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Publication from the Healthy Soils project outlining soils research through the Scottish Government Rural and Environment Science and Analytical Services (RESAS) funded Strategic Research Programme. The Soil Sentinel will be published very 4 months throughout the project duration, 2022-2027.

Migrations are essential for soil microorganisms to exploit scarce and heterogeneously distributed resources in the rhizosphere, but mobility in soil remains poorly studied due to experimental limitations. In this study, time-lapse images collected using live microscopy techniques captured collective and coordinated groups of B. subtilis cells exhibiting “crowd movement” during rhizosphere colonisation. Groups of B. subtilis cells moved through transparent soil toward plant roots and re-arranged dynamically around root tips in the form of elongating and retracting “flocks” resembling collective behaviour usually associated with higher organisms (e.g., bird flocks or fish schools). These B. subtilis flocks are likely driven by the diffusion of extracellular signalling molecules (e.g., chemotaxis and quorum sensing) and may be impacted by the physical properties of the environment. Our findings advance the understanding of bacterial migration through soil matrices and expand known behaviours for coordinated bacterial movement.

International audience; Our understanding of plant–microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants’ large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant–microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm 3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of “first contact,” before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant–microbe interactions.

Significance The lack of suitable approaches for studying root–microbe interactions, live and in situ, has severely limited our ability to understand the rhizosphere. In this study, we overcome this major limitation with an imaging system that combines transparent soils with cutting edge light sheet microscopy. The study revealed that the root cap is a point of first contact for microbes before establishment and reveals how the pore structure influences the patterns of interactions between the microbe and the plant. With the combined use of light sheet microscopy and transparent soils, we shed light on previously unseen interaction phenomena and accelerate the understanding of how rhizospheres are formed., Our understanding of plant–microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants’ large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant–microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of “first contact,” before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant–microbe interactions.

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum , reflective of distinct plant-associated lifestyles.

Whole transcriptome analysis to investigate differential gene expression and regulatory adaption can be carried out on two different technological platforms: by probe hybridisation to microarrays or by RNAseq for deep sequencing. Since there are difference in terms of their genome coverage, sensitivity and cost, there is a requirement for robust comparisons to determine the platform of choice. Here, we present datasets for the whole transcriptional response verocytoxigenic Escherichia coli (VTEC) obtained from RNA-seq and microarray platforms in response to spinach, together with a comparison between the datasets (available at Array Express: E-MTAB-3249, E-MTAB-4120, E-MTAB-7441).

Since the discovery that the plant immune system could be augmented for improved deployment against biotic stressors through the exogenous application of chemicals that lead to induced resistance (IR), many such IR-eliciting agents have been identified. Initially it was hoped that these chemical IR agents would be a benign alternative to traditional chemical biocides. However, owing to low efficacy and/or a realization that their benefits sometimes come at the cost of growth and yield penalties, chemical IR agents fell out of favour and were seldom used as crop protection products. Despite the lack of interest in agricultural use, researchers have continued to explore the efficacy and mechanisms of chemical IR. Moreover, as we move away from the approach of 'zero tolerance' toward plant pests and pathogens toward integrated pest management, chemical IR agents could have a place in the plant protection product list. In this review, we chart the rise and fall of chemical IR agents, and then explore a variety of strategies used to improve their efficacy and remediate their negative adverse effects. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The success of conventional cancer therapeutics is hindered by associated dreadful side-effects of antibiotic resistance and the dearth of antitumor drugs' selectivity and specificity. Hence, the conceptual evolution of anti-cancerous therapeutic agents that selectively target cancer cells without impacting the healthy cells or tissues, has led to a new wave of scientific interest in microbial-derived bioactive molecules. Such strategic solutions may pave the way to surmount the shortcomings of conventional therapies and raise the potential and hope for the cure of wide range of cancer in a selective manner. This review aims to provide a comprehensive summary of anti-carcinogenic properties and underlying mechanisms of bioactive molecules of microbial origin, and discuss the current challenges and effective therapeutic application of combinatorial strategies to attain minimal systemic side-effects.

Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants’ large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3 in size with as good as 5 μm resolution at a rate of up to one scan every 30 minutes. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point “first contact”, before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.

