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    CRISPR/Cas系统对金黄色葡萄球菌耐药及毒力基因的影响

    Effect of CRISPR/Cas system on drug resistance and virulence genes of Staphylococcus aureus

      摘要
    • 摘要:
      目的  了解金黄色葡萄球菌(以下简称“金葡菌”) Staphylococcus aureus中成簇的规律间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats，CRISPR)的分布情况，分析其对抗生素耐药基因和毒力基因水平转移的影响。
      方法  从公共数据库获取组装完整的金葡菌基因组575个，利用生物信息学方法，统计CRISPR结构的携带情况，菌株多位点序列分型(Multi-locus sequence typing，MLST)型别分布和菌株耐药基因、毒力基因的分布情况；对CRISPR结构阳性(CRISPR+)和CRISPR结构阴性(CRISPR−)的金葡菌耐药基因和毒力基因携带数目进行差异显著性分析。同时对实验室60株金葡菌二代测序数据进行分析，验证公共数据库分析结果。对实验室60株金葡菌中原噬菌体、接合质粒的携带情况进行统计，讨论CRISPR结构对菌株原噬菌体和接合质粒的影响。
      结果  基因组组装完整的575株金葡菌中，有62株携带CRISPR结构(CRISPR+)，513株不携带CRISPR结构(CRISPR−)；CRISPR+金葡球菌携带耐药基因、毒力基因的数目显著小于CRISPR− 金葡菌。实验室60株金葡菌中，有14株为CRISPR+，46株为CRISPR−；CRISPR+金葡菌携带更少的耐药基因和毒力基因，与公共数据库分析结果一致。对原噬菌体和接合质粒的分析结果显示，CRISPR−菌株携带更多的原噬菌体序列，与CRISPR+菌株之间差异显著(P<0.05)；接合质粒方面，CRISPR−和CRISPR+菌株无显著性差异。
      结论  CRISPR结构可能限制了金葡菌中耐药基因和毒力基因的水平转移，CRISPR−菌株更容易受到噬菌体和可移动质粒的干扰。本研究为进一步研究金葡菌耐药基因和毒力基因的传播提供了参考。

       

      Abstract:
      Objective  To understand the distribution of clustered regularly interspaced short palindromic repeats (CRISPR) in Staphylococcus aureus and analyze their effects on the horizontal transfer of antibiotic resistance gene and virulence gene.
      Method  Total 575 complete S. aureus genomes were obtained from public databases, and bioinformatics methods were used to count the CRISPR carriage, multi-locus sequence typing (MLST) types distribution of strains and the distribution of strain drug resistance genes and virulence genes. The significance analysis of the difference in the number of drug resistance genes and virulence genes was carried out between CRISPR structure-positive (CRISPR+) and CRISPR structure-negative (CRISPR−)S. aureus. The data of 60 strains of S. aureus were also analyzed by second-generation sequencing to verify the results of public database analysis. We also counted the carriage of prophages and conjugative plasmids in 60 strains of S. aureus in the laboratory, and discussed the effect of CRISPR structure on the prophages and conjugative plasmids of the strains.
      Result  Among the 575 strains with complete genome assembly, there were 62 strains with CRISPR structure (CRISPR+) and 513 strains without (CRISPR−). The number of drug resistance genes and virulence genes of CRISPR+S. aureus was less than that of CRISPR−S. aureus, and the difference was significant. Among 60 strains of S. aureus in the laboratory, there were 14 strains of CRISPR+ and 46 strains of CRISPR−. CRISPR+S. aureus carried fewer drug resistance genes and virulence genes, which was consistent with the results of the public database analysis. Analysis of prophages and conjugative plasmids showed that CRISPR− strains carried more prophage sequences, which were significantly different from CRISPR+ strains (P<0.05). For conjugative plasmids, CRISPR− and CRISPR+ strains were largely consistent with no significant difference.
      Conclusion  CRISPR structure may limit the horizontal transfer of drug resistance and virulence genes in S. aureus, and CRISPR− strains are more susceptible to be interfered by phage and removable plasmids. This study provides a reference for further research on transmission of drug resistance and virulence genes inS. aureus.

       

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