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Publisher Copyright: © 2021 The Authors. Published by American Chemical Society The global prevalence of antibiotic resistance genes (ARGs) is of increasing concern as a serious threat to ecological security and human health. Irrigation with sewage and farmland application of manure or biosolids in agricultural practices introduce substantial selective agents such as antibiotics and toxic metals, aggravating the transfer of ARGs from the soil environment to humans via the food chain. To address this issue, a hyperaccumulator (Sedum plumbizincicola) combined with biochar amendment was first used to investigate the mitigation of the prevalence of ARGs in cadmium and oxytetracycline co-contaminated soil by conducting a pot experiment. The addition of biochar affected the distribution of ARGs in soil and plants differently by enhancing their prevalence in the soil but restraining transmission from the soil to S. plumbizincicola. The planting of S. plumbizincicola resulted in an increase in ARGs in the soil environment. A structural equation model illustrated that mobile genetic elements played a dominant role in shaping the profile of ARGs. Taken together, these findings provide a practical understanding for mitigating the prevalence of ARGs in this soil system with complex contamination and can have profound significance for agricultural management in regard to ARG dissemination control.

Since the introduction of antibiotics into clinical practices in the 1940s, antibiotics have become an integral part of animal production to meet the increasing human demand for animal-derived food...

BACKGROUND Infants are at a high risk of acquiring fatal infections, and their treatment relies on functioning antibiotics. Antibiotic resistance genes (ARGs) are present in high numbers in antibiotic-naive infants' gut microbiomes, and infant mortality caused by resistant infections is high. The role of antibiotics in shaping the infant resistome has been studied, but there is limited knowledge on other factors which affect the antibiotic resistance burden of the infant gut. OBJECTIVES Our objectives were to determine the impact of early exposure to formula on the ARG load in neonates and infants born either pre- or full-term. Our hypotheses were that diet causes a selective pressure that influences the microbial community of the infant gut, and formula exposure would increase the abundance of taxa that carry ARGs. DESIGN Cross-sectionally sampled gut metagenomes of 46 neonates were used to build a generalized linear model to determine the impact of diet on ARG loads in neonates. The model was cross-validated using neonate metagenomes gathered from public databases using our custom statistical pipeline for cross-validation. RESULTS Formula-fed neonates had higher relative abundances of opportunistic pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Klebsiella oxytoca, and Clostridioides difficile. The relative abundance of ARGs carried by gut bacteria was 69% higher in the formula receiving group (fold change 1.69, (95% CI 1.12, 2.55), P = 0.013, n = 180) compared to exclusively human milk-fed infants. The formula-fed infants also had significantly less typical infant bacteria such as bifidobacteria that have potential health benefits. CONCLUSIONS The novel finding that formula exposure is correlated with a higher neonatal ARG burden lays the foundation that clinicians should consider feeding mode in addition to antibiotic use during the first months of life to minimize the proliferation of antibiotic-resistant gut bacteria in infants.Clinical trial registry number: The Institutional Review Board of Pennsylvania State University https://irb.upenn.edu/, USA (IRB #35925) approved the study.

Antibiotic resistance is one of the greatest global threats to human health, but substantial gaps in antibiotic resistance data exist in West African countries. We explored the presence of antibiotic resistance genes (ARGs) in the hospital wastewater (HWW) of nine hospitals in Benin and Burkina Faso and, for comparison, four hospitals in Finland using shotgun metagenomic sequencing. The highest sum of the relative abundance of ARGs in the 68 HWW samples was detected in Benin and the lowest in Finland. HWW resistomes and mobilomes in Benin and Burkina Faso resembled more of each other than those in Finland. Different carbapenemases were detected in varying abundances, especially in HWW from Burkina Faso and Finland. TheblaGES genes, the most widespread carbapenemase genes in the Beninese HWW, were also found in water intended for hand-washing and in a puddle at the hospital yard in Benin.mcrwere detected in the HWW of all three countries, withmcr-5being the most commonmcrgene. These and othermcrgenes were observed in very high relative abundances, even in treated wastewater in Burkina Faso and in a street gutter in Benin. The results provide evidence for public health decision-makers for the dire need to increase wastewater treatment capacity, with particular attention to HWW.ImportanceThe global emergence and increased spread of antibiotic resistance threaten the effectiveness of antibiotics and, thus, the health of the entire population. Therefore, understanding the resistomes in different geographical locations is crucial in the global fight against the antibiotic crisis. However, this information is scarce in many low- and middle-income countries (LMICs), such as those in West Africa. In this study, we describe the resistomes of hospital wastewater in Benin and Burkina Faso and, as a comparison, in Finland. Our results help to understand the hitherto unrevealed resistance in Beninese and Burkinabe hospitals. Furthermore, the results emphasize the importance of wastewater management infrastructure design to minimize exposure events between humans, HWW, and the environment, preventing the circulation of resistant bacteria and ARGs between humans (hospitals and community) and the environment.

