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This study investigates the antibiotic resistance fate in the urban water cycle, evaluating the dynamics of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) in three different full-scale wastewater treatment plants (WWTPs) and two drinking water treatment plants (DWTPs) located in the same geographical area (North-West of Italy). ARB (tetracycline-, ampicillin-, and sulfonamide-resistant bacteria) were quantified by plate counting and the abundances of selected ARGs (i.e., tetA, bla

Wastewater treatment plantsare among the main hotspots for the release of antibiotic resistance genes (ARGs) into the environment. ARGs in treated wastewater can be found in the intracellular DNA (iDNA) and in the extracellular DNA (eDNA). In this study, we investigated the fate and the distribution (either in eDNA or in iDNA) of ARGs in the treated wastewaters pre and post-disinfection by shotgunmetagenomics. The richness of the intracellular resistome was found to be higher than the extracellular one. However, the latter included different high risk ARGs. About 11% of the recovered metagenome assembled genomes (MAGs) from the extracted DNA was positive for at least one ARG and, among them, several were positive for more ARGs. The high-risk ARGbacA was the most frequently detected gene among the MAGs. The disinfection demonstrated to be an important driver of the composition of the antibiotic resistomes. Our results demonstrated that eDNA represents an important fraction of the overall ARGs, including a number of high-risk ARGs, which reach the environment with treated wastewater effluents. The studied disinfections only marginally affect the whole antibiotic resistome but cause important shifts from intracellular to extracellular DNA, potentially threating human health.

Wastewater treatment plants (WWTPs) efficiently retain microplastic particles (MPs) generated within urban areas. Among the wastewater treatment steps, disinfection has not been characterized for its potential MPs retention activity, although it has been reported that processes used to abate the bacterial load could also affect MPs concentration. For this reason, we evaluated the MPs concentration across the overall wastewater treatment process and before and after the disinfection step in four small/medium WWTPs located in the north of Italy. Most of the MPs found in the samples were fibers or fragments, smaller than 500 μm, mainly composed of polyethylene, polypropylene, or polyethylene terephthalate. The retention efficiency at the outlets was higher than 94% in all the plants analyzed. More interestingly, the disinfection treatments adopted by the different WWTPs reduced MPs concentration from a minimum of 9.1% (UV treatment) to a maximum of 67.6% (chlorination), promoting a further increase of the overall retention efficiency of the WWTPs from 0.4 to 0.7%. Quantitatively, the disinfection contributes to the MPs reduction in the outlets by retaining 0.5-6.7 million MPs per day, in WWTPs that discharge 2.7-12 million MPs per day. The results of the present work underline the importance of a careful choice of the steps that constitute the wastewater treatment, including disinfection, in order to minimize MPs discharge into the natural ecosystems.

The impact of Fenton oxidation (FO) and Air stripping (AS) pre-treatments on the bacterial community of a biological activated sludge (B-AS) process for the co-treatment of mature landfill leachate (MLL) and urban wastewater (UWW) was assessed. In this work high-throughput sequencing was used to identify changes in the composition of the bacterial communities when exposed to different landfill leachate's pre-treatments. The combination of FO and AS to increase biodegradability (BOD

The spread of antibiotic resistance is becoming a serious global health concern. Numerous studies have been done to investigate the dynamics of antibiotic resistance genes (ARGs) in both indoor and outdoor environments. Nonetheless, few studies are available about the dynamics of the antibiotic resistome (total content of ARGs in the microbial cultures or communities) under stress in outer space environments. In this study, we aimed to experimentally investigate the dynamics of ARGs and metal resistance genes (MRGs) in Kombucha Mutualistic Community (KMC) samples exposed to Mars-like conditions simulated during the BIOMEX experiment outside the International Space Station with analysis of the metagenomics data previously produced. Thus, we compared them with those of the respective non-exposed KMC samples. The antibiotic resistome responded to the Mars-like conditions by enriching its diversity with ARGs after exposure, which were not found in non-exposed samples (i.e., tet and van genes against tetracycline and vancomycin, respectively). Furthermore, ARGs and MRGs were correlated; therefore, their co-selection could be assumed as a mechanism for maintaining antibiotic resistance in Mars-like environments. Overall, these results highlight the high plasticity of the antibiotic resistome in response to extraterrestrial conditions and in the absence of anthropogenic stresses.

