Tsingke Biotechnology Co., Ltd. At the same time, the drug resistance gene control plasmid pEdit-sgRNA was obtained.

[0053] (3)pEdit-sgRNA and pCas9-sg2 plasmids were transformed into Escherichia coli S17-1 λpir. The drug resistance gene control engineering bacteria containing the pEdit-sgRNA plasmid and the control engineering bacteria containing the pCas9-sg2 plasmid were obtained by screening with the mCherry tag and the chloramphenicol resistance gene respectively.

[0054] 2. Verification of drug resistance gene control engineering bacteria

[0055] (1)The pEX18Gm-IS26 plasmid with the sequence shown in SEQ ID NO: 5 was designed and synthesized by Nanjing Tsingke Biotechnology Co., Ltd.

[0056] (2)The pEX18Gm-IS26 plasmid was transformed into Klebsiella pneumoniae ATCC13883. The Klebsiella pneumoniae ATCC13883 containing the pEX18Gm-IS26 plasmid was obtained by screening with the resistance gene tag of the transformed plasmid.

[0057] (3)The drug resistance gene control engineering bacteria, the control engineering bacteria, Escherichia coli J53 containing the pUC57-IS26 plasmid obtained in Example 1, and Klebsiella pneumoniae ATCC13883 containing the pEX18Gm-IS26 plasmid were respectively streaked on an LB plate and cultured at 37 °C for 14 h.

[0058] (4)Single colonies were picked and cultured in an LB liquid medium without antibiotics or containing the corresponding antibiotics at 37 °C for 14 h.

[0059] (5)The overnight cultured bacteria were transferred into a fresh LB liquid medium containing the corresponding antibiotics and cultured until OD 600 = 0.5;

[0060] (6)200 μL of the drug resistance gene control engineering bacteria or the control engineering bacteria, and 200 μL of Escherichia coli J53 containing the pUC57-IS26 plasmid or Klebsiella pneumoniae ATCC13883 containing the pEX18Gm-IS26 plasmid were fully mixed, dropped onto the center of a 0.2 μm filter membrane, and the filter membrane was attached to an LB medium without antibiotics and cultured at 37 °C for 24 h.

[0061] 2.1 Determination of control efficiency

[0062] The bacteria on the filter membrane were eluted with sterile PBS, serially diluted and spread on an LB plate containing the corresponding antibiotics, and cultured at 37 °C for 24 h to calculate the control efficiency.

[0063] Blocking the drug resistance of Escherichia coli J53 containing the plasmid pUC57-IS26: Escherichia coli J53 itself has sodium azide resistance. Therefore, bacteria that are resistant to sodium azide and spontaneously emit mCherry red fluorescence on the sodium azide resistance plate represent conjugant bacteria. Bacteria that are resistant to both sodium azide and ampicillin and spontaneously emit mCherry red fluorescence on the double resistance plate of sodium azide and ampicillin represent unblocked bacteria. Calculate the blocking efficiency (i.e., knockout efficiency) according to the aforementioned formula (I);

[0064] Blocking the drug resistance of Klebsiella pneumoniae ATCC13883 containing the plasmid pEX18Gm-IS26: Klebsiella pneumoniae ATCC13883 itself has ampicillin resistance. Therefore, bacteria that are resistant to ampicillin and spontaneously emit mCherry red fluorescence on the ampicillin resistance plate represent conjugant bacteria. Bacteria that are resistant to both ampicillin and gentamicin and spontaneously emit mCherry red fluorescence on the double resistance plate of ampicillin and gentamicin represent unblocked bacteria. Calculate the blocking efficiency (i.e., knockout efficiency) according to the aforementioned formula (I).

[0065] 2.2 Determination of gene copy number

[0066] (1) Extract the genomic DNA of the strain to be tested, and dilute the DNA concentration of all samples to 1 ng / μL;

[0067] (2) Use real-time fluorescence quantitative PCR to determine the copy numbers of drug resistance genes, IS26 and plasmid replication origin sites in bacteria. The internal reference gene is uidA , and the specific primers are as follows:

[0068] blaTEM-F, as shown in SEQ ID NO: 12: CTACGATACGGGAGGGCTTA;

[0069] blaTEM-R, as shown in SEQ ID NO: 13: ATAAATCTGGAGCCGGTGAG;

[0070] aacC1-F, as shown in SEQ ID NO: 14: CATCATTCGCACATGTAGGCTCGG;

[0071] aacC1-R, as shown in SEQ ID NO: 15: GCTGATGTTGGGAGTAGGTGGCTA;

[0072] IS26-F, as shown in SEQ ID NO: 16: TCGATCACTCCACGATTTACCGCT;

[0073] IS26-R, as shown in SEQ ID NO: 17: GTAGGTTTCATCCATGTGCCACGG;

[0074] ori-F, as shown in SEQ ID NO: 18: GGTGGTTTGTTTGCCGGATCAAGA;

[0075] ori-R, as shown in SEQ ID NO: 19: ACAGAGTTCTTGAAGTGGTGGCCT;

[0076] uidA-F, as shown in SEQ ID NO: 20: TCTGGCAACCGGGTGAAG;

[0077] uidA-R, as shown in SEQ ID NO: 21: TAGATATCACACTCTGTCTGGCT;

[0078] The qPCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2:

[0079] Table 1 qPCR reaction system

[0080]

[0081] Table 2 qPCR reaction conditions

[0082]

[0083] Three replicate qPCR reactions were used to obtain the average Ct value of each gene for each strain uidA and the ΔΔCT method was used to determine the fold change of each gene of interest for each sample, representing the relative copy number change of the gene.

[0084] 2.3 Determination of the minimum inhibitory concentration (MIC) of antibiotics

[0085] (1) Antibiotics were serially diluted two-fold in 96-well plates. The concentration gradients of ampicillin were 0, 2, 4, 8, 16, 32, 64, 128, 256, 512 g / L, and the concentration gradients of gentamicin were 0, 2, 4, 8, 16, 32, 64, 128 g / L;

[0086] (2) The overnight cultured test strains were diluted to OD 600 = 1 with sterile PBS and then further diluted 1000-fold with MH medium. 100 μL of each dilution was added to the 96-well plates containing antibiotics. After incubation at 37 °C for 24 hours, the OD 600 was measured using a microplate reader, and each treatment was repeated 3 times;

[0087] The results of the minimum inhibitory concentration of antibiotics for Escherichia coli J53 after treatment are as Figure 3 : As Figure 3As shown in (A), the pEdit-sgRNA has extremely high control efficiency against the drug-resistant plasmid pUC57-IS26 in Escherichia coli J53, reaching more than 99%; as Figure 3 shown in (B), the copy numbers of several key genes on the drug-resistant plasmid pUC57-IS26 were measured, and the copy numbers of the insertion sequence IS26 and the ampicillin-resistant gene bla both decreased to 0, indicating that pEdit-sgRNA completely cleared the drug-resistant genes of the bacteria. At the same time, the replication origin ori of the plasmid also decreased to 0, further indicating that the drug-resistant plasmid had been completely cleared; as Figure 3 shown in (C), the bacterial MIC results showed that pEdit-sgRNA restored the sensitivity of the originally ampicillin-resistant Escherichia coli to ampicillin;

[0088] The minimum inhibitory concentration results of antibiotics for Klebsiella pneumoniae ATCC13883 after treatment are as Figure 4 follows: as Figure 4 shown in (A), the pEdit-sgRNA also has extremely high control efficiency against the drug-resistant plasmid pEX18Gm-IS26 in Klebsiella pneumoniae ATCC13883, reaching more than 99%; as Figure 4 shown in (B), the copy numbers of several key genes on the drug-resistant plasmid pEX18Gm-IS26 were measured, and the copy numbers of the insertion sequence IS26 and the gentamicin-resistant gene aacC1 both decreased to 0, indicating that pEdit-sgRNA completely cleared the drug-resistant genes in the bacteria, and the replication origin ori of the plasmid also decreased to 0, further indicating that the drug-resistant plasmid had been completely cleared; as Figure 4 shown in (C), the bacterial MIC results showed that pEdit-sgRNA restored the sensitivity of the originally gentamicin-resistant Klebsiella pneumoniae to gentamicin.

[0089] In summary, pEdit-sgRNA has broad-spectrum drug-resistant gene control performance.

[0090] Example 3: Application of drug-resistant gene-controlled engineered bacteria in in-situ control of bacterial drug resistance in aquaculture wastewater

[0091] (1) One pig farm and one cattle farm were selected in a certain place, and aquaculture wastewater was collected. The collected aquaculture wastewater was serially diluted 10-fold, with gradients of undiluted, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 . The diluted wastewater was spread on LB plates and cultured at 37 °C for 24 h to calculate the bacterial concentration in the aquaculture wastewater;

[0092] (2) The drug-resistant gene barrier engineering bacteria and the control engineering bacteria constructed in Example 2 were streaked on LB plates and cultured at 37°C for 14 h;

[0093] (2) Pick a single clone and culture it in LB liquid medium at 37°C for 14 h;

[0094] (3) Transfer the overnight cultured bacteria into fresh LB liquid medium containing the corresponding antibiotics and culture until the OD 600 =0.5;

[0095] (4) Add drug-resistant gene-blocking engineered bacteria or control engineered bacteria according to the bacterial concentration in the aquaculture wastewater obtained in step 1. The final concentration of the engineered bacteria is 10 times the bacterial concentration in the wastewater. After fully mixing, leave it to stand at room temperature for 24 hours;

[0096] (5) Add drug-resistant gene-controlled engineered bacteria or control engineered bacteria according to the bacterial concentration in the aquaculture wastewater. The final concentration of the engineered bacteria is 10 times the bacterial concentration in the wastewater. After thorough mixing, leave it to stand at room temperature for 24 hours.

