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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

张建 燕荣帅 杨子晨 石茜 李翔 毛彤春 张一鸣

张建, 燕荣帅, 杨子晨, 等. 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[J]. 中华烧伤与创面修复杂志, 2022, 38(2): 137-146. DOI: 10.3760/cma.j.cn501120-20211130-00400.
引用本文: 张建, 燕荣帅, 杨子晨, 等. 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[J]. 中华烧伤与创面修复杂志, 2022, 38(2): 137-146. DOI: 10.3760/cma.j.cn501120-20211130-00400.
Zhang J,Yan RS,Yang ZC,et al.Analysis of genomic information and biological characteristics of a bacteriophage against methicillin-resistant Staphylococcus aureus in patients with median sternal incision infection[J].Chin J Burns Wounds,2022,38(2):137-146.DOI: 10.3760/cma.j.cn501120-20211130-00400.
Citation: Zhang J,Yan RS,Yang ZC,et al.Analysis of genomic information and biological characteristics of a bacteriophage against methicillin-resistant Staphylococcus aureus in patients with median sternal incision infection[J].Chin J Burns Wounds,2022,38(2):137-146.DOI: 10.3760/cma.j.cn501120-20211130-00400.

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

doi: 10.3760/cma.j.cn501120-20211130-00400
基金项目: 

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

详细信息
    通讯作者:

    张一鸣,Email:zhangyiming@tmmu.edu.cn

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

Funds: 

Youth Science Foundation Project of National Natural Science Foundation of China 82002051

General Program of Natural Science Foundation of Chongqing of China cstc2021jcyj-msxm0655

More Information
    Corresponding author: Zhang Yiming, Email: zhangyiming@tmmu.edu.cn
  • 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10.000 0、1.000 0、0.100 0、0.010 0、0.001 0、0.000 1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151 618 bp的线状双链DNA,序列两端有11 681 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,未预测出毒力因子或抗性基因,注释功能的噬菌体SAP23基因可分为5个组,GenBank登录号为MZ427930,噬菌体SAP23全基因组序列与共线性分析中的6个葡萄球菌噬菌体全基因组序列有5个局部共线区域,但在局部共线区域内部或外部存在差异。噬菌体SAP23属于HerelleviridaeTwortvirinae亚科Kayvirus病毒属。噬菌体SAP23的最佳感染复数为0.010 0,最佳吸附时间为10 min,潜伏期约为20 min,裂解期约为80 min;在4~37 ℃温度条件及pH值为4~9的条件中,稳定性较好。噬菌体SAP23可裂解41株MRSA中的3株。  结论  噬菌体SAP23为HerelleviridaeTwortvirinae亚科Kayvirus病毒属成员,潜伏期短,其对温度和酸碱耐受性好,可有效裂解MRSA,为不含毒力因子和抗性基因的新型烈性窄谱噬菌体。

     

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  • 1  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23及噬菌斑形态。1A.噬菌体SAP23形成透明噬菌斑 图中标尺为10 mm;1B.噬菌体SAP23头部为多面体,并有尾部 透射电子显微镜×40 000,图中标尺为40 nm

    2  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23基因注释记录图

    注:每个箭头代表1个开放阅读框,不同颜色代表编码蛋白不同功能分类;GC为鸟嘌呤胞嘧啶

    3  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23基因组与6株葡萄球菌噬菌体基因组的全基因组共线性记录图

    注:1.噬菌体SAP23,2.葡萄球菌噬菌体P108,3.葡萄球菌噬菌体812,4.葡萄球菌噬菌体VB_SavM_JYL02,5.葡萄球菌噬菌体VB_SavM_JYL01,6.葡萄球菌噬菌体vB_SauH_IME522,7.葡萄球菌噬菌体vB_ScoM-PSC1;5种颜色块代表5个局部共线区域;局部共线区域内部或外部的空白区域代表基因组之间的差异区域

    4  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23系统发生树记录图。4A.主要大衣壳蛋白氨基酸序列系统发生树;4B.全基因组序列系统发生树

    注:系统发生树中括号中编号为GenBank登录号,左侧分支数据代表可信度

    5  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23与宿主菌共培养不同时间点的噬菌体效价(x¯±s,样本数为3)

    注:PFU为菌斑形成单位;该图为经过lg处理的数据形成的描记图;坐标轴数据为未经lg处理的原始数据

    6  从1例胸骨正中切口感染患者创面标本中分离的耐甲氧西林金黄色葡萄球菌噬菌体SAP23在不同温度和pH值条件下的稳定性(x¯±s,样本数均为3)。6A.不同温度条件孵育1 h后噬菌体效价;6B.不同pH值条件下孵育1 h后噬菌体效价

    注:PFU为菌斑形成单位;图6B为经过lg处理的数据形成的描记图,坐标轴数据为未经lg处理的原始数据

    表1  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SAP23对41株耐甲氧西林金黄色葡萄球菌裂解情况

    菌株编号噬菌斑菌株编号噬菌斑菌株编号噬菌斑菌株编号噬菌斑
    DP100-CY6++NF99-G15-
    CY15-NF7-SY14-G26-
    CY14-NF70-SY19-G25-
    CY19-NF71-SY33-G31-
    CY18-NF73-SY17++G34-
    CY5-NF75-SY5-G13-
    CY11-NF8-SY13-G21-
    CY16-NF84-SY23-G17-
    CY7-NF88-SY6-G36-
    CY8-NF90+SY15-G16-
    CY20-
    注:“++”表示可见透明噬菌斑,“+”表示可见混浊噬菌斑,“-”表示未见噬菌斑
    下载: 导出CSV
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  • 收稿日期:  2021-11-30

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