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BackgroundIn the environment, microbial communities are constantly exposed to invasion by antimicrobial resistant bacteria (ARB) and their associated antimicrobial resistance genes (ARGs) that were enriched in the anthroposphere. A successful invader has to overcome the biotic resilience of the habitat, which is more difficult with increasing biodiversity. The capacity to exploit resources in a given habitat is enhanced when communities exhibit greater diversity, reducing opportunities for invaders, leading to a lower persistence. In the context of antimicrobial resistance (AMR) dissemination, exogenous ARB reaching a natural community may persist longer if the biodiversity of the autochthonous community is low, increasing the chance of ARGs to transfer to community members. Reciprocally, high microbial diversity could serve as a natural long-term barrier towards invasion by ARB and ARGs.ResultsTo test this hypothesis, a sampling campaign across seven European countries was carried out to obtain 172 environmental samples from sites with low anthropogenic impact. Samples were collected from contrasting environments: stationary structured forest soils, or dynamic river biofilms and sediments. Microbial diversity and relative abundance of 27 ARGs and 5 mobile genetic element marker genes were determined. In soils, higher diversity, evenness and richness were all significantly negatively correlated with the relative abundance of the majority (>85%) of ARGs. Furthermore, the number of detected ARGs per sample was inversely correlated with diversity. However, no such effects were found for the more dynamic, regularly mixed rivers. Conclusions: In conclusion, we demonstrate that diversity can serve as barrier towards AMR dissemination in the environment. This effect is mainly observed in stationary, structured environments, where long-term, diversity-based resilience against invasion can evolve. Such barrier effects can in the future be exploited to limit the environmental proliferation of AMR.

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.

Our study provides novel insights into the global nature of antimicrobial resistance (AMR) plasmids across the food chain. We provide compelling evidence of the globetrotting nature of AMR plasmids and the need for surveillance to sequence plasmids with a template of analyses for others to expand these data. The AMR plasmids analysed were detected in 63 countries and in samples from humans, animals and the environment. They contained a combination of known and novel AMR genes, metal resistance genes, virulence factors, phage and replicon types.

Scientists aspire to ‘discover’ new environmental phenomena; they rarely consider the existing knowledge of First Peoples. The scientific debate1 over ‘fairy circles’ (regular bare patches within arid grasslands) is a case in point. Ecologists visiting Australia used brief field observations and numerical modelling to explain ‘fairy circles’ as plants responding to dispersion of water and nutrients2,3. By contrast, Australian Aboriginal people have long understood the bare patches to be created by termites. They have used linyji (Manyjilyjarra language) in their food economies and for other domestic or sacred purposes. They transfer intergenerational knowledge of termite ecology through talk and demonstration, art and song. Aboriginal art and narratives, and scientific field measurements confirm Aboriginal knowledge that ‘fairy circles’ are pavement-nests occupied by harvester termites. Aboriginal people’s observations and uses of termites and pavements are sophisticated; linyji are real, ‘fairy circles’ are not. Some scientists are not familiar with First Peoples or their knowledge systems. However, embracing First Peoples and their knowledge can provide a deeper understanding of ecological systems than is possible from short visits to landscapes. Co-research between First Peoples and scientists leads to better understanding and management of ecological systems and supports intergenerational learning across diverse cultures.

Background We describe two complex cases in the setting of COVID-19 at the End of Life, to enhance learning for all patients. Case Presentation Maintenance of sustained comfort in two cases required multiple drugs, specifically selected for symptoms that necessitated three separate pumps delivering continuous 24-hour subcutaneous infusion. Case Management Management of sustained comfort included opioid, midazolam, anti-secretory, diclofenac for intractable temperature, phenobarbital for extreme agitation, in one, where seizure activity was present, while insomnia, was a prominent feature of another. Management of Akatasia was also required. Case Outcome Attention to each individual patient’s rapidly evolving symptoms, during the dying phase, with a thorough differential diagnosis, wa s vitally important in the context of a ‘Good Death’. This was achieved in both cases, reflected by evidence at the bedside of comfort and a minimum need for ‘as required’ drugs in the last days of life. Conclusions COVID-19 being a new illness, we need to prospectively study the symptom burden/clustering at End of Life and learn from management of this new disease for other illnesses also. Further research is required to develop protocols on; when does Midazolam dose reach tolerance and when should an alternative drug such as phenobarbital for sustained Gamma-Aminobutyric Acid effects be initiated; examine the optimal approach to sustained temperature control; be cognisant of extrapyramidal side effects of drugs used at End of Life and consider looking at a lack of need for ‘as required’ drugs in the last days of life as an outcome measure of sustained comfort.