Soil microbiomes are dynamic systems that respond to biotic and abiotic environmental factors such as those presented at seasonal scales or due to long-term anthropogenic regime shifts. These can affect the composition and function of microbiomes. Investigation of microbiomes can uncover hidden microbial roles in health and disease and discover microbiome-based interventions. Collections of soil samples are kept by various institutions in either a refrigerated or occasionally frozen state, but conditions are not optimised to ensure the integrity of soil microbiome. In this manuscript, we describe cryopreservation with a controlled rate cooler and estimate the genomic content of an exemplar soil sample before and after cryopreservation. The first hypothesis was to test the genomic integrity of the microbiome. We also enriched the soil sample with a liquid medium to estimate the growth of bacteria and compared their growth before and after cryopreservation. Sequence-based rRNA metabarcoding was used to demonstrate that the controlled rate cooler maintains intact the DNA content of the microbiome. Two methods of cryopreservation were applied and compared with control aliquots of soil. An optimised cryopreservation of soil samples is essential for the development of microbiome research in order to retain stable, functionally intact microbiomes. Our results showed that metabarcoding of 16S and ITS rRNA were useful methods to estimate successful cryopreservation. The soil microbiome after enrichment with liquid medium exhibited a similar response of cryopreserved soil and this was estimated with the comparison of the ten most abundant bacterial taxa. These findings support a successful process of cryopreservation and are promising for future use of this technology. To the best of our knowledge, this study is the first report of cryopreservation of soil using a Stirling cycle cooling approach.

Edible plants are an important vehicle in the transmission of human pathogens. Most such pathogens are food-borne, deriving from plants that are eaten raw or minimally processed. Molecular interactions between foodborne pathogens and plant hosts show similarities with those between plant pathogens and their hosts, although important distinctions are also evident. For example there are parallels between PAMP-triggered immunity to human pathogens and elicitation of defence responses in plants to their pathogens. Control of human pathogens on plants is exercised through food safety and risk management. The important overlap between crop protection and management of crop-derived human pathogens opens opportunities for the two disciplines to benefit from shared dialogue and research.

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 &deg, C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.

Food-borne illness arising from Shiga-toxigenic Escherichia coli (STEC) is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. The initial stages of the interaction involve chemotaxis, attachment and potentially, responding to the early stages of microbe perception by the plant host. We used a high-throughput positive-selection approach to identify early interaction factors of E. coli O157:H7 isolate Sakai to spinach. A bacterial artificial chromosome (BAC) clone library was quantified by microarray hybridisation, and gene loci enrichment measured using a Bayesian hierarchical model. The screen of four successive rounds of short-term (2 hour) interaction with spinach roots produced in 115 CDS credible candidates, comprising seven contiguous genomic regions. Two candidate regions were selected for functional assessment: a chaperone-usher fimbrial gene cluster (loc6) and the pO157 plasmid-encoded type two secretion system (T2SS). Interaction of bacteria with spinach tissue was reduced in the absence of the pO157 plasmid, which was appeared to involve the T2SS EtpD secretin protein, whereas loss of loc6 did not impact interactions. The T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD expressed in planta by E. coli Sakai on spinach plants. Thus, a whole genome screening approach using a combination of computational modelling and functional assays has identified a novel function for STEC T2SS in interactions with plant 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.

Current crop protection strategies against the fungal pathogen Botrytis cinerea rely on a combination of conventional fungicides and host genetic resistance. However, due to pathogen evolution and legislation in the use of fungicides, these strategies are not sufficient to protect plants against this pathogen. Defence elicitors can stimulate plant defence mechanisms through a phenomenon known as defence priming. Priming results in a faster and/or stronger expression of resistance upon pathogen recognition by the host. This work aims to study defence priming by a commercial formulation of the elicitor chitosan. Treatments with chitosan result in induced resistance (IR) in solanaceous and brassicaceous plants. In tomato plants, enhanced resistance has been linked with priming of callose deposition and accumulation of the plant hormone jasmonic acid (JA). Large‐scale transcriptomic analysis revealed that chitosan primes gene expression at early time‐points after infection. In addition, two novel tomato genes with a characteristic priming profile were identified, Avr9/Cf‐9 rapidly elicited protein 75 (ACRE75) and 180 (ACRE180). Transient and stable over‐expression of ACRE75, ACRE180 and their Nicotiana benthamiana homologs, revealed that they are positive regulators of plant resistance against B. cinerea. This provides valuable information in the search for strategies to protect Solanaceae plants against B. cinerea., Chitosan primes cell wall defences, accumulation of defence‐related hormones and expression of Avr9/Cf‐9 rapidly elicited genes against Botrytis cinerea in Solanum lycopersicum (tomato), which provides valuable information in the search for protection strategies in Solanaceous crops.

