Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis

Hicks, Linda D.; Graaf, Charlotte M. van der; Childress, Jacob; Cook, Emily; Schmidt, Karen; Rosenzweig, Frank; Kroll, Eugene

doi:10.14440/jbm.2018.218

Cited by 6 publications

(4 citation statements)

References 14 publications

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“…lusitaniae previously described either by supercontig assemblies of C. lusitaniae ATCC 42720 (8) or by chromosome separation from electrophoresis (9) and centromere mapping (10). These PacBio contigs were designated Chr 1 to Chr 8 and were sorted by their sizes (see Table S2 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae

Kannan

Asner

Trachsel

et al. 2019

mBio

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Antifungal resistance is an inevitable phenomenon when fungal pathogens are exposed to antifungal drugs. These drugs can be grouped in four distinct classes (azoles, candins, polyenes, and pyrimidine analogs) and are used in different clinical settings. Failures in therapy implicate the sequential or combined use of these different drug classes, which can result in some cases in the development of multidrug resistance (MDR). MDR is particularly challenging in the clinic since it drastically reduces possible treatment alternatives. In this study, we report the rapid development of MDR in Candida lusitaniae in a patient, which became resistant to all known antifungal agents used until now in medicine. To understand how MDR developed in C. lusitaniae, whole-genome sequencing followed by comparative genome analysis was undertaken in sequential MDR isolates. This helped to detect all specific mutations linked to drug resistance and explained the different MDR patterns exhibited by the clinical isolates.

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Section: Resultsmentioning

confidence: 99%

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae

Kannan

Asner

Trachsel

et al. 2019

mBio

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“…Five independent assemblies were obtained with the 5 isolates containing each 8 major contigs (smaller contigs corresponding to mitochondrial genomes were ignored). These 8 major contigs were likely to correspond to the 8 suggested chromosomes of C. lusitaniae previously described either by supercontigs assemblies of C. lusitaniae ATCC 42720 (8), or by chromosome separation from electrophoresis (9) and centromere mapping (10). These Pacbio contigs were designated as Chr 1 to Chr 8 and were sorted by their sizes (Table S3).…”

Section: Resultsmentioning

confidence: 71%

Comparative genomics for the elucidation of multidrug resistance (MDR) inCandida lusitaniae

Kannan¹,

Asner²,

Trachsel³

et al. 2019

Preprint

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1Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents 2 administered in combination or sequentially to the same individual. We reported earlier MDR in 3Candida lusitaniae during therapy with amphotericin B (AmB), azoles and candins. We used here 4 comparative genomic approaches between the initial susceptible isolate and 4 other isolates with 5 different MDR profiles. From a total of 18 non-synonymous SNPs (NSS) in genome comparisons 6 with the initial isolate, six could be associated with MDR. One of the SNPs occurred in a putative 7 transcriptional activator (MRR1) resulting in a V668G substitution in isolates resistant to azoles 8 and 5-fluorocytosine (5-FC). We demonstrated by gene editing that MMR1 acted by upregulation 9 of MFS7 (a multidrug transporter) in the presence of the V668G substitution. MFS7 itself 10 mediated not only azole resistance but also 5-FC resistance, which represents a novel resistance 11 mechanism for this drug class. Three other distinct NSS occurred in FKS1 (a glucan synthase that 12 is targeted by candins) in three candin-resistant isolates. Lastly, two other NSS in ERG3 and ERG4 13

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“…This powerful approach identifies lactobacilli based on data revealed from the separation of either large DNA molecules or small RE (restriction endonuclease)-digested fragments of the genome and the entire genome (Hicks et al, 2018). It can differentiate between valuable probiotic species such as L. casei, L. rhamnosus, L. helveticus, and L. johnsonii (Ben Amor et al, 2007).…”

Section: Pfgementioning

confidence: 99%

“…Agarose plugs are handled and separated case-by-case, which is timeconsuming and difficult for scale-up. Different isolates can get messed up and merged, thus leading to the misidentification of isolates (Hicks et al, 2018).…”

Section: Pfgementioning

confidence: 99%

Comparative study on biochemical and molecular identification approaches of Lactobacillus species

Senjaliya,

Georrge

2023

IFRJ

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Manufacturers’ desire to sell “healthy” food in response to the consumers’ desire to lead a healthy lifestyle has increased the use of probiotics during the past few decades. Probiotics are used in dairy products, as well as non-dairy items as a starter culture, encompassing a wide range of goods. Numerous phenotyping, physical characterisation, and genotyping techniques have been developed to identify probiotic lactobacilli to ensure quality management. These techniques are frequently precise enough to categorise probiotic strains by genus and species. Traditional microbiological methods were initially employed for genus and species identification. However, due to their numerous shortcomings as the probiotic ability is often strain-dependent, and that there is no way to differentiate between strains using simple microbiological techniques, new methods that are mostly based on the examination of nucleic acids have been developed. Therefore, the objective of the present review was to provide critical assessment on existing methods for identifying members of the genus Lactobacillus, together with newly discovered approaches. The present review aimed to give the most recent information on the scientific techniques used to measure and describe the possible probiotic properties of microorganisms. It will also emphasise molecular and non-molecular tools. Most of these tools are based on 16S ribosomal DNA sequencing, and employ PCR techniques.

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Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis

Cited by 6 publications

References 14 publications

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae

Comparative genomics for the elucidation of multidrug resistance (MDR) inCandida lusitaniae

Comparative study on biochemical and molecular identification approaches of Lactobacillus species

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