线性质粒pBSSB1 LP002基因缺陷对伤寒沙门菌致病性的影响  

Influence of linear plasmid pBSSB1 gene LP002 on the pathogenicity of Salmonella enterica serovar Typhi

作　　者：张绮思 邓予晖 张珂 王倩 董阳阳 李涛 ZHANG Qisi;DENG Yuhui;ZHANG Ke;WANG Qian;DONG Yangyang;LI Tao(Department of Laboratory Medicine,Fuwai Central China Cardiovascular Hospital,Zhengzhou,Henan 451464,China)

机构地区：[1]阜外华中心血管病医院医学检验科,河南郑州451464

出　　处：《中华实用诊断与治疗杂志》2024年第8期779-785,共7页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20230133);河南省卫生健康中青年学科带头人培养项目(HNSWJW-2022010);河南省中青年卫生健康科技创新优秀青年人才培养项目(YXKC2022043)。

摘　　要：目的构建伤寒沙门菌线性质粒pBSSB1 LP002基因缺陷株(ΔLP002),探讨LP002基因在伤寒沙门菌致病中的作用。方法采用自杀质粒介导的同源重组法制备伤寒沙门菌ΔLP002。2种菌株分别培养12 h,每隔1 h应用分光光度计检测波长600 nm处吸光度(OD_(600))值并绘制生长曲线,采用实时荧光定量PCR法检测野生株培养至OD_(600)=0.2、0.4、0.6、0.8、1.2、1.8时LP002 mRNA相对表达量,并选择合适生长期的ΔLP002和伤寒沙门菌野生株进行表型实验:采用细菌动力实验检测动力圈直径,采用生物膜形成实验检测生物膜形成量;氨苄西林、庆大霉素处理后检测活菌菌落数并绘制生存曲线,观察有无滞留菌形成,计算抗生素处理5 h时滞留率、抑菌圈直径;检测对HeLa细胞的侵袭能力及在巨噬细胞内存活能力,计算侵袭指数、增殖指数。结果经PCR筛选验证,野生株基因组扩增产物长度为1450 bp,ΔLP002基因组扩增产物长度为888 bp,敲除长度为562 bp,表明ΔLP002制备成功。培养1、2、3、4、5、6、7、8、9、10、11、12 h时,ΔLP002 OD_(600)值与野生株比较差异均无统计学意义(t=1.385、1.576、1.895、0.351、0.833、0.195、0.213、0.926、0.024、0.011、0.116、0.063,P均>0.05)。ΔLP002动力圈直径[(35.67±1.15)mm]、生物膜形成量(0.29±0.04)与野生株[(35.00±1.00)mm、0.36±0.04]比较差异均无统计学意义(t=0.756,P=0.492;t=2.196,P=0.093)。生存曲线显示,ΔLP002、野生株均存在滞留现象;ΔLP002经氨苄西林、庆大霉素处理5 h时滞留率[(0.300±0.200)%、(0.022±0.022)%]与野生株[(0.513±0.252)%、(0.063±0.044)%]比较差异均无统计学意义(t=1.143,P=0.317;t=1.464,P=0.217)。经氨苄西林、庆大霉素处理前、后ΔLP002与野生株抑菌圈直径比较差异均无统计学意义(P>0.05);ΔLP002、野生株经氨苄西林、庆大霉素处理后抑菌圈直径与处理前比较差异均无统计学意义(P>0.05)。ΔLP002侵袭指数[(28.65±4.73)%]、增殖Objective To construct a mutant strain of linear plasmid pBSSB1 gene LP002(ΔLP002)of Salmonella enterica serovar Typhi(S.Typhi)and to investigate the role of LP002 in the pathogenicity of S.Typhi.Methods The homologous recombination mediated by suicide plasmid was used to constructΔLP002.Two strains were cultured separately for 12 h.The values of optical density at 600 nm(OD_(600))were detected every hour,and the growth curves were plotted using spectrophotometer.The relative expression of LP002 mRNA was detected by real-time fluorescence quantitative PCR when the wild-type strain was cultured to OD_(600)=0.2,0.4,0.6,0.8,1.2,and 1.8.respectively.The appropriate growth stages ofΔLP002 and wild-type strain were selected for phenotype experiments.Bacterial motility experiments were used to detect the diameters of the motility circles,and biofilm formation experiment was done to detect the amount of biofilm.After treatment with ampicillin and gentamicin,the number of viable bacterial colonies was detected,and the survival curves were plotted to observe the formation of persister.The persistent rates and the diameters of bacteriostatic circles were calculated after 5 h of antibiotic treatment.The invasion ability of HeLa cells and their viability in macrophages were detected to calculate the invasion index and proliferation index.Results After PCR verification,the amplification production of the wild strain genome was 1450 bp,the length of amplification production ofΔLP002 genome was 888 bp,and the knockout length was 562 bp,which indicated thatΔLP002 was successfully constructed.At 1,2,3,4,5,6,7,8,9,10,11 and 12 h after culture,the OD_(600)values showed no significant differences betweenΔLP002 and the wild strain(t=1.385,1.576,1.895,0.351,0.833,0.195,0.213,0.926,0.024,0.011,0.116,0.063;all P values>0.05).There were no significant differences in the diameters of bacteriostatic circles and biofilm formation betweenΔLP002[(35.67±1.15)mm,0.29±0.04]and the wild strain[(35.00±1.00)mm,0.36±0.04](t=0.756,P=0.492;t

关 键 词：伤寒沙门菌 线性质粒 pBSSB1 LP002 侵袭 巨噬细胞 

分 类 号：R378[医药卫生—病原生物学]