Antibiotic resistance in aquatic ecosystems presents an environmental health issue worldwide. Urban recipient water quality is susceptible to effluent discharges with antibiotic resistance contaminants and needs to be protected, particularly for those as sources of drinking water production. Knowledge on aquatic resistome profiles in downstream of wastewater treatment plants allows a better understanding of the extent to which antibiotic resistance contaminants emerge and spread in recipient waters, but such information remains very limited in Sweden. The key objective of this study was to determine the resistome profiles of numerous antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and other genes in urban recipient water systems connected to Sweden's major drinking water reservoir. This was achieved through analysis of surface water samples for 296 genes using high-throughput quantitative PCR arrays. A total of 167 genes were detected in at least one of the samples, including 150 ARGs conferring resistance to 11 classes of antibiotics, 7 integrase MGEs and 9 other genes. There was a spatial difference in the resistome profiles with the greatest average relative abundance of resistance genes observed in the water body of Vasteras followed by Uppsala, Stockholm and Eskilstuna, as similar to the general pattern of the antibiotic sales for these regions. ARGs against beta-lactams and sulfonamides showed the highest average relative abundance in the studied water bodies, while vancomycin resistance genes were only found in the Uppsala water environment. Generally, the recipient water bodies were detected with higher numbers of genes and greater relative abundances as compared to the upstream sites. Anthropogenic pollution, i.e., wastewater discharge, in the recipient water was also reflected by the finding of intI, sul1 and crAssphage. Overall, this study provided the first quantitative assessment of aquatic environmental resistomes in Sweden, highlighting the widespread of antibiotic resistance contaminants in urban recipient waters.

The spread of antibiotic resistance genes (ARGs) has gained much attention worldwide, while the contribution of vertical gene transfer (VGT) and horizontal gene transfer (HGT) is still elusive. Here, we improved an emerging high-throughput single-cell-based technology, emulsion, paired isolation, and concatenation polymerase chain reaction (epicPCR), by lengthening the sequence of ARG in the fused ARG-16S rRNA fragments to cover the variance of both ARG and its hosts. The improved epicPCR was applied to track the hosts of a widely detected ARG, sul1 gene, in five urban wastewater treatment plants (UWTPs) during two seasons. The sul1 host bacteria were highly diverse and mostly classified as Proteobacteria and Bacteroidetes. Clear seasonal divergence of α-diversity and interaction networks were present in the host community. The consensus phylogenetic trees of the sul1 gene and their host demonstrated incorrespondence on the whole and regularity on abundant groups, suggesting the important role of both HGT and VGT, respectively. The relative importance of these two ways was further measured; HGT (54%) generally played an equal or even more important role as VGT (46%) in UWTPs. The application of the improved epicPCR technology provides a feasible approach to quantify the relative contributions of VGT and HGT in environmental dissemination of ARGs.

International audience; EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

Sulfate reduction is an important biogeochemical process in the ecosphere; however, the major taxa of sulfate reducers have not been fully identified. Here, we used epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) technology to identify the phylogeny of sulfate-reducing prokaryotes (SRP) in sediments from Tibetan Plateau saline lakes. A total of 12,519 OTUs and 883 SRP-OTUs were detected in ten lakes by sequencing of 16S rRNA gene PCR amplicons and epicPCR products of fused 16S rRNA plus dsrB gene, respectively, with Proteobacteria, Firmicutes, and Bacteroidetes being the dominant phyla in both datasets. The 120 highly abundant SRP-OTUs (> 1% in at least one sample) were affiliated with 17 described phyla, only 7 of which are widely recognized as SRP phyla. The majority of OTUs from both the whole microbial communities and the SRPs were not detected in more than one specific lake, suggesting high levels of endemism. The α-diversity of the entire microbial community and SRP sub-community showed significant positive correlations. The pH value and mean water temperature of the month prior to sampling were the environmental determinants for the whole microbial community, while the mean water temperature and total nitrogen were the major environmental drivers for the SRP sub-community. This study revealed there are still many undocumented SRP in Tibetan saline lakes, many of which could be endemic and adapted to specific environmental conditions. Electronic supplementary material The online version of this article (10.1186/s40168-019-0688-4) contains supplementary material, which is available to authorized users.

