Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli

Antimicrobial resistance (AMR) is a major threat to public health, worldwide. Currently, AMR-typing changes from phenotypic-testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g. plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suited techniques, standardized parameters and approved guidelines for a reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routine of the German National Reference Laboratory for Antimicrobial Resistances. To assess the different outcomes of the approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E. coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long read sequencing were assembled individually by unicycler, flye and in combination (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g. qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed for the detection of certain duplications and was unsuited for genome finishing. Overall, hybrid assembly led to the most comprehensive typing results especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assembly currently represents the best approach for reliable AMR typing and risk assessment