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 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 of the treatment methods had advantages and disadvantages, depending on the parameter measured. The data suggests that storage and composting are optimal for the removal of human pathogens and anaerobic digestion for the reduction in AMR genes and mobile genetic elements. The nitrogen content is increased in storage and AD and 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.Graphical abstract

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.

Background The broiler gastrointestinal microbiome is a potent flock performance modulator yet may also serve as a reservoir for pathogen entry into the food chain. The goal of this project was to characterise the effect of mannan rich fraction (MRF) supplementation on microbiome diversity and composition of the intestinum tenue and cecum of commercial broilers. This study also aimed to address some of the intrinsic biases that exist in microbiome studies which arise due to the extensive disparity in 16S rRNA gene copy numbers between bacterial species and due to large intersample variation. Results We observed a divergent yet rich microbiome structure between different anatomical sites and observed the explicit effect MRF supplementation had on community structure, diversity, and pathogen modulation. Birds supplemented with MRF displayed significantly higher species richness in the cecum and significantly different bacterial community composition in each gastrointestinal (GI) tract section. Supplemented birds had lower levels of the zoonotic pathogens Escherichia coli and Clostridioides difficile across all three intestinum tenue sites highlighting the potential of MRF supplementation in maintaining food chain integrity. Higher levels of probiotic genera (eg. Lactobacillus and Blautia) were also noted in the MRF supplemented birds. Following MRF supplementation, the cecum displayed higher relative abundances of both short chain fatty acid (SFCA) synthesising bacteria and SCFA concentrations. Conclusions Mannan rich fraction addition has been observed to reduce the bioburden of pathogens in broilers and to promote greater intestinal tract microbial biodiversity. This study is the first, to our knowledge, to investigate the effect of mannan-rich fraction supplementation on the microbiome associated with different GI tract anatomical geographies. In addition to this novelty, this study also exploited machine learning and biostatistical techniques to correct the intrinsic biases associated with microbiome community studies to enable a more robust understanding of community structure.

Enterococcus faeciumhas emerged as an important nosocomial pathogen, which is increasingly difficult to treat due to the genetic acquisition of vancomycin resistance. Ireland exhibits a recalcitrant vancomycin resistant bloodstream infection rate compared to other developed countries. A set of 28 vancomycin resistant isolates was sequenced to construct a dataset alongside 61 other publicly available Irish genomes. This dataset was extensively analysed usingin-silicomethodologies and uncovered distinct evolutionary, coevolutionary, and clinically relevant population trends. These results suggest that a stable (in terms of genome size, GC%, and number of genes), yet genetically diverse population (in terms of gene content) ofEnterococcus faeciumpersist in Ireland with acquired resistance arisingviaplasmid acquisition (vanA) or to a lesser extent, chromosomal recombination (vanB). Population analysis described five clusters with one cluster partitioned into four clades which transcend isolation dates. Pangenomic and recombination analyses revealed an open (whole genome and chromosomal specific) pangenome illustrating a rampant evolutionary pattern. Comparative resistomics and virulomics uncovered distinct chromosomal and mobilomal propensity for multidrug resistance, widespread chromosomal point-mutation mediated resistance, and chromosomal harboured arsenals of virulence factors. Comparative phagomics revealed a core prophagome of three prophages throughout the dataset. Interestingly, a potential difference in biofilm formation strategies was highlighted by coevolutionary analysis, suggesting differential biofilm genotypes betweenvanAandvanBisolates. These results highlight the evolutionary history of IrishEnterococcus faeciumisolates and may provide an insight into underlying infection dynamics in a clinical setting.

