稳定生物发光金葡菌临床株假体周围关节感染动物模型的构建

Animal model of periprosthetic joint infection established by stably luminescent staphylococcus aureus clinical strains

目的建立稳定型生物发光临床株金黄色葡萄球菌假体周围关节感染(PJI)动物模型并探讨其可行性及应用价值。方法应用噬菌体Phi11侵染带Lux发光基因的标准菌株BD1652,并通过噬菌体转导将Lux基因导入PJI临床菌株金葡菌ST1792基因组DNA,进而构建成稳定型生物发光临床菌株ST1792-Lux。通过抽纸牌,将10只健康BALB/c小鼠随机均分为实验组和对照组,使用克氏针作为两组模型动物的左膝关节假体。术中实验组左膝关节内注入10μl(105CFU)ST1792-Lux菌液,对照组注入等量0.9%氯化钠溶液,术后1、3、7、14 d,采用活体成像系统(IVIS)检测生物发光强度随时间变化;并于术后2周观察小鼠左膝关节病理学变化。以非配对t检验对ST1792-Lux和ST1792的生物膜形成能力和同一时间点实验组与对照组小鼠左膝平均生物发光强度进行比较分析。结果经临床株筛选,噬菌体Phi11转导获得生物发光菌株"ST1792-Lux",无抗生素筛选条件下连续传代培养48 h,ST1792-Lux稳定发光;Lux基因重组于ST1792基因组DNA内,较之野生株ST1792,ST1792-Lux在生长能力和不同培养条件下成膜能力(tTSB=1.16,1.29;tTSBG=0.38,0.31;P均>0.05)方面差异无统计学意义。IVIS检测示,术后每个时间点(1、3、7和14 d),实验组较对照组的左膝平均生物发光强度均明显增高(t=9.13,10.72,14.48,7.46;P均<0.05)。结论本研究成功建立了稳定生物发光临床株S.aureus PJI动物模型,其稳定性高、重复性好,可用于临床株S.aureus PJI的致病分子机制和各种干预措施有效性的体内研究。

Objective To establish an animal model of periprosthetic joint infection（ PJI） with stable luminescent staphylococcus aureus（ S. aureus） clinical strains and evaluate its application. Methods After phage Phi11 infected the standard bacterial strain BD1652 carrying Lux gene,the Lux gene was transferred to S. aureus clinical strains ST1792 from BD1652 by phage transduction,and then the stable luminescent S. aureus clinical strains ST1792-lux were obtained. Ten BALB / c healthy mice were evenly divided into experimental group and control group. Kirschner wires were used as left knee prosthesises.During the operation,ST1792-Lux（ 105CFU） in 10 μl of medium was inoculated into the cavity of the left knee in the experimental group,instead of the same volume of saline in the control group. The bacterial photon intensity in vivo was sequentially measured by in vivo imaging system（ IVIS） on days 1,3,7 and14 after the operation. The pathological examinations of left knee were determined in the second week after the surgery. Student＇s t test was used to analyze the biofilm formation ability of ST1792-Lux and ST1792 and the mean bacterial photon intensity in the left knee between the experimental group and the control group at same time after the surgery. Results The luminescent S. aureus clinical strains ST1792-Lux was achieved by Phi11 phage transduction. In the absence of antibiotics,luminescence of ST1792-Lux was stable in vitro after continuous subculture over 48 hours. The lux gene was integrated into the bacterial chromosome,and ST1792-Lux did not display growth and biofilm formation defects in vitro. Compared with the control group,the mean bacterial photon intensity in left knee were significantly increased on day 1,3,7 and 14 after the surgery in the experimental group（ t = 9. 13,10. 72,14. 48,7. 46; all P〈0. 05）.Conclusion The study has successfully established an animal model of PJI with stable luminescent S.aureus clinical strains and the model shows advantages of high stability and good repeatability which could be used to explore the pathogenic molecular mechanism of PJI caused by clinical strains S. aureus and the effectiveness of various interventions in vivo.