Spores of arbuscular mycorrhizal fungi host surprisingly diverse communities of endobacteria.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant root symbionts, which can house two endobacteria: Ca. Moeniiplasma glomeromycotorum (CaMg) and Ca. Glomeribacter gigasporarum (CaGg). However, little is known about their distribution and population structure in natural AMF populations and whether AMF can harbour other endobacteria. We isolated AMF from two environments and conducted detailed analyses of endobacterial communities associated with surface-sterilised AMF spores. Consistent with the previous reports, we found that CaMg were extremely abundant (80%) and CaGg were extremely rare (2%) in both environments. Unexpectedly, we discovered an additional and previously unknown level of bacterial diversity within AMF spores, which extended beyond the known endosymbionts, with bacteria belonging to 10 other phyla detected across our spore data set. Detailed analysis revealed that: CaGg were not limited in distribution to the Gigasporaceae family of AMF, as previously thought; CaMg population structure was driven by AMF host genotype; and a significant inverse correlation existed between the diversity of CaMg and diversity of all other endobacteria. Based on these data, we generate novel testable hypotheses regarding the function of CaMg in AMF biology by proposing that they might act as conditional mutualists of AMF.

Innovations towards achieving environmentally sustainable operating theatres: A systematic review.

INTRODUCTION: The NHS accounts for 5.4% of the UK's total carbon footprint, with the perioperative environment being the most resource hungry aspect of the hospital. The aim of this systematic review was to assimilate the published studies concerning the sustainability of the perioperative environment, focussing on the impact of implemented interventions. METHODS: A systematic review was performed using Pubmed, OVID, Embase, Cochrane database of systematic reviews and Medline. Original manuscripts describing interventions aimed at improving operating theatre environmental sustainability were included. RESULTS: 675 abstracts were screened with 34 manuscripts included. Studies were divided into broad themes; recycling and waste management, waste reduction, reuse, reprocessing or life cycle analysis, energy and resource reduction and anaesthetic gases. This review summarises the interventions identified and their resulting effects on theatre sustainability. DISCUSSION: This systematic review has identified simple, yet highly effective interventions across a variety of themes that can lead to improved environmental sustainability of surgical operating theatres. Combining these interventions will likely result in a synergistic improvement to the environmental impact of surgery.

Evidence of a selective and bi-directional relationship between arbuscular mycorrhizal fungal and bacterial communities co-inhabiting plant roots.

Arbuscular mycorrhizal fungi (AMF) provide plants with vital mineral nutrients and co-exist inside the roots alongside a complex community of bacterial endophytes. These co-existing AMF and bacterial root communities have been studied individually and are known to be influenced in structure by different environmental parameters. However, the extent to which they are affected by environmental parameters and by each other is completely unknown. The current study addressed this knowledge gap by characterising AMF and bacterial communities inside plant roots from a natural and an agricultural ecosystem. Using multivariate modelling, the relative contribution of environmental parameters in structuring the two communities was quantified at different spatial scales. Using this model, it was possible to then remove the contribution of environmental parameters and show that the co-existing AMF and bacterial communities were significantly correlated with each other, explaining up to 36% of each other's variance. Notably, this was not due to the presence of know AMF endobacteria, as removal of endobacterial reads maintained the significance of correlation. These findings provide the first empirical evidence of a selective and bi-directional relationship between AMF and bacteria co-inhibiting plant roots and indicate that a significant fraction of this covariation is due to biological and ecological interactions between them.

riboSeed: leveraging prokaryotic genomic architecture to assemble across ribosomal regions.

The vast majority of bacterial genome sequencing has been performed using Illumina short reads. Because of the inherent difficulty of resolving repeated regions with short reads alone, only ∼10% of sequencing projects have resulted in a closed genome. The most common repeated regions are those coding for ribosomal operons (rDNAs), which occur in a bacterial genome between 1 and 15 times, and are typically used as sequence markers to classify and identify bacteria. Here, we exploit the genomic context in which rDNAs occur across taxa to improve assembly of these regions relative to de novo sequencing by using the conserved nature of rDNAs across taxa and the uniqueness of their flanking regions within a genome. We describe a method to construct targeted pseudocontigs generated by iteratively assembling reads that map to a reference genome's rDNAs. These pseudocontigs are then used to more accurately assemble the newly sequenced chromosome. We show that this method, implemented as riboSeed, correctly bridges across adjacent contigs in bacterial genome assembly and, when used in conjunction with other genome polishing tools, can assist in closure of a genome.

