PDF(Pubmed)
Abstract：
Infection clusters of multidrug-resistant bacteria increase mortality and entail expensive infection control measures. Whereas whole-genome sequencing (WGS) is the current gold standard to confirm infection clusters, PCR-based assays targeting cluster-specific signatures, such as single nucleotide polymorphisms (SNPs) derived from WGS data, are more suitable to initially screen for cluster isolates within large sample sizes. Here, we evaluated four software tools (SeqSphere+, RUCS, Gegenees, and Find Differential Primers) regarding their efficiency to find SNPs within WGS data sets that were specific for two bacterial monospecies infection clusters but were absent from a WGS reference data set comprising several hundred diverse genotypes of the same bacterial species. Cluster-specific SNPs were subsequently used to establish a probe-based real-time PCR screening assay for in vitro differentiation between cluster and noncluster isolates. SeqSphere+ and RUCS found 2 and 24 SNPs for clusters 1 and 14 and 24 SNPs for cluster 2, respectively. However, some signatures detected by RUCS were not cluster specific. Interestingly, all SNPs identified by SeqSphere+ were also detected by RUCS. In contrast, analyses with the remaining tools either resulted in no SNPs (with Find Differential Primers) or failed (Gegenees). Design of six cluster-specific real-time PCR assays enabled reliable cluster screening in vitro. Our evaluation revealed that SeqSphere+ and RUCS identified cluster-specific SNPs that could be used for large-scale screening in surveillance samples via real-time PCR, thereby complementing WGS efforts. This faster and simplified approach for the surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians. IMPORTANCE Infection clusters of multidrug-resistant bacteria threaten medical facilities worldwide and cause immense health care costs. In recent years, whole-genome sequencing (WGS) has been increasingly applied to detect and to further control bacterial clusters. However, as WGS is still expensive and time-consuming, its exclusive application for screening and confirmation of bacterial infection clusters contributes to high costs and enhanced turnaround times, which many hospitals cannot afford. Therefore, there is need for alternative methods that can enable further surveillance of bacterial clusters that are initially detected by WGS in a faster and more cost-efficient way. Here, we established a system based on real-time PCR that enables rapid large-scale sample screening for bacterial cluster isolates within 7 days after the initial detection of an infection cluster, thereby complementing WGS efforts. This faster and simplified surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians.
摘要：
多重耐药细菌的感染集群会增加死亡率，并需要昂贵的感染控制措施。而全基因组测序（WGS）是目前确认感染簇的黄金标准，基于PCR的针对簇特异性特征的检测，例如来自WGS数据的单核苷酸多态性（SNP），更适合在大样本量内初步筛选集群分离株。这里,我们评估了四个软件工具(SeqSphere+，RUCS,Gegenees,和查找差异引物）关于它们在WGS数据集中发现SNP的效率，这些SNP对两个细菌单种感染簇具有特异性，但在包含相同细菌物种的数百种不同基因型的WGS参考数据集中不存在。随后使用簇特异性SNP来建立基于探针的实时PCR筛选测定,用于簇和非簇分离物之间的体外区分。SeqSphere+和RUCS在簇1中发现了2个和24个SNP，在簇2中发现了14个和24个SNP。然而,RUCS检测到的一些签名不是特定于群集的。有趣的是,SeqSphere+鉴定的所有SNP也被RUCS检测到。相比之下,用其余工具进行的分析要么没有导致SNP(用FindDifferentialPrimers)要么失败(Gegenees)。六个簇特异性实时PCR测定法的设计使得能够在体外进行可靠的簇筛选。我们的评估显示，SeqSphere+和RUCS确定了簇特异性SNP，可用于通过实时PCR在监测样本中进行大规模筛选。从而补充WGS的努力。这种更快，简化的细菌簇监测方法将改善感染控制措施，并增强对患者和医生的保护。重要性多重耐药细菌的感染集群威胁着全球的医疗设施，并造成巨大的医疗保健成本。近年来,全基因组测序（WGS）已越来越多地用于检测和进一步控制细菌簇。然而,由于WGS仍然昂贵且耗时，其用于筛选和确认细菌感染簇的独家应用导致高成本和增加的周转时间，许多医院负担不起。因此,需要能够以更快和更具成本效益的方式进一步监测最初由WGS检测的细菌簇的替代方法。这里,我们建立了一个基于实时PCR的系统，该系统可以在最初检测到感染簇之后的7天内快速大规模样品筛选细菌簇分离株，从而补充WGS的努力。这种更快和简化的细菌群监测将改善感染控制措施，并加强对患者和医生的保护。
