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Iskalni niz: "ključne besede" (next-generation sequencing) .

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1.
Genomic insights into the Mycobacterium kansasii complex : an update
Paulina Borówka, Tomasz Jagielski, Dominik Strapagiel, Marija Žolnir-Dovč, Jakobus van Ingen, Lian Pennings, Mikołaj Dziurzyński, Jarosław Dziadek, Anna Brzostek, Błażej Marciniak, Jakub Lach, Zofia Bakuła, 2020

Povzetek: Only very recently, has it been proposed that the hitherto existing Mycobacteriumkansasii subtypes (I–VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, amonophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.
Ključne besede: Mycobacteriumkansasii complex, Mycobacteriumostraviense sp. nov., non-tuberculous mycobacteria (NTM), whole genome sequencing, taxonomy
DiRROS - Objavljeno: 31.07.2020; Ogledov: 971; Prenosov: 615
.pdf Celotno besedilo (10,22 MB)

2.
Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries
Nataša Toplak, Eva Sodja, Minka Kovač, Simon Koren, Marija Žolnir-Dovč, Biljana Ilievska Poposka, Sara Truden, 2019

Povzetek: Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.
Ključne besede: drug resistant tuberculosis, next-generation sequencing, low-incidence countries, phenotypic drug susceptibility testing
DiRROS - Objavljeno: 24.07.2020; Ogledov: 1020; Prenosov: 466
.pdf Celotno besedilo (144,02 KB)

3.
Next-generation sequencing to characterize pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries
Eva Sodja, Simon Koren, Nataša Toplak, Sara Truden, Marija Žolnir-Dovč, 2021

Povzetek: Objectives. Next-generation sequencing (NGS) provide a comprehensive analysis of the genetic alterations that are most commonly linked with pyrazinamide (PZA) resistance. There are no studies reporting molecular background of PZA resistance in TB isolates from Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance, pncA, using Ion Torrent technology in comparison to phenotypic BACTEC MGIT 960 DST in clinical TB isolates from two countries of the Balkan Peninsula. Methods. Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using the AmpliSeq™ Kit for Chef DL8 and the Ion AmpliSeq TB Research Panel. Result.s Of all 61 TB isolates included, 56 TB were phenotypically resistant to any antibiotic. Among them, 38/56 (67.9%) TB isolates were phenotypically resistant to pyrazinamide and pncA mutations were detected in 33/38 cases (86.8%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS to conventional BACTEC MGIT 960 DST revealed very strong agreement (90.2%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. Conclusions. Detection of PZA resistance using NGS seems to be a valuable tool for surveillance of TB drug resistance also in the Balkan Peninsula, with great potential to provide useful information at least one weak earlier than is possible with phenotypic DST.
Ključne besede: tuberculosis, Mycobacterium tuberculosis, high-throughput nucleotide sequencing, pyrazinamide, microbial sensitivity tests, next-generation sequencing, drug susceptibility testing, Slovenia, Republic of North Macedonia
DiRROS - Objavljeno: 10.01.2022; Ogledov: 38; Prenosov: 27
.pdf Celotno besedilo (1,53 MB)

4.
Using genomic information for management planning of an endangered perennial, Viola uliginosa
Kyung Min Lee, Pertti Ranta, Jarmo Saarikivi, Lado Kutnar, Branko Vreš, Maxim Dzhus, Marko Mutanen, Laura Kvist, 2020

Povzetek: Species occupying habitats subjected to frequent natural and/or anthropogenic changes are a challenge for conservation management. We studied one such species, Viola uliginosa, an endangered perennial wetland species typically inhabiting sporadi-cally flooded meadows alongside rivers/lakes. In order to estimate genomic diversity, population structure, and history, we sampled five sites in Finland, three in Estonia, and one each in Slovenia, Belarus, and Poland using genomic SNP data with double-digest restriction site-associated DNA sequencing (ddRAD-seq). We found mono-phyletic populations, high levels of inbreeding (mean population FSNP = 0.407-0.945), low effective population sizes (Ne = 0.8-50.9), indications of past demographic ex-pansion, and rare long-distance dispersal. Our results are important in implementing conservation strategies for V. uliginosa, which should include founding of seed banks, ex situ cultivations, and reintroductions with individuals of proper origin, combined with continuous population monitoring and habitat management.
Ključne besede: nature conservation, demography, genomic diversity, population genomics, RAD sequencing, Viola
DiRROS - Objavljeno: 20.01.2022; Ogledov: 19; Prenosov: 21
.pdf Celotno besedilo (1,49 MB)
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