Antibiotic resistance in grass and soil.

Antibiotic resistance is currently one of the greatest threats to human health. The global overuse of antibiotics in human medicine and in agriculture has resulted in the proliferation and dissemination of a multitude of antibiotic resistance genes (ARGs). Despite a large proportion of antibiotics being used in agriculture, little is understood about how this may contribute to the overall antibiotic resistance crisis. The use of manure in agriculture is a traditional and widespread practice and is essential for returning nutrients to the soil; however, the impact of continuous manure application on the environmental microbiome and resistome is unknown. The use of antibiotics in animal husbandry in therapeutic and sub-therapeutic doses creates a selective pressure for ARGs in the gut microbiome of the animal, which is then excreted in the faeces. Therefore, the application of manure to agricultural land is a potential route for the transmission of antibiotic-resistant bacteria from livestock to crops, animals and humans. It is of vital importance to understand the mechanisms behind ARG enrichment and its maintenance both on the plant and within the soil microbiome to mitigate the spread of this resistance to animals and humans. Understanding this link between human health, animal health, plant health and the environment is crucial to inform implementation of new regulations and practice regarding antibiotic use in agriculture and manure application, aimed at ensuring the antibiotic resistance crisis is not aggravated.

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.

Microbial Contamination of Fresh Produce: What, Where, and How?

Promotion of healthier lifestyles has led to an increase in consumption of fresh produce. Such foodstuffs may expose consumers to increased risk of foodborne disease, as often they are not subjected to processing steps to ensure effective removal or inactivation of pathogenic microorganisms before consumption. Consequently, reports of ready-to-eat fruit and vegetable related disease outbreak occurrences have increased substantially in recent years, and information regarding these events is often not readily available. Identifying the nature and source of microbial contamination of these foodstuffs is critical for developing appropriate mitigation measures to be implemented by food producers. This review aimed to identify the foodstuffs most susceptible to microbial contamination and the microorganisms responsible for disease outbreaks from information available in peer-reviewed scientific publications. A total of 571 outbreaks were identified from 1980 to 2016, accounting for 72,855 infections and 173 deaths. Contaminated leafy green vegetables were responsible for 51.7% of reported outbreaks. Contaminated soft fruits caused 27.8% of infections. Pathogenic strains of Escherichia coli and Salmonella, norovirus, and hepatitis A accounted for the majority of cases. Large outbreaks resulted in particular biases such as the observation that contaminated sprouted plants caused 31.8% of deaths. Where known, contamination mainly occurred via contaminated seeds, water, and contaminated food handlers. There is a critical need for standardized datasets regarding all aspects of disease outbreaks, including how foodstuffs are contaminated with pathogenic microorganisms. Providing food business operators with this knowledge will allow them to implement better strategies to improve safety and quality of fresh produce.

The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli.

UNLABELLED: Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS (σ(S)). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX::gfp RpoS was shown to be essential for long-term soil survival of E. coli, since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability. IMPORTANCE: Escherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli, and their survival in it requires that they deploy a full stress protection response.

Holistic Evaluation of Field-Scale Denitrifying Bioreactors as a Basis to Improve Environmental Sustainability.

Denitrifying bioreactors convert nitrate-nitrogen (NO-N) to di-nitrogen and protect water quality. Herein, the performance of a pilot-scale bioreactor (10 m long, 5 m wide, 2 m deep) containing seven alternating cells filled with either sandy loam soil or lodgepole pine woodchip and with a novel "zig-zag" flow pattern was investigated. The influent water had an average NO-N concentration of 25 mg L. The performance of the bioreactor was evaluated in two scenarios. In Scenario 1, only NO-N removal was evaluated; in Scenario 2, NO-N removal, ammonium-N (NH-N), and dissolved reactive phosphorus (DRP) generation was considered. These data were used to generate a sustainability index (SI), which evaluated the overall performance taking these parameters into account. In Scenario 1, the bioreactor was a net reducer of contaminants, but it transformed into a net producer of contaminants in Scenario 2. Inquisition of the data using these scenarios meant that an optimum bioreactor design could be identified. This would involve reduction to two cells: a single sandy loam soil cell followed by a woodchip cell, which would remove NO-N and reduce greenhouse gas (GHG) emissions and DRP losses. An additional post-bed chamber containing media to eliminate NH-N and surface capping to reduce GHG emissions further is advised. Scenario modeling, such as that proposed in this paper, should ideally include GHG in the SI, but because different countries have different emission targets, future work should concentrate on the development of geographically appropriate weightings to facilitate the incorporation of GHG into a SI.

