Construction of Pseudomonas aeruginosa strain with deletion of pyocyanin synthesis gene PAOⅡ and study on its drug resistance
- VernacularTitle:铜绿假单胞菌缺失绿脓菌素合成基因PAOⅡ的菌株构建及其耐药性研究
- Author:
Bingfu FENG
1
;
Ling HUANG
;
Wenmin DONG
;
Gang CUI
Author Information
- Keywords: Pseudomonas aeruginosa; pyocyanin; biofilm; drug resistance; PAOⅡ genotype strain; cephalosporins; transfection; strain
- From: Journal of Clinical Medicine in Practice 2025;29(10):1-6,13
- CountryChina
- Language:Chinese
- Abstract: Objective To construct Pseudomonas aeruginosa(PA)strain with deletion of the pyocyanin synthesis gene PAOⅡ and investigate its drug resistance.Methods By analyzing the ge-nome of the PAOⅡ genotype strain of PA,the distribution of genes/gene clusters related to pyocyanin synthesis in the genome was explored.A strain with deletion of the pyocyanin synthesis gene phzM was constructed using homologous recombination-based seamless gene knockout technology.The growth and drug tolerance of the wild-type and mutant strains were compared.Results Kyoto Encyclopedia of Genes and Genomes(KEGG)clustering analysis was performed on the website https://www.kegg.jp/.The results revealed a gene cluster composed of 9 genes(including PhzA,PhzB,PhzC,PhzD,PhzE,PhzF,PhzG,phzM and PhzS),which was completely consistent with the KEGG PATHWAY:Phenazine biosynthesis-M00835 Pyocyanine biosynthesis pathway.The key gene phzM for pyocyanin synthesis was knocked out via homologous recombination.After the exchange,monoclonal strains growing on M9-citrate medium were verified using primers phzM-AF and phzM-BR.The shuttle plasmid pK18mobSacB-phzM-AB was successfully transfected into the PAOⅡ genotype strain.After gene ex-change,monoclonal strains capable of growing on antibiotic-free plates were selected,and polymerase chain reaction(PCR)was performed to obtain strains with successful scarless knockout of the phzM gene.At 3 and 6 days of culture,the biofilm formation thickness of the PAOⅡ genotype strain was greater than that of the PAOⅡ-△phzM strain,with a statistically significant difference(P＜0.05).Conclusion The biofilm synthesis capacity of the pyocyanin synthesis-deficient strain is reduced,and its resistance to cephalosporins(ceftazidime)is also decreased.