Current crop protection strategies against the fungal pathogen Botrytis cinerea rely on a combination of conventional fungicides and host genetic resistance. However, due to pathogen evolution and legislation in the use of fungicides, these strategies are not sufficient to protect plants against this pathogen. Defence elicitors can stimulate plant defence mechanisms through a phenomenon known as priming. Priming results in a faster and/or stronger expression of resistance upon pathogen recognition by the host. This work aims to study priming of a commercial formulation of the elicitor chitosan. Treatments with chitosan result in induced resistance in solanaceous and brassicaceous plants. In tomato plants, enhanced resistance has been linked with priming of callose deposition and accumulation of the plant hormone jasmonic acid (JA). Large-scale transcriptomic analysis revealed that chitosan primes gene expression at early time-points after infection. In addition, two novel tomato genes with a characteristic priming profile were identified, Avr9/Cf-9 rapidly-elicited protein 75 (ACRE75) and 180 (ACRE180). Transient and stable overexpression of ACRE75, ACRE180 and their Nicotiana benthamiana homologs, revealed that they are positive regulators of plant resistance against B. cinerea. This provides valuable information in the search for strategies to protect Solanaceae plants against B. cinerea.

Summary 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.

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.

Contamination of fresh produce with pathogenicEscherichia coli,including ShigatoxigenicE. coli(STEC), represents a serious risk to human health. Colonisation is governed by multiple bacterial and plant factors that can impact on 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 for colonisation of living plants as assessed from growth kinetics and biofilm formation in plant extracts. Fitness of STEC was compared to environmentalE. coli, 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 fastest rates of growth. Spinach extracts also supported the highest levels of biofilm formation. Saccharides were identified as the major driver of bacterial growth, although no single metabolite could be correlated with growth kinetics. The highest level ofin plantacolonisation occurred on alfalfa sprouts, though internalisation was 10-times more prevalent in the leafy vegetables than in sprouted seeds. Marked differences inin plantagrowth meant that growth potential could only be inferred for STEC for sprouted seeds. In contrast, biofilm formation in extracts related to spinach colonisation. Overall, the capacity ofE. colito colonise, grow and internalise within plants or plant-derived matrices were influenced by the isolate type, plant species, plant tissue type and temperature, complicating any straight-forward relationship betweenin vitroandin plantabehaviours.ImportanceFresh produce is an important vehicle for STEC transmission and experimental evidence shows that STEC can colonise plants as secondary hosts, but differences in the capacity to colonise occur between different plant species and tissues. Therefore, an understanding of the impact of 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 plants extracts could be related to specific plant metabolites or could predict the ability of the bacteria to colonise living plants. Growth rates for sprouted seeds (alfalfa and fenugreek) exhibited a positive relationship between plant extracts and living plants, but not for leafy vegetables (lettuce and spinach). Therefore, the detailed variations at the level of the bacterial isolate, plant species and tissue type all need to be considered in risk assessment.

International audience

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.

Metabolism is the underpinning force that sustains life. Within the rhizosphere it is a cyclic process, with substrates flowing between different compartments of the complete soil-plant-microbe system. The physiochemical and structural environment of the rhizosphere is shaped by a combination of plant genotype and soil type, both of which strongly impact the microbial community structure. External influences such as seasonality, the degree of water saturation and anthropomorphic inputs also play a role. Together these factors influence the flux of metabolites through the rhizosphere community, which in turn impacts on plant growth, development and disease. In this review, the focus is on metabolism within the bacterial population of the rhizosphere, since this group covers every type of plant-microbe interaction: from obligately symbiotic to destructively pathogenic, and includes those have little or no direct impact on plant hosts. The focus of the review is on metabolic functions that occur in the rhizosphere either during bacteria-plant interactions or bacteria-bacteria interactions and mainly covers heterotrophic metabolism of organic substrates. As such, many of the autotrophic (and phototrophic) reactions of inorganic compounds are not included.

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.

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 werepredicted 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 alternativeserovars such as S. Dublin. A key finding from both phylogeny and SVM methods was that the majority of isolates were assignedto 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 besuccessfully applied to interrogate source attribution of bacterial isolates and has the capacity to predict zoonotic potential.

