Browsing by Author "Cytryn, Eddie"
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- Antibiotic resistance genes in treated wastewater and in the receiving water bodies: a pan-European survey of urban settingsPublication . Cacace, Damiano; Fatta-Kassinos, Despo; Manaia, Célia M.; Cytryn, Eddie; Kreuzinger, Norbert; Rizzo, Luigi; Karaolia, Popi; Schwartz, Thomas; Alexander, Johannes; Merlin, Christophe; Garelick, Hemda; Schmitt, Heike; Vries, Daisy de; Schwermer, Carsten U.; Meric, Sureyya; Ozkal, Can Burak; Pons, Marie-Noelle; Kneis, David; Berendonk, Thomas U.There is increasing public concern regarding the fate of antibiotic resistance genes (ARGs) during wastewater treatment, their persistence during the treatment process and their potential impacts on the receiving water bodies. In this study, we used quantitative PCR (qPCR) to determine the abundance of nine ARGs and a class 1 integron associated integrase gene in 16 wastewater treatment plant (WWTP) effluents from ten different European countries. In order to assess the impact on the receiving water bodies, gene abundances in the latter were also analysed. Six out of the nine ARGs analysed were detected in all effluent and river water samples. Among the quantified genes, intI1 and sul1 were the most abundant. Our results demonstrate that European WWTP contribute to the enrichment of the resistome in the receiving water bodies with the particular impact being dependent on the effluent load and local hydrological conditions. The ARGs concentrations in WWTP effluents were found to be inversely correlated to the number of implemented biological treatment steps, indicating a possible option for WWTP management. Furthermore, this study has identified blaOXA-58 as a possible resistance gene for future studies investigating the impact of WWTPs on their receiving water.
- Biomarkers for monitoring antibiotic resistance in aquatic environmentsPublication . Teixeira, Ana Margarida Ribeiro; Vaz-Moreira, Ivone; Calderón-Franco, David; Weissbrodt, David Gregory; Purkrtova, Sabina; Gajdos, S.; Dottorini, G.; Nielsen, Per Halkjær; Khalifa, Leron; Cytryn, Eddie; Bartacek, Jan; Manaia, CéliaThe occurrence of antimicrobial resistance raises concerns as a human health threat that can be propagated through the environment. Wastewater discharge into the environment is an important source for antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Sewage collection and urban wastewater treatment plants (UWTPs) are major barriers that reduce environmental contamination by sewage-derived pathogens and nutrients. However, the continuous discharge of ARB and ARGs through wastewater, including when well-functioning UWTPs are available, is unavoidable. Regular and integrated antibiotic resistance monitoring in both wastewater and receiving water bodies would contribute to improve control measures. However, monitoring processes are not harmonized being the choice of suitable biomarkers a first limitation. In this study, we tested 10 selected potential antibiotic resistance biomarkers, which have been described has being associated to humans, and rare in clean environments - intI1, sul1, ermB, ermF, aph(3’’)-Ib, qacEΔ1, uidA, mefC, tetX and crAssphage. The public database MGnify (https://www.ebi.ac.uk/metagenomics/; hosted by EMBL-EBI), was screened using the filters corresponding to origin - human gut, wastewater, sewage, and fresh water. These biomarkers and the 16S rRNA gene were monitored by quantitative PCR (qPCR) tested in raw wastewater, activated sludge, treated wastewater and surface water (upstream and downstream the UWTP) samples, collected from different countries (Portugal, Czech Republic, Denmark, The Netherlands, and Israel). The abundance of the 10 potential biomarkers decreased on average by up to 2.5 log-units gene copies/mL of sample from raw wastewater to surface water, due to treatment and/or dilution in surface water. A clustering analysis of samples based on biomarkers abundance, grouped the samples according to the (waste)water type. This classification was confirmed when 12 anonymous (waste)water samples were analysed in a blind test. The tested biomarkers were observed to differentiate different types of sample, permitting the assessment of wastewater treatment efficiency or of impacts of UWTPs discharge or others in aquatic environments. The selection of suitable biomarkers that can typify different water sources and levels of ARG contamination, along with harmonized qPCR procedures, can facilitate regular and integrated legal requirements to antibiotic resistance monitoring in wastewater and related aquatic environments.
- Biomarkers for monitoring antibiotic resistance in aquatic environmentsPublication . Teixeira, A. Margarida; Vaz-Moreira, Ivone; Franco, David Calderón; Weissbrodt, David; Purkrtova, Sabina; Gajdos, Stanislav; Dottorini, Giulia; Nielsen, Per Halkjær; Khalifa, Leron; Cytryn, Eddie; Bartacek, Jan; Manaia, Célia M.
- Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environmentPublication . Teixeira, A. Margarida; Vaz-Moreira, Ivone; Calderón-Franco, David; Weissbrodt, David; Purkrtova, Sabina; Gajdos, Stanislav; Dottorini, Giulia; Nielsen, Per Halkjær; Khalifa, Leron; Cytryn, Eddie; Bartacek, Jan; Manaia, Célia M.Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3′’)-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.
- Every fifth published metagenome is not available to sciencePublication . Eckert, Ester M.; Cesare, Andrea Di; Fontaneto, Diego; Berendonk, Thomas U.; Bürgmann, Helmut; Cytryn, Eddie; Fatta-Kassinos, Despo; Franzetti, Andrea; Larsson, D. G. Joakim; Manaia, Célia M.; Pruden, Amy; Singer, Andrew C.; Udikovic-Kolic, Nikolina; Corno, GianlucaHave 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.
