Interaction of Escherichia coli with growing salad spinach plants

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this study's findings with regard to the microbiological safety of minimally processed vegetables are discussed.     

Interactions of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes plants cultivated in a gnotobiotic system

The growth and persistence of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on a diverse range of plant types over extended cultivation periods was studied. When introduced on the seed of carrot, cress, lettuce, radish, spinach and tomato all the pathogens became rapidly established shortly after germination, attaining cell densities of the order of 5.5-6.5 log cfu/g. In general, Es. coli O157:H7 and L. monocytogenes became established and persisted at significantly higher levels on seedlings (9 days post-germination) than Salmonella. Es. coli O157:H7 became internalized in cress, lettuce, radish and spinach seedlings but was not recovered within the tissues of mature plants. Internalization of Salmonella was also observed in lettuce and radish but not cress or spinach seedlings. In contrast, L. monocytogenes did not internalize within seedlings but did persist on the surface of plants throughout the cultivation period. Co-inoculation of isolates recovered from the rhizosphere of plants did not significantly affect the numbers or persistence of human pathogens. The only exception was with Enterobacter cloacae, which reduced Es. coli O157:H7 Ph1 and L. monocytogenes levels by ca. 1 log cfu/g on lettuce. With the bioluminescent phenotype of Es. coli O157:H7 Ph1, it was demonstrated that the human pathogen became established on the roots of growing plants. Scanning electron micrographs of root seedlings suggested that Es. coli O157:H7 Ph1 preferentially colonized the root junctions of seedlings. It is proposed that such colonization sites enhanced the persistence of Es. coli O157:H7 on plants and facilitated internalization within developing seedlings. The results suggest that the risk associated with internalized human pathogens in salad vegetables at harvest is low. Nevertheless, the introduction of human pathogens at an early stage of plant development could enhance their persistence in the rhizosphere. The implications of the study with regards to on-farm food safety initiatives are discussed.     

Internalization of Escherichia coli O157:H7 following biological and mechanical disruption of growing spinach plants

The internalization and persistence of a bioluminescent Escherichia coli O157:H7 Ph1 was investigated in growing spinach plants that had been either biologically or mechanically damaged. In control (undamaged) plants cultivated in soil microcosms inoculated with E. coli O157:H7 Phl, the bacterium was recovered from surface-sterilized root tissue but not from leaves. Mechanical disruption of the seminal root and root hairs of the plants did not result in the internalization of the pathogen into the aerial leaf tissue. When imprints of the root tissue were made on plates of tryptic soy agar plus ampicillin, no colonies of E. coli O157:H7 were observed around damaged tissue. The roots of growing plants were exposed to the northern root-knot nematode, Meloidogyne hapla, in the presence of E. coli O157:H7. Although this treatment caused knot formation on the roots, it did not enhance the internalization of the bacterium into the plant vascular system. Coinoculation of intact leaves with E. coli O157:H7 and the phytopathogen Pseudomonas syringae DC3000 resulted in localized necrosis, but the persistence of the human pathogen was not affected. The mechanical disruption of roots does not result in the internalization of E. coli O157:H7 into the aerial tissue of spinach, and there does not appear to be any effect of P. syringae in terms of enhancing the persistence of E. coli O157:H7 in spinach leaves.     

Small‐Molecule Proteomimetic Inhibitors of the HIF‐1α–p300 Protein–Protein Interaction

The therapeutically relevant hypoxia inducible factor HIF‐1α–p300 protein–protein interaction can be orthosterically inhibited with α‐helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C‐terminal helix of the HIF‐1α C‐TAD (C‐terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side‐chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF‐1α–p300 PPI inhibitors and the first examples of small‐molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF‐1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42‐residue HIF‐1α C‐TAD from which it is derived.     

Survival of toxigenic Escherichia coli on chamomile,peppermint, green,black, ginger,and cinnamon teas during storage and brewing

In this study, the persistence of toxigenic Escherichia coli (E. coli) on dried chamomile, peppermint, ginger, cinnamon, black and green teas stored under 4, 10, and 25°C was determined. The E. coli survival rate in ginger and cinnamon teas decreased below 0 on Day 5. In the other tested teas, E. coli survivability showed a downward trend over time, but never dropped to 0. Chamomile tea retained the greatest population of viable E. coli. Meanwhile, die-off of E. coli was higher at 25°C compared to lower temperatures. Additionally, fate of E. coli during brewing at 60, 70 and 80°C was evaluated. The E. coli population was reduced to below 2 Log colony forming units (CFU)/g after 1 min at 80°C, At the same time, the E. coli survival at 60°C was higher than that at 70°C in all tested teas. The data indicated that if E. coli survives after storage of prepared teas, it may also survive and grow after the brewing process, especially if performed using temperatures <80°C. Finally, we analyzed the correlations between temperature, time, tea varieties and E. coli survival, and successfully constructed a random forest regression model. The results of this study can be used to predict changes in E. coli during storage and fate during the brewing process. Results will form the basis of undertaking a risk assessment.     

