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Characterization of antimicrobial resistance in bacteria, including plasmids, through the development of a generic NGS-based workflow

Johannes Berbers (UGent)
(2026)
Author
Promoter
(UGent) , Sigrid De Keersmaecker and Nancy Roosens
Organization
Abstract
Door het (over)gebruik van antibiotica is de prevalentie van antimicrobiële resistentie (AMR) bij bacteriën sterk toegenomen. AMR vormt een wereldwijde bedreiging voor de volksgezondheid. Een belangrijke factor die AMR zo problematisch maakt, is hoe gemakkelijk het zich tussen bacteriën verspreidt o.a. via plasmides. AMR kan zich via (in)directe routes tussen mens, dier en omgeving verspreiden, waardoor het een One-Health probleem is. De introductie van Next-Generation Sequencing (NGS) heeft het mogelijk gemaakt om de genetische context van AMR genen in één test te bepalen. Short-read technologie is zeer nauwkeurig maar heeft moeite met repetitieve regio’s in plasmides, terwijl long-reads deze wel kunnen overbruggen maar lagere nauwkeurigheid en hogere DNA eisen hebben. Hybrid assemblies combineren de voordelen van beide. In dit project is een gebruiksvriendelijke NGS-workflow ontwikkeld, van DNA-extractie tot data-analyse. Vergelijkingen toonden aan dat hybrid assemblies van hoogwaardige DNA-extracties de meest complete resultaten geven. De workflow werd toegepast in verschillende casestudies waarbij traditionele methoden niet voldoende zouden zijn geweest om het volledige AMR-profiel en de genetische context/locatie te achterhalen, waardoor een proof-of-concept werd geleverd voor de ontwikkelde workflow. De ontwikkelde workflow maakt karakterisering van AMR genen en plasmiden mogelijk en is kosteneffectief inzetbaar voor routinematige AMR surveillance binnen een One-Health context.
Since the discovery of antibiotics, many infections that could have been deadly before are now successfully treated. However, due to the (over)use of antibiotics, the prevalence of antimicrobial resistance (AMR) in bacteria has been greatly increased. AMR is now a global public health threat and if it remains under addressed, the severe consequences could amount to up to 10 million deaths per year by 2050 and a severely impacted quality of life. A major factor that makes AMR so problematic is how easily it is spread between bacteria. AMR can transfer through (in)direct pathways between humans, animals and the environment, thus making it also a One Health problem. Eventually the AMR can be transferred to pathogens, making infections harder to treat. The most well studied mechanism of AMR transfer is through mobile genetic elements (MGE) including plasmids. Plasmids are extra-chromosomal, double-stranded DNA molecules of varying size (1 to over 300 kb) and they contain a lot of repetitive regions. Plasmids play a central role in the dissemination of AMR, due to their ability to transfer between many bacterial species. They are key carriers of clinically significant AMR genes, such as Extended Spectrum Beta-Lactamase (ESBL) genes, which confers resistance to the most commonly used antibiotics. Plasmids and other MGE can also integrate these AMR genes in the bacterial chromosome. Improvements in the detection of AMR and thereby also the surveillance of AMR, i.e. tracking changes and trends in the AMR profile of isolates, are essential to improve AMR stewardship, which aims to monitor and limit antibiotic usage depending on the prevalence of resistance toward antibiotics. At the beginning of this PhD research, the most common AMR detection methods were Minimum Inhibitory Concentration tests (phenotypic) and (q)PCRs (genotypic). The disadvantage of these methods is that they are unable to determine the full genetic context of AMR genes, i.e. are they located on the chromosome or on a plasmid. The introduction of Next-Generation sequencing (NGS) has made it possible to determine genetic context of AMR genes in a single test. Short and long-read sequencing technologies each have their own advantages and disadvantages. Short-read sequencing produces highly accurate sequencing reads, but due to their length it is difficult to bridge repetitive regions in plasmids. In contrast, long-read sequencing reads have lower accuracy but are able to completely cover repetitive regions. Moreover, long-read sequencing has higher DNA quality and quantity requirements, more complex data analysis and protocols are still changing/improving which hinders validations. It is also possible to use both technologies in hybrid assemblies to combine their advantages. NGS could be a powerful tool for AMR surveillance, however some challenges still need to be addressed. In this study, we developed a user-friendly AMR detection workflow for isolates based on NGS for both the wet-lab (handling of samples, DNA extraction and library preparation) and dry-lab (data analysis). To achieve this, several parameters were compared across this process and applied to samples of different origins/reservoirs. Furthermore, the workflow was applied to several case studies where without NGS, it would not have been possible to elucidate the full AMR profile and genetic context/location, thereby delivering a proof-of-concept for the developed workflow and strategy. To develop a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid, comparisons between commercial and classical whole genome and plasmid DNA extractions were made, 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 harbouring a large, low-copy mcr-1-carrying plasmid (>200 kb) was used. The most accurate and complete reconstructions of the chromosome and plasmids were done with hybrid assemblies from Genomic Tip 100 (anion-exchange) and MagCore (automated magnetic beads) DNA extracts of the original isolate. The Genomic Tip 100 DNA extracts produced higher quality and quantity of DNA, however the DNA extracts of the MagCore were more time-efficient when a lot of samples need to be processed and were still sufficient for long-read sequencing. 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. Then the developed NGS workflow was applied to a case study, where blaVIM-1 was detected for the first time in the Belgian food chain in an E. coli isolated from minced pork. This AMR gene confers resistance to carbapenems, which are last-resort antibiotics meaning that they are reserved for patients infected with multi-resistant pathogens. With the conventional methods it was impossible to determine if the blaVIM-1 gene was located on the chromosome or on a plasmid. With hybrid assemblies it was determined that blaVIM-1 was located on a 190kb incA/C2 plasmid along with other clinically relevant AMR genes. This plasmid showed low similarity to blaVIM-1 samples reported in Germany. However, the blaVIM-1 gene cassette showed similarity to sequences from clinical isolates, suggesting that the Belgian meat was contaminated by human handling. Next, the NGS workflow was applied to a complex genetically modified microorganism (GMM) case study. This unauthorized GM Bacillus subtilis was used for the production of vitamin B2 feed additive and isolated there from. It harboured multiple AMR genes, which were used as selection markers. With a hybrid assembly, we demonstrated both a chromosomal integration of GM plasmids with a repetitive 53 kb structure and the presence of an extra-chromosomal 38 kb plasmid. Previous studies with only short-read sequencing were unable to achieve the correct reconstruction of this GMM due to the complexity of the repetitive regions. Lastly, the developed NGS workflow was used on a larger collection of clinical isolates. Between 2013-2018, there was a sudden rise of ESBL observed in Shigella in Belgium. As it is not feasible during routine AMR surveillance to do hybrid assemblies for all isolates, the workflow was optimized so that representative isolates are selected for hybrid assemblies, while for the other isolates only short-read sequencing is done. With this optimized workflow we found the causative plasmids (IncFII) for the sudden ESBL rise, containing blaCTX-M-15 and the first detection of a chromosomal integration of ESBL genes in Shigella. Overall, this dissertation provides a NGS workflow that allows for comprehensive characterisation of all AMR genes and reconstruct plasmids in bacterial isolates of diverse originating reservoirs. In short, the recommended workflow for AMR surveillance consists of performing whole-genome DNA extractions (using anion-exchange or magnetic bead–based methods) on bacterial isolates followed by short-read sequencing, after which a selection of representative isolates is subjected to long-read sequencing to generate high-quality reference genomes for downstream analyses. This is essential for tracking the prevalence and dissemination of AMR in bacteria in a One Health context. With a cost-effective adaptation this workflow is suitable for use in routine AMR surveillance and could support public health policy against this worldwide threat.
Keywords
Antimicrobial resistance, plasmids, Next-generation Sequencing, AMR, NGS

