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Article

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

1
Epidemiology, Zoonoses and Antimicrobial Resistance, German Federal Institute for Risk Assessment (BfR), Max-Dohrn Str. 8-10, 10589 Berlin, Germany
2
Study Centre for Genome Sequencing and Analysis, German Federal Institute for Risk Assessment (BfR), Max-Dohrn Str. 8-10, 10589 Berlin, Germany
3
Unit for Veterinary Public Health and Epidemiology, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria
4
DTU Food, National Food Institute, Technical University of Denmark, Kemitorvet, Building 204, 2800 Kgs Lyngby, Denmark
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Institute of Microbiology and Epizootics, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
6
Institute of Food Safety and Food Hygiene, Working Group Meat Hygiene, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
*
Authors to whom correspondence should be addressed.
Shared senior researcher authorship.
Academic Editor: Jorge Blanco
Microorganisms 2021, 9(3), 598; https://doi.org/10.3390/microorganisms9030598
Received: 20 February 2021 / Revised: 11 March 2021 / Accepted: 12 March 2021 / Published: 14 March 2021
(This article belongs to the Special Issue Antimicrobial Resistance and Molecular Tracing of Foodborne Pathogens)
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. View Full-Text
Keywords: AMR; mobile genetic elements; qnrS; hybrid assembly; long-read sequencing; short-read sequencing AMR; mobile genetic elements; qnrS; hybrid assembly; long-read sequencing; short-read sequencing
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MDPI and ACS Style

Juraschek, K.; Borowiak, M.; Tausch, S.H.; Malorny, B.; Käsbohrer, A.; Otani, S.; Schwarz, S.; Meemken, D.; Deneke, C.; Hammerl, J.A. Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli. Microorganisms 2021, 9, 598. https://doi.org/10.3390/microorganisms9030598

AMA Style

Juraschek K, Borowiak M, Tausch SH, Malorny B, Käsbohrer A, Otani S, Schwarz S, Meemken D, Deneke C, Hammerl JA. Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli. Microorganisms. 2021; 9(3):598. https://doi.org/10.3390/microorganisms9030598

Chicago/Turabian Style

Juraschek, Katharina, Maria Borowiak, Simon H. Tausch, Burkhard Malorny, Annemarie Käsbohrer, Saria Otani, Stefan Schwarz, Diana Meemken, Carlus Deneke, and Jens A. Hammerl. 2021. "Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli" Microorganisms 9, no. 3: 598. https://doi.org/10.3390/microorganisms9030598

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