Commercial wash of leafy vegetables do not significantly decrease bacterial load but leads to shifts in bacterial species composition.

Production of leafy vegetables for the "Ready-to-eat"-market has vastly increased the last 20 years, and consumption of these minimally processed vegetables has led to outbreaks of food-borne diseases. Contamination of leafy vegetables can occur throughout the production chain, and therefore washing of the produce has become a standard in commercial processing. This study explores the bacterial communities of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia) in a commercial setting in order to identify potential contamination events, and to investigate effects on bacterial load by commercial processing. Samples were taken in field, after washing of the produce and at the end of shelf-life. This study found that the bacterial community composition and diversity changed significantly from the first harvest to the end of shelf-life, where the core microbiome from the first to the last sampling constituted <2% of all OTUs. While washing of the produce had no reducing effect on bacterial load compared to unwashed, washing led to a change in species composition. As the leaves entered the cold chain after harvest, a rise was seen in the relative abundance of spoilage bacteria. E. coli was detected after the washing indicating issues of cross-contamination in the wash water.

Dining in Blue Light Impairs the Appetite of Some Leaf Epiphytes.

Background: The phyllosphere is subjected to fluctuating abiotic conditions. This study examined the phenotypic plasticity (PP) of four selected non-phototrophic phyllosphere bacteria [control strain: Pseudomonas sp. DR 5-09; Pseudomonas agarici, Bacillus thuringiensis serovar israeliensis (Bti), and Streptomyces griseoviridis (SG)] regarding their respiration patterns and surfactant activity as affected by light spectrum and nutrient supply. Methods: The PP of the strains was examined under four light regimes [darkness (control); monochromatic light-emitting diodes (LED) at 460 nm (blue) and 660 nm (red); continuously polychromatic white LEDs], in the presence of 379 substrates and conditions. Results: Light treatment affected the studied bacterial strains regarding substrate utilization (Pseudomonas strains > SG > Bti). Blue LEDs provoked the most pronounced impact on the phenotypic reaction norms of the Pseudomonas strains and Bti. The two Gram-positive strains Bti and SG, respectively, revealed inconsistent biosurfactant formation in all cases. Biosurfactant formation by both Pseudomonas strains was supported by most substrates incubated in darkness, and blue LED exposure altered the surface activity profoundly. Blue and white LEDs enhanced biofilm formation in PA in highly utilized C-sources. Putative blue light receptor proteins were found in both Pseudomonas strains, showing 91% similarity with the sequence from NCBI accession number WP_064119393. Conclusion: Light quality-nutrient interactions affect biosurfactant activity and biofilm formation of some non-phototrophic phyllosphere bacteria and are, thus, crucial for dynamics of the phyllosphere microbiome.

Season and Species: Two Possible Hurdles for Reducing the Food Safety Risk of Escherichia coli O157 Contamination of Leafy Vegetables.

The food safety risk of Shiga toxin-producing Escherichia coli (STEC) infection per serving of leafy vegetables was investigated using a quantitative microbial risk assessment (QMRA) approach. The estimated level of E. coli O157 contamination was based on observed numbers of Enterobacteriaceae and E. coli on leafy vegetables grown and processed in southern Sweden from 2014 to 2016. Samples were collected before harvest, after washing, and at the end of shelf life. The observed counts were combined with data on the ratio of E. coli to E. coli O157 taken from earlier studies to estimate the probability of illness. The risks of STEC infection associated with species, either spinach ( Spinacia oleracea) or rocket ( Diplotaxis tenuifolia), growing season (spring or autumn), and washing (washed or not washed) were then evaluated. The results indicated that leafy vegetable species and growing season could be possible hurdles for reducing the food safety risk of STEC infection. At harvest, the probability of infection was 87% lower when consuming rocket compared with spinach and 90% lower when consuming leafy vegetables grown in spring compared with autumn. These relative risk reductions remained consistent even with other serving sizes and dose-response models. The lowest risk of STEC infection was associated with leafy vegetables early in the production chain, i.e., before harvest, while the risk increased during storage and processing. Consequently, the highest risk was observed when leafy vegetables were consumed at the end of shelf life. Washing had no effect on the food safety risk of STEC infection in this study. To improve the quality of QMRA, there is a need for additional data on the relationship between indicator organisms that can be easily enumerated (e.g., E. coli and Enterobacteriaceae) and E. coli strains that can cause STEC infection (e.g., E. coli O157) but are difficult to identify in food samples such as leafy vegetables.

Leaf mineral content govern microbial community structure in the phyllosphere of spinach (Spinacia oleracea) and rocket (Diplotaxis tenuifolia).

The plant microbiome is an important factor for plant health and productivity. While the impact of nitrogen (N) availability for plant growth and development is well established, its influence on the microbial phyllosphere community structure is unknown. We hypothesize that nitrogen impacts the growth and abundance of several microorganisms on the leaf surface. The bacterial and fungal communities of baby leaf spinach (Spinacia oleracea), and rocket (Diplotaxis tenuifolia) were investigated in a field trial for two years in a commercial setting. Nitrogen fertilizer was tested in four doses (basic nitrogen, basic + suboptimal, basic + commercial, basic + excess) with six replicates in each. Culture-independent (Illumina sequencing) and culture-dependent (viable count and identification of bacterial isolates) community studies were combined with monitoring of plant physiology and site weather conditions. This study found that alpha diversity of bacterial communities decreased in response to increasing nitrogen fertilizer dose, whereas viable counts showed no differences. Correspondingly, fungal communities of the spinach phyllosphere showed a decreasing pattern, whereas the decreasing diversity of fungal communities of rocket was not significant. Plant species and effects of annual variations on microbiome structure were observed for bacterial and fungal communities on both spinach and rocket. This study provides novel insights on the impact of nitrogen fertilizer regime on a nutrient scarce habitat, the phyllosphere.

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09.

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350-990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5-09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5-09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity.