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. 2021 Oct 15;35(10):1328-1335.
doi: 10.7507/1002-1892.202104061.

[In vivo study on the effects of intercellular adhesion operon of Staphylococcus epidermidis on the inflammation associated with bacteria-fungal mixed biofilm]

[Article in Chinese]
Guojing Zhang  1 , Zilin Wan  1 , Xiaoyan Wang  1 , Yunchao Huang  2 , Youquan Zhou  3 , Ying Chen  2 , Yujie Lei  2 , Limei Qiao  1
Affiliations

[In vivo study on the effects of intercellular adhesion operon of Staphylococcus epidermidis on the inflammation associated with bacteria-fungal mixed biofilm]

[Article in Chinese]
Guojing Zhang et al. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. .

Abstract
in English, Chinese

Objective: To study the effect of intercellular adhesion (ica) operon of Staphylococcus epidermidis on the inflammation associated with mixed biofilm of Staphylococcus epidermidis and Candida albicans on endotracheal tube material in rabbits.

Methods: The standard strains of Staphylococcus epidermidis RP62A (ica operon positive, positive group) and ATCC12228 (ica operon negative, negative group) were taken to prepare a bacterial solution with a concentration of 1×10 6 CFU/mL, respectively. Then, the two bacterial solutions were mixed with the standard strain of Candida albicans ATCC10231 of the same concentration to prepare a mixed culture solution at a ratio of 1∶1, respectively. The mixed culture solution was incubated with endotracheal tube material for 24 hours. The formation of mixed biofilm on the surface of the material was observed by scanning electron microscope. Thirty New Zealand rabbits, aged 4-6 months, were divided into two groups ( n=15), and the endotracheal tube materials of the positive group and the negative group that were incubated for 24 hours were implanted beside the trachea. The body mass of rabbits in the two groups was measured before operation and at 1, 3, and 7 days after operation. At 1, 3, and 7 days after operation, the levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and monocytechemotactic protein 1 (MCP-1) were detected by using an ELISA test kit. At 7 days after operation, the formation of mixed biofilm on the surface of the endotracheal tube materials was observed by scanning electron microscope, the inflammation and infiltration of tissues around the materials were observed by HE staining, and the bacterial infections in heart, lung, liver, and kidney were observed by plate colony counting method.

Results: Scanning electron microscope observation showed that the mixed biofilm structure was obvious in the positive group after 24 hours in vitro incubation, but no mixed biofilm formation was observed in the negative group. In vivo studies showed that there was no significant difference in body mass between the two groups before operation and at 1, 3, and 7 days after operation ( P>0.05). Compared with the negative group, the levels of MCP-1 and IL-1β at 1 day, and the levels of IL-1β, MCP-1, IL-6, and TNF-α at 3 and 7 days in the positive group all increased, with significant differences ( P<0.05). Scanning electron microscope observation showed that a large amount of Staphylococcus epidermis and mixed biofilm structure were observed in the positive group, and a very small amount of bacteria was observed in the negative group with no mixed biofilm structure. HE staining of surrounding tissue showed inflammatory cell infiltration in both groups, and neutrophils and lymphocytes were more in the positive group than in the negative group. There was no significant difference in the number of bacterial infections in heart and liver between the two groups ( P>0.05). The number of bacterial infections in lung and kidney in the positive group was higher than that in negative group ( P<0.05).

Conclusion: In the mixed infection of Staphylococcus epidermidis and Candida albicans, the ica operon may strengthen the structure of the biofilm and the spread of the biofilm in vivo, leading to increased inflammatory factors, and the bacteria are difficult to remove and persist.

目的: 探讨表皮葡萄球菌胞间黏附素基因（intercellular adhesion，ica）操纵子对气管导管材料表面表皮葡萄球菌与白假丝酵母菌混合生物膜相关炎症作用影响的体内研究。.

方法: 取表皮葡萄球菌标准株 RP62A（ica 操纵子阳性，阳性组）、ATCC12228（ica 操纵子阴性，阴性组）制备浓度为 1×10 6CFU/mL 的菌液，分别与相同浓度的白假丝酵母菌标准株 ATCC10231 菌液按照 1∶1 比例制备成混合培养菌液后，与气管导管材料片孵育 24 h，扫描电镜观察材料表面混合生物膜形成情况。取 4～6 月龄新西兰兔 30 只分为两组（ n=15），分别于气管旁植入孵育 24 h 的阳性组及阴性组气管导管材料。于术前及术后 1、3、7 d 测量两组兔体质量。术后 1、3、7 d，采用 ELISA 检测试剂盒检测血浆中 IL-1β、IL-6、TNF-α 和单核细胞趋化蛋白 1（monocytechemotactic protein 1，MCP-1）水平；术后 7 d，扫描电镜观察取出的气管导管材料片表面混合生物膜形成情况，HE 染色观察材料周围组织炎症浸润情况，平板菌落计数法观察心脏、肺、肝脏、肾脏细菌感染情况。.

结果: 扫描电镜观察示体外孵育 24 h 后阳性组可见明显混合生物膜结构，阴性组未见混合生物膜形成。体内实验显示两组术前及术后 1、3、7 d 体质量比较，差异均无统计学意义（ P>0.05）。与阴性组相比，阳性组术后 1 d IL-1β 及 MCP-1 水平，术后 3、7 d IL-1β、MCP-1、IL-6、TNF-α 水平均升高，组间比较差异有统计学意义（ P<0.05）。扫描电镜观察示阳性组可见大量表皮葡萄球菌残留以及混合生物膜结构，阴性组见极少量表皮葡萄球菌残留，未见混合生物膜结构。HE 染色示两组材料周围组织均可见炎症细胞浸润，其中阳性组中性粒细胞及淋巴细胞浸润较阴性组严重。两组心脏、肝脏细菌感染数量差异无统计学意义（ P>0.05），阳性组肺、肾脏细菌感染数量高于阴性组（ P<0.05）。.

结论: ica 操纵子在表皮葡萄球菌与白假丝酵母菌混合感染中可能由于增强了混合生物膜结构及其在体内的传播，导致炎症因子升高，细菌难以清除，感染迁延不愈。.

Keywords: Biomaterial centered infection; Candida albicans; Staphylococcus epidermidis; intercellular adhesion operon; mixed biofilm; rabbit.

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Conflict of interest statement

利益冲突：所有作者声明，在课题研究和文章撰写过程中无利益冲突。课题经费支持没有影响文章观点和对研究数据客观结果的统计分析及其报道。

Figures

图 1
图 1
Scanning electron microscope observation of the mixed biofilm formation on the surface of the endotracheal tube materials of the two groups after 24 hours in vitro incubation 体外孵育 24 h 后扫描电镜观察两组气管导管材料表面混合生物膜形成
图 2
图 2
Changes in body mass of the two groups 两组体质量变化趋势
图 3
图 3
Changes of plasma inflammatory factors in the two groups after operation 两组术后血浆炎症因子变化趋势
图 4
图 4
Scanning electron microscope observation of endotracheal tube materials in the two groups at 7 days after operation 术后 7 d 两组气管导管材料扫描电镜观察
图 5
图 5
HE staining observation of tissues surrounding endotracheal tube materials in the two groups at 7 days after operation 术后 7 d 两组气管导管材料周围组织 HE 染色观察
See this image and copyright information in PMC

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