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Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention. Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

Peracetic acid (PAA) is an organic compound used efficiently as disinfectant in wastewater treatments. Yet, at low doses it may cause selection; thus, the effect of low doses of PAA on Enterococcus faecium as a proxy of human-related microbial waste was evaluated. Bacteria were treated with increasing doses of PAA (from 0 to 25 mg L −1 min) and incubated in regrowth experiments under non-growing, limiting conditions and under growing, favorable conditions. The changes in bacterial abundance, in bacterial phenotype (number and composition of small cell clusters), and in the abundance of an antibiotic resistance gene (ARG) was evaluated. The experiment demonstrated that the selected doses of PAA efficiently removed enterococci, and induced a long-lasting effect after PAA inactivation. The relative abundance of small clusters increased during the experiment when compared with that of the inoculum. Moreover, under growing favorable conditions the relative abundance of small clusters decreased and the number of cells per cluster increased with increasing PAA doses. A strong stability of the measured ARG was found, not showing any effect during the whole experiment. The results demonstrated the feasibility of low doses of PAA to inactivate bacteria. However, the stress induced by PAA disinfection promoted a bacterial adaptation, even if potentially without affecting the abundance of the ARG.

Antibiotic resistance is a rising threat for human health. Although in clinical settings and terrestrial environments the rise of antibiotic resistant bacteria is well documented, their dissemination and spread in the marine environment, covering almost two-thirds of the Earth's surface, is still poorly understood. In this study, the presence and abundance of sulphonamide resistance gene (sul2) and class 1 integron-integrase gene (intI1), used as markers for the occurrence and spread of antibiotic resistance genes since the beginning of the antibiotic era, were investigated. Twenty-nine archived formalin-fixed samples, collected by the Continuous Plankton Recorder (CPR) survey in the Atlantic Ocean and North Sea from 1970 to 2011, were analysed using Droplet Digital PCR (ddPCR) applied for the first time on CPR samples. The two marker genes were present in a large fraction of the samples (48% for sul2 and 76% for intI1). In contrast, results from Real-Time PCR performed on the same samples greatly underestimate their occurrence (21% for sul2 and 52% for intI1). Overall, besides providing successful use of ddPCR for the molecular analysis of CPR samples, this study reveals long-term occurrence and spread of sul2 gene and class 1 integrons in the plankton-associated bacterial communities in the ocean.

The marine environment is often representing a collection site for contaminants produced by industrial processes, oil exploration and active harbours and ship transport. All of these activities can generate heavy metal contamination. In addition to heavy metals, marine sediments and bays can be influenced by effluents of wastewater treatment plants (WWTPs), a hotspot of antibiotic resistance selection and a direct source for their spread into the environment. The present study was conducted in the bay of Pula, a semi enclosed costal ecosystem experiencing very strong contamination from multiple sources. In order to detect these influences in depth microbiological and chemical characterizations of surface sediments were performed. Based on the measured heavy metal concentrations, the fifteen investigated sites clustered into three groups, with one outgroup site. Bacterial communities in the sediment samples were mainly dominated by Gammaproteobacteria and Deltaproteobacteria, except two sites close to the discharge of a WWTP, which were dominated by Epsilonproteobacteria. In all samples, metal resistance genes (arsB and czcA) were detectable by qPCR. We found a positive correlation between the relative abundance of the Ectothiorhodospiraceaeand the abundance of arsB gene copy numbers, and between Rhodospirillaceae and Sva1033 clades and the czcA gene copy numbers. Furthermore, antibiotic resistance genes against erythromycin (ermB) and β-lactams (blaTEM) were detectable in various samples throughout the bay.

