Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-08-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/9/8/503 |
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| author | Bas Berbers Pieter-Jan Ceyssens Pierre Bogaerts Kevin Vanneste Nancy H. C. Roosens Kathleen Marchal Sigrid C. J. De Keersmaecker |
| author_facet | Bas Berbers Pieter-Jan Ceyssens Pierre Bogaerts Kevin Vanneste Nancy H. C. Roosens Kathleen Marchal Sigrid C. J. De Keersmaecker |
| author_sort | Bas Berbers |
| collection | DOAJ |
| description | 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 <i>mcr-1</i>-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 <i>Salmonella</i> 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. |
| first_indexed | 2024-03-10T17:38:59Z |
| format | Article |
| id | doaj.art-cbea9fc00c55498b90995534157f81f9 |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-10T17:38:59Z |
| publishDate | 2020-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-cbea9fc00c55498b90995534157f81f92023-11-20T09:46:10ZengMDPI AGAntibiotics2079-63822020-08-019850310.3390/antibiotics9080503Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene DisseminationBas Berbers0Pieter-Jan Ceyssens1Pierre Bogaerts2Kevin Vanneste3Nancy H. C. Roosens4Kathleen Marchal5Sigrid C. J. De Keersmaecker6Transversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumBacterial Diseases, Sciensano, 1050 Brussels, BelgiumNational Reference Center for Antimicrobial Resistance in Gram-Negative Bacteria, CHU UCL Namur, 5530 Yvoir, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumDepartment of Information Technology, IDLab, Ghent University, IMEC, 9052 Ghent, BelgiumTransversal Activities in Applied Genomics, Sciensano, 1050 Brussels, BelgiumAntimicrobial 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 <i>mcr-1</i>-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 <i>Salmonella</i> 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.https://www.mdpi.com/2079-6382/9/8/503plasmidsantimicrobial resistanceconjugationDNA extractionnext-generation sequencingMiSeq |
| spellingShingle | Bas Berbers Pieter-Jan Ceyssens Pierre Bogaerts Kevin Vanneste Nancy H. C. Roosens Kathleen Marchal Sigrid C. J. De Keersmaecker Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination Antibiotics plasmids antimicrobial resistance conjugation DNA extraction next-generation sequencing MiSeq |
| title | Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination |
| title_full | Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination |
| title_fullStr | Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination |
| title_full_unstemmed | Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination |
| title_short | Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination |
| title_sort | development of an ngs based workflow for improved monitoring of circulating plasmids in support of risk assessment of antimicrobial resistance gene dissemination |
| topic | plasmids antimicrobial resistance conjugation DNA extraction next-generation sequencing MiSeq |
| url | https://www.mdpi.com/2079-6382/9/8/503 |
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