Antimicrobial resistance (AMR) and multi-drug resistance (MDR) in pathogenic bacteria are frequently mediated by plasmids. However, plasmids do not exist in isolation but rather require the bacterial host interaction in order to produce the AMR phenotype. This study aimed to utilise mass spectrometry-based proteomics to reveal the plasmid and chromosomally derived protein profile of Escherichia coli under antimicrobial stress. This was achieved by comparing the proteomes of E. coli containing the MDR pEK499 plasmid, under ampicillin, cefotaxime, imipenem or ciprofloxacin stress with the proteomes of these bacteria grown in the absence of antimicrobial. Our analysis identified statistically significant differentially abundant proteins common to groups exposed to the β-lactam antimicrobials but not ciprofloxacin, indicating a β-lactam stress response to exposure from this class of drugs, irrespective of β-lactam resistance or susceptibility. These include ecotin and free methionine-R-sulfoxide reductase. These data also identified distinct differences in the cellular response to each β-lactam. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA-cycle and several proteins associated with detoxification and a decrease in the relative abundances of proteins associated with stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the β-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial – AMR protein associations. For example, a relative increase in the amount of blaCTX-M-15 in the presence of cefotaxime and ciprofloxacin but not the other β-lactams, suggesting regulation of the blaCTX-M-15 protein production. The proteomic data from the this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobials responses in pathogens, which may well represent potential targets of AMR inhibition.

Antibiotic resistance is regarded as one of the most serious threats to human health worldwide. The rapid increase in resistance rates has been attributed to the extensive use of antibiotics since they became commercially available. The use of antibiotics as growth promotors has been banned in numerous regions for this reason. Mannan-rich fraction (MRF) has been reported to show similar growth-promoting effects to antibiotics. We investigated the effect of MRF on the microbial community, resistome and metabolic pathways within the caecum of commercial broilers at two different timepoints within the growth of the broiler, day 27 and day 34. The data indicated an overall increase in health and economic gain for the producer with the addition of MRF to the diet of the broilers. The only significant difference across the microbial composition of the samples was in the richness of the microbial communities across all samples. While all samples harboured resistance genes conferring resistance to the same classes of antibiotics, there was significant variation in the antimicrobial resistance gene richness across time and treatment and across combinations of time and treatment. The taxa with positive correlation comprised Bacilli and Clostridia. The negative correlation taxa were also dominated by Bacilli, specifically the Streptococcus genera. The KEGG-pathway analysis identified an age-related change in the metabolism pathway abundances of the caecal microflora. We suggest that the MRF-related increases in health and weight gain in the broilers may be associated with changes in the metabolism of the microbiomes rather than the microbial composition. The resistome variations across samples were correlated with specific genera. These data may be used to further enhance the development of feed supplements to reduce the presence of antibiotic resistance genes (ARGs) within poultry. While the ARGs of greatest concern to human or animal health were not detected in this study, it has identified the potential to reduce the presence of ARGs by the increase in specific genera.

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.

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.

There is a reproducibility crisis in scientific studies. Some of these crises arise from incorrect application of statistical tests to data that follow inappropriate distributions, have inconsistent equivariance, or have very small sample sizes. As determining which test is most appropriate for all data in a multicategorical study (such as comparing taxa between sites in microbiome studies), we present statsSuma, an interactive Python notebook (which can be run from any desktop computer using the Google Colaboratory web service) and does not require a user to have any programming experience. This software assesses underlying data structures in a given dataset to advise what pairwise or listwise statistical procedure would be best suited for all data. As some users may be interested in further mining specific trends, statSuma performs 5 different two-tailed pairwise tests (Student’s t-test, Welch’s t-test, Mann-Whitney U-test, Brunner-Munzel test, and a pairwise Kruskal-Wallis H-test) and advises the best test for each comparison. This software also advises whether ANOVA or a multicategorical Kruskal-Wallis H-test is most appropriate for a given dataset and performs both procedures. A data distribution-vs-Gaussian distribution plot is produced for each taxon at each site and a variance plot between all combinations of 2 taxa at each site are produced so Gaussian tests and variance tests can be visually confirmed alongside associated statistical determinants.