[0097] 1. Determination of the number of bacteria with different drug resistance in aquaculture wastewater after treatment

[0098] After 24 hours, the aquaculture wastewater was diluted 10 times in stages, with the gradient being original solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , spread on ampicillin, chloramphenicol, tetracycline, and gentamicin resistance plates respectively, culture at 37°C for 24 hours, and count the number of resistant bacteria.

[0099] 2. Determination of the absolute copy number of IS26 in treated aquaculture wastewater

[0100] (1) Streak the E. coli DH5α containing the pUC57-IS26 plasmid on an LB plate containing ampicillin and culture at 37°C for 14 h.

[0101] (2) Pick a single clone and culture it in LB liquid medium containing the corresponding antibiotics at 37°C for 14 h;

[0102] (3) Extract the plasmid, determine the plasmid concentration, and dilute it 10 times in stages (stock solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10-7 and 10 -8 ), construct the standard curve of IS26 by real-time fluorescence quantitative PCR, and the specific primers are as follows:

[0103] IS26-F2, as shown in SEQ ID NO: 22: GTAGGTTTCATCCATGTGCCACGG;

[0104] IS26-R2, as shown in SEQ ID NO: 23: TCGATCACTCCACGATTTACCGCT;

[0105] The qPCR reaction system and reaction conditions are the same as those in Example 2;

[0106] (4) Extract the DNA of the aquaculture wastewater, amplify IS26 using the above primers, reaction system and conditions, and substitute the obtained CT value into the IS26 standard curve obtained in step 3 to obtain the absolute abundance of IS26.

[0107] The measurement results of the number of different drug-resistant bacteria in the treated pig farm wastewater are as Figure 5 shown, and the measurement results of the number of different drug-resistant bacteria in the cattle farm wastewater are as Figure 6 shown. pEdit-sgRNA effectively controlled the Gentamicin, Tetracycline, Chloramphenicol, and Ampicillin-resistant bacteria in the aquaculture wastewater of pig farms and cattle farms. Among them, the number of tetracycline-resistant bacteria in the pig farm wastewater decreased particularly significantly, reaching 83.76%.

[0108] The measurement results of the absolute copy number of IS26 in the treated aquaculture wastewater are as Figure 7 shown. pEdit-sgRNA significantly reduced the copy number of the insertion sequence IS26 in the aquaculture wastewater of pig farms and cattle farms, and the reduction ratios reached 58.14% and 30.56% respectively. Based on the above results, it is suggested that the number of IS26-related drug-resistant genes is significantly reduced.

Claims

1. A drug-resistant gene control plasmid based on IS26, characterized in that, The plasmid has a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A drug-resistant gene-controlled engineered bacterium, characterized in that, The engineered bacterium is a donor bacterium containing the IS26-based drug resistance gene control plasmid as described in claim 1.

3. The drug resistance gene blocking engineered bacterium according to claim 2, wherein The donor bacterium is Escherichia coli S17-1 λpir or Escherichia coli SM10 λpir.

4. A drug-resistant gene control bactericide, characterized in that, The bacterial agent contains viable cells of the drug resistance gene control engineered bacterium as described in claim 2.

5. Use of the drug resistance gene control engineered bacterium as described in claim 2 or the drug resistance gene control bacterial agent as described in claim 4 for in-situ treatment of Gram-negative drug-resistant bacteria containing the IS26 insertion sequence in wastewater.

6. The application according to claim 5, wherein The use is for in-situ treatment of drug-resistant bacteria in wastewater from livestock and poultry farms.

7. The application according to claim 6, characterized in that, The livestock and poultry farm is a pig farm or a cattle farm.

8. The application according to claim 5, characterized in that The steps of the use include: adding the drug resistance gene control engineered bacterium or the drug resistance gene control bacterial agent into the wastewater, fully mixing and then treating for 12 - 36 h, wherein the final concentration of the drug resistance gene control engineered bacterium is 1 - 100 times the bacterial concentration in the wastewater.

9. The application according to claim 8, wherein The final concentration of the drug resistance gene control engineered bacterium is not less than 10 times the bacterial concentration in the wastewater.

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