胸骨正中切口感染患者耐甲氧西林金黄色葡萄球菌噬菌体的基因组学信息及生物学特性分析  

Analysis of genomic information and biological characteristics of a bacteriophage against methicillin-resistant Staphylococcus aureus in patients with median sternal incision infection

作　　者：张建 燕荣帅 杨子晨 石茜 李翔 毛彤春 张一鸣 Zhang Jian;Yan Rongshuai;Yang Zichen;Shi Xi;Li Xiang;Mao Tongchun;Zhang Yiming(Department of Plastic and Cosmetic Surgery,the Second Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400037,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院整形美容外科,重庆400037

出　　处：《中华烧伤与创面修复杂志》2022年第2期137-146,共10页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金青年科学基金项目(82002051);重庆市自然科学基金面上项目(cstc2021jcyj-msxm0655)。

摘　　要：目的分离提纯1株新型耐甲氧西林金黄色葡萄球菌(MRSA)的噬菌体,并对其基因组学信息和生物学特性进行分析。方法采用实验研究方法。取分离自陆军军医大学(第三军医大学)第二附属医院收治的1例胸骨正中切口感染的63岁女性患者创面的MRSA(下称宿主菌)液,采用污水共培养法和双层琼脂平板法从该院污水中分离提纯得到噬菌体,并命名为噬菌体SAP23,观察噬菌斑形态。采用磷钨酸负染法将噬菌体SAP23染色,采用透射电子显微镜观察其形态。采用十二烷基磺酸钠/蛋白酶裂解方案制备噬菌体SAP23 DNA,在Illumina NovaSeq PE150平台下进行全基因组测序,并完成序列组装、注释、系统发生树等基因组学分析。将噬菌体SAP23液分别按10.0000、1.0000、0.1000、0.0100、0.0010、0.0001感染复数与宿主菌液共培养4 h后,采用点滴法测定噬菌体效价,筛选最佳感染复数,此处及以下样本数均为3。按测得的最佳感染复数取噬菌体SAP23液与宿主菌液分别共同孵育5、10、15 min后,同前测定噬菌体效价,筛选最佳吸附时间。按测得的最佳感染复数取噬菌体SAP23液与宿主菌液按最佳吸附时间孵育后,分别于培养0(即刻)、5、10、15、20、30、40、50、60、80、100、120 min,同前测定噬菌体效价,绘制一步生长曲线。取噬菌体SAP23液分别在温度为4、37、50、60、70、80℃下,在pH值为2、3、4、5、6、7、8、9、10、11、12下孵育1 h,测定稳定性。取陆军军医大学(第三军医大学)微生物教研室储存的41株MRSA,完成噬菌体SAP23的宿主谱范围检测。结果噬菌体SAP23能在宿主菌双层琼脂板上形成透明噬菌斑。噬菌体SAP23头部是直径为(88±4)nm的多面体,其尾部长度为(279±21)nm、宽度为(22.6±2.6)nm。噬菌体SAP23基因组为全长151618 bp的线状双链DNA,序列两端有11681 bp的长末端重复序列,预测出220个开放阅读框,噬菌体可编码4个转运RNA,未预测出毒�Objective To isolate and purify a bacteriophage against methicillin-resistant Staphylococcus aureus(MRSA),and to analyze its genomic information and biological characteristics.Methods The experimental research methods were adopted.MRSA(hereinafter referred to as host bacteria)solution was collected from the wound of a 63-year-old female patient with the median sternum incision infection admitted to the Second Affiliated Hospital of Army Medical University(the Third Military Medical University).The bacteriophage,named bacteriophage SAP23 was isolated and purified from the sewage of the Hospital by sewage co-culture method and double-layer agar plate method,and the plaque morphology was observed.The morphology of bacteriophage SAP23 was observed by transmission electron microscope after phosphotungstic acid negative staining.The whole genome of bacteriophage SAP23 was sequenced with NovaSeq PE15 platform after its DNA was prepared by sodium dodecyl sulfonate/protease cleavage scheme,and genomic analysis including sequence assembly,annotation,and phylogenetic tree were completed.The bacteriophage SAP23 solution was co-incubated with the host bacterial solution for 4 h at the multiplicity of infection(MOI)of 10.0000,1.0000,0.1000,0.0100,0.0010,and 0.0001,respectively,and then the bacteriophage titer was measured by the drip plate method to select the optimal MOI,with here and the following sample numbers of 3.The bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for 5,10,and 15 min,respectively,and the bacteriophage titer was measured by the same method as mentioned above to select the optimal adsorption time.After the bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for the optimal adsorption time,the bacteriophage titers were measured by the same method as mentioned above at 0(immediately),5,10,15,20,30,40,50,60,80,100,and 120 min after culture,respectively,and a one-step growth curve was drawn.The bacteriophage SAP23 s

关 键 词：耐甲氧西林金黄色葡萄球菌 细菌噬菌体 基因组学 伤口感染 生物学特性 

分 类 号：R632[医药卫生—外科学]