A transposon carrying the carbapenemase bla NDM-1 Tn7555 and uses thereof

Abstract

The application discloses a kind of carrying carbapenamase bla NDM‑1 Transposon Tn 7555 Of gene, the nucleotide sequence of the transposon Tn 7555 It is as shown in SEQ ID NO.1.The application further provides a kind of microorganism, the microorganism contains the transposon Tn 7555 The application further provides the application of the transposon Tn 7555 , the microorganism in the preparation of detecting biological drug resistance product or the construction of drug resistance detection model.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a device carrying carbapenemase. bla NDM-1 Tn transposon of the gene 7555 And its applications. Background Technology

[0002] Carbapenem antibiotics are one of the last-resort effective drugs for the clinical treatment of multidrug-resistant Gram-negative bacterial infections, especially complicated intra-abdominal infections, bloodstream infections, and hospital-acquired pneumonia. However, the global prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has become a significant public health threat. Among them, strains producing New Delhi metallo-β-lactamases (NDMs) have become one of the most concerning CRE types due to their extremely high transmissibility and broad resistance spectrum. NDMs belong to the class B metallo-β-lactamases and can efficiently hydrolyze almost all β-lactam antibiotics, including carbapenems, and their encoding genes... bla NDM Often located on mobile genetic elements with highly efficient transmission capabilities, drug resistance spreads rapidly between bacterial species and even between different genera, posing unprecedented challenges to clinical treatment and infection control.

[0003] bla NDM Since their discovery, genes have evolved into numerous variant subtypes, exhibiting a high degree of complexity and diversity in their genetic environment. These genes are typically embedded within a sequence consisting of an insertion sequence (such as IS). Aba125 IS Aba14 In complex transposon units composed of plasmids (e.g., [missing information]), the unit itself can function as a complete, movable element for "cut-and-paste" transposition. Furthermore, this transposon unit is often captured onto different types of conjugating plasmids or chromosomes, achieving widespread dissemination across strains and species through horizontal gene transfer mechanisms such as plasmid conjugation and transformation. Therefore, analyzing [missing information]... bla NDM The precise genetic structure of a gene, especially its directly associated transposon structure, is the molecular basis for tracing its propagation path and evolutionary origin.

[0004] Current research has found bla NDM Genes can be associated with various transposon backbones or insertion sequence structures, such as those associated with Tn. 125 Related structures are common forms discovered in the early stages. However, with in-depth monitoring, novel genetic vectors with novel structures and active recombination are constantly emerging. These novel vectors often have higher mobility or broader host adaptability, which may drive... bla NDM Outbreaks or epidemics in certain regions or among specific strains. Existing technologies... bla NDM Our understanding of novel gene transposon vectors remains incomplete, particularly regarding their precise boundary definition, internal module composition, mobility potential, and distribution and role in recently clinically prevalent strains, which still lack systematic elucidation.

[0005] Therefore, identifying and elucidating novel, structurally well-defined carriers is crucial. bla NDM Gene transposons can not only enrich our understanding of the transmission mechanism of NDM resistance, but also provide key targets for establishing high-resolution molecular typing and source tracing monitoring technologies, which is of great significance for guiding precise clinical prevention and control and blocking the transmission chain of drug resistance. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a transposon Tn carrying the carbapenemase NDM gene. 7555 And its applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a carbapenemase-carrying enzyme bla NDM-1 Tn transposon of the gene 7555 The transposable Tn 7555 The nucleotide sequence is shown in SEQ ID NO. 1.

[0008] Preferably, the transposable Tn 7555 The sequence consists of two 6bp forward repeats, including a backbone region and an exogenous insertion region. The exogenous insertion region includes the insertion sequence IS. UnCu16 IS 5D IS Aeme1 and Tn 1696 -Tn 21 -Derivative transposons and insertion sequences IS Aeme17 Interrupted transposon Tn 5393c Tn 1696 -Tn 21 -Derivative transposons inserted into Tn 5393c Transposon drug resistance genes strA In the middle, strA Break it in two, Tn 1696 -Tn 21 -The left side of the derived transposon is Tn 1696 The skeletal region, with Tn on the right. 21 The skeletal region contains a complex integrin In27, which contains three variable regions, each containing a different drug resistance gene.