The rapid spread of antibiotic resistance challenges modern medicine. So far, mechanistic and quantitative knowledge concerning the spread of resistance genes mainly relies on laboratory experiments with simplified setups, e.g. two strain communities. Thus, the transferability of the obtained process rates might be limited. To investigate the role of a diverse community concerning the dissemination of the multidrug resistance plasmid RP4, an Escherichia coli harboring RP4 invaded a microbial community consisting of 21 species. Changes in the community composition as well as plasmid uptake by community members were monitored for 22 days. Special focus was laid on the question of whether the observed changes were dependent on the actual invading donor isolate and the ambient antibiotic concentration. In our microcosm experiment, the community composition was primarily influenced by the given environmental variables and only secondarily by the particular invader E. coli. The establishment of resistance within the community, however, was directly dependent on the donor identity. The extent to which ambient conditions influence the spread of RP4 depended on the E. coli donor strain. These results emphasize that even within one species there are great differences in the ability to conquer an ecological niche and to spread antibiotic resistance.

The overuse and misuse of antibiotics in animal breeding for disease treatment and growth enhancement have been major drivers of the occurrence, diffusion, and accumulation of antibiotic resistance genes (ARGs) in wastewater. Strategies to combat ARG dissemination are pressingly needed for human and ecological safety. To achieve this goal, a biochar-based polymer, magnetic biochar/quaternary phosphonium salt (MBQ), was applied in livestock wastewater and displayed a high performance in bacterial deactivation and ARG decrease. Efficient antibacterial effects were achieved by both MBQ and quaternary phosphonium salt; however, the abundance and fold change of ARGs in the MBQ treatment indicated a more powerful ARG dissemination control than quaternary phosphonium salt. The application of MBQ evidently reduced the microbial diversity and may primarily be responsible for altering the ARG profiles in wastewater. Network, redundancy, and variation partitioning analyses were further employed to reveal that the microbial community and the presence of mobile genetic elements were two critical factors shaping the pattern of the antibiotic resistome in livestock wastewater. Considered together, these findings extend the application field of biochar and have important implications for reducing ARG dissemination risks in livestock wastewater.

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.

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.

Artificial intelligence (AI) coupled with promising machine learning (ML) techniques well known from computer science is broadly affecting many aspects of various fields including science and technology, industry, and even our day-to-day life. The ML techniques have been developed to analyze high-throughput data with a view to obtaining useful insights, categorizing, predicting, and making evidence-based decisions in novel ways, which will promote the growth of novel applications and fuel the sustainable booming of AI. This paper undertakes a comprehensive survey on the development and application of AI in different aspects of fundamental sciences, including information science, mathematics, medical science, materials science, geoscience, life science, physics, and chemistry. The challenges that each discipline of science meets, and the potentials of AI techniques to handle these challenges, are discussed in detail. Moreover, we shed light on new research trends entailing the integration of AI into each scientific discipline. The aim of this paper is to provide a broad research guideline on fundamental sciences with potential infusion of AI, to help motivate researchers to deeply understand the state-of-the-art applications of AI-based fundamental sciences, and thereby to help promote the continuous development of these fundamental sciences., Graphical abstract, Public summary • “Can machines think?” The goal of artificial intelligence (AI) is to enable machines to mimic human thoughts and behaviors, including learning, reasoning, predicting, and so on. • “Can AI do fundamental research?” AI coupled with machine learning techniques is impacting a wide range of fundamental sciences, including mathematics, medical science, physics, etc. • “How does AI accelerate fundamental research?” New research and applications are emerging rapidly with the support by AI infrastructure, including data storage, computing power, AI algorithms, and frameworks.

