Optical DNA Mapping of Plasmids Reveals Clonal Spread of Carbapenem-Resistant Klebsiella pneumoniae in a Large Thai Hospital

Kk, Sriram; Sewunet, Tsegaye; Wangchinda, Walaiporn; Tangkoskul, Teerawit; Thamlikitkul, Visanu; Giske, Christian G.; Westerlund, Fredrik

doi:10.3390/antibiotics10091029

Cited by 5 publications

(4 citation statements)

References 25 publications

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“…An important difference between PCR and ODM is that, while the PCR analysis reveals the presence of a specific gene, the ODM analysis determines on which plasmid a specific gene is located. This is important, for example, for epidemiological studies of bacterial transmission [19] and plasmid conjugation [24,26]; we have also shown, previously, that detecting identical plasmids in several samples can be a proxy for ongoing clonal outbreaks [25,27].…”

Section: Antibiotic Resistance Gene Identification In Clinical Fecal Isolatesmentioning

confidence: 55%

“…By combining the ODM technique with Cas9-mediated identification of resistance genes, the method can determine the number of different plasmids in a sample, their size, on which plasmid a certain resistance gene is located and also use the ODM barcode to identify and trace plasmids [20]. Our methodology has proven to be useful in understanding the role of plasmid conjugation, for example, in hospital outbreaks and recurring urinary tract infections [19,[24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

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

Lin

Sewunet

et al. 2021

Micromachines

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Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

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Section: Antibiotic Resistance Gene Identification In Clinical Fecal Isolatesmentioning

confidence: 55%

Section: Introductionmentioning

confidence: 99%

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

Lin

Sewunet

et al. 2021

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“…It has been found to harbor various antimicrobial resistance genes, such as extended-spectrum β-lactamase, carbapenemase, aminoglycosides resistance, fluoroquinolone resistance, and tet (A) gene. In Thailand, ST147 carrying bla NDM have been reported in several studies 24 , 77 . However, the major clonal group of K. pneumoniae- CG258, comprising ST11, ST258, and ST512, was not detected in our study 4 .…”

Section: Discussionmentioning

confidence: 96%

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand

Chirabhundhu,

Luk-In,

Phuadraksa

et al. 2024

Sci Rep

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Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017–2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

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“… 35 , 36 We have used this method to identify plasmids from sequence databases and to trace plasmids during resistance outbreaks. 29 , 30 , 37 , 38 We have recently also presented a simplified version of the assay that detects resistance genes on plasmids using a microscope (Primo Star iLED) that is already present in laboratories in low- and middle-income countries for diagnosing tuberculosis. 39 …”

Section: Introductionmentioning

confidence: 99%

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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Optical DNA Mapping of Plasmids Reveals Clonal Spread of Carbapenem-Resistant Klebsiella pneumoniae in a Large Thai Hospital

Cited by 5 publications

References 25 publications

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

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

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand

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

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