This study shows that Escherichia coli can be temporarily enriched in zooplankton in natural conditions and that these bacteria can belong to different phylogroups and sequence types including environmental as well as clinical and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically the two isolates were closer to strains isolated from poultry meat than with freshwater E. coli, albeit their genomes were smaller than those from poultry. After isolation and fluorescent protein tagging of strains ED1 and ED157 we show that Daphnia sp. can take up these strains and release them alive again, thus forming a temporary host for E. coli. In a chemostat experiment we show that the association does not prolong the bacterial long-term survival, but that at low abundances it does also not significantly reduce the bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural microbiota of Daphnia, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli to Daphnia is only temporary but that the cells are viable therein and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptations to the freshwater environment.ImportanceThe contamination of freshwaters with faecal derived bacteria is of major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological as well as water safety relevance.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Seas and oceans are a global reservoir of antibiotic resistance genes (ARGs). Only a few studies investigated the dynamics of ARGs along the water column of the Black Sea, a unique environment, with a peculiar geology, biology and history of anthropogenic pollution. In this study, we analyzed metagenomic data from two sampling campaigns (2013 and 2019) collected across three different sites in the Western Black Sea at depths ranging from 5 to 2000 m. The data were processed to annotate ARGs, metal resistance genes (MRGs) and integron integrase genes. The ARG abundance was significantly higher in the deep water layers and depth was the main driver of beta-diversity both for ARGs and MRGs. Moreover, ARG and MRG abundances strongly correlated (r = 0.95). The integron integrase gene abundances and composition were not influenced by the water depth and did not correlate with ARGs. The analysis of the obtained MAGs showed that some of them harbored intI gene together with several ARGs and MRGs, suggesting the presence of multidrug resistant bacteria and that MRGs and integrons could be involved in the selection of ARGs. These results demonstrate that the Black Sea is not only an important reservoir of ARGs, but also that they accumulate in the deep water layers where co-selection with MRGs could be assumed as a relevant mechanism of their persistence.

Stochastic or deterministic processes control the bacterial community assembly in waters and their understanding is a fundamental question to correctly manage aquatic environments exposed to the release of antibiotics from anthropogenic sources. It has been suggested that microdiversity (i.e. the rare biosphere) convers freshwater communities with stability, meaning that previously rare taxa bloom when the community is disturbed. Since there might be a seed bank of similar, but not abundant, bacterial taxa in different waters, we tested whether a disturbance by an antibiotic cocktail would increase similarity in bacterial communities from different freshwater systems (a wastewater effluent and two lakes). In a continuous culture set-up in chemostats, we show that disturbance with antibiotics causes communities from different environments to become more similar. Once the antibiotic pressure is released the communities tend to become more dissimilar again. This shows that there is a similar shift in community composition even in waters from very different origins when they are disturbed by antibiotics, even at low concentrations. Antibiotics impact the bacterial communities at the cell and the community level, independently by the original degree of anthropogenic stress they are adapted to, altering the original phenotypes, genotypes, and the relations between bacteria.

The risk for human health posed by polluted aquatic environments, and especially those carrying antibiotic resistance genes (ARGs) of clinical interest, is still debated. This is because of our limited knowledge of the dynamics of antimicrobial resistance in the environment, the selection mechanisms underlying the spread of ARGs, and the ecological factors potentially favoring their return to humans. The Class 1 integron is one of the most effective platforms for the dissemination of ARGs. In this study we investigated a freshwater system consisting of a lake-river-lake continuum, determining the abundance of class 1 integrons and their associated ARGs by a modulated metagenomic approach. Bacterial abundance and community composition were used to identify the potential carriers of class 1 integrons and their associated ARGs over a period of six months. Class 1 integrons and their ARG cargoes were significantly more abundant in riverine sampling sites receiving treated wastewater. Further, class 1 integrons carried ARGs ranked at the highest risk for human health (e.g., catB genes), in particular, genes encoding resistance to aminoglycosides. Genera of potential pathogens, such as Pseudomonas and Escherichia-Shigella, were correlated with class 1 integrons. The lake-river-lake system demonstrated a clear relationship between the integrase gene of class 1 integrons (intI1) and anthropogenic impact, but also a strong environmental filtering that favored the elimination of intI1 once the human derived stressors were reduced. Overall, the results of this study underline the role class 1 integrons as proxy of anthropogenic pollution and suggest this genetic platform as an important driver of aminoglycoside resistance genes, including high risk ARGs, of potential concern for human health.