The co-occurrence of antibiotic-resistance genes (ARGs) and mobile genetic elements (MGEs) in farm environments can potentially foster the development of antibiotic-resistant pathogens. We studied the resistome of Finnish dairy and swine farms where use of antibiotics is limited to treating bacterial infections and manure is only applied from April to September. The resistome of manure, soil, and tile drainage water from the ditch was investigated from the beginning of the growing season until forage harvest. The relative ARG and MGE abundance was measured using a qPCR array with 363 primer pairs. Manure samples had the highest abundance of ARGs and MGEs, which increased during storage. Immediately following land application, the ARGs abundant in manure were detected in soil, but their abundance decreased over time with many becoming undetectable. This suggests that increases in ARG abundances after fertilizing are temporary and occur annually under agricultural practices that restrict antibiotic use. A few of the ARGs were detected in the ditch water, but most of them were undetected in the manure. Our results document the dissipation and dissemination off farm of ARGs under Finnish limited antibiotic use and suggest that such practices could help reduce the load of antibiotic-resistance genes in the environment.

Whole-cell biosensors are still the method of choice when measuring bioavailable mercury, though their implementation in environmental monitoring is limited by low sensitivity, lack of portability ...

SummaryInfants are at a high risk of acquiring infections caused by antibiotic resistant bacterial strains. Antibiotic resistance gene (ARG) load is typically higher in newborns than in adults, but it is unknown which factors besides antibiotic treatment affect the load. Our study demonstrates that inclusion of any formula in the newborn diet causes shifts in microbial community composition that result in higher ARG loads in formula-fed infants compared to infants not fed formula. The effect of formula was especially strong in premature newborns and newborns treated with antibiotics. Interestingly, antibiotics alone without formula did not have a detectable impact on the ARG load of the newborn gut. We also observed that formula-fed infants had enriched numbers of pathogenic species and were depleted in typical infant gut species such as Bifidobacterium bifidum. The results suggest infant feeding choices should include assessment of risks associated with elevated ARG abundance.

Antibiotic resistance and its environmental component are gaining more attention as part of combating the growing healthcare crisis. The One Health framework, promulgated by many global health agencies, recognizes that antimicrobial resistance is a truly inter-domain problem in which human health, animal agriculture, and the environment are the core and interrelated components. This prospectus presents the status and issues relevant to the environmental component of antibiotic resistance, namely, the needs for advancing surveillance methodology: the environmental reservoirs and sources of resistance, namely, urban wastewater treatment plants, aquaculture production systems, soil receiving manure and biosolid, and the atmosphere which includes longer range dispersal. Recently, much work has been done describing antibiotic resistance genes in various environments; now quantitative, mechanistic, and hypothesis-driven studies are needed to identify practices that reduce real risks and maintain the effectiveness of our current antibiotics as long as possible. Advanced deployable detection methods for antibiotic resistance in diverse environmental samples are needed in order to provide the surveillance information to identify risks and define barriers that can reduce risks. Also needed are practices that reduce antibiotic use and thereby reduce selection for resistance, as well as practices that limit the dispersal of or destroy antibiotic-resistant bacteria or their resistance genes that are feasible for these varied environmental domains.

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).

The use of antibiotics in aquaculture causes selection pressure for antibiotic-resistant bacteria (ARB). Antibiotic resistance genes (ARGs) may persist in ARB and the environment for long time even after stopping drug administration. Here we show monthly differences in the occurrences of genes conferring resistance to sulfonamides (i.e. sul1, sul2, sul3), and tetracyclines (tet(M)) in Japanese aquaculture seawater accompanied by records of drug administration. sul2 was found to persist throughout the year, whereas the occurrences of sul1, sul3, and tet(M) changed month-to-month. sul3 and tet(M) were detected in natural bacterial assemblages in May and July, but not in colony-forming bacteria, thus suggesting that the sul3 was harbored by the non-culturable fraction of the bacterial community. Comparison of results from Taiwanese, Japanese, and Finnish aquaculture waters reveals that the profile of sul genes and tet(M) in Taiwan resembles that in Japan, but is distinct from that in Finland. To our knowledge, this work represents the first report to use the same method to compare the dynamics of sul genes and tet(M) in aquaculture seawater in different countries. (C) 2019 Elsevier B.V. All rights reserved.

