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The article focuses on the reuse of treated urban wastewater and its challenges and opportunities addressed by the European Union (EU)-COST Action ES1403 (NEREUS). Topics include the commitment of the COST Action to provide insight on the challenges of wastewater reuse practice, the objectives of the five working groups (WGs), and the potential impact of the NEREUS.

Anemia is common in the elderly, especially in very old patients who are often frail and may be institutionalized. Senescence, the ageing process, puts the elderly at risk of developing anemia for multiple reasons, but anemia may not be attributed to senescence unless a thorough diagnostic workup has excluded other etiologies. Nutritional deficiencies are common and need to be identified and treated appropriately. Inflammatory diseases and renal failure are also frequent etiological factors and tend to be chronic. Myelodysplastic syndromes increase in frequency with age and may be difficult to diagnose and only a minority of cases respond to appropriate treatment. Anemia is associated with poor outcome and symptomatic treatment with transfusions frequently has to be considered. Red blood cell transfusion has a high therapeutic index and is likely to be effective only if anemia is symptomatic or particularly severe, as a consequence, its use has been restricted to this group. Much of the evidence on usage is limited to younger adults and specific clinical situations. Geriatricians have to deal with a large number of patients with significant anemia but with an absence of well constructed guidelines for the frail and the very old. The object of the present article is to raise awareness that anemia in the geriatric group is multi-factorial and that the patients are more than merely older than those included in most studies, that the results of ongoing trials should be appropriately interpreted and will be important in guiding future practice. [ABSTRACT FROM AUTHOR]

Antibiotic resistance genes (ARGs) are widespread in the environment, and soils, specifically, are hotspots for microorganisms with inherent antibiotic resistance. Manure and sludge used as fertilizers in agricultural production have been shown to contain vast amounts of ARGs, and due to continued applications, ARGs accumulate in agricultural soils. Some soils, however, harbor a resilience capacity that could depend on specific soil properties, as well as the presence of predatory bacteria that are able to hydrolyse living bacteria, including bacteria of clinical importance. The objectives of this study were to (i) investigate if the antibiotic resistance profile of the soil microbiota could be differently affected by the addition of cow manure, chicken manure, and sludge, and (ii) investigate if the amendments had an effect on the presence of predatory bacteria. The three organic amendments were mixed separately with a field soil, divided into pots, and incubated in a greenhouse for 28 days. Droplet digital PCR (ddPCR) was used to quantify three ARGs, two predatory bacteria, and total number of bacteria. In this study, we demonstrated that the choice of organic amendment significantly affected the antibiotic resistance profile of soil, and promoted the growth of predatory bacteria, while the total number of bacteria was unaffected.

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds., Results: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application., Conclusions: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Fungal phytopathogens cause significant reductions in agricultural yields annually, and overusing chemical fungicides for their control leads to environmental pollution and the emergence of resistant pathogens. Exploring natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We isolated and characterized a novel bacterial strain associated with the species Burkholderia cenocepacia , termed APO9, which strongly inhibits Zymoseptoria tritici , a commercially important pathogenic fungus causing Septoria tritici blotch in wheat. Additionally, this strain exhibits inhibitory activity against four other phytopathogens. We found that physical contact plays a crucial role for APO9's antagonistic capacity. Genome sequencing of APO9 and biosynthetic gene cluster (BGC) analysis identified nine classes of BGCs and three types of secretion systems (types II, III, and IV), which may be involved in the inhibition of Z. tritic i and other pathogens. To identify genes driving APO9's inhibitory activity, we screened a library containing 1,602 transposon mutants and identified five genes whose inactivation reduced inhibition efficiency. One such gene encodes for a diaminopimelate decarboxylase located in a terpenoid biosynthesis gene cluster. Phylogenetic analysis revealed that while some of these genes are also found across the Burkholderia genus, as well as in other Betaproteobacteria , the combination of these genes is unique to the Burkholderia cepacia complex. These findings suggest that the inhibitory capacity of APO9 is complex and not limited to a single mechanism, and may play a role in the interaction between various Burkholderia species and various phytopathogens within diverse plant ecosystems., Importance: The detrimental effects of fungal pathogens on crop yields are substantial. The overuse of chemical fungicides contributes not only to environmental pollution but also to the emergence of resistant pathogens. Investigating natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We discovered and examined a unique bacterial strain that demonstrates significant inhibitory activity against several phytopathogens. Our research demonstrates that this strain has a wide spectrum of inhibitory actions against plant pathogens, functioning through a complex mechanism. This plays a vital role in the interactions between plant microbiota and phytopathogens., Competing Interests: The authors declare no conflict of interest.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are important environmental contaminants. Nonetheless, what drives the evolution, spread, and transmission of antibiotic resistance dissemination is still poorly understood. The abundance of ARB and ARGs is often elevated in human-impacted areas, especially in environments receiving fecal wastes, or in the presence of complex mixtures of chemical contaminants, such as pharmaceuticals and personal care products. Self-replication, mutation, horizontal gene transfer, and adaptation to different environmental conditions contribute to the persistence and proliferation of ARB in habitats under strong anthropogenic influence. Our review discusses the interplay between chemical contaminants and ARB and their respective genes, specifically in reference to co-occurrence, potential biostimulation, and selective pressure effects, and gives an overview of mitigation by existing man-made and natural barriers. Evidence and strategies to improve the assessment of human health risks due to environmental antibiotic resistance are also discussed. Environ Toxicol Chem 2024;43:637-652. © 2022 SETAC., (© 2022 SETAC.)

