The extensive use of antibiotics in human and veterinary medicine and their subsequent release into the environment may have direct consequences for autochthonous bacterial communities, especially in ...freshwater ecosystems. In small streams and rivers, local inputs of wastewater treatment plants (WWTPs) may become important sources of organic matter, nutrients and emerging pollutants, such as antibiotic resistance genes (ARGs). In this study, we evaluated the effect of WWTP effluents as a source of ARGs in river biofilms. The prevalence of genes conferring resistance to main antibiotic families, such as beta-lactams (blaCTX-M), fluoroquinolones (qnrS), sulfonamides (sul I), and macrolides (ermB), was determined using quantitative PCR (qPCR) in biofilm samples collected upstream and downstream WWTPs discharge points in four low-order streams. Our results showed that the WWTP effluents strongly modified the hydrology, physico-chemistry and biological characteristics of the receiving streams and favoured the persistence and spread of antibiotic resistance in microbial benthic communities. It was also shown that the magnitude of effects depended on the relative contribution of each WWTP to the receiving system. Specifically, low concentrations of ARGs were detected at sites located upstream of the WWTPs, while a significant increase of their concentrations was observed in biofilms collected downstream of the WWTP discharge points (particularly ermB and sul I genes). These findings suggest that WWTP discharges may favour the increase and spread of antibiotic resistance among streambed biofilms. The present study also showed that the presence of ARGs in biofilms was noticeable far downstream of the WWTP discharge (up to 1 km). It is therefore reasonable to assume that biofilms may represent an ideal setting for the acquisition and spread of antibiotic resistance determinants and thus be considered suitable biological indicators of anthropogenic pollution by active pharmaceutical compounds.
•We evaluated the effect of WWTP effluents on small rivers.•We investigated the occurrence of antibiotic resistance genes (ARGs) in biofilms.•WWTP effluents deeply modified the characteristics of the receiving streams.•WWTP effluents favoured the spread and persistence of ARGs in biofilms.•The increase of ARGs in biofilms was noticeable far downstream of the WWTP discharge.
The study of biofilms in rivers revealed that small wastewater treatment plants can be a relevant source of antibiotic resistance genes to benthic communities of freshwater ecosystems.
Bacteriophages (phages) are the most abundant and diverse biological entities in our planet. They infect susceptible bacterial hosts into which they either multiply or persist. In the latter case, ...phages can confer new functions to their hosts as a result of gene transfer, thus contributing to their adaptation (short-term) and evolution (long-term). In this regard, the role of phages on the dissemination of antibiotic resistance genes (ARGs) among bacterial hosts in natural environments has not yet been clearly resolved. Here, we carry out a comprehensive analysis of thirty-three viromes from different habitats to investigate whether phages harbor ARGs. Our results demonstrate that while human-associated viromes do not or rarely carry ARGs, viromes from non-human sources (e.g. pig feces, raw sewage, and freshwater and marine environments) contain a large reservoir of ARGs, thus pointing out that phages could play a part on the spread of antibiotic resistance. Given this, the role of phages should not be underestimated and it should be considered when designing strategies to tackle the global crisis of antibiotic resistance.
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•Viromes from different habitat were screened for presence of antibiotic resistance genes.•Genes encoding MFS transporters and β-lactamases were found in all analyzed viromes.•Environmental bacteriophages may contain a large reservoir of resistance genes.
In this study, we quantified eleven antibiotic compounds and nine antibiotic resistance genes (ARGs) in water samples collected upstream and downstream of the discharge point from a municipal ...wastewater treatment plant (WWTP) into the Ter River. Antibiotics were analyzed by liquid chromatography coupled to mass spectrometry, whereas the concentration of ARGs in bacterial, phage and plasmid DNA fractions was determined by real-time PCR to explore their contribution to environmental antibiotic resistance. WWTP discharges resulted in higher concentrations of antibiotic residues as well as ARGs in water samples collected downstream the impact point. Specifically, genes conferring resistance to macrolides (ermB), fluoroquinolones (qnrS) and tetracyclines (tetW) showed significant differences (p<0.05) between upstream and downstream sites in the three DNA fractions (i.e. bacteria, plasmids and phages). Interestingly, genes conferring resistance to β-lactams (blaTEM, blaNDM and blaKPC) and glycopeptides (vanA) only showed significant differences (p<0.05) between upstream and downstream sites in phage and plasmid DNA but not in the bacterial DNA fraction. Our results show for the first time the extent to which phages and plasmids contribute to the mobilization of ARGs in an aquatic environment exposed to chronic antibiotic pollution via WWTP discharges. Accordingly, these mobile genetic elements should be included in further studies to get a global view of the spread of antibiotic resistance.
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•We investigated the presence of antibiotic resistance genes (ARGs) in bacterial, phage and plasmid DNA fractions.•Higher copy numbers of the target ARGs were detected in the three DNA fractions downstream of WWTP than in upstream sites.•Genes conferring resistance to β-lactams and glycopeptides only showed significant differences in phage and plasmid DNA.