Publisher Copyright: © 2018, The Author(s). The infant gut microbiota has a high abundance of antibiotic resistance genes (ARGs) compared to adults, even in the absence of antibiotic exposure. Here we study potential sources of infant gut ARGs by performing metagenomic sequencing of breast milk, as well as infant and maternal gut microbiomes. We find that fecal ARG and mobile genetic element (MGE) profiles of infants are more similar to those of their own mothers than to those of unrelated mothers. MGEs in mothers' breast milk are also shared with their own infants. Termination of breastfeeding and intrapartum antibiotic prophylaxis of mothers, which have the potential to affect microbial community composition, are associated with higher abundances of specific ARGs, the composition of which is largely shaped by bacterial phylogeny in the infant gut. Our results suggest that infants inherit the legacy of past antibiotic consumption of their mothers via transmission of genes, but microbiota composition still strongly impacts the overall resistance load.

Experimental microbial ecology and evolution have yielded foundational insights into ecological and evolutionary processes using simple microcosm setups and phenotypic assays with one- or two-species model systems. The fields are now increasingly incorporating more complex systems and exploration of the molecular basis of observations. For this purpose, simplified, manageable and well-defined multispecies model systems are required that can be easily investigated using culturing and high-throughput sequencing approaches, bridging the gap between simpler and more complex synthetic or natural systems. Here we address this need by constructing a completely synthetic 33 bacterial strain community that can be cultured in simple laboratory conditions. We provide whole-genome data for all the strains as well as metadata about genomic features and phenotypic traits that allow resolving individual strains by amplicon sequencing and facilitate a variety of envisioned mechanistic studies. We further show that a large proportion of the strains exhibit coexistence in co-culture over serial transfer for 48 days in the absence of any experimental manipulation to maintain diversity. The constructed bacterial community can be a valuable resource in future experimental work.

Whole-cell bioreporters are living organisms and thus using them for detecting environmental contaminants would reflect biological effects of these pollutants. However, bioreporters are not widely used in field studies. Many of the bioreporter field protocols are suitable for liquid samples or include pipetting steps, which is a demanding task outside the laboratory. We present a bioreporter protocol without pipetting or sample type requirements. The protocol utilizes polyester swabs, commonly used in cleanroom technology. As an example contaminant, we used tetracycline and generated test samples with known concentrations up to the maximum tetracycline residue limit of milk set by the European Union (EU) regulation. The matrices of the test samples were Milli-Q water, milk and soil. The swabs were first dipped in the bioreporter cell cultures and then to test samples and luminescence was measured after incubation. The standard deviation of measurements from ten replicate swabs was in the same range as commonly in pipetting protocols (4-19%). The test samples with lowest tetracycline concentration (5 ng mL(-1)) were distinguished from the control samples (0 ng mL(-1) tetracycline). Our results show that swabs can be used together with luminescent whole cell bioreporters, making it possible to conduct the measurements in field conditions.

Wastewater treatment plants (WWTPs) collect wastewater from various sources for a multi-step treatment process. By mixing a large variety of bacteria and promoting their proximity, WWTPs constitute potential hotspots for the emergence of antibiotic resistant bacteria. Concerns have been expressed regarding the potential of WWTPs to spread antibiotic resistance genes (ARGs) from environmental reservoirs to human pathogens. We utilized epicPCR (Emulsion, Paired Isolation and Concatenation PCR) to detect the bacterial hosts of ARGs in two WWTPs. We identified the host distribution of four resistance-associated genes (tetM, int1, qacEΔ1and blaOXA-58) in influent and effluent. The bacterial hosts of these resistance genes varied between the WWTP influent and effluent, with a generally decreasing host range in the effluent. Through 16S rRNA gene sequencing, it was determined that the resistance gene carrying bacteria include both abundant and rare taxa. Our results suggest that the studied WWTPs mostly succeed in decreasing the host range of the resistance genes during the treatment process. Still, there were instances where effluent contained resistance genes in bacterial groups not carrying these genes in the influent. By permitting exhaustive profiling of resistance-associated gene hosts in WWTP bacterial communities, the application of epicPCR provides a new level of precision to our resistance gene risk estimates., epicPCR was utilized to link antibiotic resistance genes to bacterial host species in two wastewater purification plants.