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Citation

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MLA
Berbers, Johannes. Characterization of Antimicrobial Resistance in Bacteria, Including Plasmids, through the Development of a Generic NGS-Based Workflow. Ghent University. Faculty of Sciences, 2026.
APA
Berbers, J. (2026). Characterization of antimicrobial resistance in bacteria, including plasmids, through the development of a generic NGS-based workflow. Ghent University. Faculty of Sciences, Ghent, Belgium.
Chicago author-date
Berbers, Johannes. 2026. “Characterization of Antimicrobial Resistance in Bacteria, Including Plasmids, through the Development of a Generic NGS-Based Workflow.” Ghent, Belgium: Ghent University. Faculty of Sciences.
Chicago author-date (all authors)
Berbers, Johannes. 2026. “Characterization of Antimicrobial Resistance in Bacteria, Including Plasmids, through the Development of a Generic NGS-Based Workflow.” Ghent, Belgium: Ghent University. Faculty of Sciences.
Vancouver
1.
Berbers J. Characterization of antimicrobial resistance in bacteria, including plasmids, through the development of a generic NGS-based workflow. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2026.
IEEE
[1]
J. Berbers, “Characterization of antimicrobial resistance in bacteria, including plasmids, through the development of a generic NGS-based workflow,” Ghent University. Faculty of Sciences, Ghent, Belgium, 2026.
@phdthesis{01KG2EE3TDAPS9S75VF2TME2HE,
  abstract     = {{Door het (over)gebruik van antibiotica is de prevalentie van antimicrobiële resistentie (AMR) bij bacteriën sterk toegenomen. AMR vormt een wereldwijde bedreiging voor de volksgezondheid.
Een belangrijke factor die AMR zo problematisch maakt, is hoe gemakkelijk het zich tussen bacteriën verspreidt o.a. via plasmides. AMR kan zich via (in)directe routes tussen mens, dier en omgeving verspreiden, waardoor het een One-Health probleem is.
De introductie van Next-Generation Sequencing (NGS) heeft het mogelijk gemaakt om de genetische context van AMR genen in één test te bepalen. Short-read technologie is zeer nauwkeurig maar heeft moeite met repetitieve regio’s in plasmides, terwijl long-reads deze wel kunnen overbruggen maar lagere nauwkeurigheid en hogere DNA eisen hebben. Hybrid assemblies combineren de voordelen van beide.
In dit project is een gebruiksvriendelijke NGS-workflow ontwikkeld, van DNA-extractie tot data-analyse. Vergelijkingen toonden aan dat hybrid assemblies van hoogwaardige DNA-extracties de meest complete resultaten geven. De workflow werd toegepast in verschillende casestudies waarbij traditionele methoden niet voldoende zouden zijn geweest om het volledige AMR-profiel en de genetische context/locatie te achterhalen, waardoor een proof-of-concept werd geleverd voor de ontwikkelde workflow. 
De ontwikkelde workflow maakt karakterisering van AMR genen en plasmiden mogelijk en is kosteneffectief inzetbaar voor routinematige AMR surveillance binnen een One-Health context.}},
  author       = {{Berbers, Johannes}},
  keywords     = {{Antimicrobial resistance,plasmids,Next-generation Sequencing,AMR,NGS}},
  language     = {{eng}},
  pages        = {{245}},
  publisher    = {{Ghent University. Faculty of Sciences}},
  school       = {{Ghent University}},
  title        = {{Characterization of antimicrobial resistance in bacteria, including plasmids, through the development of a generic NGS-based workflow}},
  year         = {{2026}},
}