Influence of Plant Species, Tissue Type, and Temperature on the Capacity of Shiga-Toxigenic Escherichia coli To Colonize, Grow, and Be Internalized by Plants.

Contamination of fresh produce with pathogenic Escherichia coli, including Shiga-toxigenic E. coli (STEC), represents a serious risk to human health. Colonization is governed by multiple bacterial and plant factors that can impact the probability and suitability of bacterial growth. Thus, we aimed to determine whether the growth potential of STEC for plants associated with foodborne outbreaks (two leafy vegetables and two sprouted seed species) is predictive of the colonization of living plants, as assessed from growth kinetics and biofilm formation in plant extracts. The fitness of STEC isolates was compared to that of environmental E. coli isolates at temperatures relevant to plant growth. Growth kinetics in plant extracts varied in a plant-dependent and isolate-dependent manner for all isolates, with spinach leaf lysates supporting the highest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified to be the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level of in planta colonization occurred on alfalfa sprouts, though internalization was 10 times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences in in planta growth meant that the growth potential of STEC could be inferred only for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonization. Overall, the capacity of E. coli to colonize, grow, and be internalized within plants or plant-derived matrices was influenced by the isolate type, plant species, plant tissue type, and temperature, complicating any straightforward relationship between in vitro and in planta behaviors.IMPORTANCE Fresh produce is an important vehicle for STEC transmission, and experimental evidence shows that STEC can colonize plants as secondary hosts, but differences in the capacity to colonize occur between different plant species and tissues. Therefore, an understanding of the impact that these plant factors have on the ability of STEC to grow and establish is required for food safety considerations and risk assessment. Here, we determined whether growth and the ability of STEC to form biofilms in plant extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonize living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) but not those for leafy vegetables (lettuce and spinach) exhibited a positive relationship between plant extracts and living plants. Therefore, the detailed variations at the level of the bacterial isolate, plant species, and tissue type all need to be considered in risk assessment.

A feasibility study for NOn-Traditional providers to support the management of Elderly People with Anxiety and Depression: The NOTEPAD study Protocol.

BACKGROUND: Anxiety and depression are common among older people, with up to 20% reporting such symptoms, and the prevalence increases with co-morbid chronic physical health problems. Access to treatment for anxiety and depression in this population is poor due to a combination of factors at the level of patient, practitioner and healthcare system. There is evidence to suggest that older people with anxiety and/or depression may benefit both from one-to-one interventions and group social or educational activities, which reduce loneliness, are participatory and offer some activity. Non-traditional providers (support workers) working within third-sector (voluntary) organisations are a valuable source of expertise within the community but are under-utilised by primary care practitioners. Such a resource could increase access to care, and be less stigmatising and more acceptable for older people. METHODS: The study is in three phases and this paper describes the protocol for phase III, which will evaluate the feasibility of recruiting general practices and patients into the study, and determine whether support workers can deliver the intervention to older people with sufficient fidelity and whether this approach is acceptable to patients, general practitioners and the third-sector providers. Phase III of the NOTEPAD study is a randomised controlled trial (RCT) that is individually randomised. It recruited participants from approximately six general practices in the UK. In total, 100 participants aged 65 years and over who score 10 or more on PHQ9 or GAD7 for anxiety or depression will be recruited and randomised to the intervention or usual general practice care. A mixed methods approach will be used and follow-up will be conducted 12 weeks post-randomisation. DISCUSSION: This study will inform the design and methods of a future full-scale RCT. TRIAL REGISTRATION: ISRCTN, ID: ISRCTN16318986 . Registered 10 November 2016. The ISRCTN registration is in line with the World Health Organization Trial Registration Data Set. The present paper represents the original version of the protocol. Any changes to the protocol will be communicated to ISRCTN.

Utilisation of the MVA pathway to produce elevated levels of the sesquiterpene α-copaene in potato tubers.

Potato flavour is a complex trait resulting from the presence of a combination of volatile and non-volatile compounds. The aim of this work was to investigate the effect of specifically altering the volatile content of tubers and assess its impact on flavour. Tuber-specific over-expression of a potato α-copaene synthase gene resulted in enhanced levels (up to 15-fold higher than controls) of the sesquiterpene α-copaene. A positive correlation (R(2)=0.8) between transgene expression level and α-copaene abundance was observed. No significant changes in the levels of volatiles other than α-copaene were detected. Non-volatile flavour compounds (sugars, glycoalkaloids, major umami amino acids and 5'-ribonucleotides) were also determined. Relationships between flavour compounds and sensory evaluation data were investigated. Evaluators could not detect any aroma differences in the transgenic samples compared with controls and no significant differences in taste attributes were found. Thus although successful engineering of potato tubers to accumulate high levels of the flavour volatile α-copaene was achieved, sensory analysis suggests that α-copaene is not a major component of potato flavour.

Relationships between volatile and non-volatile metabolites and attributes of processed potato flavour.

