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We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) – and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl−1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice. © 2020 The Royal Society of Chemistry., Lab on a Chip, 20 (14), ISSN:1473-0197, ISSN:1473-0189

In the past 30 years several studies have proved that the rivers flow regime is fundamental in structuring biotic communities. The condition of the ecosystem results from the occurrence of extreme events, such as floods and droughts, but also from habitat availability and from its temporal variation. Describe the relationships between macroinvertebrate community characteristics and environmental gradients of flow, temperature, nutrient supplies and habitat conditions is fundamental to understand ecological dynamics. This is the basis for predicting changes within the communities and in ecosystem functions and ultimately to properly manage and conserve the riverine ecosystems. Seven sites, along a 20-km river sector, were surveyed for macroinvertebrates and water chemistry seasonally, from 2012 to 2016. Habitat conditions were assessed along a 500 m stretch in each site. The river discharge was continuously monitored by two water level recorders and used to reconstruct various hydrological indices specific for each sampling location. During the sampling period numerous high flow events and some prolonged periods of low flow were observed. Quantile regression was used to describe the effects of potential limiting factors on macroinvertebrate community which were primarily driven by antecedent flow conditions and season while habitat conditions and water chemistry played only a minor role. Quantitative models have been developed to predict structural and functional characteristics of macroinvertebrate community as a function of antecedent flow conditions, habitat and physico-chemical water characteristics. Those models allow to identify the main drivers and predict the effect of different water management strategies to riverine ecosystem.

Nutrient transfer is a key feature of the arbuscular mycorrhizal (AM) symbiosis.Valuable mineral nutrients are transferred from the AM fungus to the plant, increasing its fitness and productivity, and, in exchange, the AM fungus receives carbohydrates as an energy source from the plant.Here, we analyzed the transcriptome of the Populus trichocarpa-Rhizophagus irregularis symbiosis using RNA-sequencing of non-mycorrhizal or mycorrhizal fine roots, with a focus on the effect of nitrogen (N) starvation. In R. irregularis, we identified 1,015 differentially expressed genes, whereby N starvation led to a general induction of gene expression. Genes of the functional classes of cell growth, membrane biogenesis and cell structural components were highly abundant. Interestingly, N starvation also led to a general induction of fungal transporters, indicating increased nutrient demand upon N starvation. In non-mycorrhizal P. trichocarpa roots, 1,341 genes were differentially expressed under N starvation. Among the 953 down-regulated genes in N starvation, most were involved in metabolic processes including amino acids, carbohydrate and inorganic ion transport, while the 342 up-regulated genes included many defense-related genes. Mycorrhization led to the up-regulation of 549 genes mainly involved in secondary metabolite biosynthesis and transport; only 24 genes were down-regulated.Mycorrhization specifically induced expression of three ammonium transporters and one phosphate transporter, independently of the N conditions, corroborating the hypothesis that these transporters are important for symbiotic nutrient exchange. In conclusion, our data establish a framework of gene expression in the two symbiotic partners under high-N and low-N conditions.

