Development of an NGS-based workflow for improved monitoring of circulating plasmids in support of risk assessment of antimicrobial resistance gene dissemination
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Development of an NGS-based workflow for improved monitoring of circulating plasmids in support of risk assessment of antimicrobial resistance gene dissemination
Berbers, Bas; Ceyssens, Pieter-Jan; Bogaerts, Pierre; Vanneste, Kevin; Roosens, Nancy H. C.; Marchal, Kathleen; De Keersmaecker, Sigrid C.J.
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.
Description:
Supplementary material: Figure S1: Mauve alignment between the reconstructed mcr-1 plasmid of assemblies 11–14. Figure S2: Visualization
of hybrid assemblies of isolates COL20160015 and S15BD05371 from Flongle runs 1 (a), 2 (b), 3 (c), 4 (d) and
5 (e). Figure S3: Visualization of MinION assemblies of isolates COL20160015 and S15BD05371 from Flongle
runs 1 (a), 2 (b), 3 (c), 4 (d) and 5 (e). Figure S4: Visualization of hybrid assemblies of isolates S16BD08730 (a),
S18BD03994 (b), S18BD00864 (c) and S18BD05011 (d). Table S1: Antimicrobial resistance genes and plasmid
replicons detected with ResFinder and PlasmidFinder in whole-genome MiSeq, MinION and hybrid assembly
of isolate COL20160015 extracted with Genomic Tip 100. Table S2: Antimicrobial resistance genes and plasmid
replicons detected with ResFinder and PlasmidFinder in whole-genome MiSeq, MinION and hybrid assembly of
isolate R274 extracted with Genomic Tip 100. Table S3: Antimicrobial resistance genes and plasmid replicons
detected with ResFinder and PlasmidFinder in whole-genome MiSeq, MinION and hybrid assembly of the
conjugated isolate R274 extracted with Genomic Tip 100 with all chromosomal reads (mapped to CP000948.1)
filtered out prior to assembly. Table S4: Quality parameters of the extracted DNA of isolate S15BD05371 used for
sequencing Table S5: The most similar sequences from the NCBI nt database to the incomplete contigs in the hybrid
assemblies. Table S6: Antimicrobial resistance genes detected with ResFinder in whole-genome MiSeq, MinION
and hybrid assembly of isolate S15BD05371 extracted with MagCore. Table S7: Antimicrobial resistance genes
detected with ResFinder in MiSeq, MinION and hybrid assembly of isolate S15BD05371 extracted with Genomic
Tip500 with plasmid extraction bu ers. Table S8: Antimicrobial resistance genes detected with ResFinder in MiSeq,
MinION and hybrid assembly of isolate S15BD05371 extracted with a phenol chloroform plasmid extraction.
Table S9: Antimicrobial resistance genes detected with ResFinder in MiSeq, MinION and hybrid assembly of
isolate S15BD05371 extracted with phenol chloroform plasmid extraction followed up by an exonuclease digestion
and amplification. Table S10: Antimicrobial resistance genes detected with ResFinder in the hybrid assembly of
isolate COL20160015 from Flongle run 1. Table S11: Antimicrobial resistance genes detected with ResFinder in the
hybrid assembly of isolate COL20160015 from Flongle run 2. Table S12: Antimicrobial resistance genes detected
with ResFinder in the hybrid assembly of isolate COL20160015 from Flongle run 3. Table S13: Antimicrobial
resistance genes detected with ResFinder in the hybrid assembly of isolate COL20160015 from Flongle run 4.
Table S14: Antimicrobial resistance genes detected with ResFinder in the hybrid assembly of isolate S15BD05371
from Flongle run 5. Table S15: Statistics of all sequencing reads that were used in assemblies of this study.