Pseudomonas aeruginosa and application thereof

Abstract

This invention discloses a pan-drug-resistant strain of *Pseudomonas aeruginosa* PAS96 and its applications. *Pseudomonas aeruginosa* PAS96 has the accession number GDMCC No: 64767. This bacterium is resistant to levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, and imipenem. This invention confirmed the resistance of *Pseudomonas aeruginosa* to the aforementioned antibiotics through Kirby-Bauer (K-B) susceptibility testing.

Description

Technical fields:

[0001] This invention belongs to the field of microbiology, specifically relating to a pan-drug-resistant strain of Pseudomonas aeruginosa and its applications. Background technology:

[0002] Pseudomonas aeruginosa (PA) is an opportunistic pathogen causing various infections in humans and a common source of infection in hospital intensive care units (ICUs). Its large genome size (6.3 Mb) provides a natural advantage in adapting to its environment and developing drug resistance. Pseudomonas aeruginosa can develop antibiotic resistance through various mechanisms, with gene mutation and horizontal migration of resistance genes being the main pathways. Drug resistance has long been the biggest obstacle to the clinical treatment of Pseudomonas aeruginosa infections.

[0003] The drug resistance mechanisms of *Pseudomonas aeruginosa* are complex, mainly manifesting as intrinsic resistance, acquired resistance, and adaptive resistance. Acquired resistance primarily involves *P. aeruginosa* cells acquiring new genetic material, enabling the bacteria to survive and resist antibiotic environments, such as through gene mutations on chromosomes or horizontal gene migration. Adaptive resistance mainly refers to the ability of *P. aeruginosa* to transiently adapt to specific antibiotic environments by regulating changes in gene or protein expression under stress conditions. Intrinsic resistance is the natural ability of *P. aeruginosa* cells to develop resistance to certain antibiotics due to their inherent structure and characteristics. This invention provides a pan-drug-resistant *P. aeruginosa* strain. Summary of the Invention:

[0004] The purpose of this invention is to provide a pan-drug-resistant strain of Pseudomonas aeruginosa, PAS96, deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, on June 19, 2024, with accession number GDMCC No: 64767.

[0005] This invention demonstrates through experiments that Pseudomonas aeruginosa PAS96 is resistant to a variety of antibiotics, including levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, and imipenem.

[0006] Therefore, the object of this invention is to provide a pan-drug-resistant strain of Pseudomonas aeruginosa PAS96.

[0007] The second objective of this invention is to provide the application of Pseudomonas aeruginosa PAS96 in drug resistance research and the development of novel antibiotics.

[0008] The antibiotics mentioned include levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, and imipenem, etc.

[0009] This invention, through Kirby-Bauer (KB) drug susceptibility testing, revealed that *Pseudomonas aeruginosa* PAS96 is resistant to levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, and imipenem, with inhibition zone diameters of 6.00, 9.11, 10.25, 8.00, 6.00, 8.98, 7.64, and 8.59 mm, respectively. Therefore, *Pseudomonas aeruginosa* PAS96 can be used for drug resistance research and the development of novel antibiotics.

[0010] Pseudomonas aeruginosa PAS96 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The deposit date is June 19, 2024, and the accession number is GDMCCNo: 64767. Detailed implementation method:

[0011] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0012] Example 1:

[0013] Pseudomonas aeruginosa PAS96 was isolated from samples from the Department of Clinical Laboratory, The First Affiliated Hospital of Sun Yat-sen University, and the bacterial species was identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) (BioMérieux, Marcy P. Etoile, France).

[0014] Pseudomonas aeruginosa PAS96 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The deposit date is June 19, 2024, and the accession number is GDMCC No. 64767.

[0015] Example 2:

[0016] Preparation of PAS96 suspension: A sample of P. aeruginosa PAS96 culture in the exponential growth phase was taken, centrifuged, washed once with PBS buffer, resuspended in PBS, and the bacterial concentration was diluted to 10⁻⁶. 8 CFU / mL was used to obtain a PAS96 bacterial suspension.

[0017] 1. KB susceptibility test of PAS96 strain

[0018] The experimental procedures for the KB antimicrobial susceptibility testing disk diffusion method should follow the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines. Pick single colonies that have grown overnight from blood agar plates to prepare a bacterial suspension, and adjust the concentration of the suspension to 1-2 × 10⁻⁶ using physiological saline. 8 CFU·mL -1 Inoculate with the bacterial suspension at the above concentration, dip a sterile cotton swab into the suspension, and spread it evenly on an MH agar plate. Place a drug sensitivity test disc flat in the center of the plate. Incubate at 37°C for 16-18 hours, and measure the diameter of the inhibition zone. Interpret the results according to CLSI 2020; the interpretation criteria are detailed in Table 1. The quality control strain used in this experiment was *Pseudomonas aeruginosa* ATCC27853.

[0019] Table 1. Antimicrobial susceptibility testing criteria for Pseudomonas aeruginosa Kirby-Bauer (KB)

[0020]

[0021]

[0022] Experimental results:

[0023] The drug susceptibility test results of *Pseudomonas aeruginosa* strain PAS96 are detailed in Table 2. The results showed that *Pseudomonas aeruginosa* PAS96 was resistant to levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, and imipenem, with inhibition zone diameters of 6.00, 9.11, 10.25, 8.00, 6.00, 8.98, 7.64, and 8.59 mm, respectively; and sensitive to polymyxin B, polymyxin E, amikacin, and tobramycin, with inhibition zone diameters of 17.90, 16.89, 22.56, and 20.45 mm, respectively. These results indicate that *Pseudomonas aeruginosa* PAS96 is a pan-drug-resistant bacterium resistant to multiple antibiotics.

[0024] Table 2. Antimicrobial susceptibility test results of Pseudomonas aeruginosa strain PAS96

[0025]

[0026]

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa PAS96, accession number: GDMCC No: 64767.

2. Use of Pseudomonas aeruginosa PAS96 according to claim 1 for the development of an antibiotic, which is levofloxacin, ciprofloxacin, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, ceftazidime, meropenem, imipenem, polymyxin B, polymyxin E, amikacin or tobramycin.

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