A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids

Kk, Sriram; Lin, Yii-Lih; Sewunet, Tsegaye; Wrande, Marie; Sandegren, Linus; Giske, Christian G.; Westerlund, Fredrik

doi:10.3390/mi12101234

Cited by 5 publications

(2 citation statements)

References 33 publications

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“…This has, for example, allowed us to trace plasmids during resistance outbreaks 14,15 and identify clonal spread of bacteria 16 . Furthermore, the analysis can be parallelized in chips with at least ten devices, to allow simultaneous analysis of multiple samples 17 .…”

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Strain-level bacterial typing directly from patient samples using optical DNA mapping

et al. 2023

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Background Identification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions. Methods In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections. Results We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids. Conclusion The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.

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Strain-level bacterial typing directly from patient samples using optical DNA mapping

et al. 2023

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“…In addition, the method can be used in primary clinical samples. Our method has been successfully applied to study clinical faecal samples 47 and identification of bacterial species from uncultured clinical urine samples. 48 …”

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Identification and characterization of plasmids carrying the mobile colistin resistance gene mcr-1 using optical DNA mapping

Wranne

Sewunet

et al. 2022

JAC-Antimicrobial Resistance

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Objectives Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1. Methods ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing. Results We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified blaCTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample. Conclusions Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited.

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Nanofluidic Lab‐On‐A‐Chip Systems for Biosensing in Healthcare

Chong,

Shen,

Palomba

et al. 2024

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Biosensing plays a vital role in healthcare monitoring, disease detection, and treatment planning. In recent years, nanofluidic technology has been increasingly explored to be developed into lab‐on‐a‐chip biosensing systems. Given now the possibility of fabricating geometrically defined nanometric channels that are commensurate with the size of many biomolecules, nanofluidic‐based devices are likely to become a key technology for the analysis of various clinical biomarkers, including DNA (deoxyribonucleic acid) and proteins in liquid biopsies. This review summarizes the fundamentals and technological advances of nanofluidics from the purview of single‐molecule analysis, detection of low‐abundance molecules, and single‐cell analysis at the subcellular level. The extreme confinement and dominant surface charge effects in nanochannels provide unique advantages to nanofluidic devices for the manipulation and transport of target biomarkers. When coupled to a microfluidic network to facilitate sample introduction, integrated micro‐nanofluidic biosensing devices are proving to be more sensitive and specific in molecular analysis compared to conventional assays in many cases. Based on recent progress in nanofluidics and current clinical trends, the review concludes with a discussion of near‐term challenges and future directions for the development of nanofluidic‐based biosensing systems toward enabling a new wave of lab‐on‐a‐chip technology for personalized and preventive medicine.

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A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids

Cited by 5 publications

References 33 publications

Strain-level bacterial typing directly from patient samples using optical DNA mapping

Strain-level bacterial typing directly from patient samples using optical DNA mapping

Identification and characterization of plasmids carrying the mobile colistin resistance gene mcr-1 using optical DNA mapping

Nanofluidic Lab‐On‐A‐Chip Systems for Biosensing in Healthcare

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