Scherff Natalie, Rothgänger Jörg, Weniger Thomas, Mellmann Alexander, Harmsen Dag
1Institute of Hygiene, University Hospital Münster, Münster, Germany.
Ridom GmbH, Münster, Germany.
Microbiol Spectr. 2024 Oct 29;12(12):e0210024. doi: 10.1128/spectrum.02100-24.
The spread of antimicrobial resistance among bacteria by horizontal plasmid transmissions poses a major challenge for clinical microbiology. Here, we evaluate a new real-time plasmid transmission detection pipeline implemented in the SeqSphere (Ridom GmbH, Münster, Germany) software. Within the pipeline, a local Mash plasmid database is created, and Mash searches with a distance threshold of 0.001 are used to trigger plasmid transmission early warning alerts (EWAs). Clonal transmissions are detected using core-genome multi-locus sequence typing allelic differences. The tools MOB-suite, NCBI AMRFinderPlus, CGE MobileElementFinder, pyGenomeViz, and MUMmer, integrated in SeqSphere+, are used to characterize plasmids and for visual pairwise plasmid comparisons, respectively. We evaluated the pipeline using published hybrid assemblies (Oxford Nanopore Technology/Illumina) of a surveillance and outbreak data set with plasmid transmissions. To emulate prospective usage, samples were imported in chronological order of sampling date. Different combinations of the user-adjustable parameters sketch size (1,000 vs 10,000) and plasmid size correction were tested, and discrepancies between resulting clusters were analyzed with Quast. When using a sketch size of 1,000 with size correction turned on, the SeqSphere pipeline agreed with the published data and produced the same clonal and carbapenemase-carrying plasmid clusters. EWAs were in the correct chronological order. In summary, the developed pipeline presented here is suitable for integration into clinical microbiology settings with limited bioinformatics knowledge due to its automated analyses and alert system, which are combined with the GUI-based SeqSphere platform. Thus, with its integrated sample database, (near) real-time plasmid transmission detection is within reach in bacterial routine-diagnostic settings when long-read sequencing is employed.
Plasmid-mediated spread of antimicrobial resistance is a major challenge for clinical microbiology, and monitoring of potential plasmid transmissions is essential to combat further dissemination. Whole-genome sequencing is often used to surveil nosocomial transmissions but usually limited to the detection of clonal transmissions (based on chromosomal markers). Recent advances in long-read sequencing technologies enable full reconstruction of plasmids and the detection of very similar plasmids, but so far, easy-to-use bioinformatic tools for this purpose have been missing. Here, we present an evaluation of an innovative real-time plasmid transmission detection pipeline. It is integrated into the GUI-based SeqSphere software, which already offers core-genome multi-locus sequence typing-based pathogen outbreak detection. It requires very limited bioinformatics knowledge, and its database, automated analyses, and alert system make it well suited for prospective clinical application.
细菌通过水平质粒传播导致的抗菌药物耐药性扩散给临床微生物学带来了重大挑战。在此，我们评估了SeqSphere（德国明斯特Ridom GmbH公司）软件中实施的一种新的实时质粒传播检测流程。在该流程中，创建了一个本地Mash质粒数据库，并使用距离阈值为0.001的Mash搜索来触发质粒传播早期预警警报（EWA）。使用核心基因组多位点序列分型等位基因差异来检测克隆传播。集成在SeqSphere+中的工具MOB-suite、NCBI AMRFinderPlus、CGE MobileElementFinder、pyGenomeViz和MUMmer分别用于表征质粒和进行可视化的成对质粒比较。我们使用已发表的具有质粒传播的监测和暴发数据集的混合组装（牛津纳米孔技术/Illumina）来评估该流程。为模拟前瞻性使用，按采样日期的时间顺序导入样本。测试了用户可调整参数草图大小（1000对10000）和质粒大小校正的不同组合，并使用Quast分析所得簇之间的差异。当使用草图大小为1000且开启大小校正时，SeqSphere流程与已发表的数据一致，并产生了相同的克隆和携带碳青霉烯酶的质粒簇。EWA按正确的时间顺序排列。总之，这里开发 的流程因其自动化分析和警报系统，结合基于图形用户界面（GUI）的SeqSphere平台，适合集成到生物信息学知识有限的临床微生物学环境中。因此，凭借其集成的样本数据库，当采用长读长测序时，在细菌常规诊断环境中实现（近）实时质粒传播检测是可行的。
质粒介导的抗菌药物耐药性传播是临床微生物学的一项重大挑战，监测潜在的质粒传播对于防止进一步传播至关重要。全基因组测序常用于监测医院内传播，但通常仅限于检测克隆传播（基于染色体标记）。长读长测序技术的最新进展能够完整重建质粒并检测非常相似的质粒，但到目前为止，缺少用于此目的的易于使用的生物信息学工具。在此，我们展示了对一种创新的实时质粒传播检测流程的评估。它集成到基于GUI的SeqSphere软件中，该软件已经提供基于核心基因组多位点序列分型的病原体暴发检测。它所需的生物信息学知识非常有限，其数据库、自动化分析和警报系统使其非常适合前瞻性临床应用。