[0009] Preferably, the variable region of the integrin27 carries a drug resistance gene cassette. dfrA12 and aadA2 ; Variable region II carries the carbapenemase gene bla NDM-1 , bla NDM-1 The gene is located in the truncated Tn 125 Within, further processed by the inserted sequence IS CR1 Mediated; variable region three by IS 26 Mediated, containing drug resistance genes bla OXA-1 , aac(6')-Ib -Hangzhou aacC2 and tmrB .

[0010] The present invention also provides a microorganism containing the transposon Tn described in this invention. 7555 .

[0011] Preferably, the microorganism is Aeromonas vaginalis (Gastromonas vulgaris). Aeromonas caviae ).

[0012] The present invention also provides the transposon Tn. 7555 The application of the aforementioned microorganisms in the preparation of products for detecting antibiotic resistance.

[0013] The present invention also provides the transposon Tn. 7555 The application of the aforementioned microorganisms in constructing drug resistance detection models.

[0014] Preferably, the drug to which the resistance is targeted is ceftazidime, piperacillin / tazobactam, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tigecycline, or ticarcillin / clavulanic acid.

[0015] This invention is the first to discover and identify a novel multidrug-resistant transposon, Tn. 7555 Its unique structural features provide new genetic evidence for elucidating the drug resistance mechanisms of multidrug-resistant strains such as Aeromonas guinea pig, provide specific targets for the development of molecular diagnostic reagents against this type of drug-resistant bacteria, help in the rapid identification and control of the spread of drug-resistant bacteria in clinical practice, and provide an important molecular model for studying the evolution and spread of multidrug-resistant bacterial regions. Attached Figure Description

[0016] Figure 1 The morphology of strain K1076 on the culture medium is shown.

[0017] Figure 2 The enzyme production results of strain K1076 are shown.

[0018] Figure 3 The results of the carbapenemase type detection for strain K1076 are shown.

[0019] Figure 4 The sequencing alignment results of the amplification products of strain K1076 are shown.

[0020] Figure 5 A schematic diagram of the structure of the new transposable Tn7555 is shown. Detailed Implementation

[0021] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0024] Example 1: Isolation and Resistance Analysis of Drug-Resistant Strains I. Isolation and Identification of Strains In 2022, the inventor isolated a strain of bacteria from the blood of a patient with a bloodstream infection and named it strain K1076.

[0025] When strain K1076 was cultured on Columbia blood agar solid medium, the colonies were smooth, moist, glossy, and had intact edges. Figure 1 Strain K1076 was identified as Aeromonas vaginalis (guinea pig) by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Aeromonas caviae ).

[0026] II. Detection of carbapenemase by a modified carbapenemase inactivation assay of Aeromonas guinea pig K1076 1. Preparation of bacterial culture Select pure culture colonies of the test strain, suspend them in sterile physiological saline, and adjust the bacterial concentration to 0.5 McFarland turbidity standard (approximately equivalent to 1.5 × 10⁻⁶). 8 (CFU / mL).

[0027] 2. Coating plate Dip a sterile cotton swab in the prepared bacterial solution and spread it evenly on the surface of an MH agar plate three times, rotating the plate about 60° each time to ensure uniform bacterial growth. Cover the plate and leave it at room temperature for several minutes to allow the moisture on the agar surface to be absorbed.

[0028] 3. Attaching paper slips and adding samples Use sterile forceps to take four meropenem paper discs and stick them onto the MH plates that have been coated with bacterial solution.

[0029] The four pieces of paper are labeled as follows: (1) Meropenem discs alone (as a control); (2) Add 10 μL of 3-aminophenylboronic acid (APB) solution to the surface of the imipenem paper; (3) Add 10 μL of ethylenediaminetetraacetic acid (EDTA) solution to the surface of the imipenem paper; (4) Add 10 μL of APB+EDTA mixed solution (or 5 μL of each) to the surface of the imipenem plate. After adding the inhibitor, wait a moment for the solution to be completely absorbed by the plate before placing it in the incubator.

[0030] 4. Cultivation Place the plate upright in an incubator at 35°C ± 2°C and incubate aerobically for 16-18 hours.

[0031] 5. Results Observation and Measurement After incubation, remove the plates. Under a black background with a translucent lamp, measure the diameter of the inhibition zone around each plate, accurate to 0.1 mm, using calipers or an electronic measuring instrument. Record the measurement results for each plate.