An in-depth evaluation of metagenomic classifiers for soil microbiomes.

BACKGROUND: Recent endeavours in metagenomics, exemplified by projects such as the human microbiome project and TARA Oceans, have illuminated the complexities of microbial biomes. A robust bioinformatic pipeline and meticulous evaluation of their methodology have contributed to the success of these projects. The soil environment, however, with its unique challenges, requires a specialized methodological exploration to maximize microbial insights. A notable limitation in soil microbiome studies is the dearth of soil-specific reference databases available to classifiers that emulate the complexity of soil communities. There is also a lack of in-vitro mock communities derived from soil strains that can be assessed for taxonomic classification accuracy. RESULTS: In this study, we generated a custom in-silico mock community containing microbial genomes commonly observed in the soil microbiome. Using this mock community, we simulated shotgun sequencing data to evaluate the performance of three leading metagenomic classifiers: Kraken2 (supplemented with Bracken, using a custom database derived from GTDB-TK genomes along with its own default database), Kaiju, and MetaPhlAn, utilizing their respective default databases for a robust analysis. Our results highlight the importance of optimizing taxonomic classification parameters, database selection, as well as analysing trimmed reads and contigs. Our study showed that classifiers tailored to the specific taxa present in our samples led to fewer errors compared to broader databases including microbial eukaryotes, protozoa, or human genomes, highlighting the effectiveness of targeted taxonomic classification. Notably, an optimal classifier performance was achieved when applying a relative abundance threshold of 0.001% or 0.005%. The Kraken2 supplemented with bracken, with a custom database demonstrated superior precision, sensitivity, F1 score, and overall sequence classification. Using a custom database, this classifier classified 99% of in-silico reads and 58% of real-world soil shotgun reads, with the latter identifying previously overlooked phyla using a custom database. CONCLUSION: This study underscores the potential advantages of in-silico methodological optimization in metagenomic analyses, especially when deciphering the complexities of soil microbiomes. We demonstrate that the choice of classifier and database significantly impacts microbial taxonomic profiling. Our findings suggest that employing Kraken2 with Bracken, coupled with a custom database of GTDB-TK genomes and fungal genomes at a relative abundance threshold of 0.001% provides optimal accuracy in soil shotgun metagenome analysis.

Comparison of soil and grass microbiomes and resistomes reveals grass as a greater antimicrobial resistance reservoir than soil.

Grasslands cover a large proportion of global agricultural landmass used to feed herbivores and ruminants and link the environment to the food chain via animals onto humans. However, most scientific studies of antimicrobial resistance and microbiomes at the environmental - animal nexus have focused on soil or vegetables rather than grasslands. Based on previous microbiome phyllosphere-soil studies we hypothesised that the microbiome and resistomes across soil and grass would have a core of shared taxa and antimicrobial resistance genes (ARGs), but that in addition each would also have a minority of unique signatures. Our data indicated grass contained a wider variety and higher relative abundance of ARGs and mobile genetic elements (MGEs) than soil with or without slurry amendments. The microbiomes of soil and grass were similar in content but varied in the composition proportionality. While there were commonalities across many of the ARGs present in soil and on grass their correlations with MGEs and bacteria differed, suggesting a source other than soil is also relevant for the resistome of grass. The variations in the relative abundances of ARGs in soil and on grass also indicated that either the MGEs or the bacteria carrying the ARGs comprised a higher relative abundance on grass than in soil. We conclude that while soil may be a source of some of these genes it cannot be the source for all ARGs and MGEs. Our data identifies grass as a more diverse and abundant reservoir of ARGs and MGEs in the environment than soil, which is significant to human and animal health when viewed in the context of grazing food animals.

Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland.

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.

Greener Operations: a James Lind Alliance Priority Setting Partnership to define research priorities in environmentally sustainable perioperative practice through a structured consensus approach.