Although the flavour of processed potatoes (Solanum tuberosum L.) is important to consumers, the blend of volatile and non-volatile metabolites that impact on flavour attributes is not well-defined. Additionally, it is important to understand how potato flavour changes during storage. In this study, quantitative descriptive analysis of potato samples by a trained taste panel was undertaken, comparing tubers from S. tuberosum group Phureja with those from S. tuberosum group Tuberosum, both at harvest and following storage. The cooked tuber volatile profile was analysed by solid phase micro extraction followed by gas chromatography-mass spectrometry analysis in sub-samples of the tubers that were assessed by taste panels. A range of non-volatile metabolites including the major umami compounds, glycoalkaloids and sugars was also measured in tuber sub-samples. Correlation and principal component analyses revealed differences between the potato cultivars and storage conditions and demonstrated associations of metabolites with the different sensory attributes.

A metabolomics study of cultivated potato (Solanum tuberosum) groups Andigena, Phureja, Stenotomum, and tuberosum using gas chromatography-mass spectrometry.

Phytochemical diversity was examined by gas chromatography-mass spectrometry in tubers of genotypes belonging to groups Andigena, Phureja, Stenotomum, and Tuberosum of the potato, Solanum tuberosum. Polar extracts (mainly amino acids, organic acids, sugars, and sugar alcohols) and nonpolar extracts (mainly fatty acids, fatty alcohols, and sterols) were examined. There was a large range in levels of metabolites, including those such as asparagine, fructose, and glucose, that are important to tuber quality, offering considerable scope for selecting germplasm for breeding programmes. There were significant differences in the levels of many metabolites among the groups. The metabolite profiles of genotypes belonging to Phureja and Stenotomum were similar and different from those of Tuberosum and the majority of Andigena genotypes. There was some agreement with the phylogeny of the groups in that Stenotomum is believed to be the ancestor of Phureja and they are both distinct from Tuberosum. Andigena genotypes could be partially distinguished according to geographical origin, Bolivian genotypes being particularly distinct from those from Ecuador. Biosynthetic links between metabolites were explored by performing pairwise correlations of all metabolites. The significance of some expected and unexpected strong correlations between many amino acids (e.g., between isoleucine, lysine, valine, and other amino acids) and between several nonpolar metabolites (e.g., between many fatty acids) is discussed. For polar metabolites, correlation analysis gave essentially similar results irrespective of whether the whole data set, only Andigena genotypes, or only Phureja genotypes were used. In contrast, for the nonpolar metabolites, Andigena only and Phureja only data sets resulted in weaker and stronger correlations, respectively, compared to the whole data set, and may suggest differences in the biochemistry of the two groups, although the interpretation should be viewed with some caution.

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes.

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44,000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene alpha-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode alpha-copaene synthase. Other potential 'flavour genes', identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5'-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.

Phytochemical diversity in tubers of potato cultivars and landraces using a GC-MS metabolomics approach.

Phytochemical diversity with respect to a range of polar (including amino acids, organic acids, sugars, and sugar alcohols) and nonpolar (including fatty acids, alkanols, and sterols) metabolites was examined within tubers from a total of 29 genetically diverse potato cultivars and Chilean landraces using a metabolomics approach by gas chromatography-mass spectrometry. From principal component analysis of the polar and nonpolar metabolite data there was insufficient variation to differentiate the majority of cultivars and landraces. Analysis of all polar metabolite profiles revealed separation of two cultivars (Glenna and Morag) from the other cultivars and landraces and a separate cluster of one landrace line, largely due to higher levels of sugars. Pentland Javelin was distinct in containing high levels of many amino acids. The two Solanum tuberosum group phureja cultivars (Inca Sun and Mayan Gold) were not particularly similar and were not separated from the S. tuberosum group tuberosum cultivars. Analysis of the nonpolar metabolite data revealed partial separation of two landrace lines and, on the basis of some minor fatty acids, Mayan Gold was distinct. The differences in metabolite profiles are considered in terms of the taxonomy and breeding history of the cultivars and possible influences from other factors such as developmental stage of the tuber. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed on all metabolites. The significance of high correlations between many amino acids and between several nonpolar metabolites is discussed.

Comparison of the calystegine composition and content of potato sprouts and tubers from Solanum tuberosum Group Phureja and Solanum tuberosum Group Tuberosum.

The calystegines detected in tubers from 17 Phureja ( S. tuberosum Group Phureja) lines and five Tuberosum ( S. tuberosum Group Tuberosum) cultivars were identified as the A 3 and B 2 structural types. Their concentration in whole tubers was of a similar order of magnitude in both species, as was the variability in the ratio of B 2 to A 3. On average, calystegine concentrations in the peel were about 13 times that found in the flesh for the five Tuberosum cultivars, and 4 times higher for four Phureja lines. Removal of the peel reduced the calystegine content by an average of over 50% in Tuberosum but by only 30% in Phureja, despite the latter having the greater proportion of peel. The calystegine content of sprouts was also determined for five Tuberosum cultivars and four Phureja lines and was found to include small amounts of four additional types, B 3, B 4, N 1, and X 2, in addition to the more abundant A 3 and B 2. Concentrations in the sprouts of Tuberosum were on average 100 times higher than that in the tuber flesh and 8 times higher than in the peel, whereas for Phureja, the equivalent values were 30 and 7 times higher, respectively. No correlation was found between sprout concentration and either flesh or peel calystegine concentration.

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein.

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.