Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus

The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.     

Can soil seed banks contribute to the restoration of dune slacks under conservation management?

Questions: Does the soil seed bank resemble the former early successional stages of a dune slack system more than the established later successional vegetation? Does it have the potential to contribute to the conservation of a highly endangered habitat? Location: Dune slacks at Newborough Warren, UK. Methods: The composition of the soil seed bank in two depth layers was determined using the seedling emergence method between March 2004 and April 2005. Long-term monitoring data on the floristic composition of the established vegetation were obtained from the national conservation agency, and additional monitoring was undertaken in 2003. Floristic composition, seed weights, seed longevity of component species and Ellenberg indicator values were used to compare the seed bank and established vegetation. Results: The soil seed bank was diverse and contained typical dune slack species, species of early successional stages and species of conservation interest. A comparison between the composition of the seed bank and historical data on the composition of the established vegetation showed that the seed bank reflects earlier successional stages more closely than the current aboveground vegetation. This study increases the scarce information currently available on the seed bank ecology of several species, including two orchid species. Conclusions: The soil seed bank can be expected to contribute to vegetation change after disturbance. Stimulation of germination from the seed bank through management may contribute to the conservation of both characteristic and threatened species typical of dune slacks.     

Identifying indicators of illegal behaviour: carnivore killing in human-managed landscapes

Managing natural resources often depends on influencing people's behaviour, however effectively targeting interventions to discourage environmentally harmful behaviours is challenging because those involved may be unwilling to identify themselves. Non-sensitive indicators of sensitive behaviours are therefore needed. Previous studies have investigated people's attitudes, assuming attitudes reflect behaviour. There has also been interest in using people's estimates of the proportion of their peers involved in sensitive behaviours to identify those involved, since people tend to assume that others behave like themselves. However, there has been little attempt to test the potential of such indicators. We use the randomized response technique (RRT), designed for investigating sensitive behaviours, to estimate the proportion of farmers in north-eastern South Africa killing carnivores, and use a modified logistic regression model to explore relationships between our best estimates of true behaviour (from RRT) and our proposed non-sensitive indicators (including farmers' attitudes, and estimates of peer-behaviour). Farmers' attitudes towards carnivores, question sensitivity and estimates of peers' behaviour, predict the likelihood of farmers killing carnivores. Attitude and estimates of peer-behaviour are useful indicators of involvement in illicit behaviours and may be used to identify groups of people to engage in interventions aimed at changing behaviour.     

The role of seasonality in lettuce consumption: a case study of environmental and social aspects

Background, aim and scope  Considerable debate surrounds the assessment of the environmental impacts and the ethical justification for providing a year-round supply of fresh produce to consumers in the developed countries of northern Europe. Society is seeking environmentally sustainable supply chains which maintain the variety of fresh food on offer throughout the year. This paper compares the environmental impacts of different supply chains providing lettuce all year round to the UK and considers consumers' meanings of—and attitudes to—available options. Lettuce has been selected as a case study as its consumption has grown steadily during the last two decades and the supply chains through cold months are protected cropping in the UK and field cropping in Spain; during warm months, lettuce is sourced from field cropping in the UK. Materials and methods  Data were collected from farms supplying each of these supply chains, and life cycle assessment methodology was used to analyse a range of impacts associated with producing (from plant propagation to harvesting and post-harvest cooling) and delivering 1 kg of lettuce to a UK Regional Distribution Centre (RDC). The downstream stages (i.e. retailing, consumption and waste management) are the same regardless of the origin of the product and were omitted from the comparison. The impacts considered included potential to induce global warming and acidification as well as three inventory indicators (primary energy use, land use and water use). Qualitative data were collected in order to assess the consumer considerations of purchasing lettuce also during winter. Results  Importation of Spanish field-grown lettuce into the UK during winter produced fewer greenhouse gas (GHG) emissions than lettuce produced in UK-protected systems at that time (0.4–0.5 vs. 1.5–3.7 kg CO₂-eq/kg lettuce in RDC). Refrigerated transport to the UK was an important element of the global warming potential associated with Spanish lettuce (42.5% of emissions), whilst energy for heating dominated the results in UK-protected cultivation (84.3% of emissions). Results for acidification were more variable and no overall trends are apparent. Results from qualitative social analysis revealed complex and multidimensional meanings of freshness and suggested that the most striking seasonal variation in vegetable/salad eating was a tendency to consume more salads in the summer and more cooked vegetables in the winter, thus suggesting that in-home consumption alone cannot explain the rise in winter imports of lettuce to the UK. Discussion  UK field-grown lettuce had the lowest overall environmental impact; however, those lettuces are only available in summer, so consumers therefore need to either accept the environmental impacts associated with eating lettuce in the winter or to switch consumption to another food product in the winter. When lettuces were field-grown in Spain and then transported by road to the UK, the overall impacts were similar to the UK field lettuces. The variation within farms of the same country employing different cultivation regimes and practices was bigger than between farms of different countries. Conclusions  This paper has explored the environmental consequences of consuming lettuce year-round in the UK. Whilst recognising the small sample size, the comparative analysis of the different supply chains does suggest that seasonality can be an important variable when defining the best choice of lettuce from an environmental point of view. Recommendations and perspectives  Further studies considering more production sites and product types are required to obtain conclusions whose general validity is clear and for different types of fresh produce. A clear distinction to be made in such studies is whether crops are produced in open fields or under protection. New characterisation methods are needed for environmental impacts derived from the use of key agricultural resources such as land and water. Social studies to investigate consumer preferences and the possibility of moving to more seasonal diets should be an integral part of these studies using samples composed of both urban and rural consumers and using a mixed methodology with both quantitative and qualitative components.