[0032] 6. Result Interpretation (1) If the diameter of the inhibition zone of imipenem containing APB solution is ≥5 mm different from the diameter of the inhibition zone of imipenem alone, the tested strain is determined to produce class A carbapenemase. (2) If the diameter of the inhibition zone of imipenem containing EDTA solution is ≥5 mm different from the diameter of the inhibition zone of imipenem alone, it indicates that the tested strain produces type B carbapenemase. (3) If APB+EDTA is added at the same time, the difference between the diameter of the inhibition zone of imipenem containing the compound inhibitor and the diameter of the inhibition zone of imipenem alone is ≥5 mm, it can be confirmed that the strain produces both type A carbapenemase and type B metallo-β-lactamase. (4) If the difference between the diameter of the inhibition zone of imipenem containing enzyme inhibitor and the diameter of the inhibition zone of imipenem alone is less than 5 mm, it can be determined that the strain does not produce class A carbapenemase or class B metallo-β-lactamase.

[0033] The enzyme production results showed that this strain produces class B metallo-β-lactamases (…). Figure 2 ).

[0034] III. Further detection of carbapenemase types produced by the strain using a carbapenemase detection kit (colloidal gold immunochromatography). 1. Add 5 drops (approximately 150 μL) of extraction buffer to the EP tube provided with the kit.

[0035] 2. Use a 1μL inoculation loop to take one loopful of bacterial sample and place it in an EP tube containing 150μL of extraction buffer.

[0036] 3. Close the EP tube.

[0037] 4. Use a vortex mixer to mix the sample for a few seconds to ensure it is evenly mixed (if the sample is viscous, use a vortex mixer to mix and shake for 3 minutes or until the sample is evenly mixed), and then let it stand at room temperature for 10 minutes before use.

[0038] 5. Open the sealed aluminum foil bag of the test card box and remove the test card box. Once opened, please perform the test immediately.

[0039] 6. Using the disposable pipette provided in the kit, take 5 drops (about 100 μL) of the prepared mixture and add it to the sample well marked "S" on the test cartridge.

[0040] 7. After adding the sample, let the test cartridge sit at room temperature for 15 minutes and then read the results.

[0041] The results are as follows Figure 3 As shown, K1076 produces NDM enzyme. Figure 3 In Chinese: C: Quality control; K: KPC enzyme; O: OXA enzyme; V: VIM enzyme; I: IMP enzyme; N: NDM enzyme.

[0042] III. Amplification and Verification of the Carbapenemase Gene NDM in Aeromonas vulgaris K1076 The supernatant obtained from the thermal lysis of Aeromonas vaginalis K1076 cells was used as a template for amplification using primers. bla NDM Genes, annealing temperature 56℃.

[0043] The primer sequences are as follows: NDM-F: 5'-GGTTTGGCGATCTGGTTTTC-3'; NDM-R: 5'-CGGAATGGCTCATCACGATC-3'.

[0044] Figure 4 Sequencing and alignment results of the amplified products showed that Aeromonas vaginalis K1076 carried... blaNDM Gene.

[0045] IV. Drug resistance test of Aeromonas vaginalis K1076 To detect drug resistance in Aeromonas vaginalis K1076. Drugs included: ceftazidime, piperacillin / tazobactam, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tigecycline, and ticarcillin / clavulanic acid.

[0046] The minimum inhibitory concentration (MIC) of Aeromonas vaginalis K1076 was determined using VITEK 2 to assess its susceptibility to antibiotics.

[0047] The results are shown in Table 1. Aeromonas vaginalis K1076 showed resistance to ceftazidime, piperacillin / tazobactam, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, and ticarcillin / clavulanic acid.

[0048] Table 1

[0049] Example 2: Obtaining and Structural Analysis of Transposon Tn7555 The whole genome of strain K1076 was sequenced using high-throughput sequencing of the strain using third-generation Nanopore combined with second-generation Illmina technology.

[0050] The sequence was submitted to Resfinder (http: / / genepi.food.dtu.dk / resfinder) to detect drug resistance genes, and a carbapenemase gene was found to be carried on the chromosome. bla NDM-1 .

[0051] Chromosomal sequences were submitted to RAST (http: / / rast.nmpdr.org / ) to predict ORF regions. Simultaneously, the sequences were submitted to ISSaga (http: / / issaga.biotoul.fr / ISsaga2 / issaga_index.php), ISfinder (https: / / isfinder.biotoul.fr / ), INTEGRALL (http: / / integrall.bio.ua.pt / ?), and CARD (https: / / card.mcmaster.ca / ) to preliminarily predict the overall distribution of insertion sequences, integrons, and drug resistance genes. This was to obtain information on the distribution and presence of mobile elements and drug resistance genes.

