摘要
目的对6株金黄色葡萄球菌（Staphylococcus aureus,S.aureus,简称金葡菌）菌株进行鉴定,为研制相关疫苗提供参考。方法将稀释的6株疫苗研制用备选金葡菌菌株49521、12602、49525、12605、55804、10832及质控菌株25923、12228菌悬液划线接种TSA平板及Columbia血平板,观察菌落形态,并涂片染色镜检;利用API Staph生化试剂盒、staphylase test血浆凝固酶检测试剂盒及30μg头孢西丁药敏纸片对菌株进行鉴定;利用PCR法鉴定菌株16S r RNA基因、荚膜基因型及mec A基因;用系列稀释的菌悬液经腹腔注射BALB/c小鼠,初步测定菌株毒力。结果6株金葡菌经平板培养,可见圆形、凸起、不透明、有光泽的菌落,Columbia血平板培养的菌落周围可见透明溶血环,镜检结果为革兰阳性球菌,呈单、双或葡萄串状排列。6株菌株的生化反应结果相同,且与质控菌株25923反应结果一致;6株菌株的凝固酶试验结果均为阳性,均对头孢西丁敏感,均未扩增出mec A基因片段,为甲氧西林敏感菌株（methicillin sensitive Staphylococcus aureus,MSSA）。6株菌株均扩增出1 544 bp的16S r RNA基因片段,与Gen Bank中的参考序列NR_075000.1的同源性均为99%;菌株49521、12605、55804、10832均扩增出340 bp的cap5基因片段,与Gen Bank中序列U81973.1的同源性为99%-100%;菌株12602、49525均扩增出171 bp的cap8基因片段,与Gen Bank中序列U73374.1的同源性均为100%。6株菌株的半数致死量（LD50）均在2.5×10^8-2.5×10^9 CFU之间。结论对6株疫苗研制用备选金葡菌进行了鉴定,为疫苗研制中菌株鉴定及不同荚膜型菌株的选择提供了参考。
Objective To identify six strains of Staphylococcus aureus（S. aureus）,and provide a reference for the development of relevant vaccines. Methods Six candidate S. aureus strains 49521,12602,49525,12605,55804 and10832 as well as quality control strains 25923 and 12228 were inoculated onto TSA plate and Columbia blood plate by streak plating,observed for the morphology of colonies,then smeared,stained and examined by microscopy. The strains were identified by API Staph kit,staphylase test kit and cefoxitinantimicrobial susceptibility test discs,of which the16 S r RNA gene,capsular genotype and mec A gene were analyzed by PCR. BALB / c mice were injected i.p. with serially diluted bacterial suspension to determine the virulence of strains. Results Six S. aureus strains cultured on plate were round and opaque colonies with processes and gloss. Transparent hemolysis rings were observed around the colonies on Columbia blood plate. Microscopy proved that the six strains were Gram positive cocci in scatter or in a chain. The results of biochemical reactions of six strains were identical,which were consistent with that of quality control strain25923. All the results of coagulase test of the six strains were positive. All the strains were sensitive to cefoxitin and methicillin sensitive S. aureus（MSSA）,in which no mec A genes were amplified. The 16 S r RNA fragments each at a length of 1 544 bp were amplified from all the strains,of which the homologies to reference NR_075000. 1 in Gen Bank were 99%. The cap5 gene fragment at a length of 340 bp was amplified from each of strains 49521,12605,55804 and10832,of which the homologies to that of U81973. 1 sequence in Gen Bank were 99% - 100%. The cap8 gene fragments at lengths of 171 bp were amplified from both strains 49525 and 12602,of which the homologies to U73374. 1 sequence in Gen Bank were 100%. The median lethal doses（LD50）of six strains were 2. 5 × 10^8 - 2. 5 × 10^9 CFU. Conclusion Six S. aureus strains as candidate strains were identified,which provided a reference for identification and selection of strains of various capsular genotypes in vaccine development.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第12期1534-1538,1542,共6页
Chinese Journal of Biologicals
关键词
金黄色葡萄球菌
荚膜基因型
毒力
Staphylococcus aureus
Capsular genotype
Virulence
