•Next-generation sequencing (NGS) to characterise pyrazinamide (PZA) resistance in TB isolates.•Comparison of NGS with conventional BACTEC MGIT 960 revealed very strong agreement.•The majority of ...PZA-resistant TB isolates harboured mutations in pncA.•The in-frame pncA mutations were randomly distributed along the entire pncA gene.•A–11G pncA promoter mutation was the most prevalent genetic alteration.
Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (DST) in clinical TB isolates from two countries of the Balkan Peninsula.
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 an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel.
Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS.
Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.
Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. ...Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012 and to investigate the occurrence of the novel SI-MRV01 MRV variant in Slovenian bats.
The detection of MRVs in bat guano was based on broad-range RT-PCR and specific bat MRV real-time RT-PCR. Subsequently, MRV isolates were obtained from cell culture propagation, with detailed molecular characterisation through whole-genome sequencing. Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33.0%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five MRV isolates of different serotypes that are designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments.
In conclusion, we have confirmed the presence of the SI-MRV01 strain in a Slovenian bat population. Moreover, according to genetic characterisation of S1 genome segment, all three MRV serotypes were present in the bat population. In this study, five independent MRV isolates were obtained and detailed whole genome analysis revealed high diversity between them. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The magic world of whiskey microbiota PRISTAVEC, Ajda; KOREN, Simon; JERŠEK, Barbara ...
Acta agriculturae slovenica,
2021, Letnik:
116, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Modern metagenomics techniques in combination with next generation sequencing are increasingly used for research of numerous environments inhabited by diverse microbiota. In the present study we ...focused on a rather unusual environment for their growth, a forgotten bottle of blended Scotch whiskey. Whiskey is a world-known popular spirit, traditionally produced in a series of steps comprising malting of barley, fermenting the malt to an alcoholic wort, distilling and at least 3-year long maturation in oak casks, followed by filtration. In the process, notably in the fermentation, microorganisms play a crucial role. However, we were primarily interested in potential microbiological and chemical changes that might have taken place over the years while the half-empty whiskey bottle was left open. We found that only a very low number of aerobic mesophilic bacteria survived in it while the ethanol content decreased from 40 % to approximately 30 %. Interestingly, the metagenomics analysis showed there was a large and diverse microbial community present in the forgotten whiskey. Among the most abundant microorganisms were members of human commensal microbiota, some potentially disease-causing and also food spoiling bacteria, in particular genus Pseudomonas. Surprisingly, we even found a non-negligible number of typically environmental bacterial species.
cleanroom strains are often exposed to sub-inhibitory concentrations of disinfectants, including didecyldimethylammonium chloride (DDAC). Consequently, they can adapt or even become tolerant to them. ...RNA-sequencing was used to investigate adaptation and tolerance mechanisms of
cleanroom strains (SE11, SE18), with
SE11Ad adapted and
SE18To tolerant to DDAC. Adaptation to DDAC was identified with up-regulation of genes mainly involved in transport (thioredoxin reductase
, the arsenic efflux pump gene ID, SE0334, sugar phosphate antiporter
), while down-regulation was seen for the Agr system (
,
,
,
, SE1543), for enhanced biofilm formation. Tolerance to DDAC revealed the up-regulation of genes associated with transporters (L-cysteine transport
; uracil permease SE0875; multidrug transporter
; arsenic efflux pump
); the down-regulation of genes involved in amino-acid biosynthesis (lysine
; histidine
; methionine
), and an enzyme involved in peptidoglycan, and therefore cell wall modifications (alanine racemase SE1079). We show for the first time the differentially expressed genes in DDAC-adapted and DDAC-tolerant
strains, which highlight the complexity of the responses through the involvement of different mechanisms.
In the last few years the advances in molecular biological methods, especially the development of next generation sequencing, have drastically changed and improved our view of microbial world. ...Progress in new molecular techniques enables us to overcome potential disadvantages of traditional microbiological techniques in fungal community identifications. It also enables us to evaluate the richness of fungal populations more efficiently and reliably. In the present study, we used the Ion Torrent PGM next generation sequencing platform to analyse fungi present in ordinary household dishwashers. The identification was based on massive parallel sequencing of the D2 LSU rRNA amplicon. The analysis revealed rich and diverse fungal communities present in our dishwashers. Interpretation of the results was based on previously published research by Zalar et al. (2011). The results of our study confirmed that the new technology in many ways surpasses classical methods used in fungal analysis by offering quicker, reliable, more sensitive and inexpensive high-throughput identification of microorganisms in entire communities.
Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP
C) and the ...disease-associated (PrP
Sc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP
Sc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP
C and PrP
Sc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP
Sc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.
The investigation of the microbial community change in the biofilm, growing on the walls of a containment tank of TRIGA nuclear reactor revealed a thriving community in an oligotrophic and ...heavy-metal-laden environment, periodically exposed to high pulses of ionizing radiation (IR). We observed a vertical IR resistance/tolerance stratification of microbial genera, with higher resistance and less diversity closer to the reactor core. One of the isolated
strains survived 15 kGy of combined gamma and proton radiation, which was surprising. It appears that there is a succession of genera that colonizes or re-colonizes new or IR-sterilized surfaces, led by
and/or
, upon which a photoautotrophic and diazotrophic community is established within a fortnight. The temporal progression of the biofilm community was evaluated also as a proxy for microbial response to radiological contamination events. This indicated there is a need for better dose-response models that could describe microbial response to contamination events. Overall, TRIGA nuclear reactor offers a unique insight into IR microbiology and provides useful means to study relevant microbial dose-thresholds during and after radiological contamination.
The formation of giant hailstones is a rare weather event that has devastating consequences in inhabited areas. This hazard has been occurring more frequently and with greater size of hailstones in ...recent years, and thus needs to be better understood. While the generally accepted mechanism is thought to be a process similar to the formation of smaller hailstones but with exceptional duration and stronger updrafts, recent evidence suggests that biotic and abiotic factors also influence the growth of these unusually large ice chunks. In this study, we improved these findings by determining the distribution of a wide variety of these factors throughout the hail volume and expanding the search to include new particles that are common in the environment and are of anthropogenic origin. We melted the concentric layers of several giant hailstones that fell to the ground over a small region in Slovenia in 2019. The samples, up to 13 cm in diameter, were analyzed for biotic and abiotic constituents that could have influenced their formation. Using 16S rRNA-based metagenomics approaches, we identified a highly diverse bacterial community, and by using scanning electron microscopy and Raman spectroscopy, we found natural and synthetic fibers concentrated in the cores of the giant hailstones. For the first time, we were able to detect the existence of microplastic fibers in giant hailstones and determine the changes in the distribution of sand within the volume of the samples. Our results suggest that changes in the composition of hail layers and their great diversity are important factors that should be considered in research. It also appears that anthropogenic microfiber pollutants were a significant factor in the formation of the giant hailstones analyzed in this study.
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•The first evidence of fibrous microplastics has been found in a hailstone.•Giant hailstones are rich in abiotic and biotic structures.•A highly diverse bacterial community structure was identified in hailstones.•Fibrous particles may affect the formation of hailstones.
The abandoned Sitarjevec mine represents a specific environment (complete darkness; 10 °C) with waters of pH 3 and it contains various ores (e.g. lead, mercury, zinc, iron, copper) and many different ...minerals (e.g. anglesite, baryte, cinnabar, pyrite, cerussite, quartz, siderite). The aim of this study was to characterize the iron oxide/hydroxide speleothems that show rapid growth of up to 5 cm/year. Their morphological and chemical characterization showed that they have a layered structure with different ferrihydrite minerals (i.e. goethite, feroxyhyte, lepidocrocite). Scanning electron microscopy-energy dispersive X-ray spectrometry confirmed that the surface layers were very porous, while for the inner parts, the bulk was compact. Bacterial populations in these speleothems and their environment were analyzed using 16S rRNA sequencing. These data revealed a great diversity of bacteria, which included 19-34 phyla across different samples. Proteobacteria were dominant in all of the samples (60-95%), although the highest bacterial diversity was seen for a water sample from the base of the speleothem, with 670 different genera and 100 different species detected. Among these there were typical iron-oxidizing bacteria, like Gallionella capsiferriformans, Sideroxydans lithotrophicus, Sphingomonas echinoides, Candidatus Nitrotoga Nitrotoga, Leptospirillum ferrooxidans, and Ferrovum myxofaciens. These iron-oxidizing bacteria were not present in other samples from the mine. Therefore, these bacteria might be involved in the structural growth of these speleothems. The specific bacterial interactions with the minerals under the environmental conditions in the mine need to be further analyzed and evaluated.
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