Abstract
Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy mcr-1-carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Salmonella Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.
Highlights
Antimicrobial resistance (AMR) genes are inherently present in bacteria
Development of an Optimized Workflow for Plasmid Reconstruction Using Next-generation sequencing (NGS) Data In Figure 1, the workflow used for this study is visualized, wherein we focused on comparing the nature of the starting isolate and the DNA extraction method
The impact of the choice of the starting isolate was evaluated, i.e., we assessed whether conjugation of the plasmid to a lab strain with known genome sequence facilitates plasmid reconstruction
Summary
Antimicrobial resistance (AMR) genes are inherently present in bacteria. the (mis)use of antibiotics increased the global prevalence of AMR; as a result, it has become one of the most prominent public health threats [1]. Plasmids are mobile genetic elements that can carry various genes beneficial for the survival of bacteria, including AMR genes. Antibiotics 2020, 9, 503 genetic content, size (1–1000 kb) and copy number (1−500). Bacteria can exchange plasmids between isolates even across the species or phyla barriers, called horizontal gene transfer (HGT) [2,3,4,5,6]. This transfer of plasmids facilitates the transmission of accompanying AMR genes, making plasmid detection and reconstruction of the utmost importance to understand the dissemination of AMR genes and to improve risk assessment
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