To gain insights into the relationships and the genetic exchange among environmental and clinical enterococci, 59 strains (29 from marine aquaculture sites and 30 from clinical settings) resistant to tetracycline, erythromycin, ampicillin and/or gentamicin were analysed for the antibiotic resistance tet(M), tet(L), tet(O), erm(A), erm(B), mef blaZ, aac(6')-Ie aph(2″)-Ia and virulence gelE, cylB, efaA and esp genes, and for the copper resistance gene tcrB. Antibiotic resistance and virulence genes were detected more frequently in clinical than in environmental enterococci; the opposite was true for copper resistance. Conjugation experiments demonstrated the transfer of antibiotic resistance genes from marine to clinical enterococci in interspecific mating and the uncommon joint transfer of tet(L) and erm(B). Enterobacterial repetitive intergenic consensus polymerase chain reaction typing evidenced a cluster (90% similarity) encompassing strains carrying multiple antibiotic resistance genes from both sets; the others marine isolates exhibited polyclonality and bore tcrB. Our results demonstrate that antibiotic-resistant marine enterococci bear antibiotic resistance genes transferable to humans and suggest that copper resistance, not observed among clinical strains, may be useful for survival in the environment, whereas virulence genes likely confer no advantage to enterococcal populations adapted to a lifestyle outside the host.

The Macrosite "IT08 Subalpine Lakes" includes a group of lakes located at the southern border of the Alps. The lake typologies are represented by large and deep lakes (Orta, Garda, Maggiore, Como and Iseo) and by a lake of smaller size and shallow depth (Candia). Topics common to all the research sites include the impact of eutrophication and climate change, the composition, structure and dynamics of biological communities (plankton, necton and littoral communities), trophic webs, paleolimnology, ecotoxicology, ecological status and remote sensing. Scientific investigations are supported by a wide availability of technological infrastructures and laboratories located in the reference institutions, and by a wide range of technological equipment for fieldwork. These facilities, funded within the individual institutes involved in the research, have allowed not only to maintain, but also to update the classical approaches used in scientific monitoring, while opening up new research fields (especially molecular ecology, microbial ecology, metabolomic profiling, antibiotic resistance genes, high frequency monitoring by sensor technology). Over the last decade, the continuity and regularity of investigations have been facilitated by the availability of both external and ordinary funds. The variety of research activities carried out on the IT08 macrosite is evidenced by the publication of numerous scientific papers, as well as contributions (oral or poster) presented at national and international conferences, and by the presence in the mass media. In this context, the scientific monitoring is a key element of LTER research, including not only data collection (basic monitoring), but also data interpretation, modeling, and experimental manipulation, with particular attention to key groups of selected variables in order to identify the most significant environmental stressors and the degree of change at the level of ecosystems, communities, species and populations.

The Lanzarote and Chinijo Islands UNESCO Global Geopark hosts one of the most extensive and diverse volcanic anchialine ecosystems in the world, consisting of water bodies with marine origin that penetrated inland through coastal crevicular systems. Marine infiltration is facilitated by the low rainfall and the permeability of the coastal terrains. Best known for Túnel de la Atlántida, Lanzarote has other types of anchialine habitats, such as pools, lakes, and even hand-made wells, all of them interconnected with the crevicular system. So far, 39 endemic stygobitic species of crustacean, annelids, and platyhelminths have been described in the island. Some of them belong to lineages previously interpreted as Tethyan vicariant relicts because they belong in ancient groups restricted to caves situated in areas along the coastline of the ancient Tethys Sea, such as Mexico, Bahamas or Western Australia. Others, instead, have clear affinities with deep-sea groups, suggesting that their ancestors might have dispersed into the island from surrounding deep-sea environments. In overall, while the anchialine habitats of Lanzarote are relatively small in comparison to other regions, the presence of so many species with such a diverse origin have puzzled zoologists and biogeographers throughout the 20th century, who have regarded the island as a model to understand the origin and evolution of similar groups in other areas of the world. The anchialine habitats in Lanzarote are subject to intense recreational use and the island itself is a major touristic destination. However, geologist and biologist working in the local government offices, as well as in UNESCO Global Geopark and Biosphere Reserve Institutions collaborate with the touristic centers and several scientist around the world with in order to implemente novel conservation policies based on the results of state-of-art research. This strategy will ensure not only a better understanding of the anchialine ecosystems in the island in the near future, but also its long term preservation.

