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中华关节外科杂志(电子版) ›› 2020, Vol. 14 ›› Issue (02) : 159 -166. doi: 10.3877/cma.j.issn.1674-134X.2020.02.006

所属专题: 文献

基础论著

红色荧光金葡菌假体感染临床株的构建
朱崇尊1, 余进龙1, 郭阁永1, 蒋峰1, 张飞洋1, 王坚镪2, 汤瑾2, 沈灏,1   
  1. 1. 200233 上海市第六人民医院骨科
    2. 200233 上海市第六人民医院检验科
  • 收稿日期:2020-01-04 出版日期:2020-04-01
  • 通信作者: 沈灏
  • 基金资助:
    国家自然科学基金(81772364,81472108); 上海市科委西医引导项目(19411962600)

Construction of clinical strain from prosthetic joint infection with red fluorescence

Chongzun Zhu1, Jinlong Yu1, Geyong Guo1, Feng Jiang1, Feiyang Zhang1, Jianqiang Wang2, Jin Tang2, Hao Shen,1   

  1. 1. Department of Orthopaedics, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200233
    2. Department of Laboratory, Shanghai Jiaotong University Affiliated Sixth People" s Hospital, Shanghai 200233, China
  • Received:2020-01-04 Published:2020-04-01
  • Corresponding author: Hao Shen
  • About author:
    Corresponding author: Shen Hao, Email:
引用本文:

朱崇尊, 余进龙, 郭阁永, 蒋峰, 张飞洋, 王坚镪, 汤瑾, 沈灏. 红色荧光金葡菌假体感染临床株的构建[J/OL]. 中华关节外科杂志(电子版), 2020, 14(02): 159-166.

Chongzun Zhu, Jinlong Yu, Geyong Guo, Feng Jiang, Feiyang Zhang, Jianqiang Wang, Jin Tang, Hao Shen. Construction of clinical strain from prosthetic joint infection with red fluorescence[J/OL]. Chinese Journal of Joint Surgery(Electronic Edition), 2020, 14(02): 159-166.

目的

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。

方法

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。

结果

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)。

结论

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。

Objective

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.

Methods

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.

Results

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).

Conclusion

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.

表1 质粒构建和鉴定所采用的引物表
图1 荧光质粒pRMC2-pts-mCherry的构建、检测以及导入工程大肠杆菌和金葡菌临床株ST1792后的荧光检测。图A 为构建荧光片段pts mcherry在pRMC2的插入位置示意图;图B为pts、mCherry片段PCR扩增后片段的大致长度;图C 为测序比对图,示插入片段后的测序;图D 为构建质粒pRMC2-pts-mCherry导入大肠杆菌和金葡菌后的筛选(Gibson系统连接片段导入DH5a和噬菌体转导ST1792后的IVIS检测结果)从左至右分别为:大肠杆菌DH5a阴性对照组,电转后大肠杆菌DH5a荧光阳性菌株,金葡菌ST1792阴性对照组,噬菌体转导后荧光阳性的金葡菌ST1792菌落
图2 金葡菌荧光株ST1792-pRMC2-pts-mCherry荧光稳定性、生长情况、生物膜形成特征的检测。图A 为体外连续传代培养ST1792-pRMC2-pts-mCherry 72 h荧光强度变化图;图B为ST1792-pRMC2-pts-mCherry 72 h荧光强度变化定量图,示无抗生素情况下传代培养72 h荧光稳定;图C 为金葡菌ST1792-pRMC2-pts-mCherry在不同条件下成膜定性和ST1792的比较,从左往右依次为:ST1792在TSBG(胰蛋白胨大豆肉汤葡萄糖)的生物膜定性、ST1792-pRMC2-pts-mCherry在TSBG内生物膜定性、ST1792在10%SF(滑液)的生物膜定性、ST1792-pRMC2-pts-mCherry在10%SF的生物膜定性,示两种菌株成膜情况相似;图D 为0.5%TSBG和10%SF中两种金葡菌生物膜的定量图,表明两种金葡菌在同一条件下成膜能力无差异(P>0.05);图E  为TSB(胰蛋白胨大豆肉汤培养基)条件下两种金葡菌生长曲线,示两者生长曲线基本一致,生长能力无差异(F=0.012, P>0.05)
图3 不同浓度的锌离子刺激巨噬细胞24 h后,对巨噬细胞吞噬功能的影响。图A 为不同时间点荧光显微镜下(×63)观察到的吞噬情况;图B 为不同培养条件对巨噬细胞吞噬率的影响,结果表明锌离子浓度>30 μmol/L对巨噬细胞的吞噬率的影响差异有统计学意义,其中:aa-30 μmol/L和0 μmol/L浓度比较(t=4.75,P<0.01),b-45 μmol/L和0 μmol/L浓度比较(t=4.086,P<0.05),c-60 μmol/L和0 μmol/L浓度比较(t=4.786,P<0.01)
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