摘要目的:分离1株耐甲氧西林金黄色葡萄球菌(MRSA)噬菌体，评估生物学特性和抗菌性能，并用于小鼠创面MRSA感染治疗。方法:苏北人民医院污水站收集污水，共培养法和双层琼脂平板法从该污水中分离MRSA噬菌体SBSS-1，观察噬菌斑形态、大小。透射电子显微镜(TEM)观察其微观形貌，并测量大小。测定其最佳感染复数(MOI)、一步生长曲线、不同pH、不同温度增殖活性。细菌活死染色、扫描电子显微镜(SEM)观察其抗菌性能。将噬菌体SBSS-1治疗创面MRSA感染小鼠，马松染色评价胶原水平，酶联免疫试剂盒检测炎性因子，免疫荧光检测血管、肉芽组织形成标志物，Image J 6.0软件分析荧光强度。两组间比较采用 t检验，多组间比较采用单因素方差分析。 结果:成功分离噬菌体SBSS-1，可形成大小为2.7~4.2 mm的透明噬菌斑。头部为正二十面体，直径(73±5) nm，连接长(201±17) nm、宽(18±5) nm的非可收缩性尾巴。属于有尾噬菌体目(Caudovirales)，长尾噬菌体科(Siphoviridae)。最佳MOI为0.1，潜伏期为20 min，裂解期为40 min，平均裂解量72 PFU/Cell；在4 ℃~50 ℃、pH 3~11，活性稳定。其可破坏MRSA膜结构，使大部分细菌呈红色荧光，并使MRSA皱缩，损伤。噬菌体SBSS-1组创面直径显著低于对照组[(0.16±0.04) cm比(0.55±0.03) cm， t＝22.06， P<0.05]；噬菌体SBSS-1组胶原沉积较对照组增多；噬菌体SBSS-1组创面创面肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-6表达水平显著低于对照组[(100.30±9.87) pg/ml比(310.00±26.46) pg/ml， t＝7.425， P<0.05；(33.33±4.67) pg/ml比(126.00±7.94) pg/ml， t＝10.060， P<0.05；(197.00±10.44) pg/ml比(711.00±45.45) pg/ml， t＝11.020， P<0.05]；噬菌体SBSS-1组小鼠创面白细胞介素-10表达水平显著高于对照组[(130.30±10.59) pg/ml比(27.33±7.54) pg/ml， t＝7.926， P<0.05]；噬菌体SBSS-1组创面细胞增殖标志物细胞核增殖抗原(Ki-67)抗原荧光强度显著高于对照组(222.70±16.76比62.67±4.70， t＝9.193， P<0.05)；噬菌体SBSS-1组创面血小板内皮细胞黏附分子荧光强度显著高于对照组(200.70±10.81比43.00±2.31， t＝14.270， P<0.05)。 结论:成功分离MRSA噬菌体SBSS-1，抗菌效果显著，能够促进MRSA感染创面愈合。

abstractsObjective:A phage strain of methicillin-resistant staphylococcus aureus (MRSA) was isolated. its biological characteristics and antibacterial performance were evaluated, then used for the treatment of MRSA infection in mice wounds. Methods:MRSA phage was isolated from sewage which collected from Northern Jiangsu People’s Hospital by sewage co-culture method and double AGAR plate method. The morphology and size of the phage plaque was observed. The morphology of phage SBSS-1 was observed by transmission electron microscope, and the size of phage SBSS-1 was measured. The optimal multiplicity of infection (MOI), one-step growth curve, tolerance at different pH or temperature were determined. The antibacterial performance of phage SBSS-1 was evaluated by bacteria live and death staining or scanning electron microscope. MRSA-Infected Wounds model of mice were prepared and then treated with phage SBSS-1. The collagen deposition was evaluated by Masson staining. The expression levels of inflammatory cytokines were detected by Elisa kit. The marker of vascularization and granulation tissue formation were detected by immunofluorescence. The fluorescence intensity was analyzed by the software of Image J 6.0. T test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups, Results:A MRSA phage was successfully isolated. Phage SBSS-1 could form transparent plaque, and the size of plaque was 2.7-4.2 mm. The head of the phage was regular icosahedron with a diameter of (73±5) nm, and connected to a non-constrictive tail with a length of (201±17) nm and a width of (18±5) nm. The phage SBSS-1 belongs to the Caudovirales family, Siphoviridae genus. The optimal multiplicity of infection of phage SBSS-1 was 0.1. The incubation period of infection was 20 min, the cleavage period was 40 min, and the average cleavage amount was 72 PFU/cell. It has good stability at the temperature between 4 ℃ and 50 ℃, at the pH values between 3 and 11. Phage SBSS-1 could destroy MRSA membrane structure and make most bacteria show red fluorescence. Phage SBSS-1 could induce MRSA shrinking and damage. The wound diameter of SBSS-1 group was obviously lower than Control group [(0.16±0.04) cm vs. (0.55±0.03) cm, t=22.06, P<0.05]; the collagen deposition of SBSS-1 group was more than Control group; the expression level of tumor necrosis factor-α, interleukin-1β and interleukin-6 in SBSS-1 group were significantly lower than those in Control group [(100.3±9.871) pg/ml vs. (310.0±26.46) pg/ml, t=7.425, P<0.05; (33.33±4.667) pg/ml vs. (126.0±7.937) pg/ml, t=10.06, P<0.05; (197.0±10.44) pg/ml vs. (711.0±45.45) pg/ml, t=11.02, P<0.05]; the interleukin-10 level of the wound in SBSS-1 group was significantly higher than that in Control group [(130.3±10.59) pg/ml vs. (27.33±7.535) pg/ml, t=7.926, P<0.05]; the fluorescence intensity of wound cell proliferation marker Ki-67 in SBSS-1 group was significantly higher than Control group (222.7±16.76 vs. 62.67±4.702, t=9.193, P<0.05); the fluorescence intensity of wound platelet endothelial cell adhesion molecules in SBSS-1 group was conspicuously more than Control group (200.7±10.81 vs. 43.00±2.309, t=14.27, P<0.05). Conclusion:Phage SBSS-1 was successfully isolated, shown excellent antibacterial performance, and it could accelerate MRSA infected wound healing.