UNLABELLED A barley field trial supplemented with bulky organic soil amendments, municipal compost or bovine slurry was sampled for Escherichia coli to test the hypothesis that E. coli isolated from the soil or from barley plants were derived from bovine slurry. A qualitative analysis showed that a total of 12% of the bulk soil cores and 16% of harvested grain samples yielded E. coli. The strongest association for positive detection of E. coli from soil was with time of year and for slurry-treated plots, with irrigation. However, E. coli were detected in plots from all treatment types and not exclusively associated with bovine slurry. Phylogroup, plasmid profiling and population genetics analysis (multilocus sequence typing) revealed extensive genetic diversity. Identical sequence types for slurry and soil isolates were detected, indicative of direct transfer into the soil, although not frequently. Host interaction assays with selected isolates showed a variation in the ability to colonize barley roots, but not in interactions with bovine cells. The work has implications in appropriate use of E. coli as a faecal indicator as isolates were widespread and diverse, reinforcing the view that some are a natural part of the microflora in agricultural systems. SIGNIFICANCE AND IMPACT OF THE STUDY Faecal deposition is considered to be the main process that introduces Escherichia coli into soil, giving rise to their use as a faecal indication species and the potential for cycling pathogens in agricultural systems. We found that bovine slurry was not the main source of E. coli in a barley trial and a high degree of diversity was present in the collection. The findings support the hypothesis that the population structure of E. coli in secondary habitats is shaped by the environment and highlight the drawbacks of its use as a faecal indicator species.

Summary 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 KH2PO4 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.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

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 colonisation. As such, we compared the ability of the VTEC strain, E. coli O157:H7 ‘Sakai’, to colonise the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum) and prickly lettuce (L. 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 analysed 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 colonisation. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 hour) transcriptional response to extracts as well as longer-term (10 days) plant colonisation or persistence. We show that propagation temperature (37 versus 18 oC) 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 utilisation. 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 uncharacterised (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

Outbreaks of enteric pathogens associated with fresh produce, in the form of raw or minimally processed fruit and vegetables, have increased over recent times. The pathogens may be bacterial, viral or protoctist parasites and many are considered to be zoonotic. Contamination of fresh produce occurs by a number of routes, in particular from irrigation water or manure. While some pathogens are vectored into the food chain by plants, others are able to colonize plants and use them as alternative hosts. Enteric bacteria normally associated with animal hosts can colonize and proliferate on or within plants, in particular in the rhizosphere. Some have the ability to internalize into plant tissue, from where they can be detected in edible foliage. Specific interactions have been demonstrated that are dependent on both the microbe and plant host. Colonization of non-animal hosts by enteric pathogens has, until recently, been largely neglected. However, alternative environmental reservoirs such as plants need to be considered in the context of transmission of enteric pathogens through the food chain.

Members of the Enterobacteriaceae have the capacity to adapt to a wide variety of environments and can be isolated from a range of host species across biological kingdoms. Bacteria that are pathogenic to animals, in particular humans, are increasingly found to be transmitted through the food chain by fruits and vegetables. Rather than simply contaminating plant surfaces, there is a growing body of evidence to show that these bacteria actively interact with plants and can colonize them as alternative hosts. This review draws together evidence from studies that investigate proven and potential mechanisms involved in colonization of plants by human pathogenic enterobacteria.

Pathogenic bacteria have a large repertoire of surface organelles involved in adherence, motility and protein export, but how individual bacteria co-ordinate surface organelle expression to prevent interference and excessive immune stimulation is unclear. Phase variation is a mechanism by which expression of surface factors is limited to a fraction of the bacterial population; however, the presence of multiple homologous surface structures controlled by related mechanisms and regulators antagonizes the independent expression achieved by phase variation. To investigate whether other mechanisms have evolved to sort out the bacterial cell surface, we examined regulatory cross-talk between multiple phase-variable pyelonephritis-associated pili (pap) operons in Escherichia coli isolates associated with urinary tract infections. Allelic variation identified in the regulatory regions and regulators acts synergistically to limit coexpression of homologous fimbrial operons. In particular, there is evidence that papI is under positive selection and PapI variants displayed differences in their capacity to activate related pap operons. Alleles of the high-affinity binding site for PapB were shown to contain a variable number of (T/A)(3) repeats occurring every 9 bp that altered the sensitivity of pap operon activation. Taken together with other examples of surface organelle cross-talk, we illustrate how this regulation could promote sequential expression.

The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC.