Although abundant and chemically peculiar, tyre wear microplastic particles (TWP) and their impact on the microbial communities in water are largely understudied. We tested in laboratory based semi-continuous cultures the impact of TWP and of polyethylene terephthalate (PET) derived particles (following a gradient of relative abundance) on the pathobiome (the group of potential human pathogenic bacteria) of a freshwater microbial community exposed to contamination by the effluent of a urban wastewater treatment plant, for a period of 28 days. We could define the modulated impact of the two types of microplastic particles: while PET does not favour bacterial growth, it offers a refuge to several potential pathogens of allochthonous origin (from the treated sewage effluent), TWP act as an additional carbon source, promoting the development and the massive growth of a biofilm composed by fast-growing bacterial genera including species potentially harmful and competitive in abating biodiversity in surface waters. Our results demonstrate the different ecological role and impact on freshwater environments of TWP and PET particles, and the need to approach the study of this pollutant not as a whole, but considering the origin and the chemical composition of the different particles.

Wastewater treatment plants (WWTPs) are among the main hotspots of antibiotic resistance genes (ARGs) in the environment. Previously, we demonstrated that, by increasing anthropogenic pollution, the antibiotic resistome persisted in the microbial community of rivers and lakes, independently by changes in community composition. In this study, we reanalysed the data to test for the relation of metal resistance genes (MRGs), plasmids, and integrons to the persistence of the antibiotic resistome. The experiment consisted in replicated co-cultures of riverine or lacustrine microbial communities and WWTP effluents in different proportions. Samples before (T0) and after a short period of incubation (TF) were collected and community metagenomic data were obtained by shotgun sequencing. The data were processed to annotate MRGs, plasmids, and integrases. The integrases stabilized in the aquatic environment following the degree of contamination with effluent water (in particular in one site), whereas MRGs and plasmids showed stochastic trajectories. These results confirm the potential correlation between integrons and anthropogenic pollution, and the reliability of intI1 as a pollution marker. Only in one site MRGs, plasmids, and ARGs were correlated, highlighting their partial contribution to the persistence of ARGs in surface waters.

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a-(Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 10(3) cells ml(-1) of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.

Urban Wastewater Treatment Plants (UWTPs) treating mixed urban sewage and industrial wastewater are among the major hotspots for the spread of Antibiotic Resistance Genes (ARGs) into the environment. This study addresses the impact of the wastewater origin on ARG dynamics in a full-scale UWTP (15,000 Population Equivalent, PE) by operating the plant with and without industrial wastewater. Composite samples (4 L) from different treatment points were characterized for their chemical composition, bacterial abundance and for the abundance of four resistance genes against tetracycline, sulfonamides, erythromycin, and quinolones (tetA, sul2, ermB, and qnrS), and of the class 1 integrons (intI1). Although the chemical composition of the outflow significantly differed when the plant operated with or without industrial wastewater, the system efficiency in the removal of bacterial cells, ARGs, and intI1 was constant. The final disinfection by peracetic acid (PAA) did not affect the removal of ARGs, independently of the wastewater origin and the chemical characteristics of the inflows. Our results demonstrated that a well-functioning small size UWTP could treat a significant amount of industrial wastewater mixed in the urban sewage without affecting the overall ARGs and class 1 integrons released into the environment.

Microplastic Particles (MPs) are ubiquitous pollutants widely found in aquatic ecosystems. Although MPs are mostly retained in wastewater treatment plants (WWTPs), a high number of MPs reaches the open waters potentially contributing to the spread of pathogenic bacteria and antibiotic resistance genes in the environment. Nowadays, a limited number of studies have focused on the role of MPs as carriers of potentially pathogenic and antibiotic resistant bacteria in WWTPs. Thus, an investigation on the community composition (by 16S rRNA gene amplicon sequencing) and the abundance of antibiotic and metal resistance genes (by qPCR) of the biofilm on MPs (the plastisphere) and of planktonic bacteria in treated (pre- and post-disinfection) wastewaters was performed. MPs resulted to be very similar in terms of type, color, size, and chemical composition, before and after the disinfection. The bacterial community on MPs differed from the planktonic community in terms of richness, composition, and structure of the community network. Potentially pathogenic bacteria generally showed higher abundances in treated wastewater than in the biofilm on MPs. Furthermore, among the tested resistance genes, only sul2 (a common resistance gene against sulfonamides) resulted to be more abundant in the plastisphere than in the planktonic bacterial community. Our results suggest that the wastewater plastisphere could promote the spread of pathogenic bacteria and resistance genes in aquatic environment although with a relatively lower contribution than the wastewater planktonic bacterial community.

