Absolute quantification of priority bacteria in aquaculture using digital PCR

Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans.publishedVersio

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.publishedVersio

Dual-polarized chipless humidity sensor tag

In this letter, a miniaturized, flexible and high data dense dual-polarized chipless radio frequency identification (RFID) tag is presented. The tag is designed within a minuscule footprint of 29 × 29 mm2 and has the ability to encode 38-bit data. The tag is analyzed for flexible substrates including Kapton® HN DuPont™ and HP photopaper. The humidity sensing phenomenon is demonstrated by mapping the tag design, using silver nano-particle based conductive ink on HP photopaper substrate. It is observed that with the increasing moisture, the humidity sensing behavior is exhibited in RF range of 4.1–17.76 GHz. The low-cost, bendable and directly printable humidity sensor tag can be deployed in a number of intelligent tracking applications

Analysis of Compound Synergy in High-Throughput Cellular Screens by Population-Based Lifetime Modeling

Despite the successful introduction of potent anti-cancer therapeutics, most of these drugs lead to only modest tumor-shrinkage or transient responses, followed by re-growth of tumors. Combining different compounds has resulted in enhanced tumor control and prolonged survival. However, methods querying the efficacy of such combinations have been hampered by limited scalability, analytical resolution, statistical feasibility, or a combination thereof. We have developed a theoretical framework modeling cellular viability as a stochastic lifetime process to determine synergistic compound combinations from high-throughput cellular screens. We apply our method to data derived from chemical perturbations of 65 cancer cell lines with two inhibitors. Our analysis revealed synergy for the combination of both compounds in subsets of cell lines. By contrast, in cell lines in which inhibition of one of both targets was sufficient to induce cell death, no synergy was detected, compatible with the topology of the oncogenically activated signaling network. In summary, we provide a tool for the measurement of synergy strength for combination perturbation experiments that might help define pathway topologies and direct clinical trials

Infective endocarditis in intravenous drug abusers: an update

Infective endocarditis despite advances in diagnosis remains a common cause of hospitalization, with high morbidity and mortality rates. Through literature review it is possible to conclude that polymicrobial endocarditis occurs mainly in intravenous drug abusers with predominance in the right side of the heart, often with tricuspid valve involvement. This fact can be associated with the type of drug used by the patients; therefore, knowledge of the patient's history is critical for adjustment of the therapy. It is also important to emphasize that the most common combinations of organisms in polymicrobial infective endocarditis are: Staphylococcus aureus, Streptococcus pneumonia and Pseudomonas aeruginosa, as well as mixed cultures of Candida spp. and bacteria. A better understanding of the epidemiology and associated risk factors are required in order to develop an efficient therapy, although PE studies are difficult to perform due to the rarity of cases and lack of prospective cohorts.This work was supported by Portuguese Foundation for Science and Technology (FCT) through the grants SFRH/BPD/47693/2008, SFRH/BPD/20987/2004 and SFRH/BPD/72632/2010 attributed to Claudia Sousa, Claudia Botelho and Diana Rodrigues, respectively

Untersuchungen zur Glykolyse und zum L Serin Stoffwechsel in Corynebacterium glutamicum

The amino acid L-serine is a central metabolite in the metabolism and of increasing importance for the industrial use. The aim of this work was to investigate if L-serine can be produced biotech nologically by Corynebacterium glutamicum and to study the influence of L-serine degradation and the provision of glycolytic precursors. To improve the availability of glycolytic precursors for the L-serine biosynthesis, a pyruvate kinase and 3-phosphoglycerate mutase deletion mutant was constructed. It could be shown that pyruvate kinase is essential for growth both on the non PTS (phosphotransferase system) sugar ribose and on the gluconeogenetic substrates acetate and citrate. Global gene expression analyses and enzyme assays with a suppressor mutant showed, that the growth of the pyruvate kinase mutant on acetate and citrate could be restored by overexpression of the mez gene encoding the malic enzyme. Further it was shown, that the pyruvate kinase mutant accumulates glycolytic precursors for L-serine biosynthesis. To inactivate the 3-phosphoglycerate mutase, the functional expressed gene was identified by sequence analysis and growth experiments. A pgm deletion mutant was only able to grow in presence of both a glycolytic and a gluconeogenetic substrate. Thereby, the non-PTS sugar maltose has been metabolised referentially to glucose that is taken up via a PTS system. It has been shown that the mutant accumulates the unphosphorylated L-serine precursor glycerate. To investigate the L-serine degradation the L-serine dehydratase encoding gene was identified in the C. glutamicum genome by sequence comparison. The subsequent deletion of the sdaA gene resulted in a 1.8-fold decreased L-serine degradation rate in comparison to the wild type but the overexpression of sdaA led to a 2-fold increased degradation rate. Interestingly, the overexpression of sdaA enabled C. glutamicum to grow on L-serine. The deletion of the sdaA gene in a C. glutamicum strain overexpressing the L-serine biosynthesis genes revealed a 960-fold gain of the L-serine accumulation. After an additional deletion of either the 3-phosphoglycerate mutase or pyruvate kinase encoding gene, a further increase of the L-serine formation was found. Further it was shown that the deletion of sdaA and the simultaneous reduction of the serine hydroxymethyltransferase activity, another L-serine converting enzyme, in a strain overexpressing the L-serine biosynthesis genes, led to a tremendous increase of L-serine accumulation up to 100 mM

The influences of phytoplankton species, mineral particles and concentrations of dispersed oil on the formation and fate of marine oil-related aggregates

The formation and fallout of oil-related marine snow have been associated with interactions between dispersed oil and small marine particles, like phytoplankton and mineral particles. In these studies, the influences of phytoplankton species, mineral particle concentration, and oil concentration on the aggregation of oil in seawater (SW) were investigated. The experiments were performed in a low-turbidity carousel incubation system, using natural SW at 13 °C. Aggregation was measured by silhouette camera analyses, and oil compound group distribution and depletion by gas chromatography (GC-FID or GC–MS). Aggregates with median sizes larger than 500 μm in diameter were measured in the presence of dispersed oil and the phytoplankton species Thalassiosira rotula, Phaeocystis globosa, Skeletonema pseudocostatum, but not with the microalgae Micromonas pusilla. When mineral particles (diatomaceous earth) were incubated at different concentrations (5–30 mg/L) with dispersed oil and S. pseudocostatum, the largest aggregates were measured at the lower mineral particle concentration (5 mg/L). Since dispersed oil rapidly dilutes in the marine water column, experiments were performed with oil concentrations of from 10 mg/L to 0.01 mg/L in the presence of S. pseudocostatum and diatomaceous earth. Aggregates larger than 500 μm was measured only at the highest oil concentrations (10 mg/L). However, oil attachment to the marine particles were also measured at low oil concentrations (≤1 mg/L). Depletion of oil compound groups (n-alkanes, naphthalenes, PAHs, decalins) were measured at all oil concentrations, both in aggregate and water phases, with biodegradation as the expected main depletion process. These results showed that oil concentration may be important for oil-related marine snow formation, but that even oil droplets at low concentrations may attach to the particles and be transported by prevailing currents.publishedVersio