- A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plantsPublication . Marano, Roberto B.M.; Fernandes, Telma; Manaia, Célia M.; Nunes, Olga; Morrison, Donald; Berendonk, Thomas U.; Kreuzinger, Norbert; Tenson, Tanel; Corno, Gianluca; Fatta-Kassinos, Despo; Merlin, Christophe; Topp, Edward; Henn, Leonie; Scott, Andrew; Heß, Stefanie; Slipko, Katarzyna; Laht, Mailis; Kisand, Veljo; Cesare, Andrea Di; Karaolia, Popi; Michael, Stella G.; Petre, Alice L.; Rosal, Roberto; Pruden, Amy; Riquelme, Virginia; Agüera, Ana; Esteban, Belen; Luczkiewicz, Aneta; Kalinowska, Agnieszka; Leonard, Anne; Gaze, William H.; Adegoke, Anthony A.; Stenstrom, Thor A.; Pollice, Alfieri; Salerno, Carlo; Schwermer, Carsten U.; Krzeminski, Pawel; Guilloteau, Hélène; Donner, Erica; Drigo, Barbara; Libralato, Giovanni; Guida, Marco; Bürgmann, Helmut; Beck, Karin; Garelick, Hemda; Tacão, Marta; Henriques, Isabel; Martínez-Alcalá, Isabel; Guillén-Navarro, Jose M.; Popowska, Magdalena; Piotrowska, Marta; Quintela-Baluja, Marcos; Bunce, Joshua T.; Polo-López, Maria I.; Nahim–Granados, Samira; Pons, Marie-Noëlle; Milakovic, Milena; Udikovic-Kolic, Nikolina; Ory, Jérôme; Ousmane, Traore; Caballero, Pilar; Oliver, Antoni; Rodriguez-Mozaz, Sara; Balcazar, Jose L.; Jäger, Thomas; Schwartz, Thomas; Yang, Ying; Zou, Shichun; Lee, Yunho; Yoon, Younggun; Herzog, Bastian; Mayrhofer, Heidrun; Prakash, Om; Nimonkar, Yogesh; Heath, Ester; Baraniak, Anna; Abreu-Silva, Joana; Choudhury, Manika; P. Munoz, Leonardo; Krizanovic, Stela; Brunetti, Gianluca; Maile-Moskowitz, Ayella; Brown, Connor; Cytryn, EddieThe 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 (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.
- High throughput analysis of integron gene cassettes in wastewater environmentsPublication . Gatica, Joao; Tripathi, Vijay; Green, Stefan; Manaia, Célia M.; Berendonk, Thomas; Cacace, Damiano; Merlin, Christophe; Kreuzinger, Norbert; Schwartz, Thomas; Fatta-Kassinos, Despo; Rizzo, Luigi; Schwermer, Carsten U.; Garelick, Hemda; Jurkevitch, Edouard; Cytryn, EddieIntegrons are extensively targeted as a proxy for anthropogenic impact in the environment. We developed a novel high-throughput amplicon sequencing pipeline that enables characterization of thousands of integron gene cassette-associated reads, and applied it to acquire a comprehensive overview of gene cassette composition in effluents from wastewater treatment facilities across Europe. Between 38 100 and 172 995 reads per-sample were generated and functionally characterized by screening against nr, SEED, ARDB and β-lactamase databases. Over 75% of the reads were characterized as hypothetical, but thousands were associated with toxin-antitoxin systems, DNA repair, cell membrane function, detoxification and aminoglycoside and β-lactam resistance. Among the reads characterized as β-lactamases, the carbapenemase blaOXA was dominant in most of the effluents, except for Cyprus and Israel where blaGES was also abundant. Quantitative PCR assessment of blaOXA and blaGES genes in the European effluents revealed similar trends to those displayed in the integron amplicon sequencing pipeline described above, corroborating the robustness of this method and suggesting that these integron-associated genes may be excellent targets for source tracking of effluents in downstream environments. Further application of the above analyses revealed several order-of-magnitude reductions in effluent-associated β-lactamase genes in effluent-saturated soils, suggesting marginal persistence in the soil microbiome.
- Inter-laboratory calibration of quantitative analyses of antibiotic resistance genesPublication . Rocha, Jaqueline; Cacace, Damiano; Kampouris, Ioannis; Guilloteau, Hélène; Jäger, Thomas; Marano, Roberto B.M.; Karaolia, Popi; Manaia, Célia M.; Merlin, Christophe; Fatta-Kassinos, Despo; Cytryn, Eddie; Berendonk, Thomas U.; Schwartz, ThomasAntibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.
- Making wavesPublication . Alygizakis, Nikiforos; Ng, Kelsey; Čirka, Ľuboš; Berendonk, Thomas; Cerqueira, Francisco; Cytryn, Eddie; Deviller, Geneviève; Fortunato, Gianuario; Iakovides, Iakovos C.; Kampouris, Ioannis; Michael-Kordatou, Irene; Lai, Foon Yin; Lundy, Lian; Manaia, Celia M.; Marano, Roberto B. M.; Paulus, Gabriela K.; Piña, Benjamin; Radu, Elena; Rizzo, Luigi; Ślipko, Katarzyna; Kreuzinger, Norbert; Thomaidis, Nikolaos S.; Ugolini, Valentina; Vaz-Moreira, Ivone; Slobodnik, Jaroslav; Fatta-Kassinos, DespoWith the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.
- Microbiome and resistome profiles along a sewage-effluent-reservoir trajectory underline the role of natural attenuation in wastewater stabilization reservoirsPublication . Leão, Inês; Khalifa, Leron; Gallois, Nicolas; Vaz-Moreira, Ivone; Klümper, Uli; Youdkes, Daniel; Palmony, Shaked; Dagai, Lotan; Berendonk, Thomas U.; Merlin, Christophe; Manaia, Célia M.; Cytryn, EddieAntibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL(-1), respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs.IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater. Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs.