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目的通过全基因组测序研究利奈唑胺(LZD)敏感株和诱导耐药耐甲氧西林金黄色葡萄球菌(MRSA)之间基因变异位点差异,了解LZD耐药基因变异位点。方法采用不同浓度梯度的LZD对遗传背景清晰的MRSA-MS4母株进行诱导,获得LZD耐药菌株MRSA-MS4-LZD100,测定最低抑菌浓度(MIC),采用普通聚合酶链反应(PCR)扩增MRSA-MS4-LZD100 23S rRNA V区及核糖体蛋白L3/L4基因,测序后与野生株比较,获得相应的突变位点;采用Illumina HiSeq 2000测序技术对样品DNA进行paired-end(PE)测序,构建Illumina PE文库,利用生物信息学完成该菌株的全基因组测序。结果经过32代诱导,获得MRSA-MS4-LZD100菌株,其LZD MIC为96μg/mL。PCR测序分析提示其V区多拷贝基因均存在G2447T突变,L3蛋白存在Gly113Val突变;全基因组包含2 744 315 bp碱基对,注释后共有2 509个基因,11个tRNA编码基因,以及2个完整的rRNA基因编码操纵子,获得PubMed全基因序列号(JXMJ00000000);共找出101个SNP和6个Small indel突变,发生于外显子的SNP占16个,SNP后导致氨基酸序列改变的蛋白质包括IstB ATP结合区域包含蛋白、凝集因子A及转座子IS1272等,而发生于外显子的Small indel占3个,Small indel突变后导致氨基酸序列改变的蛋白质包括假设蛋白、30S核糖体蛋白S1、凝集因子A。结论经LZD诱导获得LZD耐药MRSA-MS4-LZD100菌株,测序分析提示该菌株存在除23S rRNA V区及L3蛋白基因以外的突变位点,为进一步研究隐性LZD耐药机制指明了方向。
OBJECTIVE: To investigate the differences of gene mutation loci between linezolid (LZD) sensitive strains and drug-resistant methicillin-resistant Staphylococcus aureus (MRSA) by genome-wide sequencing and understand the gene mutation sites of LZD resistance. Methods MRSA-MS4-LZD100, a strain of MRSA-MS4 with clear genetic background, was induced by LZD with different concentration gradients. The minimum inhibitory concentration (MIC) of MRSA-MS4-LZD100 was determined by ordinary polymerase chain reaction The MRSA-MS4-LZD100 23S rRNA V region and the ribosomal protein L3 / L4 gene were amplified and compared with the wild-type strain after sequencing to obtain the corresponding mutation sites. Paired-end (PE) sequencing was performed on samples using Illumina HiSeq 2000 sequencing , Constructed Illumina PE library, using bioinformatics complete genome-wide sequencing of the strain. Results After 32 generations of induction, the MRSA-MS4-LZD100 strain was obtained with a LZD MIC of 96 μg / mL. PCR analysis showed that there were G2447T mutation in the multi-copy gene of V region and Gly113Val mutation in L3 protein. The genome contained 2 744 315 bp base pairs, with 2 509 genes, 11 tRNA coding genes and 2 complete (JXMJ00000000). A total of 101 SNPs and 6 Small indel mutations were found. 16 SNPs occurred in exons. The SNPs resulted in amino acid sequence alterations including IstB ATP-binding region contains protein, agglutination factor A and transposon IS1272, while small indels that occur in exons make up three. Small indel mutations lead to amino acid sequence changes including hypothetical protein, 30S ribosomal protein S1, Agglutination factor A. Conclusion The LZD-resistant MRSA-MS4-LZD100 strain was induced by LZD. Sequencing analysis indicated that there were some mutation sites other than the 23S rRNA V and L3 proteins, which indicated the direction for further studying the mechanism of LZD resistance.