Drug-resistant pseudomonas aeruginosa and application thereof

Abstract

The invention discloses a drug-resistant pseudomonas aeruginosa PA-1, the preservation number of the drug-resistant pseudomonas aeruginosa PA-1 in Guangdong Microbiological Culture Collection Center is GDMCC No: 65733. According to the drug-resistant pseudomonas aeruginosa PA-1, drug sensitive experiments show that the drug-resistant pseudomonas aeruginosa PA-1 is resistant to imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin and ceftriaxone; a quantitative dilution method experiment shows that the strain is resistant to aztreonam, imipenem, piperacillin / tazobactam, piperacillin, ceftazidime and cefepime; the drug-resistant strain can be used for screening a pseudomonas aeruginosa bacteriostatic preparation, and a new way is provided for development of drugs for treating diseases caused by pseudomonas aeruginosa.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a multidrug-resistant Pseudomonas aeruginosa and an application thereof. Background Art

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pseudomonas aeruginosa (PA) is an aerobic, oxidase-positive, motile Gram-negative bacillus that can cause a variety of infections. It can cause infections in multiple sites such as burn wounds, lungs and urinary tract, and can also cause otitis media, meningitis, sepsis and other diseases. It is also a common infection in the intensive care unit (ICU) of the hospital, and has the natural advantage of adapting to the environment and developing drug resistance. Pseudomonas aeruginosa can achieve antibiotic resistance in a variety of ways, among which gene mutation and horizontal migration of resistance genes are the main ways to produce resistance. For a long time, the problem of drug resistance has been the biggest obstacle to the treatment of clinical infections of Pseudomonas aeruginosa.

[0003] Pseudomonas aeruginosa has strong resistance, easy growth and survival characteristics, and can resist drying, ultraviolet rays and chemical disinfectants. Therefore, the resistance mechanism of PA to various antibiotics is also different. It is often the combined action of multiple resistance mechanisms, which is one of the reasons for the ineffectiveness of clinical antibacterial treatment. The resistance mechanism of PA is complex. Studies have shown that the resistance mechanism of PA mainly includes: ① The permeability of the film is reduced, thereby losing or lacking specific proteins; ② The generation of drug-resistant enzymes, mainly aminoglycoside inactivating enzymes, β-lactamase, etc.; ③ The formation of bacterial biofilms; ④ Target changes to escape the effects of antimicrobial drugs, among which the mutation of 16s ribosomal RNA methylase and topoisomerase is closely related to the resistance to aminoglycoside antibiotics; ⑤ The action of integrons leads to the spread of drug-resistant genes. Summary of the invention

[0004] The present invention provides a drug-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) PA-1, which was deposited in Guangdong Microbiological Culture Collection Center on January 9, 2025, with the deposit number GDMCC No: 65733; the deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] The invention finds that Pseudomonas aeruginosa PA-1 is resistant to imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin and ceftriaxone through drug sensitivity experiments; and finds that the bacteria is resistant to aztreonam, imipenem, piperacillin / tazobactam, piperacillin, ceftazidime and cefepime through quantitative dilution experiments.

[0006] Another object of the present invention is to use the drug-resistant Pseudomonas aeruginosa PA-1 in screening antibacterial agents for Pseudomonas aeruginosa that are resistant to imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin, ceftriaxone, piperacillin / tazobactam, ceftazidime, and cefepime.

[0007] The Pseudomonas aeruginosa PA-1 of the present invention is separated from the alveolar lavage fluid of bronchiectasis patients, is a currently popular strain of Pseudomonas aeruginosa, can be stably propagated in a culture medium, and has strong pathogenicity.

[0008] Advantages and technical effects of the present invention: The Pseudomonas aeruginosa PA-1 of the present invention is isolated from the alveolar lavage fluid of patients with bronchiectasis, is a current popular strain of Pseudomonas aeruginosa, can be stably propagated in a culture medium, and has strong pathogenicity. Experiments show that Pseudomonas aeruginosa PA-1 is resistant to multiple antibiotics, including imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin, ceftriaxone, piperacillin / tazobactam, ceftazidime, cefepime, etc.; the drug-resistant strain of the present invention can be used to screen antibacterial preparations for Pseudomonas aeruginosa, and the present invention provides a new approach for the development of therapeutic drugs for diseases caused by Pseudomonas aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The morphological diagram of isolated colonies on LB medium; Figure 2 This is the S-shaped curve of the quantitative PCR detection of Pseudomonas aeruginosa PA-1 strain; Figure 3 is the growth curve of Pseudomonas aeruginosa PA-1 strain; Figure 4 This is a microscopic observation of bronchial epithelial cells (Beas-2b); Figure 5 This is the count of bronchial epithelial cells (Beas-2b). DETAILED DESCRIPTION