The occurrence and removal of 49 antibiotics and 11 selected antibiotic resistance genes (ARGs) were investigated in 2 vertical subsurface flow (VF) constructed wetlands (1.5 m2 each): an unsaturated ...(UVF) unit and a partially saturated (SVF) unit (0.35 m saturated out of 0.8 m) operating in parallel and treating urban wastewater. Thirteen antibiotics were detected in influent wastewater, 6 of which were present in all samples. The SVF showed statistical significance on the removal of 4 compounds (namely ciprofloxacin, ofloxacin, pipemidic acid and azithromycin), suggesting that the wider range of pH and/or redox conditions of this configuration might promote the microbial degradation of some antibiotics. In contrast, the concentration of the latter (except pipemidic acid) and also clindamycin was higher in the effluent than in the influent of the UVF. Five ARGs were detected in influent wastewater, sul1 and sul2, blaTEM, ermB and qnrS. All of them were detected also in the biofilm of both wetlands, except qnrS. Average removal rates of ARGs showed no statistical differences between both wetland units, and ranged between 46 and 97% for sul1, 33 and 97% for sul2, 9 and 99% for ermB, 18 and 97% for qnrS and 11 and 98% for blaTEM.
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•The fate of antibiotics and ARGs in vertical flow constructed wetlands was studied.•Saturated and partially saturated conditions were compared.•Removal efficiency was generally low and variable for ARGs.•Average removals of ARGs showed no statistical differences between both wetlands.•The partially saturated wetland yielded significantly lower abundances of ARGs.
Members of the phylum Bathyarchaeota and the class Thermoplasmata are widespread in marine and freshwater sediments where they have been recognized as key players in the carbon cycle. Here, we tested ...the responsiveness of archaeal communities on settled plant debris and sediment from a karstic lake to different organic carbon amendments (amino acids, plant-derived carbohydrates, and aromatics) using a lab-scale microcosm. Changes in the composition and abundance of sediment and biofilm archaeal communities in both DNA and RNA fractions were assessed by 16S rRNA gene amplicon sequencing and qPCR, respectively, after 7 and 30 days of incubation. Archaeal communities showed compositional changes in terms of alpha and beta diversity in relation to the type of carbon source (amino acids vs. plant-derived compounds), the nucleic acid fraction (DNA vs. RNA), and the incubation time (7 vs. 30 days). Distinct groups within the Bathyarchaeota (Bathy-15 and Bathy-6) and the Thermoplasmata (MBG-D) differently reacted to carbon supplements as deduced from the analysis of RNA libraries. Whereas Bathyarchaeota in biofilms showed a long-term positive response to humic acids, their counterparts in the sediment were mainly stimulated by the addition of tryptophan, suggesting the presence of different subpopulations in both habitats. Overall, our work presents an in vitro assessment of the versatility of archaea inhabiting freshwater sediments towards organic carbon and introduces settled leaf litter as a new habitat for the Bathyarchaeota and the Thermoplasmata.
Mobile genetic elements (MGEs) are key agents in the spread of antibiotic resistance genes (ARGs) across environments. Here we used metagenomics to compare the river resistome (collection of all ...ARGs) and mobilome (e.g., integrases, transposases, integron integrases and insertion sequence common region “ISCR” elements) between samples collected upstream (n = 6) and downstream (n = 6) of an urban wastewater treatment plant (UWWTP). In comparison to upstream metagenomes, downstream metagenomes showed a drastic increase in the abundance of ARGs, as well as markers of MGEs, particularly integron integrases and ISCR elements. These changes were accompanied by a concomitant prevalence of 16S rRNA gene signatures of bacteria affiliated to families encompassing well-known human and animal pathogens. Our results confirm that chronic discharges of treated wastewater severely impact the river resistome affecting not only the abundance and diversity of ARGs but also their potential spread by enriching the river mobilome in a wide variety of MGEs.
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•We used metagenomics to compare the river resistome and mobilome at sites upstream and downstream of an UWWTP.•Downstream metagenomes showed a drastic increase in the abundance of ARGs and MGEs.•UWWTP discharges promote the spread of ARGs by enriching the river mobilome in a wide variety of MGEs.
Wastewater treatment plant discharges promote the spread of antibiotic resistance genes among resident microbiota by enriching the river mobilome in a wide variety of mobile genetic elements.
