Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination

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.

Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates

“…For instance, isolates E. coli 3 and K. pneumoniae 1 had a remarkably smaller genome size than Hyb ASM or Illum ASM (Supplementary Table S4). Inaccuracy in genome size using Nanopore technology has previously been reported for a conjugated test plasmid [13], and might be explained by the technology's greater sequencing error [7,48]. In addition, inaccuracy in genome size can be explained by lower MinION coverage for the mentioned isolates.…”

“…Considering the benefits and drawbacks of both short and long reads, several attempts have been made to apply a hybrid assembly approach, which uses both type of reads [3,9,[11][12][13]. For this purpose, different assemblers, such as Unicycler [3], hy-bridSPAdes [14], and MaSurCa [15], have been developed.…”

“…For instance, hybrid assembly makes the downstream analyses, mapping, and annotation of genomic features more accurate [16]. Furthermore, it has been shown that hybrid assembly provides a better resolution for studying tandem repeats as well as gene variants [17], and it is the ideal approach for predicting the plasmids and AMR genes [13].…”

Plasmid Identification and Plasmid-Mediated Antimicrobial Gene Detection in Norwegian Isolates

“…Having a less fragmented assembly where the circular plasmids are apparent makes the prediction of ARG plasmid more accurate. The current conclusion regarding the applicability of hybrid assembly for plasmid-mediated ARGs detection previously has been made [ 40 ]. Therefore, future work implanting hybrid assembly to identify ARGs in bacteria is worth investigating.…”

NGSocomial Infections: High-Resolution Views of Hospital-Acquired Infections Through Genomic Epidemiology

“…A waiting game may ensue as more cases slowly accrue in a way that will align with laboratory workflow for testing that is, by nature, not reimbursed. Nanopore sequencing also has something to offer beyond its rapid pace, since the analytical capacity of the Oxford Nanopore Flongle or Minion takes advantage of smaller batch sizes that work well with the need for rapid decision-making in the course of an ongoing hospital outbreak [ 112 , 113 ]. Certainly, more work is required to determine the cost-effectiveness of pathogen sequencing in different settings, and it is likely that hospital outbreak sequencing is one of the most cost-effective use cases for microbial WGS.…”