[0010] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial channels or prepared according to conventional methods unless otherwise specified; Example 1: Isolation and identification of Pseudomonas aeruginosa PA-1 1. The bronchiectasis lavage fluid collected from the respiratory department of Anning First People's Hospital affiliated to Kunming University of Science and Technology was streaked and inoculated on LB agar plates. The plates were cultured in a 37°C, 5% CO2 incubator for 16-20 hours. Suspicious Pseudomonas aeruginosa colonies appeared on the LB agar plates. Since they can produce blue-green water-soluble pigments (such as pyocyanin), the colonies are often blue-green strains. Suspicious colonies (smooth, slightly raised, medium-sized colonies with neat or wavy edges) were selected for separation and purification. The purified strains were as follows: Figure 1 As shown, Gram staining microscopy was performed to observe the bacterial morphology and staining results. The results showed that after Gram staining, pink or red Gram-negative rods were observed under the microscope, arranged singly, in short chains or in pairs.

[0011] 2. Molecular identification of strain PA-1 (1) Inoculate the suspected Pseudomonas aeruginosa single colony picked above into LB liquid culture medium and culture at 37°C and 5% CO2 for 8-12 h to obtain bacterial culture solution; (2) Take 200 μL of the above bacterial culture solution as test samples and extract genomic DNA respectively, using DNA as template. Pseudomonas aeruginosa nucleic acid detection kit (PCR-fluorescent probe method, Covino KWN22142) was used for quantitative detection. The S-shaped curve of strain-specific PCR quantitative detection is shown in the figure Figure 2 As shown, the CT value was 29.074, which was identified as positive for Pseudomonas aeruginosa; (3) 16S rDNA sequencing and identification The Pseudomonas aeruginosa bacterial solution identified by PCR was amplified by PCR using universal primers for 16S rDNA gene sequence. The amplification primers were: 27F (AGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). A small amount of PCR product was electrophoresed in 1.5% agarose gel, and a target band of about 1500 bp was observed, which was consistent with the expected size. The amplified PCR product was sent to Beijing Bioengineering Co., Ltd. for sequencing and the results were spliced. The sequencing results were subjected to BLAST in NCBI, and online comparison analysis showed that the strain was Pseudomonas aeruginosa The 16SrDNA sequence similarity of (MT180543.1) was 100%, so the strain was determined to be Pseudomonas aeruginosa PA-1.

[0012] Example 2: Drug sensitivity test of Pseudomonas aeruginosa PA-1 1. Colony count and growth curve determination Inoculate 4 / 1000 of the Pseudomonas aeruginosa liquid into 250 mL of LB liquid culture medium, and culture it in a constant temperature box at 37°C and 5% CO2 for 36 h. Take out 5 mL of the liquid every 2 h, and place 4 mL of the liquid in a 1 cm quartz absorption cell. Measure the absorbance OD value at 600 nm. The liquid without bacteria added at the 0th hour of culture is used as the blank solution. The remaining 1 mL of the liquid is diluted in a 10-fold gradient. Take 100 μL of the dilution and spread it evenly on the LB agar culture plate with an L-shaped glass rod (3 replicates). After culturing at 37°C and 5% CO2 for 24 h, calculate the number of colonies and take the average value. The results are shown in the figure. Figure 3 As shown, Pseudomonas aeruginosa is in the logarithmic growth phase from 9 to 12 hours, and the bacterial concentration is about 1×10 8 CFU / mL.

[0013] 2. Kirby-Bauer drug susceptibility test of PA-1 strain The experimental operation steps of the KB drug sensitivity paper diffusion method were carried out according to the method of the Clinical and Laboratory Standards Institute (CLSI) 2020 version; (1) Preparation of Pseudomonas aeruginosa (PA-1) suspension Take the exponential growth phase of Pseudomonas aeruginosa PA-1 culture medium, centrifuge, wash once with PBS buffer, resuspend in PBS, and adjust the bacterial concentration to 10 8 CFU / mL, and PA-1 bacterial suspension was obtained; (2) After dipping a sterile cotton swab into the PA-1 bacterial suspension, spread it evenly on the MH agar plate, place the drug-susceptibility paper flat in the center of the plate coated with the bacterial solution, and incubate it at 37°C for 18 h. Measure the diameter of the inhibition zone and interpret the results according to the 2020 version of the Clinical and Laboratory Standards Institute (CLSI). The results are shown in Table 1. Strain PA-1 is resistant to imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin, and ceftriaxone. Table 1 .