OBJECTIVES: To agree on the 'top 10' research priorities for environmentally sustainable perioperative practice. DESIGN: Surveys and literature review; final consensus workshop using a nominal group technique. SETTING: UK-based setting. PARTICIPANTS: Healthcare professionals, patients, carers and the public. OUTCOME MEASURES: Initial survey-suggested research questions; interim survey-shortlist of 'indicative' questions (the 20 most frequently nominated by patients, carers and the public, and healthcare professionals); final workshop-ranked research priorities. RESULTS: Initial survey-1926 suggestions by 296 respondents, refined into 60 indicative questions. Interim survey-325 respondents. Final workshop-21 participants agreed the 'top 10': (1) How can more sustainable reusable equipment safely be used during and around the time of an operation? (2) How can healthcare organisations more sustainably procure (obtain) medicines, equipment and items used during and around the time of an operation? (3) How can healthcare professionals who deliver care during and around the time of an operation be encouraged to adopt sustainable actions in practice? (4) Can more efficient use of operating theatres and associated practices reduce the environmental impact of operations? (5) How can the amount of waste generated during and around the time of an operation be minimised? (6) How do we measure and compare the short-term and long-term environmental impacts of surgical and non-surgical treatments for the same condition? (7) What is the environmental impact of different anaesthetic techniques (eg, different types of general, regional and local anaesthesia) used for the same operation? (8) How should the environmental impact of an operation be weighed against its clinical outcomes and financial costs? (9) How can environmental sustainability be incorporated into the organisational management of operating theatres? (10) What are the most sustainable forms of effective infection prevention and control used around the time of an operation (eg, personal protective equipment, drapes, clean air ventilation)? CONCLUSIONS: A broad range of 'end-users' have identified research priorities for sustainable perioperative care.

A Longitudinal and Comparative Content Analysis of Instagram Fitness Posts.

Body dissatisfaction is among the most common mental health challenges experienced by women and has been identified as a risk factor for disordered eating. Research has found that exposure to social media images depicting thin, muscular bodies, often dubbed 'fitspiration', may contribute to body dissatisfaction. Image-centred social media platforms, such as Instagram, have rising popularity among adolescents and young adults. However, little is known about the content of images produced by different fitness-related sources, such as those from fitness brands compared with individual users, and how fitness content on social media is evolving over time. This study sought to determine whether Instagram content varied between female fitness influencers and brands and how this content changed between 2019 and 2021. A longitudinal content analysis was conducted on a sample of 400 Instagram images using a coding scheme developed specifically for this project. The scheme coded images for fit ideal body depiction, fitness focus, objectification, and sexualisation. Chi-square tests indicated that female fitness influencer content was more sexualised and portrayed more of the fit ideal, while fitness brands produced more Instagram content with a fitness focus. There were no significant overall longitudinal changes for any of the four key variables. However, when looking at longitudinal changes by account type, fitness-focused influencer content increased while fitness-focused brand content decreased over time. These findings highlight discernible differences in content produced by different Instagram account types. It points future research towards the consideration of potential moderating factors, such as account type, when exploring the impact of social media images on body image and mental health.

Metagenomic and HT-qPCR analysis reveal the microbiome and resistome in pig slurry under storage, composting, and anaerobic digestion.

Direct application of pig slurry to agricultural land, as a means of nutrient recycling, introduces pathogens, antibiotic resistant bacteria, or genes, to the environment. With global environmental sustainability policies mandating a reduction in synthetic fertilisation and a commitment to a circular economy it is imperative to find effective on-farm treatments of slurry that maximises its fertilisation value and minimises risk to health and the environment. We assessed and compared the effect of storage, composting, and anaerobic digestion (AD) on pig slurry microbiome, resistome and nutrient content. Shotgun metagenomic sequencing and HT-qPCR arrays were implemented to understand the dynamics across the treatments. Our results identified that each treatment methods have advantages and disadvantages in removal pollutants or increasing nutrients. The data suggests that storage and composting are optimal for the removal of human pathogens and anaerobic digestion for the reduction in antibiotic resistance (AMR) genes and mobile genetic elements. The nitrogen content is increased in storage and AD, while reduced in composting. Thus, depending on the requirement for increased or reduced nitrogen the optimum treatment varies. Combining the results indicates that composting provides the greatest gain by reducing risk to human health and the environment. Network analysis revealed reducing Proteobacteria and Bacteroidetes while increasing Firmicutes will reduce the AMR content. KEGG analysis identified no significant change in the pathways across all treatments. This novel study provides a data driven decision tree to determine the optimal treatment for best practice to minimise pathogen, AMR and excess or increasing nutrient transfer from slurry to environment.

