摘要
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。
ObjectiveTo understand genetic mutation sites in linezolid （LZD）sensitive and inducible resistant strains of methicillinresistant Staphylococcus aureus（MRSA） using wholegenome sequencing, and realize mutation sites of LZDresistant gene.MethodsMRSAMS4 with explicit genotype and wholegenome sequences was induced by LZD of different concentration gradients, LZDresistant strain MRSAMS4LZD100 was obtained, minimum inhibitory concentration（MIC） was detected, domain V of 23S rRNA and ribosomal proteins L3/L4 gene in MRSAMS4LZD100 were amplified by polymerase chain reaction （PCR）, the sequenced products obtained the corresponding mutation site in contrast with the wildtype strain; Illumina PE library was constructed through pairedend sequencing by Illumina HiSeq 2000 technique, and whole genome sequencing was completed based on bioinformatics.ResultsMRASMS4LZD100 strain was induced after 32 passages, MIC of LZD was 96 μg/mL. Sequencing of PCR products indicated the genetic variations were G2447T mutation in multiple copies of domain V of 23S rRNA gene, and Gly113Val mutation in L3 protein respectively; the whole genome of MRSAMS4LZD100 contained 2 744 315 bp, annotation of the whole genome found a total of 2 509 genes, 11 tRNAencoding genes and 2 entire rRNAencoding operons. The data were submitted to the PubMed, and the GeneBank accession number JXMJ00000000 was assigned; a total of 101 SNPs and 6 Small indels were found, 16 of 101SNP mutations occurred in exon, of which the variant proteins with anmino acid sequence alterations included IstB ATP binding domaincontaining protein, clumping factor A, IS1272 transposase and so on; 3 of 6 Small indel mutations occurred in exon, of which the variant proteins with anmino acid sequence alterations included hypothetical protein, 30S ribosomal protein S1, and clumping factor A.ConclusionLZDresistant strain MRSAMS4LZD100 was successfully induced by LZD; beside 23S rRNA V domain and ribosomal L3 protein, the other mutant site exist in this resistant strain, which provide some direction for subsequent study of recessive LZD resistance mechanism.
作者
姚伟明
陈重
蒲彰雅
王红燕
程航
李多云
郑金鑫
邓向斌
刘晓军
邓启文
余治健
YAO Wei-ming CHEN Zhong PU Zhang-ya WANG Hong-yan CHENG Hang LI Duo- yun ZHENG Jin-xin DENG Xiang-bin LIU Xiao-jun DENG Qi-wen YU Zhi-jian(De- partment of Infectious Diseases and Key Laboratory for Research on Endogenous Infections, Nanshan District People ＇ s Hospital of Shenzhen University, Shenzhen 518052, China)
出处
《中国感染控制杂志》
CAS
北大核心
2017年第1期1-5,共5页
Chinese Journal of Infection Control
基金
深圳市重点学科资助项目(201506093)
深圳市科技创新课题(No.JCYJ20150402152130173
No.JCYJ20150402152130167)
深圳市重点学科经费资助
深圳市南山区课题资助(No.2015019
2015022)
关键词
耐甲氧西林金黄色葡萄球菌
利奈唑胺
高通量测序
全基因组序列
methicillin-resistant Staphylococcusaureus
linezolid
high-throughput sequencing
complete genomesequence