A roundtable discussion held at the fourth International Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR4) considered key issues concerning the impact on the environment of antibiotic use in agriculture and aquaculture, and emissions from antibiotic manufacturing. The critical control points for reducing emissions of antibiotics from agriculture are antibiotic stewardship and the pre-treatment of manure and sludge to abate antibiotic-resistant bacteria. Antibiotics are sometimes added to fish and shellfish production sites via the feed, representing a direct route of contamination of the aquatic environment. Vaccination reduces the need for antibiotic use in high value (e.g. salmon) production systems. Consumer and regulatory pressure will over time contribute to reducing the emission of very high concentrations of antibiotics from manufacturing. Research priorities include the development of technologies, practices and incentives that will allow effective reduction in antibiotic use, together with evidence-based standards for antibiotic residues in effluents. All relevant stakeholders need to be aware of the threat of antimicrobial resistance and apply best practice in agriculture, aquaculture and pharmaceutical manufacturing in order to mitigate antibiotic resistance development. Research and policy development on antimicrobial resistance mitigation must be cognizant of the varied challenges facing high and low income countries.

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer netw, Academy of Finland (Project 257612), National Institute of Environmental Health Sciences (Award P30-ES002109)

Low concentrations of antibiotics have numerous effects on bacteria. However, it is unknown whether ecological factors such as trophic interactions and spatial structuring influence the effects of low concentrations of antibiotics on multispecies microbial communities. Here, we address this question by investigating the effects of low antibiotic concentration on community composition and horizontal transfer of an antibiotic resistance plasmid in a 62-strain bacterial community in response to manipulation of the spatial environment and presence of predation. The strong effects of antibiotic treatment on community composition depend on the presence of predation and spatial structuring that have strong community effects on their own. Overall, we find plasmid transfer to diverse recipient taxa. Plasmid transfer is likely to occur to abundant strains, occurs to a higher number of strains in the presence of antibiotic, and also occurs to low-abundance strains in the presence of spatial structures. These results fill knowledge gaps concerning the effects of low antibiotic concentrations in complex ecological settings.

Environmental selection of antibiotic resistance may be caused by either antibiotic residues or coselecting agents. Using a strictly controlled experimental design, we compared the ability of metals (Cu or Zn) and tetracycline to (co)select for tetracycline resistance in bacterial communities. Soil microcosms were established by amending agricultural soil with known levels of Cu, Zn, or tetracycline known to represent commonly used metals and antibiotics for pig farming. Soil bacterial growth dynamics and bacterial community-level tetracycline resistance were determined using the [3H]leucine incorporation technique, whereas soil Cu, Zn, and tetracycline exposure were quantified by a panel of whole-cell bacterial bioreporters. Tetracycline resistance increased significantly in soils containing environmentally relevant levels of Cu (≥365 mg kg–1) and Zn (≥264 mg kg–1) but not in soil spiked with unrealistically high levels of tetracycline (up to 100 mg kg–1). These observations were consistent with biorepor...

Published version

Rivers, lakes and the ocean receive antibiotic resistance genes from human environments. The aquatic environments are a huge reservoir and exchange stage of antibiotic resistance genes.

Polyhydroxyalkanoates (PHAs) are macromolecules produced by bacteria as means for storing carbon and energy in intracellular granules. PHAs have physical properties similar to those of plastics and have become of interest to industry as materials for environmentally friendly bioplastic production. There is an ongoing search for new PHA-producing bacterial strains and PHA-synthesizing enzymes tolerating extreme conditions to find ways of producing PHAs at cold temperatures and high solute concentrations. Moreover, the study of PHA producers in the sea-ice biome can aid in understanding the microbial ecology of carbon cycling in ice-associated ecosystems. In this study, PHA producers and PHA synthase genes were examined under the extreme environmental conditions of sea ice and cold seawater to find evidence of PHA production in an environment requiring adaptation to high salinity and cold temperatures. Sea ice and cold estuarine water samples were collected from the northern Baltic Sea and evidence of PHA production was gathered, using microscopy with Nile Blue A staining of PHA-granules and PCR assays detecting PHA-synthesis genes. The PHA granules and PHA synthases were found at all sampling locations, in both sea ice and water, and throughout the sampling period spanning over 10 years. Our study shows, for the first time, that PHA synthesis occurs in Baltic Sea cold-adapted bacteria in their natural environment, which makes the Baltic Sea and its cold environments an interesting choice in the quest for PHA-synthesizing bacteria and synthesis genes.

