Vacuolar localisation of anthocyanin pigmentation in microgreen cotyledons of basil, cabbage and mustard greens does not impact on colonisation by Shiga-toxigenic Escherichia coli O157:H7.

Microgreens, the immature plants harvested after a few weeks of growth, are perceived as a heathy, nutritious food ingredient but may be susceptible to colonisation by human pathogens including Shiga-toxigenic Escherichia coli (STEC). Some microgreen cultivars accumulate anthocyanins or secrete essential oils which, when extracted or purified, have been reported to inhibit bacterial growth. Therefore, the impact of anthocyanins on bacterial colonisation by STEC (Sakai) was compared for three species that have pigmented cultivars: basil (Ocimum basilicum L.), cabbage (Brassica oleracea L.) and mustard greens (Brassica juncea L.). Inoculation with low concentrations of STEC (Sakai) (3 log10 colony forming units/ml (CFU/ml)) during seed germination resulted in extensive colonisation at the point of harvest, accumulating to âˆ¼ 8 log10 CFU/g FW in all cultivars. Bacterial colonies frequently aligned with anticlinal walls on the surface of epidermal cells of the cotyledons and, in basil, associated with peltate and capitate gland cells. Crude lysates of pigmented and non-pigmented basil cultivars had no impact on STEC (Sakai) growth rates, viability status or biofilm formation. Anthocyanins are located within plant vacuoles of these microgreen cultivars and did not affect colonisation by STEC (Sakai) and pigmentation therefore cannot be considered as a controlling factor in bacterial interactions.

Functional Analysis of Shiga Toxin-Producing Escherichia coli Biofilm Components in Plant Leaves.

Plants represent alternative or secondary hosts for Shiga toxin-producing Escherichia coli (STEC), enabling transmission of the pathogens through the food chain on horticultural crops. This becomes a public health concern for plants that are eaten raw or minimally processed, such as leafy salad and fruits. STEC actively interact with plants as hosts, and so to determine the mechanistic basis to the interaction, it is necessary to assess STEC gene function in planta. Here, we describe analysis of an STEC biofilm component, curli, that plays a role in STEC colony formation in plant leaves. It also serves as a suitable example of the approaches required for qualitative and quantitative assessment of functional host colonization traits.

MacConkey broth purple provides an efficient MPN estimation method for Shigatoxigenic Escherichia coli.

MacConkey broth purple provides a more efficient method for Most Probable Number estimation for Shigatoxigenic Escherichia coli (E.coli) than the process of bacterial enrichment in buffered peptone water followed by detection on MacConkey agar, since it is a single-step process that gives comparable results in plant extracts.

A high-throughput genomic screen identifies a role for the plasmid-borne type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions.

Shiga-toxigenic Escherichia coli (STEC) is often transmitted into food via fresh produce plants, where it can cause disease. To identify early interaction factors for STEC on spinach, a high-throughput positive-selection system was used. A bacterial artificial chromosome (BAC) clone library for isolate Sakai was screened in four successive rounds of short-term (2 h) interaction with spinach roots, and enriched loci identified by microarray. A Bayesian hierarchical model produced 115 CDS credible candidates, comprising seven contiguous genomic regions. Of the two candidate regions selected for functional assessment, the pO157 plasmid-encoded type two secretion system (T2SS) promoted interactions, while a chaperone-usher fimbrial gene cluster (loc6) did not. The T2SS promoted bacterial binding to spinach and appeared to involve the EtpD secretin protein. Furthermore, the T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD was expressed in planta by E. coli Sakai on spinach plants.

Dataset of Escherichia coli O157: H7 genes enriched in adherence to spinach root tissue.

A high-throughput positive-selection approach was taken to generate a dataset of Shigatoxigenic Escherichia coli (STEC) O157:H7 genes enriched in adherence to plant tissue. The approach generates a differential dataset based on BAC clones enriched in the output, after adherence, compared to the inoculum used as the input. A BAC clone library derived from STEC isolate 'Sakai' was used since this isolate is associated with a very large-scale outbreak of human disease from consumption of contaminated fresh produce; white radish sprouts. Spinach was used for the screen since it is associated with STEC outbreaks, and the roots provide a suitable site for bacterial colonisation. Four successive of rounds of Sakai BAC clone selection and amplification were applied for spinach root adherence, in parallel to a non-plant control. Genomic DNA was obtained from a total of 7.17 × 108 cfu/ml of bacteria from the plant treatment and 1.13 × 109 cfu/ml of bacteria from the no-plant control. Relative gene abundance of the output compared to the input pools was obtained using an established E. coli DNA microarray chip for STEC. The dataset enables screening for genes enriched under the treatment condition and informs on genes that may play a role in plant-microbe interactions.