Isolation Forests is an unsupervised machine learning technique for detecting outliers in continuous datasets that does not require an underlying equivariant or Gaussian distribution and is suitable for use on small datasets. While this procedure is widely used across quantitative fields, to our knowledge, this is the first attempt to solely assess its use for microbiome datasets. Here we present uniForest, an interactive Python notebook (which can be run from any desktop computer using the Google Colaboratory web service) for the processing of microbiome outliers. We used uniForest to apply Isolation Forests to the Healthy Human Microbiome project dataset and imputed outliers with the mean of the remaining inliers to maintain sample size and assessed its prowess in variance reduction in both community structure and derived ecological statistics (-diversity). We also assessed its functionality in anatomical site made available under aCC-BY 4.0 International license. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is bioRxiv preprint doi: https://doi.org/10.1101/2021.05.17.444491; this version posted May 17, 2021. The copyright holder for this preprint 2 differentiation (pre- and postprocessing) using principal component analysis, dissimilarity matrices, and ANOSIM. We observed a minimum variance reduction of 81.17% across the entire dataset and in alpha diversity at the Phylum level. Application of Isolation Forests also separated the dataset to an extremely high specificity, reducing variance within taxa samples by a minimum of 81.33%. It is evident that Isolation Forests are a potent tool in restricting the effect of variance in microbiome analysis and has potential for broad application in studies where high levels of microbiome variance is expected. This software allows for clean analyses of otherwise noisy datasets.

The waste stream of obsolete electronic equipment grows exponentially, creating a worldwide pollution and resource problem. Electrical and electronic waste (e-waste) comprises a heterogeneous mix of glass, plastics (including flame retardants and other additives), metals (including rare Earth elements), and metalloids. The e-waste issue is complex and multi-faceted. In examining the different aspects of e-waste, informal recycling in developing countries has been identified as a primary concern, due to widespread illegal shipments; weak environmental, as well as health and safety, regulations; lack of technology; and inadequate waste treatment structure. For example, Nigeria, Ghana, India, Pakistan, and China have all been identified as hotspots for the disposal of e-waste. This article presents a critical examination on the chemical nature of e-waste and the resulting environmental impacts on, for example, microbial biodiversity, flora, and fauna in e-waste recycling sites around the world. It highlights the different types of risk assessment approaches required when evaluating the ecological impact of e-waste. Additionally, it presents examples of chemistry playing a role in potential solutions. The information presented here will be informative to relevant stakeholders seeking to devise integrated management strategies to tackle this global environmental concern.

A comprehensive monitoring of a broad set of antibiotics in the final effluent of wastewater treatment plants (WWTPs) of 7 European countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway) was carried out in two consecutive years (2015 and 2016). This is the first study of this kind performed at an international level. Within the 53 antibiotics monitored 17 were detected at least once in the final effluent of the WWTPs, i.e.: ciprofloxacin, ofloxacin, enrofloxacin, orbifloxacin, azithromycin, clarithromycin, sulfapyridine, sulfamethoxazole, trimethoprim, nalidixic acid, pipemidic acid, oxolinic acid, cefalexin, clindamycin, metronidazole, ampicillin, and tetracycline. The countries exhibiting the highest effluent average concentrations of antibiotics were Ireland and the southern countries Portugal and Spain, whereas the northern countries (Norway, Finland and Germany) and Cyprus exhibited lower total concentration. The antibiotic occurrence data in the final effluents were used for the assessment of their impact on the aquatic environment. Both, environmental predicted no effect concentration (PNEC-ENVs) and the PNECs based on minimal inhibitory concentrations (PNEC-MICs) were considered for the evaluation of the impact on microbial communities in aquatic systems and on the evolution of antibiotic resistance, respectively. Based on this analysis, three compounds, ciprofloxacin, azithromycin and cefalexin are proposed as markers of antibiotic pollution, as they could occasionally pose a risk to the environment. Integrated studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and environmental risk in regular water monitoring programs, We thank all the wastewater treatment plant staff and other collaborators who made this work possible. We would also thank Virginie Keller and Andrew Johnson for their help in the data processing. Funding: This work was financed by the Water JPI through the national funding agencies supporting the consortium WaterJPI/0001/2013 STARE—“Stopping Antibiotic Resistance Evolution” (Cyprus, RPF; Germany, BMBF; Spain, MINECO; Finland, AKA; Ireland, EPA; Norway, RCN; Portugal, FCT). Authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124 and 2017-SGR-1404-Water and Soil Quality Unit). S.R.-M. acknowledges the Ramon y Cajal program (RYC-2014-16707). I.V.-M. was supported by the FCT grant SFRH/BPD/87360/2012. Laura Rodriguez de la Ballina is acknowledged for her help in the design of graphical abstract.

Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms of antibiotic resistance in the effluent of two urban WWTPs in Ireland. Effluent samples were collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out to identify AmpC or extended-spectrum β-lactamase (ESBL) producers. The presence of ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed intoEscherichia coliDH5α recipient and underwent plasmid replicon typing to identify incompatibility groups. More than 90% of isolated faecal coliforms were resistant to amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin and imipenem were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carriedblaTEM, 1.4%blaSHV-12, 0.2%blaCTX-M-1and 1%blaCTX-M-15). Plasmids extracted from 52 ESBL isolates were successfully transformed into recipientE. coli. The detected plasmid incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and are sources of ESBL-producing, carbapenem and colistin resistantEnterobacteriaceae.ImportanceAntibiotic resistant bacteria (ARB) are an emerging environmental concern with a potential impact on human health. The results provide the evidence that treated wastewater is polluted with antibiotic resistant bacteria containing mobile resistance mechanisms of importance to clinical treatment of pathogens and multi-drug resistant (MDR) faecal coliforms. They are sources of relatively high proportions of ESBL-producingEnterobacteriaceae, and include carbapenem and colistin resistantEnterobacteriaceae.The significance of this study is the identification of the role of WWTPs as a potential control point to reduce or stop the movement of ESBL, MDR and colistin resistant bacteria into the environment from further upstream sources, such as human or animal waste.

Waste water and waste water treatment plants can act as reservoirs and environmental suppliers of antibiotic resistance. They have also been proposed to be hotspots for horizontal gene transfer, enabling the spread of antibiotic resistance genes between different bacterial species. Waste water contains antibiotics, disinfectants, and metals which can form a selection pressure for antibiotic resistance, even in low concentrations. Our knowledge of antibiotic resistance in waste water has increased tremendously in the past few years with advances in the molecular methods available. However, there are still some gaps in our knowledge on the subject, such as how active is horizontal gene transfer in waste water and what is the role of the waste water treatment plant in the environmental resistome? The purpose of this review is to briefly describe some of the main methods for studying antibiotic resistance in waste waters and the latest research and main knowledge gaps on the issue. In addition, some future research directions are proposed.

Transposon-aided capture of plasmids allows for antibiotic resistance plasmids to be acquired from complex samples. It is based on the insertion of a transposon, with a known origin of replication and selectable marker, into the plasmids present in a sample which can subsequently be captured after the removal of contaminating chromosomal DNA. Here we describe isolation of bacterial cells from a complex sample, DNA extraction, transposon-aided capture of plasmids in the sample, and analysis of the captured plasmids.

The Advanced Biotechnology for Intensive-Freshwater Aquaculture Wastewater Reuse (ABAWARE) project, which is part of the European Commission’s Water Joint Programming Initiative 2016 Joint Call, aims to increase the efficiency and resilience of water use in aquaculture and minimise its negative impact on the environment and human health. This research, which forms one part of the total ABAWARE project, aimed to ascertain the impact of using microbiota and certain plant species, in conjuncture with a more traditional Recirculated Aquaculture System (RAS), as a filtering system had on the arb and G abundance in various samples taken from an aquaculture facility. Sediment and water samples were taken from the inflow, the main fish basin, after the bioactive ponds before filtration, and after filtration. The resistance genes present in these samples were detected using the Wafergen smartchip real-time qPCR system. This system allows for the simultaneous quantification of 348 distinct Antibiotic Resistance Genes for each sample. The samples also underwent microbiome analysis via 16S rRNA metagenomic sequencing. Mothur was used to analyse the sequencing data. This data informs us of the changes in the microbial population changes that are enacted by the various stages within the aquaculture facility.