Antimicrobial resistance is a growing and significant threat to global public health, requiring better understanding of the sources and mechanisms involved in its emergence and spread. We ...investigated the abundance of antibiotic resistance genes (ARGs) before and after treatment in five wastewater treatment plants (WWTPs) located in different areas of the Monastir Governorate (Tunisia). Three of these WWTPs (Frina, Sahline and Zaouiet) use a conventional activated sludge process as secondary treatment, whereas the WWTP located in Beni Hassen applies an ultraviolet disinfection step after the activated sludge process and the WWTP located in Moknine treats wastewater using naturally aerated lagoons as a secondary treatment process. The abundance of six ARGs (blaCTX-M, blaTEM, qnrA, qnrS, sul I and ermB) and the class 1 integron-integrase gene (intI1) were determined by quantitative PCR. All ARGs and the intI1 gene were detected in the wastewater samples, except the blaCTX-M gene, which was not detected in both influent and effluent samples from Sahline and Beni Hassen WWTPs, and the qnrS gene, which was not detected neither in the WWTP influent in Moknine nor in the WWTP effluent in Beni Hassen. Although the relative concentration of ARGs was generally found to be similar between samples collected before and after the wastewater treatment, the abundance of blaCTX-M, blaTEM, and qnrS genes was higher in the effluent of the Frina WWTP which, unlike other WWTPs, not only receives domestic or industrial sewage but also untreated hospital waste. To the best of our knowledge, this study quantified for the first time the abundance of ARGs in different Tunisian WWTPs, and the results agree with previous studies suggesting that conventional wastewater treatment does not efficiently reduce ARGs. Therefore, these findings could be useful to improve the design or operation of WWTPs.
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•Abundance of antibiotic resistance genes before and after treatment in five WWTPs was monitored.•Conventional wastewater treatment does not efficiently reduce resistance genes.•In some cases, the abundance of resistance genes was higher in the WWTP effluents.
Wastewater effluents increase the nutrient load of receiving streams while introducing a myriad of anthropogenic chemical pollutants that challenge the resident aquatic (micro)biota. Disentangling ...the effects of both kind of stressors and their potential interaction on the dissemination of antibiotic resistance genes in bacterial communities requires highly controlled manipulative experiments. In this work, we investigated the effects of a combined regime of nutrients (at low, medium and high concentrations) and a mixture of emerging contaminants (ciprofloxacin, erythromycin, sulfamethoxazole, diclofenac, and methylparaben) on the bacterial composition, abundance and antibiotic resistance profile of biofilms grown in artificial streams. In particular, we investigated the effect of this combined stress on genes encoding resistance to ciprofloxacin (qnrS), erythromycin (ermB), sulfamethoxazole (sul1 and sul2) as well as the class 1 integron-integrase gene (intI1). Only genes conferring resistance to sulfonamides (sul1 and sul2) and intI1 gene were detected in all treatments during the study period. Besides, bacterial communities exposed to emerging contaminants showed higher copy numbers of sul1 and intI1 genes than those not exposed, whereas nutrient amendments did not affect their abundance. However, bacterial communities exposed to both emerging contaminants and a high nutrient concentration (1, 25 and 1 mg L−1 of phosphate, nitrate and ammonium, respectively) showed the highest increase on the abundance of sul1 and intI1 genes thus suggesting a factors synergistic effect of both stressors. Since none of the treatments caused a significant change on the composition of bacterial communities, the enrichment of sul1 and intI1 genes within the community was caused by their dissemination under the combined pressure exerted by nutrients and emerging contaminants. To the best of our knowledge, this is the first study demonstrating the contribution of nutrients on the maintenance and spread of antibiotic resistance genes in streambed biofilms under controlled conditions. Our results also highlight that nutrients could enhance the effect of emerging contaminants on the dissemination of antibiotic resistance.
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•Emerging contaminants favor the spread of genes intI1 and sul1 in streambed biofilms.•Emerging contaminants and nutrients synergistically affect the spread of these genes.•Nutrients and pollutants did not change the biofilm bacterial composition.
Wastewater transport along sewers favors the colonization of inner pipe surfaces by wastewater-derived microorganisms that grow forming biofilms. These biofilms are composed of rich and diverse ...microbial communities that are continuously exposed to antibiotic residues and antibiotic resistant bacteria (ARB) from urban wastewater. Sewer biofilms thus appear as an optimal habitat for the dispersal and accumulation of antibiotic resistance genes (ARGs). In this study, the concentration of antibiotics, integron (intI1) and antibiotic resistance genes (qnrS, sul1, sul2, blaTEM, blaKPC, ermB, tetM and tetW), and potential bacterial pathogens were analyzed in wastewater and biofilm samples collected at the inlet and outlet sections of a pressurized sewer pipe. The most abundant ARGs detected in both wastewater and biofilm samples were sul1 and sul2 with roughly 1 resistance gene for each 10 copies of 16s RNA gene. Significant differences in the relative abundance of gene intI1 and genes conferring resistance to fluoroquinolones (qnrS), sulfonamides (sul1 and sul2) and betalactams (blaTEM) were only measured between inlet and outlet biofilm samples. Composition of bacterial communities also showed spatial differences in biofilms and a higher prevalence of Operational Taxonomic Units (OTUs) with high sequence identity (>98%) to well-known human pathogens was observed in biofilms collected at the inlet pipe section. Our study highlights the role of sewer biofilms as source and sink of ARB and ARGs and supports the idea that community composition rather than antibiotic concentration is the main factor driving the diversity of the sewage resistome.
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•Occurrence of antibiotic compounds in sewer biofilm and wastewater has been shown.•ARGs linked to the main antibiotic families were detected in sewage and sewer biofilm.•Sewers are likely hotspots for antibiotic resistance accumulation and spread.
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