Investigating the sRNA and mRNA transcriptional response to antibiotics in methicillin-resistant Staphylococcus aureus using Illumina RNAseq. Staphylococcus aureus

Coordinated protein-coding sequence transcriptional responses of Staphylococcus aureus to antimicrobial exposure are well described but little is known of the role of bacterial non-coding, small RNAs (sRNAs) in these responses. Here we used RNAseq to investigate the sRNA response of the epidemic multiresistant hospital ST239 S. Aureus strain JKD6009 and its vancomycin-intermediate clinical derivative, JKD6008, after exposure to four antibiotics representing the major classes of antimicrobials used to treat methicillin-resistant S. Aureus infections. These agents included vancomycin, linezolid, ceftobiprole, and tigecycline. We identified 410 potential sRNAs (sRNAs) and then compared global sRNA and mRNA expression profiles at 2 and 6 hours, without antibiotic exposure, and after exposure to 0.5 x MIC for each antibiotic, for both JKD6009 (VSSA), and JKD6008 (VISA). Overall design: Two strains were used (JKD6009, vancomycin-susceptible S. Aureus; JKD6008, in vivo derived vancomycin-intermediate S. Aureus). The complete JKD6008 genome seqeuce was used as the reference. Two time points, 2 hours and 6 hours after culture in Mueller Hinton broth. Strains were exposed to no antibiotic, or 0.5 x MIC for 10 mins for the following antibiotics; vancomycin, linezolid, ceftobiprole, tigecycline. RNA isolation procedures enriched for mRNA or sRNA. The 40 cDNA libraries were sequenced using a whole flowcell (8 lanes) in an Illumina genome analyzer GAII for 36 cycles. Data was analyzed using the BioConductor package limma, and by applying non-negative matrix factorization to determine the impact of antibiotic exposure on the sRNA and mRNA transcriptional profiles.

金黄色葡萄球菌（Staphylococcus aureus）的编码蛋白序列在抗菌药物暴露下的协调转录应答已得到充分阐释，但细菌非编码小RNA（small RNAs, sRNAs）在这类应答中所发挥的作用却鲜为人知。本研究利用RNA测序（RNAseq）技术，针对用于治疗耐甲氧西林金黄色葡萄球菌感染的主要抗菌药物类别中的四种抗生素，探究了流行型多重耐药医院分离株ST239金黄色葡萄球菌JKD6009及其万古霉素中介的临床衍生株JKD6008的sRNA应答情况。这四种抗生素分别为万古霉素、利奈唑胺、头孢吡普（ceftobiprole）与替加环素。本研究共鉴定出410条潜在sRNA，随后分别在无抗生素暴露、以及分别暴露于每种抗生素0.5倍最低抑菌浓度（MIC）的条件下，于2小时和6小时两个时间点，比较了JKD6009（万古霉素敏感株，VSSA）与JKD6008（万古霉素中介株，VISA）的全局sRNA与mRNA表达谱。
总体实验设计：本研究使用两株金黄色葡萄球菌：JKD6009为万古霉素敏感株（VSSA），JKD6008为体内衍生的万古霉素中介株（VISA）。以完整的JKD6008基因组序列作为参考基因组。实验设置两个时间点：于穆勒-欣顿肉汤（Mueller Hinton broth）中培养后2小时与6小时。菌株分别接受无抗生素暴露处理，或暴露于以下四种抗生素的0.5倍MIC环境中10分钟：万古霉素、利奈唑胺、头孢吡普、替加环素。RNA分离流程针对mRNA或sRNA进行了富集。利用Illumina基因组分析仪GAII的整条流通池（8个泳道）对40个cDNA文库进行36个循环的测序。数据分析采用BioConductor工具包中的limma包，并通过非负矩阵分解（non-negative matrix factorization）以探究抗生素暴露对sRNA与mRNA转录谱的影响。