It has been well documented that antibiotic resistance (AR) is a clinical concern that affects both human and animal health but AR in the environment and food-chain is not as well understood. AR bacteria can occur naturally in soil, water and organic fertilizers used in agriculture so there is a risk that AR can pass to humans via the food-chain. This study focuses on lettuce cultivation undergoing four treatments (Normal irrigation water+normal soil, normal irrigation water+manure, UV irrigation water+normal soil, UV irrigation water+manure)to determine the mechanisms by which the AR is transferred to the plants over the growth period of the lettuce (7 time-points – week 0 to week 6). Plasmids (n=318) have been isolated from irrigation water (n=36), soil (n=45) and lettuce (n=42) samples using the exogenous isolation method for week 0 and week 6 initially. Antibiotic susceptibility testingto amikacin, cefotaxime, ciprofloxacin, imipenem, kanamycin, tetracycline has been carried out. Multi-drug resistance profiles were established for soil taken at timepoint 0 and lettuce taken at timepoint 6. Extracted plasmid DNA was sent for metagenomic analysis to determine which genes are involved in the transfer of AR at the interfaces. The results of the sequencing showed that there are multiple AR genespresent, including Tet, Sme, Cmy, Oxa and ANT(4’)-Ib, that confer resistance to bacteria. The identification of multi-drug resistance in soil and lettuce samples is concerning and highlights the need to determine the mechanisms leading to antibiotic resistance in food.

Antibiotics are used extensively in agriculture as therapeutics, and in some countries, for prophylaxis and as growth promotors. Alarmingly, there is a potential resistance transmission pathway from animals to humans through food. We analysed the microbiome and resistome of sixteen broiler chickens, which are raised for meat production. DNA was extracted from caecal samples taken at days 27 and 34 posthatch from broilers, half of which had their diets supplemented with a mannan rich fraction. Paired end sequencing was performed using an Illumina HiSeq 4000. The data was analysed by MGnify to generate taxonomic read files. The microbiome was analysed using Calypso software and the antibiotic resistance genes (ARGs) identified using the ARGs-OAP pipeline. The main phyla detected in all samples was Firmicutes and Bacteroidetes. Clostridia was the main class detected and Clostridiales the main order. Faecalibacterium, Lactobacillus and Bacteroides were the main genus detected, which are common in the broiler caecal microbiome. There was an increase in Bifidobacterium in the treated group. Principle component analysis showed that both time points cluster together. Rarefaction analysis confirms that a sufficient sequencing depth was obtained. Tetracycline resistance comprised the greatest proportions of ARGs present, followed by the aminoglycoside, macrolide, vancomycin, beta-lactam and bacitracin classes. Further analysis of the sequences will allow for full characterisation of the resistome between treatment groups. The antibiotic resistance residues in food animals may have the potential to disseminate antibiotic resistance to the human community via the food chain.

Integrated antibiotic resistance (AR) surveillance is one of the objectives of the World Health Organization global action plan on antimicrobial resistance. Urban wastewater treatment plants (UWTPs) are among the most important receptors and sources of environmental AR. On the basis of the consistent observation of an increasing north-to-south clinical AR prevalence in Europe, this study compared the influent and final effluent of 12 UWTPs located in seven countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway). Using highly parallel quantitative polymerase chain reaction, we analyzed 229 resistance genes and 25 mobile genetic elements. This first trans-Europe surveillance showed that UWTP AR profiles mirror the AR gradient observed in clinics. Antibiotic use, environmental temperature, and UWTP size were important factors related with resistance persistence and spread in the environment. These results highlight the need to implement regular surveillance and control measures, which may need to be appropriate for the geographic regions., This work was financed by the Water JPI through the national funding agencies supporting the consortium WaterJPI/0001/2013 STARE—“Stopping Antibiotic Resistance Evolution” (Cyprus, RPF; Germany, BMBF; Spain, MINECO; Finland, AKA; Ireland, EPA; Norway, RCN; Portugal, FCT). I.V.-M. was supported by the FCT grant SFRH/BPD/87360/2012, C.N.-d.-R. by the FCT grant SFRH/BD/97131/2013, and I.H. by the FCT contract IF/00492/2013. Other funders: A grant from the Michigan State University Center for Health Impacts of Agriculture (CHIA) and the National Natural Science Foundation of China (21677149).