[0014] Example 3: Determination of the minimum inhibitory concentration of Pseudomonas aeruginosa PA-1 by quantitative dilution method 1. The preparation of Pseudomonas aeruginosa (PA-1) suspension is the same as in Example 2; 2. According to the "Implementation Standards for Antimicrobial Susceptibility Testing" of the Clinical and Laboratory Standards Institute (CLSI), set the concentration gradient of Pseudomonas aeruginosa to some common antimicrobial drugs; add 100 μL of antibiotic stock solution (e.g., aztreonam 1280 μg / mL) to the first column of wells in a 96-well plate. Starting from the first column, use a pipette to draw 100 μL of liquid and dilute it to each subsequent column in turn, and finally obtain a series of antibiotic solutions of different concentrations, for example, starting from 128 μg / mL, the dilution ratio is 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, etc. At the same time, set up negative control wells (MH broth without antibiotics and PA-1 bacterial solution) and positive control wells (MH broth containing PA-1 bacterial solution and no antibiotics); 3. Add 100 μL of bacterial solution (1×10 6 CFU / mL), so that the final volume of each well is 200 μL; the 96-well plate is sealed to avoid evaporation and cultured at 37°C for 18 hours; 4. Observation of results and determination of MIC: Observe with the naked eye or read the absorbance of each well at a wavelength of 600nm using an enzyme reader. MIC is defined as the lowest drug concentration that can inhibit the visible growth of bacteria after incubation. When the bacteria in the positive control well grow obviously (turbid), and there is no bacterial growth in the drug-containing well (clear), the drug concentration corresponding to the drug-containing well is the MIC. The results are shown in Table 2; Table 2

[0015] It can be seen from the table that strain PA-1 is resistant to aztreonam, imipenem, piperacillin / tazobactam, piperacillin, ceftazidime, and cefepime.

[0016] Example 4: Determination of the cytotoxicity of Pseudomonas aeruginosa PA-1 1. Culture of bronchial epithelial BEAS-2B cells Shake the cryotube containing 1 mL of BEAS-2B cell suspension quickly in a 37°C water bath to thaw, add it to a centrifuge tube containing 5 mL of complete medium and mix well, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in complete medium. Then add the cell suspension to a culture flask (or dish) containing 7 mL of complete medium and culture at 37°C overnight. Observe the cell growth and cell density under a microscope the next day. Figure 4 shown.

[0017] 2. Cell passaging: When the cell density reaches 80%-90%, cell culture can be carried out. The cells are passaged every 2 days. (1) Discard the original culture medium, wash three times with PBS, add 1mL of trypsin to digest for 2 minutes, and add 1mL of culture medium to stop digestion. Blow the cells to make them all fall off, aspirate them into a 15ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and take 1mL of culture medium to mix the cells. Before counting, gently break up the cell suspension to disperse the cells into single cells as much as possible; use a micropipette to draw 10μL onto the counting plate for counting, and the suspension enters the cell counting pool through capillary action and fills the entire grid; wait for the cells to settle down, select the objective lens, and place the cell counting plate on the stage of the microscope for counting. The results are as follows: Figure 5 As shown, the cell density is 1×10 6 cells / mL; (2) Cells were plated 12 h in advance and digested with 0.1% trypsin. 6 The number of cells / well was inoculated in a six-well plate, with 1 mL of cell suspension per well, and cultured at 5% CO2 and 37°C for 12 h; (3) Use an inoculation loop to pick up 3-5 Pseudomonas aeruginosa colonies and place them in LB liquid culture medium. Incubate at 37°C, 5% CO2 for 10 h. Discard the culture medium and adjust the concentration of Pseudomonas aeruginosa in the logarithmic growth phase to 1×10 7 CFU / mL and 1×10 8 CFU / mL, added to BEAS-2B cells cultured for 12 hours, 1 mL of bacterial suspension in each well, set up 3 parallels, and cultured for 12 hours and 24 hours respectively in a 5% CO2, 37°C environment; 10 μL of CCK-8 solution was directly added to each well, and a blank group was set up at the same time. PBS buffer was added to the outermost wells to reduce the impact of evaporation, and then the cells were placed in a 37°C, 5% CO2 cell culture incubator for 2 hours, and the absorbance of each well was measured at 450nm; Cell survival rate = [(experimental well - blank well) / (control well - blank well)] × 100%; Table 3

[0018] The results are shown in Table 3. The strain PA-1 had a strong virulence, and the 24 h survival rate of BEAS-2B cells was 44%.

Claims

1. A drug-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa )PA-1, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC No: 65733.

2. Use of the drug-resistant Pseudomonas aeruginosa PA-1 according to claim 1 in screening antibacterial agents for Pseudomonas aeruginosa resistant to imipenem, ceftazidime, piperacillin, ticarcillin, aztreonam, cefuroxime sodium, cefoxitin, ceftriaxone, piperacillin / tazobactam, ceftazidime, and cefepime.

Images