In natural and agricultural ecosystems, arbuscular mycorrhizal (AM) fungi play a major role in plant nutrition. In AM symbiosis, the AM fungi extract mineral nutrients from the substrate and transfer them to the host plant. Inside the roots of the host plant, the intraradical hyphae form tree like structures (arbuscules) where the nutrients are released to the plant fungal interface. In return, the AM fungi receive carbohydrates from the plants. Specialized transport systems enable nutrient uptake from the substrate and translocation across membranes. As main components of organic molecules, phosphorus (P), nitrogen (N) and carbon (C) are of particular importance for symbiotic nutrient exchanges. This work is focused on a range of genes that encode proteins contributing to transport molecules (P, N and C nutrients) across cellular membranes in the plants Populus trichocarpa (poplar) and Sorghum bicolor (sorghum), and in the AM fungus Rhizophagus irregularis. In the AM fungus R. irregularis (formerly Glomus intraradices), we identified and characterized a novel functional ammonium transporter (AMT), GintAMT3. Quantification of transcript abundances in the extraradical mycelium (ERM) and the intraradical mycelium (IRM) during symbiosis with poplar and sorghum revealed that GintAMT3 was highly expressed in the IRM of AM roots. Phylogenetic analysis showed further, that the six glomeromycotan AMTs share high sequence similarity, but are distinct to AMTs of other fungal phyla. To functionaly analyze GintAMT3, we expressed GintAMT3 in a yeast deletion mutant devoid of all AMTs. The heterologous expression revealed that GintAMT3 is a low affinity transporter. Heterologous expression of GFP tagged GintAMT3 in yeast showed that GintAMT3 is localized in the plasma membrane and the vacuolar membrane. Further, we could show that expression of GinAMT3 is dependent on the N nutrition status and the fungal C status. Taken together, our data suggested that GintAMT3 is the main export carrier for ammonium at the arbuscular site. Using mRNA sequencing, we could show that low N availability significantly increased gene expression of the AM fungus, including genes involved in cell growth and membrane biogenesis as well as genes involved in signaling and metabolic processes. High abundances of genes related to N metabolism, including glutamine synthase, aminotransferase, AMTs as well as arginases, indicated a high turnover rate of N in the symbiotic root tissue. Depending on P availability, gene expression of AM phosphate transporters (PT) and AMT changed. Induction of PT and AMT under low-P availability indicated that the AM fungus transfers more nutrients to the host plant. Further, we identified amino acids transporters and H+/oligopeptide transporters specifically induced in mycorrhizal poplar roots, indicating that amino acids are transferred between the AM fungus and the plant. In poplar, we found that root colonization and low-N conditions resulted in the down-regulation of defense gene expression, suggesting that the plant stimulated symbiotic interactions with the AM fungus. We showed that root colonization specifically induced expression of known and newly identified PT and AMT in poplar and sorghum. Specific induction of nutrient transporters upon starvation strongly indicated that they are essential components of a functional symbiosis and suggested they are located in AM roots. Furthermore, root colonization suppressed the expression of genes involved in P starvation response, indicating that root colonization efficiently alleviated P stress of the plant. Moreover, we could show that the annual sorghum is more dependent on the AM fungus than the perennial poplar, but also that more P and possibly also more N is transferred from the AM fungus to the host plant. Non-mycorrhized sorghum accumulated similar quantities of P as AM sorghum under conditions, in which only the AM fungus had access to the P source. Poplar on the other hand accumulated less P in AM plants. In addition, we observed that a subset of poplar Pht1 transporters was regulated independently on the AM fungus, but depending on the P availability of the substrate. To deepen our understanding about symbiotic C exchange, we made transcriptome analysis and qRT-PCR to investigate the role of carbohydrate transporters in AM symbiosis between R. irregularis and, poplar and sorghum, respectively. In R. irregularis, the monosaccharide transporter GintMST2 was specifically induced in the IRM independently on the nutrient condition. Interestingly, we observed the down-regulation of many carbohydrate transporters in AM roots of poplar and sorghum. However, in poplar, we identified one carbohydrate transporter, which might be involved in symbiotic C transfer. In conclusion, our data on C transport suggested that carbohydrates are taken from the plant by the AM fungus instead of actively transferred to the fungus by the host plant. Taken together, the data summarized in my thesis add to our understanding of nutrient transport in AM symbiosis under different environmental conditions and help elucidating the underlying mechanisms. Regarding climate changes and resources shortening, a precise understanding of the efficiency of AM symbiosis may help to increase the efficiency of sustainable agriculture.

In arbuscular mycorrhizal (AM) symbiosis, key components of nutrient uptake and exchange are specialized transporters that facilitate nutrient transport across membranes. As phosphate is a nutrient and a regulator of nutrient exchanges, we investigated the effect of P availability to extraradical mycelium (ERM) on both plant and fungus transcriptomes and metabolomes in a symbiocosm system. By perturbing nutrient exchanges under the control of P, our objectives were to identify new fungal genes involved in nutrient transports, and to characterize in which extent the fungus differentially modulates its metabolism when interacting with two different plant species. We performed transportome analysis on the ERM and intraradical mycelium of the AM fungus Rhizophagus irregularis associated to Populus trichocarpa and Sorghum bicolor under high and low P availability in ERM, using quantitative RT-PCR and Illumina mRNA-sequencing. We observed that mycorrhizal symbiosis induces expression of specific phosphate and ammonium transporters in both plants. Furthermore, we identified new AM-inducible transporters and showed that a subset of phosphate transporters is regulated independently of symbiotic nutrient exchange. mRNA-Sequencing revealed that the fungal transportome was not similarly regulated in the two host plant species according to P availability. Mirroring this effect, many plant carbohydrate transporters were down-regulated in P. trichocarpa mycorrhizal root tissue. Metabolome analysis revealed further that AM root colonization led to a modification of root primary metabolism under low and high P availability and to a decrease of primary metabolite pools in general. Moreover, the down regulation of the sucrose transporters suggests that the plant limits carbohydrate long distance transport (i.e. from shoot to the mycorrhizal roots). By simultaneous uptake/reuptake of nutrients from the apoplast at the biotrophic interface, plant and fungus are both able to control reciprocal nutrient fluxes., Frontiers in Plant Science, 10, ISSN:1664-462X