金黄色葡萄球菌致新西兰兔脓胸生物被膜模型的建立

Establishment of biofilm model of empyema in New Zealand rabbits induced by Staphylococcus aureus

目的:建立金黄色葡萄球菌感染的新西兰兔脓胸模型,观察其胸腔内是否有生物被膜形成。方法:选取24只健康雄性新西兰兔按随机数字表法分为实验组和对照组,各12只。通过胸腔穿刺术于右侧第六肋间隙将导管置入胸腔,实验组注射剂量为2 mL/kg的108 CFU/mL金黄色葡萄球菌,对照组使用相同剂量的Luria-Bertani(LB)肉汤。建模后持续饲养并于第4天收集胸腔留置导管进行活菌计数和结晶紫染色。收集所有脓性絮状物的匀浆液和洗涤液用于菌落计数。切取壁层胸膜组织进行苏木精-伊红(HE)染色,观察胸膜病理改变情况;肽核酸荧光原位杂交(PNA-FISH)用于观察脓性絮状物中的生物被膜形成。结果:与对照组比较,实验组患侧胸腔有脓性絮状物形成和胸膜粘连。实验组导管菌落计数显著大于对照组(P<0.01),结晶紫染色OD值显著高于对照组(P<0.05)。通过计算匀浆液和洗涤液的CFU,验证了实验组胸腔内积聚了大量金黄色葡萄球菌。HE染色结果显示,实验组胸膜明显增厚,并伴随有大量炎症细胞浸润。PNA-FISH结果证实实验组兔子胸腔脓性絮状物中有生物被膜形成。结论:金黄色葡萄球菌致新西兰兔脓胸感染模型中,胸膜腔内存在生物被膜。

Objective:To establish a New Zealand rabbit empyema model infected by Staphylococcus aureus,and observe whether there is biofilm formation in the thorax.Methods:A total of 24 healthy male New Zealand rabbits were s elected and divide into experimental group and control group according to the random number table method,12 in each group.Right side sixth intercostal space thoracentesis was used to introduce a catheter into the chest cavity.Staphylococcus aureus(108 CFU/mL)was administered to the experimental group at a dose of 2 mL/kg,whereas Luria-Bertani(LB)broth was administered to the control group at the same amount.Following the creation of the model,feeding proceeded,and on the fourth day,indwelling catheters in the chest cavity were collected for counting live bacteria and crystal violet staining.For colony counts,homogenates and washes of all purulent flocs were gathered.To evaluate the pathological changes to the parietal pleura,tissue was removed and stained with hematoxylin-eosin(HE).The peptide nucleic acid fluorescence in situ hybridization(PNA-FISH)was utilized to observe the development of a biofilm within the purulent floc.Results:In the injured thorax of the experimental group,there were purulent floc development and pleural adhesions in comparison to the control group.Both the colony count of the catheter and the absorbance value of the crystal violet staining in the experimental group were substantially greater than in the control group(P<0.05).It was determined that a significant amount of Staphylococcus aureus had accumulated in the chest cavity of the experimental group by calculating the CFU of the homogenate and washing solutions.The results of the HE staining revealed that the pleura in the experimental group had greatly thickened along with a significant infiltration of inflammatory cells.The purulent flocs in the pleural cavity of the experimental group had biofilm formation,according to the PNA-FISH data.Conclusion:A biofilm is present in the pleural cavity in the Staphylococcus aureus-induced New Zealand rabbit empyema infection model.