Horizontal transmission of a multidrug-resistant IncN plasmid isolated from urban wastewater.

Wastewater treatment plants (WWTPs) are considered reservoirs of antibiotic resistance genes (ARGs). Given that plasmid-mediated horizontal gene transfer plays a critical role in disseminating ARGs in the environment, it is important to inspect the transfer potential of transmissible plasmids to have a better understanding of whether these mobile ARGs can be hosted by opportunistic pathogens and should be included in One Health's considerations. In this study, we used a fluorescent-reporter-gene based exogenous isolation approach to capture extended-spectrum beta-lactamases encoding mobile determinants from sewer microbiome samples that enter an urban water system (UWS) in Denmark. After screening and sequencing, we isolated a ∼73 Kbp IncN plasmid (pDK_DARWIN) that harboured and expressed multiple ARGs. Using a dual fluorescent reporter gene system, we showed that this plasmid can transfer into resident urban water communities. We demonstrated the transfer of pDK_DARWIN to microbiome members of both the sewer (in the upstream UWS compartment) and wastewater treatment (in the downstream UWS compartment) microbiomes. Sequence similarity search across curated plasmid repositories revealed that pDK_DARWIN derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Furthermore, we searched for pDK_DARWIN sequence matches in UWS metagenomes from three countries, revealing that this plasmid can be detected in all of them, with a higher relative abundance in hospital sewers compared to residential sewers. Overall, this study demonstrates that this IncN plasmid is prevalent across Europe and an efficient vector capable of disseminating multiple ARGs in the urban water systems.

Insights into the circular: The cryptic plasmidome and its derived antibiotic resistome in the urban water systems.

Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes (ARGs) in different compartments of urban water systems (UWSs) in three European countries representing different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified 9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as conjugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs' plasmidome was dominated by small plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs compartments, these results should alarm and call for attention.

Extended-Spectrum ß-Lactamase and Carbapenemase Genes are Substantially and Sequentially Reduced during Conveyance and Treatment of Urban Sewage.

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-size cities in Denmark, Spain, and the United Kingdom and quantified 70 clinically important extended-spectrum ß-lactamase and carbapenemase genes along with the mobile genetic elements and microbial communities. Results from all three countries showed that sewage-especially from hospitals-carried substantial loads of ARGs (106-107 copies per person equivalent), but these loads progressively declined along sewers and through sewage treatment plants, resulting in minimal emissions (101-104 copies per person equivalent). Removal was primarily during sewage conveyance (65 ± 36%) rather than within sewage treatment (34 ± 23%). The extended-spectrum ß-lactamase and carbapenemase genes were clustered in groups based on their persistence in the UWS compartments. The less-persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while the more persistent groups appeared horizontally transferred and correlated significantly with total cell numbers and mobile genetic elements. This documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based antibiotic resistance surveillance.

Antibiotic resistance contamination in four Italian municipal solid waste landfills sites spanning 34 years.

Municipal solid waste landfill is now recognized as a significant reservoir of antibiotics and antibiotic resistance genes (ARGs). This study investigates the contamination of antibiotics resistance, in 10 leachate samples collected from four MSW landfills in north Italy spanning 34 years, including ARGs as well as mobile genetic element (MGEs). Antibiotics (0-434740 ng/L) and ARGs (5.56-6.85 × 105copies/µL leachate) were found in leachate. Abundances of the measured ARGs were found to be clustered into two groups with different changing tendencies with landfilling age in different landfills. Even though some antibiotics were banned or limited in Italy, they were found to still occur in landfills and drive the long-term contamination of ARGs indirectly, indicating the persistence of antibiotic resistance. What's more, the complexity of antibiotic resistance in leachate was found to synthetically relate to antibiotics, metals, microbes and MGEs presenting that Mn, SMX and EFC influence positively (p < 0.01) the contamination of tetW, tetQ, tetM, tetA, ermB, and cat, contributing importantly in new leachate. This study discusses the AR pollution of leachate in Italy where antibiotics are used the most in Europe, less reported in literatures. Our results suggest that a full-scale view for landfill antibiotics resistance should be considered with history of landfills, use of antibiotics and different phase in landfills, with both "relative static" and "dynamic tracking" perspective to focus on the principal antibiotic-resistance pollutants for leachate treatment, and raise the attention for landfill post-closure care and landfill mining.

Fate of antibiotics and antibiotic resistance genes in a full-scale restaurant food waste treatment plant: Implications of the roles beyond heavy metals and mobile genetic elements.

Is our food safe and free of the crisis of antibiotics and antibiotic resistance (AR)? And will the derived food waste (FW) impose AR risk to the environment after biological treatment? This study used restaurant FW leachates flowing through a 200 tons-waste/day biological treatment plant as a window to investigate the fate of antibiotics and antibiotic-resistance genes (ARGs) during the acceptance and treatment of FW. Sulfonamides (sulfamethazine, sulfamethoxazole) and quinolones (ciprofloxacin, enrofloxacin, ofloxacin) were detected during FW treatment, while tetracyclines, macrolides and chloramphenicols were not observable. ARGs encoding resistance to sulfonamides, tetracyclines and macrolides emerged in FW leachates. Material flow analysis illustrated that the total amount of antibiotics (except sulfamethazine) and ARGs were constant during FW treatment processes. Both the concentration and total amount of most antibiotics and ARGs fluctuated during treatment, physical processes (screening, centrifugation, solid-liquid and oil-water separation) did not decrease antibiotic or ARGs concentrations or total levels permanently; the affiliated wastewater treatment plant appeared to remove sulfonamides and most ARGs concentrations and total amount. Heavy metals Ni, Co and Cu were important for disseminating antibiotics concentrations and MGEs for distributing ARGs concentrations. Humic substances (fulvic acids, hydrophilic fractions), C-associated and N-associated contents were essential for the distribution of the total amounts of antibiotics and ARGs. Overall, this study implied that human food might not be free of antibiotics and ARGs, and FW was an underestimated AR pool with various determinants. Nonetheless, derived hazards of FW could be mitigated through biological treatment with well-planned daily operations.

Co-occurrence of mobile genetic elements and antibiotic resistance genes in municipal solid waste landfill leachates: A preliminary insight into the role of landfill age.

Since municipal solid waste (MSW) landfill harbours miscellaneous wastes, pollutants and microorganisms, it gradually becomes a huge potential reservoir for breeding antibiotic resistance genes (ARGs). The objective of this study was to determine the prevalence and diversity of ARGs associated with various mobile genetic elements (MGEs) in MSW landfill leachates. The relationship of ARGs with leachate characteristics was also studied to explore the influence of landfill age. Seven sulfonamides (sulfapyridine, sulfadiazine, sulfathiazole, sulfamethoxazole, sulfamerazine, sulfamethazine and sulfaquinoxaline), three encoded ARGs (sul-I, sul-II and sul-III) and four types of MGEs (plasmids, transposons, integrons and insertion sequences) were quantified in leachates with landfill ages ranging from 3 months-6 years. ARGs increased to an absolute concentration of 106 copies/µL and were positively correlated (p < 0.05) to MGEs. Significant correlations (p < 0.05) were also discovered among ARGs and the increasing humic acids, heavy metals (Zn, Cu and Co) and antibiotics (except for sulfathiazole and sulfaquinoxaline), implying landfilling might contribute to the enrichment of ARGs in the long-term. Non-target full scans revealed the role of persistent unknown compounds in stimulating the ARGs dissemination. Overall, this study demonstrates the exacerbation of ARGs pollution in landfill environment and a detailed delineation of the complex inter-relationships between ARGs and the substances harbouring in landfills is badly required.