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 (

The Fenton process activated by Zero Valent Iron (ZVI-Fenton) is shown here to effectively remove antibiotics reserved for hospital settings (specifically used to treat antibiotic-resistant infections) from wastewater, thereby helping in the fight against bacterial resistance. Effective degradation of cefazolin, imipenem and vancomycin in real urban wastewater was achieved at pH 5, which is quite near neutrality when compared with classic Fenton that works effectively at pH 3–4. The possibility to operate successfully at pH 5 has several advantages compared to operation at lower pH values: (i) lower reagent costs for pH adjustment; (ii) insignificant impact on wastewater conductivity, because lesser acid is required to acidify and lesser or no base for neutralization; (iii) undetectable release of dissolved Fe, which could otherwise be an issue for wastewater quality. The cost of reagents for the treatment ranges between 0.04 and 0.07 $ m−3, which looks very suitable for practical applications. The structures of the degradation intermediates of the studied antibiotics and their likely abundance suggest that, once the primary compound is eliminated, most of the potential to trigger antibiotic action has been removed. Application of the ZVI-Fenton technique to wastewater treatment could considerably lower the possibility for antibiotics to trigger the development of resistance in bacteria.

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., Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? This Essay reveals that every fifth published metagenome is not available to science, and asks whether data accessibility should be considered a mandatory component of manuscript submission.

The efficiency of a new Advanced Oxidation Process (AOP), namely the photo Fenton like process UV-C/H

Lakes are exposed to anthropogenic pollution including the release of allochthonous bacteria into their waters. Antibiotic resistance genes (ARGs) stabilize in bacterial communities of temperate lakes, and these environments act as long-term reservoirs of ARGs. Still, it is not clear if the stabilization of the ARGs is caused by a periodical introduction, or by other factors regulated by dynamics within the water column. Here we observed the dynamics of the tetracycline resistance gene (tetA) and of the class 1 integron integrase gene intI1 a proxy of anthropogenic pollution in the water column and in the sediments of subalpine Lake Maggiore, together with several chemical, physical and microbiological variables. Both genes resulted more abundant within the bacterial community of the sediment compared to the water column and the water-sediment interface. Only at the inset of thermal stratification they reached quantifiable abundances in all the water layers, too. Moreover, the bacterial communities of the water-sediment interface were more similar to deep waters than to the sediments. These results suggest that the vertical distribution of tetA and intI1 is mainly due to the deposition of bacteria from the surface water to the sediment, while their resuspension from the sediment is less important.

The development of new methods and technologies for microbial characterization as well as their increasing availability at more affordable costs, has made evident the limitations of the conventional and routinely applied (typically cultivation based) methods to exhaustively and fully characterize the actual effect of disinfection process in urban wastewater treatment plants (UWWTPs). Such problem is even more relevant and of concern if microbial challenges, such as the occurrence of pathogens as well as the spread of antibiotic resistance, are taken into account. Such threats move scientists to investigate new and more effective disinfection processes from one side and new methods, techniques and approaches to characterize disinfection process efficiency from the other side. In this opinion paper, the limitations of routine detection methods are discussed according to the relevant and updated scientific literature to explain how research oriented methods and technologies (namely, quantitative real-time PCR, flow cytometry, metagenetics and metagenomics) can allow a better evaluation of disinfection processes. After a short introduction to the main disinfection processes, the application of different microbial characterization methods is discussed according to distinct challenges, such as pathogens inactivation or antibiotic resistance dissemination, when wastewater safety is of concern (for example in reuse scenarios). The routine and research oriented techniques can be successfully used in complementary way to evaluate disinfection process efficiency. Recommendations for UWWTPs managers for internal control of disinfection process are proposed.

