A neonatal MRSA outbreak investigation using rapid whole genome sequencing

Background: Isolates of methicillin-resistant Staphylococcus aureus (MRSA) belonging to a single lineage are often indistinguishable using current typing techniques. Whole genome sequencing (WGS) may provide improved resolution to define transmission pathways and characterize outbreaks. Methods: We investigated a putative MRSA outbreak on a neonatal intensive care unit. Using rapid high-throughput sequencing technology with a clinically relevant turnaround time, we retrospectively sequenced the DNA from 7 isolates associated with the outbreak, and another 7 MRSA isolates associated with carriage or bacteremia in the same hospital. Results: A phylogenetic tree was constructed by comparing single nucleotide polymorphisms in the core genome to a reference genome (EMRSA-15, sequence type 22). This demonstrated a distinct clustering of the outbreak isolates, and clear separation between these and the non-outbreak isolates. A previously missed transmission event was detected between two patients with bacteremia that were not part of the outbreak. We created an artificial ‘resistome’ of antibiotic resistance genes and demonstrated concordance between this and phenotypic susceptibility testing, as well as a ‘toxome’ consisting of toxin genes. One outbreak isolate was a hypermutator with a comparatively higher number of SNPs, highlighting the difficulty in imposing a simple cut-off of SNPs between isolates to decide whether they are part of a recent transmission chain. Conclusions: WGS can provide clinically relevant data within a timescale that can influence patient management. Automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation.

背景：隶属于同一进化分支的耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）分离株，在当前常用的分型技术下往往难以区分。全基因组测序（whole genome sequencing, WGS）可提供更高的分辨能力，以明确传播路径并解析暴发疫情。
方法：本研究针对某新生儿重症监护病房内一起疑似MRSA暴发疫情展开调查。我们采用具备临床相关检测周转时长的快速高通量测序技术，对与该暴发相关的7株MRSA分离株，以及同医院内另外7株与定植或菌血症相关的MRSA分离株进行了回顾性DNA测序。
结果：以参考基因组EMRSA-15（序列型22）为参照，通过比对核心基因组中的单核苷酸多态性（single nucleotide polymorphism, SNP）构建了系统发育树。结果显示，暴发相关分离株呈现出显著的聚类特征，且与非暴发相关分离株界限清晰。研究还发现了此前未被察觉的两例菌血症患者间的传播事件，这两名患者均不属于本次暴发队列。我们构建了人工耐药基因组（resistome）集合，涵盖各类抗生素耐药基因，证实其与表型药敏试验结果具有一致性；同时构建了包含毒素基因的毒素基因组（toxome）。另有1株暴发相关分离株为高突变株，其单核苷酸多态性数量相对更高，这表明仅通过设定分离株间的简单SNP截断值来判断是否属于近期传播链存在难度。
结论：全基因组测序可在可影响患者诊疗的时限内提供具有临床价值的数据。自动化数据解读以及出具具有临床意义的报告，仍是其临床应用面临的障碍。