Human disease outbreaks caused by pathogenic Escherichia coli are increasingly associated with the consumption of contaminated fresh produce. Internalization of enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strains into plant tissues may present a serious threat to public health. In the current study, the ability of the fluorescing Shiga toxin-negative E. coli O104:H4 strain C227/11ϕcu/pKEC2 to adhere to and to internalize into the roots of Lactuca sativa and Valerianella locusta grown in diluvial sand (DS) and alluvial loam (AL) was investigated. In parallel, the soil microbiota was analyzed by partial 16S rRNA gene sequencing. The experiments were performed in a safety level 3 greenhouse to simulate agricultural practice. The adherence of C227/11ϕcu/pKEC2 to the roots of both plant varieties was increased by at least a factor three after incubation in DS compared to AL. Compared to V. locusta, internalization into the roots of L. sativa was increased 12-fold in DS and 108-fold in AL. This demonstrates that the plant variety had an impact on the internalization ability, whereas for a given plant variety the soil type also affected bacterial internalization. In addition, microbiota analysis detected the inoculated strain and showed large differences in the bacterial composition between the soil types.

Antibiotic resistance reservoirs within food-producing animals are thought to be a risk to animal and human health. This study describes the minimum natural resistome of pig faeces as the bacteria are under no direct antibiotic selective pressure. The faecal resistome of 257 different genes comprised 56 core and 201 accessory resistance genes. The genes present at the highest relative abundances across all samples were tetW, tetQ, tet44, tet37, tet40, mefA, aadE, ant(9)-1, ermB and cfxA2. This study characterized the baseline resistome, the microbiome composition and the metabolic components described by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in healthy pig faeces, without antibiotic selective pressures. The microbiome hierarchical analysis resulted in a cluster tree with a highly similar pattern to that of the accessory resistome cluster tree. Functional capacity profiling identified genes associated with horizontal gene transfer. We identified a statistically significant positive correlation between the total antibiotic resistome and suggested indicator genes, which agree with using these genes as indicators of the total resistomes. The correlation between total resistome and total microbiome in this study was positive and statistically significant. Therefore, the microbiome composition influenced the resistome composition. This study identified a core and accessory resistome present in a cohort of healthy pigs, in the same conditions without antibiotics. It highlights the presence of antibiotic resistance in the absence of antibiotic selective pressure and the variability between animals even under the same housing, food and living conditions. Antibiotic resistance will remain in the healthy pig gut even when antibiotics are not used. Therefore, the risk of antibiotic resistance transfer from animal faeces to human pathogens or the environment will remain in the absence of antibiotics.

There is little known about the wastewater treatment plant (WWTP) effluent microbiome, its structure and dynamics. Here, we provide a study of the microbiome of effluent leaving conventional WWTPs and entering the water environment. DNA was extracted from WWTP effluent samples collected in 2015 and 2016. The bacterial communities were studied using Illumina MiSeq 16S rRNA gene amplicon sequencing and analysed using Calypso software. The Proteobacteria, Bacteroides, Actinobacteria, Firmicutes, Tenericutes and Verrucomicrobia phyla dominated the microbiomes. The bacterial community composition at high taxonomic levels is consistent between the tested WWTP effluents, and in agreement with previous studies of WWTP effluents in different global locations. The analysed microbiomes of the WWTPs shared high similarities with human faecal microbiome and contained potential human pathogens. The bacterial phyla/class composition of the bacterial communities varied greatly in both WWTP effluents in October 2015. The bacterial diversity was slightly different between studied WWTPs. Two main bacterial clusters were detected in all samples during all sampling periods. In conclusion, this work highlights the need for a better understanding of the bacterial communities in WWTP effluent. This data should be considered when analysing the risk posed by WWTP effluent to the environment and to human health.