Microorganisms in coastal sediments are fundamental for ecosystem functioning, and regulate processes relevant in global biogeochemical cycles. Still, our understanding of the effects anthropogenic perturbation and pollution can have on microbial communities in marine sediments is limited. We surveyed the microbial diversity, and the occurrence and abundance of metal and antibiotic resistance genes is sediments collected from the Pula Bay (Croatia), one of the most significantly polluted sites along the Croatian coast. With a collection of 14 samples from the bay area, we were able to generate a detailed status quo picture of a site that only recently started a cleaning and remediation process (closing of sewage pipes and reduction of industrial activity). The concentrations of heavy metals in Pula Bay sediments are significantly higher than in pristine sediments from the Adriatic Sea, and in some cases, manifold exceed international sediment quality guidelines. While the sedimentary concentrations of heavy metals did significantly influence the abundance of the tested metal resistance genes, no strong effect of heavy metal pollution on the overall microbial community composition was observed. Like in many other marine sediments, Gammaproteobacteria, Bacteroidota and Desulfobacterota dominated the microbial community composition in most samples, and community assembly was primarily driven by water column depth and nutrient (carbon and nitrogen) availability, regardless of the degree of heavy metal pollution.

Macroalgal competition can indirectly influence the health of corals and their response to changing environmental conditions by altering their associated bacterial community. However, the effect of macroalgae on the composition of epilithic microbial biofilms, an important determinant of coral recruitment, is poorly known. In the back-reefs of Moorea (French Polynesia), we evaluated how the experimental removal of either the canopy of the seaweed Turbinaria ornata or that of the entire macroalgal assemblage influenced the composition of the bacterial biofilm and coral recruitment on macroalga-free substrata. The number of bacterial colonies on culture plates inoculated with dilutions of 9 d old biofilm from canopy removal sites was smaller compared with control sites. After 3.5 mo, the diversity of bacterial operational taxonomic units (OTUs) was lower at both canopy and total macroalgal removal sites. Total macroalgal removal sites had a lower relative abundance of several bacterial families, including Rhodobacteraceae, Erythrobacteraceae, Cyanobacteria Family IV and Family VIII, Flavobacteriaceae and Verrucomicrobiaceae. After 8 mo, coral recruitment was generally low, but greater at total macroalgal removal sites. The relative abundance of Cyanobacteria, Sphingo - bacteria and Verrucomicrobia was negatively correlated with coral recruitment and explained ~70% of variation in coral recruit density. Our study shows that the removal of T. ornata and understory macroalgae influences the composition of epilithic bacterial assemblages and coral recruitment. Thus, eradication campaigns are unlikely to sustain long-term reductions in the abundance of T. ornata and, hence, increase coral recruitment, when plant holdfasts and understory macroalgae are left in place.

The oceans are increasingly polluted with plastic debris, and several studies have implicated plastic as a reservoir for antibiotic resistance genes and a potential vector for antibiotic-resistant bacteria. Bioplastic is widely regarded as an environmentally friendly replacement to conventional petroleum-based plastic, but the effects of bioplastic pollution on marine environments remain largely unknown. Here, we present the first evidence that bioplastic accumulates antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in marine sediments. Biofilms fouling ceramic, polyethylene terephthalate (PET), and polyhydroxyalkanoate (PHA) were investigated by shotgun metagenomic sequencing. Four ARG groups were more abundant in PHA: trimethoprim resistance (TMP), multidrug resistance (MDR), macrolide-lincosamide-streptogramin resistance (MLS), and polymyxin resistance (PMR). One MRG group was more abundant in PHA: multimetal resistance (MMR). The relative abundance of ARGs and MRGs were strongly correlated based on a Mantel test between the Bray-Curtis dissimilarity matrices (R = 0.97, p < 0.05) and a Pearson's analysis (R = 0.96, p < 0.05). ARGs were detected in more than 40% of the 57 metagenome-assembled genomes (MAGs) while MRGs were detected in more than 90% of the MAGs. Further investigation (e.g., culturing, genome sequencing, antibiotic susceptibility testing) revealed that PHA biofilms were colonized by hemolytic Bacillus cereus group bacteria that were resistant to beta-lactams, vancomycin, and bacitracin. Taken together, our findings indicate that bioplastic, like conventional petroleum-based plastic, is a reservoir for resistance genes and a potential vector for antibiotic-resistant bacteria in coastal marine sediments.