Variability in growth responses of non-O157 EHEC isolates in leafy vegetables, sprouted seeds and soil extracts occurs at the isolate level.

Foods of plant origin are recognised as a major source of foodborne pathogens, in particular for Shigatoxigenic Escherichia coli (STEC). Most work for STEC and plant-based fresh produce has focused on the most prevalent outbreak serogroup, O157. However, non-O157 STEC is an emerging hazard, and as such it is important to characterise aspects within this group that reflect their ability to colonise alternative hosts and habitats relevant to horticultural production. Growth kinetics were quantified for a diverse set of clinical enterohaemorrhagic E. coli isolates in extracts made from different tissues of spinach, lettuce or sprouted seeds, or from soil, to represent association with ready-to-eat fresh produce production. For leafy vegetables, spinach apoplast supported the fastest rates of growth and lettuce root extracts generated the slowest growth rates. Growth rates were similar for the majority of isolates in fenugreek or alfalfa sprouted seed extracts. Monosaccharides were the major driver of bacterial growth. No correlations were found for growth rates between different serotypes or for Shigatoxin gene carriage. Thus, growth rates varied in a plant-dependent and isolate-dependent manner, for all plant or soil extracts tested, indicative of isolate-specific differences in metabolic flexibility. These findings are relevant for risk assessment of non-O157 STEC.

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.

Quantification and colonisation dynamics of Escherichia coli O157:H7 inoculation of microgreens species and plant growth substrates.

Microgreens are edible plants used in food preparation for their appealing flavours and colours. They are grown beyond the point of harvest of sprouted seeds, and normally include the cotyledons and first true leaves. Their method of production is similar to sprouted seeds, which is known to be favourable for growth of microbial pathogens, although there is little data on the potential of food-borne pathogens such as Shigatoxigenic Escherichia coli (STEC) to colonise these plants. We found colonisation of nine different species of microgreen plants by STEC (isolate Sakai, stx-), with high levels of growth over five days, of approximately 5 orders of magnitude, for plants propagated at 21 °C. STEC (Sakai) formed extensive colonies on external tissue, with some evidence for internalisation via stomatal pores. Several factors impacted the level of colonisation: (1) plant tissue type such that for broccoli microgreens, the highest levels of STEC (Sakai) occurred on cotyledons compared to the true leaf and hypocotyl; (2) the route of contamination such that higher levels occurred with contaminated irrigation water compared to direct seed contamination; (3) inoculation dose, although only at low levels of inoculation (3 log10) compared to medium (5 log10) or high (7 log10) levels; (4) environmental factors, including to some extent humidity, but also plant growth substrate types. It was also evident that a starvation response was induced in STEC (Sakai) in low-nutrient plant irrigation medium. Together these data show that microgreens represent a potential hazard of contamination by food-borne pathogens, and to mitigate the risk, they should be considered in the same manner as sprouted seeds.

Patchy promiscuity: machine learning applied to predict the host specificity of Salmonella enterica and Escherichia coli.

Salmonella enterica and Escherichia coli are bacterial species that colonize different animal hosts with sub-types that can cause life-threatening infections in humans. Source attribution of zoonoses is an important goal for infection control as is identification of isolates in reservoir hosts that represent a threat to human health. In this study, host specificity and zoonotic potential were predicted using machine learning in which Support Vector Machine (SVM) classifiers were built based on predicted proteins from whole genome sequences. Analysis of over 1000 S.enterica genomes allowed the correct prediction (67 -90 % accuracy) of the source host for S. Typhimurium isolates and the same classifier could then differentiate the source host for alternative serovars such as S. Dublin. A key finding from both phylogeny and SVM methods was that the majority of isolates were assigned to host-specific sub-clusters and had high host-specific SVM scores. Moreover, only a minor subset of isolates had high probability scores for multiple hosts, indicating generalists with genetic content that may facilitate transition between hosts. The same approach correctly identified human versus bovine E. coli isolates (83 % accuracy) and the potential of the classifier to predict a zoonotic threat was demonstrated using E. coli O157. This research indicates marked host restriction for both S. enterica and E. coli, with only limited isolate subsets exhibiting host promiscuity by gene content. Machine learning can be successfully applied to interrogate source attribution of bacterial isolates and has the capacity to predict zoonotic potential.