[0052] By comparing data using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the specific location of the drug resistance movement element on the chromosome was determined, the key integrase gene of the transposon was found, and the repetitive sequences at both ends of the transposon were found using BLAST. Further in-depth analysis of the internal structure of the transposon was conducted, and each gene inside the transposon was annotated. The inserted sequences, transposons, and integrons were annotated separately. A script was used to call Inkscape to draw a fine structural map of the transposon. Novel transposable Tn 7555 See structural diagram Figure 5 Its sequence is shown in SEQ ID NO. 1.

[0053] Novel transposable Tn 7555 This is a novel carbapenemase resistance-associated transposon. It is 70.15 kb in length, with 6 bp orthogonal repeats at both ends (base sequence CCCCAT), including a backbone region and exogenous insertion regions. The exogenous regions consist of four sequences, one of which is the insertion sequence IS. UnCu16 IS 5D , ISAeme1 and being Tn 1696 -Tn 21 -Derivative transposons and insertion sequences IS Aeme17 Interrupted transposon Tn 5393c Tn 1696 -Tn 21 -Derivative transposons inserted into Tn 5393c Transposon drug resistance genes strA In the middle, strA Break it in two, Tn 1696 -Tn 21 -The left side of the derived transposon is Tn 1696 The skeletal region, with Tn on the right. 21 Within the skeletal region, a complex integrin In27 is inserted. This integrin contains three variable regions, each containing a different drug resistance gene. Variable region one (VR1) carries a drug resistance gene cassette. dfrA12 and aadA2 The variable region 2 (VR2) mainly carries the carbapenemase gene. bla NDM-1 This gene is located in the truncated Tn 125 Within, further processed by the inserted sequence IS CR1 Mediated by. Variable region 3 (VR3) is mainly composed of IS. 26 The mediated substance contains drug resistance genes. bla OXA-1 , aac(6')-Ib -Hangzhou aacC2 and tmrB.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A carbapenemase-carrying enzyme bla NDM-1 Tn transposon of the gene 7555 Its characteristics are, The transposable Tn 7555 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The transposon Tn according to claim 1 7555 Its characteristics are, The transposable Tn 7555 The sequence consists of two 6bp forward repeats, including a backbone region and an exogenous insertion region. The exogenous insertion region includes the insertion sequence IS. UnCu16 IS 5D IS Aeme1 and Tn 1696 -Tn 21 -Derivative transposons and insertion sequences IS Aeme17 Interrupted transposon Tn 5393c Tn 1696 -Tn 21 -Derivative transposons inserted into Tn 5393c Transposon drug resistance genes strA In the middle, strA Break it in two, Tn 1696 -Tn 21 -The left side of the derived transposon is Tn 1696 The skeletal region, with Tn on the right. 21 The skeletal region contains a complex integrin In27, which contains three variable regions, each containing a different drug resistance gene.

3. The transposon Tn according to claim 2 7555 Its characteristics are, The variable region of the integrin In27 carries a drug resistance gene cassette. dfrA12 and aadA2 ; Variable region II carries the carbapenemase gene bla NDM-1 , bla NDM-1 The gene is located in the truncated Tn 125 Within, further processed by the inserted sequence IS CR1 Mediated; variable region three by IS 26 Mediated, containing drug resistance genes bla OXA-1 , aac(6')-Ib -Hangzhou aacC2 and tmrB .

4. A microorganism, characterized in that, The microorganism contains the transposon Tn as described in any one of claims 1 to 3. 7555 .

5. The microorganism according to claim 4, characterized in that, The microorganism in question is Aeromonas vaginalis.

6. The transposon Tn as described in any one of claims 1 to 3 7555 The use of the microorganisms as described in any one of claims 4 to 5 in the preparation of products for detecting drug resistance.

7. The application according to claim 6, characterized in that, The drugs targeted by the resistance are ceftazidime, piperacillin / tazobactam, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tigecycline, or ticarcillin / clavulanic acid.

8. The transposon Tn as described in any one of claims 1 to 3 7555 The application of the microorganisms as described in any one of claims 4 to 5 in constructing drug resistance detection models.

9. The application according to claim 8, characterized in that, The drugs targeted by the resistance are ceftazidime, piperacillin / tazobactam, cefoperazone / sulbactam, cefepime, aztreonam, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tigecycline, or ticarcillin / clavulanic acid.

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