In the last decade, the study of the origin and fate of plastic debris received great attention, leading to a new and broad awareness of the hazard represented by these particles for the environment and the biota. At the same time, the scientific consideration on the leading role of the environment regarding the spread of antibiotic resistant bacteria (ARB) increased. Both, microplastic particles (MPs) and ARB share pollution sources and, in aquatic systems, MPs could act as a novel ecological niche, favouring the survival of pathogens and ARB. MPs can host a specific microbial biofilm, referred to as plastisphere, phylogenetically different from the surrounding planktonic microbial community and from the biofilm growing on other suspended particles. The plastisphere can influence the overall microbiome of a specific habitat, by introducing and supporting different species and by increasing horizontal gene transfer. In this review we collect and analyse the available studies coupling MPs and antibiotic resistance in water, highlighting knowledge gaps to be filled in order to understand if MPs could effectively act as a carrier of ARB and antibiotic resistance genes, and pose a real threat to human health.

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.

Notwithstanding the fundamental role that environmental microbes play for ecosystem functioning, data on how microbes react to disturbances are still scarce, and most factors that confer stability to microbial communities are unknown. In this context, antibiotic discharge into the environment is considered a worldwide threat for ecosystems with potential risks to human health. We therefore tested resilience of microbial communities challenged by the presence of an antibiotic. In a continuous culture experiment, we compared the abundance, composition and diversity of microbial communities undisturbed or disturbed by the constant addiction of tetracycline in low (10 µg/L) or intermediate (100 µg/L) concentration (press disturbance). Further, the bacterial communities in the three treatments had to face the sudden pulse disturbance of adding an allochthonous bacterium (Escherichia coli). Tetracycline, even at low concentrations, affected microbial communities by changing their phylogenetic composition and causing cell aggregation. This, however, did not coincide with a reduced microbial diversity, but was mainly caused by a shift in dominance of specific bacterial families. Moreover, the less disturbed community (10 µg/L tetracycline) was sometimes more similar to the control and sometimes more similar to heavily disturbed community (100 µg/L tetracycline). All in all, we could not see a pattern where the communities disturbed with antibiotics were less resilient to a second disturbance introducing E. coli, but they seemed to be able to buffer the input of the allochthonous strain in a similar manner as the control.

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 Tunel de la Atlantida, 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 platyhelminthes 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 lineages, 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 have started close collaborations with the touristic centers and independent scientist from several universities and research centers around the world with the goal of implementing 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.

Treated wastewater discharged into the environment acts as a disturbance of the natural microbial communities in terms of taxonomic composition and of functional gene pool, including antibiotic resistance genes. We tested whether stochastic and heterogeneous site-specific trajectories or generalities, potentially driven by deterministic processes, control the fate of allochthonous bacteria from anthropogenic sources and the persistence of their functional traits in freshwater. Finding generalities would allow the identification of wastewater treatments that could be effective in abating determinants of antibiotic resistance. We analysed the short-term response of native bacterial communities in waters exposed to the disturbance of wastewater at different dilutions, using a metagenomic approach that revealed both microbial community composition and the scope and abundance of the resistome that can pose indirect risks to human health. We found that the taxonomic composition of the communities after the disturbance was driven by case-specific stochastic processes, whereas the resistome had a deterministic trajectory, rapidly stabilising its functional traits with higher proportions of wastewater effluents, regardless of differences in taxonomic composition, richness of antibiotic resistance genes and of bacterial taxa, phenotypic features of the bacterial communities, and type of wastewater treatment. The observed deterministic proliferation of resistomes in freshwater bodies receiving wastewater effluents, suggests that this process may contribute to the global propagation of antibiotic resistance, and thus calls for new legislations promoting alternative tertiary treatments for the wastewater reuse, and targeting bacterial functional traits and not only bacterial abundances.