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This manuscript reports the complete genome sequence of a Salmonella enterica subsp. enterica serovar Typhimurium strain (designated "Bnaya"), isolated from a dead dairy calf with severe diarrhea in Israel. The isolate exhibited multi-drug resistance, which is highly unusual in bovine Salmonella spp. in Israel, prompting further investigation., Competing Interests: The authors declare no conflict of interest.

Scarcity of freshwater for agriculture has led to increased utilization of treated wastewater (TWW), establishing it as a significant and reliable source of irrigation water. However, years of research indicate that if not managed adequately, TWW may deleteriously affect soil functioning and plant productivity, and pose a hazard to human and environmental health. This review leverages the experience of researchers, stakeholders, and policymakers from Israel, the United-States, and Europe to present a holistic, multidisciplinary perspective on maximizing the benefits from municipal TWW use for irrigation. We specifically draw on the extensive knowledge gained in Israel, a world leader in agricultural TWW implementation. The first two sections of the work set the foundation for understanding current challenges involved with the use of TWW, detailing known and emerging agronomic and environmental issues (such as salinity and phytotoxicity) and public health risks (such as contaminants of emerging concern and pathogens). The work then presents solutions to address these challenges, including technological and agronomic management-based solutions as well as source control policies. The concluding section presents suggestions for the path forward, emphasizing the importance of improving links between research and policy, and better outreach to the public and agricultural practitioners. We use this platform as a call for action, to form a global harmonized data system that will centralize scientific findings on agronomic, environmental and public health effects of TWW irrigation. Insights from such global collaboration will help to mitigate risks, and facilitate more sustainable use of TWW for food production in the future., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hila Korach-Rechtman reports a relationship with Kando Environmental Services LTD that includes: employment., (© 2023 The Author(s).)

Food safety of leafy greens is an emerging public health issue as they can harbor opportunistic human pathogens (OHPs) and expose OHPs to consumers. Protists are an integral part of phyllosphere microbial ecosystems. However, our understanding of protist-pathogen associations in the phyllosphere and their consequences on public health remains poor. Here, we examined phyllosphere protists, human pathogen marker genes (HPMGs), and protist endosymbionts from four species of leafy greens from major supermarkets in Xiamen, China. Our results showed that Staphylococcus aureus and Klebsiella pneumoniae were the dominant human pathogens in the vegetable phyllosphere. The distribution of HPMGs and protistan communities differed between vegetable species, of which Chinese chive possessed the most diverse protists and highest abundance of HPMGs. HPMGs abundance positively correlated with the diversity and relative abundance of phagotrophic protists. Whole genome sequencing further uncovered that most isolated phyllosphere protists harbored multiple OHPs which carried antibiotic resistance genes, virulence factors, and metal resistance genes and had the potential to HGT. Colpoda were identified as key phagotrophic protists which positively linked to OHPs and carried diverse resistance and virulence potential endosymbiont OHPs including Pseudomonas nitroreducens, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia. We highlight that phyllosphere protists contribute to the transmission of resistant OHPs through internalization and thus pose risks to the food safety of leafy greens and human health. Our study provides insights into the protist-OHP interactions in the phyllosphere, which will help in food safety surveillance and human health., (© 2023. ISME Publications B.V.)

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Antibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL -1 , respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs. IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater., Competing Interests: The authors declare no conflict of interest.

Bacillus cereus sensu lato (Bcsl) strains are widely explored due to their capacity to antagonize a broad range of plant pathogens. These include B. cereus sp. UW85, whose antagonistic capacity is attributed to the secondary metabolite Zwittermicin A (ZwA). We recently isolated four soil and root-associated Bcsl strains (MO2, S-10, S-25, LSTW-24) that displayed different growth profiles and in-vitro antagonistic effects against three soilborne plant pathogens models: Pythium aphanidermatum (oomycete) Rhizoctonia solani (basidiomycete), and Fusarium oxysporum (ascomycete). To identify genetic mechanisms potentially responsible for the differences in growth and antagonistic phenotypes of these Bcsl strains, we sequenced and compared their genomes, and that of strain UW85 using a hybrid sequencing pipeline. Despite similarities, specific Bcsl strains had unique secondary metabolite and chitinase-encoding genes that could potentially explain observed differences in in-vitro chitinolytic potential and anti-fungal activity. Strains UW85, S-10 and S-25 contained a (~500 Kbp) mega-plasmid that harbored the ZwA biosynthetic gene cluster. The UW85 mega-plasmid contained more ABC transporters than the other two strains, whereas the S-25 mega-plasmid carried a unique cluster containing cellulose and chitin degrading genes. Collectively, comparative genomics revealed several mechanisms that can potentially explain differences in in-vitro antagonism of Bcsl strains toward fungal plant pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Moshe, Gupta, Sela, Minz, Banin, Frenkel and Cytryn.)

