Flow cytometry, fluorescent probes, and flashing bacteria

Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, Lactococcus lactis , Lactobacillus plantarum , cheese, milk, probiotic In food industry there is a perceived need for rapid methods for detection and viability assessment of microbes. Fluorescent staining and flow cytometry provide excellent tools for microbial analysis. This thesis describes fluorescent techniques for assessment of the physiological state of lactic acid bacteria.Lysis of lactic acid bacteria plays a crucial role in cheese manufacturing. It is generally considered that lysis results in leakage of intracellular enzymes in the cheese curd and, thus, plays an important role in ripening and flavor formation. Bac Light (Molecular Probes) was applied for monitoring the lysis process of Lactococcus lactis MG1363 in a buffered suspension with high osmolarity to mimic cheese conditions. The Bac Light kit combines the nucleic acid dyes propidium iodide (PI) and SYTO 9. PI is commonly used to determine membrane integrity based on dye exclusion. When used in combination with the permeant SYTO 9, membrane-damaged cells are stained by PI (red) while the intact cells are stained by SYTO 9 (green). Lysis was induced with mutanolysin and followed in time using fluorescence microscopy and flow cytometry. Also, enzyme assays and plate counts were performed. The results demonstrated a transient permeable cell status that has a significant role in the lysis process. Furthermore, permeable cells were demonstrated in ripening cheese with confocal scanning laser microcopy and Bac Light.Viability assessment by conventional plate counting requires long incubation times and provides limited information. Flow cytometric assessment of the viability of lactic acid bacteria was investigated and compared with plate counts. The esterase substrate carboxyfluorescein diacetate (cFDA) and the impermeant nucleic acid dyes PI and TOTO-1 were tested using exponential phase at 70°C heat-killed cultures of a Lactococcus , a Streptococcus , three Lactobacillus , two Leuconostoc , an Enterococcus , and a Pediococcus species. The combination of cFDA and TOTO-1 gave the best results. The intact and membrane-damaged subpopulations were distinguished well. Sorting and plating showed that cFDA stained the culturable and TOTO-1 the nonculturable cells. The assay was applied to cultures exposed to deconjugated bile salts or to hydrochloric acid and results corresponded well with plate counts.Subsequently, flow cytometry with cFDA and TOTO-1 staining was applied to Lactobacillus plantarum WCFS 1 suspended in milk. To facilitate flow cytometry clearing of the milk was required. A procedure based on a milk clearing solution was optimized to increase the signal-to-noise-ratio and flow cytometry enumerations were accurate to a lower limit of 10 5cells/ml.Finally, the novel assay was applied to starter cultures for cheese and yogurt and to the probiotic products Yakult, Mona Vifit, and Orthiflorplus. Flow cytometry in combination with plate counts revealed three populations: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed between the tested products.In conclusion, the development of flow cytometry for bacteria is an important asset for microbiological research. The rapid novel methods described in this thesis provide possibilities for examination of fermentation processes and food products

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase Implications for NADPH recognition and structural stability

AbstractPhe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH

PLATFORM policy brief No. 3. The role of the ERA-NET instrument in fostering inclusiveness

In the spirit of building an inclusive European Research Area, this policy brief provides recommendations for the European Commission (EC), ERA -NET initiatives and the so -called lower performing countries (LPCs) on how LPCs can become an integral and emp owered part of the ERA -NET community

A randomized crossover study comparing different tacrolimus formulations to reduce intrapatient variability in tacrolimus exposure in kidney transplant recipients

A high intrapatient variability (IPV) in tacrolimus exposure is a risk factor for poor long-term outcomes after kidney transplantation. The main objective of this trial was to investigate whether tacrolimus IPV decreases after switching patients from immediate-release (IR)-tacrolimus to either extended-release (ER)-tacrolimus or LifeCyclePharma (LCP)-tacrolimus. In this randomized, prospective, open-label, cross-over trial, adult kidney transplant recipients on a stable immunosuppressive regimen, including IR-tacrolimus, were randomized for conversion to ER-tacrolimus or LCP-tacrolimus, and for the order in which IR-tacrolimus and the once-daily formulations were taken. Patients were followed 6 months for each formulation, with monthly tacrolimus predose concentration assessments to calculate the IPV. The IPV was defined as the coefficient of variation (%) of dose corrected predose concentrations. Ninety-two patients were included for analysis of the primary outcome. No significant differences between the IPV of IR-tacrolimus (16.6%) and the combined once-daily formulations (18.3%) were observed (% difference +1.7%, 95% confidence interval [CI] -1.1% to -4.5%, p = 0.24). The IPV of LCP-tacrolimus (20.1%) was not significantly different from the IPV of ER-tacrolimus (16.5%, % difference +3.6%, 95% CI -0.1% to 7.3%, p = 0.06). In conclusion, the IPV did not decrease after switching from IR-tacrolimus to either ER-tacrolimus or LCP-tacrolimus. These results provide no arguments to switch kidney transplant recipients from twice-daily (IR) tacrolimus formulations to once-daily (modified-release) tacrolimus formulations when the aim is to lower the IPV.Personalised Therapeutic

Report of the joint workshop "Smart Mitigation of GHG in livestock production", 29th and 30th November 2016, in Potsdam, Germany

FACCE ERA-GAS (ERA-NET Cofund for Monitoring & Mitigation of Greenhouse gases from Agri- and Silvi-culture), together with the ERA-NET SusAn, (Sustainable Animal Production Systems) and ERA-NET ICT-AGRI 2 (Information and Communication Technologies and Robotics for Sustainable Agriculture) organized a joint workshop on 29-30 November in Potsdam to identify promising approaches to reduce GHG emissions in livestock production. The joint workshop, the first of its kind involving three ERA-NETs, had close to 70 participants from 22 different countries. The three ERA-NETs have already identified a number of potential areas of synergy. This workshop explored one of those areas in detail: Comparison of animal production systems with respect to GHGs. Particular attention was paid to the following two sub-topics: (1) Production technology and management (e.g. housing systems; optimal field and grazing management), and (2) Breeding, physiology, feed & nutrition.The outputs of the workshop will help to set the research priorities for future joint calls and other activities between the three ERA-NETs