The efficiency of a new Advanced Oxidation Process (AOP), namely the photo Fenton like process UV- C/H 2 O 2 /IDS-Cu, in removing determinants of antibiotic resistance and pathogenic bacteria was compared to a consolidated AOP (namely UV-C/H 2 O 2 ) in a secondary treated municipal WasteWater (WW). A re- ductionist experimental laboratory-based approach was applied on real WW and the parameters were collected by an alternative integrated approach using (i) flow cytometry to enumerate bacteria and test for the fitness of the bacterial communities and (ii) molecular analyses to define the community com- position (16S rRNA amplicon sequencing) and the abundances of Antibiotic Resistance Genes (ARGs) and of the class 1 integron ( int I1 gene) (by quantitative PCR). The same approach was applied also to post- treatment regrowth tests (24 h) to define the potential persistence of the tested parameters. These exper- iments were performed in both, human pathogens favorable conditions (HPC, in rich medium and 37 °C) and in environmental mimicking conditions (EMC, original WW and 20 °C). UV-C/H 2 O 2 /IDS-Cu process re- sulted to be more effective than the UV-C/H 2 O 2 in inactivating bacterial cells in the EMC post-treatment regrowth experiments. Both AOPs were efficiently abating potential human pathogenic bacteria and ARGs in the HPC regrowth experiments, although this trend could not be detected in the measurements taken immediately after the disinfection. In comparison with the UV-C/H 2 O 2 , the UV-C/H 2 O 2 /IDS-Cu process did not apparently offer significant improvements in the abatement of the tested parameters in the WW effluent but, by evaluating the results of the regrowth experiments it was possible to extrapolate more complex trends, suggesting contrasting efficiencies visible only after a few hours. This study offers a de- tailed view on the abatement efficiency of microbiological/genetic parameters for the UV-C/H 2 O 2 /IDS-Cu process, calling for technical adjustments for this very promising technology. At the same time, our re- sults clearly demonstrated the inadequacy of currently applied methodologies in the evaluation of specific parameters (e.g. determinants of antibiotic resistance and pathogenic bacteria) in WW.

Superstition has it that tossing coins into wells or fountains brings good luck, thereby causing a potential accumulation of microbially contaminated metal particles in the water. Here, we characterized the microbiota and the resistance profile in biofilm on such coins and their surrounding sediments. The study site was a tidal marine lake within a touristic center located in a natural reserve area. Notwithstanding the fact that coin-related biofilms were dominated by typical marine taxa, coin biofilms had specific microbial communities that were different from the communities of the surrounding sediment. Moreover, the communities were different depending on whether the coin were made mainly of steel or of copper. Sequences affiliated with putative pathogens were found on every third coin but were not found in the surrounding sediment. Antibiotic resistance genes (ARGs) were detected on most of the coins, and interestingly, sediments close to the area where coins accumulate had a higher frequency of ARGs. We suggest that the surface of the coins might offer a niche for ARGs and faecal bacteria to survive, and, thus, tossed coins are a potential source and vector for ARGs into the surrounding environment.

We carry out a mesocosms experiment to assess the impact of high-quality treated wastewater intended for agricultural reuse (HQWR) on freshwater bacteria seldom exposed to anthropogenic pollution. Effects were assessed by comparing the abundance and composition of bacterial communities as well as their resistance profile under control (source water from an unpolluted lake) and treatment conditions (source water mixed 1:1 with HQWR, with and without 5 μg L−1 of cefotaxime). We investigated the effect of the different conditions on the abundance of genes encoding resistance to β-lactams and carbapenems (blaTEM, blaCTX-M, blaOXA, and blaKPC), fluoroquinolones (qnrS), tetracyclines (tetA), sulfonamides (sul2), macrolides (ermB), arsenic and cadmium (arsB and czcA, respectively), and on the gene encoding the Class 1 integron integrase (intI1). Bacterial communities exposed to HQWR showed a significant higher abundance of tetA, arsB, czcA, and intI1 genes, whereas those exposed to Cefotaxime-amended HQWR did not. Genes conferring resistance to carbapenems, β-lactams, fluoroquinolones, and macrolides were below detection limit in all treatments. Besides, the higher availability of nutrients under treatment conditions favored bacterial growth in comparison to those exposed to control conditions. Particularly, Acinetobacter spp. and Pseudomonas spp. were significantly enriched after 22 days of treatment exposure. The presence of cefotaxime (a third generation cephalosporine) in the feeding medium caused an enrichment of bacterial communities in sequences affiliated to Acinetobacter thus suggesting that these resistant forms may possess resistance genes other than those studied here (blaCTX-M, blaOXA, and blaKPC). Although derived from a mesocosm experiment in continuous cultures, our results call attention to the need of refined regulations regarding the use of reclaimed water in agriculture since even high-quality treated wastewater may lead to undesired effects on receiving bacterial communities in terms of composition and dissemination of antibiotic resistance genes.