质粒及限制酶分析和外膜蛋白测定对新生儿血培养不动杆菌阳性的研究

Study on amotile bacteria of positive blood culture in new-born:the analysis of plasmid and restriction enzyme and determination of outer membrane protein

研究目的探讨新生儿血培养不动杆菌高阳性率的原因及败血症的诊断。研究方法疑诊新生儿败血症者，在不同部位取双份血作细菌培养，用K—B纸片扩散法做药敏试验。用改良Birnboim法快速提取质粒，并进行质粒分析，用HindⅢ和EcoRⅠ两种限制酶对35株流行株的质粒DNA进行酶切。用SDS—聚丙烯酰胺梯度凝胶电泳法测定外膜蛋白（OMP）。结果血培养不动杆菌阳性患儿51例，而污染者高达35例（68．6％），败血症仅16例（31．4％）。54．8％（57／104）菌株对12种以上药物耐药。189株细菌中165株（87．3％）含质粒，经HindⅢ酶切为6个DNA片段（1．9、2、4、5、8．5、18Kb），经EcoRⅠ酶切为6个DNAH段（2、2．6、3．2、6．3、22Kb），表明来自同一克隆。流行株含10条主要OMP，而非流行株含11条OMP，多1条25Kd带。具有上述质粒谱、限制酶谱和OMP谱的不动杆菌仅见于空气及治疗盘针头。结论新生儿血培养不动杆菌高阳性率的主要原因是污染。确诊败血症必须通过细菌培养、质粒分析、质粒DNA限制酶分析及OMP测定并结合病史、临床表现。

Objective To search for the reasons of high positive rate of amotile bacteria and the diagnosis of septicemia in new-born Methods The blood was drawn from the different site of the new-born with septicemia and carricd out blood culture. The drug sensitivity test had been done by the method of paper stripdiffusion. The plasmids of bacteria were extracted rapidly by medified Birnboim method and the plasmid analyss was carried out. The plasmids's DNA of 35 epidemic strain was cut off by both restriction enzyme of Hind Ⅲ and EcoR Ⅰ. The outer membrane protein (OMP) was determined by SDS-polyacrylamide gel electrophoresis.Results There are 51 patients with positive blood culture amotile bacterium,of them, pollution; 35 cases (68．6%), septicemia: only 16 cases (31．4%),54．8% (57/104) strains bacteria have drug resistance to more of 12 drugs. 87．3% (165/189) strains bacteria have plasmids. They are cut off as 6 DNA fragments (1．9,2,4,5, 8．5 and 18Kb) by Hind Ⅲ restrietion enzyme. and as 5 DNA fragments (2,2．6,3．2, 6．3 and 22 Kb) by EcoR Ⅰrestrietion enzyme, it is showed that they come from a same clone. The epidemic strain include 10 slips OMP, but non-epidemic strain have 11 slip OMP, increase a 25Kd belt. The amotile bacteria with above-mentioned plasmid spectrum, restriction enzyme spectrum and OMP spectrum are only seen in the air, therapeutic dish and syringe needle.Conclusion The pollution is an important reason of amotile bactorium high positiye rate in new-born.Diagnosing septicemia should depend on bacteria culture, plasmid analysis restriction enzyme analysis of plasmid DNA, oMP determination and combining medical history and clinical manifestation.