Freshwater bodies are critical components of terrestrial ecosystems. The microbial communities of freshwater ecosystems are intimately linked water quality. These microbes interact with, utilize and recycle inorganic elements and organic matter. Here, we present three metagenomic sequence datasets (total of 182.9 Gbp) from different freshwater environments in Israel. The first dataset is from diverse freshwater bodies intended for different usages - a nature reserve, irrigation and aquaculture facilities, a tertiary wastewater treatment plant and a desert rainfall reservoir. The second represents a two-year time-series, collected during 2013-2014 at roughly monthly intervals, from a water reservoir connected to an aquaculture facility. The third is from several time-points during the winter and spring of 2015 in Lake Kinneret, including a bloom of the cyanobacterium Microcystis sp. These datasets are accompanied by physical, chemical, and biological measurements at each sampling point. We expect that these metagenomes will facilitate a wide range of comparative studies that seek to illuminate new aspects of freshwater microbial ecosystems and inform future water quality management approaches., (© 2022. The Author(s).)

Farmers apply broiler chicken litter to soils to enrich organic matter and provide crops with nutrients, following varying periods of stockpiling. However, litter frequently harbors fecal-derived microbial pathogens and associated antibiotic resistance genes (ARGs), and may be a source of microbial contamination of produce. We coupled a cutting-edge Loop Genomics long-read 16S rRNA amplicon-sequencing platform with high-throughput qPCR that targeted a suite of ARGs, to assess temporal (five time points over a 60-day period) and spatial (top, middle and bottom layers) microbiome and resistome dynamics in a broiler litter stockpile. We focused on potentially pathogenic species from the Enterobacteriaceae , Enterococcaceae and Staphylococcaceae families associated with food-borne disease. Bacterial diversity was significantly lower in the middle of the stockpile, where targeted pathogens were lowest and Bacillaceae were abundant. E. coli was the most abundant Enterobacteriaceae species, and high levels of the opportunistic pathogen Enterococcus faecium were detected. Correlation analyses revealed that the latter was significantly associated with aminoglycoside ( aac (6')-Ib(aka aac A4), aad A5), tetracycline ( tet G), vancomycin ( van C), phenicol ( flo R) and MLSB ( mph B) resistance genes. Staphylococcaceae were primarily non-pathogenic, but extremely low levels of the opportunistic pathogen S. aureus were detected, as was the opportunistic pathogen S. saprophyticus , which was linked to vancomycin ( vanSA , vanC1 ), MLSB ( vatE, ermB ) and tetracycline ( tetK ) resistance genes. Collectively, we found that stockpile microbiomes and resistomes are strongly dictated by temporal fluctuations and spatial heterogeneity. Insights from this study can be exploited to improve stockpile management practice to support sustainable antimicrobial resistance mitigation policies in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Background: Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae)., Results: Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28-76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and β-lactam resistance genes was identified., Conclusions: Within a "farm-to-fork, one health" perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract., (© 2021. The Author(s).)

Aquaculture facilities such as fishponds are one of the most anthropogenically impacted freshwater ecosystems. The high fish biomass reared in aquaculture is associated with an intensive input into the water of fish-feed and fish excrements. This nutrients load may affect the microbial community in the water, which in turn can impact the fish health. To determine to what extent aquaculture practices and natural seasonal cycles affect the microbial populations, we characterized the microbiome of an inter-connected aquaculture system at monthly resolution, over 3 years. The system comprised two fishponds, where fish are grown, and an operational water reservoir in which fish are not actively stocked. Clear natural seasonal cycles of temperature and inorganic nutrients concentration, as well as recurring cyanobacterial blooms during summer, were observed in both the fishponds and the reservoir. The structure of the aquatic bacterial communities in the system, characterized using 16S rRNA sequencing, was explained primarily by the natural seasonality, whereas aquaculture-related parameters had only a minor explanatory power. However, the cyanobacterial blooms were characterized by different cyanobacterial clades dominating at each fishpond, possibly in response to distinct nitrogen and phosphate ratios. In turn, nutrient ratios may have been affected by the magnitude of fish feed input. Taken together, our results show that, even in strongly anthropogenically impacted aquatic ecosystems, the structure of bacterial communities is mainly driven by the natural seasonality, with more subtle effects of aquaculture-related factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marmen, Fadeev, Al Ashhab, Benet-Perelberg, Naor, Patil, Cytryn, Viner-Mozzini, Sukenik, Lalzar and Sher.)

Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring γ-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.

