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目的研究不同氧浓度对铜绿假单胞菌生长及毒力的调节作用,阐明氦氧饱和高气压潜水富氧环境中铜绿假单胞菌感染高发机制。方法以铜绿假单胞菌PAO1(ATCC15692)为研究对象,主要设置实验对照组(20%O_2)、富氧实验组(40%O_2)、低氧实验组(2%O_2)共三个暴露组。用半固体培养基、丛集运动培养基观察细菌生长及运动能力,绿脓菌素测定培养基结合光密度法测定绿脓菌素含量。用HE染色结合CCK-8方法,观察细菌培养上清对靶细胞A549的损伤效应。结果与对照组相比,富氧环境生长的细菌克隆厚、中心明显隆起,运动生长轨迹不明显;低氧环境生长的细菌克隆中心凹陷、平铺生长,细菌运动生长轨迹显著。相较于实验对照组,低氧实验组绿脓菌素分泌能力降低(0.38±0.08 vs 0.16±0.03,F=10.22,P=0.033),富氧浓度组绿脓菌素分泌能力增加(0.38±0.08 vs 0.66±0.06,F=24.00,P=0.008)。细胞毒性检测显示,与空白对照组(F=93.55,P=0.001)和实验对照组(F=14.69,P=0.019)相比,富氧暴露生长细菌分离上清与细胞共培养8 h后,靶细胞出现明显损伤效应。结论富氧浓度可通过调节铜绿假单胞菌生长、运动及毒力,诱导铜绿假单胞菌致病能力。
Objective To study the regulatory effect of different oxygen concentrations on the growth and virulence of Pseudomonas aeruginosa and to elucidate the mechanism of the high incidence of Pseudomonas aeruginosa infection in the oxygen-rich environment of helium-oxygenated and high-pressured diving. Methods Pseudomonas aeruginosa PAO1 (ATCC15692) was used as the research object. The experimental group (20% O_2), oxygen enriched experimental group (40% O_2) and hypoxic experimental group (2% . The growth and motility of bacteria were observed by using semi-solid medium and clustered exercise medium, and the pyocyanin content was determined by pyocyanin determination medium combined with optical density method. The effect of bacterial culture supernatant on target cell A549 was observed by HE staining combined with CCK-8 method. Results Compared with the control group, the bacterial colonies grown in oxygen-enriched environment were thick, the center was obviously uplifted, and the locomotor growth path was not obvious. The growth of bacteria in the hypoxia environment was significant. Compared with the experimental control group, pyocyanin secretion ability in hypoxia experimental group decreased (0.38 ± 0.08 vs 0.16 ± 0.03, F = 10.22, P = 0.033), pyocyanin secretion increased in hypoxia group (0.38 ± 0.08 vs 0.66 ± 0.06, F = 24.00, P = 0.008). Cytotoxicity assay showed that compared with the blank control group (F = 93.55, P = 0.001) and the experimental control group (F = 14.69, P = 0.019) Target cells showed obvious damage effect. Conclusion Oxygen enrichment can induce P. aeruginosa pathogenicity by regulating the growth, movement and virulence of Pseudomonas aeruginosa.