Rocío Arazo del Pino (Köln / DE), Tessa Burgwinkel (Köln / DE), Linda Casselman (Köln / DE), Samuel Chorlton (Vancouver / CA), Vivien Persy (Köln / DE), Janine Zweigner (Köln / DE), Paul G. Higgins (Köln / DE), Kyriaki Xanthopoulou (Köln / DE)
Introduction
Based on data from the National Reference Center for Gram-negative bacteria, the prevalence of NDM-5 carbapenemase in Germany has increased in recent years. In the present study, we investigated the clonality and genetic background of NDM-5 positive Escherichia coli isolates within a German hospital.
Materials
Four isolates obtained from four patients between February and September 2023 were subjected to whole-genome sequencing (WGS) using the MiSeq (Ilumina) and MinION (Nanopore). Species identification, core-genome MLST (cgMLST), 7-loci MLST, resistome, and plasmidome analysis relied on assemblies generated using Unicycler (Ubuntu) and BugSeq (https://bugseq.com/), an automated bioinformatics platform. Furthermore, plasmids were annotated using BAKTA and compared by BRIG.
Results
The four E. coli isolates (AML1585, AML1672, AML1679, and AML1698) were tested carbapenem-resistant using VITEK (bioMérieux) by the routine diagnostic laboratory. The isolates differed by up to 2302 alleles based on cgMLST and were therefore considered unrelated. However, all isolates shared antimicrobial resistance genes, i.e., blaNDM-5, aadA2, dfrA12, and sul1, which were encoded on multireplicon plasmids, containing the IncFll, IncFIB and IncFIA replicons (Table 1). The BugSeq platform grouped pAML1585 and pAML1679 as closely related (plasmid cluster A). Plasmid cluster A was confirmed using BRIG. Furthermore, plasmid analysis confirmed that pAML1585 and pAML1679 shared not only the plasmid backbone but also antimicrobial resistance genes including the NDM-5 suggesting a mobile genetic element transfer between the two isolates. The NDM-5 encoding plasmids pAML1672 and pAML1698 were unrelated to plasmid cluster A and were considered as unique.
Summary
Our findings revealed the presence of the carbapenemase NDM-5 encoded on a plasmid in four unrelated E. coli isolates. Two NDM-5-encoding plasmids formed a cluster confirmed by the BugSeq platform and our in-house workflow. These data underline the crucial contribution of WGS in tracking plasmid spread as well as a user-friendly application of bioinformatics pipelines for analysing bacterial genomes and plasmids.