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Outcome of different sequencing and assembly approaches on the detection of plasmids and localization of antimicrobial resistance genes in commensal Escherichia coli

Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 43 Epidemiology, Zoonoses and Antimicrobial Resistance, Germany
Juraschek, Katharina;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Study Centre 4SZ - Genome Sequencing and Analysis Germany
Borowiak, Maria;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Study Centre 4SZ - Genome Sequencing and Analysis Germany
Tausch, Simon H.;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Study Centre 4SZ - Genome Sequencing and Analysis Germany
Malorny, Burkhard;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 43 Epidemiology, Zoonoses and Antimicrobial Resistance, Germany; University of Veterinary Medicine Vienna, Unit for Veterinary Public Health and Epidemiology, Austria
Käsbohrer, Annemarie;
Affiliation
Technical University of Denmark, DTU Food, National Food Institute, Denmark
Otani, Saria;
Affiliation
Freie Universität Berlin, Institute of Microbiology and Epizootics, Department of Veterinary Medicine, Germany
Schwarz, Stefan;
Affiliation
Freie Universität Berlin, Institute of Food Safety and Food Hygiene, Working Group Meat Hygiene, Department of Veterinary Medicine, Germany
Meemken, Diana;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Study Centre 4SZ - Genome Sequencing and Analysis Germany
Deneke, Carlus;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 43 Epidemiology, Zoonoses and Antimicrobial Resistance, Germany
Hammerl, Jens Andre

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 suitable techniques, standardized parameters and approved guidelines for 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 routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different 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 or Flye or a combination of both (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 to detect certain duplications and was unsuitable for genome finishing. Overall, the 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 assemblies currently represents the best approach for reliable AMR typing and risk assessment.

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