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- 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.
- 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.
- Antibiotic resistance in wastewater treatment plants: tackling the black boxPublication . Manaia, Célia M.; Rocha, Jaqueline; Scaccia, Nazareno; Marano, Roberto; Radu, Elena; Biancullo, Francesco; Cerqueira, Francisco; Fortunato, Gianuário; Iakovides, Iakovos C.; Zammit, Ian; Kampouris, Ioannis; Vaz-Moreira, Ivone; Nunes, Olga C.Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.