| 1. |
- Goyal, Gaurav, 1983, et al.
(författare)
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A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several beta-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings.
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| 2. |
- Goyal, Gaurav, 1983, et al.
(författare)
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CRISPR/CAS9 BASED DNA-COMBING ASSAY FOR DETECTING ANTIMICROBIAL RESISTANCE GENES ON PLASMIDS
- 2021
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Ingår i: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. ; , s. 801-802
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Konferensbidrag (refereegranskat)abstract
- We present a method based on CRISPR/Cas9 excision and DNA combing to detect anti-microbial resistance (AMR) genes on bacterial plasmids. The assay is inexpensive, simple, fast, and also provides information on the number and size of plasmids in a sample. We demonstrate detection of the gene encoding for the New Delhi metallobeta-lactamase 1 (blaNDM-1) enzyme, known to make bacteria resistant to a broad range of beta-lactam antibiotics.
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| 3. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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A parallelized nanofluidic device for high-throughput optical dna mapping of bacterial plasmids
- 2021
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 12:10
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Tidskriftsartikel (refereegranskat)abstract
- Optical DNA mapping (ODM) has developed into an important technique for DNA anal-ysis, 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 demon-strate 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 plas-mids in clinical diagnostics.
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| 4. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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High diversity of bla NDM-1 -encoding plasmids in Klebsiella pneumoniae isolated from neonates in a Vietnamese hospital
- 2022
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 1872-7913 .- 0924-8579. ; 59:2
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: The carbapenemase-encoding gene blaNDM-1 has been reported in Vietnam during the last 10 years, and blaNDM-producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of blaNDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of blaNDM-1-encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study. Methods: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the blaNDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients. Results: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding blaNDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding blaNDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same blaNDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques. Conclusions: The plasmids encoding the blaNDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis.
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| 5. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Identification and characterization of plasmids carrying the mobile colistin resistance gene mcr-1 using optical DNA mapping
- 2023
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Ingår i: JAC-Antimicrobial Resistance. - : Oxford University Press (OUP). - 2632-1823. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- 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 (similar to 30 kb) plasmids. We further identified bla(CTX-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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| 6. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Multiplexed optical DNA mapping to identify plasmids and their resistance genes in fecal samples
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 856-857
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Konferensbidrag (refereegranskat)abstract
- Multi-drug resistant bacteria are a major health threat worldwide. Resistance is frequently horizontally transferred among bacteria via mobile genetic elements, such as plasmids. We have developed a one-step staining protocol to reveal sequence-correlated fluorescence profiles along stretched plasmids. In this report, we demonstrate an automation approach to increase the throughput of nanofluidic optical DNA mapping(ODM). Using plasmid samples derived from patients' feces, the results demonstrate an improved throughput, both regarding the number of plasmids analyzed per sample as well as the number of samples analyzed. By combining the ODM with Cas9/CRISPR, plasmids containing the antibiotic resistance gene are readily identified.
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| 7. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Optical dna mapping of plasmids reveals clonal spread of carbapenem-resistant klebsiella pneumoniae in a large thai hospital
- 2021
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1 .
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| 8. |
- Lin, Yii Lih, 1978, et al.
(författare)
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Nanofluidic optical DNA mapping for rapid identification of antibiotics resistant plasmids
- 2018
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Ingår i: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. ; 3, s. 1822-1825
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Konferensbidrag (refereegranskat)abstract
- Bacterial resistance to antibiotics has become a major threat to health worldwide. In 2014, the World Health Organization (WHO) classified antibiotic resistance as one of the major threats to human health.1 As the resistance genes are frequently transmitted via mobile genetic elements, such as plasmids, a method to rapidly detect and identify plasmids is needed. This paper reports an improved nanofluidic optical mapping strategy to identify plasmids and locate the antibiotic resistance genes via the Cas9/CRISPR technique. We used the assay to analyze clinical samples from a potential nosocomial outbreak in an Ethiopian hospital, tracing the transmission routes among wards.
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| 9. |
- Lin, Yii Lih, 1978, et al.
(författare)
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Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: A case study of an outbreak in a rural Ethiopian hospital
- 2020
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 75:10, s. 2804-2811
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements.We believe that this method has the potential to be a tool of pivotal importance for MDR control. Methods: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. Results: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. Conclusions: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low-and middle-income countries.
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| 10. |
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