Fresh produce, in the form of raw or minimally processed fruits and vegetables, is recognized to be an important source of food-borne disease. Sporadic cases or outbreaks can arise from bacterial, viral, or parasitic contamination. Importantly, while the latter both require animal hosts for proliferation and cannot grow on or within crop plants, bacteria can do so and are able to use plants as secondary hosts. This type of interaction means that human pathogenic bacteria need to undergo adaptation to the plant host, which presents an environment quite distinct to animals or humans. Important differences lie in both physiochemical and biological properties, e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature. There is now good published evidence to describe different aspects of the interactions between plants and human pathogenic bacteria, and the subsequent effects on the outcome of colonization. However, in comparison to the interactions between the pathogens and their animal hosts, for which we know a great deal about both host and microbial factors, this area is relatively new with many important knowledge gaps. Research in this area is of most relevance to food safety, since the results can be and indeed, are already applied by public health agencies and food producers. It also reveals fascinating aspects of the bacterial life-cycle that were hitherto under appreciated. One of the important features of plant host colonization is the adaptation of pathogens to the host defense response. Innate immunity is the first line of defense with both physical barriers and biological recognition of human pathogens, and the different layers of the response are reviewed in Melotto et al. (2014). An intriguing aspect is the finding that some human pathogens are able to subvert the plant defense, shown using Salmonella enterica and Arabidopsis as an example (Garcia and Hirt, 2014). Perception of human pathogens is based on recognition of PAMPs (e.g., flagellin) similar to those in phytopathogens and other plant-associated bacteria; perhaps unsurprising given that the mechanisms of recognition tend to be based on evolutionary conserved proteins. What was unexpected was the fact that some Salmonella serovars encode flagellin variants that are no longer recognized by the plant, presumably as a result of evolutionary selection (Garcia and Hirt, 2014). There are good parallels in microbial suppression of the innate response of animal and plant hosts, for example via effectors secreted through the type 3 secretion (T3S) apparatus. Despite structural differences in the apparatus between human- and phyto-pathogens, the general function of host subversion is shared. Commonalities (and differences) in which pathways in animal and plant hosts are targeted by human pathogens are highlighted in Brunner and Fraiture (2014), showing where there is evolutionary conservation in the cellular hubs. Detailed examination of the function for one of the T3S effectors from S. enterica, SpvC, shows that it targets a signaling pathway that is shared in plant and animal hosts (Neumann et al., 2014). This effector is a phosphothreonine lyase able to de-phosphorylate activated MAP kinases, therefore attenuating the immune response in Arabidopsis. Examination of mechanisms of colonization shows that although some aspects are broadly the same, there are important differences in the interaction between S. enterica and either plant or animal hosts (Wiedemann et al., 2015). To better understand the molecular basis of the interaction, it is necessary to determine the protein-protein interactions (PPIs) between the pathogen and the plant host. An important comparison of PPIs between Salmonella and Arabidopsis proteins to known interactions between bacterial and animal proteins was presented in Schleker et al. (2015). In order to characterize the interactions on a global scale, an approach was used that combines several new as well as previously described algorithms predicting new PPIs (Kshirsagar et al., 2015). However, while this approach yields candidates worthy of further investigation, it also serves to highlight the paucity of reported functional data. To further explore the molecular interactions, methods must be developed that are appropriate to the bacteria-plant system, for example for the analysis of bacterial gene expression. An improved method for the extraction of RNA from mixed samples was presented in Holmes et al. (2014), which allows the analysis of expression patterns in bacteria and the plant host at the same time. Colonization of plant hosts by human pathogens rarely, if ever, occurs in isolation and successful colonization is dependent on the ability of the bacteria to compete with the native microflora. Microbiome approaches show that crops not obviously associated with human pathogens may harbor potential pathogens. As shown with an example of grapevines, crop plants can support the endophytic growth of human pathogen, in this case Propionibacterium acnes, which probably arose from human contamination in the first place (Yousaf et al., 2014). Application of bulky organic fertilizers, such as manure, to crops may increase the chances of transmission of bacteria into the food chain. However, the potential for transmission clearly shows some specificity, not least from differences between species and sub-species of bacteria and the host plants (Hofmann et al., 2014). A combination of axenic system with susceptible plants (e.g., spinach) revealed that 400 bacteria in one ml are sufficient to successfully colonize the crop plant. This collection of articles helps to highlight the underpinning mechanisms, but also shows the complexity of interactions between human pathogenic bacteria and plant hosts. It is becoming clear that it is not possible to apply a broad set of rules to these interactions: there is a great deal of specificity that depends on multiple factors. This is particularly important in the consideration of how to address issues related to food safety, for practices applied at the pre-harvest stage or for prevention of transmission and contamination during post-harvest processing.