Differences in internalization and growth of Escherichia coli O157:H7 within the apoplast of edible plants, spinach and lettuce, compared with the model species Nicotiana benthamiana.

Internalization of food-borne bacteria into edible parts of fresh produce plants represents a serious health risk. Therefore, internalization of verocytotoxigenic E. coli O157:H7 isolate Sakai was assessed in two species associated with outbreaks, spinach (Spinacia oleracea) and lettuce (Lactuca sativa) and compared to the model species Nicotiana benthamiana. Internalization occurred in the leaves and roots of spinach and lettuce throughout a 10 day time-course. The plant species, tissue type and inoculum dose all impacted the outcome. A combination of low inoculum dose (~102 CFU) together with light microscopy imaging highlighted marked differences in the fate of endophytic E. coli O157:H7 Sakai. In the fresh produce species, bacterial growth was restricted but viable cells persisted over 20 days, whereas there was > 400-fold (~2.5 Log10 ) increase in growth in N. benthamiana. Colony formation occurred adjacent to epidermal cells and mesophyll cells or close to vascular bundles of N. benthamiana and contained components of a biofilm matrix, including curli expression and elicitation, extracellular DNA and a limited presence of cellulose. Together the data show that internalization is a relevant issue in crop production and that crop species and tissue need to be considered as food safety risk parameters.

Alternate thermoregulation and functional binding of Escherichia coli type 1 fimbriae in environmental and animal isolates.

Type 1 fimbriae (T1F) are well characterised cell surface organelles expressed by Escherichia coli and required for adherence to mannosylated host tissue. They satisfy molecular Koch's postulates as a virulence determinant and a host-adapted role has been reinforced by reports that T1F expression is repressed at submammalian temperatures. Analysis of a group of 136 environmental and animal E. coli isolates that express T1F at 37°C showed that 28% are also capable of expression at 20°C, in a phase variable manner. The heterogeneous proportions varied widely, and although growth temperature impacted the total proportion expressing T1F, there was no direct correlation between growth at 37°C and 20°C, indicative of differences in thermoregulation of the genetic switch (fimS) that controls phase variation. Specificities of the adhesin (FimH) also varied between the isolates: most bound to α-(1-3) mannan and yeast extracts as expected, but some recognised ß-(1-4)-mannans and N-linked glycoproteins from plants, and T1F from two of the isolates mediated binding to plant roots. The results expand our view of a well-described adherence factor to show alternative expression profiles and adhesin specificities, which in turn may confer an advantage for certain isolates in alternative hosts and habitats.

Corrigendum: Whole-Transcriptome Analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

[This corrects the article on p. 1088 in vol. 7, PMID: 27462311.].

Whole-Transcriptome Analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonization. As such, we compared the ability of the VTEC strain, E. coli O157:H7 'Sakai,' to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analyzed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonization. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 h) transcriptional response to extracts as well as longer-term (10 days) plant colonization or persistence. We show that propagation temperature (37 vs. 18°C) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilization. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling. A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

Bacterial flagella: twist and stick, or dodge across the kingdoms.

The flagellum organelle is an intricate multiprotein assembly best known for its rotational propulsion of bacteria. However, recent studies have expanded our knowledge of other functions in pathogenic contexts, particularly adherence and immune modulation, e.g., for Salmonella enterica, Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli. Flagella-mediated adherence is important in host colonisation for several plant and animal pathogens, but the specific interactions that promote flagella binding to such diverse host tissues has remained elusive. Recent work has shown that the organelles act like probes that find favourable surface topologies to initiate binding. An emerging theme is that more general properties, such as ionic charge of repetitive binding epitopes and rotational force, allow interactions with plasma membrane components. At the same time, flagellin monomers are important inducers of plant and animal innate immunity: variation in their recognition impacts the course and outcome of infections in hosts from both kingdoms. Bacteria have evolved different strategies to evade or even promote this specific recognition, with some important differences shown for phytopathogens. These studies have provided a wider appreciation of the functions of bacterial flagella in the context of both plant and animal reservoirs.

Escherichia coli common pilus (ECP) targets arabinosyl residues in plant cell walls to mediate adhesion to fresh produce plants.

Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7.

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Flagella interact with ionic plant lipids to mediate adherence of pathogenic Escherichia coli to fresh produce plants.

Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2 PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A.thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E.coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.