不同浓度耐甲氧西林金黄色葡萄球菌制备慢性骨髓炎模型对比研究

Comparative study of different concentrations of methicillin-resistant Staphylococcus aureus in the preparation of chronic femoral osteomyelitis models

目的探讨采用耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)建立兔股骨慢性骨髓炎模型的合适浓度。方法取成年新西兰大白兔48只随机分为6组,每组8只,B、C、D、E、F组分别于股骨髁上2 cm处注射1×10~9、1×10~8、1×10~7、1×10~6、1×10~5 CFU/m L MRSA,A组注射无菌生理盐水作为对照。术后4周行实验动物大体观察,取伤口分泌物行细菌学检查;术前及术后2、4周检测血清C反应蛋白含量;术后4周行X线片及CT检查并行Norden影像学评分;术后4周处死动物取材后行标本大体观察及评分,并行HE染色观察及组织学评分。结果 B组因感染严重死亡5只动物,C组死亡2只,D、E、F组均无动物死亡。大体观察A组切口愈合良好,无软组织肿胀;B～D组大部分可见切口红肿及窦道形成,股骨增粗,骨质破坏,破坏程度随浓度降低而降低;E、F组切口可见感染征象,感染程度不及D组。细菌学观察示B、C、D、E组切口窦道形成动物均培养出阳性结果,随着浓度降低,切口窦道形成动物数目逐渐降低;F组无窦道形成遂未做细菌学培养。术前各组间C反应蛋白含量差异无统计学意义(P>0.05);术后2、4周B～F组C反应蛋白含量均显著高于A组(P<0.05);术后4周C反应蛋白含量B组>C组>D组>E组>F组>A组,各组间比较差异均有统计学意义(P<0.05)。影像学检测示A组无软组织肿胀及骨质破坏;B、C组可见骨质破坏,大量死骨形成,软组织肿胀严重;D、E组可见骨质破坏,死骨形成程度不及C组;F组可见少量骨感染。B～F组Norden评分显著高于A组,B、C组显著高于D、E、F组,D、E组显著高于F组(P<0.05);B、C组间及D、E组间比较差异无统计学意义(P>0.05)。B～F组标本大体观察评分显著高于A组,B、C组显著高于D、E、F组(P<0.05);D、E、F组间比较差异无统计学意义(P>0.05)。HE染色示A组骨小梁结构清晰,结构排列整齐;B～F组可见骨小梁破坏,炎性细胞浸润且程度逐渐降低。B～F组组织学评分显著高于A组,B组显著高于C～F组,C、D组显著高于F组(P<0.05);C、D、E组间以及E、F组间比较差异均无统计学意义(P>0.05)。结论采用MRSA制备兔股骨慢性骨髓炎模型的最佳菌液浓度为1×10~6～1×10~7 CFU/m L。

ObjectiveTo investigate the appropriate concentration of methicillin-resistant Staphylococcus aureus （MRSA） in establishing chronic femoral osteomyelitis model in rabbits.MethodsForty-eight adult New Zealand white rabbits were randomly divided into 6 groups with 8 rabbits in each group. Animals in groups B, C, D, E, and F were injected 1×109, 1×108, 1×107, 1×106, 1×105 CFU/mL MRSA on the location of 2 cm of the femoral supracondyle, respectively, and group A was injected with aseptic saline as a control. The general observation were performed at 4 weeks after operation, and the wound secretions were taken for bacteriological examination. The serum C-reactive protein content was detected at preoperation and 2 weeks and 4 weeks after operation. The X-ray, CT scan, and Norden imaging scoring were performed at 4 weeks after operation. At 4 weeks after operation, the animals were sacrificed, and the specimens were observed and evaluated by general scores; and the HE staining and histological score were also performed.ResultsFive rabbits died of severe infection in group B, 2 died in group C, and no rabbit died in groups D, E, and F. General observation showed that the incision healed without soft tissue swelling in group A; most animals had visible incision swelling and sinus formation, femoral thickening, bone destruction, and damage decreased with the decreasing of the concentration of liquid bacterial in groups B-D; the infection signs were seen in groups E and F, and the degree of infection were less than that of group D. Bacteriological examination showed that fistula formation animal in groups B, C, D, and E were cultured with positive results, and with the decrease of concentration, the number of animal fistula formation decreased gradually; and bacteriological culture did not be performed in group F because of no sinus formation. There was no significant difference in the content of C-reactive protein between groups before operation （P〉0.05）. The contents of C-reactive protein in groups B-F were significantly higher than those in group A at 2 and 4 weeks after operation （P〈0.05）. At 4 weeks after operation, the content of C-reactive protein was in the order of groups B, C, D, E, F, and A in turn from high to low, showing significant differences between groups （P〈0.05）. Imaging examination showed that there was no soft tissue swelling and bone destruction in group A; bone destruction, massive sequestrum formation, and soft tissue swelling were found in groups B and C; bone destruction was observed in groups D and E, and the degree of sequestrum formation was not as good as that in group C; and there was a small amount of bone infection in group F. The Norden scores in groups B-F were significantly higher than that in group A, and in groups B and C than those in groups D, E, and F, and in groups D and E than that in group F （P〈0.05）; there was no significant difference between groups B and C, and between groups D and E （P〉0.05）. The specimens general observation scores in groups B-F were significantly higher than that in group A, while in groups B and C than those in groups D, E, and F （P〈0.05）; there was no significant difference between groups D, E, and F （P〉0.05）. HE staining showed that the structure of bone trabecula in group A was clear and the structure was arranged neatly; in groups B-F, trabecular bone destruction and inflammatory cell infiltration were seen and the degree gradually decreased. The histological scores in groups B-F were significantly higher than that in group A, and in group B than those in groups C-F, in groups C and D than that in group F （P〈0.05）; there was no significant difference between groups C, D, and E, and between groups E and F （P〉0.05）.ConclusionThe optimal MRSA concentration of rabbit model of chronic osteomyelitis of femur is between 1×106 and 1×107 CFU/mL.