The genus Flavobacterium is characterized by the capacity to metabolize complex organic compounds and a unique gliding motility mechanism. Flavobacteria are often abundant in root microbiomes of various plants, but the factors contributing to this high abundance are currently unknown. In this study, we evaluated the effect of various plant-associated poly- and mono-saccharides on colony expansion of two Flavobacterium strains. Both strains were able to spread on pectin and other polysaccharides such as microcrystalline cellulose. However, only pectin (but not pectin monomers), a component of plant cell walls, enhanced colony expansion on solid surfaces in a dose- and substrate-dependent manner. On pectin, flavobacteria exhibited bi-phasic motility, with an initial phase of rapid expansion, followed by growth within the colonized area. Proteomic and gene expression analyses revealed significant induction of carbohydrate metabolism related proteins when flavobacteria were grown on pectin, including selected SusC/D, TonB-dependent glycan transport operons. Our results show a positive correlation between colony expansion and the upregulation of proteins involved in sugar uptake, suggesting an unknown linkage between specific operons encoding for glycan uptake and metabolism and flavobacterial expansion. Furthermore, within the context of flavobacterial-plant interactions, they suggest that pectin may facilitate flavobacterial expansion on plant surfaces in addition to serving as an essential carbon source., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kraut-Cohen, Shapiro, Dror and Cytryn.)

Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria , while the remaining were not associated with known bacteria. One Streptomyces -derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs. IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions., (Copyright © 2020 Dror et al.)

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp ( Hypophthalmichthys molitrix ) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes ( sul1, sul2, dfrA1, tetA and blaTEM ) and class-1 integrons ( int1 ). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 10 3 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Wastewater (WW) reuse is expected to be increasingly indispensable in future water management to mitigate water scarcity. However, this increases the risk of antibiotic resistance (AR) dissemination via irrigation. Herein, a conventional (chlorination) and an advanced oxidation process (heterogeneous photocatalysis (HPC)) were used to disinfect urban WW to the same target of Escherichia coli <10 CFU/100 mL and used to irrigate lettuce plants ( Lactuca sativa ) set up in four groups, each receiving one of four water types, secondary WW (positive control), fresh water (negative control), chlorinated WW, and HPC WW. Four genes were monitored in water and soil, 16S rRNA as an indicator of total bacterial load, intI1 as a gene commonly associated with anthropogenic activity and AR, and two AR genes bla OXA-10 and qnrS . Irrigation with secondary WW resulted in higher dry soil levels of intI1 (from 1.4 × 10 4 copies/g before irrigation to 3.3 × 10 5 copies/g after). HPC-treated wastewater showed higher copy numbers of intI1 in the irrigated soil than chlorination, but the opposite was true for bla OXA-10 . The results indicate that the current treatment is insufficient to prevent dissemination of AR markers and that HPC does not offer a clear advantage over chlorination.

Antibiotic or antimicrobial resistance (AR) facilitated by the vertical and/or horizontal transfer of antibiotic resistance genes (ARGs), is a serious global health challenge. While traditionally associated with pathogens in clinical environments, it is becoming increasingly clear that non-clinical environments may also be reservoirs of ARGs. The recent improvements in rapid and affordable next generation sequencing technologies along with sophisticated bioinformatics platforms has the potential to revolutionize diagnostic microbiology and microbial surveillance. Through the study and characterization of ARGs in bacterial genomes and complex metagenomes, we are now able to reveal the genetic scope of AR in single bacteria and complex communities, and obtain important insights into AR dynamics at species, population and community levels, providing novel epidemiological and ecological perspectives. A suite of bioinformatics pipelines and ARG databases are currently available for genomic and metagenomic data analyses. However, different platforms may significantly vary and therefore, it is crucial to choose the tools that are most suitable for the specific analysis being conducted. This review provides a detailed account of available bioinformatics platforms for identification and characterization of ARGs and associated genetic elements within single bacterial isolates and complex environmental samples. It focuses primarily on currently available ARG databases, employing a comprehensive benchmarking pipeline to identify ARGs in four bacterial genomes (Aeromonas salmonicida, Bacillus cereus, Burkholderia sp. and Escherichia coli) and three shotgun metagenomes (human gut, poultry litter and soil) providing insight into which databases should be used for different analytical scenarios., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? Here, we reveal that metagenomes from no fewer than 20% of the papers found in our literature search, published between 2016 and 2019, were not deposited in a repository or were simply inaccessible. The proportion of inaccessible data within the literature has been increasing year-on-year. Noncompliance with Open Data is best predicted by the scientific discipline of the journal. The number of citations, journal type (e.g., Open Access or subscription journals), and publisher are not good predictors of data accessibility. However, many publications in high-impact factor journals do display a higher likelihood of accessible metagenomic data sets. Twenty-first century science demands compliance with the ethical standard of data sharing of metagenomes and DNA sequence data more broadly. Data accessibility must become one of the routine and mandatory components of manuscript submissions-a requirement that should be applicable across the increasing number of disciplines using metagenomics. Compliance must be ensured and reinforced by funders, publishers, editors, reviewers, and, ultimately, the authors., Competing Interests: The authors have declared that no competing interests exist.

