Evaluation of a Real-time Plasmid Transmission Detection Pipeline

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 (EWA). Clonal transmissions are detected using cgMLST allelic differences. The integrated tools pyGenomeViz and MUMmer were used for visual pairwise plasmid comparisons. We evaluated the pipeline using published hybrid assemblies (Oxford Nanopore Technology R9.4.1/Illumina) of 81 carbapenem-resistant isolates from Addenbrooke's Hospital. 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 size correction were tested and discrepancies between resulting clusters were analyzed with Quast. The SeqSphere + pipeline agreed with the published data and produced the same clonal (n=7) and carbapenemase-carrying plasmid (n=9) clusters. EWAs also were in the correct chronological order. A reduced sketch size of 1,000 led to one missing cluster but could be compensated using size correction. Among 44 potential transmission events, 12 were clonal, 18 single plasmid and 14 co-transmissions of two or more plasmids to the same recipient. 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 integrated into the GUI-based SeqSphere + platform.