Bioluminescent Saccharomyces cerevisiae yeast- based bioreporters were used to monitor bisphenol A and other estrogenic chemicals in thermal paper samples collected mainly from Finland on two occasions in 2010/2011, and 2013. The bisphenol A-targeted (BPA-R) and the human oestrogen receptor (hERα) bioreporters were applied to ana- lyse both non-treated and extracted paper samples. Bisphenol A was readily bioavailable to the yeast bioreporters on the non-treated paper samples without any pre-treatment. Detect- ed concentrations ranged from a detection limit of 9-142 μg/g to over 20 mg/g of bisphenol A equivalents in the thermal papers. Low bisphenol A likeactivities were detected in many samples, and were considered to be caused by residual bisphenol A or other types of bisphenols, such as bisphenol S. Most of the thermal paper samples were toxic to the yeast bioreporters. The toxicity did not, however, depend on the bisphenol A concentration of the samples. The yeast bioreporters were demonstrated to be a robust and cost- efficient method to monitor thermal paper samples for their bisphenol A content and estrogenicity. Thermal paper was considered as a potential BPA source for both human expo- sure and environmental emission.

The first Saccharomyces cerevisiae yeast bioreporter for analysis of a single endocrine disrupting compound, bisphenol A (BPA), was developed. The bioreporter contains mutated human estrogen receptor α (hERα), called bisphenol A-targeted receptor (BPA-R). The BPA-R bioreporter was characterized with mixtures of estrogenic chemicals and tested with spiked influent wastewater samples. The detection limit for BPA was 4.2-fold lower (0.107 μM, i.e., 24 μg L(-1)), while that of the native hormone 17β-estradiol (E2) (1 μM, i.e., 272 μg L(-1)) was 166,000-fold higher compared to the wild type hERα bioreporter. The BPA-R bioreporter responded only to BPA in a chemical cocktail and spiked concentrated wastewater samples with high concentrations of other estrogenic chemicals. As a conclusion, wastewater and other environmental water samples can be concentrated and specifically analyzed for BPA without risk of the mixture effect caused by other estrogenic chemicals. The BPA-R bioreporter is a robust and cost-efficient choice for high-throughput monitoring of BPA and its bioavailability in complex samples.

Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.

The quality of sewage sludge-based products, such as composts and growth media, is affected by the contamination of sewage sludge with, potentially, hundreds of different substances. Therefore, it is difficult to achieve the reliable environmental quality assessment of sewage sludge-based products solely based on chemical analysis. In the present work, we demonstrate the use of the kinetic luminescent bacteria test (ISO 21338) to evaluate acute toxicity and the Vitotox™ test to monitor genotoxicity of sewage sludge and composted sewages sludge. In addition, endocrine-disrupting and dioxin-like activity was studied using yeast-cell-based assays. The relative contribution of industrial waste water treated at the Waste Water Treatment Plants led to elevated concentrations of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins and -furans (PCDD/F) in sewage sludge. The effect of elevated amounts of organic contaminants could also be identified with biotests able to demonstrate higher acute toxicity, genotoxicity, and potential for endocrine-disruptive properties. Additional extraction steps in kinetic luminescent bacteria test with DMSO and hexane increased the level of toxicity detected. Composting in a pilot-scale efficiently reduced the amounts of linear alkylbenzensulphonates (LASs), nonylphenols and nonylphenolethoxylates (NPE/NPs) and PAH with relative removal efficiencies of 84%, 61% and 56%. In addition, decrease in acute toxicity, genotoxicity and endocrorine-disrupting and dioxin-like activity during composting could be detected. However, the biotests did have limitations in accessing the ecotoxicity of test media rich with organic matter, such as sewage sludge and compost, and effects of sample characteristics on biotest organisms must be acknowledged. The compost matrix itself, however, which contained a high amount of nutrients, bark, and peat, reduced the sensitivity of the genotoxicity tests and yeast bioreporter assays.