Reuse of municipal wastewater is a growing global trend, but currently there is lack of consensus regarding the potential dissemination of antibiotic resistance elements by treated wastewater irrigation. We tracked intI1, a proxy for anthropogenic pollution, and an assemblage of antibiotic resistance genes associated with mobile elements and/or wastewater (bla GES , bla OXA2 , bla OXA10 , bla TEM , bla CTX-M-32 and qnrS) in treated wastewater effluents, effluent stabilization reservoirs, and along irrigation water-soil-crop continua in experimental lysimeters and large-scale commercial fields. While several of the targeted antibiotic resistance genes were profuse in effluents, there was almost no correlation between gene abundance in irrigation water and those detected in soil, and no evidence of systematic gene transfer to irrigated soil or crops. In contrast, soil intI1 abundance correlated strongly to irrigation water levels in lysimeters and sandy field soils, but this was not the case for clay-rich soils or for most of the analyzed crops, suggesting that intI1 may not always be a reliable marker for tracking the impact of treated wastewater irrigation. We hypothesize that "ecological boundaries" expedited by biotic and abiotic factors constrain dissemination of antibiotic resistance elements, and assert that a more holistic perception of these factors is crucial for understanding and managing antibiotic resistance dissemination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

There is increasing public concern regarding the fate of antibiotic resistance genes (ARGs) during wastewater treatment, their persistence during the treatment process and their potential impacts on the receiving water bodies. In this study, we used quantitative PCR (qPCR) to determine the abundance of nine ARGs and a class 1 integron associated integrase gene in 16 wastewater treatment plant (WWTP) effluents from ten different European countries. In order to assess the impact on the receiving water bodies, gene abundances in the latter were also analysed. Six out of the nine ARGs analysed were detected in all effluent and river water samples. Among the quantified genes, intI1 and sul1 were the most abundant. Our results demonstrate that European WWTP contribute to the enrichment of the resistome in the receiving water bodies with the particular impact being dependent on the effluent load and local hydrological conditions. The ARGs concentrations in WWTP effluents were found to be inversely correlated to the number of implemented biological treatment steps, indicating a possible option for WWTP management. Furthermore, this study has identified bla OXA-58 as a possible resistance gene for future studies investigating the impact of WWTPs on their receiving water., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d-leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the β-lactamase PenP. This β-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to β-lactam resistance under different conditions. To test whether β-lactam resistance and β-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five β-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental β-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of β-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between β-lactams and β-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The β-lactams are the largest group of clinically applied antibiotics, and resistance to these is primarily associated with β-lactamases. There is increasing understanding that these enzymes are ubiquitous in natural environments and henceforth, elucidating the global diversity, distribution, and mobility of β-lactamase-encoding genes is crucial for holistically understanding resistance to these antibiotics. In this study, we screened 232 shotgun metagenomes from ten different environments against a custom-designed β-lactamase database, and subsequently analyzed β-lactamase homologs with a suite of bioinformatic platforms including cluster and network analyses. Three interrelated β-lactamase clusters encompassed all of the human and bovine feces metagenomes, while β-lactamases from soil, freshwater, glacier, marine, and wastewater grouped within a separate "environmental" cluster that displayed high levels of inter-network connectivity. Interestingly, almost no connectivity occurred between the "feces" and "environmental" clusters. We attributed this in part to the divergence in microbial community composition (dominance of Bacteroidetes and Firmicutes vs. Proteobacteria , respectively). The β-lactamase diversity in the "environmental" cluster was significantly higher than in human and bovine feces microbiomes. Several class A, B, C, and D β-lactamase homologs ( bla CTX-M , bla KPC , bla GES ) were ubiquitous in the "environmental" cluster, whereas bovine and human feces metagenomes were dominated by class A (primarily cfxA ) β-lactamases. Collectively, this study highlights the ubiquitous presence and broad diversity of β-lactamase gene precursors in non-clinical environments. Furthermore, it suggests that horizontal transfer of β-lactamases to human-associated bacteria may be more plausible from animals than from terrestrial and aquatic microbes, seemingly due to phylogenetic similarities.