Coliphage as an indicator of fecal contamination in hydroponic cucumber (Cucumis sativus L) greenhouses

A preliminary study has been performed to evaluate the utility of somatic and F⁺‐specific coliphage as quality indicators of irrigation water used in hydroponic cucumber greenhouse operations. Samples of incoming water, waste hydroponic solution, cucumber fruit and plant roots derived from two greenhouses were screened for bacterial fecal indicators (generic Escherichia coli, fecal coliforms, total coliforms and Clostridium perfringens) and coliphage. Bacterial fecal indicators were present in incoming water and in spent hydroponic solution with coliphage only being sporadically recovered. However, both somatic and F⁺‐specific coliphage were consistently recovered from the roots of cucumber plants along with bacterial fecal indicators. Despite the heavy contamination of plant roots, the cucumber fruits were within acceptable microbiological limits. F⁺‐specific coliphage was recovered from 1 out of 25 cucumbers tested along with generic E coli. In contrast, no somatic coliphage was recovered from cucumbers despite coliforms being present on 15 out of 25 units. In conclusion, coliphage represents a poor index of hydroponic irrigation water quality but presence on cucumber fruit and roots can be used to highlight the presence of fecal indicator bacteria within greenhouse operations. Copyright © 2005 Society of Chemical Industry     

Pyrene Tags for the Detection of Carbohydrates by Label-Assisted Laser Desorption/Ionisation Mass Spectrometry**

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming the limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed a series of pyrene-based LDI reagents (LALDI tags) that can be used for labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and demonstrated the use of LALDI tags for the detection of lactose. We have also exemplified the potential of LALDI tags for labelling carbohydrates in biological samples by direct detection of lactose in cow's milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.     

Why doesn’t every family practice rainwater harvesting? Factors that affect the decision to adopt rainwater harvesting as a household water security strategy in central Uganda

ABSTRACT

This article investigates the reasons householders do, and don’t, adopt domestic rainwater harvesting (DRWH). Using a mixed-methods research approach, we collected data in three districts in central Uganda. Factors that emerged as important with respect to uptake of DWRH to address water shortage, especially at the household scale, include the work of intermediary organizations, finance mechanisms, life course dynamics and land tenure.     

The effect of different processing parameters on the efficacy of commercial post-harvest washing of minimally processed spinach and shredded lettuce

The effect of different processing parameters on the efficacy of commercial post-harvest biocidal washes to decrease the bacterial loading on spinach and lettuce has been evaluated. Sampling was performed at two spinach processors (Facility A & B) and a shredded lettuce producer (Facility C). Aerobic colony counts (ACC) and coliform counts were determined on samples taken at pre- and post-wash. In parallel, the heterotrophic plate count (HPC) and coliform levels in wash water was also determined. Processing parameters measured were the temperature of leafy greens (pre- and post-washing) and wash water. The sanitizer levels (peroxyacetic acid, oxidation-reduction potential), pH, conductivity and turbidity were also measured. The wash process in Facility B had a residence time of 50 s for the spinach, maintained a constant hypochlorite concentration and continuously re-charged the tanks with fresh water. In contrast, Facility A had a short residence time (15 s) did not maintain a constant sanitizer (peroxyacetic acid) concentration or re-charge tanks with fresh water. Despite the differences in processing operations there was no statistical difference between the log count reductions (LCR) obtained in ACC and coliform counts although counts were only reduced by <0.6 log cfu/g. The carriage of Escherichia coli on pre-wash spinach was 19% and 25% in Facility A and B respectively. There was a high prevalence (57% positive) of E. coli in the wash water of Facility A with none being recovered in water samples taken from Facility B. Yet, the carriage of E. coli on post-wash spinach was the same in the two facilities (7%). Lettuce harboured a lower level of both ACC and coliforms with LCR being significantly greater than spinach. In general, the LCR in ACC and coliforms could be positively correlated to bacterial counts of pre-washed leafy greens and conductivity (solids content) of the wash water. A negative correlation was found between LCR and water temperature. Interestingly, within the ranges measured the LCR was independent of the bacterial loading of the water. The results of the study confirmed the limited efficacy of biocidal washes to remove field acquired contamination. Although it is thought maintaining a low microbial loading in the wash water and maintaining sanitizer concentration is key the current study suggests high conductivity and low temperature of the wash water enhances the LCR achieved.