1,721,066 research outputs found
A Microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens
No full textA major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays
Benefits and challenges of upcoming microbial plant protection applications sustaining planetary health
No full textPlant disease outbreaks pose severe risks to global food security. Due to climate change, new diseases are expected to emerge, and the current use of chemical pesticides poses risks to environmental and human health. In the last decade, alternative plant protection agents of microbial origin have been developed, which also raise great expectations in the industry. Current products primarily represent individual microbial strains, either fungi or bacteria, which occasionally fail under field conditions due to various factors while their regulatory status differs globally. Recently, more diverse applications have started to emerge, ranging from microbial consortia, phages and protists to microbiome modulation or soil translocation. Integrated solutions, incorporating artificial intelligence are also proposed. In this review, we discuss the opportunities and challenges of these solutions, providing specific examples and discuss the regulatory needs for their market entry as well as their relevance for improving food security and planetary health.Authors received funding by the Horizon Europe project RATION (“Risk assessment of low-risk pesticides”, HORIZON-CL6-2022-FARM2FORK-01, HORIZON-RIA, no. 101084163).Peer reviewe
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Optimization of the PCR for microarray-based detection of pathogens
Full text linkDas von Kostic et al. entwickelte mikrobielle diagnostische Mikroarray ermöglicht die spezifische Detektion von Pathogenen in Lebensmitteln und Wasser mit Hilfe des phylogenetisch robusten gyrB Gens als diagnostischen Marker. Das System ist allerdings nicht sensitiv genug, wobei die Amplifikation des gyrB Gens der limitierende Faktor ist. Im Rahmen dieser Arbeit, sollte daher die gyrB PCR optimiert werden. Die folgenden Ansätze wurden dafür verwendet: i) nested PCR mit Primern komplementär zum sequencing tag der ursprünglichen, degenerierten Primer, die 1995 von Yamamoto und Harayama publiziert wurden, und ii) Design neuer Primer. Die nested PCR lieferte keine reproduzierbaren Ergebnisse, daher wurden neue Primer spezifisch für die folgenden Spezies designed: Salmonella spp., E. coli, Y. enterocolitica, Y. pseudotuberculosis, S. aureus, C.jejuni, C. lari, C. coli, C. upsaliensis, C. perfringens and C.difficile. Die Primer wurden individuell und als Primermix mit gDNA der einzelnen Stämme sowie mit gDNA-Mischungen getestet. Der Primermix mit einer Primerkonzentration von je 150nM lieferte die besten Ergebnisse, wobei die Nachweisgrenze von S. Typhimurium um mindestens zwei Log-Stufen niedriger war. Experimente mit einer gDNA-Mischung von vier verschiedenen Organismen haben gezeigt, dass nach Amplifikation mit den neuen Primern alle bei einer Konzentration von 50ng/µl nachgewiesen werden konnten, während nur zwei Organismen nach Amplifikation mit den alten Primern detektiert wurden. Außerdem wurden Experimente mit gespickten Lebensmittelproben durchgeführt, die die Anwendbarkeit der Primer für die Lebensmittelanalyse bestätigten.The microbial diagnostic microarray (MDM) that was developed by Kostic et al. (2007, 2010) allows the highly specific detection of food- and water-borne pathogens using the phylogenetically robust gyrB gene as diagnostic marker. However, the system lacks sensitivity; the amplification of the gyrB gene being the limiting factor. Thus, the gyrB PCR should be optimized in the course of this project. Different approaches were tried in order to do so: i) a nested PCR targeting the sequencing tags of the original, highly degenerate gyrB primers that were published by Yamamoto and Harayama (1995) and ii) new primers were designed. The nested approach failed to yield reproducible results in the initial experiments and had to be discontinued. Therefore, new species-specific primers were designed for the set of selected species (Salmonella spp., E. coli, Y. enterocolitica, Y. pseudotuberculosis, S. aureus, C. jejuni, C. lari, C. coli, C. upsaliensis, C. perfringens and C. difficile) and tested individually and as primer mix using both single strain gDNA and gDNA mixes. It was found that a primer mix with a concentration of 150nM each performed best, improving the limit of detection (LOD) of the MDM by at least two log steps for S. Typhimurium. Tests with a mix containing gDNA from four different organisms showed that all of them could be detected at concentration of 50ng/µl when amplified with the new primers, while only two could be detected after amplification with the old primers. Furthermore, experiments with spiked food samples were conducted, showing that the new primers are suitable for the application in food analysis.<br /
Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops
No full textRhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium.sponsorship: We thank IWT (Agency for Innovation by Science and Technology) to support this research (project IWT-LA: 120761). (IWT (Agency for Innovation by Science and Technology)|IWT-LA: 120761)status: Publishe
Antimicrobial drimane sesquiterpenes and their effect on endophyte communities in the medical tree Warburgia ugandensis
No full textMetabolite profiles (GC–MS), drimane sesquiterpenes, sugars and sugar alcohols, were compared with bacterial and fungal endophyte communities (T-RFLP, DNA clones, qPCR) in leaves and roots of the pepper bark tree, Warburgia ugandensis (Canellaceae). Ten individuals each were assessed from two locations east and west of the Great Rift Valley, Kenya, Africa, which differed in humidity and vegetation, closed forest versus open savannah. Despite organ- and partially site-specific variation of drimane sesquiterpenes, no clear effects on bacterial and fungal endophyte communities could be detected. The former were dominated by gram-negative Gammaproteobacteria, Pseudomonadaceae and Enterobacteriaceae, as well as gram-positive Firmicutes; the fungal endophyte communities were more diverse but no specific groups dominated. Despite initial expectations, the endophyte community of the pepper bark tree did not differ from other trees that much
miCROPe 2019 – emerging research priorities towards microbe-assisted crop production
Full text linkThe miCROPe 2019 symposium, which took place from 2 to 5 December 2019 in Vienna, Austria, has unified researchers and industry from around the world to discuss opportunities, challenges and needs of microbe-assisted crop production. There is broad consensus that microorganisms—with their abilities to alleviate biotic and abiotic stresses and to improve plant nutrition—offer countless opportunities to enhance plant productivity and to ameliorate agricultural sustainability.