Biochar can enhance plant growth and reduce diseases, but frequently the optimal doses for these two benefits do not coincide. An approach is needed that will extend the range of biochar doses resulting in a concurrence of maximum benefits for both plant productivity and disease suppression. A biochar-amended growth medium was pre-conditioned by pre-planting fertigation in order to enhance the indigenous microbial community structure and activity. Cucumber plant performance and resistance against damping-off caused by Pythium aphanidermatum were monitored. Soil microbial activity, as well as bacterial and fungal community structure, were assessed by high-throughput 16S rRNA and ITS1 gene amplicon sequencing. Pre-conditioning enhanced the efficacy of biochar for improving plant performance and suppressing soilborne disease through enriching the medium in beneficial soil microorganisms, increasing microbial and fungal diversity and activity, and eliminating biochar phytotoxic compounds. The pre-conditioning process brought dose-response curves for both growth and disease resistance into sync, resulting in maximum benefits for both. These findings suggest that pre-conditioning should be incorporated as an important stage during biochar application in soil and soilless media., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Plasmids harboring qnr genes confer resistance to low fluoroquinolone concentrations. These genes are of significant clinical, evolutionary and environmental importance, since they are widely distributed in a diverse array of natural and clinical environments. We previously extracted and sequenced a large (∼185 Kbp) qnrB -harboring plasmid, and several small (∼8 Kbp) qnrS -harboring plasmids, from Klebsiella pneumoniae isolates from municipal wastewater biosolids, and hypothesized that these plasmids provide host bacteria a selective advantage in wastewater treatment plants (WWTPs) that often contain residual concentrations of fluoroquinolones. The objectives of this study were therefore to determine the effect of residual fluoroquinolone concentrations on the growth kinetics of qnr plasmid-harboring bacteria; and on the copy number of qnr plasmids and expression of qnr genes. Electrotransformants harboring either one of the two types of plasmids could grow at ciprofloxacin concentrations exceeding 0.5 μg ml -1 , but growth was significantly decreased at concentrations higher than 0.1 μg ml -1 . In contrast, plasmid-free strains failed to grow even at 0.05 μg ml -1 . No differences were observed in plasmid copy number under the tested ciprofloxacin concentrations, but qnr expression increased incrementally from 0 to 0.4 μg ml -1 , suggesting that the transcription of this gene is regulated by antibiotic concentration. This study reveals that wastewater-derived qnr plasmids confer a selective advantage in the presence of residual fluoroquinolone concentrations and provides a mechanistic explanation for this phenomenon.

The use of reclaimed wastewater (RWW) for the irrigation of crops may result in the continuous exposure of the agricultural environment to antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In recent years, certain evidence indicate that antibiotics and resistance genes may become disseminated in agricultural soils as a result of the amendment with manure and biosolids and irrigation with RWW. Antibiotic residues and other contaminants may undergo sorption/desorption and transformation processes (both biotic and abiotic), and have the potential to affect the soil microbiota. Antibiotics found in the soil pore water (bioavailable fraction) as a result of RWW irrigation may be taken up by crop plants, bioaccumulate within plant tissues and subsequently enter the food webs; potentially resulting in detrimental public health implications. It can be also hypothesized that ARGs can spread among soil and plant-associated bacteria, a fact that may have serious human health implications. The majority of studies dealing with these environmental and social challenges related with the use of RWW for irrigation were conducted under laboratory or using, somehow, controlled conditions. This critical review discusses the state of the art on the fate of antibiotics, ARB and ARGs in agricultural environment where RWW is applied for irrigation. The implications associated with the uptake of antibiotics by plants (uptake mechanisms) and the potential risks to public health are highlighted. Additionally, knowledge gaps as well as challenges and opportunities are addressed, with the aim of boosting future research towards an enhanced understanding of the fate and implications of these contaminants of emerging concern in the agricultural environment. These are key issues in a world where the increasing water scarcity and the continuous appeal of circular economy demand answers for a long-term safe use of RWW for irrigation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microorganisms carried by dust storms are transported through the atmosphere and may affect human health and the functionality of microbial communities in various environments. Characterizing the dust-borne microbiome in dust storms of different origins or that followed different trajectories provides valuable data to improve our understanding of global health and environmental impacts. We present a comparative study on the diversity of dust-borne bacterial communities in dust storms from three distinct origins (North Africa, Syria and Saudi Arabia) and compare them with local bacterial communities sampled on clear days, all collected at a single location: Rehovot, Israel. Storms from different dust origins exhibited distinct bacterial communities, with signature bacterial taxa. Dust storms were characterized by a lower abundance of selected antibiotic resistance genes (ARGs) compared with ambient dust, asserting that the origin of these genes is local and possibly anthropogenic. With the progression of the storm, the storm-borne bacterial community showed increasing resemblance to ambient dust, suggesting mixing with local dust. These results show, for the first time, that dust storms from different sources display distinct bacterial communities, suggesting possible diverse effects on the environment and public health.

Biochar, in addition to sequestering carbon, ameliorating soil, and improving plant performance, can impact foliar and soilborne plant diseases. Nevertheless, the mechanisms associated with suppression of soilborne diseases and improved plant performances are not well understood. This study is designed to establish the relationships between biochar-induced changes in rhizosphere microbial community structure, taxonomic and functional diversity, and activity with soilborne disease suppression and enhanced plant performance in a comprehensive fashion. Biochar suppressed Fusarium crown and root-rot of tomato and simultaneously improved tomato plant growth and physiological parameters. Furthermore, biochar reduced Fusarium root colonization and survival in soil, and increased the culturable counts of several biocontrol and plant growth promoting microorganisms. Illumina sequencing analyses of 16S rRNA gene revealed substantial differences in rhizosphere bacterial taxonomical composition between biochar-amended and non-amended treatments. Moreover, biochar amendment caused a significant increase in microbial taxonomic and functional diversity, microbial activities and an overall shift in carbon-source utilization. High microbial taxonomic and functional diversity and activity in the rhizosphere has been previously associated with suppression of diseases caused by soilborne pathogens and with plant growth promotion, and may collectively explain the significant reduction of disease and improvement in plant performance observed in the presence of biochar.

