应用16S rDNA克隆文库法分析B6-Co小鼠角膜炎细菌组成

Bacterial composition of B6-Co mice with bacteria keratitis analyzed with 16S rDNA clone library

目的鉴定B6-Co突变系小鼠角膜的病原菌组成,并与人类细菌性角膜炎病原菌组成进行比较分析。方法选取10 d龄B6小鼠和B6-Co小鼠各6只为实验动物,提取小鼠角膜组织刮取物细菌基因组DNA,通过PCR扩增构建16S rDNA克隆文库,采用随机测序法分析B6-Co小鼠角膜细菌组成,B6小鼠作为对照。结果 B6-Co角膜混浊小鼠中共鉴定出20种细菌,隶属于15个属,包括葡萄球菌属、假单胞菌属、链球菌属、微球菌属、棒状杆菌属等。葡萄球菌属和假单胞菌属为优势群,葡萄球菌属丰度为19.7%~53.1%,假单胞菌属的丰度为17.4%~32.8%。其中葡萄球菌属主要是表皮葡萄球菌,丰度为13.8%~20.9%;缓慢葡萄球菌,丰度为14.0%~30.9%;金黄色葡萄球菌,丰度为7.4%~20.3%。此外,棒状杆菌和微球菌也比较常见。10 d龄B6小鼠未检测出细菌。结论 B6-Co突变系小鼠角膜细菌组成与人类角膜炎病原菌相似,是研究人类角膜炎诊断方法、发病机理及临床用药的很好的动物模型。

Objective To investigate the bacterial composition in the eyes of/36- Co mice with bacteria keratitis, and compare with that in humans with bacteria kerati- tis. Methods Six 10-day-old C57BL/6 （ B5 ） and C57BL/5-Corneal opacity （BS-Co） mice, respectively, were chosen as experimental animals. Total bacterial genomic DNA was extracted from corneal scrapings of B5 and BS-Co mice. 16S rDNA clone libraries from PCR products were constructed. The bacterial composition from corneal scrapings of B6-Co mice with bacteria keratitis was determined by sequence analysis. B5 mice were used as controls. Result~ The microbiota analysis detected 20 species in the eyes of BS-Co mice with bacterial keratitis, including 15 genera such as Staphylococcus, Pseudomonas,Streptococcus, Corynebacterium and Mierococcus. The dominant genera were Staphylococcus and Pseudomonas and accounted for 19. 7% - 53. 1% and 17.4% -32. 8%, respectively. Staphylococcus included Staphylococcus epidermidis, Staphylococcus lentus and Staphylococcus aureus, which accounted for 13. 8% - 20. 9% , 14.0% - 30.9% and 7.4% - 20.3% , respectively. In addition, Corgnebacterium and Micrococcus were also frequently detected in the eyes of BS-Co mice. No bacteria was detected in 10-day-old B5 mice controls. Conclusion The bacterial composition in the eyes of BS-Co mice is similar to that in humans with bacteria keratitis,which suggests that BS-Co mouse is a favorable model to study diagnostic methods,pathogenesis and clinical medication of bacteria keratitis in humans.