Effect of contrasting phosphorus levels on nitrous oxide and carbon dioxide emissions from temperate grassland soils.

Agricultural practices such as repeated fertilization impact carbon (C), nitrogen (N) and phosphorus (P) cycling and their relationships in the plant-soil continuum, which could have important implications for the magnitude of greenhouse gas emissions. However, little is known about the effect of C and N additions under contrasting soil P availability status on nitrous oxide (N2O) and carbon dioxide (CO2) emissions. In this study, we conducted a field-based experiment that investigated the impact of long-term (23 years) P management (no (P0, 0 kg P ha-1), low (P15, 15 kg P ha-1) and high (P45, 45 kg P ha-1) P inputs) on N2O and CO2 emissions following two C + N application events in two managed grassland ecosystems with loam and sandy loam soils. The magnitude of fluxes varied between the soil P availability levels. Cumulative N2O emission was significantly higher in P0 soils (1.08 ± 0.09 g N2O-N m-2) than P45 soils (0.63 ± 0.03 g N2O-N m-2), with the loam soil (1.04 ± 0.04 g N2O-N m-2) producing significantly higher emissions than the sandy loam soil (0.88 ± 0.05 g N2O-N m-2). We conclude that P-limitation stimulates N2O emissions, whereas P-enrichment promotes soil respiration in these temperate grassland sites. Our findings inform effective nutrient management strategies underpinning optimized use of N and P inputs to agricultural soils as mitigation measures for both food security and reducing greenhouse gas emissions.

Survival of Escherichia coli and Listeria innocua on Lettuce after Irrigation with Contaminated Water in a Temperate Climate.

Microbial disease outbreaks related to fresh produce consumption, including leafy green vegetables, have increased in recent years. Where contamination occurs, pathogen persistence may represent a risk for consumers' health. This study analysed the survival of E. coli and L. innocua on lettuce plants watered with contaminated irrigation water via a single irrigation event and within stored irrigation water. Separate lettuce plants (Lactuca sativa var. capitata) were irrigated with water spiked with Log10 7 cfu/mL of each of the two strains and survival assessed via direct enumeration, enrichment and qPCR. In parallel, individual 20 L water microcosms were spiked with Log10 7 cfu/mL of the individual strains and sampled at similar time points. Both strains were observed to survive on lettuce plants up to 28 days after inoculation. Direct quantification by culture methods showed a Log10 4 decrease in the concentration of E. coli 14 days after inoculation, and a Log10 3 decrease in the concentration of L. innocua 10 days after inoculation. E. coli was detected in water samples up to 7 days after inoculation and L. innocua was detected up to 28 days by direct enumeration. Both strains were recovered from enriched samples up to 28 days after inoculation. These results demonstrate that E. coli and L. innocua strains are able to persist on lettuce after a single contamination event up until the plants reach a harvestable state. Furthermore, the persistence of E. coli and L. innocua in water for up to 28 days after inoculation illustrates the potential for multiple plant contamination events from stored irrigation water, emphasising the importance of ensuring that irrigation water is of a high quality.

Long-term persistence and leaching of Escherichia coli in temperate maritime soils.

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 +/- 0.77 CFU ml(-1)), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.

Characterization of environmentally persistent Escherichia coli isolates leached from an Irish soil.

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.

Easy phylotyping of Escherichia coli via the EzClermont web app and command-line tool.

The Clermont PCR method for phylotyping Escherichia coli remains a useful classification scheme even though genome sequencing is now routine, and higher-resolution sequence typing schemes are now available. Relating present-day whole-genome E. coli classifications to legacy phylotyping is essential for harmonizing the historical literature and understanding of this important organism. Therefore, we present EzClermont - a novel in silico Clermont PCR phylotyping tool to enable ready application of this phylotyping scheme to whole-genome assemblies. We evaluate this tool against phylogenomic classifications, and an alternative software implementation of Clermont typing. EzClermont is available as a web app at www.ezclermont.org, and as a command-line tool at https://nickp60.github.io/EzClermont/.