However, microbe-assisted cultivation approaches face challenges that need to be addressed before a breakthrough of such technologies can be expected. Following up on the miCROPe symposium and a linked satellite workshop on breeding for beneficial plant–microbe interactions, we carved out research priorities towards successful implementation of microbiome knowledge for modern agriculture. These include (i) to solve context dependency for microbial inoculation approaches and (ii) to identify the genetic determinants to allow breeding for beneficial plant–microbiome interactions. With the combination of emerging third generation sequencing technologies and new causal research approaches, we now stand at the crossroad of utilising microbe-assisted crop production as a reliable and sustainable agronomic practice
Development and evaluation of molecular assays for microbial water quality assessment
Full text linkAlternative DNA-basierter molekulare Methoden ermöglichen eine schnellere und spezifischere Analyse in der Wasserqualitätsbewertung mit erhöhtem Probendurchsatz im Vergleich zu zeitintensiven Kultivierungsverfahren. Die Verwendung der real-time (RT-PCR) in Kombination mit der Behandlung von Propidium Monoazide (PMA), zur Differenzierung von DNA aus lebenden und toten Bakterienzellen, wurde für alle in der Trinkwasserverordnung (TVO, BGBl. II Nr. 304/2001) definierten mikrobiellen Parameter (E .coli, coliforme, Enterococcus spp., P. aeruginosa und Keimzahlbestimmung (KBE)) etabliert. In der Entwicklungsphase wurde das Potential der Lebend/Tot Differenzierung, durch Verwendung von 10 µM PMA anhand der signifikanten (3log10) oder kompletten Unterdrückung von DNA aus hitze-getöteten E. coli und P. aeruginosa Zellen in einer Probe mit abundanter Wassermikroflora demonstriert. Das Anwendungspotenzial der PMA-RT-PCR wurde in einer Evaluierung zu Referenzverfahren und RT-PCR ohne PMA mit einer Vielzahl an Trinkwasserproben und Prozesswasserproben getestet und resultierte in einer hohen Spezifität für die Detektion von E. coli, Enterococcus spp., P. aeruginosa, jedoch zeigte sich die Limitation der Sensitivität und der Detektion von coliformen Keimen und der KBE. Zudem wurde die KBE und deren Kultivierungsparameter, definiert in EN ISO 6222:1999 und EPA, auf die Bakterienökologie durch 16S rRNA Sequenzanalyse betrachtet. Eine signifikante Auswirkung auf das präsente Bakterienspektrum konnte durch die Kultivierungsparameter beobachtet werden, wobei signifikante Effekte durch den Parameter der Temperatur (p< 0.01) statistisch bewertet werden konnten. Zusammenfassend lässt sich sagen, dass die KBE Bestimmung bezugnehmend auf deren Aussage des Qualitätszustands einer einzeln analysierten Wasserprobe überdacht werden sollte. Kultivierungsmethoden als standardisierte Verfahren sind derzeit in der Wasserqualitätsbewertung noch nicht durch molekulare Methoden zu ersetzen. Gelingt es die Sensitivität zu verbessern, birgt sich eine gute Verwertungschance in der PMA-RT-PCR Methode durch die hohe Spezifität und die Gewährleistung der schnelleren Aussage um ein rasches Eingreifen im Kontaminationsfall zu ermöglichen.Alternative DNA-based molecular assay enable a rapid, specific and high-throughput analysis in water quality assessment in comparison to laborious cultivation-based techniques. Real-time (RT)-PCR in combination with the treatment of propidium monoazide (PMA) was developed for the detection of microbial parameters specified by the Austrian Drinking Water Regulation (BGBl. II Nr. 304/2001), i.e. E. coli, coliforms, Enterococcus spp., P. aeruginosa and heterotrophic plate count (HPC). The PMA treatment was included into RT-PCR analysis to allow for discrimination between DNA targets from living and dead organism, which is prerequisite for quality assessment, as only viable cells are determined by standardized techniques and may pose a health risks. In the proof of principle study the live/dead discrimination potential of PMA-RT-PCR was demonstrated. Treatment with 10 µM PMA resulted in the significant (3 log10) or complete suppression of false positive signals arising from heat-killed E. coli and P. aeruginosa from samples with an abundant background water microflora. The application potential of PMA-RT-PCR in comparison to conventional reference methods and RT-PCR without PMA was approved in an extended evaluation with a set of drinking and process water samples. Best performance due to high specificity of PMA-RT-PCR was achieved for E. coli, Enterococcus spp. and P. aeruginosa. Challenges remained in the sensitivity and the establishment of molecular assays for the detection of coliforms and total bacterial count. Further the HPC used as quality parameter was assessed in detail for the composition of culturable HPC community under different cultivation conditions, outlined in EN ISO 6222:1999 and EPA, by 16S rRNA gene sequence analysis. HPC communities revealed significant differences in microbial composition accordingly to cultivation parameter applied, with significant effects of temperature (p< 0.01). Summarizing the findings, the significance of HPC in water quality assessment should be reconsidered for isolated assessment of a single water sample. At present cultivation-based methods for water quality assessment cannot be replaced by molecular assays, but given the careful optimization for improving sensitivity and refinements in PMA-RT-PCR, molecular assays represent a promising and valuable detection method due to the high specificity and rapid analysis, to allow immediate actions in case of a determined contamination
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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