Abstract
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.
Highlights
Antimicrobial resistance (AMR) in food- and livestock-associated bacteria can represent an important threat to public health and needs to be monitored [1,2]
Commensal Escherichia (E.) coli were chosen as indicator organisms, as they belong to the common intestinal microbiota of livestock and reflect trends in the development of antimicrobial resistances associated with the lifestyle of animals [4]
Due to the broad diversity of determinants associated with decreased susceptibilities of isolates against specific antimicrobial classes, whole-genome sequencing (WGS) provides deeper insight into the genetic basis of antimicrobial resistances, possible routes of transmissions and important clonal lineages, which are useful for risk assessment [6]
Summary
Antimicrobial resistance (AMR) in food- and livestock-associated bacteria can represent an important threat to public health and needs to be monitored [1,2]. Due to the broad diversity of determinants associated with decreased susceptibilities of isolates against specific antimicrobial classes, whole-genome sequencing (WGS) provides deeper insight into the genetic basis of antimicrobial resistances, possible routes of transmissions and important clonal lineages, which are useful for risk assessment [6]. A WGS-based monitoring will prospectively provide a uniform basis for the identification of dissemination paths of genetic elements, supporting the fight against resistance development in livestock-associated and foodborne commensals and pathogens [8]. As plasmids are commonly implicated in the dissemination of AMR, it is important to correctly determine whether resistance genes are fixed on the chromosome or located on mobile genetic elements (MGEs) [11]
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