Antibiotics are considered to be one of the major medical breakthroughs in history. Nonetheless, over the past four decades, antibiotic resistance has reached alarming levels worldwide and this trend is expected to continue to increase, leading some experts to forecast the coming of a 'post-antibiotic' era. Although antibiotic resistance in pathogens is traditionally linked to clinical environments, there is a rising concern that the global propagation of antibiotic resistance is also associated with environmental reservoirs that are linked to anthropogenic activities such as animal husbandry, agronomic practices and wastewater treatment. It is hypothesized that the emergence and dissemination of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) within and between environmental microbial communities can ultimately contribute to the acquisition of antibiotic resistance in human pathogens. Nonetheless, the scope of this phenomenon is not clear due to the complexity of microbial communities in the environment and methodological constraints that limit comprehensive in situ evaluation of microbial genomes. This review summarizes the current state of knowledge regarding antibiotic resistance in non-clinical environments, specifically focusing on the dissemination of antibiotic resistance across ecological boundaries and the contribution of this phenomenon to global antibiotic resistance., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The 'biochar effect' depicts a phenomenon in which biochar soil amendment enhances plant performance by promoting growth and suppressing disease. Although this phenomenon has been observed in numerous studies, the mode of action that explains it is currently unknown. In order to elucidate mechanisms responsible for the 'biochar effect', we comprehensively monitored tomato plant development and resistance to the foliar fungal pathogen Botrytis cinerea, in biochar-amended and nonamended soils using native biochar and washed biochar, striped of labile chemical constituents. We concomitantly assessed bacterial community succession in the rhizosphere by high-throughput 16S rRNA gene amplicon sequencing and carbon-source utilization profiling. Biochar had little impact on plant physiological parameters. However, both native and washed biochar treatments were characterized by higher rhizosphere bacterial diversity and enhanced carbohydrate and phenolic compound utilization rates coupled to stimulation of bacteria known to degrade phenolic compounds. This study indicates that the 'biochar effect' is at least partially dictated by increased diversity and changes in metabolic potential in the rhizosphere microbiome, which is primarily triggered by the recalcitrant carbon backbone of the biochar and tightly bound compounds. It corresponds to the growing consensus that soil amendments which enhance microbial diversity have important benefits to ecosystem functioning., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The genus Aeromonas is ubiquitous in aquatic environments encompassing a broad range of fish and human pathogens. Aeromonas strains are known for their enhanced capacity to acquire and exchange antibiotic resistance genes and therefore, are frequently targeted as indicator bacteria for monitoring antimicrobial resistance in aquatic environments. This study evaluated temporal trends in Aeromonas diversity and antibiotic resistance in two adjacent semi-intensive aquaculture facilities to ascertain the effects of antibiotic treatment on antimicrobial resistance. In the first facility, sulfadiazine-trimethoprim was added prophylactically to fingerling stocks and water column-associated Aeromonas were monitored periodically over an 11-month fish fattening cycle to assess temporal dynamics in taxonomy and antibiotic resistance. In the second facility, Aeromonas were isolated from fish skin ulcers sampled over a 3-year period and from pond water samples to assess associations between pathogenic strains to those in the water column. A total of 1200 Aeromonas isolates were initially screened for sulfadiazine resistance and further screened against five additional antimicrobials. In both facilities, strong correlations were observed between sulfadiazine resistance and trimethoprim and tetracycline resistances, whereas correlations between sulfadiazine resistance and ceftriaxone, gentamicin, and chloramphenicol resistances were low. Multidrug resistant strains as well as sul1, tetA , and intI1 gene-harboring strains were significantly higher in profiles sampled during the fish cycle than those isolated prior to stocking and these genes were extremely abundant in the pathogenic strains. Five phylogenetically distinct Aeromonas clusters were identified using partial rpoD gene sequence analysis. Interestingly, prior to fingerling stocking the diversity of water column strains was high, and representatives from all five clusters were identified, including an A. salmonicida cluster that harbored all characterized fish skin ulcer samples. Subsequent to stocking, diversity was much lower and most water column isolates in both facilities segregated into an A. veronii -associated cluster. This study demonstrated a strong correlation between aquaculture, Aeromonas diversity and antibiotic resistance. It provides strong evidence for linkage between prophylactic and systemic use of antibiotics in aquaculture and the propagation of antibiotic resistance.

Integrons are extensively targeted as a proxy for anthropogenic impact in the environment. We developed a novel high-throughput amplicon sequencing pipeline that enables characterization of thousands of integron gene cassette-associated reads, and applied it to acquire a comprehensive overview of gene cassette composition in effluents from wastewater treatment facilities across Europe. Between 38 100 and 172 995 reads per-sample were generated and functionally characterized by screening against nr, SEED, ARDB and β-lactamase databases. Over 75% of the reads were characterized as hypothetical, but thousands were associated with toxin-antitoxin systems, DNA repair, cell membrane function, detoxification and aminoglycoside and β-lactam resistance. Among the reads characterized as β-lactamases, the carbapenemase bla OXA was dominant in most of the effluents, except for Cyprus and Israel where bla GES was also abundant. Quantitative PCR assessment of bla OXA and bla GES genes in the European effluents revealed similar trends to those displayed in the integron amplicon sequencing pipeline described above, corroborating the robustness of this method and suggesting that these integron-associated genes may be excellent targets for source tracking of effluents in downstream environments. Further application of the above analyses revealed several order-of-magnitude reductions in effluent-associated β-lactamase genes in effluent-saturated soils, suggesting marginal persistence in the soil microbiome.

