实用临床医药杂志2025，Vol.29Issue(10)：1-6,13,7.DOI:10.7619/jcmp.20245328
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
Construction of Pseudomonas aeruginosa strain with deletion of pyocyanin synthesis gene PAOⅡ and study on its drug resistance
摘要
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.关键词
铜绿假单胞菌/绿脓菌素/生物被膜/耐药性/PAOⅡ基因型菌株/头孢类抗生素/转染/菌株Key words
Pseudomonas aeruginosa/pyocyanin/biofilm/drug resistance/PAOⅡ genotype strain/cephalosporins/transfection/strain分类
临床医学引用本文复制引用
冯炳富,黄玲,董文敏,崔刚..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[J].实用临床医药杂志,2025,29(10):1-6,13,7.基金项目
国家自然科学基金项目(32200098) （32200098）