The Lanzarote and Chinijo Islands UNESCO Global Geopark hosts one of the most extensive and diverse volcanic anchialine ecosystems in the world, consisting of water bodies with marine origin that penetrated inland through coastal crevicular systems. Marine infiltration is facilitated by the low rainfall and the permeability of the coastal terrains. Best known for Túnel de la Atlántida, Lanzarote has other types of anchialine habitats, such as pools, lakes, and even hand-made wells, all of them interconnected with the crevicular system. So far, 39 endemic stygobitic species of crustacean, annelids, and platyhelminths have been described in the island. Some of them belong to lineages previously interpreted as Tethyan vicariant relicts because they belong in ancient groups restricted to caves situated in areas along the coastline of the ancient Tethys Sea, such as Mexico, Bahamas or Western Australia. Others, instead, have clear affinities with deep-sea groups, suggesting that their ancestors might have dispersed into the island from surrounding deep-sea environments. In overall, while the anchialine habitats of Lanzarote are relatively small in comparison to other regions, the presence of so many species with such a diverse origin have puzzled zoologists and biogeographers throughout the 20th century, who have regarded the island as a model to understand the origin and evolution of similar groups in other areas of the world. The anchialine habitats in Lanzarote are subject to intense recreational use and the island itself is a major touristic destination. However, geologist and biologist working in the local government offices, as well as in UNESCO Global Geopark and Biosphere Reserve Institutions collaborate with the touristic centers and several scientist around the world with in order to implemente novel conservation policies based on the results of state-of-art research. This strategy will ensure not only a better understanding of the anchialine ecosystems in the island in the near future, but also its long term preservation.

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 ��-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 (

The Macrosite "IT08 Subalpine Lakes" includes a group of lakes located at the southern border of the Alps. The lake typologies are represented by large and deep lakes (Orta, Garda, Maggiore, Como and Iseo) and by a lake of smaller size and shallow depth (Candia). Topics common to all the research sites include the impact of eutrophication and climate change, the composition, structure and dynamics of biological communities (plankton, necton and littoral communities), trophic webs, paleolimnology, ecotoxicology, ecological status and remote sensing. Scientific investigations are supported by a wide availability of technological infrastructures and laboratories located in the reference institutions, and by a wide range of technological equipment for fieldwork. These facilities, funded within the individual institutes involved in the research, have allowed not only to maintain, but also to update the classical approaches used in scientific monitoring, while opening up new research fields (especially molecular ecology, microbial ecology, metabolomic profiling, antibiotic resistance genes, high frequency monitoring by sensor technology). Over the last decade, the continuity and regularity of investigations have been facilitated by the availability of both external and ordinary funds. The variety of research activities carried out on the IT08 macrosite is evidenced by the publication of numerous scientific papers, as well as contributions (oral or poster) presented at national and international conferences, and by the presence in the mass media. In this context, the scientific monitoring is a key element of LTER research, including not only data collection (basic monitoring), but also data interpretation, modeling, and experimental manipulation, with particular attention to key groups of selected variables in order to identify the most significant environmental stressors and the degree of change at the level of ecosystems, communities, species and populations.

The beta lactamase gene blaCTX-M, responsible of the resistance to cephalosporins, has been detected in microbes from hospitals to open waters. We studied the seasonality and stability of blaCTX-M in Lake Maggiore over 3 years and the role of potential inputs of allochthonous bacteria and/or antibiotic pollution in promoting its occurrence. blaCTX-M was mainly present from January to July in the pelagic microbial community and the gene occurrence was significantly related to low water temperature. To evaluate its temporal stability in the bacterial community over a short period, we measured blaCTX-M daily over the course of 6 days. The gene was below the limit of quantification except for one sampling when its abundance peaked, suggesting a point contamination. The bacterial community of the lake in which blaCTX-M was detected suggests that at least two distinct bacterial populations contained the gene. The occurrence of known blaCTX-M containing genera and the occurrence of the gene, however, did not overlap. Furthermore, the experimental addition of cefotaxime to lake water incubations did not promote abundance of the gene. These data imply that blaCTX-M was present in the environmental microbial community. Increases of gene abundances were likely caused by environmental parameters other than antibiotic contamination.