The aim of this study was to determine the phylogenetic diversity of ceftriaxone resistance and the presence of known extended-spectrum β-lactamase (ESBL) genes in culturable soil resistomes. Libraries of soil bacterial isolates resistant to ceftriaxone were established from six physicochemically diverse soils collected in Hawaii (USA) and Israel. The phylogenetic affiliation, ceftriaxone and multidrug resistance levels, and presence of known ESBL genes of the isolates were determined. The soil bacterial isolates were phylogenetically grouped with the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Ceftriaxone minimum inhibitory concentrations (MICs) largely followed the phylogeny structure and higher levels of ceftriaxone resistance corresponded to higher multidrug resistance. Three distinct blaTEM variants were detected in soil bacterial isolates belonging to nine different genera. In conclusion, the culturable soil resistomes for ceftriaxone exhibited high phylogenetic diversity and multidrug resistance. blaTEM was the only known ESBL detected in the soil resistomes, and its distribution in different phylogenetic groups suggests its ubiquitous presence and/or possible horizontal gene transfer within the soil microbiomes., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

We evaluated the impact of Saharan dust storms on the local airborne microbiome in a city in the Eastern Mediterranean area. Samples of particles with diameter less than 10 μm were collected during two spring seasons on both dusty and nondusty days. DNA was extracted, and partial 16S rRNA gene amplicons were sequenced using the Illumina platform. Bioinformatic analysis showed the effect of dust events on the diversity of the atmospheric microbiome. The relative abundance of desert soil-associated bacteria increased during dust events, while the relative abundance of anthropogenic-influenced taxa decreased. Quantitative polymerase chain reaction measurements of selected clinically significant antibiotic resistance genes (ARGs) showed that their relative abundance decreased during dust events. The ARG profiles on dust-free days were similar to those in aerosol collected in a poultry house, suggesting a strong agricultural influence on the local ambient profiles. We conclude that dust storms enrich the ambient airborne microbiome with new soil-derived bacteria that disappear as the dust settles, suggesting that the bacteria are transported attached to the dust particles. Dust storms do not seem to be an important vector for transport of probed ARGs.

The presence of antibiotic drug residues, antibiotic resistant bacteria, and antibiotic resistance genes in agroecosystems has become a significant area of research in recent years and is a growing public health concern. While antibiotics are used in both human medicine and agricultural practices, the majority of their use occurs in animal production where historically they have been used for growth promotion, in addition to the prevention and treatment of disease. The widespread use of antibiotics and the application of animal wastes to agricultural lands play major roles in the introduction of antibiotic-related contamination into the environment. Overt toxicity in organisms directly exposed to antibiotics in agroecosystems is typically not a major concern because environmental concentrations are generally lower than therapeutic doses. However, the impacts of introducing antibiotic contaminants into the environment are unknown, and concerns have been raised about the health of humans, animals, and ecosystems. Despite increased research focused on the occurrence and fate of antibiotics and antibiotic resistance over the past decade, standard methods and practices for analyzing environmental samples are limited and future research needs are becoming evident. To highlight and address these issues in detail, this special collection of papers was developed with a framework of five core review papers that address the (i) overall state of science of antibiotics and antibiotic resistance in agroecosystems using a causal model, (ii) chemical analysis of antibiotics found in the environment, (iii) need for background and baseline data for studies of antibiotic resistance in agroecosystems with a decision-making tool to assist in designing research studies, as well as (iv) culture- and (v) molecular-based methods for analyzing antibiotic resistance in the environment. With a focus on the core review papers, this introduction summarizes the current state of science for analyzing antibiotics and antibiotic resistance in agroecosystems, discusses current knowledge gaps, and develops future research priorities. This introduction also contains a glossary of terms used in the core reivew papers of this special section. The purpose of the glossary is to provide a common terminology that clearly characterizes the concepts shared throughout the narratives of each review paper., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

Various culture-based methodologies are used in assessment of antibiotic resistance in samples collected in agroecosystems. Culture-based methods commonly involve isolating target bacteria on general or selective media and assessing growth in response to specific concentrations of antibiotics. The advantages of culture-based methods are multifold. In particular, isolation of bacteria is key to understanding phenotypic characteristics of isolates and their resistance patterns, and most national and international antibiotic resistance monitoring projects are isolate based. This review covers current knowledge of bacterial groups and antibiotics commonly targeted in resistance studies using bacterial culture and discusses the range in methods used, data interpretation, and factors supporting and confounding the use of culture-based methods in assessment of antibiotic resistance. Gaps in knowledge related to study design and resistance databases are discussed. Finally, a case is made for the integration of culture-based and molecular methods to better inform our understanding of antibiotic resistance in agroecosystems., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)