Translational Arrest Due to Cytoplasmic Redox Stress Delays Adaptation to Growth on Methanol and Heterologous Protein Expression in a Typical Fed-Batch Culture of Pichia pastoris

Results

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.

Conclusion

Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.     

Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility.

Intact cell mass spectrometry (ICMS) rapidly analyses the surface composition of microorganisms providing rapid, discriminatory fingerprints for identification and subtyping of important nosocomial pathogens such as methicillin resistant Staphylocccus aureus (MRSA). In this study, ICMS using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF/MS) was assessed for the identification and subtyping of MRSA. An intra- and inter-laboratory reproducibility study was carried out and the effects of culture media (an important source of variation for ICMS) were also studied. Several media used for the cultural identification of MRSA were examined using a panel of well-characterised staphylococcal isolates (n=26). Six MRSA isolates were analysed over a 1-month period for intra-laboratory reproducibility on the same instrument and three different culture media. Spectra were consistent for each isolate between the four experiments on the same culture medium. Individual isolates produced different spectral profiles on different culture media. Spectra from organisms grown on Columbia blood agar contained more peaks (approximately 120) compared to Columbia agar (approximately 50) and methicillin mannitol salt agar (approximately 25). All 26 staphylococcal isolates were subjected to an inter-laboratory study on two MALDI instruments. For each isolate, the overall spectral profile was the same for each of the two instruments but the baseline threshold values was adjusted due to instrument differences in detector sensitivities. Differences between certain regions of the spectra reproducibly identified isolates belonging to the two major MRSA strains (EMRSA phage group 15 and 16). These results demonstrate ICMS with appropriate media selection is a rapid and reproducible technique for identification and discrimination of MRSA.     

The sleeping policeman: understanding issues of enforcement and compliance in conservation

Rules governing human behaviour are at the heart of every system of natural resource management. Without compliance, however, rules are meaningless so effective enforcement is essential if conservation is to be successful. There is a large body of theory concerning enforcement and compliance with rules spread over several disciplines, including psychology, economics and sociology. However, there have been few attempts to extend this theory to conservation applications and there is little practical guidance for managers and conservation planners on the optimal design of enforcement programmes. We review approaches to understanding why individuals break rules and how optimal policy choices can reduce rule-breaking, highlighting research which has specifically dealt with natural resources. Because of the difficulty of studying rule-breaking behaviour directly, modelling approaches have been particularly important and have been used to explore behaviour at the individual, group and institutional levels. We illustrate the application of models of enforcement and compliance to conservation using the African elephant Loxodonta africana as a case study. Further work is needed to create practical tools which can be applied to the design of enforcement measures in conservation. Particular challenges include understanding the importance of violations of rationality assumptions and incorporating intertemporal choice in models of decision making. In conclusion, we argue that a new field of robust theory and practice is urgently needed to ensure that issues of enforcement and compliance do not undermine conservation initiatives.