Evaluation of enrichment-free PCR-based detection on the rfbE gene of Escherichia coli O157--application to municipal wastewater

Escherichia coli O157 strains have emerged as important human enteric pathogens. Strains that express the O-antigen 157 are commonly associated with severe clinical manifestations, including bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. E. coli O157 strains may be transmitted in a variety of ways, including food, water and person-to-person or animal-to-person contact. Fecal contamination is one source of environmental contamination and is responsible for the presence of these pathogens in the environment. We used a specific and sensitive PCR assay based on the rfbE gene to detect low levels of these pathogens in wastewater. The set of primers used was designed to amplify an intragenic segment of the rfbE gene. The amplification assay detected 200 CFU of E. coli O157 in pure water. The prevalence of E. coli O157 in the effluents of 44 wastewater treatment plants was determined (7%).     

Impact of microparticles on UV disinfection of indigenous aerobic spores

Numerous studies have shown that the efficacy of ultraviolet (UV) disinfection can be hindered by the presence of particles that can shield microorganisms. The main objective of this study was to determine to what extent natural particulate matter can shield indigenous spores of aerobic spore-forming bacteria (ASFB) from UV rays. The extent of the protective shielding was assessed by comparing the inactivation rates in three water fractions (untreated, dispersed and filtered on an 8 microm membrane) using a collimated beam apparatus with a low-pressure lamp emitting at 254 nm. Levels of inactivation were then related to the distribution and abundance of particles as measured by microflow imaging. Disinfection assays were completed on two source waters of different quality and particle content. A protocol was developed to break down particles and disperse aggregates (addition of 100mg/L of Zwittergent 3-12 and blending at 8000 rpm for 4 min). Particle size distribution (PSD) analysis confirmed a statistically significant decrease in the number of particles for diameter ranges above 5 microm following the dispersion protocol and 8 microm filtration. The fluence required to reach 1-log inactivation of ASFB spores was independent of particle concentration, while that required to reach 2-log inactivation or more was correlated with the concentration of particles larger than 8 microm (R(2)>0.61). Results suggest that natural particulate matter can protect indigenous organisms from UV radiation in waters with elevated particle content, while source water with low particle counts may not be subject to this interference.     

Parameter estimation and prediction uncertainties for multi-response kinetic models with uncertain inputs

Error-in-variables model (EVM) methods are used for parameter estimation when independent variables are uncertain. During EVM parameter estimation, output measurement variances are required as weighting factors in the objective function. These variances can be estimated based on data from replicate experiments. However, conducting replicates is complicated when independent variables are uncertain. Instead, pseudo-replicate runs may be performed where the target values of inputs for repeated runs are the same, but the true input values may be different. Here, we propose a method to estimate output-measurement variances for use in multivariate EVM estimation problems, based on pseudo-replicate data. We also propose a bootstrap technique for quantifying uncertainties in resulting parameter estimates and model predictions. The methods are illustrated using a case study involving n-hexane hydroisomerization in a well-mixed reactor. Case-study results reveal that assumptions about input uncertainties can have important influences on parameter estimates, model predictions and their confidence intervals.     

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.     

Correlated changes in skeletal muscle connective tissue and flesh texture during starvation and re‐feeding in brown trout (Salmo trutta) reared in seawater

Quantitative and qualitative changes to muscle and collagen were analysed following starvation and re‐feeding of brown trout (Salmo trutta) reared in seawater. Fish were submitted to starvation for 2 months followed by re‐feeding for 1 month and compared with a control group continuously fed. Classical effects of starvation on growth and morphometrics traits were observed with only a partial recovery of these parameters after 1 month of re‐feeding. Muscle composition of starved fish was significantly affected (lower dry matter content and higher post‐mortem pH) compared with control fish and was partially recovered in re‐fed fish compared with continuously fed fish. Muscle structure and composition were affected with thinner muscle fibre and higher connective tissue content for the starved fish but similar thickness of myosepta compared with the control group. No difference was observed after 1 month of re‐feeding. Characteristics of the connective tissue were significantly affected by starvation (more high weight molecular collagen form, higher thermal stability of skin collagen). These differences remained significant after the re‐feeding period. Starved fish showed also higher mechanical resistance of the raw flesh compared with the control group, but no difference in rheological measurements was observed after 1 month of re‐feeding. The changes in texture and their relationships with muscle composition, muscle structure and collagen characteristics are discussed. Copyright © 2004 Society of Chemical Industry     

Optimisation of the secondary ball mill using an on-line ball and pulp load sensor – The Sensomag

The ball load and pulp load have a significant influence on the ball mill product size and production capacity. To improve the circuit performance at industrial scale these variables must be tweaked to levels where the plant can get grind and capacity benefits. In most grinding circuits the influence of these variables are not quantified because it is difficult to obtain precise measurements of the pulp load for an industrial scale mill and the conventional method of obtaining ball load measurements that involves crash stops is not attractive. A comprehensive investigation was performed on an industrial scale mill to quantify the effects of both ball and pulp load. A wide range of ball and pulp loads were tested and the findings are reported in this paper. The Sensomag, a sensor developed by Magotteaux, was used to obtain ball and pulp load measurements during the experimental work.     

Fracture toughness of coated TiCN–WC–Co cermets with graded composition

Mixed TiCN–WC–Co cermets are developed to improve at the same time toughness and resistance to deformation of materials for cutting tool applications. Moreover, graded materials joining optimum properties according to the functional part of the tool are elaborated. To this end, TiCN–WC–Co cermets are interesting because they develop a WC–Co layer at the surface during the sintering. This tough layer at the surface limits the crack propagation that can lead to the rupture of the tool. Such materials show a good resistance to the deformation in the bulk and a good toughness at the surface, where the cracks are initiated upon machining. Cutting tools are often coated by CVD to improve the wear resistance. This paper proposes a method to measure the toughness K_IC at high temperature by using this CVD coating for initial crack formation. The coating thickness is the precrack length of traditional K_IC measurements. Samples are fractured by three point bend tests. The rupture stress is measured by Weibull statistics. This method is particularly interesting for graded structure materials where the influence of surface layers on toughness must be estimated. The comparison between cermets with and without WC–Co layer shows an improvement of 28% of the toughness when the layer is present. The possible bias of internal stresses on the results is discussed.