摘要
目的：对不完全溶血表型的金黄色葡萄球菌（金葡菌）的溶血素及毒力基因进行分析，研究该类金葡菌的生物学特点。方法连续收集2013年至2014年苏州大学附属第二医院住院患者各类样本分离的不完全溶血表型金葡菌50株，选取同期住院患者样本分离得到的完全溶血表型金葡菌作为对照，接种于四种不同商品化绵羊血琼脂平板培养，比较溶血表型。采用实时荧光定量PCR 技术分析标准菌株、完全和不完全溶血金葡菌四种溶血素基因（hla、hlb、hlc 和 hld）mRNA 的表达，采用2－ΔΔCt法计算其相对表达量，使用 t 检验比较各菌株溶血素基因 mRNA 相对表达量的差异。采用免疫印迹技术分析α溶血素的表达。应用微量肉汤稀释法检测不完全溶血表型金葡菌的耐药特性；运用 PCR 技术检测 mecA、pvl 和 tst 基因的携带情况。结果50株不完全溶血金葡菌在不同商品化血琼脂平板上均出现可稳定遗传的不完全溶血现象。以标准菌株四种溶血素 mRNA 表达量为1，不完全溶血表型菌株α、β、γ、δ溶血素 hla、hlb、hlc 和 hld mRNA 的相对表达量分别为0．02、7．51、0．06和0．12，二者相比差异具有统计学意义（t ＝8．46，－56．40，8．12和7．61，P 值均＜0．05），且α溶血素的表达量降低。药敏结果显示，50株不完全溶血表型菌株均为耐甲氧西林金黄色葡萄球菌（MRSA），仅3株传代后使替考拉宁和利奈唑胺的最低抑菌浓度发生改变，但均未影响药敏结果的判断。 mecA、pvl 和 tst 基因在不完全溶血表型金葡菌中均呈阳性。结论不完全溶血表型金葡菌是一类β溶血素高表达而α、γ、δ溶血素表达量下降的 MRSA，该类金葡菌携带毒力基因 pvl 和 tst，提示其潜在的毒力较强，应引起临床重视。
Objective To analyze hemolysin and virulence -related genes in incomplete hemolytic Staphylococcus aureus.Methods Fifty strains of incomplete hemolytic Staphylococcus aureus were isolated from patients admitted in the Second Affiliated Hospital of Soochow University during 2013 and 2014, and the isolates with complete hemolytic phenotype were also collected at the same period as the control strains . All the strains were inoculated and subcultured on four kinds of sheep blood agar plates supplied by different manufacturers to compare their hemolytic phenotype .The relative mRNA expressions of hemolysin genesnbsp;（hla, hlb, hlc, hld） in standard strain, complete and incomplete hemolytic phenotype strains were detected by real-time quantitative polymerase chain reaction （RT-qPCR）, and valued by 2 -△△Ct method.t test was used to compare mRNA expressions of hemolysin genes .Western blot was performed to analyze the expression of α-hemolysin.Antibiotic susceptibility test of incomplete hemolytic strains was performed using broth microdilution method.Resistant gene mecA and virulence genes pvl, tst were detected by PCR.Results The steady and hereditary incomplete hemolysis was observed in 50 strains of incomplete hemolytic Staphylococcus aureus on the sheep blood agar plates from different suppliers .Taking mRNA expression of hla, hlb, hlc, hld in standard strain as 1, the relative mRNA expressions of hemolysin genes in incomplete hemolytic strains were 0.02, 7.51, 0.06 and 0.12 respectively, there were statistical differences between standard strain and incomplete hemolytic strains （t =8.46, -56.40, 8.12 and 7.61, all P 〈0.05）.And the expression of α-hemolysin was decreased in incomplete hemolytic strains .All the strains were identified as methicillin resistant Staphylococcus aureus （MRSA）.Three strains exhibited different minimum inhibitory concentrations of teicoplanin and linezolid after subcultured , but the differences had no impact on the final results of antibiotic susceptibility test .
出处
《中华临床感染病杂志》
2016年第3期236-242,共7页
Chinese Journal of Clinical Infectious Diseases
基金
苏州市科技研究计划（SYS201551）
江苏省普通高校研究生实践创新计划项目（SJLX15_0580）
苏州大学大学生创新创业训练计划项目（2014xj056）
关键词
金黄色葡萄球菌
不完全溶血
溶血素
耐药
毒力
Staphylococcus aureus
Incomplete hemolysis
Hemolysin
Drug resistance
Virulence
