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Contamination of leafy vegetables with Escherichia coli O157:H7 is a growing problem, as reported outbreaks are increasing. However, establishment of this human pathogen in the phyllosphere is not completely understood. Using laser scanning confocal microscopy, we demonstrated that E. coli O157:H7gfp+ can invade plant tissue of Swiss chard and wild rocket leaves and that the bacterium is more sensitive to surface sanitization of Swiss chard leaves. Damage to leaf tissue promoted leaf invasion, but the nature of the damage (abiotic or biotic) and plant species had an impact.

Aims Greenhouse trials were conducted with different cultivars of baby leaf spinach, rocket and Swiss chard and inoculation of Escherichia coli O157:H7 gfp+, to determine whether plant species and cultivar have an impact on the establishment of this strain. Methods and Results Three cultivars each of spinach, rocket and Swiss chard were spray inoculated with E. coli O157:H7 gfp+ at doses of log 7 CFU per ml. Due to the different lengths of growing period spinach and Swiss chard were spray inoculated three times and rocket five times, with final inoculation performed 3 days prior to harvest. After a growing period of 26–33 days, E. coli O157:H7 gfp+ was recovered from the leaf surface in mean populations between log 1 and 6 CFU per gram. The lowest occurrence of E. coli O157:H7 gfp+ was found on rocket leaves and the highest on spinach. There was no significant difference in the establishment of E. coli O157:H7 gfp+ between cultivars, but there were differences between plant species. Indigenous phyllosphere bacteria were pure cultured and identified with 16S rRNA gene sequencing. Conclusions Despite the same high inoculation dose of E. coli O157:H7 gfp+ on leaves, the establishment rate differed between plant species. However, plant cultivar did not affect establishment. Pantoea agglomerans dominated the identified bacterial isolates. Significance and Impact of the Study As previous studies are inconclusive on choice of model plant species and cultivar, we studied whether plant species or cultivar determines the fate of E. coli O157:H7 gfp+ on leafy vegetables. The findings indicate that plant species is a key determinant in the establishment of E. coli O157:H7 gfp+.

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The influence of organic nitrogen sources on the establishment of E. coli O157:H7 and the occurrence of 17 other potentially human pathogenic bacteria on baby leaf salads was evaluated. Greenhouse-grown rocket 18and Swiss chard were spray-inoculated with gfp-tagged E. coli O157:H7 twice a week from when their first 19 true leaves reached a length of 2 cm until three days before harvest. Analysis of nitrogen content in leaves 20 revealed differences between treatments. Untreated plants had the lowest values, followed by plants 21 fertilized with pig hair pellets and chicken manure. The same pattern was seen for the growth medium at 22 the day of harvest. The applied strain showed similar establishment (measured using culture-dependent 23 methods) irrespective of treatment, but Swiss chard hosted significantly more E. coli O157:H7 than rocket. 24Differences in the risk of infection were found for the crops, with plants fertilized with pig hair pellets 25showing a slightly higher risk. No relationship was found for total nitrogen content in leaves and 26 colonization with E. coli O157:H7 gfp+. Isolating dominant Enterobacteriaceae from leaves, some strains 27 showed conformity to bacterial species that have previously been identified to influence the establishment 28 of E. coli O157:H7 in the phyllosphere. In this study, no inhibitory effects were observed in vitro. 29Colonies showing matching characteristics for Listeria spp. and Salmonella spp. were detected in a range 30 from below the detection limit to 4 log CFU g -1 fresh weight on rocket, and from 3.5 to up to 5.5 log CFU 31 g -1 fresh weight and 1 to and 5 log CFU g -1 fresh weight, respectively, on Swiss chard. However, 32 presumptive Listeria spp. and Salmonella spp. could not be confirmed by sequencing of the 16S rRNA gene. 33 34 Keywords: culture-dependent methods, Enterobacteriaceae, food safety, human pathogenic bacteria, 35 organic fertilizers, phylogenetic analysis, risk assessment 36 37

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Background: The Cancer Genome Atlas (TCGA) network (United States National Cancer Institute) identified four molecular endometrial cancer (EC) subtypes using an extensive multi-method approach. The aim of this study was to determine the four TCGA EC molecular subtypes using a single-method whole-exome sequencing (WES)-based approach provided by MH Guide (Molecular Health, Heidelberg, Germany). Methods: WES and clinical data of n = 232 EC patients were obtained from TCGA. The four TCGA EC molecular subtypes designated as (i) Mutated Polymerase ε (POLE), (ii) Microsatellite Instability (MSI), (iii) Copy Number (CN) low and, (iv) CN-high were determined using the MH Guide software. The prognostic value of the subtypes determined by MH Guide were compared with the TCGA classification. Results: Analysis of WES data using the MH Guide software led to the precise identification of the four EC molecular subtypes analogous to the TCGA classification. Both approaches displayed high concordance in terms of prognostic significance. Conclusions: The multi-method-based TCGA EC molecular subtypes can reliably be reproduced by the single-method-based MH Guide approach. The easy-to-implement single-method MH Guide approach represents a promising diagnostic tool.

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