建立红色荧光标记金黄色葡萄球菌(金葡菌)假体感染(PJI)临床株，探究工具菌的生物学特性及和初步应用可行性。

通过分子克隆手段构建mCherry红色荧光表达质粒，通过电转化和噬菌体转导构建红色荧光标记金葡菌PJI临床株。通过生长曲线检测，体外成膜和生物量分析和荧光强度测量鉴定工具菌生物学特性。采用RAW264.7细胞株、工具菌建立共培养模型。采用该模型示踪不同锌离子浓度处理的巨噬细胞对于荧光PJI金葡菌吞噬能力。使用独立样本t检验、重复测量方差分析对定量数据进行统计。

使用Gibson系统成功构建了荧光表达质粒pRMC2-pts-mCherry，并且使用噬菌体Phage11将该质粒成功导入金葡菌假体感染临床株ST1792。较之于野生株ST1792，荧光金葡菌ST1792-pRMC2-pts-mCherry两者的生长曲线没有统计学差异(F＝0.012，P>0.05)。在0.5%葡萄糖胰蛋白胨大豆肉汤培养基(TSBG)内，两者的生物膜光密度(OD)值分别为(3.700±0.003)和(3.715±0.042)(t＝0.6056，P>0.05)；10%滑液中生物膜OD值为(3.682±0.084)和(3.648±0.095)(t＝0.474，P >0.05)。使用动物活体成像系统(IVIS)检测荧光强度，24 h荧光强度为(1.95± 0.04)、48 h荧光为(1.94±0.13)、72 h荧光强度为(1.95±0.04)，荧光强度72 h稳定(F＝2.944，P>0.05)。通过荧光显微镜观察以及菌落形成单位(CFU)计数评价发现，使用锌离子浓度30～60 μmol/L不含抗生素的培养基处理RAW264.7 24 h，可以促进其吞噬金葡菌，其中30 μmol/L吞噬率为(44±4)%(t ＝4.75，P<0.01)、45 μmol/L吞噬率为(45±6)%(t＝4.086，P<0.05)、60 μmol/L离子处理的巨噬细胞，其吞噬率为(38±4)%(t ＝4.786，P <0.01)。

本研究成功构建红色荧光临床PJI金葡菌，并验证其用于PJI相关体外吞噬研究的可行性。

To establish a prosthetic joint infection(PJI) clinical strain of staphylococcus aureus(S.aureus) labeled with red fluorescence and explore the biological characteristics and feasibility of application in vitro.

mCherry red fluorescent shuttle plasmid was constructed by molecular cloning, and a PJI clinical strain of fluorescenent staphylococcus aureus was constructed by electrical transformation and phage transduction. Growth curve, fluorescenence intensity, biofilm formation and biomass assay were used to evaluate the biological characteristics of the tool bacteria.RAW264.7 cell and tool bacteria were used to establish the co-culture model, which was used to trace the phagocytosis ability of macrophages against against S. aureus treated with different concentrations of zinc ion. The independent sample t test and repeated measures analysis of variance were used to analyze the quantitative data.

The fluorescent shuttle plasmid pRMC2-pts-mCherry was successfully constructed using Gibson system, and the plasmid was successfully introduced into the clinical S. aureus strain ST1792 using phage 11. Compared with the wild strain ST1792, the growth curve of S. aureus ST1792-pRMC2-pts-mCherry was not statistically different(F=0.012, P>0.05). In 0.5% glucose tryptone peptone soybean broth (TSBG), the biofilm optical density(OD) values of the two were (3.700±0.003) and (3.715±0.042) (t=0.6056, P>0.05); 10% synovial fluid, the median biofilm OD values were (3.682±0.084) and (3.648±0.095) (t=0.474, P>0.05). IVIS was used to detect the fluorescence intensity. The fluorescence intensity at 24h was (1.95±0.04), the fluorescence at 48h was (1.94±0.13), the fluorescence intensity at 72h was (1.95±0.04), and the fluorescence intensity was stable at 72 hours(F=2.944, P>0.05). Through fluorescence microscope observation and colony forming units counts, it was found that when RAW264.7 was treated with antibiotics-free culture medium with a zinc ion concentration of 30-60 μmol/L for 24 h com promote its phagocytosis of S. aureus: 30 μmol/L phagocytic rate was(44±4)% (t =4.75, P <0.01), 45 μmol/L phagocytic rate was(45±6)% (t=4.086, P <0.05). 60 μmol/L phagocytic rate of macrophage was(38±4)% (t=4.786, P<0.01).

In this study, the fluorescent clinical S. aureus is successfully constructed, and its feasibility for the study of PJI related phagocytosis in vitro is verified.